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Proceedings of the AACR Volume 58 | April 2017 Part B: Abstracts 3063­5947 TABLE OF CONTENTS

BIOINFORMATICS AND SYSTEMS BIOLOGY: SEQUENCING ANALYSIS AND ALGORITHMS MODELING AND ALGORITHMS SYSTEMS BIOLOGY APPROACHES TO CANCER DIAGNOSTICS AND DISEASE MANAGEMENT CANCER CHEMISTRY: DRUG DESIGN AND OPTIMIZATION STRATEGIES DRUG SCREENING INNOVATION CHEMICAL BIOLOGY AND STRUCTURE BIOLOGY APPROACH TO STUDY CANCERS CLINICAL RESEARCH: CLINICAL LABORATORY AND IMAGING CORRELATES INNATE EFFECTORS IN IMMUNITY TO CANCER LIQUID BIOPSIES 3: CIRCULATING TUMOR CELLS, EXTRACELLULAR VESICLES, AND EXOSOMES PREDICTIVE BIOMARKERS 3 DIAGNOSTIC BIOMARKERS EPIGENETICS AND DNA REPAIR IMMUNOMODULATORY AGENTS AND THERAPEUTICS PROGNOSTIC BIOMARKERS 1 RADIOBIOLOGY AND RADIOTHERAPY LIQUID BIOPSIES 4 CLINICAL AND TRANSLATIONAL ENDOCRINE ONCOLOGY INNATE IMMUNITY TO GENERATE ADAPTIVE IMMUNITY

LIQUID BIOPSIES 5: CFDNA, MICRORNA, AND PROTEIN PROGNOSTIC BIOMARKERS 2 ENDOCRINOLOGY: NUCLEAR RECEPTORS AND ENDOCRINE ONCOLOGY THERAPIES EPIDEMIOLOGY: SURVIVORSHIP RESEARCH BIOMARKERS OF ENDOGENOUS OR EXOGENOUS EXPOSURES, EARLY DETECTION, AND BIOLOGIC EFFECTS FAMILIAL AND HEREDITARY CANCERS MOLECULAR AND GENETIC EPIDEMIOLOGY HEALTH DISPARITIES, SCREENING, AND RISK PREDICTION NUTRITION AND ANTHROPOMETRIC FEATURES EXPERIMENTAL AND MOLECULAR THERAPEUTICS: DETERMINANTS OF DRUG SENSITIVITY AND RESISTANCE DIFFERENTIATION THERAPY DRUG DELIVERY TECHNOLOGY AND ANTIBODY TECHNOLOGY MOLECULAR MEDICINE MOLECULAR TARGETED THERAPIES 1 NOVEL MECHANISMS 1 NOVEL MOLECULAR TARGETS 2 ASSAY TECHNOLOGY COMBINATION THERAPIES AND APPROACHES TO SENSITIZE CANCER CELLS TO DRUGS MECHANISTIC UNDERSTANDING OF NOVEL ANTICANCER THERAPIES MOLECULAR TARGETED THERAPIES 2 NEW TARGETS AND NEW DRUGS NOVEL MECHANISMS 2 TARGETING PROTEIN KINASES AND DNA REPAIR NOVEL APPROACHES FOR EXPERIMENTAL THERAPEUTICS NOVEL SMALL MOLECULAR TARGETS AND APPROACHES ANTICANCER PRECISION CLINICAL PHARMACOLOGY

EPIGENETICAGENTS GENE­AND VECTOR­BASED THERAPY NOVEL CHEMOTHERAPIES NOVEL ONCOGENES DRUG DELIVERY TECHNOLOGY PRECLINICAL ANDTUMOR SUPPRESSORS AS THERAPEUTIC TARGETS RADIOTHERAPEUTICS TRANSPORT AND METABOLISM IMMUNOLOGY: BITES DENDRITIC BISPECIFICS AND CHECKPOINTS I CELLS AS CRITICAL IMMUNE TARGETS IMMUNOCONJUGATES AND ANTIBODIES CLINICAL NFLAMMATION IN THE TUMOR MICROENVIRONMENT I IMMUNOTHERAPY, VIRUSES, AND BACTERIA ADOPTIVE NNATE IMMUNE RESPONSE TO CANCER ENZYMES CELLULAR THERAPY FOR CANCER I AND HORMONES AND METABOLISM IN TUMOR IMMUNITY MMUNE CHECKPOINTS AND IMMUNOSURVEILLANCE MOLECULAR AND CELLULAR BIOLOGY / GENETICS: AUTOPHAGY AND CANCER CELL GROWTH SIGNALING PATHWAYS 6 EPIGENETICS3 THYROIDCANCERS GENOMICLANDSCAPE OF BREAST, PROSTATE, OVARIAN, MELANOMA, AND M GENOTYPE­PHENOTYPE ASSOCIATIONS ONCOGENES ICRORNAS AND OTHER NONCODING RNAS AS TUMOR SUPPRESSORS OR 2 MITOSIS,TELOMERES, AND PROLIFERATION CONTROL NONCODING RNAS AND CANCER: EXPRESSION, FUNCTION, AND THERAPEUTICS ONCOGENIC TRANSCRIPTION FACTORS REGULATION CELLDEATHINCANCER OF METABOLIC PATHWAYS IN CANCER

CELL GROWTH SIGNALING PATHWAYS 7 EPIGENETICS GENOMIC LANDSCAPE METABOLIC 4 OF HEAD AND NECK AND GI CANCERS M REGULATION AND CANCER THERAPY 1 ONCOGENES ICRORNA PROFILING IN CANCER POST­TRANSCRIPTIONAL AND TUMOR SUPPRESSORS 2 T AND TRANSLATIONAL REGULATION IN CANCER EXPLOITING UMOR HYPOXIA AND THE UNFOLDED PROTEIN RESPONSE TRANSCRIPTIONAL METABOLIC VULNERABILITIES TO TREATCANCER CELL REGULATION IN CANCER CELLS DEVELOPMENT GROWTH SIGNALING PATHWAYS 8 EPIGENETICS OF HIGH THROUGHPUT ANALYTIC METHODS METABOLIC GENOMIC ANALYSES 5 OF CIRCULATING TUMOR MATERIAL AND RNA MICRORNAREGULATION AND CANCER THERAPY 2 OXIDATIVE DIAGNOSTICS AND THERAPEUTICS IN CANCER REGULATION STRESS,AUTOPHAGY, AND SENESCENCE TUMORSUPPRESSORS2 OF ONCOGENIC TRANSCRIPTION PREVENTION RESEARCH: STRATEGIES POPULATION AND BIOMARKERS FOR CLINICAL PREVENTION BIOMARKERS SCIENCE IN CANCER PREVENTION RESEARCH CHEMOPREVENTION AND INTERVENTION STUDIES AND CANCER TUMOR BIOLOGY: CELL PEDIATRIC CULTURE AND ANIMAL MODELS OF CANCER 4 TARGETINGCANCER 2: EPIGENETICS AND STEM CELLS T CANCER STEM CELLS TUMOR CELL DISSEMINATION AND THE PREMETASTATIC NICHE TUMOR EVOLUTION AND HETEROGENEITY 2 TUMOR MICROENVIRONMENT 5 UMOR MICROENVIRONMENT 6

CELLS CARCINOGENESIS, CANCERSTEM CELL MODELS, ANDTUMOR BIOLOGY CULTURE AND ANIMAL MODELS OF CANCER 5 METASTASIS SUPPRESSORS AND PROMOTERS PEDIATRIC CANCER 3: GENETICS AND GENOMICS THERAPEUTIC INTERVENTION OF CANCER AND METASTASIS TUMOR MICROENVIRONMENT 7 MICROENVIRONMENTAL MODELS CUES IN IMMUNE ESCAPE AND THERAPY RESISTANCE T FOR TREATMENT RESISTANCE AND DRUG DISCOVERY UMOR STEM CELL BIOLOGY CARCINOGENESIS AND HUMAN TUMORS CELL EFFECTS CULTURE AND ANIMAL MODELS OF CANCER 6 PEDIATRICOF TUMOR­MICROENVIRONMENT CROSSTALK ON METASTASIS CANCER 4: IMMUNOLOGY, SIGNALING, AND THE TUMOR MICROENVIRONMENT RADIOPROTECTORS, T RADIOSENSITIZERS, AND RADIATION RESISTANCE UMOR CELL ADHESION AND DRUG RESISTANCE TUMOR MICROENVIRONMENT 8 TUMOR MICROENVIRONMENT 9 RECENT TRENDS IN REGULATORY SCIENCE

Tuesday, April 4, 2017 EXPERIMENTAL AND MOLECULAR THERAPEUTICS: Determinants of Drug Sensitivity and Resistance #3063 Predicting efficacy of c­Met targeting therapy in autocrine and paracrine tumor cell lines. Veronica S. Hughes, Dietmar W. Siemann. Univ. of Florida, Gainesville, FL. Tumor cells frequently harbor abnormalities in signaling pathways, leading to increased migration, invasion, survival, angiogenesis, and proliferation. C­Met is one such commonly aberrant pathway. Because of its effects on tumor cells, c­Met has been a target of interest for anti­ metastatic therapies. Clinical trials of c­Met inhibition select patients based on total Met expression, or amplification of the Met gene. However, c­Met inhibitors target the activated pathway. We therefore predict that phospho­Met will be a better marker for patient sensitivity to, and hence selection for, c­Met inhibitor trials. C­Met is activated by the endogenous ligand Hepatocyte Growth Factor (HGF). In order to mimic the paracrine signaling during in vitro experiments, many studies add exogenous HGF at concentrations of 25­50 ng/mL. However, these concentrations may not be representative of in vivo conditions. HGF serum levels of healthy humans is 0.4 ng/mL. While HGF levels do rise in cancer patients, it is unusual to see serum levels higher than 1 ng/mL. Conditioned media from several cell lines (MDA­MB­231, PC­3, DU145, LNCaP, OS156, OS521, U87, U118, RKO, KHT, SCC7, and RIF) was collected to analyze for secretion of HGF in conditioned media. These cells were then examined for basal phosphorylation of Met. The cell lines that secreted HGF also had higher levels of phospho­Met in comparison to non­HGF­secreting cell lines. Importantly, levels of total Met did not predict for levels of phospho­Met. In migration and invasion assays, the HGF­secreting cell line KHT was found to be sensitive to the c­Met inhibitor BMS­777607.The non­HGF­secreting cell line DU145 only became sensitive to the inhibitor when high, non­physiologic exogenous

levels of HGF (25­50 ng/mL) were added.These results suggest that autocrine activation of the c­Met pathway may be a factor in predicting sensitivity to c­Met inhibition. This may indicate the existence of a subset of patients most likely respond to c­Met inhibition therapy. Furthermore, consideration of exogenous HGF concentration should be considered when conducting preclinical trials of c­Met inhibitors, as addition of exogenous HGF may change the apparent efficacy of the inhibitor. #3064 Retinoic acid: An effective therapy for basal­like breast cancer. Krysta Mila Coyle, Cheryl A. Dean, Dejan Vidovic, Ian C. Weaver, Carman A. Giacomantonio, Paola Marcato. Dalhousie Univ., Halifax, Nova Scotia, Canada. We have identified a novel strategy to identify breast cancer patients who will benefit from an existing anti­cancer agent, retinoic acid (RA). While RA has not yet achieved success in the treatment of breast cancers, we hypothesized that it can be an effective therapy for a subset of triple­ negative breast cancer (TNBC) patients. TNBC is among the most aggressive breast cancers, and lacks targeted therapies. TNBCs can be further subtyped into basal­like and claudin­low, which differ in gene expression and drug sensitivities. Understanding the molecular basis of these subtypes will lead to the development of more effective treatment options for TNBC. To test this hypothesis, we performed tumor growth assays on TNBC cell lines and patient­derived xenografts (PDXs). We found that RA treatment decreased the tumor growth of four basal­like TNBC cell lines (MDA­MB­468, HCC70, SUM149, HCC1937). In contrast, RA increased the tumor growth of two claudin­low TNBC cell lines (MDA­MB­231, MDA­MB­436). Gene expression and methylation analysis of these affected cell lines revealed subtype­specific expression of RA­inducible genes due to silencing by DNA methylation, e.g. of the RA­inducible tumor­suppressor gene RARRES1. RARRES1 is silenced by methylation in claudin­low cell lines, but is hypomethylated and expressed in basal­like cells. Use of the subtype­specific expression and methylation profiles allowed us to accurately predict the response of 4 PDXs to RA treatment. Continued classification of TNBCs into these two subtypes will enable clinical use of RA, in part due to the subtype­ specific hypomethylation of RA­inducible tumor suppressor genes including RARRES1. We have identified additional subtype­specific biomarkers which can predict the response of patient tumors to RA

treatment, thus identifying a novel targeted therapy strategy for TNBCs. #3065 High throughput screen identifies HSP90 as a potential therapeutic target in models of advanced PCa, with notable efficacy in the absence of PTEN and p53. Keith H. Jansson,1 John K. Simmons,1 Caitlyn Fuller,1 John Tucker,1 Ross Lake,1 Supreet Agarwal,1 Michael Beshiri,1 Lei Fang,1 Lauren Stahl,1 JuanJuan Yin,1 Aian Neil Alilin,1 Paul G. Hynes,1 Jacob Cawley,1 Rajarshi Guha,2 Xiaohu Zhang,2 R. Mark Simpson,1 Craig Thomas,2 David Proia,3 Kathleen Kelly1. 1National Cancer Institute, Bethesda, MD; 2NCATS, MD; 3C4 Therapeutics, Cambridge, MA. Castrate resistant prostate cancer (CRPC) remains the primary cause of mortality for prostate cancer (PCa) patients. Several therapies are currently approved for use against CRPC, consisting mostly of androgen receptor (AR) signaling inhibitors and toxic anti­microtubule chemotherapeutics that do not score long­term durable responses. Recent deep­sequencing analysis studies have elucidated the complex and heterogeneous landscape of CRPC, highlighting the necessity to develop effective targeted therapies against specific CRPC subtypes or therapies effective against numerous subtypes of CRPC. PTEN and p53 are two of the most frequently altered genes in CRPC, and are associated with therapy resistance and a poor clinical prognosis. The objective of this study was to identify and subsequently validate therapeutic targets against models of advanced PCa, specifically in the absence of PTEN and p53. Using non­biased high throughput screening technology, we identified HSP90 inhibitors to be potent and efficacious against a model of PTEN/p53 null PCa. HSP90 is a critical regulator of prostate cancer cell signaling homeostasis, and recently developed second generation compounds targeting HSP90 are effective against genotypically heterogeneous panels of cancer cell lines and have favorable safety parameters in the clinic. Screening results and subsequent validation efforts in vitro identified ganetespib as the most potent HSP90 inhibitor, with efficacy that translated into established human PCa cells lines. Ganetespib also displayed strongly inhibitory activity against PTEN/p53 null progenitor cells, a plastic subpopulation of PCa believed to be partly responsible for therapy resistance. In vivo, ganetespib completely

inhibited progression of PIN to invasive adenocarcinoma in the Pten/Tp53 null PCa mouse model, leading to a significant reduction in tumor weight. Expanding from the mouse model to clinically predictive PDX­derived LuCaP models of human PCa, we found that ganetespib displayed a range of activity against a genotypically diverse array of 11 LuCaP organoids ex vivo. Furthermore, the PTEN null/p53 altered LuCaP 136 displayed a robust response to ganetespib in vivo, with ganetespib causing a significant reduction in tumor growth. Mechanistic interrogation in vitro, ex vivo, and in vivo revealed ganetespib induced effects to be multifactorial and model specific, with unifying trends being inactivation of PI3K signaling and modulation of cell cycle regulatory proteins. In all, these data indicate that inhibition of HSP90 is a novel therapeutic to treat advanced PCa via multifactorial perturbation of growth regulatory pathways. #3066 Inhibition of hsp90 by auy922 preferentially kills mutant KRAS colon cancer cells by activating Bim through ER stress. Chun Yan Wang, Su Tang Guo, Jia Yu Wang, Xu Guang Yan, Margaret Farrelly, Yuan Yuan Zhang, Fen Liu, Hamed Yari, Ting La, Fu Xi Lei, Lei Jin, Chen Chen Jiang, Xu Dong Zhang. university of Newcastle, Newcastle, Australia. Colon cancer is one of the most common and deadly malignancies (1). Despite recent advances in early diagnosis and the development of molecularly targeted therapy, the overall survival of patients with metastatic colon cancers remains disappointing (1). This is often associated with resistance of colon cancer cells to systemic therapies resulting from oncogenic mutations of KRAS that drive activation of multiple downstream signalling pathways important for cell survival and proliferation. Here we report that mutant KRAS colon cancer cells are nevertheless more susceptible to apoptosis induced by the heat shock protein 90 (HSP90) inhibitor AUY922 than those carrying wild­type KRAS. Although AUY922 inhibited HSP90 activity with the comparable potency in colon cancer cells irrespective of their KRAS mutational statuses, those with activating mutations of KRAS were markedly more sensitive to AUY922­induced apoptosis. This was associated with upregulation of the BH3 only proteins Bim, Bik, and PUMA. However, only Bim appeared essential, in that knockdown of Bim abolished, whereas knockdown of Bik or PUMA only moderately attenuated

apoptosis induced by AUY922. Mechanistic investigations revealed that endoplasmic reticulum (ER) stress was responsible for AUY922­induced upregulation of Bim, which was inhibited by a chemical chaperone or overexpression of GRP78. Conversely, siRNA knockdown of GRP78 or XBP­1 enhanced AUY922­induced apoptosis. In addition, AUY922 inhibited the growth of mutant KRAS colon cancer xenografts through activation of Bim that was similarly associated with ER stress. Taken together, these results suggest that AUY922 is a promising drug in the treatment of mutant KRAS colon cancers, and that agents that enhance the apoptosis­inducing potential of Bim may be useful to improve the therapeutic efficacy. #3067 Galectin­3, a target for KRAS­addicted lung cancer. Laetitia Seguin, Maria F. Camargo, Hiromi I. Wettersten, Shumei Kato, Tami von Schalscha, Kathryn C. Elliott, Sara M. Weis, David A. Cheresh. UC San Diego, La Jolla, CA. KRAS­mutant cancers are notoriously therapy­resistant. While responsible for oncogenesis, mutant KRAS or the sustained activation of KRAS­driven effectors may or may not be required throughout the course of cancer progression. Thus, understanding which tumors remain dependent on oncogenic KRAS could lead to the identification of unique vulnerabilities and opportunities to treat them. We previously discovered that the carbohydrate­binding Galectin­3 (Gal­3) brings together KRAS with integrin αvβ3 at the cell membrane in epithelial cancer cells to promote tumor progression. Here, we show that by directly binding to the cell surface receptor integrin αvβ3, Gal­3 drives addiction to oncogenic KRAS by enhancing macropinocytosis and reducing mitochondrial reactive oxygen species (ROS). This pathway drives increased expression of SOD2, a superoxide dismutase that clears mitochondrial ROS to protect against cell death. Targeting Gal­3 with a clinically active drug decreases macropinocytosis and increases ROS to eradicate KRAS­ addicted lung cancer in patient­derived xenografts and spontaneous KRAS­driven lung cancer in mice. Our work reveals Gal­3 as a druggable target for KRAS­addicted lung cancer, and indicates integrin αvβ3 as a biomarker to identify this dependence. #3068 Proteotoxic stress associated with mTORC1 activation in ovarian

carcinoma: proteasome inhibition as a therapeutic strategy. M. Herman Chui, Patricia Shaw, Robert Rottapel. University of Toronto, Toronto, Ontario, Canada. Genetic profiling studies of high grade serous ovarian carcinoma have revealed recurrent alterations in the mTORC1 signalling network (e.g. mutations/copy number alterations in PTEN, TSC1, TSC2, and PIK3CA) and pathway activation, detected by phospho­4E­BP1, has been associated with poor prognosis. We sought to characterize functionally the role of mTORC1 signalling and its therapeutic implications in ovarian cancer. Treatment of ovarian cancer cell lines with rapamycin resulted in inhibition of mTORC1 signalling and decreased rate of protein synthesis. However, irrespective of PTEN mutation status, only mild cytostatic effects were achieved even with high concentrations of rapamycin. We next examined the phenotypic consequences of mTOR activation, using siRNA directed against TSC2. Surprisingly, we observed striking growth inhibition in the majority of ovarian cancer cell lines, whether grown under adherent monolayer culture or in 3­dimensional spheroid culture conditions. While mTORC1 pathway activation was confirmed biochemically, knockdown of TSC2 also resulted in activation of the unfolded protein response (UPR), with elevated levels of phospho­EIF2α and ATF4, consistent with the accumulation of misfolded proteins in the endoplasmic reticulum. From a therapeutic standpoint, the resulting burden on the ubiquitin­ proteasome system should render these cells particularly sensitive to proteasome inhibition. We show that treatment with the proteasome inhibitor, bortezomib, causes increased accumulation of detergent­ insoluble poly­ubiquinated proteins and formation of larger and more abundant cytoplasmic protein aggregates in siTSC2­transfected compared to scrambled siRNA­transfected ovarian carcinoma cells. This was accompanied by a more pronounced UPR stress response, including induction of pro­apoptotic CHOP, and suppression of autophagy, resulting in marked cytotoxicity. Conversely, we show that inhibition of protein synthesis by cycloheximide renders tumor cells resistant to bortezomib. Increased resistance to bortezomib was also noted when cells were grown as spheroids, a condition associated with suppression of mTORC1 signalling and decreased protein synthesis. This resistant phenotype of tumour spheroids however was ameliorated with TSC2 knockdown. Our findings demonstrate that protein homeostasis is finely­ tuned in ovarian cancer and that mutations in mTORC1 pathway

components do not necessarily imply “oncogene addiction”. In early­ stage clinical trials, bortezomib has achieved notable responses in a few patients. Ovarian carcinomas with genetic alterations causing increased mTORC1 signalling may be particularly amenable to treatment with proteasome inhibitors. #3069 The S­phase checkpoint function of MGMT provides an unexpected rational route to profoundly increase the efficacy of anticancer antimetabolites in cell culture and xenograft models. AGM Mostofa, Debasish Basak, Hanumantha Madala, Surendra R. Punganuru, Kalkunte S. Srivenugopal. Texas Tech Univ. Health Sciences Ctr., Amarillo, TX. MGMT is an anti­mutagenic DNA repair protein highly expressed not only in brain tumors, but also breast cancers, melanoma and colon cancers, and confers drug resistance. Recently, we showed that human MGMT has non­repair functions with vital roles in cell cycle; MGMT underwent PCNA­dependent degradation in the S­phase along with p21cip1 and CDT1 proteins. MGMT inactivation by O6­benzylguanine (BG) or a pool of shRNAs in asynchronous or G1/S­phase synchronized cells led to the activation of a checkpoint resulting in a significant inhibition of DNA synthesis and greatly diminished progression of cells into S­phase. We hypothesized that the replication stress caused by MGMT inactivation will provide a means to combine the S­phase specific drugs for synergistic cytotoxicity. We screened the sensitivity of a panel of colon cancer and glioblastoma cell lines with this combination using MTT and clonogenic growth assays. Annexin V/PI and acridine orange/ EtBr cell stainings were used to quantify apoptosis. MGMT inhibition/depletion significantly reduced the IC50 (5 to 10­fold) for different antimetabolites (5­FU, gemcitabine, Ara­C) and enhanced the overall apoptotic cell death (>5­fold). Nevertheless, the synergistic effects strongly depended on time and administration schedule of these two agents. Thus, a pretreatment of BG or MGMT shRNA followed by 5­FU for or a simultaneous treatment of the two were far less cytotoxic than the antimetabolite first followed by BG or shRNA as in the post­treatment mode. While the pre or simultaneous MGMT inhibition significantly prolonged the antimetabolite­induced S­phase arrest, the BG­post­ treatments resulted in the abrogation of antimetabolite­induced cell cycle block and rapid progression into premature mitosis and apoptosis. We

observed an increased activation of checkpoint kinases (pChk1 or pChk2) during pre or simultaneous treatments. In contrast, the post­MGMT inhibition interfered with S­phase checkpoint activity and had an abnormal CDK2 activation. Finally, we verified the therapeutic potentiation of antimetabolites by BG using the HT29­luc2 subcutaneous xenografts developed in nude mice. BG (60mg/Kg), 5­FU (30 mg/Kg), gemcitabine (100 mg/Kg) and Ara­C (40 mg/Kg/dose) were administered through i.p. route once a week for 7 weeks. Tumor growth revealed a 50 to 70% more reduction in combination groups (antimetabolites+BG) compared with drugs alone. No significant changes in mouse body weight, liver functions, and whole blood counts were noted. A marked downregulation of ki­67 and increased levels of apoptotic markers (cleaved caspase­3, cleaved PARP) in was evident in tumor tissues. Collectively, our data suggest that MGMT inhibition is a rational strategy not only for alkylating agents but also for antimetabolites as well (supported by CPRIT RP130266 & RP170207 grants to KSS). #3070 Lenalidomide and SMAC mimetic LCL161 show combination activity in cells harboring loss of chromosome 5q. Nafeeza Hafeez, Alex Gaither, Dale Porter. Novartis, Cambridge, MA. LCL161 is a small molecule SMAC mimetic that binds to cIAP1, cIAP2, and XIAP and sensitizes cancer cells to TNF­alpha as well as other inducers of cell death. An siRNA screen was performed in the SK­OV­3 cancer cell line to identify genes that when knocked down prevent LCL161­mediated cell death. As expected, siRNAs targeting RIPK1 rescued cells from LCL161­induced death. Unexpectedly, siRNAs targeting GPR68 also rescued cells from LCL161­induced death. Since knockdown of GPR68 has been reported to rescue myelodysplastic syndrome (MDS)­derived cells harboring loss of chromosome 5q from lenalidomide treatment, we tested for combination activity with lenalidomide plus LCL161 in 7­day in vitro proliferation assays. Interestingly, combination activity was observed in the SET­2 cell line harboring cytogenetic features of MDS including loss of chromosome 5q. These data suggest that, in combination, LCL161 may improve responses to lenalidomide in subsets of MPN and MDS patients and potentially additional contexts where lenalidomide is active. #3071

Exploring the molecular mechanism underlying SETD2­PI3Kβ synthetic­lethal interaction in renal cell carcinoma (RCC). Esteban Terzo,1 Jeanne Chiang,2 W. Kimryn Rathmell1. 1Vanderbilt University Medical Center, Nashville, TN; 2University of North Carolina, Chapel Hill, NC. Epigenetic regulators are emerging as major drivers of renal cell carcinoma (RCC). SETD2, a tumor suppressor commonly mutated in RCC, is the sole enzyme responsible for tri­methylating histone H3 on lysine 36 (H3K36me3) of actively transcribed genes. We recently discovered that SETD2 also trimethylates the non­histone target α­tubulin on lysine 40 (α­TubK40Me3) of mitotic microtubules. We have demonstrated that mutations altering SETD2’s ability to trimethylate H3K36 and α­TubK40 are associated with genomic instability in cancer progression. We hypothesized that defective SETD2 methylating activity gives rise to deleterious cell signaling cascades that promote the tumorigenic features of SETD2 loss. Preliminary data show that RCC­ derived 786­0 cells that are deleted for SETD2 (SETD2­KO) or harbor pathogenic mutations are more sensitive to treatment with the PI3Kβ­ specific inhibitor TGX­221 than their wild type counterparts. Phosphatidylinositol 3kinases (PI3Ks) are a family of lipid kinases that coordinate signals from growth factors, cytokines and other environmental cues, translating them into intracellular signals controlling diverse signaling pathways. Several members of the PI3K/AKT signaling pathway, including the PI3K α and β isoforms, are frequently mutated in a variety of cancers, making this pathway a prime drug target for anti­ cancer therapy. These pathways control numerous biological processes, such as cell proliferation, growth, and motility. Examination of structurally variant SETD2 mutants suggests that the loss of SETD2 methylating activity mediates this interaction, and future studies will examine the functional consequences of inhibiting this pathway. In summary, these data suggest a synthetic­lethal type of interaction between SETD2 loss (and specifically loss of methylating activity) and an aberrant PI3K/AKT pathway. #3072 A clinical pharmacodynamic biomarker assay that distinguishes potentially repairable, cytotoxic drug­induced DNA double strand breaks (DSBs) from DSBs associated with apoptotic cell death.

Angie B. Dull,1 Deborah Wilsker,1 Robert J. Kinders,1 Ralph E. Parchment,1 David Evans,1 Beverly A. Teicher,2 James H. Doroshow2. 1Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD; 2National Cancer Institute, Bethesda, MD. The effectiveness of certain classes of cytotoxic cancer therapeutics likely depends on whether drug­induced DNA damage is successfully repaired or not, with the latter situation leading to mutations and strand breaks. However, double strand breaks (DSBs) also occur independently of genotoxic insults during apoptotic cell death caused by many drug classes, as well as natural biological processes. We have developed an immunofluorescent confocal microscopy assay that uses a biomarker profile suitable for individual cell analysis designed to distinguish between DSBs caused by apoptosis and those caused by direct DNA damage from cytotoxic drug action. γ­H2AX is an established biomarker for DSBs and activated cleaved caspase 3 is an executioner caspase important for apoptosis, which leads to nuclear condensation, DNA fragmentation, plasma membrane blebbing, and subsequent cell death. Our assay defines the DSBs of apoptosis by co­localized γ­H2AX and cleaved caspase 3 in individual cells, while defining the DSBs from early drug effects of DNA damaging chemotherapeutics by γ­H2AX induction in the absence of cleaved caspase 3. Building on our published findings that topotecan strongly induces γ­H2AX and DSBs within 1­4 hours in vitro and in vivo, we observed exposure­dependent increases in γ­H2AX /cleaved caspase 3 double positive cells at later time points, both in an HT29 in vitro spheroid model and an MDA­MB­231 xenograft model. Fit­for­purpose studies in the MDA­MB­231 xenograft model treated with birinipant, a SMAC mimetic and IAP deregulator that does not directly produce lethal DSBs, demonstrated a dose­dependent increase in cellular co­localization of γ­H2AX/cleaved caspase 3 consistent with birinipant induced apoptosis and the established mechanism of action of this compound. Clinical feasibility was established in a canine clinical trial using formalin­fixed paraffin­embedded (FFPE) 18­gauge needle biopsies: two novel indenoisoquinolines, indotecan (LMP400) and indimitecan (LMP776), increased tumor cell co­localization of γ­ H2AX/cleaved caspase 3 in tumor samples obtained on day 5 of qdx5 treatment. This PD biomarker assay of early and late DSB response to drug exposure could have important applications for elucidation of

mechanisms of action of anticancer drugs and the development of investigational agents. Funded by NCI Contract No. HHSN261200800001E. #3073 Potential predictive biomarkers of clinical responses for a novel CDC7­selective inhibitor TAK­931. Kenichi Iwai,1 Tadahiro Nambu,1 Osamu Kurasawa,1 Noriko Uchiyama,1 Ryo Dairiki,1 Yukiko Yamamoto,1 Satoru Nishizawa,1 Mengkun Zhang,2 Yuko Ishii,2 Huifeng Niu,2 Akihiro Ohashi1. 1Takeda Pharmaceutical Company, Fujisawa, Japan; 2Takeda Pharmaceuticals International, Cambridge, MA. Cell division cycle 7 (CDC7) is a serine/threonine kinase, which plays important roles in initiation of DNA replication by phosphorylating MCM2. Kinase activity of CDC7 is controlled by its binding protein DBF4 in a cell­cycle dependent manner. Here we developed a potent CDC7 inhibitor TAK­931 (IC50< 0.3 nM) as a cancer therapeutic drug candidate, which exhibits a time­dependent ATP­competitive kinetics to its ATP­binding pocket. The selectivity studies using the 308 kinases revealed >120­fold selectivity of TAK­931 for CDC7 kinase inhibition compared to other kinase inhibitions. Treatment with TAK­931 suppressed the cellular MCM2 phosphorylation at Ser40, resulting in a delayed S phase progression, checkpoint activation, apoptosis, and potent growth suppression in various cancer cell lines. Furthermore, oral administration of TAK­931 as a single agent caused a significant antitumor activity in multiple xenograft models which include both cell line­based xenografts and patient­derived xenograft (PDX) models. These results demonstrate that TAK­931 is a highly potent and selective inhibitor of CDC7 kinase, and causes a potent antiproliferation both in vitro and in vivo studies using various cancer cells. Next, to identify potential predictive biomarkers to guide patient­selection strategies, in vitro cell panel screening of TAK­931 using was tested for its ability to antiproliferation in 246 cell lines, which includes both solid and hematological cancer cells. TAK­931 inhibited proliferation of multiple cancer cell lines, with mean concentration producing a half­maximal response (EC50) values ranging from 29.1 nM to > 30 μM (median = 554.5 nM). While the wide range of TAK­931 antiproliferative spectrum was observed, neither doubling speed nor CDC7 expression profile did

predict tTAK­931 sensitivity in cancer cell lines. A correlative study of the tumor genetic mutations in relation to antiproliferative activity that KRAS mutant cancer cells were more sensitive to TAK­931 compared to KRAS non­mutant cell lines (p60% TGI. Our findings suggest that the KRAS­mutant pancreatic tumors could be the potential candidate for the TAK­931 target indication. #3074 Selective lethality of cisplatin in pancreatic cancer is dependent on mitotic functions of BRCA2. Barbara Orelli, Hans C. Maurer, Carmine Palermo, Steven A. Sastra, Kenneth P. Olive. Columbia Univ. Irving Comp. Cancer Ctr., New York, NY. Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy with a 5­year survival rate of just 8%. Mutations in BRCA2 are among the most common known germline alterations that predispose to PDA. While the sensitivity of BRCA2­mutant breast and ovarian tumors to platinum compounds has long been recognized, the recent approval a PARP inhibitor in BRCA2­mutant ovarian cancer confers hope that such tumors might be targeted with greatly reduced toxicity. To test whether PDA exhibits a similar genetic susceptibility to these agents, we carried out preclinical intervention studies in Kras/p53­mutant genetically engineered mouse models (KPC mice) with or without the additional mutation of BRCA2 (KPCB2 mice). Mice were enrolled based on 3D ultrasound imaging criteria to assure similar initial tumor burden, and tumor growth was monitored longitudinally by ultrasound. To our surprise, loss of Brca2 failed to sensitize pancreatic tumors to Parp inhibition and did not provide additional benefit in combination with cisplatin treatment. By contrast, cisplatin induced substantial regressions

in most KPCB2 tumors, leading to a >3­fold increase in overall survival, while having no effect on the growth or survival of mice with wild­type BRCA2. More surprisingly, the efficacy of cisplatin was not shared with oxaliplatin, even at higher doses. While BRCA2 is primarily known for its role in homology­directed repair, a network biology analysis of the function of BRCA2 in human pancreatic cancer led us to investigate the role of BRCA2 in mitosis. Wild type BRCA2 protein localizes to the midbody­ a structure formed during the final stages of telophase­ and plays and poorly understood role in the final abscission of cytoplasm between two dividing cells. Timelapse video microscopy of KPCB2 tumor cells treated once weekly for 2 hours with cisplatin revealed the frequent induction of endoreduplication and eventual formation of polyploid giant cells subsequent death. Similar cells were found throughout KPCB2 tumors during regression in response to cisplatin treatment, but were largely absent from tumors in other treatment arms. Mechanism studies found that BRCA2 deficient PDA cells are susceptible to replicative stress following cisplatin treatment that selectively leads to the formation of radial chromosomes that cannot be resolved during mitosis. These studies argue for the consideration of cisplatin­based (rather than oxaliplatin­based) regimens for the treatment of BRCA2­mutant pancreatic tumors. #3075 Analysis of microRNA profiles involved in the resistance to trifluridine. Kenta Tsunekuni,1 Jun Koseki,2 Masamitsu Konno,2 Ayumu Asai,2 Norihiro Nishida,2 Hugh Colvin,2 Koichi Kawamoto,2 Yuichiro Doki,2 Masaki Mori,2 Hideshi Ishii3. 1Taiho Pharmaceutical, Tokushima, Japan; 2Osaka University, Osaka, Japan; 3Osaka University, Tokushima, Japan. Background: Trifluridine (FTD) is a key component of the novel oral antitumor drug trifluridine/tipiracil, which was approved for the treatment of patients with metastatic colorectal cancer refractory to standard chemotherapies. A comprehensive analysis of miRNA profiles was performed in cell lines resistant to FTD established by ourselves, in order to explore the underlying mechanisms of resistance to the drug. Method: We established subline resistance to FTD through continuous administration and increasing dose of the drug for 5 months using the

colorectal cancer cell line DLD­1. Total RNA was extracted at intervals whilst establishing FTD resistant sublines, and miRNA expression was analyzed by microarray. The expression of miRNA that was significantly downregulated in the FTD resistant subline was knocked down to test its involvement in resistance to FTD. Cell viability was evaluated by crystal violet cytotoxicity test. Results: The established FTD­resistant sublines showed more than 22­ fold higher resistance to FTD and no cross­resistance to 5­FU. miRNA and mRNA clustered in the genome locus located in chromosome 9, 9p22.32 were downregulated in the FTD­resistant cell line, one of which was the miRNA let­7d­5p, which is one of the let­7 family and is known to target oncogenes and several key components of the cell cycle and cell proliferation. Anti­let­7d­5p treated DLD­1 was less sensitive to FTD than compared to control. The IC50 values of FTD were 16.8 µM and 7.6 µM for the anti­let­7d­5p treated cells and control respectively. On the other hand, overexpression of let­7d­5p in DLD­1 using let­7d­5p mimic increased the sensitivity to FTD compared to control. The IC50 values of FTD were 3.7 µM and 13.9 µM in the let­7d­5p overexpression cells and control respectively. 5FU sensitivity was only altered slightly in anti­ let­ 7d­5p treated or let­7d­5p mimic treated cells. These data suggest that let­ 7d­5p is more relevant to sensitivity for FTD than that of 5FU. Conclusion: Let­7d­5p expression level influenced FTD sensitivity more effectively than 5FU. It suggests that the let­7d­5p is a potential predictive marker for trifluridine/tipiracil treatment in the clinical setting. #3076 A novel nanoparticle formulation of doxorubicin is clearly differentiated from free doxorubicin in overcoming resistance mechanisms in chemo­resistant tumors. Véronique Trochon­joseph, Christelle Zandanel, Caroline Lemarchand, Vincent Hayes, Séverine Rochas, Yamina Rayah, Jean­Louis Labernardière, Graham Dixon. onxeo, paris, France. Background and Aims. Chemo­resistance in hepatocellular carcinoma (HCC) tumor cells can be mediated by several mechanisms including P­ gp efflux pumps and drug sequestration by the autophagy process. This study investigates the potential of doxorubicin loaded nanoparticle (NP) to reverse chemo­resistance by these mechanisms compared to free doxorubicin. We present the preclinical evaluation of anti­tumor effects of NP as monotherapy and in combination with standard agents used in

treatment of HCC, pancreatic and sarcoma cancers. A phase III clinical study comparing NP to Best Standard of Care (Relive study) in patients with advanced HCC is in the final stages of recruitment with preliminary results expected in 2H 2017. Methods. Tumor cell lines were incubated with drugs in cell proliferation assay. In vivo efficacy of NP alone (4­8 mg/kg) or in combination with current and investigational treatments for pancreatic cancer (e.g. Gemcitabine, Erlotinib, Abraxane) and HCC (Sorafenib, Regorafenib and Lenvatinib) were performed in mouse tumor models using tumor weight as primary endpoint. In all experiments NP was compared to administration of free doxorubicin. Doxorubicin quantification in tumor and organs to asses PK and biodistribution was also performed using an LC/MS based method. Autophagy was measured by cell proliferation in the presence of inhibitors e.g.. Concanamycin A, Hydroxychloroquine sulfate added 30 min before incubation with the test compound. Results. NP showed a dose­dependent inhibition of cell proliferation in all resistant cancer cell lines tested with a superior activity compared to free doxorubicin and other tested drugs. In contrast to free doxorubicin, NP showed consistent anti­proliferative activity in the absence/presence of inhibitors of P­gp pumps and autophagy. In a range of in vivo models, NP was preferentially taken up by the tumor tissue and significantly reduced tumor growth when compared with free doxorubicin and with at least equivalent reduction in tumor growth compared to current treatments. Furthermore NP administered in combination with current treatments significantly increased the inhibitory effect of each drug without additional toxicity (as measured by no change in body weight). The results comparing efficacy of NP alone and in combination in HCC, pancreatic and sarcoma cancer models will be presented. Conclusions. These results demonstrate that NP is clearly differentiated from free doxorubicin, in 1) overcoming resistance mechanisms linked to efflux and autophagy, and 2) having a superior biodistribution profile both of which results in significantly enhanced activity on chemo­ resistant tumors. NP also provides an opportunity to combine with other agents, enhancing activity without increasing toxicity. The implications of these results on the further development of NP will be discussed. #3077 Tumor cells with acquired resistance to EGFR inhibitors and overexpression or activation of AXL, MET and FGFR1 are insensitive to single­agent treatment targeting AXL, MET or FGFR.

Jordi Bertran­Alamillo,1 Miguel Angel Molina­VIla,1 Cristina Teixidó,1 Jordi Codony­Servat,1 Ana Giménez­Capitán,1 Carles Codony­Servat,1 Silvia García­Román,1 Erika Aldeguer,1 Sonia Rodríguez,1 Rafael Rosell2. 1Pangaea­Biotech, Barcelona, Spain; 2Institut Catala d'Oncologia, Badalona, Spain. Background: Aberrant activity of the MET, FGFR1 and AXL receptors has been associated with the development of resistance to first, second and third generation EGFR tyrosine kinase inhibitors (TKI) in EGFR­ mutated non­small cell lung cancer (NSCLC) patients. Methods: We obtained 6 resistant lines by treating EGFR­mutated (exon 19), TKI sensitive PC9 cells with increasing concentrations of gefitinib or erlotinib. The p.T790M resistance mutation emerged in two cell lines (GR1, GR4), which remained sensitive to osimertinib, a third generation EGFR TKI. Six new cell lines to resistant to “second line” osimertinib were generated from GR1 and GR4 by exposure to increasing concentrations of the inhibitor. Finally, six more cell lines resistant to “first line” osimertinib were derived from the PC9 parental cells. All resistant cell lines were genotyped for selected genes (including EGFR) and characterized for AXL, MET and FGFR1 expression and activation by Q­RT­PCR, immunohistochemistry and Western blotting. The effects of AXL (BGB324), MET (crizotinib, capmatinib) and FGFR1 (nindetanib) inhibitors on the parental and the 18 resistant cell lines were analyzed by MTT and, in some cases, by colony formation. AXL was stably silenced in some of the resistant cell lines. Results: All cell lines resistant to “first line” gefitinib, erlotinib and osimertinib maintained the exon 19 EGFR sensitizing mutation. In contrast, three of the resistant cell lines to “second line” osimertinib lost the exon 19 and the p.T790M mutations. In two more, the p.T790M dropped to low allelic fractions (1% and 0.03%). Regardless of the EGFR status, AXL overexpression was the most common event related to EGFR TKI resistance in our panel of 18 cell lines, with FGFR1 and MET overexpression or activation as less frequent events. In proliferation assays, the IC50 of the EGFR TKI resistant cell lines for BGB324 (AXL inhibitor) was indistinguishable from the IC50 of the parental, EGFR TKI sensitive cell line. Similar results were obtained in the case of capmatinib, crizotinib (MET inhibitors) and nintedanib (FGFR inhibitor). Stable silencing of AXL on some of the AXL­overexpressing resistant cell lines had no effects in terms of doubling times, morphology of cells or

sensitivity to EGFR TKIs. In combination experiments, the effect of BGB and MET inhibitors was found to be additive. Conclusions: In tumor cell line models of acquired resistance to EGFR TKIs, overexpression or activation of AXL, MET and FGFR1 was not associated to sensitivity to single­agent treatment with AXL, MET or FGFR inhibitors. Multitargeted approaches might be more effective in this setting. #3078 The role of extracellular vesicles in chemotherapy resistance in gastric cancer. Edson Cassinela, Michele C. Landemberger, Gabriela P. de Oliveira, Vilma Regina Martins. A.C. Camargo Cancer Center, Sao Paulo, Brazil. Gastric adenocarcinoma (GAd) is the fourth most common cancer and the second cause of cancer death in the world. Chemotherapy has been largely used to treat this disease however acquired drug resistance is a common event and is associated to the development of new more aggressive clones of tumor cells. Herein, a gastric adenocarcinoma cell line (AGS) resistant to the 5­fluorouracil (rAGS­FU) was generated in order to establish mechanisms associated to acquire drug resistance. In the presence of 5­FU (10μM) resistant cells show a 2­3 times higher proliferation ratio than parental cells, a plating efficiency of 38% compared to 1% of parental cells and a higher invasive phenotype. Remarkable, when compared to parental cells, rAGS­FU resistant cells secreted 2­3 times more extracellular vesicles, EVs (85­175nm). When parental cells were treated with EVs from rAGS­5­FU resistant cells they acquired the same phenotype of the latter cells regarding higher proliferation, plating efficiency and invasion. The proteomic analysis of parental and rAGS­FU cells showed 306 differentially expressed proteins, while 66 differentially expressed proteins were found when EVs secreted from these cells were compared. Our data suggest that cells resistant to chemotherapy have a more aggressive phenotype and are able to transfer these characteristics to non­resistant cells. These findings point to mechanisms of chemoresistance mediated by components that are carried from cell to cell by EVs, what may indicate novel approaches that can be addressed to impair drug resistance and improve treatment response. Supported by FAPESP #3079

Activation of CCN1 signaling in solid tumor cells diminishes response to a histone deacetylase inhibitor: A dark side of HDACIs. Arnab Ghosh, Priyanka Ghosh, Gargi Maity, Sushanta K. Banerjee, Snigdha Banerjee. VA Medical Center, Kansas City, MO. Background and Objective: Histone deacetylase (HDAC) inhibitors are clinically proven epigenetic­based drugs for hematological cancers. However, the impact of these inhibitors on solid tumors is disappointing and debatable. Recent studies have shown that the solid tumor cell lines are less sensitive to a HDAC inhibitor Vorinostat (suberoylanilide hydroxamic acid; SAHA) as compared to hematological cell lines. However, it remains elusive why solid tumor cell lines responded weakly to HDAC inhibitors. Previously, it has been reported that CCN1/Cyr61 plays critical role in invasive front and drug resistance in breast and pancreatic cancer. Thus, our goal is to determine whether CCN1 signalling impair activity of SAHA in breast and pancreatic cancer cells. Methods: To test this objective, various breast cancer cell lines (MCF­7, ZR­75­1, MDA­MB­231, HCC­70) and pancreatic cancer cell lines (BxPC­3, AsPC­1 and Panc­1) were treated with SAHA with different does and times. Levels of CCN1 and its downstream signalling molecules were determined in SAHA­treated or untreated cell extracts using Western blotting. In addition, cell viability, migration and sphere formation were examined in parental and CCN1­depleted cells in the presence or absence of SAHA. Results and Conclusions: Here we have shown that CCN1 was upregulated in various breast and pancreatic cancer cell lines following SAHA treatment via epigenetic mechanism. Distribution of CCN1 in the nucleus and cytoplasm was also drastically altered in SAHA­treated cells. Normally, CCN1 expression was detected in the cytoplasm. However, following SAHA treatment, CCN1 expression was located predominantly in the nucleus. Functionally, CCN1 had dual outcome in these cells. The cell viability assay indicated that CCN1­positve breast cancer or pancreatic cancer cells were more sensitive to SAHA as compared to CCN1­negative cells. CCN1 ablation by neutralizing antibody treatment in MDA­MB­231 cells significantly suppressed the inhibitory effect of SAHA in MDA­MB231 cells. However, aggressive phenotype such as migration towards SDF­1 and sphere­formation were markedly elevated in SAHA treated cells as compared to untreated cells. These effects of SAHA were rescued by CCN1­depletion. Collectively, these studies indicat that CCN1 activation limits the response to SAHA in solid tumor

cells and thus suggesting that combination therapy of SAHA and CCN1­ inhibitor could be an ideal therapeutic approach to make HADAC inhibitor sensitive to solid tumors. #3080 Y­box binding protein 1 is crucial in acquiring drug resistance in advanced renal cell carcinoma. Ninadh M. D'Costa, Peter Raven, Zheng Tan, Werner Struss, Sebastian Frees, Claudia Chavez­Munoz, Alan I. So. University of British Columbia, Vancouver, British Columbia, Canada. Objective: Investigating the role of Y­box binding protein 1 (YB1) in drug resistant advanced kidney cancer. Background: Renal cell carcinoma (RCC) is the 6th most common malignancy with approximately 1,800 deaths in 2015 and 2.3% annual increase in Canada. Despite the partial or total surgical removal of kidney in patients with localized RCC, metastatic patients are treated with tyrosine kinase inhibitors (TKIs) in a purely palliative approach. However, TKI­resistance (Sunitinib) is developed after a median time of 10­14 months. Therefore, identifying the factor(s) responsible for TKI­ resistance development and disease advancement in RCC is imperative. It is now widely recognized that evolutionarily conserved Y­box binding protein 1 (YB1) is essential for cell growth and survival. Upregulation of YB1 in numerous cancer types was found to be positively correlated with tumor growth, metastasis and drug­resistance development. YB1 is also involved in intercellular communication through its secretion in the tumor microenvironment by cell­surface transporters, ABC­transporters. Summary of the data: Endothelial cells (HUVEC) were co­cultured with Caki­1DC (Sunitinib­resistant, developed in our lab) and Caki­1WT (Sunitinib­sensitive) RCC cell­lines, and increased migration of HUVEC was observed with Caki­1DC compared to Caki­1WT. A drastic increase in YB1 and its downstream target ABCB1 in Caki­1DC compared to Caki­1WT cells was also detected. Consistent with previous reports, we observed granular structures in the Caki­1DC cells that support potential secretion of YB1 into the tumor microenvironment. Moreover, blocking ABCB1 reverted the Caki­1DC cells to being drug­sensitive. Experimental procedures: Caki­1WT and Caki­1DC were co­cultured with HUVEC cells followed by scratch assay to test for HUVEC cell migration and associated secretory factors. Western blot and qPCR were used to determine the protein and mRNA levels respectively between the

two cell­lines. Immuno­histochemical staining was carried out on the RCC tumors from Sunitinib­sensitive and resistant mouse models (developed in our animal facility). Granular structures were observed using immunofluorescence staining against YB1. Presto­blue was used for cell biomass assay following different drug treatments. Summary: The molecular function of YB1 in RCC and its potential in targeted therapy is not well understood. Therefore, understanding the function of YB1 in metastatic RCC and in drug­resistance development is of vital importance. Our data suggests that inhibition of YB1 may slow disease progression and, possibly, revert the drug resistance mechanism. The results from this study have the potential to introduce YB1 inhibitors in conventional RCC chemotherapy, alone or in combination, to improve survival in advanced kidney cancer patients. #3081 Precision medicine for patients with advanced small cell lung cancer treated with novel therapeutic agents in a phase I clinical trials unit. Joline S. Lim, Samuel J. Harris, Malaka Ameratunga, Raghav Sundar, Joo Ern Ang, Dearbhaile Collins, Maxime Chénard­Poirier, Alvaro I. Garces, Stan B. Kaye, Juanita Lopez, Udai Banerji, Johann S. de Bono, Timothy A. Yap. Royal Marsden Hospital, London, United Kingdom. Introduction Small cell lung cancer (SCLC) is aggressive and relapse is inevitable. Novel therapies in the context of phase I (Ph1) clinical trials should be considered in this setting. Next generation sequencing (NGS) identifying putative driver mutations may aid therapy allocation in such trial settings. Methods Retrospective analysis of characteristics and clinical outcomes of patients (pts) with advanced SCLC referred to the Phase I Clinical Trials Drug Development Unit at the Royal Marsden Hospital between 1992 and 2016, using electronic patient records Results 85 pts with advanced SCLC were included, of which 45 pts were allocated to ≥1 trial each (48 allocations). Of these, 21 (46.7%) pts started ≥1 trial, with a total of 24 trials commenced. Of the 64 pts who were unable to commence trials, 43 (67.2%) had rapid disease progression, 11 (17.2%) sought other therapies, 5 (7.8%) declined Ph1 trials, and 5 pts did not enrol for other reasons. Pts had a median of 2 previous lines of treatment (range:1­3). 13 pts had tumor molecular profiling with high coverage targeted NGS, yielding 22 distinct mutations; Aberrations in DNA damage repair (DDR) pathway were most common: TP53 (53.8%),

FANC (30.8%), ATM (15.4%), ATR, BARD1 and CHEK2 (7.7% each). All pts found to have ≥1 DDR pathway mutation had platinum sensitive disease (disease progression ≥90 days from last platinum dose) in first line setting. Other mutations found include ALK, APC, AR, AXIN1, DDB2, ERBB2, mTOR, PI3K, NOTCH, NTRK17, PDGFR, RB1, RET, STK11, FKT3 and VGFR2. Pts were most commonly treated with novel inhibitors against PARP (n=9), PI3K/AKT (n=3), PD­1 (n=2) and BCL (n=2). Therapies were generally well tolerated with no dose limiting toxicities observed. Of 9 pts who received PARP inhibitors, 1 achieved a RECIST partial response (PR) and remained on trial for 16 weeks; 4 achieved RECIST stable disease (SD) for a median of 17 weeks (range:10.9­31.1 weeks). 1 exceptional responder whose tumor harbored multiple somatic aberrations (TP53, FANCF, AR, ERBB2, NOTCH2) was enrolled on 3 sequential phase I trials ­ she achieved durable RECIST SD on a PARP inhibitor (31.1 weeks), RECIST PR with a PD­1 inhibitor (38.7 weeks), and durable RECIST SD with carboplatin/ATR inhibitor (27.9 weeks). Platinum sensitivity in the first line setting predicted for improved disease control (RECIST PR/SD 73.3% vs 0%, p 1 µM), but was sensitive to crizotinib (IC50 = 98 nM). The receptor tyrosine kinase assay revealed the activation of MET and EGFR in ABC­14. Quantitative RT­PCR and FISH confirmed MET gene amplification. Quantitative RT­PCR also indicated the overexpression of an EGFR ligand, EGF, TGF­α but no EGFR mRNA overexpression. The combination of crizotinib (dual ALK/MET­TKI) and an EGFR­TKI, gefitinib, showed an additive inhibitory effect on cell growth compared with each drug alone in vitro. ABC­17 showed resistance to both alectinib and crizotinib; consistently showed no activation of MET and no MET gene amplification. Interestingly, ABC­17 showed metastatic ability in the lung in NOG mouse PDX models. Conclusion The mechanism of rapid resistance to alectinib may be complicated and heterogeneous. Crizotinib combined with gefitinib, which inhibits the ALK, EGFR, and MET pathways, may represent one potent strategy against alectinib resistance. Further next­generation sequencing of clinically relevant samples should provide deeper insights into its resistance. (This work was supported by KAKEN 16K19454 and KAKEN 15H04830.) #3165 3D culture may better represent trastuzumab resistance associated with PIK3CA mutation than 2D culture. Takashi Tatara,1 Toru Mukohara,2 Rina Tanaka,1 Yohei Shimono,1 Masanori Toyoda,1 Naomi Kiyota,1 Midori Hirai,1 Yoshihiro Kakeji,1 Hironobu Minami1. 1Kobe University Graduate School of Medicine, Kobe, Japan; 2Kobe University Hospital, Kobe, Japan. Background: It is becoming clear that presence of PIK3CA mutations is associated with lower pathological complete response rate in patients with HER2­overexpressing breast cancer when treated with trastuzumab­based

chemotherapy in neo­adjuvant settings. On the other hand, in in vitro studies using traditional 2­dimentional (2D) cell culture, differential cellular or biochemical response to trastuzumab between PIK3CA­mutant (mt) and ­wild­type (wt) cells has not been clearly demonstrated. Further, while tumor shrinkage is occasionally observed in breast cancer patients who are treated with trastuzumab as a single agent, cyto­toxic effect of trastuzumab is not simulated in 2D culture models. Recently, many studies reported 3­dimensional (3D) cell culture mimics in vivo environment better than 2D culture. Therefore, we hypothesized that 3D culture better represents clinically­observed trastuzumab resistance associated with PIK3CA mutation than 2D culture, and decided to comparatively investigate cellular and biochemical response to trastuzumab in HER2­amlified PIK3CA­mt and ­wt cell lines cultured in 2D and 3D environments. Method: HER2­amplified breast cancer cell lines, BT474 (PIK3CA­wt), and UACC893 and MDA­MB361 (PIK3CA­mt) were seeded (day 0) and allowed to grow in 2D and 3D (NanoCluture Plate®, ORGANOGENIX, Kanagawa, Japan) cell culture plates. On day 3, trastuzamab (10 µg/ml) and/or BKM120 (1 and 5 µM), a PI3K inhibitor, were added. The effect of the drugs on cell growth was evaluated with WST­8 assay on days 3 through 7. Apoptosis and cell signaling were evaluated using Western blot on day 6 and days 3 through 5, respectively. Result: In PIK3CA­wt BT474, treatment with trastuzumab led to decrease in cell number, indicating cyto­toxic effect, only in 3D culture but not in 2D culture. In PIK3CA­mt UACC893 and MDA­MB­361 cell lines, treatment with trastuzumab resulted in no cellular reduction either in 2D or 3D cultures. Consistently, increase in cleaved PARP, indicative for apoptosis, was observed only in 3D­cultured BT474 but not in 2D­ cultured BT474 or two PIK3CA­mt cell lines. Furthermore, in BT474, greater decrease in phosphorylation of AKT (p­AKT) was observed in 3D culture than in 2D culture. In PIK3CA­mutant cell lines, trastuzumab did not change level of p­AKT regardless of cell culture conditions. In PIK3CA­mutant UACC893, combined treatment with trastuzumab and BKM120 resulted in greater increase in expression of cleaved PARP than either drug alone. Conclusion: Trastuzumab­induced inhibition of PI3K/AKT pathway and resultant apoptosis in HER2­overxpressing PIK3CA­wt cells may be observed in 3D culture, which may be simulating cyto­toxic effect of trastuzumab by itself observed in clinic. Furthermore, 3D cell culture represents the resistance to trastuzumab associated with PIK3CA

mutation better than 2D cell culture. #3166 Loss of PARP1 in human cancer cells confers resistance to PARP inhibition via activation of innate immune signaling. Rajib Ghosh, Sanchita Roy, Sonia Franco. Johns Hopkins Univ. School of Medicine, Baltimore, MD. Purpose: PARP inhibitors (PARPi) are FDA­approved for the treatment of ovarian cancer and in clinical trials for the treatment of other malignancies. PARPi block the catalytic site of PARP1, a poly(ADP­ ribose) polymerase that catalyzes the repair of DNA single­strand breaks (SSBs) and double­strand breaks (DSBs). Although cancer cells harboring defects in Homologous Recombination (HR) are particularly sensitive to PARPi, disease progression is eventually observed due to the emergence of resistant cells. To address the mechanisms leading to PARPi resistance and identify novel preventive strategies, we have generated and characterized multiple human cancer cell lines genetically engineered to acquire PARPi resistance via depletion of PARP1. Experimental Procedures: We used CRISPR/Cas9D10A (“double nickase”) to introduce DSBs within exon 2 of PARP1 in human cancer cell lines HCT116 and HEK293T. After allowing for repair via endogenous error­prone pathways, single cell­derived subclones were screened by immunoblotting with an antibody to PARP1. PARP1 “knock out” (KO) clones were further analyzed for proliferation, cell cycle distribution and sensitivity to DNA damaging agents, including ionizing radiation (IR) and the PARPiolaparib. Radiation response was assessed using proliferation and clonogenic assays, immunofluorescence (IF) for γ­H2AX and immunoblotting for ATM substrates phospho­KAP1 (Ser824) and phospho­CHK2 (Thr68). Finally, differential gene expression between PARP1­proficient and deficient HCT116 and HEK293T cells was analyzed by RNA­Seq. Findings: Although genetic depletion of PARP1 initially led to significant death of HCT116 and HEK293T mass cultures, surviving cells could be isolated to establish stable PARP1 KO clones. Relative to PARP1­ proficient controls, HCT­116PARP1­/­ and HEK293TPARP1­/­ cells exhibited decreased proliferation and clonogenic capacity in vitro and decreased growth in vivo. In addition, loss of PARP1 led to radiosensitivity and persistent activation of cell cycle checkpoints, delayed kinetics of γ­H2AX foci resolution and persistent ATM signaling

after IR. In contrast, PARP1 loss induced resistance to the PARPi olaparib in both lines. PARP1 reconstitution using either stable or transient approaches resulted in only a partial rescue of these phenotypes. RNA­Seq analyses revealed increased expression of innate immune signaling and inflammatory cascades upon PARP1 loss, including type I/III interferons and their inducible genes. Conclusions: PARP1 KO human cancer cells may represent a valuable preclinical model to investigate mechanisms underlying resistance to PARP inhibitors observed in the clinical setting. Loss of PARP1 triggers activation of “viral mimicry” in cancer cells, suggesting that drugs targeting this pathway may synergize with PARPi to kill cancer cells and prevent the emergence of resistance. #3167 MIF­induced stat3 activation promotes resistance to MEK blockade in KRAS mutant colorectal cancer cells. Seul­Ki Cheon,1 Hwang­Phill Kim,1 Ye­Lim Park,1 Si Hyun Lee,1 Jun­ Kyu Kang,1 Yoojoo Lim,2 Sang­Hyun Song,1 Sae­Won Han,2 Tae­You Kim2. 1Seoul National University, Seoul, Republic of Korea; 2Seoul National University hospital, Seoul, Republic of Korea. Although MEK blockade has been highlighted as a promising anti­tumor drug, it has poor clinical efficacy in KRAS mutant colorectal cancer. Several feedback systems have been described in which inhibition of one intracellular pathway leads to activation of a parallel signaling pathway, thereby decreasing the effectiveness of single­MEK targeted therapies. In this study, we describe a feedback mechanism in which MEK inhibition leads to activation of macrophage migration inhibitory factor (MIF)­ induced stat3 signaling pathway in KRAS mutant colorectal cancer (CRC) cells. We found that KRAS mutant CRC cells with refametinib, MEK inhibitor, induced MIF secretion and resulted in activation of Stat3. MIF knockdown by siRNA partially restored sensitivity to refametinib in KRAS mutant cells. In addition, combination with refametinib and 4IPP, a MIF inhibitor, effectively reduced the activity of stat3 and MAPK, more than single agent treatment. As a result, combined therapy was found to exhibit a synergistic growth inhibitory effect against refametinib­resistant cells by downregulating MIF expression. These results reveal that MIF­ induced stat3 activation evoked an intrinsic resistance to refametinib. Our results provide the basis for a rational combination strategy against

KRAS mutant colorectal cancers, predicated on the understanding of cross­talk between the MEK and MIF pathways. #3168 Overcoming acquired drug resistance by TPX­0005, an ALK, ROS1 and pan­TRK inhibitor. Wei Deng, John Huang, Dayong Zhai, Evan Rogers, Jean Cui. TP Therapeutics, Inc., San Diego, CA. The inevitable development of clinical drug resistance presents a common challenge for targeted cancer therapy. In non­small cell lung cancer (NSCLC) patients with ALK and ROS1 rearrangements, the emergence of mutations in the targeted oncogenes was identified as one of the mechanisms that confer drug resistance. In particular, a group of mutations known as solvent front mutations, such as ALKG1202R and ROS1G2032R, render common resistance to ALK and ROS1 inhibitors. Similar solvent front mutations, such as TRKAG595R and TRKCG623R, have also been identified in cancer patients with rearrangement of NTRK family genes who developed resistance to TRK­targeted therapies. TPX­ 0005, a compact three­dimensional macrocyclic molecule, was designed to completely locate at the adenine binding site of ATP in order to efficiently target the active kinase conformation and systematically circumvent the steric interference from various clinical resistant mutations, especially the solvent front mutations. TPX­0005 is an orally available and potent ATP­competitive inhibitor against ALK, ROS1, TRKA, TRKB and TRKC recombinant kinases and their corresponding clinical resistant mutants. TPX­0005 demonstrated potent anti­ proliferative activity in the range of sub­nanomolar to low nanomolar in a number of human cancer cell lines and engineered stable cell lines expressing the targeted oncogenes or their solvent front mutants, accompanied by inhibition of target phosphorylation and concomitant inactivation of downstream effectors such as ERK, AKT and STAT3. In patient derived xenograft tumor models, TPX­0005 treatment resulted in significant regression of tumors harboring the oncogenic ALK, ROS1 and TRKC fusions. Moreover, in a series of mouse xenograft tumor models, TPX­0005 exhibited marked anti­tumor activity not only in tumors harboring the wildtype oncogenic targets but also in tumors harboring the oncogenes with the solvent front mutations via inhibition of the target phosphorylation. Taken together, these pre­clinical studies have

demonstrated the potent activities of TPX­0005 against not only wild type oncogenic ALK, ROS1, TRK fusions but also their corresponding solvent front mutations, which will bring in a new therapy for cancer patients resistant to currently available ALK, ROS1, and TRK inhibitors in clinic. #3169 SerpinB2 enhances invadopodia­like structure protrusions and is down­regulated in acquired gefitinib­resistant non­small cell lung cancer cells. Song Yi Bae,1 Donghwa Kim,2 Hyen Joo Park,2 Woong Sub Byun,2 Ji­ Young Hong,2 Hye­Jung Lee,2 Sang Kook Lee2.1MIT, Cambridge, MA; 2Seoul National University, Seoul, Republic of Korea. Non­small cell lung cancer (NSCLC) is a major type of lung cancer which accounts for approximately 80­85% of all lung cancers. The targeted therapies have significantly improved the survival of advanced NSCLC patients, but the failure of targeted therapy due to the resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR­ TKIs), such as gefitinib, is now considered a major problem. SerpinB2 is a component of the urokinase plasminogen activator (uPA) system and has been recognized as a biomarker for the progression and metastasis of lung cancer. We found that SerpinB2 is down­regulated in gefitinib­ resistant (H292­Gef) cells compared to gefitinib­sensitive (H292) cells. The low SerpinB2 levels in H292­Gef cells were also associated with an enhancement in invasiveness and increase in the length of invadopodia­ like structures in the cells. The effect on invasiveness and gefitinib sensitivity was confirmed by knockdown and overexpression of SerpinB2. In addition, an antitumor agent yuanhuadine (YD) was used to test the possibility to overcome the resistance through the up­regulation of SerpinB2. YD effectively elevated SerpinB2 levels and suppressed invasive properties in H292­Gef cells. Collectively, these findings demonstrate the prospective role of SerpinB2 as a novel biomarker for acquired gefitinib resistance and a potential target for NSCLC treatment. Acknowledgement This study was funded by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1D1A1A02062012).

Novel Mechanisms 1

#3170 MCAM modulates small cell lung cancer chemoresistance via PI3k/Akt/Sox2 signaling pathway. Satyendra C. Tripathi,1 Johannes F. Fahrmann,1 Muge Celiktas,1 Mitzi Aguilar,1 Kieren D. Marini,2 Mohit K. Jolly,3 Hiroyuki Katayama,1 Hong Wang,1 Eunice N. Murage,1 Jennifer B. Dennison,1 D. Neil Watkins,4 Herbert Levine,3 Edwin J. Ostrin,1 Ayumu Taguchi,1 Samir M. Hanash1. 1MD Anderson Cancer Center, Houston, TX; 2Hudson Institute of Medical Research, Australia; 3Rice University, Houston, TX; 4Garvan Institute of Medical Research, Australia. Despite favorable responses to initial therapy SCLC relapse occurs within a year exhibiting a multidrug resistant phenotype. Due to limited accessibility of patient tissues for research purpose, SCLC patient derived xenografts (PDXs) have provided the best opportunity to address this limitation. We sought to identify novel mechanisms involved in SCLC chemoresistance. Through in­depth proteomic profiling, we identified MCAM as a markedly upregulated surface receptor in chemoresistant SCLC cell lines that exhibited a mesenchymal phenotype and in chemoresistant PDXs compared to matched treatment­naïve tumors. MCAM is a cell membrane protein whose expression has been implicated in multiple human cancers. MCAM expression is also detected in lung adenocarcinoma; however, its expression and role in SCLC is still not been explored. MCAM knockdown in chemoresistant cells reduced cell proliferation and decreased the IC50 inhibitory concentration of chemotherapeutic drugs. MCAM was found to modulate sensitivity of SCLC cells to chemotherapeutic drugs through up­regulation of MRP1/ABCC1 expression and of the PI3K/AKT pathway in a SOX2 dependent manner. Metabolomic profiling revealed that MCAM can modulate glutamic acid and lactate production in chemoresistant cells with a distinct metabolic phenotype sustaining low oxidative phosphorylation. MCAM may serve as a novel therapeutic target to overcome chemoresistance in SCLC. #3171 Analysis of "drug addiction" mechanisms in the drug­resistant ROS1­rearranged cancer cells.

Hayato Ogura,1 Jun Adachi,2 Takeshi Tomonaga,2 Naoya Fujita,1 Ryohei Katayama1. 1The Cancer Chemotherapy Ctr., JFCR, Tokyo, Japan; 2Lab. of Proteome Research, National Institute of Biomedical Innovation, Health and Nutrition, Osaka, Japan. ROS1­rearranged non­small­cell lung cancer (NSCLC) is observed in approximately 1% of lung cancer patients. Crizotinib, ALK/ROS1/cMET tyrosine kinase inhibitor (TKI), showed high effect against ROS1­ rearranged NSCLC in the clinical trials and is approved in US and EU. However, the cancers inevitably relapse due to the acquired resistance such as ROS1­G2032R mutation. We previously reported that cabozantinib could overcome those secondary mutations mediated crizotinib­resistance. Based on this finding, we tried to establish cabozantinib­resistant ROS1 mutant cells by ENU mutagenesis screening. Surprisingly, we found out a few CD74­ROS1 mutant clones that only grew in the presence of low­dose cabozantinib. Namely, these mutant clones were “addicted” to cabozantinib. When the fluorescent labeled cabozantinib addicted ROS1 mutant cells were co­cultured with non­ addicted CD74­ROS1­WT­BaF3 cell in the presence or absence of low­ dose cabozantinib, the drug­addicted cells became dominant in the low­ dose cabozantinib, and CD74­ROS1­WT cells became dominant in the absence of it. To analyze the molecular mechanism of “drug­addiction” in the addicted CD74­ROS1 mutant cells, we performed comprehensive analysis such as inhibitor screening, cDNA microarray, and phosphoproteome analysis. As the result, we observed that the expression and phosphorylation of CD74­ROS1 in the addicted cells were excessively upregulated within 24 hours after removal of cabozantinib or other ROS1 inhibitors, such as crizotinib or lorlatinib. We also observed that the apoptosis was induced in these cells upon ROS1 inhibitor removal, whereas ROS1 mediated growth signaling were simultaneously activated. Consistent with this, cDNA microarray analysis revealed that the expression of both cell survival and cell death related genes were changed by ROS1 inhibitor removal. Furthermore, we identified that several proteins were highly phosphorylated by excessive ROS1 activation originated from ROS1 inhibitor removal. These observations suggest that these drug­addicted cells were died by excessive oncogenic signaling, and the appropriate oncogene signaling by low­dose TKI made them survive and grow without inducing apoptosis. Thus, the excessive oncogene signaling has “double­edged sword” characteristics for cancer cell viability and those characteristics could be a new therapeutic target.

#3172 Inhibition of coiled coil domain containing protein 69 (CCDC69) enhance platinum­mediated apoptosis in ovarian cancer cells. Long Cui, Joseph Kwong, Chi Chiu Wang. The Chinese University of Hong Kong, Hong Kong, Hong Kong. Background and Objective:Molecular mechanisms of chemo­resistance in ovarian cancer are poorly understood. To identify gene involved in ovarian cancer chemo­resistance, 135 ovarian cancer patients with intact chemo­response information from The Cancer Genome Atlas (TCGA) database were included. Our analysis revealed that the level of CCDC69 mRNA is differentially expressed between chemo­sensitive group and chemo­resistant group. Moreover, there was a significant negatively correlation between CCDC69 promoter methylation and mRNA expression. The aim of the study is to examine the role of CCDC69 in the underlying mechanism of chemo­resistance. Methods: The expression levels of CCDC69 were detected in chemo­ sensitive ovarian cancer A2780, chemo­resistant A2780cis and SKOV3 cell lines using quantitative RT­PCR and immunoblots. Promoter methylation status of CCDC69 were investigated by bisulfite sequencing. Silencing CCDC69 in A2780cis and SKOV3 cells were performed by Small­interfering RNAs (siRNAs) and CRISPR­Cas9. Cell viabilities after cisplatin treatment were evaluated by MTT and colony formation assays. Apoptosis was assessed by Annexin V/PI staining and caspases 3/8 activity. Cell cycle distributions and mitochondrial membrane potential (ΔΨm) were measured by flow cytometry. Relevant pathway proteins were determined by immunoblotting assays. Results: Heavy CpG methylation (73.1% and 74.3%) was found in A2780 and A2780cis cells. Restoration in the expression of CCDC69 were found in A2780 and A2780cis cells after 5­Aza­dC treatment. In fact, the expression levels of CCDC69 were about 3­4 fold higher in chemo­ resistant A2780cis cells than its parental chemo­sensitive A2780 cells. Inhibition of CCDC69 in chemo­resistant A2780cis cells by si­RNA significantly increased sensitive to cisplatin treatment (p100 fold resistance in 4T1 cells. Western blot analysis showed enhanced expression of HER2, β­ catenin and downstream molecules such as TCF/LEF, c­Myc, Cyclin D in these resistant cells. We have recently demonstrated that penfluridol, an anti­psychotic drug, suppresses the growth of triple negative metastatic breast cancer cells (Ranjan and Srivastava, Cancer Res 2016; 76(4): 877­ 890), giving us the rationale to evaluate whether penfluridol inhibits HER2 and β­catenin signaling. Our current results showed that penfluridol treatment not only suppressed HER2 but also inhibited β­ catenin expression. We also observed down regulation of LEF­1/TCF, Cyclin D1 and c­Myc expression with penfluridol treatment in paclitaxel sensitive as well as resistant cells resulting in reduced survival of cells. Our results further showed that penfluridol treatment synergistically enhanced the growth suppressive effects of paclitaxel in MCF­7 and 4T1 paclitaxel resistant cells. Treatment of paclitaxel resistant 4T1 cells with 1.5μM of penfluridol in combination with 50nM of paclitaxel resulted in 65% of cell growth suppression whereas either treatment alone was not cytotoxic at all. We also observed an enhanced down regulation of proteins involved in paclitaxel resistance such as HER2, β­catenin, c­Myc and Cyclin D1 as well as increase in apoptotic markers such as Cl­PARP and Cl­Caspase3 when paclitaxel treatment was combined with penfluridol in resistant cells. Taken together, our results provided a novel insight into the mechanism of resistance to paclitaxel and also opened new avenues for application of penfluridol in cancer therapeutics. Further detailed mechanistic and in vivo studies are in progress. (Supported in part by RO1 grant CA129038, awarded by National Cancer Institute, NIH).

#3177 KITENIN leads to resistance against temozolomide through enhancement of cancer stemness factors in mouse glioma model. Kyung­Hwa Lee, Eun­Jung Ahn, Shin Jung, Jae­Hyuk Lee, Kyung­Keun Kim, Kyung­Sub Moon. Chonnam National University Hwasun Hospital & Medical School, Hwasun­gun, Republic of Korea. Background: Recently, a new research spotlighted the role of KITENIN (KAI1 COOH­terminal interacting tetraspanin) on glioma invasiveness and progression, associated with the up­regulation of EMT (epithelial­ mesenchymal transition) and cancer stemness markers. In this study, furthermore, it is investigated whether KITENIN leads to resistance against TMZ through enhancement of cancer stemness factors using mouse glioma model. Materials and Methods: TMZ­resistant T98G cell line (T98G/TR) was developed. EMT and cancer stemness markers and KITENIN expression were assessed by Western blotting and qRT­PCR. With KITENIN modulated U251 (knockdown) and GL261 (overexpression) cells, cell viability and apoptotic cell death factors after TMZ treatment were evaluated. Biological role of KITENIN on TMZ­resistance was investigated using magnetic resonance imaging (MRI) and immunofluorescence analysis for tumor sections of orthotopic glioma model. Using human glioma samples and primary cells, the mechanistic link between KITENIN and cancer stemness factors was confirmed. Results: T98G/TR cells showed increased expression of the KITENIN and EMT and cancer stemness factors. Also, In vitro assays revealed that KITENIN knockdown inhibited cell viability against TMZ treatment, whereas KITENIN overexpression promoted their viability, via apoptotic cell death pathway. In orthotopic glioma models, mice implanted with KITENIN­overexpressed cells showed resistance to TMZ on MRI and histopathological examination. The expression of KITENIN/ALDH1 or KITENIN/CD44 were co­localized and significantly higher in tumor sections of mouse transplanted with KITENIN­overexpressed cells than in tumor sections of mouse transplanted with control cells. Genetic down­ regulation of KITENIN for KITENIN­overexpressed cell line and primary glioma cells led decreased expression of ALDH1 and CD44. In human glioma samples, high expression of KITENIN was closely related with high expression of ALDH1. Conclusion: KITENIN affects TMZ­resistance in malignant gliomas through induction of ALDH1 and CD44. Therefore, it could be suggested

that KITENIN is therapeutic target to overcome TMZ­resistance of malignant gliomas. #3178 The p53 tumor suppressor protein paradoxically drives chemo­ resistance in human medulloblastoma cells through suppressing the mTOR AKT pathway. Aisha Naeem, Muhammad U. Choudhry, Maria L. Avantaggiati, Olga C. Rodriguez, Chris Albanese. Georgetown Univ., Washington, DC. Medulloblastoma (MB) accounts for approximately 25% of childhood brain tumors with 70% of the cases occurring in children under 10. Prognosis for children less than three years old is considerably worse and, due to its destructive effects on the developing nervous system, irradiation is largely avoided in this age group. The p53 pathway is considered a key determinant of anti­tumor responses in many tumors; however, its role in the regulation of cell survival, chemo­sensitivity and chemo­resistance in MB is much less well defined. It has been shown that p53 pathway defects, mutations and nuclear levels increase significantly from MB diagnosis to relapse and correlate with an adverse prognosis. We recently reported on the highly novel and unexpected finding that both the genetic (sh­RNA) and chemical silencing of p53 led to a significant increase in cell death by the drug VMY in MB cell lines with elevated basal p53 (e.g. in D556 cells which express wild type p53 and in DAOY cells that contain mutant p53) as measured by colony forming assays, DNA degradation assays and annexin­V staining, suggesting a surprising commonality in the p53 signaling in both cell lines despite the differences in their p53 status. Equally surprising was the observation that the silencing of p53 in D556 cells enhanced cell death by the clinically used drugs doxorubicin and vincristine. Conversely, we now find that suppressing p53 with sh­RNA in D283 cells, which express much lower levels of wild type p53 compared to D556, resulted in the more classical chemoresistance profile, suggesting a form of p53 ‘addiction’ in D556 and DAOY cells. Mechanistically, we observed that suppressing p53 with sh­RNA in D556 cells treated with VMY or doxorubicin significantly increased the levels of phosphorylated Chk1, S6K, gH2AX, and MDM2. Importantly, phospho­mTOR levels were also significantly increased in the D556/p53 sh­RNA cells and the suppression of mTOR enhanced chemoresistance. These somewhat paradoxical findings suggest that activated Akt/mTOR may induce MB cell death and that suppression of

mTOR by p53 enhances chemoresistance. Our data provide new mechanistic insights into the role of p53 in primitive neuroectodermal tumors and may provide new approaches for enhancing the clinical outcome of patients with MB. #3179 The effect of eribulin resistant mechanism in breast cancer on microenvironment. Wataru Goto, Shinichiro kashiwagi, Koji Takada, Katsuyuki Takahashi, Tsutomu Takashima, Satoru Noda, Naoyoshi Onoda, Shuhei Tomita, Kosei Hirakawa, Masaichi Ohira. Osaka City University, Abeno­ku, Osaka, Japan. Backgorund: Erbulin, a nontaxane, synthetic microtuble dynamics inhibitor, is currently in clinical use for advanced or metastatic breast cancer. Eribulin treatment results in improved tumor perfusion in breast cancer and induces the remodeling of tumor vasculature and a reversing effect on the epithelial to mesenchymal transition (EMT) of cancer cells. Although eribulin significantly increased overall survival by monotherapy, most breast cancer cells acquire resistance to eribulin after a series of treatments. The development of resistance to chemotherapy appears to have become a major clinical problem of breast cancer. In this study, we established two eribulin­resistant cell lines, and investigated the mechanisms underlying the development of eribulin resistance in breast cancer cells. Material and Methods: Eribulin­resistant breast cancer cell lines were obtained by culturing MDA­MB­231 and MCF­7 cells with stepwise increases in the concentration of eribulin for more than 6­months. The relative eribulin resistance of each eribulin­resistant cell line was determined using a MTT assay. To confirm the expression of proteins related to drug resistance, we examined multidrug resistance protein 1 (MDR1), end­binding 1 (EB1) and E­cadherin by western blot analysis. To investigate the effect of eribulin, a flow cytometry analysis for cell­ cycle distribution was performed. In addition, we produced MDA­MB­ 231, MDA­MB­231/Eribulin, MCF­7 and MCF­7/Eribulin subcutaneous xenografts, and analysed protein expression by immunohistochemical study of surgically resected tissues from xenografts. Results: Compared to parental cell lines, eribulin­resistant cell lines had a higher resistance to eribulin. Eribulin­resistant cell lines acquired resistance to not only eribulin, but also paclitaxel, vinorelbine,

doxorubicin, and fluorouracil. After eribulin treatment, MDA­MB­ 231/Eribulin cell lines showed no morphological change, and expression of E­cadherin did not increase. Also, cell cycle distribution and expression of EB1 did not exhibit significantly differences from parental cell lines. On the other hand, PD­L1 was overexpressed in eribulin­ resistant cell lines compared to parental cell lines. Additionally, eribulin­ resistant cell lines exhibited increased level of MDR1 protein. Conclusion: Our established eribulin­resistant cell lines may suppress eribulin effect of inhibiting microtube, EMT, and immune evasion mechanism. These should be useful tools for identifying new mechanism of eribulin resistance. #3180 Chemotherapeutical agent triggers transient resistance of cancer cells by promoting ABCB1 intercellular transfer. Xiaokun Wang,1 Dongjuan Qiao,2 Likun Chen,1 Liyan Huang,1 Meng Xu,1 Zhen Chen,1 Fang Wang,1 Zhesheng Chen,3 Musheng Zeng,1 Li­ Wu Fu1. 1Sun Yat­Sen Univ. Cancer Ctr., Guangzhou, China; 2Department of Chemistry, College of Life Science and Technology, Jinan University, Guangzhou, China; 3Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, NY. Multidrug resistance remains a major impediment to successful chemotherapy. In clinical chemotherapy, some patients often develop chemoresistance quite rapidly, the underlying mechanism remains elusive. Herein, we report that a transient exposure to chemotherapeutic agents can acutely promote the intercellular transfer of ABCB1 through stimulating Rab8B­mediated secretion of microvesicles (MVs) containing ABCB1 in drug­resistant donor cells, and promoting parallelly the endocytic recycling of ABCB1 in recipient tumor cells via the downregulation of Rab5. Sensitive recipient cells take up these ABCB1­ incorporated MVs by a clathrin­ and dynamin 2­ dependent endocytic pathway, thereby acquiring a transient resistance to chemotherapeutic agents which are ABCB1 substrates. More fascinatingly, ABCB1 molecules can locally or distantly transfer to recipient tumor cells and respond to chemotherapeutical drug in the xenograft tumor models. In some cases of patients with NSCLC, a variable increase of ABCB1 surface expression was also detected in the peripheral blood monocyte

subpopulations after they received the first chemotherapy. Our findings proposed a novel molecular mechanism of how sensitive cancer cells rapidly acquire the emergency resistance to chemotherapeutic agents by stimulating intercellular transfer of ABCB1. The precise modulation of this process may provide a valid therapeutic strategy to alleviate the MDR phenotype for successful treatment. #3181 A novel topoisomerase IIα 90 kDa isoform in etoposide resistant human leukemia K562 cells produced as a result of alternative RNA processing. Ragu Kanagasabai,1 Lucas Serdar,1 Soumendrakrishna Karmahapatra,1 Corey A. Kientz,1 Mary K. Ritke,2 Terry S. Elton,1 Jack C. Yalowich1. 1Ohio State Univ. College of Pharmacy, Columbus, OH; 2University of Indianapolis, Indianapolis, IN. DNA topoisomerase IIα (TOP2α) is a prominent target for anticancer drugs whose clinical efficacy is often limited by chemoresistance. We previously characterized acquired resistance to etoposide (VP­16) in a cloned human K562 leukemia cell line, K/VP.5, containing reduced TOP2α. In the present study, using an antibody specific for the amino­ terminus of TOP2α, immunoassays indicated the existence of two TOP2α isoforms, 170 and 90 kDa, present in K562 leukemia cells and in the etoposide resistant K/VP.5 cells. TOP2α/90 expression was dramatically increased in etoposide­resistant K/VP.5 compared to parental K562 cells. We hypothesized that TOP2α/90 was the translation product of novel alternatively processed pre­mRNA, confirmed by 3’­RACE, PCR, and sequencing. TOP2α/90 mRNA includes retained intron 19 which harbors an in­frame stop codon, and two consensus poly(A) sites. The processed transcript is polyadenylated. TOP2α/90 mRNA encodes a 90,076 Da translation product missing the carboxyl­terminal 770 amino acids of TOP2α/170, replaced by 25 unique amino acids through translation of the exon 19/intron 19 ‘readthrough’. Immunoassays, utilizing antisera raised against these unique amino acids, confirmed that TOP2α/90 is expressed in both cell types, with overexpression in K/VP.5 cells. Immunodetection of Complex of Enzyme­to­DNA (ICE) and single cell gel electrophoresis (Comet) assays demonstrated that K562 cells transfected with a TOP2α/90 expression plasmid, exhibited reduced etoposide­mediated TOP2α­DNA covalent complexes and decreased etoposide­induced DNA

damage, respectively, compared to similarly treated K562 cells transfected with empty vector. Since TOP2α/90 lacks the active site tyrosine (Tyr805) of full length TOP2α, these results strongly suggest that TOP2α/90 exhibits dominant­negative properties. . In separate studies the TOP2α/90 mRNA splice variant was found to be expressed in most human tissues suggesting that this novel protein isoform may play a role in both intrinsic chemosensitivity as well as in acquired resistance. Further studies are underway to characterize the mechanism(s) by which TOP2α/90 plays a role in acquired resistance to etoposide and other TOP2α targeting agents. In addition, future studies will be directed to examine the RNA processing mechanism(s) operational that suppress intron 19 splicing in TOP2α pre­mRNA. #3182 Cytoskeletal modulation results in increased tumor survival and drug resistance through attenuation of p53 dependent apoptosis. Victoria E. Wang,1 John Doench,2 David Root,2 Rene Bernards,3 Jeffrey Settleman,4 Frank McCormick1. 1UCSF, San Francisco, CA; 2Broad Institute, Cambridge, MA; 3Netherland Cancer Institute, Amsterdam, Netherlands; 4Genentech, South San Francisco, CA. One of the major challenges to eradicating cancer involves the evolution of drug resistant clones and persistence of residual disease that escapes our current limit of detection but may proliferate upon cessation of therapy. These microscopic foci of residual diseases often exhibit stem­ cell like properties and are intrinsically more resistant to drug therapy. A better understanding of the mechanisms underlying the innate drug resistance of these cellular populations may lead to improved treatment strategies, resulting in more durable remissions and ultimately improved patient survival. We utilize functional genomic platforms to identify mechanisms responsible for the persistence of drug tolerant cells. A genome wide shRNA enrichment screen was performed using the c­Met addicted cell line GTL­16 to identify genes whose knockdown conferred DTP survival. Inhibition of the RhoA­ROCK­myosin pathway promotes drug resistance in a variety of tumor models, including those treated with either targeted therapy or conventional chemotherapy. Furthermore, pharmacological validation using multiple small molecule inhibitors of ROCK1 phenocopied both myosin heavy chain (MYH9) and light chain (MYL12)

shRNA knockdown. More recently, utilizing a combination of gene expression and biochemical approaches, we have identified attenuation of p53 induced apoptosis to be a key event in mediating survival of these drug tolerant persisters. Reactivation of p53 using nutlin results in increased cell death. These findings demonstrate that modulation of cytoskeleton is an important, but underappreciated, mechanism of drug resistance across many tumor types. These downstream effectors may serve as novel therapeutic targets for intervention and also biomarkers to stratify patients and their response to treatment. #3183 Proteomic characterization of aromatase inhibitor resistant mammospheres reveal the presence of a novel nuclear chaperone. Syreeta L. Tilghman,1 Jamal Pratt,2 Shawn D. Llopis,2 A. Michael Davidson,1 Rashidra R. Walker,1 Patrick Carriere,3 Ian R. Davenport,2 Wensheng Zhang,2 Karen Zhang2. 1Florida A&M University, Tallahassee, FL; 2Xavier University of Louisiana, New Orleans, LA; 3Morehouse School of Medicine, Atlanta, GA. Postmenopausal women with early­stage metastatic estrogen­dependent breast cancer are generally treated with aromatase inhibitors (AIs) (e.g., letrozole). However, acquired resistance remains a major clinical obstacle. Previously, our group revealed a global proteomic signature of a letrozole­resistant cell line (LTLT­Ca) associated with hormone independence, enhanced cell motility and epithelial to mesenchymal (EMT). Given recent evidence suggesting a convergence of EMT and cancer stem cells (CSC), we chose to utilize a two­dimensional (2D) vs three­dimensional (3D) culture system to compare the proteome of LTLT­Ca cells, as 3D culture not only enriches for CSC, but more accurately recapitulates the tumor microenvironment, morphology, function and response to therapy compared to conventional 2D culture. We hypothesize utilizing a novel systems biology approach may reveal previously unconsidered molecular changes that could aid in understanding complex signaling networks and be exploited as therapeutic targets. To address this hypothesis ovariectomized immunocompromised female mice were inoculated in the mammary fat pad with LTLT­Ca or letrozole sensitive cells (AC­1) and intratumoral putative CSC marker expression was assessed by immunohistochemistry.

Results indicate LTLT­Ca tumors were CD44high/CD24low while AC­1 tumors were CD44­/CD24low. Mammosphere formation assays were conducted and LTLT­Ca cells formed mammospheres at a 3.4­fold higher index than AC­1 cells. A quantitative proteomic analysis of whole cell lysates from LTLT­Ca (2D adherent cells) versus LTLT­Ca (3D mammospheres) was conducted. Results identified significant protein expression changes within a panel of 1010 proteins; 173 were upregulated and 186 downregulated (p1.20). Additionally, functional enrichment analyses were performed and 19 gene ontology (GO) terms and one KEGG pathway (hsa03010:Ribosome) were over­ represented (BH adjusted p­value < 0.01) by the cognate genes. Notably, there was a 35.04­fold increase in midasin (MDN1), a nuclear chaperone protein required for maturation and nuclear export of pre­60S ribosome subunit. Increased MDN1 expression was strongly correlated with highly tumorigenic breast cancer spheres. Additionally, Kaplan­Meier survival plots demonstrate that increased MDN1 levels were positively correlated with decreased relapse free survival in estrogen receptor negative breast cancer, and is constitutively expressed as breast tumors progress from atypical ductal hyperplasia to ductal carcinoma in situ to invasive, metastatic breast cancer. The TGCA database was interrogated and MDN1 was frequently amplified or mutated in breast tumors. Taken together our study for the first time implicates a role for ribosomal assembly in AI resistant cells enriched for CSCs and underscores a potential for MDN1 in the progression and responsiveness to therapy. #3184 Autophagy regulation by vacuolar­ATPases: A contributing mechanism of chemo­resistance in ovarian cancer. Arpita Kulshrestha, Gajendra K. Katara, Safaa A. Ibrahim, Alice Gilman­ Sachs, Kenneth D. Beaman. Rosalind Franklin University of Medicine and Science, North Chicago, IL. In ovarian cancer (OVCA), the main obstacle in the clinical usefulness of platinum based drugs is the eventual emergence of chemo­resistant tumors. Exposure of cancer cells to platinum drugs such as cisplatin elicits a stress response that leads to the induction of coping mechanisms favoring cancer cell survival. Autophagy induction is the primary protective mechanism in cancer cells that also contributes to cisplatin resistance. A tightly regulated pH is imperative for autophagy and the role of major pH regulators is therefore vital to this process. Vacuolar

ATPases (V­ATPases) are the multi­subunit proton pumps that regulate intra/extra­cellular pH in cancer. Its `a2` isoform (V­ATPase­a2) is located at the plasma membrane and early/late endosomal compartments. We previously found that this isoform is overexpressed on cisplatin resistant cells (cis­R) and its inhibition sensitizes cis­R cells to platinum drugs. In continuation with our efforts, the present study was performed to understand what alterations in the autophagy process occur upon V­ ATPAse­a2 inhibition and how these contribute to cisplatin sensitization. Expression profiling of ovarian cancer cells lines (A2780­cis R and TOV­ 112D­cis R) revealed that upon a2 inhibition, the basal levels of autophagosome membrane protein, LC3B were enhanced compared to control cis­R cells as determined by flow cytometry and western blot analysis. Upon cisplatin treatment, the LC3B levels were further enhanced in a2 inhibited cis­R cells compared to control cells. Further, we observed a significantly increased expression of the autophagy protein, beclin­1 in these cells as compared to control cells. In addition, the inhibition of V­ATPase­a2v increased the protein levels of SQSTM1/p62, a known substrate for the autophagy process. The enhanced LC3B and P62 levels suggest an accumulation of the autophagosomes due to an altered autophagy flux upon V­ATPase­a2v inhibition. The study provides a rationale for the utility of V­ATPase­a2 inhibitors in combination with standard drugs as a novel strategy to improve the treatment efficacy of the chemoresistant ovarian cancer. #3185 Adipocyte­derived hydrogen peroxide promotes chemoresistance to daunorubicin in leukemia cells. Jean­Hugues Parmentier,1 Christina M. Dieli­Conwright,2 Steven D. Mittelman1. 1Children's Hospital of Los Angeles, Los Angeles, CA; 2University of Southern California, Los Angeles, CA. Background: Adipocytes protect acute lymphoblastic leukemia (ALL) cells from several chemotherapeutic drugs. This protection is associated with an oxidative stress response in adipocytes. Since adipocytes are known to release reactive oxygen species, and anthracyclines are sensitive to inactivation mediated by hydrogen peroxide (H2O2), we investigated whether adipocyte release of H2O2 into the micro­ environment might contribute to drug resistance of ALL cells. Methods: Pre­B lymphoblast/myeloblast BV173 cells in complete media

(CM) were co­cultured for 3 days in transwells over 3T3­L1 fibroblasts or adipocytes. Viable cells were determined by Trypan blue exclusion for BV173 cells. Leukemia Conditioned Media (LCM) was prepared from BV173 cultured for 48 hrs. Total antioxidant capacity was measured by ABTS­based assay. H2O2 released in the extracellular compartment of adipocytes was monitored with Amplex Red and ROS­Glo. Results: BV173 cells were protected from DNR in presence of 1 mM H2O2 (9.34±2.15x104 vs. 3.33±1.01x104 viable cells with and without H2O2, p=0.03). Interestingly, this protection was only observed when pyruvate was present in the culture media at equimolar concentrations with H2O2 (DNR: 4.14±2.14x104; DNR+pyruvate: 4.17±1.53x104; DNR+H2O2: 3.77±2.74x103; DNR+pyruvate+H2O2: 3.21±0.59x105, p=0.02). Adipocytes released H2O2 into the media measured with Amplex Red fluorescence in a time­dependent manner significantly more than fibroblasts (1.96­fold, p=0.001). LCM potentiated adipocyte H2O2 released (1.5­fold over CM, p=0.05). Human adipose tissue biopsies from breast cancer survivors also released H2O2 (4.37x103 RFU for biopsies vs. 0.65x103 RFU for HBSS, 90 min, n=5). Co­culture of adipocytes and BV173 cells increases the survival of BV173 to DNR treatment (1.55±0.34x105 vs. 6.88±2.91x103 viable cells with and without adipocytes, p=0.006). Adipocyte­mediated protection of BV173 cells was reversed in a dose­dependent manner by adding the H2O2­inactivating enzyme catalase to the extracellular media (7.28±1.44x104 vs. 15.58±3.48x104 viable cells with and without 500 U/ml catalase, p=0.01). Conclusion: These data show that adipocytes release H2O2, which in the presence of pyruvate, protects ALL cells from DNR. Further work is needed to determine whether adipocytes secrete significant amounts of H2O2 in the bone marrow micro­environment to contribute to anthracycline resistance. #3186 Taxane resistance in prostate cancer: A role for miRNA 181a. Cameron M. Armstrong, Chengfei Liu, Wei Lou, Allen C. Gao. UC Davis, Sacramento, CA.

Introduction: Docetaxel (DTX) is one of the primary drugs used for treating castration resistant prostate cancer (CRPC). Unfortunately, over time patients invariably develop resistance to DTX therapy and their disease will continue to progress. The mechanisms by which resistance develops are still incompletely understood. This study seeks to determine the involvement of miRNAs, specifically miR­181a, in DTX resistance in CRPC. Methods: Total RNA from parental C4­2B prostate cancer cells and DTX resistant C4­2B cells (C4­2B TaxR) was submitted for small RNA deep sequencing. Data was analyzed to ascertain which miRNAs expressions were most altered in C4­2B TaxR cells compared to parental cells. Having identified an increase in miR­181a in resistant cells, its expression was modulated in C4­2B and C4­2B TaxR cells by transfecting them with miR­181a mimics or antisense, respectively. Following transfection, cell number was determined after 48 h with or without DTX. Cross resistance to cabazitaxel induced by miR­181a was also determined. Western blots were used to determine ABCB1 protein expression and rhodamine assays used to assess activity. Phospho­p53 expression was assessed by western blot and apoptosis was measured by ELISA in C4­2B TaxR cells with inhibited miR­181a expression with or without DTX. Results: miR­181a is significantly upregulated in C4­2B TaxR cells compared to parental C4­2B cells as analyzed by small RNA sequencing. Overexpression of miR­181a in C4­2B cells confers DTX and cabazitaxel resistance. Knockdown of miR­181a in C4­2B TaxR cells re­sensitizes them to treatment with both DTX and cabazitaxel. miR­181a knockdown alone induced apoptosis in C4­2B TaxR cells which is further enhanced by DTX. We next assessed if miR­181a altered expression or activity of ABCB1, which is overexpressed/active in C4­2B TaxR cells and promotes resistance to DTX by pumping the drug out of cells. We found that miR­181a does not impact ABCB1 expression or activity. Since we previously demonstrated that phospho­p53 can modulate DTX sensitivity, we determined if miR­181a can alter p53 expression in C4­2B TaxR cells. Knockdown of miR­181a in C4­2B TaxR cells induced phospho­ p53 expression, suggesting that miR­181a induced resistance to DTX is mediated by inhibition of phospho­p53 expression. Conclusions: Overexpression of miR­181a in C4­2B TaxR cells contributes to their resistance to DTX and inhibition of miR­181a expression can restore treatment response. This is due, in part, to modulation of p53 phosphorylation and induction of apoptosis.

#3187 Role of exosome secretion in the survival of enzalutamide­resistant prostate cancer cells: Syntaxin 6 as a novel therapeutic target. Taylor C. Peak, Nicole P. Kasica, Gati K. Panigrahi, Sierra L. Patterson, Ravi Singh, Ashok K. Hemal, Rhonda L. Bitting, Gagan Deep. Wake Forest Baptist Medical Center, Winston­Salem, NC. Prostate cancer (PCa) is the most commonly diagnosed malignancy in the United States and remains the second leading cause of cancer deaths among American men. PCa deaths are typically the result of metastatic castration­resistant PCa (mCRPC). Recently, enzalutamide (Enz), an oral AR inhibitor, was approved for treating patients with mCRPC, and has significantly improved patient survival. But, invariably all PCa patients eventually develop resistance against Enz and die from mCRPC. Therefore, novel strategies aimed at overcoming Enz­resistance are urgently needed to improve the survival of PCa patients. Exosomes (extracellular vesicle, 40­150 nm in size) have previously been shown to communicate oncogenic signals and to promote growth and metastasis; however, their role in drug resistance has not been fully elucidated. Therefore, we set out to better understand exosomes role in the mechanism underlying Enz­resistance in PCa cells. We isolated exosomes secreted by Enz­sensitive (S) and ­resistant (R) PCa cells by ultracentrifugation. Complete characterization of these exosomes followed using nanoparticle tracking analysis (NTA), transmission electron microscopy, proteomics, and western blotting. MTT and trypan blue exclusion assays were used to evaluate cell viability. Results showed that Enz­resistant C4­2B­R cells secreted significantly higher amount (2.67 fold) of exosomes compared to sensitive C4­2B­S cells, but without any significant change in exosome size. Proteomics analysis revealed significantly lesser number and amounts of proteins loaded in exosomes secreted by C4­2B­R cells compared to C4­2B­S cells. Western blotting validated these results, and in general less loading was observed for several exosomal biomarker proteins (e.g. CD63, Lox, TSG101, and MMP9). Of note, AR­V7, a variant of the androgen receptor, was upregulated in C4­2B­R cells as well in their exosomes. In order to better understand the biological significance of increased exosome production with concomitant decreased protein expression, we administered C4­2B­ R­derived exosomes to C4­2B­S cells. Interestingly, exosomes from resistant cells significantly reduced the survival of C4­2B­S cells. This suggested not only that the resistant cells were utilizing exosomes to

secrete Enz, but that these exosomes were being taken up by the sensitive PCa cells, thus leading to enhanced sensitive cell death. The inhibition of exosome biogenesis in C4­2B­R cells using GW4869 (20 µM) and dimethyl amiloride (1 µg/mL) strongly decreased their cell viability, and also increased their sensitivity towards Enz treatment. We also observed strong upregulation of syntaxin 6 in Enz­resistant C4­2B­R cells. Together, these results identified a unique mechanism underlying drug­ resistance, and also offer a novel target to treat Enz­resistant CRPC. #3188 ER­stress induced translational regulation by RGS2 leads to tumor­ promoting microenvironment and chemoresistance. Hye­Young Min, Shin­Hyung Park, Ho Jin Lee, Jaebeom Cho, Myungkyung Noh, Seung Yeob Hyun, Ho­Young Lee. Seoul National Univ., Seoul, Republic of Korea. Lung cancer is the leading cause of the cancer­related death worldwide. Despite extensive efforts for the cure of lung cancer, the effectiveness of currently available therapeutic regimens has been marginal and limited, and recurrence is still an inevitable consequence of anticancer therapies. Therefore, mechanistic understanding on the progression and relapse of lung cancer and finding out relevant targets are essential for development of efficacious therapeutic strategies. We have investigated mechanisms of chemoresistance by using subsets of lung cancer cell lines carrying acquired resistance to chemotherapies. These chemoresistant sublines of NSCLC cells exhibited a prominent downregulation of in vitro tumor activities, including proliferation, metabolism, and protein synthesis, but displayed increased tumor growth in vivo. Additional studies revealed that the chemoresistant sublines experienced increased secretion of various soluble factors that led to increases in proliferation and stemness of drug­naïve cancer cells and recruitment of macrophages, establishing tumor microenvironment (TME) prone to tumor recurrence. We further demonstrate a pivotal role of regulator of G protein signaling 2 (RGS2) in tumor­promoting cell­cell communications, ultimately leading to chemoresistance. RGS2 disrupted chemotherapy­induced apoptosis and secretion of soluble factors involved in cellular communications in TME by regulating unfolded protein responses, a typical cellular event associated with downregulation of protein synthesis. Mechanistically, RGS2 induced proteasome­mediated protein degradation of a component of eukaryotic translation initiation factor complexes through direct

interaction, resulting in blockade of the CAP­dependent protein translation. RGS2 expression was elevated in tumors derived from patients with lung cancer compared with normal counterparts and significantly associated with poor clinical outcomes. These findings suggest that RGS2 is a novel biomarker responsible for chemoresistance by establishing tumor­promoting TME and can be a potential therapeutic target for the treatment of lung cancer with chemoresistance. #3189 Neuronal transcription factor BRN2 is an androgen suppressed driver of neuroendocrine differentiation in prostate cancer. Jennifer L. Bishop,1 Daksh Thaper,1 Sepideh Vahid,1 Ravi S. Munuganti,1 Paul Ahn,1 Alastair Davies,1 Kirsi Ketola,1 Ka Mun Nip,1 Dong Lin,1 Yuzhuo Wang,1 Himisha Beltran,2 Amina Zoubeidi1. 1Vancouver Prostate Ctr., Vancouver, British Columbia, Canada; 2Weill Cornell Medical College, New York, NY. Neuroendocrine prostate cancer (NEPC) is an incurable, rapidly progressing and lethal disease. NEPC is increasingly recognized as a highly therapy resistant tumor variant that evolves from castration­ resistant prostate cancer (CRPC) in a subset of patients treated with potent androgen receptor (AR) pathway inhibitors like enzalutamide (ENZ). Importantly, mechanisms by which the AR directly controls the emergence of NEPC from CRPC under the selective pressure of ENZ remain elusive. As the AR is the cornerstone therapeutic target in men with CRPC, understanding its contribution to the development of NEPC is critical to better implement current standard­of­care therapies such as ENZ, and to identify novel therapeutic targets for this incurable disease. Hallmarks of NEPC are resistance to ENZ and the loss or reduced activity of the AR. Thus, we hypothesized that a consequence of ENZ treatment and resistance in CRPC is relief of AR­mediated suppression of factors that drive NEPC. To investigate this, we developed a model of prostate cancer that mimics clinical progression to CRPC and ENZ resistance (ENZR CPRC). Mirroring what is observed in some patients who progress on ENZ, 25% of our ENZR CRPC tumors and derived cell lines showed strong reduction in classic activity of the AR and a NEPC phenotype. By interrogating NEPC­like ENZR CRPC and ENZ­treated CRPC tumors and cell lines with RNA­Seq and mechanistic in vitro and in vivo studies, as well as human tumors with RNA­Seq and IHC, we

identified the master neural transcription factor BRN2 as a central and clinically relevant driver of NEPC differentiation. Specifically, we found AR binding in the BRN2 enhancer directly represses BRN2 transcription and this release drives NEPC marker expression and aggressive growth of ENZR CRPC. Our data also reveal a striking overlap of AR and SOX binding motifs in the enhancer region of BRN2 creating a competitive binding scenario between AR and SOX2, another cell­fate determining transcription factor associated with NEPC. We discovered that upregulation of BRN2 further enhances SOX2 expression and that a BRN2­SOX2 interaction contributes to NEPC differentiation. Importantly, we found BRN2 is highly expressed in human NEPC and metastatic CRPC, especially in patients with low AR activity, highlighting its clinical relevance to disease that is difficult to treat with mainstay therapies. These data underscore the consequences of potent AR inhibition in CRPC, revealing a novel mechanism of AR­dependent control of NEPC via direct suppression of BRN2. This work uncovers BRN2 as a clinically relevant potential therapeutic target to prevent emergence of NEPC from ENZR CRPC. #3190 Using a genome­wide shRNA screen to investigate the mechanism of stroma­conferred gemcitabine resistance in pancreatic ductal adenocarcinoma. Graham D. Mills, Nicolas Erard, Frances M. Richards, Gregory J. Hannon, Duncan I. Jodrell. Cancer Research UK Cambridge Institute, Cambridge, United Kingdom. The purpose of this study is to identify mechanisms of resistance to gemcitabine in pancreatic ductal adenocarcinoma (PDAC) using an in vitro coculture system. The inherent resistance of PDAC to first line treatments such as gemcitabine has been attributed to the dense fibrotic stroma. Targeting stromal components, in particular cancer­associated fibroblasts (CAFs), the activated form of pancreatic stellate cells (PSCs), is a promising avenue for therapeutic intervention. We have demonstrated previously the induction of gemcitabine resistance when co­culturing KRAS G12D; p53 R172H; Pdx­cre (KPC) mouse PDAC­derived tumour cells with αSMA+ fibroblast­like cells from PDAC tumour (FLCs), in comparison to PDAC cells co­cultured with PSCs or in monoculture. This resistance­inducing effect is specific to cells of mesenchymal phenotype, is transient in nature and a product of cell­cell contact. To investigate the

exact mechanism by which PDAC cells become resistant to gemcitabine in coculture, we undertook a genome­wide shRNA depletion viability screen, infecting KPC mouse PDAC cells with a pooled shERWOOD UltramiR shRNA library both in mono­ and co­culture with FLCs, in the presence and absence of gemcitabine. Next­generation sequencing and differential expression analysis, using DESeq2, identified shRNAs depleted through 6 cycles of gemcitabine treatment in coculture, indicating they target genes driving the resistance effect. Pathway analysis of differential expression using MetaCore implicated DNA damage repair and protein modification pathways in the gemcitabine resistance effect. Depletion of p97, ATR, or Chk1 all sensitised PDAC cells to gemcitabine in coculture, corroborating findings with translational value in previous studies. Further evaluation is ongoing using validation shRNA dropout assays in combination with targeted literature reviews to collate a candidate list of targetable genes with translational value in preventing and combating chemotherapy resistance in patients with PDAC. #3191 Daytime blue­enriched LED light­induced circadian amplification of the nighttime melatonin signal increases sorafenib sensitivity in human hepatocellular carcinoma via enhanced suppression of the Warburg effect. Robert T. Dauchy, David T. Pointer, Aaron E. Hoffman, Melissa A. Wren­Dail, Shulin Xiang, Lin Yuan, David E. Blask, Victoria P. Belancio, Steven M. Hill. Tulane Univ. School of Medicine, New Orleans, LA. Over 36,000 people in the United States will be diagnosed with hepatocellular carcinoma (HCC) in 2016, the second leading cause of cancer death worldwide. Metabolic pathways within the liver and in HCC are highly regulated by the central circadian clock in the suprachiastmatic nucleus (SCN). The SCN drives nighttime production of the circadian anti­cancer hormone melatonin by the pineal gland in rats and humans. We have shown that the nighttime circadian melatonin signal suppresses the Warburg effect (aerobic glycolysis) in human breast cancer xenografts and that blue­enriched light (460­480 nm) from LEDs at daytime (bLAD), amplifies the nighttime circadian melatonin signal by 7­fold in rats over cool white fluorescent (CWF) lighting. Here we tested whether daytime exposure of tissue­isolated HepG2 xenograft­bearing male nude

rats to bLAD amplifies the nighttime melatonin signal to increase tumor sensitivity to nighttime administered sorafenib (FDA­approved multi­ kinase inhibitor) treatment via enhancing suppression of the Warburg effect. Animals were randomized to 6 subgroups (n = 4): CWF Groups I, 12 h light:12 h dark (LD,12:12) (CWF + vehicle) and II (CWF + sorafenib); bLAD Groups III (LD,12bLAD:12) (bLAD + vehicle), IV (bLAD + sorafenib), V (bLAD + S20928 MT1/MT2 receptor blocker), and VI (bLAD + S20928 + sorafenib). Drug treatments began when tumors were 2.5 g estimated weight. Plasma nighttime melatonin levels were 7­fold higher in Groups III­VI, compared to Groups I and II. Tumor latency­to­onset of growth and growth rates were markedly delayed and decreased, respectively, in Group III compared to Group I. Sorafenib induced tumor regression at a rate that was 2­fold higher in the bLAD­ vs CWF­exposed rats. Tumor glucose uptake and lactate production (Warburg effect) at the mid­dark phase were significantly reduced in CWF­exposed rats receiving sorafenib vs vehicle. In vehicle­treated bLAD­exposed rats, the Warburg effect was significantly decreased vs CWF­exposed rats receiving vehicle. In bLAD + sorafenib­treated rats, the Warburg effect was reduced by an additional 51% (glucose uptake) and 89% (lactate production), respectively, vs CWF + sorafenib­treated rats. Melatonin receptor blocker S20928 completely prevented the effects of bLAD and bLAD + sorafenib on the Warburg effect and made these tumors completely resistant to sorafenib­induced tumor regression. These findings are the first to show in vivo that: 1) nighttime circadian sorafenib therapy inhibited the Warburg effect and induced HepG2 tumor regression under CWF lighting conditions, and 2) the bLAD­amplified nocturnal circadian melatonin signal increased tumor sensitivity to sorafenib­induced regression by enhancing a melatonin receptor­mediated suppression of the Warburg effect. #3193 Treatment of high Trop­2­expressing triple­negative breast cancer (TNBC) with sacituzumab govitecan (IMMU­132) overcomes homologous recombination repair (HRR) rescue mediated by Rad51. Thomas M. Cardillo, Ali A. Mostafa, Diane L. Rossi, Donglin Liu, Chien­Hsing Chang, Robert M. Sharkey, David M. Goldenberg. Immunomedics, Inc., Morris Plains, NJ. Purpose: IMMU­132 is an antibody­drug conjugate composed of a humanized anti­Trop­2 IgG conjugated via a cleavable linker to SN­38, a

topoisomerase I inhibitor and active component of irinotecan. It is currently under clinical investigation in a range of Solid tumors (NCT01631552). We investigated the hypothesis that IMMU-132, through its targeting of Trop-2 in Solid tumors, will be superior to irinotecan in overcoming RadS1-mediated HRR repair of DNA breaks in TNBC tumors with high Trop-2 expression. Methods: RadS1 and DNA-breaks (Y-H2A.X) were determined by Western blot. Cells with different Trop-2 levels were exposed to IMMU 132 for 24 h (25 - 100 nM SN-38 equivalents), including squamous cell lung carcinoma (SK-MES-1; "30,000 Trop-2/cell) and TNBC (HCC1806, ~90,000 Trop-2/cell and MDA-MB-231, -30,000 Trop-2/cell). Also, two Trop-2-transfectants of MDA-MB-231, designated C13 and C39 (4- and 25-fold higher Trop-2 levels, respectively), were likewise exposed to IMMU-132. Mice bearing MDA-MB-231, C13, or C39 tumors were treated with irinotecan (MTD, 40 mg/kg; q2dx5) or IMMU-132 (0.5 mg; 9 Jg SN-38 equivalent, twice wkly x 4). Tumors were measured and mice weighed twice weekly. Study Survival endpoint was tumor progression to >1.0 cm”. Results: SK-MES-1 and HCC1806 are sensitive to IMMU-132 therapy whereas MDA-MB-231 is resistant. IMMU-132 mediated a >2-fold

increase in RadS1 levels in MDA-MB-231 cells, but had no effect in SK MES-1 or HCC1806. At 25 nM IMMU-132, there were lower levels of DNA breaks detected in MDA-MB-231 relative to SK-MES-1 and

HCC1806 (2-fold increase in MDA-MB-231 vs. 23-fold). At higher concentrations of IMMU-132 (100 nM), all 3 cell lines demonstrated similar levels of DNA breaks (~5-fold above background), suggesting that higher levels of SN-38 can overcome RadS1-mediated repair. Both the C13 and C39 clones had a similar response as parental MDA-MB-231 upon IMMU-132 exposure. Mice bearing MDA-MB-231, C13, or C39 tumors treated with irinotecan demonstrated significant improvements in median survival times (MST) compared to saline (P70d vs. 28d for C39; P-0.0007), supporting the hypothesis that IMMU-132 is able to deliver more SN-38 to tumors with high Trop-2 than can be achieved by irinotecan, and can thus Overcome RadS1-mediated HRR.

Conclusion: IMMU-132, with its unique SN-38—delivery platform, has the

potential to provide clinical benefit both to chemo­sensitive solid tumors with low Trop­2 expression, as well as to chemo­resistant tumors with high Trop­2 expression. #3194 A new mechanism of drug resistance in cancer: extracellular ATP­ induced resistance through ATP internalization and upregulation of protein phosphorylation in Akt and ERK pathways. Xuan Wang, Yunsheng Li, Yanrong Qian, Yanyang Cao, Xiaozhuo Chen. Ohio University, Athens, OH. The opportunistic uptake of extracellular molecules has been named as a key emerging hallmark of cancer metabolism[1]. Extracellular ATP (eATP) levels of various cancer types are 103 to 104 times higher than those in their corresponding normal tissues[2]. However, the biological significance of the high ATP concentrations is not clear. We recently reported that cancer cells uptake extracellular ATP via different endocytoses to enhance growth, survival, and drug resistance to tyrosine kinase inhibitors (TKIs)[3 ,4]. We hypothesized that eATP induces resistance to TKIs by endocytoses­mediated internalization of eATP, which competes with TKIs for the ATP­binding site of receptor TKs (RTKs), phosphorylating and activating RTKs and RTK­mediated signaling pathways. In contrast to previous reports that synthesized intracellular ATP (iATP) elevation contributed acquired drug resistance, here we report a novel intrinsic drug resistance in which eATP was internalized and substantially increased iATP levels and promoted cancer cell drug resistance to the TKI sunitinib in human NSCLC A549 cells. The iATP level elevation and drug resistance were mediated primarily by macropinocytosis. The resistance was reduced when macropinocytosis was suppressed by inhibitors or an siRNA knockdown of a key micropinocytosis protein PAK1. Intracellularly, the elevated iATP upregulated phosphorylation of PDGFR and proteins/enzymes in the PDGFR­mediated Akt­mTOR and Raf­ERK signaling pathways, resulting in reduced apoptosis triggered by sunitinib. Furthermore, both in vitro and in A549 tumors, eATP partially restored phosphorylation levels of PDGFR and PDGFR­mediated proteins/enzymes suppressed by sunitinib. The resistance cannot be accounted for by the overall purinergic receptor­mediated signaling, glycolysis, or mitochondrial OXPHOS. These results strongly suggest that the eATP­elevated intracellular ATP

levels reversed the inhibition of TKIs by using the mechanism we hypothesized, linking for the first time the ATP­rich tumor microenvironment with cancer drug resistance. All these findings significantly expand our understanding of the roles of extracellular ATP in cancer, and offer new anti­drug resistance targets. 1. Pavlova N, Thompson C. Cell Metab. (2016) 23:27­47. 2. Falzoni et al., Interface Focus.(2013) 3: 20120101. 3. Qian Y, Wang X, et al., Cancer Lett. (2014)351: 242­5. 4. Qian Y, Wang X et al., Mol Cancer Res (2016) 14:1087­96 #3195 DYRK1B inhibitors prevent pharmacologic quiescence and sensitize lung cancers to EGFR inhibitors. Maria Vilenchik,1 Alexandra Kuznetsova,1 Michael Frid,1 Yuriy Gankin,1 Marc Duey,1 Neal Rosen2. 1Felicitex Therapeutics Inc., Newton, MA; 2Memorial Sloan Kettering Cancer Center, New York, NY. Many oncoproteins that activate signaling dysregulate cell growth by activating the cyclinD/cdk4, cdk6 complex and abrogate in the G1 checkpoint (mutant RAS, BRAF, PIK3CA, EGFR etc.) Inhibition of these oncoproteins inhibits cyclinD expression and kinase activity as well as RB phosphorylation and results in G0/G1 arrest and apoptosis. It has been posted that cells arrested in G0/G1 are protected from cell death. DYRK1B kinase plays an important role in physiologic quiescence by maintaining the viability of cells arrested in the G0/G1 phases of the cell cycle. DYRK1B regulates the G0/G1 transition by phosphorylating cyclin D1 and p27, whereby destabilizing cyclin D1 and stabilizing p27. The balance between these two proteins is essential for maintenance of cells in G0 (quiescent) state. DYRK1B is sparsely expressed in healthy tissues yet is overexpressed in patient samples with variety of neoplasms, including NSCLC. Cancer and normal cells exhibit differential sensitivity to DYRK1B inhibition. Whereas DYRK1B appears essential for survival of cancer cells either endogenously expressed or upregulated, its inhibition in healthy cells was not cytotoxic and had no effect on the cell cycle distribution. We hypothesized that if DYRK1B is required for maintenance of quiescence, its inhibition would enhance the effects of inhibitors of oncogenic signaling. We used a selective, ATP competitive DYRK1B inhibitor to test this idea. EGFR inhibitors are effective in non­ small cell lung cancers with mutant EGFR and induce G0/G1 block and

some apoptosis. These drugs significantly extend survival, but acquired resistance almost always supervenes. In vitro, the anti­proliferative response of lung tumors with mutant EGFR to EGFR inhibitors was shown to be primarily due to the entry of cells into a quiescent (G0) state associated with an induction of DYRK1B expression. Moreover, addition of a DYRK1B inhibitor reversed the quiescent state in cells exposed to EGFR inhibitors. Combined therapy of EGFR TKIs and the DYRK1B inhibitor significantly enhanced the antitumor response compare to either drug alone. Specifically, it is shown that treatment with osimertinib results in an increase of cells in G0 state: 63% as compared to non­treated cells, 29%. The induction of quiescence is prevented when cells are treated with the combination of osimertinib and DYRK1B inhibitor and massive apoptosis is observed: 62% compared to 12% with osimertinib alone. It is important to note that co­treatment with DYRK1B inhibitor not only prevented the entry of cancer cells into quiescence but also depleted pre­existent reservoir of quiescent cancer cells. The results suggest that inhibition of EGFR causes tumor cells to enter into a DYRK1B­dependent quiescent state in which DYRK1B is required for their survival. Inhibition of DYRK1B dramatically enhances the antitumor activity of EGFR inhibition and may improve outcome in patients. #3196 Genetic and pharmacologic approaches to overcome epithelial to mesenchymal mediated chemoresistance in breast cancer. Michael J. Crowley, Nasser Altorki, Vivek Mittal, Dingcheng Gao. Weill Cornell Medical College, New York, NY. Development of resistance to conventional chemotherapy remains a major barrier to effective treatment of breast cancer. We and others have recently demonstrated in breast and pancreatic cancer models that epithelial­mesenchymal transition (EMT) may not be a critical mediator of cancer metastasis, however, it contributes to cancer drug resistance (Fischer et al. Nature 2015; Zeng et al Nature 2015). Importantly, blocking EMT through miR­200 family abrogated chemoresistance, indicating potentials for clinical translation. Using our novel EMT lineage tracing system, we have performed both genetic (CRISPR/Cas9 mediated genome­wide targeted mutagenesis) and pharmacological (high­ throughput small molecule libraries) screens, to identify potential candidates to overcome EMT­mediated chemoresistance in breast cancer

metastasis. The identified molecules provide not only novel mechanistic insights but also attractive anti­metastatic strategies for breast cancer treatment and the design of future clinical trials. #3197 Splicing factors and the role of Navitoclax in drug­resistant ovarian cancer. Helen M. Coley,1 Laura Lattanzio,2 Ornella Garrone,2 Marco Merlano,2 Daniela Vivenza,2 Cristiana Lo Nigro,2 Nelofer Syed,3 Alistair Thompson,4 Bhavya Rao,4 Tim Crook5. 1Univ. of Surrey, Guildford Surrey, United Kingdom; 2Ospedale Carle, Cuneo, Italy; 3Imperial College, London, United Kingdom; 4Dundee Medical Centre and University, Dundee, United Kingdom; 5Southend Hospital, Southend, United Kingdom. Alternative RNA splicing allows processing of a pre­mRNA into various mature mRNAs. In this way multiple protein products can be derived from a single gene. In cancer it is known that specific splice variants may facilitate malignant transformation and therapeutic resistance. Our current studies focus on the problem of drug resistance in ovarian cancer and we have approached this in a variety of ways. Following sequencing analysis of a panel of ovarian cancer cell lines we identified the RNA splicing factors TCERG and SRSF2 as being reduced in drug resistant ovarian cancer cell lines, compared with their drug sensitive counterpart. TCERG is important in regulating the splicing of variant forms of the BClx gene: BClxL (long form) and BClxs (short form). There are clear implications for drug resistance whereby low TCERG results in reduced BClx splicing to yield the pro­apoptotic BClxs form. In the SKOV­3 human ovarian cancer cell line panel both paclitaxel resistant SKOV­3TaxR and SKOV­ 3CR carboplatin resistant cells showed reduced levels of TCERG mRNA using qPCR analysis. In addition, qPCR analysis for BClxl and BClxs levels indicated increased ratio of long:short form with drug resistance: SKOV­3 parent line ratio 1.0; SKOV­3TaxR 1.8; SKOV­3CR 1.65. Western immunoblotting showed relatively decreased levels of Bax protein in drug resistant versus drug sensitive cell line in line with a reduced apoptotic reduced. We then explored the use of Navitoclax (ABT0263), the small­molecule Bcl­2 family protein inhibitor for Bcl­xL, Bcl­2 and Bcl­w in our cell line panel. Navitoclax in combination with

paclitaxel for 48h gave rise to significantly increased apoptotic response in drug­resistant versus drug sensitive ovarian cancer cell line counterparts, using the Annexin V assay with PI, indicative of re­ sensitisation to paclitaxel. Synergistic effects were also seen for the parental cell line but the effects were more marked for the resistant cell lines. Our future work will also involve the use of clinical biopsies of ovarian cancer cases. #3198 Using a genome­wide CRISPR­Cas9 knockout library to identify therapeutic combinations in oral cancer. Megan Ludwig, Andrew Birkeland, Sai Nimmagadda, Sue Foltin, Aditi Kulkarni, Hui Jiang, Thomas Carey, Chad Brenner. University of Michigan, Ann Arbor, MI. Oral squamous cell carcinoma (OSCC) has remained a disease with poor survival for decades with few novel treatment options. We anticipate great potential for personalized targeted therapies, facilitated by recent characterizations of the mutational landscape of OSCCs. Combinations of targeted therapies may have greater efficacy through inhibiting compensatory pathways. For example, targeting EGFR has had limited success. To identify synergistic combinations with EGFR­targeted therapy, we introduced Genome­scale CRISPR­Cas9 Knock­Out (GeCKO) libraries into OSCC cell lines. CRISPR­Cas9 generates individual genetic knockouts through targeted gene editing. We used a pool of CRISPRs targeting over 18,000 genes to perform genome­scale screening for drivers of sensitivity to EGFR­targeted therapy. Upon selection of the OSCC GeCKO pool, we identified gene knockouts in the FGFR pathway that increased sensitivity to the EGFR inhibitor gefitinib. Using resazurin viability assays we tested combinations of EGFR and FGFR inhibitors in 14 OSCC cell lines. Six/14 (43%) of the cell lines were responsive to the combination, indicating that the FGFR pathway is an alternate mechanism of resistance to EGFR­targeted therapy in some tumors. In complement, we again used CRISPR­Cas9 to generate a syngeneic EGFR knockout (KO) OSCC cell line. The parent OSCC cell line is responsive to the EGFR and FGFR dual inhibition, and the EGFR KO derivative retains sensitivity to FGFR­targeted monotherapies. We expect that this EGFR KO cell line will be a useful tool in further evaluating the compensatory mechanism of the FGFR pathway. OSCC remains a common and frequently lethal cancer with great potential for

the development of personalized targeted therapies. Here, we describe the use of genome­wide CRISPR­Cas9 library to discover the synergistic combination of EGFR and FGFR inhibition. Further investigation suggests the FGFR pathway is a common compensatory mechanism to EGFR inhibition. We hope to use this approach to identify additional compensatory mechanisms of resistance to targeted therapies with the eventual goal of translating these findings to clinic. #3199 Diverse non­FLT3 molecular mechanisms of crenolanib resistance. Haijiao Zhang,1 Anna M. Schultz,1 Samantha Savage,1 Daniel Bottomly,1 Beth Wilmot,1 Shannon K. McWeeney,1 Christopher Eide,1 Hoang Ho,2 Yee L. Lam,2 Richard Sweat,2 Jaime Faulkner,1 Evan Lind,1 Jeffrey W. Tyner1. 1Oregon Health & Science University Knight Cancer Institute, Portland, OR; 2Arog Pharmaceuticals, Inc., Dallas, TX. FMS­like tyrosine kinase 3 (FLT3) activating mutations are primary molecular targets for the treatment of acute myeloid leukemia (AML) due to high prevalence and unfavorable prognosis. Several type II FLT3 inhibitors have shown clinical benefits but acquisition of secondary FLT3 mutations was reported as a common mechanism of resistance. Previous studies showed that crenolanib, a type I FLT3 inhibitor, had clinical activity without acquisition of secondary FLT3 mutations. To identify the molecular mechanisms associated with crenolanib sensitivity and resistance, we performed exome sequencing on crenolanib treated patients with FLT3­mutant multiply relapsed or refractory AML. Baseline mutational analysis revealed distinct mutational profiles in patients with prior FLT3 inhibitor exposure, especially in the following pathways: NRAS, IDH1, WT1 and RUNX1. Further analysis of patients who had poor response to crenolanib showed mutations in other cell signaling genes such as NRAS and PTPN11. Variant allele frequency (VAF) analysis showed that these mutations sometimes occurred in subclones independent of the FLT3 mutation­ bearing clone or sometimes were acquired by the FLT3 mutation­bearing clone. To characterize the influence of these mutations on crenolanib sensitivity, we transduced genes of interest into cell lines harboring FLT3 activating mutations and treated these cells with crenolanib at various concentrations. Significantly increased crenolanib IC50 and IC90 were observed in NRAS G12V MOLM14 and PTPN11 A72D/FLT3 D835

Ba/F3 cells relative to the respective control NRAS WTMOLM14 and PTPN11 WT/FLT3 D835 Ba/F3 cells. Notably, addition of trametinib restored crenolanib sensitivity and demonstrated synergistic cytotoxic effects on cells with FLT3 and NRAS or PTPN11 mutations. In addition, increases in TET2 nonsense/frameshift mutations were observed in patients who did not respond to crenolanib. VAF analysis demonstrated that TET2 mutations co­occurred with FLT3 mutations. We also observed that bone marrow cells from FLT3­ITD knock­in/TET2 knock­out mice are resistant to crenolanib at low concentrations, but remain sensitive to azacytidine at the same level as FLT3­ITD knock­ in/TET2 WT cells. The remaining patients exhibited a diverse spectrum of secondary mutations associated with chromatin modifiers, cohesion, spliceosomes and transcription factors which mostly expanded during treatment, suggesting an elaborate genetic/epigenetic mechanism of resistance to crenolanib. Our data suggest that comprehensive sequencing should be carried out on patient samples prior to treatment to identify and pre­emptively target problematic clones. In addition, even with high VAF FLT3 mutations, although FLT3 inhibitor monotherapy provide some clinical benefit, combining agents targeting cooperative lesions will be imperative to eradicate both the dominant clone and resistant subclones and improve patient responses.

Novel Molecular Targets 2 #3200 Targeting PHF5A for the treatment of glioblastoma and other Myc­ driven cancers. Andrew J. Mhyre,1 Shanon Turnbaugh,1 Shelli M. Morris,1 Hu Xin,2 Patrick J. Paddison,1 Marc Ferrer,2 James M. Olson1. 1Fred Hutchinson Cancer Research Center, Seattle, WA; 2National Institutes of Health, Bethesda, MD. Glioblastoma multiforme (GBM) is one of the most aggressive and invasive types of brain cancer, but targeted treatment options remain elusive. The standard of care (surgery chemotherapy and radiation) falls far short of where it should be with two­year survival rates less than 10%. Using stem cell isolates from GBM patients, we found that perturbing PHF5A, a component of the spliceosome machinery, was lethal and caused hundreds of genes to be mis­spliced. These mis­splicing events included both exon skipping and intron inclusions. In contrast, similar levels of PHF5A suppression in normal control stem cells and astrocytes failed to induce cell death and mis­splicing, indicating that PHF5A plays a specific role in the cancer biology. Moreover, when normal astrocytes were transformed with the Myc oncogene, they became sensitive to PHF5A perturbation. Taken together, these results suggested that specifically inhibiting PHF5A would be an effective therapy for glioblastoma and other Myc­driven cancers. Specifically targeting PHF5A would also likely result in reduced side­effects seen with general spliceosome inhibitors. Unfortunately, there are currently no known inhibitors that target PHF5A. In order to discovery novel PHF5A inhibitors, we created a mini­gene mis­splicing reporter assay that was sensitive to both general spliceosome inhibitors and PHF5A perturbation. In a 96­well assay format, the assay was robust with a 200­fold assay window and Z’ values over 0.8. Following miniaturization to a 1536­well format, we conducted a high throughput screening (HTS) campaign testing 450,000 small molecule compounds. The initial hits were retested and counter­screened yielding 381 confirmed actives and we are further interrogating these actives in secondary and tertiary assays. Future efforts will focus on developing an SAR of the lead and backup series and identifying potential liabilities that will be addressed, if necessary, in further lead optimization efforts. We

are enthusiastic about the potential of developing a targeted PHF5A inhibitor as a novel and effective therapy for patients and their families fighting GBM and other Myc­driven cancers. #3201 Therapeutic potential of anti­angiogenic multimodal biomimetic peptide in hepatocellular carcinoma. Mustafa A. Barbhuiya, Adam C. Mirando, Brian W. Simons, Ghali Lemtiri­Chlieh, Jordan J. Green, Aleksander S. Popel, Niranjan B. Pandey, Phuoc T. Tran. Johns Hopkins University School of Medicine, Baltimore, MD. Hepatocellular carcinoma (HCC) related mortality ranks second worldwide and stands one step behind lung cancer. Due to the asymptomatic nature of the cancer, early diagnosis is a major problem. Currently only liver transplantation is curative for early stage hepatocellular cancer. There are other multidisciplinary treatment options like radiation and chemotherapy available for advanced patients. However, chemotherapy resistance is a common problem in the treatment of liver cancer. Newer therapeutics are needed for better management of advanced HCC. Angiogenesis and lymphangiogenesis are processes that are vital for tumorigenesis and metastasis. HCC is known to be a hypervascular tumor and many pro­angiogenic proteins are found significantly overexpressed in HCC. We explored the therapeutic potential of the anti­angiogenic and anti­lymphangiogenic, biomimetic peptide SP2043 developed by our group in HCC. Hepatocellular carcinoma cell lines HuH­7, Hep3b and HepG2 showed significant disruption of cell adhesion and migration following treatment. Furthermore, SP2043 was found to impair microvascular endothelial cell (MEC) tube formation induced by HepG2 tumor conditioned media and to significantly inhibit HepG2 tumor xenograft growth compared to untreated controls. The peptide drug was also found to improve the survival of autochthonous Myc induced HCC in a transgenic mouse model from two weeks to four weeks. We discovered the peptide to be a multi­modal anti­angiogenic drug that also inhibits IGF1R and MET signaling pathways in HCC cell lines. We also found that SP2043 treatment reduced the microvascular density in both subcutaneous and autochthonous liver tumors with reduced tumor cell proliferation and increased apoptosis. This study shows the therapeutic potential of SP2043 in the treatment of hepatocellular carcinoma.

#3202 Pharmacologic modulation of Sigma1 induces autophagic degradation of programmed death­ligand 1 in cancer cells. Christina M. Maher, Jeffrey D. Thomas, Charles G. Longen, Derick A. Haas, Halley M. Oyer, Jane Y. Tong, Felix J. Kim. Drexel University College of Medicine, Philadelphia, PA. Emerging evidence suggests that Sigma1 (also known as sigma1 receptor) is a unique ligand­operated integral membrane chaperone or scaffolding protein that contributes to cellular protein homeostasis. Previously, we found that treatment of various cancer cell lines with some prototypic small molecule modulators of Sigma1 can engage endoplasmic reticulum (ER) associated protein homeostasis pathways including the unfolded protein response and autophagy. Programmed death­ligand 1 (PD­L1) is a type 1 integral membrane glycoprotein that is processed and transported through the ER and secretory pathway of tumor cells. PD­L1 expressed at the surface of tumor cells can act as a T­cell inhibitory checkpoint molecule that inactivates tumor infiltrating immune cells that express PD­ 1, its cognate receptor. Here, we show that Sigma1 physically associates with PD­L1. In triple negative breast and androgen­independent prostate cancer cells, PD­L1 protein levels are suppressed by both RNAi mediated knockdown of Sigma1 and pharmacological modulation of Sigma1. We observe decreased cell surface and intracellular levels of PD­L1 by flow cytometry and biochemical subcellular fractionation respectively, which corresponds with a dose­responsive decrease in functional PD­L1/PD­1 interaction in a co­culture of cancer cells and T­cells. Inhibitors of autophagy block this suppression of PD­L1 protein levels, suggesting PD­ L1 is degraded away by autophagy after Sigma1 modulation. Through confocal microscopy, we show that Sigma1 modulation results in colocalization of PD­L1 and GFP­LC3, a marker of autophagosomes. From these conclusions, we hypothesize that autophagic degradation of nascent PD­L1 after Sigma1 modulation plays a key role in preventing the transport of functional PD­L1 to the plasma membrane. Together, these data demonstrate that Sigma1 modulators have the potential to act as novel therapeutic agents in PD­1/PD­L1 blockade strategies. #3203 Targeting tumor hypoxia with prodrug conjugates of potent small­ molecule inhibitors of tubulin polymerization.

Kevin G. Pinney,1 Mary Lynn Trawick,1 Ralph P. Mason,2 Li Liu,2 David J. Chaplin,3 Blake A. Winn,1 Laxman Devkota,1 Tracy E. Strecker,1 Jeni Gerberich,2 Alex Winters,2 Yifan Wang,1 Matthew T. MacDonough1. 1Baylor University, Waco, TX; 2University of Texas Southwestern Medical Center, Dallas, TX; 3Mateon Therapeutics, Inc., South San Francisco, CA. Regions of pronounced hypoxia in malignant tumors represent an opportunity for selectivity in drug activation, and hence targeting and delivery. In this study, a series of bioreductively activatable prodrug conjugates (BAPCs) of small­molecule inhibitors of tubulin polymerization were synthesized and evaluated in biochemical and biological studies. The BAPCs are designed to be biologically inert until selectively cleaved under hypoxic conditions, by reductase enzymes found in the tumor, to release potent effector anticancer agents. BAPCs were evaluated in the following assays: (1) cytotoxicity using the regular sulforhodamine B assay (normoxic conditions) to confirm their reduced cytotoxicity in cancer cells in culture compared to the effector agents; (2) cleavage under anoxic conditions by the reductase enzyme, NADPH cytochrome P450 oxidoreductase (POR), that is implicated in the bioreductive cleavage of compounds with nitrothiophene and nitroimidazole triggers; and (3) differential cytotoxicity under hypoxic versus normoxic conditions in cancer cell lines with the established bioreductive compound tirapazamine (TPZ) as the control. The POR assay was modified by the addition of protocatechuate 3,4­dioxygenase to ensure anoxic conditions, and Triton X­100 to facilitate solubilization of the BAPCs. A series of unsubstituted, methyl, and gem­dimethyl nitrothiophene­ and nitroimidazole­triggered prodrug conjugates (BAPCs) of two experimental anticancer agents (KGP03 and KGP18) were synthesized. KGP03 and KGP18 were inspired by the natural product combretastatin A­4 (CA4). A unique attribute of the selected anticancer agents is that both bind tubulin at the colchicine site, inhibit tubulin polymerization into microtubules, and thus function as pronounced cytotoxic, anti­proliferative agents in human cancer cell lines and as vascular disrupting agents (VDAs). The assays were validated with a series of nitrothiophene analogues of CA4 (Thomson et al., Mol. Cancer Ther. 2006 5:2886). In our evaluation of this series, the gem­dimethyl nitrothiophene analogue of CA4 was efficiently cleaved by POR and gave an average differential hypoxia cytotoxicity ratio (HCR: GI50 values of

normoxic/hypoxic conditions) of 41 in the human A549 lung carcinoma cell line. The corresponding prodrug of combretastatin A­1 had an HCR = 26. The monomethyl nitroimidazole BAPCs of KGP03 and KGP18 produced positive HCRs in our initial assays. In preliminary studies, the CA4 BAPC demonstrated antivascular activity in an orthotopic syngeneic breast tumor mouse model (4T1/BALB/c). In the POR assay, the gem­ dimethyl nitrothiophene analogue of KGP03 was completely cleaved in 24 h. Biological evaluation of these BAPCs indicates the occurrence of selective, hypoxic activation resulting in release of the potent inhibitors of tubulin polymerization (CA4, CA1, KGP03, KGP18). #3204 Tryptophan 318/324, the target of C­terminal binding protein (CtBP) inhibitors, plays a critical role in CtBP enzymatic activity, oligomerization and transcriptional coregulation. Martin M. Dcona,1 Benjamin L. Morris,1 Priyadarshan K. Damle,1 Zaid Nawaz,1 Michael J. Dennis,1 Sahib J. Singh,1 William E. Royer,2 Keith C. Ellis,1 Steven R. Grossman1. 1Virginia Commonwealth University, Richmond, VA; 2University of Massachussetts, Worcester, MA. C­terminal Binding Proteins (CtBP) 1 and 2 constitute a family of oncogenic transcriptional co­regulators overexpressed in tumor tissues that are associated with worse prognostic outcome and aggressive tumor characteristics in multiple cancer types. Specifically, CtBP has been found to repress expression of genes responsible for apoptosis and EMT (eg. BIK and CDH1) and promote expression of genes that partake in the migration of cancer cells and those that are responsible for enhanced drug resistance (eg. TIAM1 and MDR1). CtBP2 is also critically required for colon cancer stem cell self­renewal. CtBP is unique among transcription co­regulators in harboring a conserved D­isomer specific 2­hydroxyacid dehydrogenase (D2DH) domain, which reduces an alpha­keto acid substrate to an alpha­hydroxy acid in the presence of NADH. The presence of NADH also facilities oligomerization of CtBP, leading to assembly of higher order complexes of CtBP with both DNA binding transcription factors and histone modifying enzymes that then leads to modulation target genes. We have identified hydroxyimino­3­ phenylpropanoic acid and its 4­chloro derivative (HIPP; 4­Cl­HIPP) as potent substrate competitive inhibitors of the CtBP dehydrogenase (IC50’s=240nM, 180 nM), which also disrupt CtBP oligomerization,

promoter localization, and transcriptional regulation. Co­crystallization of HIPP and CtBP1/2 indicated a strong π­π interaction between the HIPP phenyl ring and the indolyl ring in tryptophan W318/324 of CtBP1/2, indicating that this tryptophan is critical to CtBP interaction with HIPP inhibitors, dehydrogenase function, and quaternary structure. Of note W318/324, though conserved in CtBP1/2, is unique among D2DH, suggesting that a better understanding of W318/324 role and function in CtBP structure and function is critical to optimizing design of inhibitors that targeting this evolutionary unique residue among dehydrogenases. To further elucidate the mechanism of action in catalysis and oligomerization, as well as functional importance for transcription of CtBP2 W324, we analyzed enzyme kinetics, oligomerization, transcriptional co­regulatory activity and cell migration in a series of CtBP2 W324 mutants overexpressed in breast and colon cancer cell lines with concomitant knockdown (siRNA) or knockout (CRISPR/Cas9) of CtBP2. Our data demonstrates that mutation of W324 abrogated dehydrogenase activity, oligomerization, transactivation of the validated CtBP target gene TIAM1 and induction of migration. In summary, our findings suggest that the W324 residue is critical for CtBP2’s function and its unique conservation in CtBP1/2 vs. other dehydrogenases will allow the development of high specificity CtBP W318/324 inhibitors to limit potential toxicity due to off target inhibition of related metabolic dehydrogenases. #3205 A nanomolar potency small­molecule compound against castration­ resistant and bone metastatic prostate cancer. Kenza Mamouni,1 Lajos Gera,2 Xin Li,1 Daqing Wu1. 1Augusta University, Augusta, GA; 2University of Colorado Denver, Aurora, CO. A standard treatment for prostate cancer (PCa) is androgen deprivation therapy (ADT) that suppresses androgen receptor (AR) signaling axis. Although initially responsive, most patients receiving ADT eventually develop metastatic castration­resistant prostate cancer (CRPC), with more than 90% of them exhibiting bone metastases. A mechanism by which CRPC cells evade ADT is the expression of constitutively active AR variants (AR­Vs), such as the well­characterized AR­V7. Recently we developed GH501, a flurbiprofen­modified small­molecule compound, and investigated its anti­cancer activity and mechanism of action in pre­ clinical models of CRPC. At nanomolar range, GH501 effectively

induces cell cycle arrest and apoptosis in CRPC cells regardless of their resistance status to enzalutamide treatment. RNA­seq analysis of GH501 combined with Western blotting analysis identified key targets implicated in CRPC progression, including the full­length AR, AR­V7 variant, and other important genes implicated in CRPC progression. Importantly, low doses of GH501 effectively inhibit the skeletal growth of CRPC in a xenograft model without obvious in vivo toxicities. These preclinical results indicate that GH501 is a promising small­molecule compound that can be further developed for the treatment of lethal prostate cancer. #3206 A potent and selective CSF­1R inhibitor, DCR­0064, inhibits colony stimulating factor 1 signaling in vitro and in vivo. Chu­Bin Liao, Shao­Zheng Peng, Chen­Hsuan Ho, Chao­Pin Lee, Jia­ Ming Chang, Hung­Jyun Huang, Sian­Yi Ciou, Yu­Chih Pan, Yu­Kai Chen. Development Ctr. for Biotechnology, Xizhi City, Taiwan. Tumor­associated macrophages (TAMs) are major components of leukocytic infiltrate of tumors. It has been reported that high TAMs density is associated with poor clinical prognosis. Within the tumor microenvironment, TAMs rely on signaling through CSF­1/CSF­1 Receptor kinase axis to promote tumor growth, angiogenesis, and metastasis. Therefore, decrease of TAMs density in tumors through inhibition of CSF­1R kinase is considered a potential target for drug development. In this study, we report that a novel small molecule DCR­ 0064 inhibits CSF­1R kinase with IC50 value below 10 nM and possesses specificity for CSF­1R over other tyrosine or serine/threonine kinases. DCR­0064 selectively inhibits CSF­1 induced growth of M­NFS­60 myeloid cells and effectively suppresses intracellular CSF­1R activation with IC50 value less than 100 nM. The potency and selectivity of DCR­ 0064 is superior to the current lead CSF­1R inhibitor, PLX3397, which is in phase III clinical development for treating tenosynovial giant cell tumor. The selectively growth inhibitory activity of DCR­0064 is consistent with inhibition of CSF­1R signaling. Oral administration of DCR­0064 reduces TAMs density in vivo in mice. DCR­0064 is being developed as a candidate for preclinical development. #3207 Targetting proteasome function by inhibition of the proteasome deubiquitinase USP14.

Stig T. Linder,1 Padraig D'Arcy,2 Xiaonan Zhang1. 1Karolinska Inst. Cancer Center, Stockholm, Sweden; 2Linköping University, Linköping, Sweden. The 20S proteasome core particle has evolved as an important target for anti­cancer drug development. We previously identified the small molecule b­AP15 as a novel class of proteasome inhibitors that function by abrogating the deubiquitinating (DUB) activity of the 19S regulatory particle. An optimised lead of b­AP15 (VLX1570) has been approved by the FDA for clinical studies and is currently in clinical trials for relapsed multiple myeloma. VLX1570 has also shown promising activity in Ewings sarcoma and paediatric leukemias. b­AP15 and VLX1570 bind to the proteasome DUB ubiquitin­specific protease­14 (USP14) in vitro and in exposed cells (CETSA). Binding and inhibition of enzymatic activity is believed to be due to targeting Cys114 in the active site. The cell death response to b­AP15/VLX1570 is distinct from that of 20S proteasome inhibitors. Thus, the sensitivity to these compounds is unaffected by overexpression of BCL2­family proteins and unaffected by p53 status. We here show that b­AP15 induces direct effects on mitochondria, possible explaining this phenomenon. We also provide additional understanding of drug­target interactions using a series of compounds identified by screening. Our findings demonstrate promising antiproliferative activities of USP14 inhibitors in vitro and in vivo. #3208 AZD1775 modulates kinetochore component Wee1­CDC2 and NF­κB signaling as potential therapeutic targets in head and neck squamous cell carcinoma. Zhengbo Hu,1 Anthony Saleh,2 Jianghong Chen,2 Carlson Sophie,2 Yi An,2 Clavijo Paul,2 Ru Wang,2 Shaleeka Cornelius,2 Zhong Chen,2 Carter Van Waes2. 1The First Hospital of Shaoguan, Guangdong, Shaoguan, China; 2NIDCD/NIH, Bethesda, MD. Recent findings in The Cancer Genome Atlas (TCGA) analysis of head and neck squamous cell carcinomas (HNSCC) revealed frequent alterations affecting the cell cycle and NF­κB pathway, which are critically involved in cancer cell proliferation and survival. We hypothesized that common mechanism(s) could coordinate cell cycle and

NF­κB cell survival signaling in HNSCC. Recently, we established an NF­κB β−lactamase reporter UM­SCC­1 cell line and performed genome­wide RNAi screening to explore this hypothesis. We identified genes involved in the cell cycle and related to components of the kinetochore that co­modulate NF­κB activity. Among these targets, WEE1 kinase is currently under investigation as a therapeutic target of inhibitor AZD1775 in HNSCC. We found that WEE1­CDC2 axis protein levels were higher in a panel of HNSCC lines than in normal human oral keratinocytes (HOK). To further investigate the potential cross­talk between WEE1 cell cycle regulation and NF­κB activation, we showed that treatment of HNSCC cells with AZD1775 decreased NF­κB transcriptional activation and decreased TNFα­induced phosphorylation of IKKs and RELA subunit. Furthermore, AZD1775 inhibited NF­κB regulated proteins modulating the cell cycle and survival, including p21, CyclinD1, BCL2 and BCL­XL. These changes also correlate with alteration in cell cycle and survival detected by flow cytometry, as tumor cells treated by AZD1775 exhibited increased G2/M phase and sub­G0 fragmented DNA, and decease in G1/S phases. WEE1 inhibitor AZD1775 inhibited tumor cell proliferation and colony formation in a dose­ dependent manner, and exhibited combinatory effects with cisplatin chemotherapy in tumor xenografts. Thus, we identified the novel cross­ talk between WEE1­CDC2 and NF­κB signaling, potentially implicating decreased NF­κB prosurvival signaling in the anti­tumor activity of Wee1 inhibitors. (Supported by NIDCD/NIH intramural projects ZIA­DC­ 000016, 73, and 74; NIDCD core facility: advanced imaging, ZIC DC000081). #3209 Polo­like kinase 1 as a new molecular target for small cell lung carcinoma. Noro Rintaro, Masahiro Seike, Fenfei Zou, Akihiko Miyanaga, Kaoru Kubota, Akihiko Gemma. Japan, Tokyo, Japan. No target therapies are presently available in the treatment of small­cell lung cancer of Lung (SCLC). Therefore there is a need to develop new therapeutic agents. The protein kinases are a family of genes that play critical roles in various signaling pathways. Some cancer cells show addiction to constitutive activation of certain signaling pathways for proliferation and survival. To identify new drug targets for SCLC, we screened a panel of small interfering RNAs (siRNAs) that target 720

genes encoding human protein kinases and related proteins using SBC5 SCLC cell (SN38 (irinotecan hydrochloride) resistant cell). PLK1 inhibition suppressed cell proliferation strongest among 20 significant promising total genes using different 5 SCLC cells as a varidation study). PLK1 mRNA expression was significantly higher than other pathological phenotypes among 20 celllines and the 200 clinical samples consist of three independent cohorts. The patients with high PLK1 expresssion was significantly associated with poor prognosis in SCLC patients. Furthermore we investigated the change of SN 38 sensitivity and preventing from SN38 resistance after knockdown PLK1. Our results indicated that PLK1 inhibitor as BI 2536 and Volasertib, was a promising molecular target therapy for pharmacologic intervention in SCLC in both monotherapy and the combination therapy with SN38. #3210 Targeting PTP4A3 phosphatase in ovarian cancer with the potent noncompetitive inhibitor JMS­631­053. John S. Lazo,1 Paula Pekic,1 Alex Cheung,1 Kelley McQueeney,1 Joseph Salamoun,2 Peter Wipf,2 Charles N. Landen,1 Elizabeth R. Sharlow1. 1Univ. of Virginia, Charlottesville, VA; 2Univ. of Pittsburgh, Pittsburgh, PA. PTP4A3 is a highly attractive molecular target for ovarian cancer (OvCa). Elevated levels of PTP4A3 mRNA and protein in human ovarian tumors correlate with disease progression, poor prognosis and poor survival. Genetic depletion of PTP4A3 in OvCa cell lines diminishes their ability to migrate and reduces their in vivo tumorigenicity while PTP4A3 overexpression increases tumor cell migration, invasion, and dissemination. Together, these data suggest PTP4A3 is a novel molecular target for OvCa; however, the lack of potent and selective PTP4A3 small molecule inhibitors has hindered PTP4A3’s definitive validation in OvCa and in other cancers. To drive the pharmacological validation of PTP4A3, nM in vitro), specific, noncompetitive, we 4,6(5H,7H)­dione), developed JMS­631­053 a potent (Ki=3 (7­imino­2­phenylthieno[3,2­c]pyridine­ PTP4A3 inhibitor. JMS­631­053 inhibited cellular migration, invasion, and colony formation in soft agar. Potent OvCa cell­based effects were observed by profiling JMS­631­053 against a panel of 8 OvCa cell lines using a 2D drug susceptibility assay, with EC50 values as low as 600 nM.

Likewise, JMS­631­053 killed OvCa 3D spheroids, including those derived from high grade serous OvCa cell lines, with EC50 values as low as 300 nM. The OvCa drug resistant cell lines (i.e., A2780CP20 and HeyA8MDR) retained responsiveness to JMS­631­053 (using 2D and 3D culturing conditions) suggesting that inhibition of PTP4A3 may be a viable therapeutic strategy for chemoresistant OvCa. Preliminary data also suggests that JMS­631­053 synergizes with cisplatin when used in combination studies. Hence, JMS­631­053 will be a valuable chemical tool for further validation of PTP4A3 as an OvCa target as well as provide potential leads for future drug discovery. #3211 Inhibition of autophagy potentiates cytotoxicity of CX­5461 treatment in chemoresistant epithelial ovarian cancer. Robert Cornelison,1 Danielle C. Llaneza,1 Yulia Petrova,1 Zachary C. Dobbin,2 David A. Schneider,2 Charles N. Landen1. 1University of Virginia School of Medicine, Charlottesville, VA; 2University of Alabama at Birmingham, Birmingham, AL. Epithelial ovarian cancer remains a deadly diagnosis with poor prognosis and patients succumbing to chemoresistant disease in 80% of cases. Mechanisms of chemoresistance are numerous, but we have identified ribosomal biogenesis basal machinery as being a common upregulated signaling network in response to chemotherapeutic insult. Tumors treated with chemotherapy showed dramatic increases in size and morphology of nucleoli, suggesting global changes in ribosomal synthesis with stress. We next sought to use the RNA Polymerase I inhibitor CX­5461 to identify biologic changes in targeting ribosomal machinery that may allow resensitization to chemotherapy. CX­5461 induced autophagy, senescence and mitotic catastrophe in ovarian cancer cell lines Overall, 13 ovarian cancer lines examined were highly sensitive to Pol I inhibition by MTT, with IC50s ranging from 25nM to 2μM, and chemoresistant lines were generally more sensitive to CX­5461. In p53 mutant cells CX­ 5461 appears to be primarily cytostatic with mitotic catastrophe being a relatively rare event accompanying cytokinesis failure. This could be indicative of the cells successfully using autophagy to decrease ribosome requirements and evade treatment. When treated in combination with chloroquine as an autophagy inhibitor IC50s decreased to 550nM in HeyA8 (from 2μM), 70nM for HeyA8MDR (from 191nM), 110nM in

SKOV3 (from 595nM) and 60nM for SKOV3TR­ip2 (from 109nM) with abundant multinucleated cells, suggesting synergy between ribosomal synthesis and autophagy inhibitors in vitro. Chloroquine administration appears to push cell to mitotic catastrophe after co­administration with CX­5461, thereby enhancing cytotoxicity over cytostatic endpoints after treatment, even in multi­drug resistant cell populations. Inhibition of autophagy appears to enhance efficacy of RNA Polymerase I inhibition in both sensitive and chemoresistant populations, prompting a potential new approach to chemoresistant disease. #3212 ONC201 shows efficacy in BRCA­deficient cancer cells and synergy with PARP inhibitors in glioblastoma, breast, prostate, and ovarian cancers. Marie D. Baumeister,1 Ozan C. Küçükkase,2 Varun V. Prabhu,3 David T. Dicker,4 Josh E. Allen,3 Wafik S. El­Deiry4. 1Lewis Katz School of Medicine, Philadelphia, PA; 2Boğaziçi University, Istanbul, Turkey; 3Oncoceutics Inc, Philadelphia, PA; 4Fox Chase Cancer Center, Philadelphia, PA. BRCA1 and BRCA2 are involved in control of DNA repair by homologous recombination (HR). Germline mutations in these genes substantially increase lifetime risk of developing breast, ovarian and other cancers. BRCA­deficient tumors show increased sensitivity to therapies that target defective HR. Sporadic tumors lacking germline BRCA mutations but sharing the molecular features of BRCA­mutant tumors may respond to these types of therapies as well. PARP inhibitors represent a form of targeted therapy for HR­deficient tumors that have been approved in ovarian cancer and are being tested in clinical trials in breast cancer, metastatic castration­resistant prostate cancer, and glioblastoma. Our lab has previously identified a small molecule called ONC201 in a screen for compounds capable of inducing the tumor necrosis factor­related apoptosis­inducing ligand (TRAIL) gene. Our lab has previously reported that ONC201 induces an integrated stress response that involves ATF4/CHOP and inactivates pro­survival kinases Akt and ERK. The latter leads to decreased phosphorylation of transcription factor FOXO3a, its nuclear translocation, and induction of transcription of the target gene TRAIL. A first­in­human clinical trial showed that the compound is well tolerated, achieves a therapeutic

pharmacokinetic profile, exhibits biological activity. It has since entered multiple phase I/II trials. ONC201 has anti­proliferative and pro­apoptotic effects in a wide range of tumor types including BRCA­deficient breast and ovarian cancers (n=10), with GI50 values in the low micromolar range. Treatment with ONC201 induces surface TRAIL and inhibits Akt activity in BRCA deficient breast and ovarian cancers. PARP inhibitors have been previously shown to upregulate DR5 through transcription factor CHOP, sensitizing solid tumors to TRAIL. Resistance to PARP inhibitors can occur through PI3K/Akt pathway activation, and PI3K/MEK blockade improves their anti­tumor effects. We observed synergy between ONC201 and PARP inhibitors olaparib and rucaparib in BRCA­deficient breast and ovarian cancer cell lines in cell viability assays with combination indices (CI) ranging from 0.4 ­ 0.8. Robust synergy was also observed in prostate cancer and glioblastoma cells. The mechanisms of the observed synergy are currently under investigation. These results indicate that ONC201 possesses single agent activity in BRCA­deficient cancer cells and that the combination of ONC201 with PARP inhibitors represents a promising synergistic therapeutic approach that could be exploited in multiple solid tumors. #3213 Antagonism of D2­like dopamine receptors plays a role in Onc201’s anticancer effects. Christina Leah B. Kline, Amriti Lulla, Jessica Wagner, David Dicker, Marie Baumeister, Sophie Oster, Wafik El­Deiry. Fox Chase Cancer Center, Philadelphia, PA. ONC201/TIC10 is a first­in­class small molecule inducer of TRAIL that causes early activation of the integrated stress response and inactivates both Akt and ERK. Its promising safety profile and broad spectrum efficacy in vitro has been confirmed in Phase I/II trials in several advanced malignancies. Biochemical and reporter assays have shown that ONC201 is a selective and competitive antagonist of the D2­like receptors, specifically, dopamine receptor D2 (DRD2) and dopamine receptor D3 (DRD3) with a KD value of ~3 µM. The theme that dopamine and dopamine receptors are important in cancer has emerged in the literature. We hypothesize that ONC201’s interaction with DRD2 is critical for ONC201’s anticancer effects. Co­treating HCT116 and RKO colorectal cancer cells with ONC201 and dopamine or the selective D2­ like receptor agonist sumanirole partially abrogated ONC201­induced

ATF4/CHOP expression and apoptosis. Knocking down DRD2 expression using siRNA negated ONC201’s effects on viable cell count. Overexpressing DRD2 in a cancer cell line that has very low levels of DRD2, increased ONC201­induced PARP cleavage. Quantitative RT­ PCR analyses showed that cells that have acquired resistance to ONC201 did not express detectable mRNA levels of the D2­like receptors. To further determine the anti­tumor potential of targeting the D2­like receptor, we treated different cancer cell lines with other D2­like receptor antagonists. Similar to ONC201, the D2­selective antagonist L­741,626 decreased cell viability and induced apoptosis in a number of cancer cell lines. In contrast to ONC201, however, L­741,626 has a poor therapeutic index. Our findings show that the ability of ONC201 to inhibit D2­like receptors contributes to ONC201’s antiproliferative and pro­apoptotic activity. Ongoing work is aimed at elucidating the mechanisms by which antagonism of D2­like receptors can promote apoptotic cell death, especially with regard to ATF4/CHOP/DR5 and Akt/ERK/Foxo3a/TRAIL, which have been shown to be stimulated in ONC201­treated and ­sensitive tumor cells. #3214 Systematic drug repurposing for faster cures of pediatric cancer identifies that Digoxin prolongs survival in a PDOX model of group 4 medulloblastoma. Lei Huang,1 Sarah G. Injac,2 Hong Zhao,1 Qi Lin,2 Mari Kogiso,2 Chris T. Man,2 Xiao­Nan Li,2 Ching C. Lau,3 Stephen T. Wong1. 1Houston Methodist Research Institute, Houston, TX; 2Texas Childrens Hospital, Houston, TX; 3Connecticut Children’s Medical Center, University of Connecticut School of Medicine, Hartford, CT. Background: Medulloblastoma (MB) is the most common malignant brain tumor of childhood. While the current standard care for MB leads to long term survival in approximately 75% of patients; recurrent and refractory MB continues to have dismal outcomes. In addition, long term survivors face significant treatment­related sequelae especially poor neurocognitive outcomes. Drug repositioning for known drugs is a promising potential strategy not only for drug discovery but also for accelerated translation into the clinical setting. To systematically explore thousands of known drugs available, we integrated computational biology and empirical biology methods to find old drugs for new indications in

MB. Results: A non­parametric, bootstrapping based simulated annealing (NPBSA) algorithm was employed to identify driver signaling pathways for over 1,800 patients with Group 3 and Group 4 MB through an integrative analysis on their mRNA expression, DNA­copy number, DNA­methylation and DNA­seq profiles. Then, drug functional networks were constructed based on gene expression profiles under drug treatment as well as chemical structures and were clustered into drug modules with potential mechanisms of action. By evaluating targeted effects of 1,309 drugs from connectivity map database within each drug module in driver signaling pathways, we identified a group of known cardiac glycosides that top ranked among the total drug candidates for the Group 3 and 4 MB subtypes. In addition to traditional chemotherapeutic agents, members of the cardiac aminoglycoside family were repeatedly identified as potential therapeutic agents for MB. These findings were validated in multiple MB­derived cell lines which showed high rates of growth inhibition by cardiac aminoglycosides compared to controls. To evaluate if this growth inhibition in vitro correlated to prolonged survival in vivo, an extensively characterized patient­derived orthotopic­xenograft (PDOX) model of Group 4 MB (ICb­1078MB) was treated with digoxin (2 mg/kg i.p.) for 2 cycles of 14 days. Digoxin treatment significantly prolonged survival to 113 days from a median of 92 days in untreated controls (p=0.001). Histological evaluation of recurrent tumors following digoxin treatment demonstrated changes in the pattern of tumor spread, vascularity and necrosis compared to untreated controls. Conclusions: Leveraging big data in the domains of pharmacogenomics and the notion of drug functional networks and driver signaling pathways represents a powerful tool to repurpose known drugs for new indications in pediatric cancers. Using this integrative biology approach, we identified the cardiac aminoglycosides family generally and Digoxin, specifically, as potential novel agents in the treatment of pediatric medulloblastoma #3215 Therapeutic potential of dual­function small molecules for breast cancers. Nicole Nicholas,1 Alfredo Velena,1 Scott Grindrod,2 Mira Jung1. 1Georgetown University, Washington, DC; 2Shuttle Pharmaceuticals INC, Rockville, MD.

Epigenetic silencing of tumor suppressors is an important mechanism in tumor initiation and progression. A tumor suppressor gene, BRCA1, is part of complex molecules in the repair of DNA double­strand breaks (DSBs), and loss of BRCA1 contributes to both sporadic and inherited breast tumor progression. Furthermore, recent studies have shown that the ataxia­telangiectasia mutated (ATM) activity is abnormal in breast cancer. Therefore, we tested the hypothesis that the dual­functional drug that fused the HDAC inhibitor with an ATM activator may enhance killing of breast cancer (BC) cells by increasing ATM activation. In addition, the expression of ATM may enhance the synergistic antitumor effects of HDAC inhibitors in combination with radiotherapy. To determine the HDAC inhibitory potency, SP­1­303 was screened against a panel of Class I and Class II HDAC enzymes in vitro and identified as a pan­HDAC inhibitor (IC50=120 nM). Western blot confirmed that SP­1­ 303 increased acetylated histone H3/H4, α­tubulin, and phosphorylation of ATM within 30 min ­ 4 h in a time­dependent manner. Using two ER positive BC cell lines (MCF7 and T­47D), two triple negative breast cancer (TNBC) cell lines (MDA­MB­231 and HCC 1937), and one normal breast epithelial cell line as a control (MCF10A), the effects of SP­1­303 on cell growth and cell death were examined by performing cytotoxicity assays and flow cytometry. SAHA and Tamoxifen were used as controls. SP­1­303 conferred the values of cytotoxicity (IC50) for the ER positive BC cells in a range of 0.25­0.4 μM, while the values are in a range of 1­3 μM for TNBC and 12 μM for normal breast epithelial cells, respectively. Flow cytometry (FACS) analyses demonstrated significant increases in G1 arrest (81%) in MCF7 cells and a further enhancement in the G1 phase (91%) in combination with radiation. The data implies the mechanism of action from this drug—cancer cells are being arrested in the G1 phase of the cell cycle, a radiation sensitive phase, suggesting that SP­1­303 may have a synergistic effect with radiation on cell killing. Taken together, SP­1­303 had the largest impact on ER positive breast cancer over TNBC. These data imply that an increased cell death is occurring in ER positive breast cancers from these dual­action molecules through HDAC inhibition and increased ATM activation, suggesting that SP­1­303 alone or its combination with radiotherapy may improve clinical efficacy. #3216 Attenuation of pancreatic tumor growth by a small molecule tubulin inhibitor.

Vivek K. Kashyap, Bilal B. Hafeez, Qinghui Wang, Saini Setua, Andrew Massey, Aditya Ganju, Murali M. Yallapu, Duane D. Miller, Wei Li, Meena Jaggi, Subhash C. Chauhan. University of Tennessee Health Science Center, Memphis, TN. Introduction: Pancreatic cancer (PanCa) is one of the most fatal cancers and is ranked as the fourth common cause of cancer­related deaths among both men and women in the US. The management of PanCa is exceptionally difficult due to the extremely poor response to available chemotherapeutic drugs. Microtubules are dynamic structures composed of α­β­tubulin heterodimers that are essential in cell division and are important targets for several clinical drugs (paclitaxel, docetaxel and vinblastine). However, clinical use of these tubulin­targeting drugs have toxicity and drug resistance issues in cancer patients. Thus, identification of more potent non­toxic inhibitors of β­tubulin is urgently required for cancer therapy purposes. In this study, we have identified a synthetic compound (ABI­231) which is a potent inhibitor of β­tubulin and evaluated its therapeutic efficacy against PanCa in vitro, and in vivo model systems. Methods: ABI­231 ((2­(1H­indol­3­yl)­1H­imidazol­4­yl) (3, 4, 5­ trimethoxyphenyl))­ methanone was synthesized and characterized in our department. Effect of ABI­231 on proliferation, migration and invasion of human PanCa cells (ASPC1, HPAFII, and PANC1) was performed by in vitro functional assays (MTS, wound healing, and Boyden chamber migrations). Effect of ABI­231 on the expression of β­tubulin isoforms was determined and compared with other clinical inhibitors of β­tubulin by Western blot, and qRT­PCR. Moreover, the effect of ABI­231 on the expression of β­tubulin III in PanCa cells was determined by confocal microscopy. Therapeutic efficacy of ABI­231 against PanCa was evaluated in an ectopic xenograft mouse model. Results: ABI­231 treatment inhibited cell proliferation, invasion, migration and colony formation abilities of PanCa cells in a dose­ dependent manner (1­100 nM) compared to vehicle treated group. Aberrant expression of β­tubulin III is involved in aggressiveness and drug resistance of various type of cancers including PanCa. ABI­231 effectively inhibited the protein levels and mRNA expression of total β­ tubulin (TBB), TBB1, TBB2c, TBB3 and TBB4 in PanCa cells via destabilization. Our confocal microscopy results further showed inhibition of β­tubulin in ABI­231 treated PanCa cells. Upregulation of micro RNA 200c (miR­200c) has been shown to inhibit the expression of

β­tubulin III in cancer cells. ABI­231 treatment of PanCa cells showed significant (p 2.0, p­value G; pC134W) is a defining feature of GCT and is used as a robust marker for diagnosis. However, other than the FOXL2 mutation the pathogenesis and the driving pathways remain unknown. Determining secondary genetic events in GCTs is essential to understanding and improving prognosis. In a pilot study, we completed an analysis of whole genome sequencing of ten GCTs and matched normal cases to generate a comprehensive catalogue of coding and non­coding events. We identified a TERT promoter mutation (c.228C>T) in 50% of these cases. TERT is normally inactivated in somatic tissues; however, this promoter mutation has been shown to re­activate transcription of TERT. We validated this TERT mutation in an international cohort of 300 GCTs and found it was present in approximately 25% of cases overall. These TERT promoter mutations have been used to revise the molecular classification of other cancer types such as gliomas. In GCT, we found that this TERT mutation was correlated with a significantly worse survival outcome in patients with primary GCT (p40,000 cases diagnosed every year and >10,000 deaths. Recent multi­ platform genomic studies have revealed a very complex picture of the disease, with no clear subcategories linking to the histopathological markers currently used in clinical practice. The multifocal and heterogeneous nature of the disease suggests that single biopsy sites may be missing valuable subclones which contribute to the etiology of the disease. To address this, we obtained fresh core biopsies from multiple sites (4­8) in the prostate from eight high­risk patients undergoing prostatectomies at the Christie NHS Foundation Trust. The tissues were immediately processed by the pathology department and each core divided in two, one for genomic analysis and one for parallel disease modeling (patient derived xenografts (PDXs), patient­derived cell lines and organoids). The cores were cryo­sectioned and H&E analysis performed at the top, middle and bottom of each core. These were reviewed by a pathologist and tissue was micro dissected prior to simultaneous DNA and RNA extraction of normal and tumor tissue. Blood for germline DNA and plasma for circulating free DNA (cfDNA) was also obtained. Our study comprises eight patients with two or more tumor sites (2­4), some bifocal, which were analyzed using whole exome sequencing (WES), copy number aberration (CNA) profiling, transcriptomic analysis and methylation profiling. The most frequent aberration identified was loss of 8p (NKX3­1) in 6/8 patients (12/24 tumor cores). Loss of 13q (RB1) was observed in 5/8 patients (8/24 tumor cores), but never in all

cores from a single patient, suggesting a late event. Loss of 5q (CHD1) was identified in 4/8 patients (11/24 tumor cores). Loss of 6q (MAP3K7), 10q (PTEN), amplification of 8q (MYC) and TMPRSS2­ERG fusion were identified in 3/8 patients. Some copy number events were only observed in either one patient or one tumor core demonstrating extensive inter and intra­patient heterogeneity. SPOP was the only gene affected by recurrent mutations across patients, but different amino acids were affected within and between patients. Mutations in PTEN, TP53, APC, BRAF and ERCC3, were also identified, among many others. The most significantly overexpressed gene was ERG, seen in patients with the TMPRSS2­ERG fusion. Changes in gene expression differed between cores from the same patients, reflecting the heterogeneity at the DNA level. These data will be analyzed in conjunction with the results from disease modeling to investigate the functional impact of these changes and cfDNA analysis is underway to understand which tumor clones are entering the bloodstream. The multifocal and genomically heterogeneous nature of prostate cancer is highlighted by this data and is likely to impact on precision medicine approaches for this disease. We will correlate the molecular profiling of our patient tumors with their clinical data in order to identify targets for further validation. #3394 CGP identifies largely non­overlapping high tumor mutational burden and HRD genomic alterations in 721 clinically advanced prostate acinar adenocarcinoma cases. Ninad Dewal,1 Yuting He,1 Richard J. Lee,2 Alexa B. Schrock,1 Jon Chung,1 Christopher Hoimes,3 Zachary R. Chalmers,1 Garrett M. Frampton,1 James X. Sun,1 Primo N. Lara,4 Neeraj Agrawal,5 Paul Matthew,6 Philip J. Stephens,1 Vincent A. Miller,1 Jeffrey S. Ross,1 Siraj M. Ali1. 1Foundation Medicine, Inc., Cambridge, MA; 2Massachusetts General Hospital, Boston, MA; 3Case Western Reserve University School of Medicine, Cleveland, OH; 4University of California, Davis Comprehensive Cancer Center, Sacramento, CA; 5University of Utah School of Medicine, Salt Lake City, UT; 6Tufts University School of Medicine, Boston, MA. Background: Effective therapies for the management of castrate resistant

prostate cancer are lacking. We performed comprehensive genomic profiling (CGP) on advanced prostate carcinomas (PC) in the course of clinical care to identify genomic alterations that could suggest benefit from targeted, immune­ and PARP inhibitor therapeutic strategies. Methods: DNA was extracted from 40 microns of FFPE specimen from 721 clinically advanced PC cases. CGP was performed using a hybrid­ capture, adaptor ligation based next generation sequencing assay to a mean coverage depth of over 500X. All four classes of genomic alterations (GA) ­ base substitutions, insertions and deletions, gene fusions, and copy number alterations (amplifications and losses) ­ were identified. Results: Of the 721 PC patients ­ men with median age of 65 (range 34­ 88) ­ CGP was performed on 335 (46.5%) prostate specimens and 386 (53.5%) specimens from metastatic sites. The most common genes altered were TP53 (N=336, 46.6%), PTEN (N=254, 35.2%), TMPRSS2­ERG (N=214, all fusions, 29.7%), AR (N=173, 24%), and MYC (N=105, 14.6%). Median tumor mutational burden (TMB) for this series was 3.6 mut/Mb (range 0 ­ 305). AR and MYC amplifications were enriched in metastatic tumors compared to primary tumors (p < 0.001 for both). Alterations characteristic of homologous recombination deficiency (HRD) were found in >15% of cases, including homozygous deletions and truncating mutations in BRCA2 in 79 (11%) samples, as well as truncating mutations in ATM in 41 (5.7%) samples, with only 3 samples possessing alterations in both genes. Cases with these HRD­relevant alterations possessed overall higher genomic loss of heterozygosity (LOH) content than did those without (p = 0.02). HRD and non­HRD cases had median TMB of 4.8 versus 2.7 mut/Mb, respectively, and mean TMB of 10.4 versus 4.0 mut/Mb (p < 0.01). Only 2.6% of non­HRD cases were TMB­high (20 mut/Mb or more), versus 8.4% of HRD cases, a small but significant enrichment in the latter. Conclusions: CGP for advanced PC cases identifies largely non­ overlapping TMB­high and HRD positive cases, suggesting benefit from immunotherapeutics and PARP inhibitors respectively. Further investigation will assess whether HRD pathway alterations result in genomic LOH in the context of a hypermutated tumor, and enrichment or exclusivity with other GA in prostate carcinoma. #3395 Alternative splicing of the MEAF6 gene promotes neuroendocrine prostate cancer development.

Ahn R. Lee, Yinan Li, Ning Xie, Colin C. Collins, Xuesen Dong. University of British Columbia, Vancouver, British Columbia, Canada. In response to the selection pressures exerted by potent androgen receptor (AR) pathway inhibitors (ARPI), adenocarcinoma prostate cancer (PCa) cells can undergo an adaptive process of cellular phenotype reprogramming termed neuroendocrine trans­differentiation. With this AR­bypass mechanism of survival emerges a lethal treatment­resistant PCa subtype called treatment­induced neuroendocrine PCa (t­NEPC). t­ NEPC is becoming a major clinical issue as it is estimated to affect >25% of advanced­stage PCa patients with the level of incidences predicted to rise as a result of the extensive applications of ARPI in the clinic. This underscores the gravity of our aims to delineate the molecular underpinnings of t­NEPC to inform future therapies that prevent or mitigate t­NEPC development. In this study, we have identified a splice variant of the MYST/Esa1­ associated factor 6 (MEAF6) gene, MEAF6­1, that is highly expressed in t­NEPC tumor biopsies as well as neuroendocrine cell lines of prostate and lung cancers. We show that the neuronal RNA splicing factor, SRRM4, stimulates MEAF6­1 splicing. Enhanced MEAF6­1 expression in prostate adenocarcinoma cell lines does not induce neuroendocrine trans­differentiation of these cells. Rather, it stimulates cell proliferation, anchorage­independent cell growth, invasion, and xenograft tumor growth. Gene microarray identified that these MEAF6­1 actions are in part mediated by the ID1 and ID3 genes. These findings suggest that the MEAF6­1 variant does not induce neuroendocrine differentiation of prostate cancer cells, but facilitates t­NEPC progression through accelerating proliferation of cells that have acquired neuroendocrine phenotypes. #3397 Post­mortem examination of an aggressive case of medullary thyroid cancer characterised by catastrophic genomic abnormalities. Darran P. O'Connor,1 Sudipto Das,1 Deirdre Kelly,2 Bruce Moran,3 Kathleen Han,2 Niall Mulligan,2 Ciara Barrett,2 Patrick Buckley,1 Peter McMahon,2 J McCaffrey,2 Henrik van Essen,4 Kate Connor,1 Bauke Ylstra,4 Diether Lambrechts,5 William M. Gallagher,3 Catherine M. Kelly2. 1Royal College of Surgeons in Ireland, Dublin, Ireland; 2Mater

Misericordiae University Hospital, Dublin, Ireland; 3University College Dublin, Dublin, Ireland; 4VU University Medical Center, Amsterdam, Netherlands; 5Vesailus Research Center, Leuven, Belgium. Catastrophic genomic alterations may drive aggressive cancer phenotypes. We describe a diagnostically challenging and rapidly fatal case of medullary thyroid carcinoma (MTC) occurring in a young, morbidly obese man presenting with diffuse bone marrow involvement and disseminated intravascular coagulation. Whole­exome (WES) and shallow whole­genome sequencing (sWGS) were carried out for the primary tumour, adjacent normal, bone marrow tissues, multiple metastases and blood samples derived from the patient. We identified three germline single nucleotide polymorphisms (SNPs) within the RET proto­oncogene that remained undetected using routine hospital genetic testing procedures. One variant (L769L) has been previously reported to be associated with aggressive MTC presentation yet remains untested for in the routine diagnosis of MTC. Supported by findings from both WES and sWGS, we report for the first time in thyroid cancer on the occurrence of a “chromothripsis­like pattern”, which involved shattering of chromosome 4 leading to complete abrogation of normal chromosomal function, along with dramatic widespread copy number aberrations across both primary tumour and bone marrow samples. Based on the somatic variants identified, we describe the evolutionary pathway of the case, showing that bone marrow metastasis occurred separately to other metastatic sites. The presence of disease­associated SNPs within the RET proto­oncogene supports their inclusion as part of routine genetic testing for MTC cases. The copy number aberrations and chromothripsis­like pattern affected a much broader range of genes than single mutations and may have led to the widespread chromosomal instability evident, possibly contributing to the rapid fatal course of the case. These results provide a rationale for the application of comprehensive genomic analysis of cancers presenting with unusual and aggressive phenotypes to facilitate more appropriate therapeutic options and diagnoses. #3398 The genomic and transcriptomic analysis of nine widely invasive follicular thyroid carcinomas (wiFTC) in Korean patients. Angela Byuri Cho,1 Seong­Keun Yoo,1 Min­Hwan Sohn,1 Jong­Yeon Shin,1 Su­jin Kim,2 Eun Kyung Lee,3 Young Joo Park,2 Jeong­Sun Seo1.

1Seoul National Univ. Genomic Medicine Institute, Seoul, Republic of

Korea; 2Seoul National Univ. Hospital, Seoul, Republic of Korea; 3National Cancer Center, Goyang­si, Gyeonggi­do, Republic of Korea. Widely invasive follicular thyroid carcinoma (wiFTC) can be characterized from minimally invasive follicular thyroid carcinoma (miFTC) as it exhibits poor prognosis with frequent distant metastasis. We have reported that miFTC shows similar mutational and transcriptional spectrum when compared to follicular adenoma (FA). Here, we performed whole­exome and transcriptome sequencing on wiFTC samples to define their aggressive biological features by comparing with 30 miFTCs and 26 FAs. Well­known miFTC and FA driver genes, NRAS (66.67%), HRAS (11.11%), and EIF1AX (11.11%), were identified in wiFTC. TERT mutation was found in all samples which can explain its aggressive feature. Chromosome 17p/q amplification was found in most wiFTC samples as well as 22q deletion that is frequently aberrant in H/K/NRAS mutated thyroid cancers. Most of wiFTCs were classified as RAS­like (66.67%) and a few were BRAF­ like (22.22%) and NBNR (11.11%) molecular subtypes. From differentially expressed gene analysis, we found 1,832 and 1,384 up­ regulated and down­regulated genes when wiFTC was compared to miFTC and FA together. wiFTC had many overlapped down­regulated genes as poorly differentiated and anaplastic thyroid carcinoma which also supports its aggressiveness. Through weighted correlation network analysis we have established gene expression networks, highly associated with wiFTC, that have SIRT6 and TGFBR2 as the central nodes. In this study, we demonstrated the distinctive mutational and transcriptional characteristics of wiFTC hence found the underlying molecular mechanism for its aggressiveness. #3399 Genomic analysis of melanoma evolution following a 30 year disease­ free interval. Jerry J. Miller,1 Kristopher A. Lofgren,2 Sarah R. Hughes,1 Steven E. Cash,2 David R. Meier,2 Paraic A. Kenny2. 1Gundersen Health System, La Crosse, WI; 2Gundersen Medical Foundation, La Crosse, WI. The rate of ultra­late recurrence (beyond 10­15 years) of cutaneous

melanoma has been estimated to be between 2.0­6.9% from large case series. Two major factors complicate the interpretation of these data. Firstly, the risk of second primary melanoma is approximately 5% creating uncertainty about whether at least some of these late onset tumors might be independent of the original lesion. Secondly, in the majority of cases, the original pathology specimen is no longer available for comparative analysis. Accordingly, putative late recurrences are difficult to unambiguously distinguish from a new primary melanoma. We identified a patient with a second melanoma diagnosed after a 30 year disease­free interval, and sought to determine if this new lesion was a recurrence of the original melanoma. We report the genomic sequence analysis of the exomes of two melanoma lesions isolated from the same individual in 1985 and 2015, and their comparison to each other and to the germline DNA of the patient. Identification of many shared somatic mutations between these lesions prove a lineal relationship spanning 30 years. Unlike prior reports of ultra­late melanoma recurrence, the availability of the original tumor and the use of comprehensive genomic analysis allowed us to confirm that the second lesion is truly a recurrence. We demonstrate the acquisition of numerous additional mutations during the three decade asymptomatic period. This is, to our knowledge, the longest disease­free interval that has been rigorously confirmed in melanoma or any other solid tumor type. These data highlight the low but very long­lasting risk of recurrence in this patient population.

Genotype­Phenotype Associations #3400 Progression of epidermal growth factor receptor (EGFR)­ independent colorectal cancer. Carolina Mantilla Rojas,1 Yu Ming,2 David Threadgill1. 1Texas A&M Univ., College Station, TX; 2University of North Carolina, Chapel Hill, NC. When combined with standard of care treatment for colorectal cancer (CRC), the use of monoclonal antibodies or small molecule inhibitors against epidermal growth factor receptor (EGFR) show modest efficacy in the clinic. Early detection efforts and more effective therapies have reduced mortality, yet CRC remains the second mostly deadly cancer in the United States with approximately 50,000 deaths expected this year. Thus, it is imperative to better understand the mechanisms governing molecular progression of CRC. Primary and secondary resistance to anti­ EGFR therapies occurs in approximately 80% of the patients with CRC. Mutations in Kras explain some non­responding CRCs, but even in cancers lacking Kras mutations, little is known about which cancers are likely to respond to EGFR targeted treatment, suggesting an alternative and EGFR­independent CRC progression mechanism. In this study, we used a conditional Egfr allele (Egfrf) within the ApcMin/+ mouse model and identified EGFR­independent tumors with faster growth rates than those developing in EGFR­wild type mice. To assess aggressiveness of EGFR­independent tumors, we used a metastatic CRC mouse model containing conditionally inactivated Apc alleles (Apcf/f) in combination with a conditionally active allele of Kras (KrasLSL­G12D). It has been reported that delivery of Cre recombinase­expressing adenovirus to the distal colon of these mice results in tumors that progress to carcinoma within 20 weeks, and liver metastases develop in approximately 20% of mice at 24 weeks. We discovered a 10% increase in the penetrance of tumors arising in the absence of EGFR (Egfrf/f, Apcf/f, KrasLSL­G12D/+). Endoscopic analysis suggests an increase in tumor multiplicity in EGFR­ deficient tumors when compared with tumors developing in EGFR­wild­ type mice. Biweekly colonoscopies confirmed that colonic tumors have a faster growth rate in the absence of EGFR. High­frequency abdominal ultrasound suggests liver metastasis at 16 weeks in 20% of mice lacking

EGFR. These findings demonstrate the existence of an EGFR­ independent mechanism by which CRC can arise and progress. Moreover, tumors lacking EGFR grow larger than those developing under normal EGFR activity and may be a more aggressive form of CRC. We also have evidence that ERBB3 and ERBB4, related EGFRs, mediate compensatory and alternative pathways, suggesting an important role for these receptors in the progression of EGFR­independent CRC. This study will advance our understanding of ERBB family biology during colonic tumorigenesis, ultimately contributing to better therapies for CRC. #3401 Role of membrane­associated guanylate kinase inverted 2 in advanced and castration­resistant prostate cancers. Bao Le, Kimberly Hammer, Katya Frantskevich, Irene Ong, Wei Huang, Paul Marker. University of Wisconsin­Madison, Madison, WI. Background: Prostate cancer is the most common non­cutaneous malignancy for men in the U.S. Patients with advanced and/or metastatic prostate cancer are commonly treated with androgen deprivation therapy. Unfortunately, incurable castration resistant prostate cancer commonly develops 2­3 years after initial treatment. Methods: To identify candidate cancer genes involved in the development of castration resistance, the Sleeping Beauty (SB) transposon system and androgen­sensitive LNCaP cell line were used in a forward mutagenesis screen designed to model the transition from androgen sensitive to androgen insensitive prostate cancer. MAGI­2 was identified in the screen as a common insertion site gene, potentially involved in the transition to androgen independence. Clinical significance of MAGI­2 in prostate cancer was further supported by human TCGA data. Functional assays were used to confirm the involvement of MAGI­2 in prostate cancer progression. Results: MAGI­2 expression was prognostic for patient survival outcome. Ectopic expression of MAGI­2 in 22Rv1 cells resulted in a decrease in cell proliferation and colony formation in in vitro studies. At the molecular level, overexpression of MAGI2 induced a suppression of known androgen responsive genes. MAGI­2 overexpression also limited the growth of grafts compared to empty vector grafts in in vivo xenograft models. Conclusion: MAGI­2 was identified in a forward mutagenesis screen modeling the development of castration resistant prostate cancer. Human

protein expression data from patient tissues supported a possible role for MAGI­2 in prostate cancer. Functional studies confirmed the potential involvement of MAGI­2 in prostate cancer progression. Future studies are seeking to elucidate the functional role and mechanism­of­action for MAGI­2 in prostate cancer. Ultimately, this may identify MAGI­2­ regulated signaling pathways as therapeutic targets for prostate cancer. #3402 The impact of germline single nucleotide polymorphisms (SNPs) in ERBB­family genes and genes associated with homologous recombination deficiency (HRD) on response to taxotere, platinum and trastuzumab (TCH) based therapy in the treatment of HER2­ positive breast cancer patients. Stephen F. Madden,1 Sinead Toomey,1 Simon Furney,2 Malgorzata Milewska,1 Joanna Fay,1 Elaine W. Kay,1 John Crown,3 Susan Kennedy,3 Bryan T. Hennessy,1 Alex J. Eustace1. 1Royal College of Surgeons in Ireland, Dublin, Ireland; 2University College Dublin, Dublin, Ireland; 3St Vincent's University Hospital, Dublin, Ireland. BACKGROUND: We have shown that ERBB (EGFR, ERBB2, ERBB and ERBB4) germline single nucleotide polymorphisms (SNPs) have a negative impact on the outcome of trastuzumab treated HER2­positive breast cancer (BC) patients. Currently TCH (taxotere, platinum and trastuzumab) based therapy is used to treat early stage HER2­positive BC. We investigate the importance of germline SNPs in ERBB genes and those genes involved in homologous recombination deficiency (HRD), on how patients respond to TCH therapy. PATIENTS AND METHODS: ERBB/HRD SNPs were identified in a panel of 32 HER2­positive BC patients by next generation sequencing (NGS). Agena MassArray analysis confirmed the genotype of these SNPs in a further 157 women. Kaplan­Meier estimates and Cox regression analysis identified that both ERBB/HRD SNPs were associated with relapse free survival (RFS) in patients who received a TCH based treatment versus those who received alternate therapies. Protein extracted from formalin fixed paraffin embedded tumours (n=60), was run on an RPPA platform to measure expression and phosphorylation of proteins (69 antibodies). Logistical regression identified protein levels associated with the presence/absence of ERBB/HRD SNPs that were significantly associated with RFS.

RESULTS: Ten ERBB/HRD SNPs were profiled in 157 trastuzumab treated HER2­positive BC patients. The minor alleles of the ERBB2 (rs1136201), ERBB3 (rs2229046) and BARD1 (rs2070096) SNPs significantly associated with a worse RFS in patients who received TCH based therapy relative to those who had the reference allele (ERBB2: HR=2.67 (CI=1.05­6.78), p=0.04; ERBB3 rs2229046: HR=4.95 (CI=1.91­12.79), p=9.75x10­4; BARD1: HR=3.27 (CI =1.16­9.17), p=0.02). The impact of ERBB/HRD SNPs on RFS was not observed in patients who did not receive TCH treatment. The minor allele of the RNF8 rs2284922 SNP is associated with a worse RFS (RNF8: HR=12.42 (CI =2.00­77.19), p=6.89x10­3) relative to those who had the reference allele only in patients who did not receive TCH treatment. RPPA analysis identified that patients who received TCH therapy and had the minor allele of the ERBB3 SNPs were significantly associated with the expression of HER2, p27 and MEK1/2 (rs2229046; minor allele associated with low expression of p27(p=7.22x10­3) and with high expression of HER2 (p=6.53x10­3); rs773123, minor allele associated with low expression of p27(p=5.38x10­4) and with high expression of MEK1/2 (p=6.24x10­3). CONCLUSIONS: The presence of germline ERBB/HRD SNPs may play an important role in how a patient responds to TCH based therapy, and clinical assessment of these SNPs by targeted genetic screening of patients' blood may allow for stratification of patients prior to treatment. #3403 Telomere shortening in pancreatic cancer is correlated to KRAS mutation. Yoko Matsuda,1 Naotaka Izumiyama,1 Mutsunori Fujiwara,2 Naoshi Ishikawa,1 Junko Aida,1 Kaiyo Takubo,1 Toshiyuki Ishiwata,1 Tomio Arai1. 1Tokyo Metropolitan Geriatric Hospital & Inst. of Gerontology, Tokyo, Japan; 2Japanese Red Cross Medical Center, Tokyo, Japan. Background: Pancreatic cancer is characterized by genomic complexity and chromosomal instability, and atypical mitotic figures are morphological features of this phenotype. Previously we have reported that approximately 30% of total mitosis in pancreatic cancer was atypical including multipolar, lag­type, ring and asymmetrical mitosis, and

anaphase bridges, and the number of total mitosis and atypical mitosis in pancreatic cancers was correlated with aggressive phenotype and prognosis (Pancreatology, 2016). In the present study, we clarified the relation between atypical mitotic figures, telomere length, and genetic abnormality in the pancreatic cancer. Methods: We surveyed the mitotic figures of the normal epithelium, pancreatic intraepithelial neoplasias (PanINs), and pancreatic cancers using surgically resected pancreatic cancer specimens (n=40). Telomere length was analyzed using quantitative fluorescent in situ hybridization technique. We also analyzed mutations of Kras codon 12 and 13 by PCR and microsatellite instability by immunohistochemical staining of MLH1, MSH2, MSH6 and PMS2. Results: Pancreatic cancer and duodenal epithelium showed significantly higher mitotic indices as compared with the duct, acinar cells, and PanINs. Normal mitosis was also higher in pancreatic cancers and the duodenal epithelium, while atypical mitosis was significantly elevated only in pancreatic cancers. Number of total mitosis and atypical mitosis were negatively correlated with telomere length, suggesting that telomere shortening plays important roles in cancer proliferation and chromosomal instability. In comparison with normal ducts, telomere length was decreased in PanIN­1, ­2 and ­3 and cancer. Furthermore, telomere length was gradually shorter among PanIN grades. Most of pancreatic cancers harbored mutations in Kras codon 12, and pancreatic cancer cases with Kras mutation showed shorter telomere length as compared with cases without Kras mutation. All pancreatic cancer cases in the present study were microsatellite stable. Conclusion: Our data strongly suggest that telomere shortening occurs in the early stages of pancreatic carcinogenesis and progresses with precancerous development. Telomere shortening and chromosomal instability in the duct epithelium plays key roles on carcinogenesis of the pancreas. #3404 Loss of Abi1 abrogates lung metastasis in the PyMT mouse model of breast cancer. Angelina Regua,1 Isabelle Bichindaritz,2 Tiffany Caza,1 Julie White,3 Robert Adamiecki,1 Mira Krendel,1 Gennady Bratslavsky,1 Leszek Kotula1. 1Upstate Medical University, Syracuse, NY; 2SUNY Oswego, Oswego, NY; 3Memorial Sloan­Kettering Cancer Center, New York, NY.

This study aims to elucidate the role of Abelson interactor 1 (Abi1), a key protein in the WAVE regulatory complex, in mammary carcinogenesis and metastasis. Breast cancer is the second­leading cause of mortality in women in the United States with an estimated ~200,000 new cases and over 40,000 deaths this year. Despite current treatment modalities, breast cancer patients often relapse after only a few years of treatment thus emphasizing the need for better therapeutic targets. Abi1 is an adaptor protein mainly associated with the WAVE (Wiscott­Aldrich syndrome protein family verprolin homologous) regulatory complex and Arp2/3 (Actin­related proteins 2 and 3)­mediated actin cytoskeleton remodeling. Our bioinformatic and gene expression analyses of human tumor data indicates that Abi1 is frequently upregulated in invasive breast cancers, is associated with poor survival, and may promote an aggressive breast tumor phenotype. Downregulation of Abi1 also abrogates motility and invasion of breast cancer cells, most likely through inactivation of both Src and PI3 kinase as well as certain matrix metalloproteases. We therefore hypothesize that Abi1 positively regulates breast tumor progression and invasion through dysregulation of these cell signaling pathways. To determine the role of Abi1 in breast tumor progression, we used a Cre­lox system to conditionally delete Abi1 in the mammary tissue of Polyoma Middle T (PyMT) breast cancer mice. Abi1 knockout (KO) and control mice were palpated bi­weekly to determine tumor latency and tumors were measured with a caliper to determine total tumor burden. We are currently analyzing changes in tumor kinetics as a result of conditional Abi1 knockout in the mammary epithelium of PyMT mice. Our preliminary studies of indicate slowed tumor growth in Abi1 KO PyMT mice. Western blot analyses of Abi1 KO mammary tumors indicate concomitant loss of WAVE complex proteins supporting our previous findings that WAVE complex integrity is dependent on Abi1. Most interestingly, Abi1 null PyMT mice exhibit significantly reduced incidence of lung metastasis, supporting our hypothesis that Abi1 promotes invasion by breast cancer cells. In summary, Abi1 loss leads to reduction of lung metastasis in PyMT mice, possibly through inactivation of key cell signaling and proliferation pathways such as Src and PI3 kinase. This work will establish Abi1 as a potential prognostic marker and therapeutic target in metastatic breast cancer. #3405 Screening for two recurrent BRCA1 mutations in Tunisian women

with triple negative breast cancer. Wijden Mahfoudh,1 Inchirah Bettaib,1 Sallouha Gabbouj,1 Noureddine Bouaouina,2 Lotfi Chouchane,3 Abdelfattah Zakhama1. 1Faculty of Medicine of Monastir, MONASTIR, Tunisia; 2Department of Radiation Oncology, CHU Farhat Hached, SOUSSE, Tunisia; 3Department of Genetic Medicine, Weill Cornell Medical College, Doha, Qatar. Triple­negative breast cancers (TNBC) lack expression of oestrogen, progesterone and HER2 receptors. Reports to date indicate that up to 20 % of TNBC patients harbour germline BRCA mutations; however, the prevalence of mutations may vary with ethnic group and with geographic region. In the Tunisian population, as yet a limited number of BRCA germline mutations have been reported. Tow recurrent mutations were found in exon 5 and 20 in BRCA1 gene (c.211dupA and c.5266dupC). To investigate the contribution of BRCA1 gene mutations to TNBC in Tunisia, we screened the exons 5 and 20 of BRCA1 gene in 20 TNBC patients by direct sequencing. In our study, we identified the c.5266dupC mutation in BRCA1 exon 20 in 2 of 20 triple­negative patients with a prevalence of 10%. Our study is the first investigation on the role of BRCA1 gene in TNBC in Tunisia. In this study, we show that targeting only the exon 20 in BRCA1 gene allows detection of a substantial percentage of mutations in Tunisian TNBC patients. #3406 Hereditary risks of male breast cancer in a multi­gene panel testing cohort. Elizabeth C. Chao,1 Mary Pritzlaff,2 Summerour Pia,2 Rachel McFarland,3 Shuwei Li,2 Jill Dolinsky,2 David Goldgar,4 Hermela Shimelis,5 Fergus Couch,5 LaDuca Holly2. 1UC Irvine, Newport Beach, CA; 2Ambry Genetics, Aliso Viejo, CA; 3UC Irvine, Irvine, CA; 4University of Utah, Newport Beach, CA; 5Mayo Clinic, Rochester, MN. While the population­based risk for breast cancer in males remains relatively low (1:1000), inherited predisposition can significantly raise this to as high as 10%, in men who carry a mutation in the BRCA2 gene. Lifetime breast cancer risks of 1­2% have also been reported in men who carry a mutation in BRCA1. These risks, as well as elevated risks of

prostate, pancreatic, and melanoma cancers, are important to discuss in families diagnosed with Hereditary Breast and Ovarian Cancer Syndrome, which is often viewed as being clinically relevant only to the women in an affected family. However, beyond the BRCA1/2 genes, limited data is available on hereditary predisposition to male breast cancer. We analyzed clinical histories and molecular results from multi­ gene panel testing for hereditary cancer predisposition in a cohort of 715 men affected by breast cancer. A total of 708 male breast cancer patients were eligible for inclusion in the final analysis. Molecular testing included analysis of 5 to 59 genes for DNA coding sequence and copy number variants by next­generation sequencing and microarray. Genetic variants identified were classified according to a 5­tier system using previously validated algorithms1,2. Only those variants classified as pathogenic or likely pathogenic were included in the analyses as positive for a mutation. Overall, a mutation was detected in 18% of these men. Four subjects carried a mutation in two different breast cancer predisposition genes. BRCA2 and CHEK2 were the most frequently mutated genes. The risk of breast cancer was significantly elevated compared to public controls in individuals with a mutation in BRCA2 (odds ratio (OR)=13.9; p=1.92x10­16), CHEK2 (OR=3.8; p=6.24x10­24) and PALB2 (OR=6.6, p=0.01). Average age was similar amongst men with (63.5±2.7 years) and without (62.3±1.2 years) mutations, as were clinical and family histories of additional cancers. The high overall diagnostic yield suggests the utility of testing all male breast cancer patient regardless of age or family history by multigene panel testing, and provides data to support risk­based counseling and medical recommendations for screening and/or prevention in male mutation carriers. 1.1. LaDuca et al. Genet Med. 2014 Nov;16(11):830­72. 2. Richards et al. Genet Med. 2015 May;17(5):405­24 #3407 Side effects of BRAF inhibitors mimic RASopathies. Alicia Sfecci,1 Alain Dupuy,2 Monica Dinulescu,1 Catherine Droitcourt,1 Henri Adamski,1 Smail Hadj­Rabia,3 Sylvie Odent,4 Marie­Dominique Galibert,4 Lise Boussemart4. 1CHU de Rennes, Rennes, France; 2Rennes 1 University, Rennes, France; 3Hôpital Universitaire Necker­Enfants malades, Paris, France; 4Université de Rennes 1, Rennes, France.

Recent advances in targeted anticancer therapies have substantially improved the prognosis of several cancers but they are not free of side effects. These side effects are, however, clearly distinct from those induced by classical cytotoxic chemotherapies, likely because targeted therapies are designed to interfere with specific oncogenic signaling pathways and not to inhibit cell proliferation in general. We, therefore, evaluated whether specific side effects of BRAF inhibitor therapies would resemble symptoms seen in patients with RASopathies, in which the MAPK pathway is affected and which includes Costello, Noonan and Cardio­Facio­Cutaneous syndromes. To this end, we collected a cohort of 18 patients with genetically confirmed RASopathies that were followed between 2012 and 2016 at the University Hospital of Rennes, France and that were evaluated based on their personal medical history and a clinical examination. Strikingly, RASopathy patients, particularly those with Cardio­Facio­Cutaneous syndrome, showed symptoms resembling those that we and others have previously seen in genetically characterized patients undergoing BRAF inhibitor treatment. These symptoms included keratosis pilaris, wavy hair, sparse eyelashes/eyebrows, poor hair growth, palmo­plantar hyperkeratosis in areas of pressure, verrucous papillomas, nevi efflorescence, and increased cancer risk. Nevertheless, BRAF­ inhibitor treatment can also lead to side effects not typically found in patients with RASopathies, including acneiform dermatitis and vemurafenib­specific phototoxicity, which may even increase under a combined BRAF/MEK inhibition. These results suggest that the side effects of BRAF inhibition that are similar to symptoms in patients with RASopathies are due to direct targeting of the MAPK pathway in BRAF­ non­mutated (wild type) cells while those that are dissimilar represent off­target effects that do not affect the MAPK pathway. We hope that this type of comparative analysis may lead to a better understanding of the multiple effects of targeted therapies and perhaps prompt modifications in the targeted therapy approach to minimize these side effects. #3408 Telomere length and TERT promoter mutations in cutaneous melanoma. Sivaramakirishna Rachakonda,1 Barbara Heidenreich,1 Eduardo Nagore,2 Rajiv Kumar1. 1German Cancer Research Center, Heidelberg, Germany; 2Instituto Valenciano de Oncologia, Valencia, Spain.

Telomeres at chromosomal ends are comprised of multiple short repeat sequences. In humans TTAGGG repeats account for telomere length ranging 10­15 kb. Telomere sequences are mainly double stranded that end in a single stranded G­rich tail of 150­200 nucleotides. Telomeres in somatic cells undergo gradual shortening due to inherent limitations of DNA replication and limited levels of specialized enzyme telomerase that adds the repeats at chromosomal ends to maintain homeostasis. We previously reported somatic mutations in the core promoter of the telomerase reverse transcriptase (TERT) gene that lead to increased transcription of catalytic subunit and tumor specific telomerase reactivation. Telomere length per se is associated with risk in different cancers. In this study, we measured leukocyte telomere length using real­ time PCR in 1469 melanoma patients and compared with that in 1158 matched healthy controls. The melanoma patients had statistically significantly longer telomeres than matched controls (t­test; P 6X10^­10). Mendelian randomization, carried out using two polymorphisms represented by rs1317082 and rs7726159 that associated with telomere length in genome wide association studies, showed association between increased telomere length and melanoma risk with an odds ratio of 2.3 (95% confidence interval 1.8­2.8). When measured in blood tissues from a melanoma family with the germline ­57A>C TERT promoter mutation, the carriers had longer telomeres (median 1.12) than the non­carriers (median 0.87). The TERT promoter mutations create binding motifs for E­twenty six (ETS) transcription factors and in stem cell the presence of the promoter mutations resulted in continued TERT expression and telomerase activity following differentiation into adult cells. Individuals with the germline TERT promoter mutations develop melanoma with an early age of onset and rapid progression to metastases; two individuals in the family who lived past median age of onset developed several other malignancies, besides melanoma. Thus, dysregulated telomerase leads to a severe phenotype. In an analogy, we hypothesize that association of longer rather than shorter telomeres with an increased risk of melanoma reflects stochastic increased telomerase levels due to common genetic variation. In contrast, the telomere length was shorter in tumors from unrelated melanoma patients with (121) than without (170) somatic TERT promoter mutations (P1X10^­5), which reflects the selection of the mutations at telomere crisis. Thus, a dynamic but controlled system evolved around telomere homeostasis when dysregulated leads to an increased cancer risk and affects tumor progression.

#3409 Age of cancer onset differentiated by sex and TP53 codon change in Li­Fraumeni Syndrome patient population. Lauren Erdman,1 Ben Brew,2 Jason Berman,3 Adam Shlien,1 Andrea Doria,1 David Malkin,1 Anna Goldenberg1. 1University of Toronto/Hospital for Sick Children, TORONTO, Ontario, Canada; 2Hospital for Sick Children, TORONTO, Ontario, Canada; 3Dalhousie University/IWK Health Centre, TORONTO, Nova Scotia, Canada. Introduction:Li­Fraumeni Syndrome (LFS) is a highly penetrant autosomal dominantly inherited cancer predisposition disorder. Germline mutations of the TP53 tumor suppressor gene cause >80% of LFS and confer an increased risk of a range of early onset cancers, as well as of second tumors even in the absence of a family history of cancer. For this reason, we previously reported the implementation of a comprehensive life­long clinical surveillance protocol for individuals with a germline TP53 mutation for early tumor detection. Here, we set out to build a predictive model of age of onset of cancer in LFS patients to inform this screening protocol aiming to make it more targeted. We identify characteristics that differentiate the age of cancer onset consistently, across multiple LFS patient cohorts. Methods:The LFS cohort at Toronto’s Hospital for Sick Children (SickKids) (n = 171 patients) was used as a discovery set to identify factors that distinguish age of onset among LFS patients. This project focused specifically on patient characteristics such as sex and mutations within TP53, both as they appear on the genome and manifest in the protein, as predictors for age of onset. These predictors were tested in an exponential parametric survival model. Findings from the SickKids discovery set were tested for replication in the International Agency for Research on Cancer (IARC) TP53 database (n = 2374 patients). Results:In the discovery cohort, female sex was associated with a 1.53 fold later age of cancer onset than in males (p = 0.019). This did not replicate in the IARC TP53 set with 0.99 fold earlier onset for females than males (p = 0.843). However, in the discovery set, there appears to be a point at which female and male age of onset converges at 43 years. Controlling for onset before vs after 43 years in our replication set shows 1.12 (p=0.0204) times later age of cancer onset in females than in males which is the same direction and significance as in our discovery set. The

discovery cohort also showed 2.23 (p = 0.08) later cancer onset for individuals with a germline Arginine to Cysteine (Arg>Cys) codon change (model significance p = 0.047). This finding replicated in the IARC TP53 data set which showed individuals with an Arg>Cys codon change having onset 1.29 (p = 0.043) times later than those with a TP53 mutation that did not result in this codon change. Conclusions:Our study identified two LFS patient characteristics, sex and TP53 Arg>Cys codon change, which consistently differentiate age of cancer onset within the LFS patient population. Females under the age of 43 when compared to males under the age of 43 appear to have later tumor onset, an effect which disappears after the age of 43. Individuals with an Arg>Cys TP53 codon change are expected to have later onset cancer than those with TP53 mutations that do not result in this change. Future work will disentangle these findings further and build a more comprehensive predictive model of cancer onset in LFS patients. #3410 Phenotypic characterization of allelic variation within the HEAT repeats of the mechanistic target of rapamycin. Patricia A. Wiley,1 Joy Gary,2 Zaw Phyo,1 Shuling Zhang,1 Amanda Sciorillo,3 Dorian Fraizer4. 1National Cancer Institute, Bethesda, MD; 2Center for Disease Control and Prevention, GA; 3Marymount University, Arlington, VA; 4University of Maryland, Balitimore County, Baltimore, MD. The mammalian target of rapamycin (mTOR), a protein in the phosphoinositide 3­kinase (PI3K) pathway important to cell cycle regulation, is frequently activated in cancer. This signaling pathway regulates cell growth and metabolism, and when dysregulated in cancer, it contributes to tumor angiogenesis and growth. mTOR contains two distinct complexes, MTORC1, and MTORC2. MTORC1 plays a role in biosynthesis, while MTORC2 helps with cellular metabolism. It has been shown that mTOR knockout models are embryonically lethal to mice. However, lower levels of functional mTOR have been shown to be beneficial and slow cancer progression. Allelic variation occurs within inbred strains of mice, including BALB/c mice. One allelic variant in mTOR, R628C, which contributes to tumor susceptibility has been identified in BALB/c mice. This single amino acid change occurs in the HEAT repeats of mTOR. HEAT repeats have been shown to facilitate

binding partner interactions, dimerization, and localization. Studies on mutations found within the HEAT repeats in several proteins have altered protein function and emphasize the structural importance of HEAT repeats. The goal of this study is to further elucidate the functional significance of HEAT repeats in TOR function and how allelic variation in the HEAT repeats may contribute to tumor susceptibility. Characterization of this allelic variant has shown stress inputs, such as irradiation, lead to a more severe phenotype and reduced mTORC1/2 signaling. Subsequent in vitro studies using a tetracycline inducible expression system (T­RExTM) have shown differential binding to mTORC1/2 complex partners in cells expressing the Balb/c allele. Additional studies are being performed to assess other complex binding partners, dimerization state and localization of the allelic variant. Our findings will model mTOR signaling activation mechanisms in humans to aid in development of new cancer treatments. #3411 Rare BRCA2 K3326X increases susceptibility to sporadic pancreatic ductal adenocarcinoma: a PANDoRA study. Ofure M. Obazee,1 Gabriele Capurso,2 Angelo Andriulli,3 Pavel Soucek,4 Ewa Małecka­Panas,5 Juozas Kupcinskas,6 Rudolf Kaaks,7 Maria Gazouli,8 Thilo Hackert,9 Aldo Scarpa,10 Giulia M. Cavestro,11 Claudio Pasquali,12 Hermann Brenner,13 Daniele Campa,14 Raffaele Pezzilli,15 Andrea Mambrini,16 Beatrice Mohelnikova­Duchonova,17 Ugo Boggi,18 Jakob Izbicki,19 Pavel Vodicka,20 Elzbieta Iskierka­Jazdzewska,21 Federico Canzian1. 1German Cancer Research Centre (DKFZ), Heidelberg, Germany; 2Digestive and Liver Disease Unit, Pancreatic Disorders Clinic, S. Andrea Hospital, University Sapienza, Rome, Italy; 3Division of Gastroenterology and Research Laboratory, Department of Oncology, IRCCS Scientific Institute and Regional General Hospital “Casa Sollievo della Sofferenza”, San Giovanni Rotondo, Italy; 4Laboratory of Pharmacogenomics, Biomedical Centre, Faculty of Medicine in Plzen, Charles University in Prague, Plzen, Czech Republic; 5Department of Digestive Tract Diseases, Medical University of Lodz, Lodz, Poland; 6Department of Gastroenterology, Lithuanian University of Health Sciences, Kaunas, Lithuania; 7German Cancer Research

Centre (DKFZ), Division of cancer epidemiology, Heidelberg, Germany; 8Department of Basic Medical Sciences, Laboratory of Biology, Medical School National and Kapodistrian University of Athens, Michalakopoulou 176, Athens, Greece; 9Klinik für Allgemein­, Viszeral­ und Transplantationschirurgie, Im Neuenheimer Feld 110, Heidelberg, Germany; 10ARC­Net, Applied Research on Cancer Centre, University of Verona, Verona, Italy; 11Gastroenterology and Gastrointestinal Endoscopy Unit, Vita­Salute San Raffaele University, IRCCS San Raffaele Scientific Institute, Milan, Italy; 12Department of Surgery, Oncology and Gastroenterology ­DiSCOG, University of Padova, Padova, Italy; 13Division of Clinical Epidemiology and Aging Research, German Cancer Research Centre (DKFZ), Heidelberg, Germany; 14Dipartimento di Biologia, Università di Pisa, Pisa, Italy; 15Pancreas Unit, Department of Digestive System, Sant’Orsola­Malpighi Hospital, Bologna, Italy; 16Massa Carrara Oncological Department, Azienda USL Toscana Nord Ovest, Carrara, Italy; 17Department of Oncology, Palacky University Olomouc and University Hospital Olomouc, Olomouc, Czech Republic; 18Division of General and Transplant Surgery, Pisa University Hospital, Pisa, Italy; 19Department of General­, Visceral­ and Thoracic­ Surgery, University Medical Center, Hamburg, Germany; 20Institute of Experimental Medicine, Czech Academy of Science, Prague and Institute of Biology and Medical Genetics, 1st Medical Faculty, Charles University, Prague, Czech Republic; 21Department of Hematology, Medical University of Lodz, Poland, Lodz, Poland. Background: The incredibly poor outlook of pancreatic cancer patients underscores an urgent need for early diagnostic markers. Pancreatic cancer ranks third most­frequent among BRCA1/2­deficient cancers with germline mutations detected in T) which introduces a premature stop codon thus truncating the protein, has previously been implicated in familial PDAC, but not in sporadic cases. A frameshift pathogenic mutation c.6503delTT (rs11571658, p.Leu2092Profs) reported to occur in tandem with K3326X in breast and ovarian cancer

families, has also been speculated to influence risk associations due to linkage disequilibrium between both variants. Method and results: K3326X was genotyped in 2,969 sporadic cases and 4,700 controls using Taqman chemistry and fidelity of genotypes assessed based on concordance of internal replicates and negative controls. K3326X was observed in 1.2% of cases and 0.8% of controls. Odds ratios (ORs) and associated 95% confidence intervals (CIs) were estimated by multivariate unconditional logistic regression with adjustment for age, sex and region of origin. We also performed a stratified analysis based on age at diagnosis to estimate the risk association between K3326X and early­onset pancreatic cancer. To rule out the likely shared effect of the c.6503delTT mutation on sporadic PDAC risk, we also sequenced DNA from carriers of K3326X in this study. We found K3326X to be associated with increased risk of developing sporadic PDAC ((OR = 1.71, 95% CI = 1.18 ­ 2.49), P = 0.005). This risk was considerably higher among cases aged 50 years and younger (OR = 2.13, 95% CI = 1.10 ­ 4.11, P = 0.03). Furthermore, carriers of K3326X did not bear the c.6503delTT mutation thus confirming that the observed risk effect was not influenced by the latter. Conclusion: These robust associations implicate K3326X in the etiology of sporadic and early­onset PDAC, and therefore warrant replication as well as functional studies to elucidate the role of K3326X in DNA repair mechanisms. #3412 Functional knockout of ATRX or DAXX permits the alternative lengthening of telomeres (ALT) mechanism in prostate cancer cells. Mindy K. Graham. Johns Hopkins School of Medicine, Baltimore, MD. A key hallmark of cancer is unlimited replication, which requires cancer cells to evade replicative senescence induced by short telomeres. The majority of cancers overcome this critical replication barrier by upregulating the telomerase enzyme, a telomere­specific reverse transcriptase. However, a subset of cancers lack telomerase, and telomeres are maintained by employing the Alternative Lengthening of Telomeres (ALT) pathway, which is dependent on homologous recombination. Across a variety of tumor types, our laboratory and others have reported a robust correlation between ALT and recurrent cancer­ associated somatic inactivating mutations in the ATRX­DAXX chromatin remodeling complex. In a previous comprehensive cancer survey of ALT,

we reported that ALT was highly prevalent in some tumor types (e.g. astrocytomas, sarcomas, pancreatic neuroendocrine tumors), but we did not observe any ALT­positive primary prostate cancer (out of 1,176 analyzed). However, we subsequently found a subset of metastatic prostate cancers that harbor ALT, suggesting that mutations giving rise to ALT in this disease are unique to metastatic prostate cancer. Here, we have created the first prostate cancer cell lines exhibiting ALT, with the explicit purpose of molecularly characterizing ALT in prostate cancer and identifying promising therapeutic targets for men with ALT­ positive lethal metastatic prostate cancer. Inactivating mutations in either ATRX or DAXX using the CRISPR/Cas9n system were introduced into the genetically well­characterized, telomerase­positive (ALT­negative) prostate cancer cell lines, LAPC­4 and CWR22Rv1. Resulting mutant subclones were compared to their parental (or empty vector) counterparts. In these new models, abolishing ATRX expression was sufficient to induce the ALT phenotype in both LAPC­4 and CWR22Rv1, as assessed by multiple biomarkers of ALT, including the presence of bright telomeric FISH foci, ALT­associated PML bodies (ABPs), and extrachromosomal telomere c­circles. Interestingly, abolishing DAXX expression induced ALT in only a subset (3/5) of LAPC­4 DAXX KO clones, and a subset (4/19) of CWR22Rv1 DAXX KO clones. We have successfully activated the ALT telomere maintenance phenotype in two prostate cancer cell lines through CRISPR­mediated targeted gene deletion. We are currently utilizing these isogenic cell lines to further characterize and elucidate the underlying biology of cancers harboring ALT, with the goal of pharmacologically targeting the molecular features unique to the ALT phenotype (e.g. ATR inhibition). The identification of ALT­specific drugs may pave the way for the development of new targeted treatments for the subset of men with ALT­positive lethal metastatic prostate cancer, and more broadly, other ALT­positive cancers. ALT is easily detected in clinical tissue samples, and thus would serve as a predictive biomarker for personalized medicine. #3413 Identification of a novel cancer predisposition variant associated with risk of CMM and other cancers. Craig C. Teerlink,1 John G. Zone,1 Sancy A. Leachman,2 Lisa A. Cannon­Albright1. 1Univ. of Utah Health Sciences Ctr., Salt Lake City, UT; 2Oregon Health and Science University, Portland, OR.

Introduction: In a search for the responsible CMM predisposition gene/variant, whole exome sequencing was performed on a pair of related CMM cases (cousins) from an extended high­risk Utah CMM pedigree showing evidence of linkage to chromosome band 1q41. Methods: Standard bioinformatics tools were used to identify rare or novel variants shared in the 2 related CMM cases in a CMM pedigree showing 1q41 linkage. Sanger sequencing and a Taqman assay were used to confirm the original variants, and to confirm segregation of the variant in other cancer­affected relatives. Taqman assay of the variant in independent Utah CMM cases (n=491) and controls (n=207) was performed. Results: A novel variant in SLC30A10 (rs 550245711, Chr1q41:220091798 C­T (G­A ­strand) SLC30A10: NM_018713: exon3:c.G757A:p.V253M) was shared by the 2 related CMM cases (cousins) in the 1q41 linked pedigree. A combination of Sanger sequencing and Taqman assay in 5 additional related CMM cases in the pedigree identified 4 who carried the variant. Two untested pedigree­ member parents of variant carriers (obligate carriers) were diagnosed with cancer of another site. A combination of Sanger sequencing and Taqman assay in 12 additional relatives diagnosed with cancers of other sites (small intestine, thyroid, breast, bladder, and endometrial) who ranged from closely (first degree), to distantly (fifth­degree), related to a carrier CMM case identified 4 additional cancer­affected carriers (small intestine, breast, and endometrial cancers). None of 491 independent CMM cases or 207 controls was observed to carry the variant. ExAc frequency for the variant in Non­Finnish Europeans = 5/66,062. The Cosmic ID is COSM3710592 and the variant has been confirmed as a somatic mutation in an oral squamous cell carcinoma. The variant is predicted to be damaging in 7 out of 8 algorithms considered, and has a GERP score of 6.08. Conclusions: A novel variant in SLC30A10, an important and evolutionarily conserved manganese and zinc transporter, was identified in a region of linkage in an extended CMM high­risk pedigree. The variant is shared by 6 CMM cases, 2 stomach cancer cases, 1 small intestine cancer case, 2 breast cancer cases and 1 endometrial cancer case within a 5 generation pedigree originally studied for a significant excess of CMM. Functional validation studies are underway. #3414

Novel MGMT variant association in Mexican patients with astrocytoma. Liliana Gomez­Flores­Ramos,1 Talia Wegman­Ostrosky2. 1Institute of Biomedical Research, UNAM. National Cancer Institute, INCAN, Mexico City, Mexico; 2National Cancer Institute, NIH, Bethesda, MD. Introduction: Astrocytomas are the most common and lethal brain tumors with a median survival of 15 months. Different biomarkers have arisen to classify and treat this type of tumors; one of these markers is MGMT. The methylation status of this gene is a prognostic and predictive factor. The MGMT (O­6­Methylguanine­DNA Methyltransferase) is involved in the cellular response to the effects of O6­methylguanine (O6­MeG) in DNA. Repairs alkylated guanine in DNA by transferring the alkyl group at the O­6 position to a cysteine residue in the enzyme. The dysregulation of this methylation is critical in the development of certain cancers. Objective: To identify germinal MGMT gene variations in patients with astrocytoma. Material and methods: 55 randomly selected Mexican patients diagnosed with astrocytoma between 2008­2014 were consented to perform the molecular analysis of the whole gene and 5´UTR and 3´UTR regions of MGMT at Ion Torrent platform. Each amplified region had a depth of minimum 1500x. Ion Reporter software was used to analyze the genetic variants and using Integrative Genomic Viewer we confirmed that each variant had a Phred higher than 25. Genotypic frequencies were compared with HapMapMex and Mexican ancestry population from the 1000 Genomes project. Results. In lymphocytes DNA, eight genetic variants were found, and two of them were statistically significant risk factors: rs7896488 alternative allele A with a frequency of 15% in patients with astrocytoma, and only 4% of the control group (OR=5.53, IC 1.96­15.59 p = 0.0007) and rs2308326 ancestral allele C, where 93% of the patients were carriers of allele C in contrast with 73% of controls (OR=5.39, IC 2.29­12.69 p = 0.000019). These variants are non­coding single nucleotide polymorphisms, rs7896488 is a 3' UTR genetic variant, while rs2308326 is an intronic variant. Conclusion: Our study found two new risk SNV for Astrocytoma in a Mexican population. #3415 RNF43 somatic mutations in endometrioid ovarian cancers occur in

the setting of synchronous endometrioid endometrial cancers. Defne L. Levine,1 Fanny Dao,2 Narciso Olvera,2 Katherine LaVigne,1 David B. Solit,1 Petar Jelinic2. 1Memorial Sloan Kettering Cancer Center, New York, NY; 2NYU Perlmutter Cancer Center, New York, NY. Introduction: Somatic frameshift mutations in RNF43 have previously been filtered out in cancer genome sequencing projects given that they occur in homopolymer tracts and resemble polymerase slippage errors. Two RNF43 hotspot mutations have been identified and validated in endometrioid endometrial cancer (EEC) and appear to negatively regulate Wnt signaling. These frameshift mutations occur at codons R117 and G659. Due to shared clinical and morphologic features of EEC and endometrioid ovarian cancer (EmOC), we determined the frequency of RNF43 somatic mutations in EmOC. Methods: We reviewed the clinical and pathologic features of EmOC samples diagnosed from 2006 to 2015, retrieved from laboratory databases and institutional archives. DNA was extracted from formalin­ fixed, paraffin­embedded tumor samples using standard protocols. Sanger and next­generation sequencing (NGS) were used to screen for hotspot mutations at codons R117 and G659 with custom designed primers. NGS at each hotspot was covered with a minimum sequencing depth of 400X. Results: Forty­seven EmOC patients with available tumor specimens were identified and included in the analysis. The median age at diagnosis was 55 years old (range 34­84). The majority of patients had FIGO stage I or stage II disease (n=38, 81%). Thirteen (28%) patients had synchronous endometrial endometrioid or mixed histology tumors. Two (4.3%) RNF43 somatic mutations at codon G659 were identified in both Sanger and NGS from the EmOC tumor specimens. No mutations at codon R117 were identified. The allele fractions of the G659 mutations were 4.3% and 2.8% seen in 33 and 18 reads with a coverage of 775X and 646X, respectively. A review of pathology reports indicated that both mutated samples had synchronous EECs. These two mutated samples represent 15.4% of cases diagnosed with synchronous EmOC and EEC in this cohort. Conclusions: RNF43 somatic mutations are uncommon in EmOCs and associated with synchronous EECs. Recent massively parallel sequencing data suggests that EmOCs in the setting of synchronous EECs are clonally related and disseminated cells are related to nearby anatomic structures. Our data supports that RNF43 somatic mutations in EmOC are

clonally expanded from EECs likely through trans­tubal migration. #3416 Clarifying the biological significance of the CHK2 K373E somatic mutation discovered in The Cancer Genome Atlas database. Masayoshi Higashiguchi,1 Izumi Nagatomo,1 Takashi Kijima,1 Osamu Morimura,1 Kotaro Miyake,1 Toshiyuki Minami,2 Shohei Koyama,1 Haruhiko Hirata,1 Kota Iwahori,1 Takayuki Takimoto,1 Yoshito Takeda,1 Hiroshi Kida,1 Atsushi Kumanogoh1. 1Osaka University Graduate School of Medicine, Osaka, Japan; 2UC San Diego Moores Cancer Center and Sanford Consortium for Regenerative Medicine, University of California, San Diego, CA. Identification of somatic mutations that contribute to cancer development leads not only to more precise understanding of cancer pathogenesis, but also development of novel molecular targeted therapies. Analyses of cancer genomes using high­throughput sequencing technology are currently conducted as international collaborative research projects, and the results of those analyses are deposited in public databases, allowing everyone in the world to access to these data. To identify a somatic mutation that plays an important role in cancer pathogenesis, we counted the occurrences of each somatic mutation found in the TCGA lung adenocarcinoma dataset and became interested in the CHK2K373E mutation (c1117A>G), which was present in 31 of 542 patients. The K373E mutation impaired CHK2 autophosphorylation at Thr383 and Ser516. In vitro kinase assay revealed that the K373E mutation markedly attenuates CHK2 kinase activity. Growth curves showed that wild­type CHK2 substantially suppressed cell proliferation, but this effect was lost in the K373E mutant in HCT­15 harboring tetracycline­inducible CHK2. Clonogenic assays revealed that wild­type CHK2 promoted survival after ionizing radiation and this pro­survival function was impaired in K373E CHK2. The results of western blotting and RT­PCR suggested that the p53­independent induction of p21 by CHK2 might underlie these effects. Therefore, we identified a somatic mutation that contributes to cancer pathogenesis, using information in a public database. This kind of attempt is expected to lead to discovering new 'driver' mutations in the future. #3417

The impact of p53 codon 72 SNP upon aging and longevity in mouse models. Yuhan Zhao, Lihua Wu, Xuetian Yue, Cen Zhang, Jun Li, Jianming Wang, Zhaohui Feng, Wenwei Hu. Rutgers Cancer Institute of New Jersey, New Brunswick, NJ. p53 has dual functions on longevity. p53 plays a crucial role in tumor suppression to prevent early death due to cancer. However, it has been suggested by animal models that constitutively increased p53 activity accelerates the decline of stem/progenitor cells’ self­renewal function during aging process, which leads to a reduced lifespan. In humans, the role of p53 in aging and longevity has not been well established. As a haplo­insufficient gene, p53 is under the tight regulation in cells. Attenuation of p53 function contributes greatly to tumorigenesis. p53 codon 72 single nucleotide polymorphism (SNP) with either an arginine (R72) or a proline (P72) at codon 72 is a naturally occurring common SNP that can influence the activity of p53. The P72 allele is weaker than the R72 allele in inducing apoptosis and suppressing cellular transformation. Individuals with the P72 allele have increased cancer risk compared to the R72 allele. However, it is unclear whether the change of p53 activity in humans by functional SNPs could impact upon longevity. A perspective study with an aging human population showed that P72 allele is associated with longer survival despite its increased risk for cancer development. These findings strongly suggest that p53 activity is reversely associated with aging, and SNPs in the p53 pathway could impact upon the life span in humans. In this study, we employed a mouse model system with knock­in of human p53 gene (Hupki) carrying either R72 or P72 SNP to investigate the impact of p53 codon 72 SNP upon longevity and its underlying mechanism. Mice with p53 P72 allele showed weaker transcriptional activity than the R72 allele toward a subset of p53 target gene, suggesting that these mice retain the function of p53 codon 72 SNP in human. We found that although mice with p53 P72 have increased cancer risk compared to mice with p53 R72, mice with p53 P72 that escaped tumor development showed longer lifespan compared to mice with p53 R72 that do not develop tumor. Mice with p53 P72 displayed a delay in aging process compared to mice with p53 R72; mice with p53 P72 have slower reduction in bone density, dermal thickness and wound healing ability during aging process. We compared the effects of p53 codon 72 SNPs on stem cell population and function as a possible mechanism that contributes to their difference in longevity.

Compared to mice with p53 R72, mice with p53 P72 allele have lower number of long­term stem/progenitor cells and better self­renewal function during aging process. Consistently, aging mice with p53 P72 allele exhibited better long­term stem cell ability of engraftment and repopulation than aging mice with p53 R72 allele as evaluated by bone marrow transplantation assay. Taken together, results from this study demonstrate that p53 codon 72 SNP has a direct impact on aging and longevity, and strongly support the role of p53 in regulation of stem/progenitor cells’ function and longevity. #3418 Additional mutation in PTPN11 gene promotes tumorigenesis of the NF1 gene mutated cells. Yoshimi Arima, Ritsuko Harigai, Ryo Sato, Toshiki Takenouchi, Kenjiro Kosaki, Hideyuki Saya. Keio University, School of Medicine, Tokyo, Japan. The NF1 tumor suppressor gene encodes neurofibromin and is a functional Ras GTPase­activating protein (RasGAP) involved in negatively regulating the Ras signal by accelerating the conversion of activated Ras­GTP to inactive Ras­GDP. NF1 gene germline mutations cause Neurofibromatosis type 1 (NF1, von Recklinghausen disease). We hypothesized that additional genetic alterations promote the malignancy of NF1­associated tumors. To test our hypothesis, we inoculated a GFP­ labeled human NF1­deficient cell line, sNF96.2­GFP, which has a frame­ shift mutation (c.3683delC, p.Asn1229MetfsTer11) in the NF1 gene, into the renal sub­capsules of immunodeficient mice. A subclonal cell line, the A­1 cell, was established from the developed tumor. We found that A­1 cells show much higher tumorigenic activity and phosphorylation status of MEK and Akt than the parental sNF96.2­GFP cells. We analyzed the genomic DNA of both the sNF96.2 and the A­1 cells by using the next­ generation sequencing and our medical exome panel of 4813 genes, which are known to be responsible for most human genetic disorders. We identified 18 heterozygous variants within coding regions of 17 genes that were present in the A­1 cells, but not in the original sNF96.2 cells. We found a single base substitution (c.1508G>T, p.Gly503Val) in the PTPN11 gene, which encodes the tyrosine phosphatese SHP­2, and is associated with the regulation of the Ras signaling pathway. It is critical to note that constitutional gain­of­function mutations in the PTPN11 gene cause Noonan Syndrome in humans due to activation of the Ras pathway.

To determine the role of PTPN11 mutation in NF1­associated tumors, we established a cell line overexpressing PTPN11mut in sNF96.2­GFP cells. We inoculated parental cells and PTPN11mut cells into the subcutaneous of nude mice, and we found that PTPN11mut cells show much higher tumorigenic activity than the parental sNF96.2­GFP cells. Our data suggests that this additional gene mutation in PTPN11 promotes the malignant characteristics of NF1­associated tumors. #3419 Uncovering the functional relevance of FBXW7 mutations in endometrial cancer. Mary Ellen Urick, Bo Hong, Meghan L. Rudd, Daphne W. Bell. NIH, Bethesda, MD. The goal of this study is to elucidate the functional consequences of FBXW7 mutations in the context of endometrial carcinoma (EC). EC is a heterogeneous disease, consisting of multiple histological subtypes associated with distinct clinical outcomes. Endometrioid endometrial cancer is the most commonly diagnosed subtype and is associated with an overall favorable prognosis. Less common but more clinically aggressive subtypes that contribute disproportionately to patient deaths include clear cell and serous ECs. We previously reported that FBXW7 is somatically mutated in 10% of endometrioid, 13% of clear cell and 29% of serous ECs. FBXW7 functions as a substrate­recognition protein within a SKP/CUL1/F­Box ubiquitin ligase complex, which targets numerous proteins for ubiquitin­mediated proteosomal degradation. Although FBXW7 is a known tumor suppressor, the functional consequences of these mutations have not yet been fully elucidated in the context of EC. As a means to this end, we utilized shRNA and Western blotting to identify proteins that are upregulated following FBXW7 depletion in ARK1 serous EC cells. Among proteins that were consistently upregulated were cyclin E1 and cMYC. We treated EC cells harboring endogenous FBXW7 mutations and those with wildtype FBXW7 with a proteasome inhibitor to confirm that EC cells regulate cyclin E1 and cMYC through proteasome degradation. Furthermore, we identified three EC cell lines that harbor endogenous mutations in FBXW7 and show that these cell lines exhibit delayed degradation of these substrates compared to EC cells expressing wildtype FBXW7. Finally, we generated expression constructs for wildtype FBXW7α and six recurrent mutants

found in EC. We show that transient exogenous overexpression of five out of six recurrent FBXW7 mutant constructs in two different serous EC cell lines resulted in increased levels of phosphorylated cMYC and cyclin E1 compared to wildtype FBXW7α. These results suggest that not all recurrent FBXW7 mutations are functionally equivalent. Our findings begin to reveal the molecular consequences of FBXW7 mutations in EC and ongoing studies are aimed at elucidating additional functional consequences of these aberrations in the context of EC. #3420 Mutational status, expression and functional behaviors of FAM134B in colorectal cancer. Farhadul Islam,1 Vinod Gopalan,1 Riajul Wahab,1 Katherine Ting­wei Lee,1 Afraa Mamoori,1 Cu­tai Lu,2 Robert A Smith,3 Alfred K­Y Lam1. 1Griffith University, Gold Coast, Southport, Australia; 2Gold Coast Hospital, Gold Coast, Southport, Australia; 3Queensland University of Technology, Brisbane, Australia. Background: Family with sequence similarity 134B (FAM134B) is an ER­autophagy regulator and involved in the pathogenesis of neuronal disorders, vascular diseases and carcinomas. In colorectal carcinomas, FAM134B plays important role in the pathogenesis and associated with aggressiveness of the disease. However, the frequency of mutations, expression pattern and functional roles in cell have never been studied in colorectal cancer. Objectives: To investigate FAM134B mutations in tissues samples from patients with colorectal cancer and cell lines. Also, the expression of FAM134B at protein and mRNA levels were examined. In addition, functional roles of FAM134B in colon cancer were studied. Methods: Mutations in FAM134B sequence in eighty­eight cancer tissues and matched non­cancer samples was studied by high­resolution melt curve analysis followed by Sanger sequencing. FAM134B expression was studied and quantified in cell lines and cancer tissues samples using immunofluorescence, immunocytochemistry, Western blot and real­time PCR. In vitro functional assays were performed to unveil the molecular roles of FAM134B in colon cancer pathogenesis followed by shRNA­ mediated silencing in cells. Mouse xenotransplantation model was used to confirm the functional behavior of FAM134B in colon cancer. Results: In this study, 46.5% (41/88) patients with colorectal cancer were

identified as FAM134B mutations positive. Thirty­one novel pathogenic mutations were detected. Of the 31 mutations, 8 novel frameshift mutations caused nonsense­mediated mRNA decay and associated with gender of the patients, presence of metachronous cancer, size, T staging, presence of distant metastases and positivity of microsatellite instability (MSI) in the cancer (p < 0.05). FAM134B expression in cancer cells derived from advanced stages (stage III; SW48 and stage IV; HCT116) of colon cancer was significantly (pT (S768I) and chr7:55259515T>G (L858R), are found approximately in 30% of Asian (Japanese) patients. In functional prediction results, these sites are exonic and nonsynonymous mutations. The REVEL scores of three EGFR mutations are 0.824, 0.765 and 0.961, and gerp++ scores are 5.5, 5.85 and 5.71, respectively. Both REVEL and gerp++ show high scores for severe damage of protein structure in the mutations. According to the results, researchers can infer that the three mutations are pathogenic variants implying the nucleotide positions with a higher constraint. #3574 A survey of mutations in biomedical literature using a machine based approach. Takahiko Koyama, Kahn Rhrissorrakrai, Laxmi Parida. IBM Research, Yorktown Heights, NY. Introduction: Being able to characterize mutations for both pathogenicity and drug response is indispensable to the analysis of tumor genomics and the development of therapeutic options. While a great deal of data has been deposited in various structured, genomic databases, a large portion of insights are primarily and often times solely found in biomedical literature. Medline contains about 26 million literature citations; a number that is unrealistic for a human to read. Thus machine based approaches are needed to comprehensively capture the landscape of reported mutations. Method: An automated pattern matching method is utilized to extract mutations

from Medline abstracts as presented in Human Genome Variation Society (HGVS) format and RefSNPs (rs) number. A typical HGVS protein mutation is described as [reference amino acid][position][new amino acid], as in p.His1047Arg, His1047Arg, or simply H1047R in HGVS format. This method identifies and consolidates all mentioned protein mutations and their alternate formulations. Result: Over 300,000 unique abstract­mutation pairs were identified including 90,000 unique mutations. Well known cancer mutations such as BRAF V600E, JAK2 V617F and EGFR L858R are among the most frequent appearing in oncology literature. At the other end, 51,000 mutations are mentioned in just a single abstract, 16,000 mutations in two abstracts, 7,600 in three abstracts, and so forth. Conclusion: The number of mutations appearing in Medline abstracts represents just a small portion of the 2 million unique coding mutations contained in the COSMIC database. While we expect the actual coverage of mutations by literature to be more comprehensive if this approach is extended to the full text body, the number would likely remain small compared with the total reported COSMIC mutations. One of the great challenges in oncology is characterizing variants of unknown significance (VUS), and by first extracting all reported mutations, even those mentioned in only one article, and their specific biological context, we can begin to identify broader patterns in mutations’ pathogenicity and their impact on drug response. #3575 Frequency of imaging findings suspicious for and suggestive of cancer between three different hospitals within a single health system. Lauren F. Comisar, Hanna M. Zafar, Darco Lalevic, Christopher Pizzurro, Charles E. Kahn, Mitchell Schnall, Tessa S. Cook. University of Pennsylvania, Philadelphia, PA. Outpatient imaging rates have risen over the past two decades. Imaging findings representing possible cancer are commonly detected on these exams and require follow­up. Our health system utilizes a standardized coding scheme, similar to the breast imaging and reporting data system (BI­RADS), to classify the malignant potential of masses on all abdominal and pelvic imaging exams; two of these categories are

suspicious (for masses that clearly represent malignancy) and suggestive (for masses that may represent malignancy). These codes are used at three hospitals in a single health system: a university hospital, a community hospital, and a Level 1 trauma center. Our objective was to evaluate the frequency of imaging findings suspicious for or suggestive of malignancy in the liver, kidneys, pancreas, and adrenals between outpatients and inpatients at these three hospitals. Over 1600 suspicious and suggestive masses were discovered in one year: 843 in the liver, 499 in the kidneys, 152 in the pancreas, and 137 in the adrenal glands. Masses suspicious for cancer were unevenly distributed by patient location. In the liver and kidneys, suspicious masses were more common in outpatients at all three hospitals; in the pancreas and adrenal glands, suspicious masses were more common in inpatients. Suspicious masses were unevenly distributed by modality. The proportion of MRI exams where at least one organ was coded as suspicious was higher than the proportion of CT exams. Masses suspicious for cancer were unevenly distributed across the three hospitals. Liver masses suspicious for cancer were twice as likely to be discovered at the university hospital compared to the community hospital and the Level 1 trauma center (present in 2% vs. 0.7% vs. 0.6% of all livers evaluated, respectively). Adrenal masses suspicious for cancer were also more commonly detected at the university hospital (present in 0.3% vs. 0.1% vs. 0.1% of adrenal glands evaluated, respectively). A similar trend was observed for renal and pancreatic lesions. Masses coded as suggestive of malignancy (rather than clearly suspicious for malignancy) were more frequently detected at the university hospital. This was particularly evident in adrenal lesions where 0.3% of adrenal glands evaluated at the university hospital were suggestive of malignancy compared to 0.06% at the community hospital and 0.03% at the Level 1 trauma center. A similar trend was observed for pancreatic lesions. In conclusion, this novel database of standardized codes for abdominal masses provides insight into how masses suspicious for and suggestive of cancer are coded variably by organ, patient location, and modality between three different hospitals within the same urban academic health system. Further investigation will be needed to determine the influence of patient case mix, referral patterns, and radiologist characteristics that contribute to these observed differences. #3576 Accurate identification of somatic mutations in cancer patient specimens in the lack of normal tissue by targeted high­throughput

sequencing. Francisco M. De La Vega,1 Sean Irvine,2 David Ware,3 Kurt Gaastra,3 Yannick Pouiliot,4 Len Trigg3. 1Stanford University, Stanford, CA; 2Real Time Genomics, Inc., Hamilton, New Zealand; 3Real Time Genomics, Hamilton, New Zealand; 4TOMA Biosciences, Foster City, CA. Tumor molecular profiling is rapidly becoming the standard clinical test for selecting targeted therapies in refractory cancer patients. DNA extracted from patient samples is enriched for cancer genes and sequenced to identify actionable somatic mutations therein. A major challenge arises when tumor­derived data is analyzed in the absence of normal tissue data, as it is common in clinical scenarios. The distinction between somatic and germline variants become difficult, leaving clinicians to resort to crude heuristic filtering. We present here a variant calling software, developed under quality system regulation protocols, capable of accurately identifying somatic mutations from targeted next­ generation sequencing data. A novel Bayesian Network approach models the distribution of reads harboring germline and somatic mutations, estimates the contamination from normal tissue in the sample, scores somatic mutations, and imputes germline variants, without matching normal tissue data. This approach also allows joint analysis of multiple specimens from the same patient (e.g. FFPE and ctDNA), when available, improving the limit of detection. To improve specificity, our caller can also utilize prior information from different databases including somatic mutations, germline variation, and healthy controls data, in a principled fashion. We validated our method by analyzing data from the TOMA OS­ Seq 131 cancer gene panel using the Illumina platform. Sample inputs ranging from 2­600ng of DNA were sequenced to a depth of >1000X, achieving on target rates ≤73% and uniformity ≥ 3.2 fold 80 penalty. Through adaptors with molecular barcodes we measured a median duplicate rate 30% in 8/15 cases of CO; 4/8 of PA, and 2/2 of KI. To determine whether DC­HIL+ MDSC migrate into cancer sites, lesional specimens from CO patients (n=5) with high blood counts of DC­HIL+ MDSC were immunohistochemically stained for CD14 and DC­HIL expression: DC­HIL was absent from all cancer cells, but present strongly in many CD14+ cells surrounding the cancers. This outcome contrasts starkly with melanoma, in which DC­HIL was expressed highly by the cancer cells surrounded by only a few DC­HIL+ CD14+ cells. Finally we evaluated in vivo effects of blocking DC­HIL using a mouse model of MC38 colon cancer, which is DC­HILneg. MC38 tumor challenge induced DC­HIL+ Gr1lowLy6Chigh MDSC in the tumor site. Intravenous infusion of anti­DC­HIL mAb into mice with pre­ established MC38 tumor significantly inhibited tumor growth by 50­70%, reduced blood levels of MDSC, and enhanced cytotoxic T­lymphocyte response. Our findings support DC­HIL blockade as a potential treatment for metastatic cancers, with high blood levels of DC­HIL+ MDSC as a prognostic marker for the best responders. #3661 Breaking down the barrier restricting infiltration and differentiation of APC in the tumor microenvironment with a first­in­class antibody targeting Semaphorin4D, and rational combination therapies. Elizabeth E. Evans,1 Holm Bussler,1 Crystal Mallow,1 Christine Reilly,1 Sebold Torno,1 Maria Scrivens,1 Cathie Foster,1 Alan Howell,1 Stephen R. Comeau,1 Leslie Balch,1 Alyssa Knapp,1 John E. Leonard,1 Terrence L. Fisher,1 Siwen Hu­Lieskovan,2 Antoni Ribas,2 Ernest S. Smith,1 Maurice Zauderer1. 1Vaccinex, Rochester, NY; 2UCLA, Los Angeles, CA. Purpose: We expand mechanistic findings in preclinical studies to demonstrate that antibody blockade of Semaphorin 4D (SEMA4D, CD100) reduces expansion of MDSC and shifts the balance of myeloid cells within the TME to facilitate tumor rejection. Efficacy is further

enhanced when combined with various immunotherapies. Design of Phase 1b/2 combination trials of VX15/2503, a humanized IgG4 antibody targeting SEMA4D, with immune checkpoint inhibition will be presented. Methods: Anti­SEMA4D antibodies were evaluated alone and in combination with other immunotherapies in various preclinical models. Anti­tumor activity and immune response was characterized by immunohistochemistry, flow cytometry, functional assays, and cytokine, chemokine and gene expression analysis. A Phase I trial for single agent VX15/2503 was completed, and several 1b/2 combination immunotherapy trials are planned. Results: SEMA4D restricts migration of monocytes and promotes expansion of suppressive myeloid cells in vitro. Strong expression of SEMA4D at the invasive margins of actively growing tumors in vivo restricts the infiltration and modulates polarization of leukocytes in the TME. Antibody blockade of SEMA4D facilitated recruitment of activated DCs and T lymphocytes in preclinical models. M­MDSCs were significantly reduced in tumor and blood following treatment. A significant shift towards increased Th1 cytokines (IFNγ, TNFα) and CTL­recruiting chemokine CXCL9, with concurrent reduction in Treg­, MDSC­ and M2­macrophage promoting chemokines (CCL2, CXCL1, CXCL5) was observed. Accordingly, Teff:Treg ratio (3x, p
2Seattle Genetics, Bothell, WA.

B cell maturation antigen (BCMA) has recently emerged as an attractive therapeutic target in multiple myeloma. BCMA has restricted expression on plasma cells with little to no expression on other normal tissues, but is upregulated on the surface of multiple myeloma cells. BCMA can regulate proliferation and survival of myeloma cells via binding to its ligands APRIL and BAFF and induce downstream signaling pathways. Thus, several approaches to target BCMA are currently under clinical investigation, including chimeric antigen receptor (CAR) T cell therapies, bispecific antibodies and antibody drug conjugates. The Antibody­Coupled T cell Receptor (ACTR) technology is a universal, engineered T cell therapy consisting of the extracellular domain of human CD16 and the intracellular T cell co­stimulatory and signaling domains. ACTR is designed to engage the Fc domain of therapeutic antibodies opsonized to target cells to mediate anti­tumor activity. Previous work has demonstrated ACTR T cell activity in combination with rituximab, trastuzumab, and hu14.18 K322A against CD20, Her2, and GD2 expressing cell lines, respectively (Kudo et al. Cancer Res 2014; 74:93­103). Currently ACTR is being evaluated in Phase I clinical trials with rituximab to treat relapsed refractory B cell lymphoma. Here we demonstrate a humanized afucosylated anti­BCMA antibody, SEA­BCMA, binds to ACTR expressing T cells with high affinity and mediates T cell activation, potent cytotoxicity, cytokine release and proliferation across a wide range of BCMA expressing myeloma cells. ACTR activity was specific to SEA­BCMA ­ opsonized target cells, dose dependent and had no activity on BCMA negative tumor lines. Furthermore, the SEA­BCMA antibody has additional properties that might contribute to a therapeutic effect, including blocking the binding of ligands to BCMA and driving natural killer cell mediated ADCC effects. These preclinical studies demonstrate a promising multi­faceted activity of ACTR T cells in combination with the anti­BCMA antibody, SEA­ BCMA, for clinical consideration in multiple myeloma patients. #4606 Preclinical characterization of a novel fully human IgG1 anti­PD­L1 mAb CK­301. Leonid Gorelik,1 George Avgerinos,1 Yune Kunes,2 Wayne A. Marasco3.

1Checkpoint Therapeutics, New York, NY; 2TG Therapeutics, New York,

NY; 3Dana­Farber Cancer Institute, Boston, MA. Antibodies targeting Programmed Death­1 (PD­1), or its ligand, PD­L1, have demonstrated remarkable efficacy in subsets of cancer patients, with inhibition of the interaction between PD­1 on T­cells and PD­L1 on tumor cells leading to the recovery of anti­tumor immune response and immune­mediated eradication of tumors. However, not all patients respond to existing PD­1 and PD­L1 targeting agents and relapses to therapy still occur. Therefore, there exists a need to identify additional therapeutics and approaches to engage the immune system to enhance the efficacy of current anticancer therapies. Using phage and yeast display approaches, we have discovered and optimized a novel, fully human, PD­ L1 specific IgG1 antibody, CK­301, which exhibits subnanomolar binding affinity for PD­L1. CK­301 blocks binding of PD­L1 to both PD­ 1 and B7­1 in enzyme­linked immunosorbent assays (ELISA) and cell­ based competition assays. Using an assay measuring inhibition of a nuclear factor of activated T­cells (NFAT) reporter caused by PD­L1 binding to PD­1, we demonstrate that CK­301 completely reverses reporter inhibition at concentration of less than 1 µg/ml, IC50 of the dose response curve is 80ng/ml. CK­301 enhances IFN­gamma secretion in allogeneic mixed lymphocyte reaction (MLR) using primary human T­ cells and immature dendritic cells. CK­301 can also trigger antibody­ dependent cell­mediated cytotoxicity (ADCC) and complement­ dependent cytotoxicity (CDC) mediated killing of PD­L1+ cell lines, including lymphoma cells. CK­301 has similar subnanomolar affinity for cynomolgus monkey PD­L1 as for human PD­L1, hence we chose Macaca fascicularis for pre­clinical toxicology and safety pharmacology studies. Single­dose administration of CK­301 to monkeys up to the highest tested dose of 100 mg/kg was shown to be safe and demonstrated linear dose­dependent pharmacokinetic (PK) properties over the dose range from 1 to 100 mg/kg with a half­life of 15 days at 100 mg/kg. A first­in­human Phase 1 study of CK­301 is planned to commence in mid­ 2017. #4607 Specific inhibition of PGE2­EP4 signaling by E7046 promotes anti­ tumor activity of checkpoint blockade agents through boosting cytotoxic T cell activity.

Diana I. Albu,1 David Verbel,1 Yuan Huang,1 Donna Kolber­Simonds,1 Zichun Wang,1 Xulong Wang,1 Zoltan Dezso,2 Christy Ingersoll,1 Kuan­ Chun Huang,1 Janna Hutz,1 Mary Woodall­Jappe,3 Xingfeng Bao1. 1Eisai Andover Innovative Medicines Institute, Andover, MA; 2Nurix Inc., San Francisco, CA; 3Eisai Oncology Business Group, Eisai Inc., Andover, MA. Purpose: Immunotherapies targeting immune checkpoint receptors have shown great promise for a subset of cancer patients; however, robust and safe combination therapies are still needed. In the tumor microenvironment, prostaglandin E2 receptor type 4 (EP4) signaling has been implicated in both protumoral myeloid cell differentiation and cytotoxic T cell exhaustion. We evaluated the combination of the EP4 antagonist E7046 (clinical trial NCT02540291) with anti­PD1 or anti­ CTLA4 in preclinical tumor models, and also interrogated the relationship between PGE2 pathway activation and cancer patient survival. Materials/Methods: Mouse syngeneic tumor models CT­26 and 4T1 were used for pharmacological investigation. GMP grade E7046 was administered to tumor­bearing animals by oral gavage. Co­culture of EG7­OVA and OT1 cells in an antigen­specific cytotoxic T cell (CTL) activation assay provided mechanistic insights. For translational validation, transcripts of five major genes involved in PGE2 synthesis, transport and degradation were compared between malignant and normal tissues across all TCGA tumor types, and correlation of their expression with overall survival was assessed. Results: In the CT26 tumor model, the combination of E7046 and anti­ PD1 resulted in significantly more tumor­free animals compared with either agent alone. In the 4T1 tumor model, the combination of E7046 and anti­CTLA4 was also more effective in suppressing tumor growth and tumor rejection compared with anti­CTLA4 alone, and was accompanied by a markedly increased accumulation of GZMB+CD8T+ CTLs in the treated tumors. Consistent with those findings, addition of anti­PD1 antibody promoted OVA­specific CTL activation in vitro while addition of PGE2 strongly inhibited it, as measured by IFNγ secretion. Inclusion of E7046 dose­dependently reversed the PGE2­induced suppressive activity in the presence of anti­PD1 antibody. Among major human PGE2 pathway genes, TCGA analysis showed that PTGES1 was

upregulated and HPGD downregulated across a broad range of tumor types. In contrast, COX1, COX2 and PGT showed less difference between malignant and normal tissues. Importantly, these differences of one or multiple PGE2 pathway genes were strongly associated with patient survival in certain cancer types. Conclusions: A subset of human cancer types displays upregulated PGE2 pathway that is associated with a poorer prognosis. PGE2­EP4 signaling potently suppresses antigen­specific CTL activation in the presence of PD1 signaling blockade. The combination of EP4 antagonist E7046 with either anti­PD1 or anti­CTLA4 demonstrated superior anti­tumor activity compared with anti­PD1 or anti­CTLA4 alone. This increased activity was accompanied by increased CTL activation. #4608 A 5T4x CD3 bispecific DART® molecule with extended half­life for T­cell immunotherapy of cancers. Ling Huang,1 Gurunadh Chichili,1 Ralph Alderson,1 Francine Chen,2 Jennifer Brown,1 Hua Li,1 Valentina Ciccarone,1 Jim Tamura,1 Daorong Liu,1 Liqin Liu,1 Syd Johnson,1 Ezio Bonvini,1 Paul Moore1. 1MacroGenics, Inc., Rockville, MD; 2MacroGenics, Inc., South San Francisco, MD. Introduction: 5T4 (trophoblast glycoprotein), an oncofetal antigen involved in embryo development, is expressed on the cell surface of multiple cancers. Several 5T4­directed interventions have been reported, including a cancer vaccine and an antibody­drug conjugate. A superantigen­driven redirected cell killing modality was also developed, but obstacles, such as immunogenicity, remain to a successful 5T4 therapeutic molecule. We have developed an Fc­bearing 5T4 x CD3 DART® bispecific protein designed to redirect T cells to target 5T4­ expressing tumors. Methods: 5T4x CD3, a humanized Fc­bearing DART molecule, was stably expressed in CHO cells and purified to homogeneity via a standard antibody­purification platform. In vitro characterization and functional studies were performed with 5T4­positive tumor cell lines and human T cells. In vivo studies were performed in immune­deficient tumor­bearing mice co­implanted with activated human T cells or reconstituted with human peripheral blood mononuclear cells (PBMCs). Pharmacokinetic

studies were performed in both human FcRn transgenic mice and cynomolgus monkeys. Results: 5T4x CD3 demonstrated bispecific binding properties to both human and cynomolgus monkey antigens. In redirected cytolysis studies, 5T4x CD3 mediated lysis of 5T4­positive pancreatic, lung, renal, triple­ negative breast, and ovarian cancer cell lines with EC50 values ranging between 0.03 and 0.08 ng/mL. 5T4x CD3 displayed favorable pharmacokinetics with a prolonged circulating half­life in human FcRn transgenic mice and cynomolgus monkeys. Tumor clearance studies in NOD/SCID mice implanted subcutaneously with activated human T cells and tumor cells demonstrated robust inhibition of tumor growth upon intravenous administration of 5T4 x CD3 at doses as low as 0.8 μg/kg, but not with a CD3­binding control DART protein. In addition, human PBMC­reconstituted NOD/SCID/IL2 gamma­chain null mice were implanted intradermally with renal and pancreatic tumor cell lines or orthotopically with a triple­negative breast cancer line and, after tumor establishment, were treated with 5T4 x CD3. Anti­tumor activity was observed at doses as low as 4 μg/kg. Immunohistochemical analysis of tumor xenografts confirmed tumor clearance was associated with T­cell recruitment into the tumor mass. Conclusions: In summary, 5T4 x CD3 displays robust antitumor activity against several cancer cell lines in vitro and in vivo together with a favorable pharmacokinetic profile and merits further consideration as a potential treatment for 5T4­positive cancers. #4609 Agenus’ next generation cancer vaccine platforms. Mohamed Uduman,1 Mithun Khattar,1 Bishnu Joshi,1 Antoine Tanne,1 Benjamin Morin,1 Armen Karapetyan,1 Elise Drouin,1 Sandra Craig,1 Paisley Myers,1 Erin Jeffery,1 Nicholas Wilson,1 Amy Yang,1 Victor H. Engelhard,2 Mark Cobbold,3 Donald F. Hunt,4 Dennis Underwood,1 Shiwen Lin,1 Mark Findeis,1 Jeffrey Raizer,1 John Goldberg,1 Jennifer S. Buell,1 Robert Stein,1 Daniel L. Levey,1 John Castle1. 1Agenus Inc., Lexington, MA; 2University of Virginia School of Medicine, Charlottesville, VA; 3Massachusetts General Hospital Cancer Center, Charlestown, MA; 4University of Virginia, Lexington, MA. Most cancer cells carry mutations unique to the patient’s individual tumor

and shared biochemical signatures that are not present in healthy cells. Agenus has three vaccine platforms designed to treat cancers based on the unique needs of a given patient. Our vaccine platforms are designed to educate the patient’s immune system to recognize tumor­specific aberrations, or neo­antigens, and mount an anti­tumor immune response. Agenus’ Prophage™ vaccine platform is an individualized vaccine made from the patient’s own tumor tissue. Heat shock proteins (gp­96) that naturally chaperone and bind tumor­derived peptides are extracted from the patient’s tumor and constitute the vaccine. Some of these peptides are neo­antigens. Agenus has completed Phase 2 clinical trials with Prophage™ vaccine in newly diagnosed glioblastoma (ndGBM), and has previously reported that there was improved progression­free and overall survival with Prophage™ vaccine compared to standard of care. Agenus’ AutoSynVax™ vaccines are uniquely designed and manufactured for each patient based on NGS profiling of the patient’s tumor from a biopsy. Leveraging the Agenus Immunogenic Mutation (AIM™) workflow, we are able to generate a synthesis­ready blueprint for an optimal immunogenic and personalized neo­antigen vaccine. The AIM™ platform provides a robust and efficient approach to computational vaccinology designed to deliver a set of likely immunogenic peptides, agnostic to vaccine format, followed by generation of a format­specific blueprint ready for vaccine synthesis and manufacture. The synthesized neo­epitopes are complexed to recombinant heat shock protein 70 (HSC70) and are administered along with our QS­21 Stimulon® adjuvant. HSC70 is known to transport epitopes and play a role in displaying them to T cells. While the first two of Agenus’ vaccine platforms are highly individualized, our PhosphoSynVax™ vaccine is an off­the­shelf vaccine format targeting a novel class of tumor neo­antigens arising from post translational modifications (PTMs). Due to dysregulated cell signaling pathways in cancer, self­peptides can be aberrantly phosphorylated, a number of which are subsequently presented on HLA molecules. Using mass­spectrometry, we have identified a library of over a thousand HLA phospholigands. Many of these are tumor specific and found in multiple patients across multiple indications, enabling pre­manufacture of PhosphoSynVax™ vaccines for ready use. Upon testing the HSP plus synthetic peptide vaccine format in murine models, we have demonstrated effective tumor control in a therapeutic setting and also effective immune memory in a long­term prophylactic setting. Given Agenus’ diverse portfolio we have the opportunity to

combine our immune education strategies with immunomodulatory antibodies to increase therapeutic efficacy. #4610 Towards understanding the cellular uptake patterns of nano­ particles among different immune cell lines. Noha Ismail,1 Ashish Kulkarni,2 Siva Kumar,2 Vineeth krishna,2 Shiladitya Sengupta2. 1The American Univ. in Cairo/Brigham and Women’s Hospital – Harvard Medical School/Harvard­MIT Health Sciences and Technology, Cairo, Egypt; 2Brigham and Women’s Hospital – Harvard Medical School/Harvard­MIT Health Sciences and Technology, Cambridge, MA. Today with the privilege of being able to synthesize extremely small particles in the nano­range and make use of nanoscience and nanotechnology, we can reach superior properties than the bulk scale. Nanoparticles have their own physicochemical properties that make them promising in the field of cancer immunotherapy in form of drug delivery, diagnostic and theranostic modalities. Manipulation of the immune response by therapeutic intervention is becoming of great interest owing to the significant role of immunity in the general health and disease control. APCs like Dendritic cells and macrophages are important targets for the particulate delivery system due to their ability to trigger cascade of events on both levels cellular and humoral immune response specially DCs. Successful targeting of DCs and macrophages will have a great impact on T­cell activation and priming. The design of successful particulate system that can elicit preferential targeting towards specific immune cell in the tumor microenvironment is becoming crucial. This might represent a novel avenue for enhancing the antitumor immunity. Liposomes are made of phospholipid bilayer with a composite nature. Their properties are highly linked to their physicochemical characteristics such as size, surface charge and composition. These physicochemical properties can trigger certain immune response.The hypothesis here whether passive targeting via particulate system can offer a platform for preferential targeting for critical immune cells residing in spleen and tumor. Moreover, test whether there will be differences in uptake according to certain preference towards surface charge in specific time frame. In the current study, three sets of fluorescently labelled nano­ liposomes were engineered as a model for different surface charges, the

cationic DOTAP NP, anionic DOPG NP and near neutral DOPC NP with mean diameter of ~ 200 nm. Physical stability of the NPs was evaluated by monitoring the changes in size and zeta potential. B16 melanoma cancer model was induced subcutaneously in C57BL/6 black mice, divided into four groups each of five mice. CD11c Dendritic Cells (DCs), CD11b macrophages, CD90.2 T­cells and CD49b Natural Killer (NK) cells were isolated from the tumors and spleens of each group. The three sets of NPs were tested against the isolated cell lines. The cellular uptake (internalization) was assessed by normalizing the fluorescence of the cells against their protein concentration, then all samples were acquired to flow cytometry, and shifts in fluorescence histograms on horizontal axis were monitored against PE channel on the vertical axis. Results reveal the presence of preferential internalization of specific surface charge over others in some cell lines in different time frames. For the first time differences in the internalization pattern are reported in the same immune cell line isolated from two different contexts tumor and spleen. #4611 A 3D in vitro culture­based method to visualize and quantify effects of immuno­modulatory drugs. Lidia Daszkiewicz, Kuan Yan, Maarten Klop, Leo Price. OcellO, Leiden, Netherlands. BACKGROUNDUnleashing the full potential of cancer immunotherapy is hampered by a lack of effective in vitro testing methods. 2D cultured cells poorly reflect the in vivo situation and lack the tumour microenvironment whereas humanized animal models are slow, have low throughput and are expensive. 3D cultures, on the other hand, more faithfully reproduce the organization of a tissue, recapitulating the cell­ cell connections and cellular heterogeneity found in the tumour and allow for a cost­efficient testing of drug candidates. To address the unmet need of immunotherapy cancer drug developers for robust assays that incorporate the human immune system, with functional read­outs in a high throughput (384well plate) format, we developed an in vitro platform based on 3D co­cultures of cancer tumoroids and immune cells. This assay is designed to test the capacity of small molecules, bi­specific antibodies, checkpoint modulators, vaccines and combinations of thereof to enhance anti­cancer immune responses. METHODSCancer cells of different sources were grown in a hydrogel to form tumoroids and then HLA­matched PBMCs pre­conditioned via different protocols to achieve

activated or exhausted T cells, were added in the presence or absence of immune­modulatory drugs. T cell infiltration and subsequent tumoroid killing was quantified using 3D high­content imaging. After ‘optical sectioning’ 3D image stacks were reconstituted and morphometric analysis was performed with OMiner software. RESULTS AND CONCLUSIONS Addition of immune checkpoint inhibitors, pembrolizumab and ipilimumab, led to enhanced T cell infiltration and cytotoxicity of exhausted T cells. These results showed that automated 3D image and data analysis enables discrimination of immune­tumour cell interactions depending on activation status of T cells and permits the visualization of T cell re­gain of function in the presence of checkpoint modulators. A 3D environment allows the different cell types to engage in a more realistic setting than when cells are grow in a monolayer and enables the quantification of spatially resolved information, which is of an utmost importance when studying tumor­immune cell relationships. This new and innovative platform empowers immunotherapy drug developers with a powerful tool to evaluate drug performance. Furthermore, drug combinations can be studied to determine synergistic effects with other checkpoint inhibitors of standard of care compounds in an efficient manner. #4612 Development and validation of a screening platform for the identification of novel immuno­oncology targets. Ariane Scoumanne,1 Virginie Rabolli,2 Lea Legrand,3 Murielle Martini,4 marie­claire Letellier,5 Stefano Crosignani,6 Christophe Quéva,3 Michel Detheux,7 Sandra Cauwenberghs,8 Jakub Swiercz9. 1Ariane Scoumanne, Gosselies, Belgium; 2Virginie Rabolli, Gosselies, Belgium; 3iTeos Therapeutics, Gosselies, Belgium; 4Murielle Martini, Gosselies, Belgium; 5Marie­Claire Letellier, Gosselies, Belgium; 6Stefano Crosignani, Gosselies, Belgium; 7Michel Detheux, Gosselies, Belgium; 8Sandra Cauwenberghs, Gosselies, Belgium; 9Jakub Swiercz, Gosselies, Belgium. Although immune therapy of cancer, including immune­checkpoint blockade have demonstrated therapeutic benefit in patients with various advanced cancers, further understanding of human immune pathology triggered by the tumor microenvironment, is essential to improve these therapeutic approaches.

In order to shed light on novel immune suppressive mechanisms in tumor, iTeos Therapeutics developed a target discovery and drug repurposing platform based on phenotypic screening assays. We established a co­ culture assay combining tumor immune suppressive cells and T­cells. This assay is flexible to allow the screening of chemicogenomics, shRNA and cDNA libraries. Multi­parameter readouts are combined to assess both T cell activation and proliferation, through high content imaging of T cell clusters formation, complemented with detection of IFNγ secretion and tumor cell death, as assessed using a cytotoxicity assay. The 96­well format of the assay allows medium­throughput testing of up to 3000 samples/screen. From the technical point of view we were able to adapt the assay to low level of automation, making it affordable to the biotech start­ups and academic laboratories. As a proof­of­concept we evaluated the assay for its ability to detect metabolic immune­oncology targets in A549 cells, a lung cancer immune suppressive cell line. A549 express indoleamine­2,3­dioxygenase 1 (IDO1), an enzyme expressed in many cancers that mediates local T­cell suppression through depleting the essential amino acid tryptophan. The assay conditions were validated with an IDO1 inhibitor as positive control and subsequently scaled up for automation. A commercially available small molecule library of 1900 compounds, with a high percentage of clinically tested drugs was screened. The library was tested at two different concentrations (0.3μM and 3μM), with two independent T­cell donors and spiked with IDO1 inhibitor as control. Combined analysis of T­cell activity and tumor killing led to the identification of 42 compounds with activity on multiple, potential immune suppressive pathways, including metabolism, epigenetics, autophagy, TGFβ, Wnt/β­ catenin and TNFα/NF­κB signaling. #4613 Cytotoxicity of selenium trastuzumab and bevacizumab immunoconjugates against triple negative breast cancer cells. Soni Khandelwal, Lauren Gollahon, Julian Spallholz, Mallory Boylan, Maria Del Mar Garcia­Hernandez. Texas Tech University, Lubbock, TX. Breast cancer (BC) is the second leading cause of death among women with an estimated 246,660 new cases of invasive breast cancer expected to be diagnosed this year. Of the different subtypes, 15­20% are classified as triple negative breast cancer (TNBC) because they lack three key receptors; progesterone, estrogen and HER2. These receptors are

considered essential for “targeted therapies”. Thus, systemic chemotherapy is the only TNBC drug treatment option. In this study, two clinical monoclonal antibodies; Herceptin® and Avastin® were covalently labeled with redox selenium and used to treat two TNBC cell lines (MDA­MB­231 and MDA­MB­468) in culture. TNBC cell lines were treated with vehicle control, increasing concentrations of selenium as Selenite (ranging from 2 to 20 µg); Se­Herceptin® or Se­Avastin® and compared to native antibodies with equal concentrations of protein. Cell counts and viability were analyzed using Beckman ViCell Counter and Trypan Blue exclusion over a seven day period. Selenite, Se­Avastin® and Se­Herceptin® were observed to be more cytotoxic over dose and time to both TNBC cell lines in comparison to control cells and cells treated with native antibodies alone. Morphological changes were observed under phase contrast microscopy in all selenium treated cell lines. In contrast, visible alterations in morphology were absent in native Herceptin® and Avastin® treated cells compared to control cells. We believe this is the first report of selenium Antibody­Drug Conjugates (ADCs) being demonstrated as cytotoxic to these TNBC cell lines, suggesting a potential strategy to design more effective treatments of TNBC resistant to chemotherapy. #4614 Upregulation of memory T cell population and enhancement of Th1 response by lenvatinib potentiate antitumor activity of PD­1 signaling blockade. Yu Kato. Eisai.Co.,Ltd., Tsukuba­Shi, Ibaraki, Japan. Introduction: Lenvatinib (LEN) selectively inhibits the kinase activity of VEGFR1­3, FGFR1­4, KIT, PDGFRα, and RET, which are involved in tumor angiogenesis and tumor cell proliferation in several cancer types. Currently, Phase 1b/2 clinical trials of the combination of LEN and pembrolizumab (a monoclonal antibody [mAb] that blocks the interaction between PD­1 and its ligands) are ongoing for selected types of cancer including renal cell carcinoma, melanoma and non­small cell lung carcinoma. We have reported that tumor associated macrophage and regulatory T cell population were downregulated by treatment of LEN. In this study, mechanism of LEN and PD­1 mAb combination was investigated by flow cytometory and RNA­seq analyses. Experimental Procedure: We examined antitumor activity of combination treatment of LEN (10mg/kg, qd) and anti­mouse PD­1 mAb

(500μg/mouse, twice weekly) in Ct26 and B16F10 syngeneic mouse models. In order to investigate tumor­specific memory T cell response, re­inoculation of fresh CT26 cells into tumor­free mice was conducted. For immune cell population analyses, tumor or spleen samples were analyzed by flow cytometry. Changes of molecular immune response in tumors were examined by RNAseq followed by weighted gene co­ expression network analysis (WGCNA). Summary of data: Combination of LEN with PD­1 mAb showed more potent anti­tumor growth activity in CT26 and B16F10 models compared with either treatment alone. Notably, complete tumor regression was detected in a portion of mice that received combination treatment only. Re­inoculation of fresh CT26 cells into these tumor­free mice did not grow tumor; in contrast, all naïve animals grew tumor aggressively in the same experimental condition. RNAseq analysis of treated CT26 tumors indicated that combination of LEN with PD­1 mAb synergistically up­ regulates IFN signaling­related genes. Flow cytometory analysis revealed that LEN increased memory T cell population compared with vehicle treatment. Conclusions: The results indicate that the combination of LEN with PD­1 mAb was more effective than either single agent alone in multiple syngeneic tumor models and was accompanied with a potent antitumor immune response, especially the up­regulation of memory T cell and Th1 activation by LEN. #4615 Development of an antibody­drug conjugate with broad anticancer activity. Siang­Yo Lin,1 Zoltan Szekely,1 Chen­Yong Lin,2 Joseph R. Bertino,1 Gulam Mohmad Rather1. 1Rutgers Cancer Instutute of NJ, New Brunswick, NJ; 2Georgetown Medical School, Georgetown, MD. Treatment for patients with advanced solid tumors that include triple negative breast cancer (TNBC), non­small cell lung cancer (NSCLC), and castrate resistant prostate cancer (CRPC), as well as Mantle Cell Lymphoma (MCL), while increasing survival is not curative. We have identified a membrane bound protease, “activated” matriptase, as an attractive target antigen for highly selective antibody delivery of cytotoxins as activated matriptase expression is restricted to epithelial tumors and some B­cell lymphomas. We generated a novel ADC by

linking M69, a mouse antibody specific to activated matriptase, to monomethyl auristatin E (MMAE) via a PEGylated, releasable di­peptide linker as a proof­of­principle prototype. Both in cell lines and in human xenograft models of TNBC, NSCLC, CRPC and MCL, the conjugate was found to exhibit potent anticancer activity against all of these tumor types without toxicity. Encouraged by these results, we are also exploring the use of this ADC against gastric and pancreatic cancer, and in combination with chemotherapy and immunotherapy. As this is a mouse antibody, we are also generating a chimeric antibody for toxicity studies in primates.

Inflammation in the Tumor Microenvironment #3688 Imprime PGG, a novel innate immune therapeutic in phase 2 clinical development, induces mobilization of monocytes and focalized recruitment of innate immune cells to tumor sites. Steven Leonardo, Nadine Ottosson, Keith Gorden, Takashi Kangas, Xiaohong Qiu, Ross Fulton, Benjamin Harrison, Adria Jonas, Richard Walsh, Katie Ertelt, Jamie Lowe, Richard Huhn, Jeremy Graff, Nandita Bose, Mark T. Uhlik. Biothera Pharmaceuticals, Inc., Eagan, MN. Immune checkpoint inhibitors (CPI) have shown compelling clinical efficacy in multiple tumor types, though only in a minority of treated patients. Significant research and clinical development are focused on expanding CPI efficacy. Imprime PGG is a novel, IV administered 1,3/1,6 β­glucan PAMP (pathogen­associated molecular pattern) that activates innate immune effector cells to enhance tumor killing, to repolarize the suppressive myeloid cells of the tumor microenvironment and to activate the antigen presentation capability of dendritic cells, macrophages and monocytes. In multiple preclinical models, Imprime enhances the anti­tumor efficacy of CPIs. Imprime is now in multiple phase 2 clinical studies in combination with the CPI, pembrolizumab. We sought to understand more precisely how Imprime activates the innate immune system to enable a concerted innate and adaptive anti­cancer immune response. Using multispectral fluorescence IHC we now show that Imprime induces focalized recruitment of innate immune cells to tumor bearing tissue. In the B16F10 experimental metastasis model, Imprime dosed in combination with the tumor­targeting antibody TA­99 can nearly completely repress the outgrowth of pulmonary metastases across a 19 day time course. At 24h post­Imprime treatment, the presence of Ly6G+ neutrophils was evident throughout the lung tissue. At later time points (72h and beyond) the formation of immune cell clusters was readily evident in lungs from Imprime treated mice and rarer in control mice or mice treated only with TA­99. These immune cell clusters were predominately localized to arterioles near B16 tumor sites and comprised of multiple immune cell subtypes including macs, B cells, T cells as well as a monocyte population that are CD11b+, Ly6G­ and F4/80­ and strongly positive for MHCII. Consistent with these preclinical findings, IV administration of Imprime to healthy human volunteers increased neutrophil and monocyte mobilization into peripheral blood 2­3 fold 4h

post infusion. Imprime treatment also resulted in a significantly increased subset of CD16+ monocytes that are known to have higher antigen presentation capability and express higher levels of the activation markers CD86, PD­L1, and HLA­DR (MHCII). Furthermore, RNA expression profiling of whole blood from Imprime­treated volunteers shows increased expression of the CCL3, CCL4, IL­1β and TNF­α, functional mediators produced by these monocyte populations. Together, these data show that Imprime drives the concerted activation of multiple innate immune subtypes and promotes the appearance of unique monocyte populations that may be critical for an Imprime­induced anti­cancer immune response. #3689 Jak3 mutations in colorectal neoplasia­Preliminary data on a not so silent minority. Martin Tobi,1 Xiaoping Zhao,2 Darshana Jhala,3 Rebecca Rodriguez,4 Fadi Antaki,5 Edi Levi,5 Paula Sochacki,5 John Lieb,3 MaryAnn Rambus,6 Tapan Ganguly,7 Martin Bluth,8 Michael J. Lawson9. 1Saginaw VA Medical Ctr and Medical Disciplines, Central Michigan University College of Medicine, Saginaw, MI; 2Detroit VAMC R&D, Central Michigan University College of Medicine, Detroit, MI; 3Philadelphia VAMC and the University of Pennsylvania School of Medicine, Philadelphia, PA; 4Philadelphia VAMC, Philadelphia, PA; 5Detroit VA Medical Ctr and Wayne State University School of Medicine, Detroit, MI; 6Detroit VA Medical Ctr, Detroit, MI; 7University of Pennsylvania School of Medicine, Philadelphia, PA; 8Wayne State University School of Medicine, Detroit, MI; 9Kaiser Permanente Medical Center, Sacramento, CA. As a tumorigenesis model, colorectal cancer is associated with multiple gene mutations accumulating progressively but also has mutations that may alter disease course and provide a therapeutic target. Good examples of this are EGFR and VEGF. In melanoma, PD­1 interactions involving the immunobiome are therapeutically important. We conducted a study to detect potential mutations that might enhance therapy in colorectal cancer guided by expression of p87, a product of innate immune system Paneth cells. Methods: Adnab­9 immunohistochemistry or ELISA was used to

define significant p87 Adenoma­associated antigen field effects (FE) in 10 patients with >1cm large high grade dysplastic adenomas (LHiGDA) and 3 with smaller high grade dysplastic adenomas (SHiGDA). We postulated that SHiGDA are not immunologically recognized by host defenses leading to negative outcomes. We used Ion Torrent™ sequencing (ITS) to find mutations in DNA (QiaAmp kit) extracted from 4 normal­appearing colonic segments taken from 1 patient in each group. Novel mutations found on ITS would then be sought using PCR with appropriate primers and subsequent sequencing of the PCR product circulating DNA extracted from available serum samples. These samples were taken from the Large­ and SHiGDA groups described above and from 17 patients undergoing colonoscopy for diverse indications. Results: p87 FE were found in 40% of 10 LHiGDA and 0% of 3 SHiGDA patients. The ITS in the 2 representative patients showed unique mutational fields in: KRAS, APC, p53 in the LHiGDA and Jak3, PIK3Ca, p53, APC in the SHiGDA patient, both of whom lacked p87 FE. PCR using the Jak3 primers used in the ITS and subsequent sequencing revealed the same non­synonymous mutation in the serum of a FAP patient after colonic resection and an additional colonic segment of the selected ShiGDA patient but not in his serum. Other Jak3 mutations were found in 1 of 8 LHiGDA, 1 (the selected patient) of 3 SHiGDA, 1 of 7 patients with FAP and 1 of 10 colonoscopy patients with a family history of colorectal cancer with a FE and an untoward outcome. The selected SHiGDA patient subsequently contracted and died of NSCLC adenocarcinoma. The positive LHiGDA and FAP patients had a severely dysplastic anal condyloma and severe pancreatitis, respectively. Overall, the non­synonymous mutation occurring in the non­FAP SHiGDA patient occurred in the absence of p87 FE. Conclusions: In this pilot study we demonstrate the presence of Jak3 mutations likely associated with the lack of p87 expression in patients with high grade dysplastic adenomas and 1 FAP patient. Most of these patients had a clinical course which may have differed from their group members suggesting an altered immune system milieu. If confirmed in SHiGDA and FAP, Jak 3 mutations, associated with the SCID and late onset combined immunodeficiency, may allow for intervention with currently available medications to potentially avert a deleterious clinical outcome. #3690 The leukocyte chemoattractant chemerin modulates PTEN via CMKLR1 in human tumors.

Keith R. Rennier, Ping Wang, Robert Crowder, Russell K. Pachynski. Washington University School of Medicine in St.Louis, St. Louis, MO. Background: Recent data in preclinical models has shown that phosphatase and tensin homolog (PTEN) loss correlated with decreased tumor immune cell infiltration as well as decreased response to T cell­ based immunotherapy. Chemerin (RARRES2) is a recently identified endogenous leukocyte chemoattractant shown to recruit innate immune cells through its G­protein coupled receptor CMKLR1. Chemerin/RARRES2 is commonly downregulated in prostate and other cancers (e.g. sarcoma) compared to their normal tissue counterparts. Methylome­wide studies in multiple tumor types have identified RARRES2 as being one of the most hypermethylated genes, potentially leading to decreased chemerin expression. Our previous preclinical studies showed that forced overexpression of chemerin in tumors was capable of recruiting immune effector cells, including T cells, into the tumor microenvironment and suppressing tumor growth. Methods: In order to study the effects of chemerin overexpression on tumor cell intrinsic processes we exposed prostate and sarcoma tumor lines to exogenous recombinant chemerin in vitro. Evaluation of PTEN was performed at both the mRNA and protein levels, using both quantitative PCR and Western blotting, in comparison with normal prostate epithelia RWPE­1 as well as PTEN­null PC3 cells as controls. in vitro invasion assays were performed to investigate the functional impact of chemerin exposure on tumor intrinsic activity. Knockdown of CMKLR1 using siRNA was performed to determine its role in tumor response. Results: Using both prostate and sarcoma tumor lines, we found exogenous chemerin was able to significantly upregulate PTEN expression at both the mRNA and protein levels in a dose­response manner. Exposure to chemerin did not result in increased apoptosis or altered in vitro proliferation. Importantly, chemerin treatment significantly decreased in vitro tumor invasion. Knockdown studies showed CMKLR1 abrogation resulted in restored tumor migration, suggesting a link between this GPCR and PTEN expression and activity. Conclusions: For the first time, to our knowledge, we have shown a link between chemerin and PTEN expression and activity in both prostate and sarcoma tumor lines. Our collective study shows chemerin’s ability to upregulate PTEN activity and to mitigate tumor cell migration via CMKLR1. This work has functional implications on both tumor cell

intrinsic and extrinsic responses to chemerin­based immunotherapeutic strategies. Further studies are needed to investigate the interaction between chemerin and PTEN signaling, and its connection to oncogenic signaling pathways manipulated in malignant tumors. Additionally, future in vivo studies using these tumor lines will help further elucidate the chemerin­PTEN axis and its role in tumor immunosurveillance. We hypothesize that increased chemerin­driven PTEN activity may help facilitate an improved immunotherapy response and functional ability to attack cancer cells. #3691 Analysis of germline BRCA1/2 mutation associated breast and ovarian tumors reveals distinct pathways of immunosuppression. Adam Kraya, Kara N. Maxwell, Brandon M. Wenz, Bradley Wubbenhorst, Nicole Lunceford, Amanda Barrett, Jennifer J. Morrissette, Michael D. Feldman, Susan M. Domchek, Robert H. Vonderheide, Katherine L. Nathanson. University of Pennsylvania, Philadelphia, PA. BRCA1 and BRCA2 are essential homologous recombination (HR) repair proteins; germline mutations confer elevated risk for breast and ovarian cancer. Defects in BRCA can lead to immune­related effects including depletion of peripheral T­cell pools and increased somatic mutational burden. We investigated the immunophenotypic properties of breast and ovarian cancers associated with germline BRCA1/2 mutations. We determined neoantigen load in 16 breast and 26 ovarian tumors with germline BRCA1/2 mutations and 50 non­BRCA tumors for each disease using whole exome sequencing data from the TCGA. Although neoantigen load (IC50 10% of all TANs) directly correlated with the presence of IFN­γ and GM­CSF in the autologous tumor tissue. Using bone marrow­derived immature granulocytes, which were found to have prolonged survival in vitro, we discovered that these APC­like hybrid neutrophils originate from CD11b+CD15+CD10­CD16­/low/int neutrophil progenitors in the presence of IFN­γ and GM­CSF or in tumor­conditioned media. Functionally, the APC­like hybrid neutrophils are superior to canonical neutrophils in their ability to: 1) stimulate antigen non­specific autologous T cell responses 2) directly stimulate antigen­specific autologous memory T cell responses, 4) augment NY­ESO­1 specific effector T cell responses by providing a co­stimulatory signals through the OX40L, 4­1BBL CD86, CD54 molecules, and 5) cross present tumor­associated antigen as IgG­immune complex. In summary, we provide the first evidence of two subsets of TANs in lung cancer. All TANs had an activated phenotype and could support (rather than inhibit) T cell functions to some degree. However, we identified a subset of TAN in early­stage lung tumors that can undergo a unique differentiation process resulting in formation of specialized subset of APC­like hybrid neutrophils. These hybrid neutrophils may provide new opportunities to boost the efficacy of vaccines based on cytotoxic T lymphocyte induction

#3708 RAS­MAPK signal is required for enhanced PD­L1 expression in human lung cancers. Hidetoshi Sumimoto, Atsushi Takano, Koji Teramoto, Yataro Daigo. Shiga University of Medical Science, Otsu, Japan. Ectopic programmed cell death ligand 1 (PD­L1) expression in non­small cell lung cancers (NSCLCs) is related to immune evasion by cancer, and it is a molecular target of immune checkpoint therapies. Since the precise mechanisms responsible for ectopic PD­L1 expression remains obscure, we analyzed the molecular mechanisms of ectopic PD­L1 expression in human lung cancers, focusing on the MAPK signal. Because we found a higher frequency of EGFR/KRAS mutations in NSCLC cell lines with high PD­L1 expression (p < 0.001), we evaluated the relationships between downstream signals and PD­L1 expression, particularly in three KRAS­mutant adenocarcinoma cell lines. The MEK inhibitor U0126 (20 μM) significantly decreased the surface PD­L1 levels by 50­60% compared with dimethyl sulfoxide (p < 0.0001). Phorbol 12­myristate 13­ acetate stimulation (100 nM, 15 min) increased (p < 0.05) and two ERK2 siRNAs as well as KRAS siRNAs decreased (p < 0.05) PD­L1 expression. Post­transcriptional mechanism by miR­200s appears not to be in general under the downstream of MAPK in the PD­L1 expression. The transcriptional activity of the potential AP­1 site (+4785 to +5056 from the transcription start site) in the PD­L1 gene was demonstrated by luciferase assays, which was inhibited by U0126. The chromatin immunoprecipitation assay demonstrated the binding of cJUN to the AP­ 1 site. Two STAT3 siRNAs decreased PD­L1 expression by 10­32% in two of the three KRAS­mutant lung adenocarcinoma cell lines (p < 0.05), while the PI3K inhibitor LY294002 (40 μM) did not change the expression level. Supervised cluster analysis and gene set enrichment analysis between the PD­L1­high and ­low NSCLCs revealed a correlation between PD­L1 expression and genes/pathways related to cell motility/adhesion. These results indicate that MAPK signaling is the dominant downstream signal responsible for ectopic PD­L1 expression at a transcriptional level, in which STAT3 is also involved to some extent. Furthermore, MAPK signaling may control the expression of PD­L1 and several genes related to enhanced cell motility. Our findings suggest that MAPK is important for determining PD­L1 expression, which could be useful for targeted therapies against lung cancers.

#3709 Deciphering the impact of tumor genetics on immune cell infiltration in major solid cancer types. Jan Budczies,1 Michael Bockmayr,1 Frederick Klauschen,1 Abrecht Stenzinger,2 Carsten Denkert1. 1Charité ­ Universitätsmedizin Berlin, Berlin, Germany; 2Heidelberg University Hospital, Heidelberg, Germany. With the advent of immune therapies including inhibition of the PD­ 1/PD­L1 axis there is an urgent need to identify cancer patients that benefit from these therapies and to overcome mechanisms of resistance. Here, we analyzed the impact of tumor genetics on the composition of tumor microenvironment across 21 solid cancer types from the TCGA project. Using specific mRNA markers of 10 cell populations (MCPcounter), we estimated the content of T cells, CD8+ T cells, cytotoxic lymphocytes, B lineage cells, NK cells, monocytic lineage cells, myeloid dendritic cells, neutrophils, fibroblasts and endothelial cells in tumor tissues. Immune cell compositions were correlated with mutational load, the activity of 27 mutational signatures (MutSigs) and PD­L1 mRNA expression. Correlation strength was assessed using Spearman’s rho statistics. Multiple testing was addressed using the Benjamini­ Hochberg method and FDR control at 5%. MutSig 1 (clock­like, age­ related) showed significant positive correlations with immune cell infiltrations in low­grade gliomas, negative correlations in breast cancer, melanoma, stomach cancer, lung and prostate adenocarcinoma, but no significant correlations in the remaining 15 cancer types. Of the two APOBEC­related signatures, MutSig 2 showed positive significant correlations with immune cell infiltrates in cervical cancer, bladder cancer, lung adenocarcinoma, head and neck cancer and thyroid cancer, MutSig 13 in breast and cervical cancer. In particular, correlation of MutSig 2 with cytotoxic lymphocytes was significant in cervical cancer, bladder cancer and lung adenocarcinoma (R = 0.29, R= 0.17 and R = 0.17), the corresponding correlation of MutSig 13 in breast cancer (R = 0.17). MutSig 4 (tobacco smoke­related) showed a positive significant correlation with CD8+ T cells and NK cells in lung adenocarcinoma (R = 0.15 and R = 0.14). In contrast, correlation of MutSig 4 with immune cell infiltrates was non­significant or negative in head and neck cancer. In colorectal cancer, MutSig 6 (defective mismatch repair) showed significant positive correlations with NK cells, cytotoxic lymphocytes,

monocytic lineage cells and neutrophils infiltration (R = 0.45, R = 0.37, R = 0.23 and R = 0.19). PD­L1 mRNA expression and immune cell infiltrates correlated positively in all 21 cancer types. In particular, correlation with monocytic lineage cells was positive and significant in all cancer types and strong (R > 0.50) in 12 cancer types. Correlation with T cells was positive and significant in 18 cancer types and strong in 10 cancer types. In summary, pan­cancer analysis of DNA and RNAseq data showed that specific mutational signatures contribute to cancer cell immunogenicity, while the ubiquitous clock­like mutational process did not positively correlate with immune cell infiltrates in 20 of 21 cancer types. Moreover, PD­L1 mRNA expression strongly correlated with infiltrates of immune cells in the majority of cancer types. #3710 Components of HLA class I molecules influence pancreatic cancer cell proliferation and migration. Bailee Sliker,1 Cassie Liu,1 Brittany Poelaert,1 H Carlo Maurer,2 Kenneth P. Olive,2 Joyce C. Solheim1. 1University of Nebraska Medical Center, Omaha, NE; 2Columbia University Medical Center, New York, NY. Human leukocyte antigen (HLA) class I molecules are composed of a heavy chain with three isoforms (­A, ­B, and –C) and a light chain (beta 2­microglobulin or β2m). These molecules are best known for their role within the immune system, in which they present antigens to cytotoxic T cells. These molecules have been found in some tumor models to be down regulated as a form of immune escape; however, recent studies in breast, gastric, and lung cancer have correlated high expression levels of HLA class I with a poorer patient prognosis. β2m has also been found to be high in expression in many cancers, and it has been implicated in promoting renal cancer progression by increasing proliferation and migration. Whether any components of the HLA class I molecule contribute to the pathogenesis of other cancers, such as pancreatic cancer, has not been elucidated. Our recent results show that human pancreatic cancer cell lines have high total and cell­surface expression of β2m, HLA­A, and HLA­B, with the exception of one pancreatic cancer cell line that we found exhibits low expression of HLA­B. Furthermore, differential gene expression analysis performed on laser capture­ microdissected human pancreatic cancer patient samples revealed that HLA­B expression is not reduced in pancreatic ductal adenocarcinoma

cells, compared to precursor (PanIN) epithelial or stromal cells. Together, our results indicate that pancreatic cancer cells have not selectively down regulated HLA class I molecule expression. This observation suggests that HLA class I molecules might provide some benefit to pancreatic cancer cells that counterbalances any risk of immune attack caused by HLA class I molecules. To examine whether the components of the HLA class I molecule actually confer potential advantages to pancreatic cancer cells, we transfected siRNA specific for β2m, HLA­A heavy chain, or HLA­B heavy chain into several pancreatic cancer cell lines and evaluated the effects on their proliferation and migration. Down­ regulation of either β2m or HLA­B expression slowed the proliferation of one pancreatic cancer cell line, whereas down­regulation of HLA­A expression had the inverse effect on the same cell line. Two other pancreatic cancer cell lines had an opposite phenotype: proliferating more slowly when HLA­A expression was reduced, but more rapidly when HLA­B expression was decreased. Our investigation of the impact of down regulating HLA class I molecule components on the migration of pancreatic cancer cells showed the same trends with each cell line as we had observed in our proliferation analyses. In conclusion, we found that pancreatic cancer cells express substantial levels of HLA class I molecule components, and that these components significantly influence the proliferation and migration of pancreatic cancer cell lines in a manner that varies for HLA­A versus HLA­B, and that also varies in pattern among the types of pancreatic cancer cell lines. #3711 R­Ras is required for T cell trafficking in high endothelial venues and implicated in anti­tumor immunity. Andrew M. Chan,1 Xiaocai Yan,2 Wai Nam Liu,1 Mingfei Yan1. 1Chinese University of Hong Kong, Hong Kong, Hong Kong; 2The Medical College of Wisconsin, Milwaukee, WI. R­Ras belongs the RAS superfamily of small GTP­binding proteins implicated in cell adhesion signaling. Unlike classical Ras proteins, R­ Ras is not activated by growth factor receptors. Rather, its GTPase activity is modulated by molecules implicated in cell­cell adhesion including semaphorins and notch. Using an R­Ras knockout mouse strain, we have previously reported that R­Ras is required for murine Dendritic cell functions. Further characterization of this mouse strain revealed

reduced cellularity of peripheral lymph nodes (PLNs) by 40% with T cell regions in the paracortex in which high endothelial venules (HEV) resided were less prominent when compared with wild­type (WT) mice. Consistently, immunohistological analysis for MECA­79, a marker of mature HEV, showed a 3­ to 4­fold decrease in staining intensity. This is correlated with a 30­40% reduction in the number of CD62L+ CD4 and CD8 T cells in PLNs. Furthermore, R­Ras null T cells had reduced proliferative and homing capacity when compared with WT. More importantly, we demonstrated for the first time that the chemokine CCL21 was able to stimulate the GTP­loading of R­Ras within 15 seconds of addition. Indeed, R­Ras null T­cells displayed reduced binding to soluble ICAM­1 as well as to 2H­11 endothelial cells. To explore the role of R­Ras in anti­tumor immunity, B16/F10 melanoma cells were injected subcutaneously to WT and R­Ras null mice. In addition, a mouse breast tumor model was generated ineither WT or R­Ras­null genetic background. Tumor development and the number of infiltrating immune cells were analyzed to examine if R­Ras was involved in either anti­ or pro­tumor immune responses. This work was supported by NIH CA78509 (AMC), MH59771 (AMC). MY was supported by Hong Kong PhD Fellowship Scheme (PF12­13584) and AMC was supported by the Lo Kwee­Seong Seed Fund, and a General Research Fund Grant, 14120915, from Hong Kong University Grants Committee. #3712 Mechanistic link between phosphatidylinositol­4,5­bisphosphate 3­ kinase catalytic subunit alpha (PIK3CA) activity and PDL1 expression in head and neck squamous cell carcinoma (HNSCC). Rebecca C. Hoesli, Nicole L. Michmerhuizen, Vivek Nair, Chloe Matovina, Elizabeth Leonard, Matthew E. Spector, Carol R. Bradford, Mark E. Prince, Andrew C. Birkeland, J Chad Brenner. University of Michigan, Ann Arbor, MI. Introduction: HNSCC is an immunosuppressive disease, with multiple functional and quantitative defects contributing to immune evasion and tumor escape. One of the identified areas for therapy development is the Programmed Death­1 (PD­1)/Programmed Death Ligand 1 (PD­L1) pathway, as this pathway has been hypothesized to allow cancer cells to evade the immune system by promoting T cell anergy and apoptosis. Pembrolizumab, a PD­L1 inhibitor, has recently been approved for the treatment of recurrent/metastatic HNSCC. As regulation of PD­L1

expression could play an important role in the effectiveness of therapy, we further explored the regulation of PD­L1 expression in HNSCC cells. Specifically, we targeted our investigation by evaluating the effects on expression of one of the most frequently mutated genes in HNSCC, PIK3CA. We evaluated this with and without interferon­γ, which has previously been shown to affect PD­L1 expression, possibly through activation of the STAT1 pathway. Methods: HPV+ and HPV­ HNSCC cell lines were grown in cell culture and treated with selective and non­selective PI3K inhibitors, in combination with interferon­γ. After 72 hours, cells were harvested and flow cytometry was used to measure the expression of PD­L1. Protein expression of various pathway intermediaries was evaluated via western blot to better delineate the mechanism of PD­L1 upregulation. Results: Treatment with selective PI3K inhibitors in combination with interferon­γ in several cell lines significantly increased expression of PD­ L1, beyond the increase noted after treatment with interferon­γ alone. Maintenance of STAT1 phosphorylation correlated with upregulation of PD­L1 expression, while total STAT1 expression remained stable. The majority of the cell lines maintaining STAT1 phosphorylation were HPV+, but a few HPV­ cell lines also maintained this phosphorylation with a correlating upregulation in PD­L1 expression. Additionally, mutation in PIK3Ca despite HPV­status was noted to maintain phosphorylation of STAT1 with upregulation of PD­L1 expression. Conclusions: Treatment with PI3K inhibitors in combination with interferon­γ significantly upregulated PD­L1 expression in several cell lines, suggesting a possible synergistic effect. Since PD­L1 expression correlated with maintenance of phosphorylation of STAT1, these results suggest a pivotal link between PIK3CA signaling, STAT1 activity and PD­L1 expression in PIK3CA aberrant HNSCC. #3713 Higher numbers of cancer stem cells in the peripheral blood of children with B­ALL upon conventional chemotherapy. Mohamed L. Salem. Tanta University, Tanta, Egypt. Background: Acute lymphocytic leukemia (ALL) is biologically and clinically considered as a heterogeneous neoplasm of lymphoid progenitor cells in the bone marrow (BM). 15­20% of children with ALL who achieve an initial remission, will show relapse. One potential mechanism behind this relapse could be the emergence of cancer stem

cells (CSCs) which are considered the driving force of tumourigenesis due to their ability of self­renewal as well as the emergence of immune regulatory cells including myeloid­derived suppressor cells (MDSCs) and regulatory T cells (Treg). Aim: the main aim of this study was to analyze the numbers of CSCs and correlate these numbers with the numbers of blast cells, MDSCs and Treg cells in children with B­ALL before and after induction of chemotherapy. Materials and Methods: CSCs were defined as CD45dimCD19+CD10+CD34+CD38­, MDSCs were defined as Lin­HLA­ DR­CD33+CD11b+ and Treg cells were defined as CD4+CD25+CD127­. The frequencies of these cells were analyzed in the peripheral blood of B­ ALL patients before (n=10) and after (n=10) induction of chemotherapy using flow cytometry. Results: Significant increases in the numbers of CSCs were shown in B­ ALL patients after induction of chemotherapy as compared to newly diagnosed patients (7.6± 8.3 vs. 2.7± 2.4, P30%) tissues compared with neoadjuvant (viable cells25%) amounts of huCD45 cells in all groups and models. PDX models being sensitive towards checkpoint inhibitor treatment (responders) displayed a higher percentage of DAB+ nuclei in huCD45 IHC stains than non­responder models as determined by image analysis. Irrespective thereof, in responders as well as non­responders the treatment with checkpoint inhibitors enhanced the percentage of DAB+ nuclei. Whole­slide image analysis of the H&E stains revealed an increase of the stromal compartment proportion in the tumor tissue under treatment with checkpoint inhibitors in responder models. In non­responder models the ratio between tumor and stroma was not influenced by drug treatment. The use of PDX based humanized mouse models in a SMT format allows screening approaches in complex mouse models. The combination with a comprehensive image analysis tool enables additional read­outs to quantify antitumoral activity of immune modulatory compounds. The latter can be used to identify possible biomarkers in the preclinical setting. Moreover, the translation and validation of these biomarker candidates in a clinical setting is self­ evident as primary material needed for these types of analyses is easily accessible. #4816 Circulating tumor cell monitoring, isolation, and culture from a

patient with metastatic triple­negative breast cancer for drug screening and creation of a patient­derived xenograft model. Arturo B. Ramirez,1 C Anthony Blau,2 Timothy J. Martins,2 Elisabeth Mahen,2 Lacey E. Dobrolecki,3 Michael T. Lewis,3 Jackie L. Stilwell,1 Eric P. Kaldjian1. 1RareCyte, Inc., Seattle, WA; 2University of Washington, Seattle, WA; 3Baylor College of Medicine, Houston, TX. Background. Enumeration, phenotyping and single cell genomics of circulating tumor cells (CTCs) provide three types of information to guide cancer therapy. In some instances, a fourth type is possible: functional analysis in vitro, or in in vivo patient­derived xenografts (PDXs). We used a density­based rare cell separation and analysis system to collect CTCs from the blood of a patient with metastatic triple­negative breast cancer (TNBC) for in vitro culture and high­throughput drug screening and to generate a PDX model. Methods: The patient was enrolled in the ITOMIC­001 study (University of Washington) and after informed consent, CTCs were evaluated prior to initial cisplatin treatment and tracked longitudinally using the AccuCyte – CyteFinder system (RareCyte). Samples containing high numbers of CTCs were placed into 3 different culture media. Cells grown in culture were tested against a panel of anti­cancer drugs and injected into mice to form a PDX model. Results: Nine CTC evaluations were performed over 9.5 months. CTCs were verified by expression of epithelial (cytokeratin and/or EpCAM) and nuclear stains without CD45 expression. After initial treatment with cisplatin, the CTC count per 7.5 mL rose from 4 to 19 cells at 3 months, consistent with the lack of a clinical response, and decreased after LE 011 (CDK4/6 inhibitor) and then glembatumumab vedotin (anti­gpNMB) to 8 and 4 cells at 5 and 7 months respectively. At 9 months the CTC count rose to > 13,000 and 5 days later to > 80,000 shortly before her death. At autopsy there was massive infiltration of the liver and pulmonary vasculature by tumor cells. Cultures in all media showed initial growth, but only one (RPMI + 10% serum) was sustained, forming semi­adherent 3D tumor clusters. 6 million cells were harvested and a drug screen using 160 anti­cancer agents was performed. The CTC line showed sensitivity to several agents. Cells were also injected into the mammary fat pad of immunodeficient mice. In at least one mouse, macroscopic tumors were observed. The CTC cell line has grown continually in culture for over a year. Aliquots of this cell line have been frozen and thawed with no

noticeable effect on cell growth. Conclusions: Using a density­based rare cell collection system, we have established a CTC cell line from a TNBC patient with extremely high CTC counts. The line was used to perform a screen for agents active against the tumor cells and to create a PDX model. As in vitro techniques advance, smaller number of CTCs may be effectively cultured and thus allow this approach to be used in real time to find effective drug regiments for individualized cancer therapy. #4817 Regression of malignant ascites via PAPP­A inhibition in ovarian cancer patient­derived xenograft model. Valentina Zanfagnin, Laurie K. Bale, Marc A. Becker, Xiaonan Hou, Diogo Torres, Cheryl A. Conover, Saravut J. Weroha. Mayo Clinic, Rochester, MN. Malignant ascites is one of the most common causes of morbidity in end stage ovarian cancer patients with negative impact on quality of life. Novel non­invasive palliative therapeutic options are lacking for such patients. The zinc metalloprotease, pregnancy­associated plasma protein­A (PAPP­ A), plays a key role in the insulin­like growth factor (IGF) pathway, promoting ovarian cancer cellular transformation, growth and invasiveness. Furthermore, patient primary malignant ascites is known to contain high levels of PAPP­A by ELISA. Preliminary data shows that inhibition of PAPP­A through a neutralizing monoclonal PAPP­A antibody (mAb­PA) inhibits the accumulation and promote the regression of ascites in an ovarian cancer patient­derived xenograft (PDX) models. In the current study, we investigated whether mAb­PA can promote ascites regression in an additional ovarian PDX model with measurable ascites. Patient derived ascites xenograft (PDAX) models were defined as SCID mice that developed ascites after intraperitoneal heterotransplantation of patient solid tumor collected at the time of primary cytoreduction. Ascites from these models (n=51) was screened for human PAPP­A protein by ELISA and models were divided into two groups by the relative concentration of PAPPA­A: high (n=18) and low (n=33). PAPP­A High PDAX model PH438 was re­established intraperitoneal in 20 SCID mice by ascites injection (0.1 ml per mouse). When ascites area reached a threshold of >0.60 cm2 by ultrasound, mice were treated with

60mg/Kg of mAb­PA (n=10) or IgG2a control (n=10) on day one and three. On day four, a second ultrasound measurement was obtained and the mice were euthanized. Ascites burden was measured at necropsy. Personnel involved with the acquisition of ultrasounds measurements, subsequent ascites harvests, and post hoc analyses were blinded to the treatments. The ratio of mean ascites (mAb­PA/IgG2a in grams) collected at necropsy was 2.04/2.51, indicating that the mAb­PA arm had less ascites compared to control. These data were consistent with pre­ and post­treatment ultrasound measurements of ascites burden; a statistically significant (p=0.0283 by paired ttest) reduction of ascites burden of 52.3% was observed, compared to the starting baseline, indicating that mAb­PA treatment causes ascites regression. Ascites weight at necropsy was compared to the area of greatest fluid echogenicity by ultrasound and the Pearson correlation R value achieved was of 0.859 (p 0.05). Similarly, knockdown of each of the test genes did not affect cell proliferation in HCC1806 (± CREB3L1) cells, except for a significant decrease in proliferation in HCC1806 CREB3L1­deficient cells with Gene 1 knockdown (p=0.02). Preliminary results indicate that knockdown of Genes 1, 2, and 4 in HCC1806 CREB3L1­deficient cells reduces cancer cell properties, including cell migration and anchorage­independent growth (p < 0.05). Conclusions: Promising candidates will be tested in follow­up mouse xenograft studies. Encouraging results in these pre­clinical models would provide a strong rationale for the development and testing of inhibitors to one or more of these potential targets as a new therapeutic for the 30% of breast cancers that are CREB3L1­deficient. #4853 NRP2b, a unique isoform of NRP2, promotes aggressive lung cancer

phenotypes. Anastasios Dimou,1 Patrick Nasarre,1 Joyce Nair­Menon,1 Federico Cappuzzo,2 Lorenza Landi,2 Armida D'Incecco,2 Hidetaka Uramato,3 Takeshi Yoshida,4 Eric Haura,5 Monica Gooz,1 Kent Armeson,1 Robert Gemmill,1 Harry Drabkin1. 1Medical University of South Carolina, Charleston, SC; 2Istituto Toscano Tumori, Leghorn, Italy; 3Kanazawa Medical University, Uchinada, Japan; 4Kinki University Faculty of Medicine, Charleston, SC; 5Moffitt Cancer Center, Tampa, FL. Neuropilins (NRPs) 1 and 2 are highly­related receptors for class 3 semaphorins, and interact with heparin­binding growth factors and their receptors (e.g. HGF­MET, VEGF­VEGFR2 etc.). We previously reported that NRP2 is upregulated by TGFβ and is required for its pro­tumorigenic activity. Here, we show that this upregulation preferentially involves NRP2b, a largely uninvestigated isoform encoding a divergent, yet highly­conserved C­terminus. Importantly, using a panel of lung cancer cell lines and orthotopic metastasis model, NRP2b promoted migration, invasion, metastasis and tumorsphere formation, whereas the prototype receptor, NRP2a had opposite effects. TGFβ­mediated resistance to gefitinib in EGFR mutated tumors also was dependent on NRP2b expression. In addition, NRP2b, but not NRP2a, linked MET activation by HGF to AKT phosphorylation. Mechanistically, in co­ immunoprecipitation assays, we found that NRP2a robustly interacted with PTEN, while the interaction with NRP2b was weak. At the clinical level, NRP2b expression was commonly upregulated in patient lung cancer samples and this upregulation was a significant poor­prognostic factor. Collectively, these data indicate that NRP2b plays an important role in lung cancer invasion, metastasis and EGFR inhibitor resistance. Furthermore, isoform­specific interactions between NRP2 and PTEN may be responsible for the observed differences in MET signaling in response to HGF. #4854 Evaluating HDAC6 as a causal factor in metastatic breast cancer to develop immunotherapy. Debarati Banik,1 Melissa Hadley,1 Jennifer Kim,1 Tessa Knox,1 Jayakumar Nair,1 Alan Kozikowski,2 Sida Shen,2 Charu Vyas,1 Ashleyn

Donohue,1 Eduardo Sotomayor,1 Alejandro Villagra1. 1The George Washington University, Washington, DC; 2University of Illinois at Chicago, Chicago, IL. Histone deacetylases (HDACs), originally described as histone modifiers, have recently been demonstrated to modify a variety of other proteins involved in diverse cellular processes unrelated to the chromatin environment, including the modulation of proteins related to cell cycle/apoptosis and immune regulation. In contrast to the well­ documented effects of HDAC inhibitors (HDACi) in the control of cell cycle and apoptosis, their role in immune­biology is incompletely understood. We have found that the pharmacological or genetic abrogation of a single HDAC, i.e. HDAC6, inactivates the STAT3 pathway and modulates the expression of immuno­regulatory proteins, including the down­regulation of PD­L1, PD­L2 and B7­H4, important negative regulators of immune function, often over­expressed in cancer cells; including breast cancer. HDAC6 has been also involved in a number of structural functions related to cellular motility, shape and intracellular transport through the regulation of the acetylation of numerous targets, including tubulin and cortactin. This function is strongly suggestive of HDAC6 being a key player in metastatic cancer progression. In our initial studies we observed that the selective HDAC6 inhibitor Nexturastat A is capable of reducing the tumor growth in a highly aggressive murine mammary carcinoma that mimics human triple negative breast cancer (TNBC), under both orthotopic and subcutaneous conditions of implantation. Additionally, we observed that the size and number of secondary tumor nodules in lungs were significantly diminished after the HDAC6i treatment. In order to boost the anti­tumor T­cell response, we also tested check­point inhibitors against the tumor (such as anti PD­1 and CTLA4 antibodies). While each of the standalone treatments showed a certain degree of success in reducing tumor growth and enhancing intra­tumoral IFNγ, we demonstrated that HDAC6i improves anti­tumor immune responses when combined with immune check­point blockade. #4855 Loss of USP18 represses invasion and metastasis of lung cancer. Lin Zheng, Lisa Mustachio, Yulong Chen, Xi Liu, Jason Roszik, Jonathan Kurie, Ethan Dmitrovsky. UTMD Anderson Cancer Center, Houston, TX.

Metastasis is a major cause of human lung cancer mortality. Uncovering novel targets and mechanisms involved in regulating metastasis is critical for developing effective ways to improve lung cancer survival. It is reported that ubiquitin and ubiquitin­like pathways can regulate invasion and metastasis. We previously found that USP18 (Ubiquitin Specific Peptidase 18) is substantially upregulated in several cancers, including lung cancer. Engineered repression of the deubiquitinase USP18 decreased growth, increased apoptosis and augmented chemotherapeutic agent response of lung cancer cells. Our prior work showed that loss of USP18 can destabilize specific oncogenic proteins and this results in the marked reduction of lung cancer cell growth and in vivo tumorigenicity. This study sought to explore the precise role of USP18 expression in invasion, migration and metastasis of lung cancer. In murine (KC2 and 344SQ) and human (A549 and H1299) lung cancer cells with varying degrees of metastatic potential, knock­down of USP18 by different short hairpin RNAs (shRNAs) decreased cell growth and increased cell death as compared to vector control transfectants. The findings from wound­ healing migration and Transwell invasion assays established that USP18 knock­down in all studied lung cancer cell lines conferred substantial reduction of migration and invasion versus vector control transfected cell lines (p < 0.001). In mouse models, USP18 knock­down of lung cancer cell lines displayed a lower number of lung cancer metastasis in vivo. To discern engaged mechanisms, Reverse Phase Protein Arrays (RPPAs) were performed to interrogate over 300 growth­regulatory proteins in murine (344SQ and KC2) and human (A549 and H1299) lung cancer cells following USP18 knock­down. Using RPPAs, potential targets identified were differentially expressed between USP18 knock­down and vector control transfected lung cancer cells. Highlighted species included Myc, eEF2K (Eukaryotic Elongation Factor 2 Kinase), Programmed Cell Death 4 (PDCD4), Hes Family BHLH Transcription Factor 1 (Hes) and Polo Like Kinase 1 (PLK1), among other species. In addition, phosphorylated Acetyl­CoA Carboxylase (ACC), a key component of the 5'­adenosine monophosphate­activated protein kinase (AMPK) pathway, was significantly affected in lung cancer cell lines following USP18 knock­down (p < 0.05). Ingenuity Pathway Analysis and functional validation are now underway to uncover specific pathways directly involved in the observed reduction of lung cancer metastases. Taken together, this study implicates the deubiquitinase USP18 as a molecular target to combat lung cancer metastases.

#4856 High mRNA expression of splice variant SYK short correlates with poor hepatic metastasis free survival in untreated lymph node negative colon cancer patients. Robert R. Coebergh van den Braak, Anieta S. Sieuwerts, Zarina S. Lalmahomed, Sandra Bril, Annemieke M. Timmermans, Vanja de Weerd, Michelle van der Vlugt ­ Daane, Anne van Galen, Shanshan Xiang, Katharina Biermann, John A. Foekens, John W. Martens, Jan N. IJzermans. Erasmus University Medical Center, Rotterdam, Netherlands. Introduction: In lymph node negative (LNN) colon cancer 20% of the patients will develop recurrence of disease. Identification of these patients is an unmet need. SYK, a protein kinase, has been ascribed both a tumor promoter and suppressor role in epithelial cancers. The prognostic value of SYK and its splice variants, largely uknown in colorectal cancer however, was explored in a clinically well­defined cohort of colon cancer patients. Methods: Total mRNA expression of SYK [SYK(T)] and of its two splice variants SYK short(S) and SYK long(L) were measured using RT­qPCR in a clinically well­defined prospectively collected cohort of 240 colon cancer patients (n=160 untreated lymph node negative [LNN] and n=80 adjuvant treated lymph node positive [LNP] patients) selected from the MATCH­cohort. mRNA expression levels were related to microsatellite instability (MSI), mRNA expression of epithelial (EPCAM), stromal (BGN, FAP, INHBA) and infiltrate markers (VEGFA, CD45), known CRC mutations (n=238), and disease free (DFS), hepatic metastasis free (HFS) and overall survival (OS). Results: Overall increased SYK levels were associated with stage I/II, a left­sided located primary tumor and MicroSatellite Stability (MSS). However, these associations and their interrelation differed significantly between SYK(T), SYK(S) and SYK(L) expression implicating an added value for measuring mRNA expression of the splice variants next to SYK(T). SYK(T), SYK(S) and SYK(L) levels all showed a significant positive correlation with the expression of EPCAM, FAP was weakly negatively associated with SYK(S) and VEGFA was weakly positively correlated with SYK(T) and SYK(S). This suggests a higher expression of SYK in epithelial­rich, stromal­poor tumors. SYK(T) and SYK(S) expression was significantly lower in tumors with a BRAF or PTEN mutation (mt) compared to wild type (wt) tumors. Although others reported differential expression of SYK between KRAS­dependent and KRAS­independent

cell lines and KRAS mt versus wt tumors in 221 TCGA­samples (p=0.008), we observed no significant differences for expression of SYK(T), SYK(S) and SYK(L) between KRAS­mutant (mt) and KRAS­ wild type (wt) tumors. In the LNN group, using univariate Cox regression analysis increasing mRNA expression of SYK(T) (HR=2.05 95%CI=1.01­4.17 p=0.047) and SYK(S) (HR=1.83 95%CI=1.09­3.05 p=0.021) was associated with worse HFS, which remained significant for SYK(S) when correcting for the number of assessed lymph nodes (HR=1.83; 95% CI=1.08­3.12; p=0.026 and HR=1.27; 95%CI=1.009­ 1.60; p=0.042). No other significant associations between SYK(T), SYK(S) and SYK(L), and DFS, HFS and OS were observed. Conclusion: In our untreated LNN colon cancer cohort SYK(S) is a pure prognostic marker for HFS. These results may help to identify LNN patients at overall low risk to develop liver metastases. #4857 TAK1 mediated IL1 expression as autocrine signaling to promote breast cancer metastasis. Oihana Iriondo, Mostafa Elhodaky, Yarong Liu, Grace Lee, Julie E. Lang, Pin Wang, Min Yu. Univ. of Southern California, Los Angeles, CA. TGFβ­activated kinase 1 (TAK1), a serine/threonine kinase from the MAPKKK family, is regulated by different cytokines such as IL1, TGFβ, TNFα and BMPs, and it is therefore a key player in the cellular responses induced by changes in the microenvironment. TAK1 regulates cell survival, differentiation and inflammatory responses by activating other intracellular kinases such as p38, JNK or IKK. TAK1 activation is thought to influence the progression of several types of cancer, including lymphomas and pancreatic, colon, liver and breast cancer. In this study, we aimed to investigate the mechanisms by which TAK1 regulates breast cancer progression. (5Z)­7­Oxozeanol is a compound that inhibits the kinase activity of TAK1. Treatment of mice injected with MDA­MB­231 cells with (5Z)­7­Oxozeanol containing nanoparticles reduced metastasis formation, while growth of the primary tumor was not affected. TAK1 involvement in metastasis was confirmed by xenotransplantation experiments done with MDA­MB­231 cells overexpressing wild type or dominant negative forms of TAK1. Using these stable cell lines, we confirmed that TAK1 is required for p38 activation induced by several cytokines. Interestingly, cells cocultured with macrophages showed a TAK1­dependent increase in the expression levels of IL1α and IL1β,

which can lead to TAK1 activation themselves. These cytokines were also detected in cancer cells that colonized the mouse lung, suggesting that the lung microenvironment could promote the initiation of a positive feedback loop by cancer cells, promoting the growth of metastatic lesions. Disruption of this positive autocrine feedback loop may be a potential therapeutic approach to suppress breast cancer metastasis to the lung. #4858 AK4 promotes colorectal cancer progression and metastasis. Yau Hei Yu, Joanna Hung Man Tong, Yi Pan, Raymond Wai Ming Lung, Ka Fai To, Anthony Wing Hung Chan. The Chinese University of Hong Kong, Hong Kong. Metastatic colorectal cancer (CRC) is a fatal disease with a poor prognosis. It is crucial to understanding the underlying pathogenesis to battle this dismal condition. We previously identified that AK4, a gene on chromosome 1p31.3 encoding a member of adenylate kinase family enzymes, was progressively up­regulated from normal colon, primary CRC to metastasis by mRNA gene expression microarray of paired CRC samples. This study was aimed to validate the overexpression of AK4 and explore in­vitro effects in CRC. We firstly examined the mRNA expression level of AK4 in a panel of CRC cell lines. AK4 mRNA expression was higher in CRC cell lines (n=8) than normal colon cell line, and significantly higher in advanced stage cell lines (n=4, Dukes’ C and D) than early stage ones (n=4, Dukes’ A and B) (P=0.03). We further evaluated expression level of AK4 in two different cohorts of clinical samples. The first cohort consisted of 18 paired primary CRCs and its corresponding normal colonic mucosa, and 27 metastatic CRC (including 15 cases with paired primary CRC and normal colonic mucosa). AK4 mRNA level was significantly higher in metastatic CRC than normal colon (P 1.5), hypo­methylation (β­ value≅0.3), and no expression of TERT. Intriguingly, while groups 1­3 show telomeric loss (TBrpm ≅ 0.7­0.9), 4S tumors show conservation of telomere abundance between tumor and blood (TBrpm ≅ 1); hence no indication of telomere crisis, perhaps due to early timing of biopsy at diagnosis. Conclusion. We have used ASE status and gene body methylation of TERT in order to understand and validate mechanisms underlying TERT activation; we extend MYCN­driven TERT activation to tumors with high MYC/MYCN activity in the absence of MYCN amplification. Our telomere analysis suggests that different ALT mechanisms might take place in NBLs. Defining the mechanism of 4S NBL spontaneous regression requires further investigation. #4882 Genomic mechanisms of disease progression in pediatric medullary thyroid cancer (MTC). Ira L. Kraft,1 Srivandana Akshintala,2 Keith J. Killian,1 Robert B. Hufnagel,3 John W. Glod,1 Claudia Derse­Anthony,4 Yuelin Zhu,1 Holly S. Stevenson,1 Diana Bradford,1 Maria J. Merino,1 Frank M. Balis,5 Elizabeth Fox,5 Brigitte C. Widemann,1 Jack F. Shern,1 Paul S. Meltzer1.

1National Cancer Institute, Center for Cancer Research, Bethesda, MD; 2New York University, Langone Medical Center, New York, NY; 3National Eye Institute, Bethesda, MD; 4Clinical Research

Directorate/Clinical Monitoring Research Program, Leidos Biomedical Research, Inc., NCI Campus at Frederick, Frederick, MD; 5Children's Hospital of Philadelphia, Philadelphia, PA. Background: Multiple Endocrine Neoplasia (MEN) 2B is a rare hereditary disorder characterized by medullary thyroid cancer (MTC) in early childhood, pheochromocytoma, and mucosal neuromas. Patients with advanced MTC are treated with rearranged during transfection (RET) targeting tyrosine kinase inhibitors (TKIs) such as vandetanib. Despite initial responses, many patients progress on TKI therapy and mechanisms of resistance are yet to be elucidated. We analyzed tumor samples from seven children with MEN2B and MTC enrolled in a natural history study (NCT01660984). Methods: DNA samples from tumor and adjacent normal tissue from paraffin embedded blocks or unstained slides were analyzed by a custom capture next­generation sequencing panel. Bioinformatics analyses identified point mutations, insertions, deletions and copy number variations. Results: Seven patients (median age at study enrollment 14 years, range 11­17 years) with the RET p.Met918Thr germline mutation were included in analysis. Tumor samples were available for three patients pre­ TKI (four samples), two patients at progression on TKI (three samples), and two patients both, pre­TKI and at progression (five samples). Pre­ TKI samples exhibited few tumor specific mutations or copy number variations and 4/5 patients had loss of chromosome 1p. Progression for one patient was associated with acquisition of a previously unidentified p.Leu790Phe mutation within the kinase domain of the RET gene. Loss of heterozygosity and increase in copy number variations were noted in 4/5 samples at tumor progression. Two patients had copy number loss of chromosome 14 and three had copy number gain of chromosome 1q. Recurrent, somatic, non­synonymous mutations were not identified in the sample set. Conclusion: In children with MEN2B and MTC, we identified increase in copy number variations and a somatic mutation within the RET gene as potential mechanisms of drug resistance. Our data imply that a common genetic mechanism for progression on TKI may not exist within this

small sample set and highlight the need for serial collection of tumor tissue. Further, whole genome aneuploidy may provide rationale for the evaluation of cytotoxic chemotherapy in patients who experience progressive disease on TKI therapy. Analysis of additional samples and whole exome DNA and RNA sequencing are ongoing. #4883 Targeting tumor­amplified ODC1 with difluoromethylornithine (DFMO) inhibits global protein translation and has antitumor activity in neuroblastoma. Andrea T. Flynn,1 Annette Vu,1 Kangning Liu,1 Elizabeth Scadden,1 Edward Attiyeh,1 Murray Norris,2 Michelle Haber,2 Michael Hogarty1. 1Children's Hospital of Philadelphia, Philadelphia, PA; 2Children's Cancer Institute, Sydney, Australia. MYC genes are predominant oncogenic drivers in neuroblastoma, a lethal pediatric tumor, often via amplification of MYCN. MYC genes coordinately deregulate programs that link cell cycle entry with requisite biomass and energy creation. Polyamine synthesis is one such deregulated program that supports protein synthesis. Ornithine decarboxylase (Odc, encoded by ODC1) is the rate­limiting enzyme in polyamine synthesis, a direct MYC target, and a bonafide oncogene. In addition to hyperactivated MYCN driving polyamine signaling, ODC1 itself is co­amplified in a subset of high­risk tumors. We studied 30 neuroblastoma cell lines and 916 primary tumors via SNP­array. ODC1 amplification (confirmed by qPCR) was found exclusively in tumors with MYCN amplification, though amplification peaks were distinct (ODC1 maps 5.5 Mb telomeric to MYCN). ODC1 amplification was identified in 33 of 256 (13%) MYCN amplified primary tumors and 4 of 13 (31%) MYCN­amplified cell lines. Difluoromethylornithine (DFMO) is an FDA­approved irreversible inhibitor of the oncogenic Odc enzyme, and we have shown DFMO inhibits tumor progression and synergizes with chemotherapy in complementary murine models of neuroblastoma. We postulated DFMO inhibits protein translation as its principal mechanism of anti­tumor activity. Polyamines support protein translation via effects on both eIF5A and eIF4F­complex activities, yet their relative contributions remain poorly defined. Using the puromycin­incorporation assay we show a marked decrease in global protein translation following DFMO exposure (up to 95% reduction), with translation inhibition

largely correlated with MYCN amplification status in the tumor cells. Studies to define the dose­response and kinetics of DFMO­mediated inhibition in tumors with distinct MYCN and ODC1 genotypes are ongoing, as are studies of eIF5A­hypusination (by IEF) and eIF4F­ complex status (polyamine­dependent 4E­BP phosphorylation). Moreover, eIF5A is required to resolve ribosome stalling at polyproline stretches, so we characterized the human proteome for polyproline motifs (PPP of >3) to define an eIF5A­dependent translatome. Candidate genes of diverse polyproline content are being assessed for sensitivity to DFMO to define whether ribosome pausing and selective translation inhibition contributes to DFMO effects. Elucidating mechanisms of DFMO activity and correlating this with genomic status (MYCN and/or ODC1 amplified) will identify opportunities for drug synergy and provide a responder hypothesis to test in pivotal Phase 2 and 3 clinical trial of DFMO for neuroblastoma. #4884 Focal 22q11.22 deletions combined with IKZF1 alterations are associated with worse clinical outcome in acute lymphoblastic leukemia. Julia A. Meyer,1 Clint C. Mason,1 Luke Maese,1 Deqing Pei,2 Cheng Cheng,2 Ching­Hon Pui,2 Mel Greaves,3 Richard Aplenc,4 Charles G. Mulligan,2 Elizabeth Raetz,1 Rodney R. Miles,1 Karen R. Rabin,5 Joshua D. Schiffman1. 1University of Utah, Salt Lake City, UT; 2St. Jude Children's Research Hospital, Memphis, TN; 3Institute of Cancer Research, London, United Kingdom; 4Children's Hospital of Philadelphia, Philadelphia, PA; 5Baylor College of Medicine, Houston, TX. Introduction: Prognostic biomarkers in childhood acute lymphoblastic leukemia (ALL) are vital for risk­stratification and intensifying therapy for children at high risk for remission induction failure or relapse. Copy number alterations in genes such as IKZF1 and VPREB1 have been shown to correlate with poor outcome in ALL, highlighting genetic alterations as prognostic markers (NEJM 360:470, 2009, Leukemia 28(1):216­20, 2014). A second focal deletion in chromosome 22q11.22, 200 kilobases (Kb) in length, occurs more frequently and in the same IGLL region as VPREB1 and is distinct from deletions associated with

physiologic IGLL rearrangement. We further investigated this novel genomic lesion, 22q11.22, and the prevalence of co­occurrence with IKZF1. Methods: 22q11.22 deletions were characterized in a compiled childhood ALL cohort (N=832) and correlated with available clinical outcome using multiple previously published studies (Clinical outcome total N=730; Utah Cohort [N=56], TARGET P9906 cohort [N=215], St. Jude Children's Research Hospital cohort [SJCRH, N=236], Children’s Hospital of Philadelphia cohort [CHOP, N=160], and Down Syndrome cohort [DS, N=63]). Microarray data was analyzed (Utah = Molecular Inversion Probe 330K [Affymetrix]; TARGET = SNP 500K [Affymetrix]; SJCRH = SNP 500K/6.0 [Affymetrix], CHOP = 850K, 610K, and Omni Quadv1 [Illumina], DS = SNP 500K, MIP 330K [Affymetrix]) by Nexus Copy Number (BioDiscovery, Inc.). Results: ALL patients that harbored copy number deletion 22q11.22 were present in about 30­45% of each cohort: Utah = 42.8%, TARGET = 29%, SJCRH = 40%, CHOP = 34%, DS = 46.7%. The majority of deletions, 93%, had a common recurring region just under 12 Kb in length. The 12 Kb deleted segment encodes no known genes. Patients that harbored a combined deletion in both IKZF1 and 22q11.22 (IKZF1+22q) were present in about 10­15% of each cohort: Utah = 12.5%, TARGET = 18%, SJCRH = 8.4%, CHOP = 6.3%, DS = 14.4%. IKZF1+22q conferred worse event­free survival (N=730, P=0.0062) compared to those with only IKZF1 deletions and worse overall survival (N=507, P=0.0365). Additionally, those patients with IKZF1+22q losses had a median decrease in event­free survival compared to those patients with neither deletion (normal cases): TARGET (high risk cohort) = 0.63 years, P= 90% from metastasis, it remains one of the most challenging process for targeted therapeutic interventions. Identification and characterization of chemically active and bioavailable compounds from medicinal plants targeting the cell­ signaling pathways of Cancer Stem Cells (CSCs), tumor cells and their microenvironment that abet metastasis is of great clinical significance. Hence, MCF­7; a breast cancer cell line, was treated with fractionated alkaloidal extracts from Rhazya stricta with previously documented anti­ cancer activity in lung and pancreatic cancer cell lines. RNA­seq. analysis was performed for samples treated with compounds that actively inhibited cell migration in scratch assay to further substantiate their anti­metastatic role at the cellular and molecular level. Genomic data corroborated the presence of anti­metastatic compounds that actively down­regulated genes involved in promoting metastasis (CDC6), cell migration (KIF23, DLGAP5), invasion and CSCs’ expansion (PTTG1). We also found down­regulation of GAS1 expression, an enhancer of Hedgehog signaling pathway. To our knowledge, this study, first of its kind, sets precedence in exploiting the anti­metastatic potential of these compounds. Further

proof of concept studies in mouse models of breast cancer to establish precise mechanism­of­action(s), would certainly open up avenues for the development of new chemical entities (NCEs), targeting metastasis regulating gene(s) with precision. #4908 Neuropilin­1 is up­regulated in the adaptive response of prostate tumors to androgen targeted therapies and is prognostic of metastatic progression and patient mortality. Marianna Volpert,1 Brian Tse,2 Ellca Ratther,1 Nataly Stylianou,1 Mannan Nouri,3 Melanie Lehman,1 Stephen McPherson,1 Mani Roshan­ Moniri,3 Mandeep Takhar,4 Nicholas Erho,4 Mohamed Alshalafa,4 Elai Davicioni,4 Robert Jenkins,5 Ashley Ross,6 Jeffrey Karnes,5 Robert Den,7 Ladan Fazli,3 Martin Gleave,3 Elizabeth Williams,1 Paul Rennie,3 Ralph Buttyan,3 Pamela Russell,1 Colleen Nelson,1 Brett Hollier1. 1Queensland University of Technology, Brisbane, Australia; 2Translational Research Institute, Brisbane, Australia; 3University of British Columbia, Vancouver, British Columbia, Canada; 4GenomeDx Biosciences, Vancouver, British Columbia, Canada; 5Mayo Clinic, Rochester, MN; 6Johns Hopkins Medical Institutions, Baltimore, MD; 7Thomas Jefferson University, Philadelphia, PA. Aims: Androgen­targeted therapies (ATTs) are the mainstay treatment for metastatic prostate cancer (PCa). However, ATTs promote adaptation of tumour cells and lead to castration resistant disease (CRPC). We have recently identified the cell surface receptor, Neuropilin­1 (NRP1) as increased during EMT and in CRPC. However, the role of NRP1 in the prostate epithelium is poorly understood. This study aims to determine whether the inhibition of NRP1 will be a feasible therapeutic strategy for blocking PCa metastasis and therapy resistance. Methods: qPCR and western blotting were used to assess NRP1 expression in PCa cell lines. NRP1 expression in CRPC was assessed using a murine LNCaP xenograft model of castration. NRP1 was knocked down with shRNA sequences from the pLKO.1 lentiviral construct. For metastasis assays, PC3 cells were microinjected into the zebrafish yolk sac and metastatic dissemination imaged 5 days later. NRP1 expression in radical prostatectomy (RP) samples from Mayo Clinic (545 patients) and

Johns Hopkins Medical Institutions (JHMI; 188 patients) cohorts was quantified by Affymetrix exon arrays and multivariable analysis performed. Wound scratch migration and invasion assays were performed with the WoundMaker™ tool and IncuCyte™ FLR imaging systems. Results: NRP1 levels were elevated in humanCRPC xenografts, metastatic and castrate resistant clinical PCa samples (p 50% (RT­ PCR) to 5% (nanoString) for PBS control group samples. Using the nanoString assay, mean KRAS mRNA knockdown was 52% and 68% (p50μm apart within four NSCLC tissues and was found to be ≤21%. Taken together, these data validate the use of the KRAS nanoString assay for use in clinical FFPE tissues, and demonstrate the level of knockdown of KRAS mRNA to be >50% at a dose of AZD4785 that causes regression in a preclinical PDX model. #5096 Targeted ablation of essential oncogenes in rhabdomyosarcoma with CRISPR/Cas9 gene therapy. Michael Phelps, Heechang Yang, Eleanor Chen. University of Washington, Seattle, WA. Targeted therapy seeks to exploit unique vulnerabilities in cancer cells not present in normal tissue. The development of effective therapies targeting essential tumor proteins has had significant challenges particularly for tumor oncogenes many of which have proven “undruggable”. Targeting these critical tumor oncogenes has been one of the foremost challenges in cancer research. Current sequencing technology facilitates rapid identification of novel cancer mutations yet there are no effective therapeutic strategies aimed at targeting essential cancer genes at the genome level. To overcome the challenges associated with targeting “undruggable” cancer proteins, we have employed high efficiency CRISPR/Cas9 genome editing technology to destroy cancer cells by directly targeting the genomic DNA of essential cancer genes. We used pediatric rhabdomyosarcoma to demonstrate the potential of CRISPR/Cas9 gene therapy in solid tumor preclinical xenograft mouse models. Rhabdomyosarcoma is an aggressive pediatric sarcoma characterized by myogenic differentiation arrest. The two major subtypes of rhabdomyosarcoma (RMS), embryonal (ERMS) and alveolar (ARMS) are defined largely by either activation of the RAS pathway (ERMS) or the presence of a fusion oncogene between PAX3 and FOXO1 (ARMS). CRISPR/Cas9 was first used to target critical exons in the activated RAS or PAX3/FOXO1 oncogenes unique to each RMS subtype, using inducible or viral CRISPR delivery systems. Targeting these essential genes resulted in significant regression of tumor xenografts due to a combination of cell death and differentiation. By precisely targeting the activating mutations in the RAS oncogene or the fusion site in the PAX3/FOXO1 fusion gene we further demonstrate that tumor­specific CRISPR/Cas9­mediated gene therapy can also be achieved. This strategy disrupts the essential cancer oncogenes with minimal impact to normal cells that do not harbor the unique cancer mutations. Our results highlight the utility of exploiting essential cancer genetic mutations for personalized therapy. This novel approach to cancer therapy provides a potential alternative strategy for targeting “undruggable” oncogenes while establishing a foundation for further development of CRISPR/Cas9 gene therapy for cancer treatment

#5097 An industrial method of manufacturing a novel RNAi anticancer drug, DFP­10825, for the treatment of peritoneal disseminated gastric cancer. Tatsuhiro Ishida,1 Hidenori Ando,1 Masakazu Fukushima,2 Cheng­Long Huang,3 Hiromi Wada3. 1Tokushima University, Tokushima, Japan; 2Delta­Fly Pharma, Inc., Tokushima, Japan; 3Kyoto University, Kyoto, Japan. We have developed an RNAi based anticancer drug, DFP­10825 [a cationic liposome and short­hairpin RNAi molecule for thymidylate synthase (TS shRNA)] and here established an industrial method of manufacturing the formulation. The therapeutic efficacy of the DFP­10825 was examined in a MKN45 human gastric carcinoma xenograft mouse model (peritoneal disseminated gastric cancer model). The mice were received intraperitoneal injection with 5 doses of DFP­10825 or control formulation (short­hairpin RNA for luciferase) (1 mg shRNA/kg/day) once every 3 days. The treatment with DFP­10825 obviously prolonged survival time of the mice compared to control formulation and significantly suppressed the target mRNA level in the peritoneal disseminated tumors. We recently reported that DFP­10825 showed a significant therapeutic efficiency in the human malignant pleural mesothelioma orthotopic xenograft model. Accordingly, DFP­ 10825 may lead a clinical benefit to patients with pulmonary malignant mesothelioma or disseminated peritoneal tumor. In addition, we established an industrial method of manufacturing DFP­10825, which provides a large scale preparation. Water solution of TS shRNA was gently mixed at an optimal ratio with ethanol solution of cationic lipids, composed of DC­6­14 (a cationic lipid), DOPE and DOPC. Then, the mixture was lyophilized under optimal condition. The freeze­dried cake provided a ready for injection formulation by a one­step hydration with saline. Physicochemical properties of the obtained DFP­10825 such as particle size, zeta­potential, and size distribution were almost comparable with those of conventionally prepared DFP­10825, which prepared with conventional preparation method, mixing TS shRNA solution and cationic liposomes. Our findings in this study demonstrate that intraperitoneal chemotherapy with a simple freeze­ dried RNAi medicine, DFP­10825, showing an equivalent pharmaceutical and therapeutic potency with conventionally prepared DFP­10825, meets a criteria to use for clinical settings and will have a clinical benefit to treat patients with disseminated peritoneal and pulmonary cancers. #5098 NG­348: a novel oncolytic virus designed to mediate anti­tumour activity via the potent and selective polyclonal activation of tumor­infiltrating T­cells. Brian R. Champion, Matthieu Besneux, Nalini Marino, Darren Plumb, Prithvi Kodialbail, Sam Illingworth, Rochelle Lear, Alice C. Brown. PsiOxus Therapeutics Ltd, Abingdon, United Kingdom. NG­348 is a transgene­modified variant of enadenotucirev, a chimeric oncolytic group B adenovirus with potent and selective anti­tumor activity against a range of epithelial cancer cells. Enadenotucirev has a blood stability profile that enables systemic dosing and has been administered intravenously to over 90 cancer patients. These studies have demonstrated that IV dosed enadenotucirev is delivered to tumors and subsequent virus activity is associated with CD8+ T­cell infiltration in tumor cell nests, consistent with

immune stimulation within the tumor. NG­348 encodes two immunomodulatory proteins in its genome: full­length human CD80, and a membrane anchored single chain variable fragment of the mouse anti­human CD3ε monoclonal antibody OKT3. Together these membrane proteins provide both T­cell receptor (signal 1) and costimulatory (signal 2) activation signals required to polyclonally activate tumor­infiltrating T­cells. When expressed on the surface of NG­348 infected tumor cells the transgenes therefore enhance the potency of the virus by driving local T­cell immune responses selectively in the tumor microenvironment. The expression of both transgenes encoded in the NG­348 virus is controlled by the endogenous virus major late promoter. This restricts expression of the proteins to the surface of cells permissive to virus infection (i.e. tumor cells) and prevents off­target expression in the cells from healthy tissues. Using co­cultures of human T­cells with human tumor cell lines, we have shown that NG­348 infected tumor cells potently activate both CD4 and CD8 T­cells. This was demonstrated by analysis of activation marker expression (CD25, CD69), intracellular and secreted cytokines (IL­2, TNF, IFNγ) and induction of T­cell mediated tumor cell death by apoptosis (prior to oncolytic death by the virus). Treatment of different human non­tumor cells (e.g. fibroblasts, T­cells, PBMCs) with NG­348 did not lead to transgene protein expression or activation of T­cells in co­ cultures. NG­348 has also been shown to activate human T­cells in vivo, using a human tumor xenograft model system in immunodeficient mice reconstituted with human PBMCs. Collectively these data indicate that following delivery to tumor tissues of patients, NG­ 348 oncolytic virus can selectively replicate and express it’s payload of T­cell activating ligands. NG­348 should therefore stimulate potent antigen­independent, polyclonal activation of T­cells already present in the tumor, as well as those recruited into the tumor in response to the virus infection, to drive effective anti­tumor immunity. NG­348 is currently in preclinical development with a first phase I study planned to initiate in Q4 2017.

Novel Chemotherapies #5099 Development of novel antimetabolite, DFP­11207, with self­toxicity protection and its promising preclinical and clinical profiles. Masakazu Fukushima,1 Kenzo Iizuka,1 Kokoro Eshima,1 Chung Zhang,1 Cheng Jin,1 Kiyoshi Eshima,1 Jaffer Ajani2. 1Delta­Fly Pharma, Inc., Tokushima, Japan; 2University of Texas, Houston, TX. Background: For over 50 years, 5­fluorouracil (5­FU) has played a critical role in the systemic chemotherapy of various gastrointestinal cancers including gastric, colorectal and pancreatic cancer which are the leading causes of cancer death globally. Although systemic cytotoxics such as 5­FU can modestly prolong overall survival in the adjuvant and advanced settings, but at the cost of unpleasant toxicities. Therefore better drug formulations are desirable. Methods: To this end, we developed a new conceptual fluoropyrimidine, DFP­11207 that is engineered to reduce toxicity without loss of antitumor activity as well in addition to providing sustained concentrations of the active anticancer moiety. DFP­11207 contains three components in one formulation: 1­ethoxymethyl­5­fluorouracil (EM­FU) as a prodrug of 5­ FU, 5­chloro­2,4­dihyroxypyridine (CDHP) as a potent inhibitor of 5­FU degradation and cytrazinic acid (CTA) as a gastrointestinal regulator of 5­ FU phosphorylation. Results: In in vitro metabolism studies using cell­free extracts from plasma, liver and tumors or intact tumor cells, DFP­11207 was rapidly hydrolyzed to 3 components and subsequently EM­FU was specifically converted to 5­FU by liver microsomes, and CDHP and CTA strongly inhibited 5­FU degradation and phosphorylation, respectively. Following consecutive oral administration to human tumor­bearing nude rats, DFP­ 11207 attained favorable antitumor efficacy and long­sustained PK profiles with lack of GI­ and myelo­toxicities. Next, in investigational clinical study in patients with solid tumors, 12 patients were treated at 8 unique dose levels of DFP­11207, ranging from 40 to 440mg/m2/day by each 1 pt. MTD and RD of daily and 28­day consecutive DFP­11207 was found to be 440 (n=2) and 330 mg/m2 (n=6), respectively. The main AEs were nausea,, anemia, neutropenia, febrile neutropenia but these events were very mild, and no thrombocytopenia was observed as expected. Furthermore, review of the preliminary PK data, DFP­11207 at 330

mg/m2 resulted in a desirable low but efficacious (~15­30 ng/ml) steady­ state plasma concentration of 5­FU. Interestingly, some of patients heavily treated with therapeutic drugs had a stable­disease for long periods. Conclusion: Our preclinical and early clinical data with DFP­11207 suggest that it’s a promising compound for the treatment of gastrointestinal cancers and can overcome the shortcomings of all other oral fluoropyrimidines. #5100 AB61, a new potent nucleoside cytostatic: Molecular mechanisms of action and preclinical activity. Petr Dzubak,1 Marian Hajduch,1 Pavla Perlikova,2 Gabriela Rylová,1 Petr Naus,2 Tomas Elbert,2 Eva Tloustova,2 Aurelie Bourderioux,2 Lenka Slavetinska,2 Kamil Motyka,3 Dalibor Dolezal,1 Pawel Znojek,1 Alice Nova,1 Monika Harvanova,1 Michal Siler,1 Jan Hlavac,1 Michal Hocek2. 1Palacky University in Olomouc, Faculty of Medicine and Dentistry, Olomouc, Czech Republic; 2Institute of Organic Chemistry and Biochemistry of the CAS, Prague, Czech Republic; 3Palacky University in Olomouc, Faculty of Natural Sciences, Olomouc, Czech Republic. 7­(2­Thienyl)­7­deazaadenosine (AB61) showed nanomolar cytotoxic activities against various cancer cell lines but only mild (micromolar) activities against normal fibroblasts. The selectivity of AB61 was found to be due to inefficient phosphorylation of AB61 in normal fibroblasts. The phosphorylation of AB61 in the leukemic CCRF­CEM cell line proceeds well and it was shown that AB61 is incorporated into both DNA and RNA, preferentially as a ribonucleotide. It was further confirmed that a triphosphate of AB61 is a substrate for both RNA and DNA polymerases in enzymatic assays. Gene expression analysis suggests that AB61 affects DNA damage pathways and protein translation/folding machinery. Indeed, the formation of large 53BP1 foci was observed in nuclei of AB61­treated U2OS­GFP­53BP1 cells indicating DNA damage. Random incorporation of AB61 into RNA blocked its translation in an in vitro assay and reduction of reporter protein expression was also observed in mice after 4­hour treatment with AB61. AB61 also significantly reduced tumor volume in mice bearing SK­ OV­3, BT­549, HT­29 and MDA­MB231 xenografts. The results indicate that AB61 is a promising

compound with the unique mechanism of action and deserves further development as an anticancer agent. This work was supported by the Ministry of Education of the Czech Republic (LO1304). #5101 Defining exposure­PD and efficacy relationships with the novel liver­ targeting nucleotide prodrug MIV­818 for the treatment of liver cancers. Mark Albertella,1 Biljana Rizoska,1 Alastair Kyle,2 Andrew Minchinton,2 Annelie Linqvist,1 Sanja Juric,1 Susanne Sedig,1 Karin Tunblad,1 Fredrik Öberg,1 Björn Classon,1 Anders Eneroth,1 John Öhd,1 Richard Bethell1. 1Medivir AB, Huddinge, Sweden; 2Cabenda, Vancouver, British Columbia, Canada. Background: Many systemic chemotherapeutics have failed to show efficacy in hepatocellular carcinoma (HCC), often because systemic toxicity prevents efficacious liver levels of the drug from being reached. MIV­818, a nucleotide prodrug of troxacitabine­monophosphate (TRX­ MP) has been designed as a novel approach to deliver high levels of the chain­terminating nucleotide troxacitabine­triphosphate (TRX­TP) to the liver after oral dosing while minimizing systemic exposure. We investigated MIV­818 and troxacitabine using in vivo models in order to identify therapeutic levels of TRX­TP required in the tumors. Methods: MIV­818 or troxacitabine were administered to nude mice with subcutaneous Hep3B or Huh7 xenografts. LC­MS/MS was used to assess MIV­818 and its metabolites. Effects on tumor growth, plasma AFP, inhibition of proliferation and induction of DNA damage were examined and correlated with exposures to TRX­TP in the tumor. Quantitative immuno­fluorescent histology was used to assess DNA damage (pH2AX) proliferation (BrdU), and hypoxia (pimonidazole). Results: Compared to the parent nucleoside troxacitabine, MIV­818 has increased potency of inhibition of HCC cell line growth, increased conversion to its active metabolite TRX­TP and in vitro properties optimized for oral bioavailability and liver targeting, including permeability and intestinal stability. MIV­818 also shows strong synergistic anti­proliferative activity with sorafenib in a number of HCC cell lines in vitro. Pronounced tumor growth inhibition of 70­100% and extensive tumor growth delays of up to 26 days were observed in the Hep3B xenograft model following a five day period of dosing. 12­32­fold induction of

DNA damage was seen throughout the tumor sections, consistent with the expected mechanism of action, and with associated inhibition of proliferation. Clear PD responses were apparent even in hypoxic regions of the tumor, indicating effective distribution of TRX­TP even far from blood vessels. DNA damage persisted for up to 7 days after the final dose, demonstrating long­lasting effects and indicating that intermittent dosing is likely to be effective. Similar dosing regimens given to Huh7 xenograft models resulted in significant tumor growth inhibition and induction of DNA damage. Intratumoral TRX­TP exposures across both models were correlated with anti­tumor effects including DNA damage induction, proliferation inhibition and tumor growth inhibition. Conclusions: We have identified TRX­TP exposures required for pronounced anti­tumor effects to give a comprehensive understanding of PK­PD­efficacy relationships for the active metabolite of MIV­818. These data could be used to guide dosing and dose selection in clinical studies. MIV­818 is currently in preclinical development in preparation for the initiation of clinical trials in patients with advanced HCC and other liver cancers. #5102 TRXE­009­1 has pan­acting anticancer activity and potently inhibits colony formation of prostate cancer epithelial cell explants. Eleanor I. Ager,1 Dominika Butler,2 Alex Stevenson,3 Eiffe Eiffe,1 Andrew Heaton,1 Brian Gabrielli,3 Norman J. Maitland,2 David Brown1. 1Novogen, Hornsby, Australia; 2University of York, York, United Kingdom; 3University of Queensland, Woolloongabba, Australia. Background: TRXE­009­1 is a novel small molecule from the super benzopyran (SBP) drug family under development as an intravenous cytotoxic agent. Cantrixil (TRXE­002­1), our lead SBP candidate targeting ovarian cancer, recently achieved Investigational New Drug (IND) status and is undergoing safety assessment in a multi­center Phase I clinical trial. Our aim was to investigate pathways associated with response to TRXE­009­1 and identify cancer types for clinical translation. Methods: Eurofins Oncopanel240 with multiplex analysis and bioinformatic assessment was used to identify cytotoxic and pathway response across a panel of cancer types. Western blot and live cell imaging were used to investigate protein levels and cell cycle progression. Results: TRXE­009­1 demonstrated broad cytotoxic activity across a

large panel of cell lines and was particularly active against kidney (RCC), liver (HCC), head and neck cancer, lung cancer, sarcoma, prostate cancer, osteosarcoma, and neuroblastoma. Further, TRXE­009­1 combined synergistically with two targeted agents used extensively in the clinical treatment of RCC patients. Cleaved caspase 3/7 and G2/M arrest were strongly associated with TRXE­009­1 activity. Live imaging confirmed G2/M arrest and implicated cytoskeleton disruption in TRXE­009­1 induced cell death. When compared to 43 reference oncology compounds, TRXE­009­1 clustered with a known epigenetic modifier. Additional analysis indicated that, compared to the least sensitive lines, the most sensitive cancer cell lines were more likely to be positive for LIN28B, a stemness marker associated with aggressive cancers. This is in line with historical studies suggesting that SBPs may preferentially target cancer­ stem like cells. Using a panel of prostate cancer epithelial explants in a colony forming assay, we confirmed strong activity against colony­ initiating cells. Studies are current to confirm protein changes associated with SBP activity and to further investigate the impact of TRXE­009­1 on the cytoskeleton. In vivo studies previously established activity of TRXE­ 009­1 against melanoma, prostate, and brain cancer and additional studies are current to optimize the delivery formulation to be used in preclinical safety studies prior to clinical progression. Conclusion: Given that cancer stem­like cells are typically more chemoresistant and are thought to support tumor recurrence, the activity of TRXE­009­1 against this subpopulation of cancer cells makes it a particularly promising candidate for further development. #5103 Development of MJ­66 as a novel anticancer agent for the treatment of malignant gliomas. Mann­Jen Hour,1 Tai­Lin Chen,1 Po­Wu Gean,2 Hong­Zin Lee1. 1School of Pharmacy, China Medical University, Taichung, Taiwan; 2Department of Pharmacology, College of Medicine, National Cheng­Kung University, Tainan, Taiwan. Malignant glioma is the most common aggressive adult primary tumor of the central nervous system. Treatments of malignant gliomas include surgery, radiotherapy and adjuvant temozolomide (TMZ) chemotherapy. However, inherent­ and acquired resistance to TMZ present major obstacles to successful treatment, and the prognosis of patients with malignant gliomas remains very poor.

MJ­66, a synthetic qunazoline compound, was identified in our study as a potent anti­proliferative agent especially on human glioma with IC50 of approximately 60 nM. In intracranial glioma xenograft model, MJ­66 (0.14 mg/kg in saline, i.p., q.d., 10 days) significantly inhibited tumor growth and increased the survival of the experimental mice, however, did not decrease the body weight of mice. Currently, there is a profound unmet medical need for a new drug in the treatment of malignant brain tumor. The MJ­66 demonstrated superior efficacy to TMZ that are the only first­line­drug in clinical use. MJ­66 is suggested to be a promising anticancer candidate. #5104 A novel N­mustard­quinoline conjugate is a potent agent against colorectal cancer. Tai­Lin Chen,1 Yan­Bo Chen,2 Chia­Ning Shen,2 Tsann­Long Su,3Te­ Chang Lee3. 1Institute of Biomedical Sciences, Academia Sinica and Institute of Biochemistry and Molecular Biology, National Yang­Ming University, Taipei, Taiwan; 2Genomics Research Center, Academia Sinica, Taipei, Taiwan; 3Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan. Colorectal cancer (CRC) is the third leading cancer worldwide. The prognosis of patients diagnosed with CRC at the stage IV is poor with a 5­year survival rate less than 10%. Combination chemotherapy with platinum­based drugs and 5­fluorouracil (5­FU) remains the first line treatment, while the chemotherapy combined target therapeutic agents were decreasing the mortality of CRC patients. However, the efficacy of platinum­based drugs has often been limited because of the substantial risk for severe toxicities, including nephrotoxicity or neurotoxicity. Therefore, there is an unmet need to develop a novel drug for improving the treatment of advance colorectal cancer. We have previously designed and synthesized several series of DNA­directed alkylating agents with potent antitumor activity. Recently, one derivative of N­mustard­ quinoline conjugates with hydrazinecarboxamide as a linker, designated as SL­1, was selected to explore its anti­colorectal cancer (CRC) activity because it was highly cytotoxic to a panel of CRC cell lines in culture. Flow cytometric analysis showed that treatment of CRC cells with SL­1 induced G2/M arrest and triggered apoptosis. We further demonstrated that SL­1 preferentially targeted on the selective guanine sequence by

sequencing gel electrophoresis. By the aid of comet assay, SL­1 in combination with 5­FU resulted in increased DNA tail moments and induced more DNA damage. Evaluation of the potency of antitumor activity both in culture and in xenograft models, we observed that SL­1 was more potent than 5­FU and oxaliplatin against SW620, RKO and RKO­E6 (an oxaliplatin resistant­subline) cells. Notable, SL­1+5­FU were more efficacious than oxalipatin+5­FU in suppression the growth of RKO or SW620 in xenograft models. We further found that SL­1 enhanced 5­FU and induced more apoptosis by c­caspase 3 and TUNNEL staining. Additionally, histopathological examination showed that SL­1 by itself has no obvious kidney and liver toxicity in ICR mice comparing to cisplatin, oxaliplatin or 5­FU. Taken together, SL­1 alone or in combination with 5­FU may warrant our further development as a potential antitumor agent for the treatment of CRC patients. #5105 Structure­activity relationship study of pyrrolo[2,3­d]pyrimidines as microtubule targeting antitumor agents. Aleem Gangjee,1 Tasdique M. Quadery,1 Susan L. Mooberry2. 1Duquesne University, Pittsburgh, PA; 2University of Texas Health Science center at San Antonio, San Antonio, TX. Microtubules are cytoskeleton protein polymers composed of αß­tubulin heterodimers which play pivotal roles in cell division, cellular transport and cell signaling. Interfering with microtubule dynamics is a well­ established strategy for the treatment of cancer. However, clinical utility of microtubule targeting agents (MTAs) in cancer chemotherapy is often limited by the emergence of drug resistance. Expression of P­ glycoprotein (Pgp) or the βIII isotype of tubulin, are two clinically established drug resistance mechanisms. We previously reported that compound AG346 binds to the colchicine site of tubulin and has microtubule depolymerization activity in A­10 cells (EC50 = 365 nM). Molecular modeling and docking studies of this compound in the X­ray crystal structure of tubulin (PDB: 402B) suggested that the pyrrole NH can be substituted with alkyl chains of optimal length to obtain an altered binding conformation which can facilitate interaction with the hydrophobic pocket formed by amino acid Val181 of side chain A, and Lys352 and Thr314 of side chain B. We designed and synthesized a variety of pyrrole NH­alkyl substituted compounds and determined that

the compound AG473 shows a 43­fold improved microtubule depolymerization potency (EC50 = 8.4 nM) in A­10 cells, compared to the lead compound. In addition, this compound inhibited the growth of human melanoma cancer cell line MDA­MB­435 in culture with an IC50 of 7.2 nM, which is a 5­fold improvement in potency over the parent compound. These analogs also circumvented resistance mediated by Pgp and βIII­tubulin. These attributes provide the impetus for further preclinical development of these compounds as microtubule targeted antitumor agents in vivo and these studies are currently underway. #5106 In vitro and in vivo evaluation of WAC­224, a novel quinolone class of topoisomerase II inhibitor for cancer therapy. Taichi Ueshima, Tomonori Yamaguchi, Kenji Itoh, Naoki Kashimoto, Tatsuya Hirano, Rumiko Shimabara, Yohei Kawakubo, Masayuki Sato, Junpei Yamashita, Akira Yazaki, Koichi Tamura. Wakunaga Pharmaceutical Co., Ltd, Akitakata­shi, Hiroshima, Japan. Introduction: DNA topoisomerases (Topo) are classical but still attractive targets for drug therapy in multiple types of cancers. Topo inhibitors, such as Etoposide and Daunorubicin, have been effectively used; however, their clinical use is often limited by drug resistance in cancer cell population. Therefore, the development of a novel chemical class of Topo inhibitors has been desired to overcome drug resistance. Recently, Vosaroxin (QINPREZO) is identified as a first­in­class anti­cancer quinolone derivative targeting Topo II, which is under development for relapsed or refractory acute myeloid leukemia (AML). In this study, we evaluated the in vitro and in vivo activities of WAC­224, a novel quinolone derivative for Topo II inhibition, for various cancer cell lines including multi­drug resistant cells and in mouse xenograft models, respectively. Materials and Methods: In vitro anti­proliferative activities against over 20 cell lines were determined using WST cell proliferation reagent. Apoptosis was measured by Annexin­V staining. In vivo anti­tumor activity was determined in immunodeficient mice bearing multi­drug resistant human uterine sarcoma MES­SA/DX5. Results: WAC­224 accomplished extensive anti­proliferative activities for cancer cell lines including multi­drug resistant ones with the EC50 range of 0.001 to 1 microM. Especially, in MV4­11 AML cell line, WAC­224 showed a potent and superior anti­proliferative activity

compared with Doxorubicin (EC50: 1.4 nM for WAC­224 v.s. 2.5 nM for Doxorubicin). Molecular and cellular mechanisms of WAC­224 were defined as inhibition of human Topo II, induction of G2/M phase cell cycle arrest and apoptosis. Additionally, significant antitumor effects of WAC­224 were confirmed in mouse xenograft models. Furthermore, WAC­224 showed no toxic effect on small intestinal crypts in mice. Conclusion: This study demonstrated that WAC­224 has strong in vitro activities against broad cancer cell types along with the potent in vivo efficacy and well tolerability, without gastrointestinal toxicity. These results indicate that WAC­224 is promised to provide a new therapeutic option for various cancers including multi­drug resistant and hematological ones. #5107 Fisetin treatment reduces tumor growth and metastasis by modulating PI3K/AKT and MEK/ERK pathways in a BRAFV600E/PTENNULL mouse model of melanoma. Harish C. Pal, Mary Katherine Montes de Oca, Pooja Sharma, Ross L. Pearlman, Farrukh Afaq. University of Alabama at Birmingham, Birmingham, AL. Melanoma is the deadliest form of skin cancer due to its propensity to metastasize. It accounts for about 80% of skin cancer­related deaths. A substitution of valine for glutamic acid at position 600 results in the BRAFV600E mutation found in approximately 50% of melanomas. The BRAFV600E mutation drives activation of MEK/ERK signaling and also cooperates with the PI3K/AKT pathway, thus enhancing tumor initiation, progression, metastasis, and drug resistance. Fisetin (3,3',4',7­ tetrahydroxyflavone), a naturally occurring flavonoid found in fruits, vegetables, nuts, and wine, exhibits anti­proliferative, anti­invasive, and anti­tumorigenic properties. Earlier, we showed that fisetin treatment downregulates the PI3K/AKT pathway and reduces phosphorylation of MEK/ERK in melanoma. In this study we determined the effects of fisetin on tumor growth and metastasis in a genetically engineered transgenic mouse model of metastatic melanoma. Melanocyte specific Cre activity was induced in six­week­old BRAFV600E/PTENNull/CreTg mice by topical application of 4­hydroxytamoxifen on shaved backs once per day for 3 consecutive days. Mice were observed daily until a highly pigmented measurable tumor appeared. Size of the tumors was measured

twice weekly and treatment with fisetin (45mg/kg b.wt. & 90mg/kg b.wt.; orally 5 times/week) was started when tumor size reached 100 mm3. Measurement and quantification of tumor volume showed that fisetin administration inhibited melanoma growth in these mice as compared to the control group. Hematoxylin and eosin staining revealed that fisetin treatment reduced pigmented cells in the ear and skin. In addition, fisetin treatment reduced the metastasis of melanoma cells into the spleen and draining lymph nodes. Furthermore, evaluation of tumor tissues revealed that fisetin treatment reduced the (i) expression of PI3Kp110α and p85, (ii) phosphorylation of AKT at Ser473 and Thr308, (iii) phosphorylation of mTOR at Ser2481, and (iv) expression of raptor and rictor. In addition, fisetin treatment reduced the phosphorylation of MEK and ERK when compared to control tumor samples. Fisetin treatment also resulted in (i) cleavage of caspase­3, (ii) inhibition of Bcl2 and Bcl­xL, (iii) induction of Bax, and (iv) inhibition of PCNA and Ki67 expression. Further, fisetin treatment reduced the expression of mesenchymal marker proteins (N­ cadherin and vimentin) with concomitant increase in epithelial marker proteins (E­cadherin and desmoglein). These data suggest the ability of fisetin to exhibit anti­proliferative, pro­apoptotic, anti­tumorigenic, and anti­metastatic potentials in a mouse model of melanoma. We suggest that fisetin could be used as an adjuvant chemotherapy to prevent drug resistance and improve the therapeutic efficacy of anti­melanoma drugs for the management of melanoma. #5108 Potent small molecule compounds that selectively inhibit proliferation of ABC­DLBCL cell lines. Leena Khare Satyam,1 Dinesh Chikkanna,1 Vinayak Khairnar,1 Manoj Pothuganti,1 Sunil Panigrahi,1 Anirudha Lakshminarasimhan,1 Narasimha Rao,1 Wesley Balasubramanian,1 Sandeep Patil,1 Sreevalsam Gopinath,1 Gunta Upendra,1 Jwala Nagaraj,1 Kiran Aithal,1 Vijay Ahuja,2 Sanjeev Giri,2 Chetan Pandit,1 Murali Ramachandra1. 1Aurigene Discovery Technologies Ltd., Bangalore, India; 2Aurigene Discovery Technologies Ltd., Hyderabad, India. Diffuse large B cell lymphoma (DLBCL), which accounts for 25% of all lymphomas cases, has been classified into molecular subtypes including germinal center B cell like (GCB) DLBCL, activated B cell­like (ABC)

DLBCL, and primary mediastinal B cell lymphoma (PMBL). Among these subtypes, patients with ABC­DLBCLs have the worst prognosis because of the high chemo­resistance, and require effective therapies. Mucosa­Associated Lymphoid Tissue Lymphoma Translocation 1 (MALT1) protease activity is linked to the pathogenesis of ABC­DLBCL. Therefore, a focused library of covalent compounds selected based on molecular docking on the reported crystal structure was screened for selective sensitivity to ABC­DLBCL, but not GCB­DLBCL cell lines. Optimization of initial hits resulted in the identification of lead compounds with an anti­proliferative EC50 of 10 over cellular EC50 for up to 8 hours. The lead compounds showed dose­dependent tumor growth inhibition in a xenograft model upon oral dosing. In summary, we have identified novel and potent MALT1 inhibitors capable of selectively inhibiting proliferation of DLBCL cell lines with optimized drug­like properties including oral bioavailability. The data presented here strongly support further development of these compounds for DLBCL and other indications. #5109 Beyond malaria: Second career of artesunate as cancer drug. Thomas A. Efferth. Johannes Gutenberg Univ. Mainz, Mainz, Germany. More than a decade ago, we initiated a research program on the molecular pharmacology of phytochemicals derived from Chinese medicinal herbs. Bioactive plant extracts have been fractionated by chromatographic techniques. We isolated bioactive compounds and elucidated their chemical structures by nuclear magnetic resonance and mass spectrometry. A promising compounds was artemisinin from Artemisia annua L. and its semisynthetic compound artesunate. Artemisinin and

artesunate are anti­malarial drugs. Our data indicated profound activity against cancer cells, but also against various viruses, Schistosoma, Trypanosoma, and even plant crown gall tumors. To elucidate the molecular mode of actions against cancer, we applied molecular biological and pharmacogenomic approaches in vitro and in vivo. Different signaling pathways were identified not only in cancer cells but also in cells infected with viruses, e.g. HCMV, HSV1 and others. To translate the experimental results in cell lines and animals to the bedside, we report on the compassionate use of artesunate in single cancer patients as well as on our efforts to initiate several clinical phase I/II trials in veterinary tumors as well as in human cervix or colorectal carcinoma. These pilot studies indeed indicate that artesunate is not only useful as antimalarial drug, but also exerts activity against cancer and viral diseases. Clinical results will also be presented that not only artesunate as semisynthetic chemical derivative of artemisinin, but also herbal extracts from Artemisia annua are active in veterinary and human tumor patients. Artesunate represents an illustrative example for the therapeutic potential of medicinal herbs and drugs derived from traditional Chinese medicine.References 1. Efferth et al.: Journal of Molecular Medicine 2002;80:233­42. 2. Efferth: Drug Resistance Updates 2005;8:85­97. 3. Efferth et al.: Molecular Cancer Therapeutics 2008;7:152­61. 4. Li et al.: Cancer Research 2008;68:4347­51. 5. Shapira et al.: Clinical Infectious Diseases 2008;46:1455­7. 6. Efferth et al.: Clinical Infectious Diseases 2008;47:804­11. 7. Krishna et al.: Ebiomedicine 2014;2:82­90. 8. Saeed et al.: Pharmacological Research 2016;110:216­226. #5110 A novel nucleoside analog therapeutically active against plasma cell malignancies and other ADK­expressing cancers including colon and pancreatic adenocarcinomas. Jouliana Sadek,1 Utthara Nayar,2 Jonathan Reichel,3 Jennifer Totonchy,4 Shizuko Sei,5 Robert Shoemaker,5 David Warren,1 Olivier Elemento,1 Kenneth Kaye,6 Ethel Cesarman1. 1Weill Cornell Medicine, New York, NY; 2Dana­Farber Institute, Boston, MA; 3Memorial Sloan Kettering Cancer Center, New York, NY; 4Chapman University, CA; 5National Cancer Institute, Frederick, MD; 6Harvard Medical School, Boston, MA. A number of nucleoside analogues are used successfully for the treatment

of several cancers, and in particular leukemias and lymphomas, but these have distinct efficacies for different tumor types, and many malignancies do not respond to currently available nucleoside analogues or other forms of chemotherapy. A high throughput screen conducted in our lab to search for inhibitors of primary effusion lymphoma (PEL) identified the nucleoside analog 6­ethylthioinosine (6­ETI) as a potent and selective inhibitor of PEL, a largely incurable malignancy of B cell origin with plasmacytic differentiation. 6­ETI induced necrosis and ATP­depletion accompanied by S­phase arrest, DNA damage and inhibition of DNA synthesis. To understand 6­ETI mechanism of selectivity, RNA­seq analysis of in vitrogenerated drug­resistant PEL clones revealed inactivating mutations and loss of expression of adenosine kinase (ADK) as the mechanism of resistance. In vitro assays showed that 6­ETI is a pro­drug that gets phosphorylated and activated by adenosine kinase (ADK) into its active form. We found high ADK expression in PEL cell lines and primary specimens of PEL, multiple myeloma (MM) and plasmablastic lymphoma (PBL) patient samples. 6­ETI was effective at killing multiple myeloma cell lines, primary MM specimens, and had a remarkable anti­tumor response in a disseminated multiple myeloma and PEL xenograft mouse models. Thus, ADK expression can serve as a predictive biomarker to help identify patients that are most likely to respond to 6­ETI treatment. To further assess the spectrum of activity and sensitivity of 6­ETI, we examined ADK expression in other cancer subtypes and found that colorectal and pancreatic adenocarcinomas also overexpress ADK and are highly sensitive to killing by 6­ETI at the low nanomolar concentration. We also found high ADK expression in primary colon and pancreatic adenocarcinoma patient specimens. We compared 6­ETI to other FDA­approved purine analogs and failed to find other compounds with similar potency or selectivity profile. Herein, we report the identification of a novel purine analog, 6­ethylthioinosine, as an effective therapeutic with exquisite sensitivity to plasma cell malignancies and other ADK­expressing cancers. We have successfully used RNASeq­based “resistome” analysis to identify its mechanism of specificity and discovered a new biomarker that can potentially impact patient care and the treatment of some of the most aggressive tumors. #5111 Norcantharidin impairs tumor growth in vivo and inhibits stemness of triple­negative breast cancer cells. Damian E. Berardi, Guido Cicuttin, Maria A. Taruselli, Stefano M.

Cirigliano, Elisa D. Bal de Kier Joffé, Alejandro J. Urtreger, Laura B. Todaro. Angel H. Roffo Inst. of Oncology, Buenos Aires, Argentina. Triple­negative breast cancer (TNBC) is characterized by an abundance of treatment­resistant cancer stem cells (CSC). The absence of a molecular target, coupled with its highly aggressiveness, leads to the lack of an effective therapy for TNBC. Norcantharidin (NCTD) is a synthetic demethylated small­molecule analog of the naturally occurring cantharidin isolated from blister beetles (Mylabris phalerata Pall). Unlike the conventional chemotherapeutics, NCTD toxicity is higher to cancer cells than normal ones, making this small molecule promising for cancer treatment. The aims of this work were: A) To study the effect of NCTD on 4T1 cell line proliferation in vitro. B) To analyze the effect of NCTD on 4T1 derived CSC on self­renewal and clonogenic capacity. C) To evaluate the effect of NCTD on 4T1 tumor growth in vivo. We employed the well­known 4T1 triple­negative breast cancer cell model, which presents a huge proportion of CSC. We observed that NCTD treatment during 96 h significantly reduced 4T1 cell proliferation in vitro. In addition, the IC50 value of NCTD was 27.35 ± 2.83 μM. Related to CSC, NCTD pre­treatment for 96 h impaired CSC self­renewal (Number of secondary mammospheres: Control: 276±39; NCTD: 163±18; p≤0.05) as well as the clonogenic capacity (Number of colonies: Control: 359±38; NCTD: 122±11; p≤0.05). By q­PCR, we observed that NCTD treatment for 48 h significantly induced an increase of Gli­1 and Smooth in CSC, keys member of Sonic Hedgehog pathway. Finally, we performed an in vivo assay, where 4T1 cells were orthotopically inoculated on mammary gland of BALB/c mice, and NCTD was i.p. inoculated twice a week (5mg/kg). We observed that NCTD treatment significantly reduced tumor growth in vivo. Our data suggest that NCTD treatment reduces tumor growth both in vitro and in vivo, possibly through the direct effect on CSC self­renewal and clonogenic capacity, by modulating Sonic Hedgehog pathway. #5112 Evaluation of FF­10502­01, a new pyrimidine nucleoside analogue, in pancreatic (PANC) patient­derived xenograft (PDX) models compared to gemcitabine and in combination with nab­paclitaxel. Takeaki Suzuki,1 Linda J. Paradiso,2 Jill Ricono,3 Jonathan Nakashima,3

Yoshihide Iwaki,4 Shinji Mima,4 Takayuki Yamada,4 Chihaya Kakinuma,4 Hiroyuki Iwamura,4 Shinichi Watanabe4. 1FUJIFILM Pharmaceuticals, U.S.A., Inc., Cambridge, MA; 2Strategia Therapeutics, Inc., Houston, TX; 3Crown Bioscience, San Diego, CA; 4FUJIFILM Corporation, Tokyo, Japan. Introduction FF­10502­01 is a synthetic pyrimidine nucleoside analogue structurally similar to gem with a substitution of sulfur for oxygen in the pentose sugar. In previous studies, FF­10502­01 demonstrated preclinical efficacy across multiple solid tumors, including PANC cancer. In this study, we investigated the anti­tumor effect of FF­10502­01 in PANC PDX models. Methods 10 PANC PDX tumors were sourced from primary (2) or metastatic sites (8). 7 demonstrated high resistance (HR) to gem, 1 intermediate, and 2 low. In the dose­finding study, 3 PDX models were studied in 9 grps of NOD­SCID mice (n=10/grp), treated with 240 or 480 mg/kg FF­10502­ 01, 3 or 6 mg/kg nab­pac or 240 mg/kg gem, alone and in combination for 28d, followed by 28d observation. The definitive study consisted of 7 PDX models. 6 grps (n=10/grp) were treated with 240 or 480 mg/kg FF­ 10502­01, 6 mg/kg nab­pac or 240 mg/kg gem, alone and/or in combination for 28d, followed by 28d observation. After subcutaneous transplantation, animals were left undisturbed for 7d. Animals were monitored weekly and tumor volume measured with calipers. Average tumor volume (mm3) for each group at randomization into treatment grps ranged from 184.34 – 199.51 (SD ± 20.94 – 30.40). Statistical significance was determined using one­way ANOVA and Tukey’s test. Results At clinically relevant doses, FF­10502­01, alone and in combination with nab­pac demonstrated greater tumor growth suppression than vehicle­ treated animals (p≤0.0001). In 7 models, FF­10502­01/nab­pac demonstrated higher tumor growth suppression than gem/nab­pac (p≤0.5, p≤0.01 or p≤0.001), irrespective of resistance to gem. In 3 models, there was no difference, but these models were highly responsive to gem/nab­ pac, thus minimizing the effects of FF­10502­01/nab­pac. Despite statistical insignificance, FF­10502­01/nab­pac still demonstrated greater tumor growth inhibitory activity to gem/nab­pac. In HR gem models, FF­ 10502­01 was superior to gem (p≤0.0001, p≤0.001, p≤0.05) in 3 of 7, and FF­10502­01/nab­pac was superior to gem/nab­pac in 6 of 7 (p nab­pac

(p≤0.0001, p≤0.0001, p≤0.01, p≤0.05). FF­10502­01/nab­pac also was more tolerable than gemcitabine/nab­pac, as demonstrated by less weight loss. Conclusions FF­10502­01 is a new pyrimidine nucleoside analogue with demonstrated preclinical efficacy in solid tumors, including PANC cancer. In PANC PDX models, FF­10502­01 alone and in combination with nab­pac demonstrated higher efficacy and better tolerability than gem alone or gem/nab­pac. FF­10502­01 is in Phase 1 clinical development. #5113 Usnic acid, lichen secondary metabolite, inhibits glioblastoma progression through the reduction of epithelial­mesenchymal transition and glioma stemness factors. Kyung­Hwa Lee, Se­Jeong Oh, Shin Jung, Kyung­Keun Kim, Jae­Hyuk Lee, Kyung­Sub Moon. Chonnam National University Hwasun Hospital & Medical School, Hwasun­gun, Republic of Korea. Background: Usnic acid (UA), an active compound mainly found in lichens, has shown some anti­tumoral activities for lung and breast cancers. The therapeutic role of UA in glioblastoma (GM) have not yet been determined, nor has the definitive relationships of UA with EMT and cancer stem cells. Methods: We tested the anti­tumoral activities of UA against glioblastoma (GM) progression and further investigated the mechanistic link with epithelial­mesenchymal transition (EMT) and cancer stemness factors. The targeting and anti­tumor effect of UA was also checked in orthotopic mouse glioma model. Results: In vitro assay, we found that UA increased apoptotic cell death and inhibited the invasion/migration of GM cells. Sphere and colony forming abilities were also decreased in treated GM cells. UA decreased the expression of the EMT markers (N­cadherin, ZEB1, ZEB2, SNAIL and SLUG) and the cancer stemness markers (CD133, ALDH1 and CD44). In orthotopic mouse glioma models, UA localized in GBM and significantly decreased tumor growth and progression to lead longer survival. Conclusion: Taken together, these findings showed that UA prevent GBM invasiveness and progression, through the down­regulation of EMT and cancer stemness markers.

#5114 Design, synthesis, and biological evaluation of tricyclic thieno[2,3­ d]pyrimidines as microtubule targeting antitumor agents. Farhana Islam,1 Aleem Gangjee,1 Xin Zhang,1 Xilin Zhou,1 Susan L. Mooberry2. 1Duquesne Univ. School of Pharmacy, Pittsburgh, PA; 2University of Texas Health Science Center at San Antonio, San Antonio, TX. Microtubules are dynamic structures that, together with actin microfilaments and intermediate filaments, constitute the cellular cytoskeleton. Besides their well­known roles in cell division, their functions involve maintenance of cell shape and morphology, cellular motility, and trafficking of organelles and vesicles. Recently, we reported a compound AG 370 as a microtubule targeting agent, that circumvented the Pgp and βIII­tubulin mediated drug resistance mechanisms that limit the efficacy of paclitaxel, docetaxel, and the vinca alkaloids. Molecular modeling and docking studies of the parent compound (AG 370) in the colchicine binding site (PDB: 402B) suggest that the C­5 and C­6 of parent compound are oriented towards the hydrophobic pockets with the side chain of Ala316, Val315, Leu255 and Met259. However, this binding pocket in tubulin is relatively large. To further explore the hydrophobic pocket, an additional cyclohexene ring was introduced at C­ 5 and C­6 of AG370. In the molecular modeling study, the resulting tricyclic scaffold showed hydrophobic interactions with the amino acids of the colchicine binding site. The 2­Me substituent of the tricyclic scaffold was replaced with a 2­H and a 2­NH2 group and 4­position of the scaffold was replaced with appropriate anilines. The 2­amino­ N4­ methoxyphenyl moiety (AG61) was found to be the most potent analog in the tricyclic series in both antiproliferative assay in human melanoma cancer cell line (MDA­MB­435 cell line, IC50 = 9.0 ± 0.2 nM) and in microtubule depolymerization assay in A­10 cells (EC50=19 nM). Thus, we identified tricyclic thieno[2,3­d]pyrimidines as a novel structural scaffold with potent antiproliferative activity as well as microtubule depolymerizing activity. These analogs are selected for preclinical development. #5115 Establishing an experimental paradigm to study the interphase effects of microtubule targeting agents.

April L. Risinger, Nicholas F. Dybdal­Hargreaves, Roma Kaul, Allison D. Clark, Susan L. Mooberry. UT Health Science Ctr. at San Antonio, San Antonio, TX. Microtubule targeting agents (MTAs) are highly effective anticancer drugs. While these drugs were traditionally classified as antimitotics, compelling evidence suggests that the ability of MTAs to interrupt microtubule­dependent trafficking and signaling in interphase cells contributes to their anticancer efficacies. Previous studies of the interphase effects of MTAs on oncogenic signaling pathways have led to an important reevaluation of their mechanisms of anticancer actions. These effects have been primarily reported in cells that have been treated with MTAs for extended periods of time. However, MTAs rapidly alter microtubule dynamics which results, within a few hours, in global changes to gene expression and cellular signaling. We propose that evaluating the effects of MTAs on oncogenic pathways at times and concentrations that are associated with early microtubule disruption will allow analysis of the initiating events that link the direct action of MTAs on microtubule structure and dynamics to effects on interphase signaling that contribute to anticancer efficacy. The concentrations and treatment times that were optimal for the study of clinically used MTAs on cellular trafficking and signaling events were first determined. Our experimental paradigm of a 2 h treatment of breast cancer cells with clinically relevant concentrations of MTAs eliminated contributions due to mitotic accumulation and changes in gene expression associated with longer treatments. While all MTAs disrupt microtubule dynamics, the differences among MTAs in their rapid downstream effects on cellular signaling have not been systematically evaluated. A goal of this project was to identify differences in the effects of diverse MTAs on interphase signaling pathways that may underlie their differential efficacy in patient populations. This short term treatment paradigm led to the identification of profound differences among MTAs in their ability to disrupt Src­ dependent E­cadherin re­localization, canonical and non­canonical TGF­ β signaling, and β­catenin localization. These findings demonstrate the ability of diverse MTAs to rapidly impact interphase oncogenic signaling and trafficking pathways. This experimental design sets forth a method to evaluate the initial effects of diverse MTAs to gain critical insight into their differential abilities to inhibit key oncogenic signaling pathways. These types of studies might, in the future, help facilitate the rational

selection of specific MTAs for patients depending on tumor characteristics. Funding for this work was provided by Eisai Inc. #5116 Identification of new treatment options for Merkel cell carcinoma using high throughput chemical screening. Tara Gelb,1 Daniel Urban,2 Kenneth Daily,1 Ying Xiao,1 Min Shen,2 Matthew Hall,2 Isaac Brownell1. 1National Cancer Institute, Bethesda, MD; 2National Center for Advancing Translational Sciences Chemical Genomics Center, Rockville, MD. Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer with limited treatment options. Approximately 80% of MCC tumors have Merkel cell polyomavirus (MCPyV) DNA integrated into the host genome, and viral oncogenes are thought to drive carcinogenesis. In contrast, MCPyV­negative (MCPyV­) tumors have higher rates of proto­oncogene mutations. Transcriptome differences between MCPyV+ and MCPyV­ tumors further suggests divergent underlying pathologies. In order to develop efficacious treatments for MCC and elucidate the pathophysiologies underlying MCPyV+ and MCPyV­ MCC, we screened the effects of mechanistically annotated drug libraries on MCC cell viability (CellTiter­Glo). We screened ~4,500 compounds including the NCGC Pharmaceutical Collection of approved and investigational drugs and the oncology­focused MIPE (Mechanism Interrogation PlatE) library against six MCC cell lines (3 MCPyV+ and 3 MCPyV­). We ran follow­ up screens on the above cell lines as well as non­MCC control cells to identify agents that specifically reduced viability in MCC. DNA topoisomerase inhibitors, proteasome inhibitors, and PI3K inhibitors potently and efficaciously reduced viability in all MCC cell lines tested. Moreover, using hierarchical clustering we found that viral status impacted pharmacological responses. For example, a subset of HDAC inhibitors were more potent in MCPyV+ than MCPyV­ MCC cells. In contrast, a dual RasGAP/ERK inhibitor more potently reduced viability in MCPyV­ MCC cells relative to MCPyV+ MCC cells. Together these results identify existing drugs that can be repurposed for treating MCC. Moreover, they help elucidate the distinct pathophysiology driving MCPyV+ and MCPyV­ MCC. #5117

Triplatin preferably suppress lung metastasis of breast cancer, and peritoneal carcinomatosis of colon and pancreatic cancer. Eriko Katsuta,1 Erica J. Peterson,2 Samantha J. Katner,2 Nicholas P. Farrell,2 Kazuaki Takabe1. 1Roswell Park Cancer Institute, Buffalo, NY; 2Virginia Commonwealth University, Richmond, VA. Background: Since metastatic spread is often directly associated with the poor outcome, an effective treatment for metastatic lesion is expected to improve overall survival. This is particularly the case in peritoneal carcinomatosis of pancreatic cancer where there is no effective treatment. Recent clinical trials demonstrated that Cisplatin is effective in certain metastatic breast cancer, however with severe side effects. Polynuclear platinum analog, Triplatin, was developed to overcome the severe toxicity. Methods: Murine cancer cell lines, 4T1­luc2 and E0771 (breast), Panc02­ luc (pancreas) and CT26­luc (colon) were used. Cell proliferation was quantified by CCK8 assay. Syngeneic orthotopic implantation of 4T1­ luc2 cells was used for metastatic breast cancer model. For mastectomy model, primary tumors were surgically removed 8 days after inoculation. Syngeneic intraperitoneal injection of CT26­luc cells and Panc02­luc cells were used for peritoneal carcinomatosis models of colon and pancreatic cancer respectively. Triplatin (0.3 mg/kg) or vehicle was administered intraperitoneally every four days three times and tumor burden was quantified by bioluminescence imaging. Results: Triplatin suppressed cell growth of both breast cancer and pancreatic cancer in a dose dependent manner in vitro (IC50 of 4T1­luc2, E0771 and Panc02­luc cells were 0.08, 0.51 and 0.07 μM, respectively). Triplatin did not suppress the growth of 4T1­luc2 primary breast tumor, however, ex vivo results showed that lung metastases were significantly reduced to 14% of the control (p = 0.034) in orthotopic model. This result was reproduced in post­mastectomy “adjuvant therapy” model where Triplatin treatment was began after primary implanted tumor was removed, which suppressed lung metastases down to 1.9% of control by Day21 (p = 0.038), and significantly prolonged survival (p = 0.007). This result led us to use Triplatin in CT26­luc colon cancer carcinomatosis model, where Triplatin suppressed total tumor burden to 1.9% of control by Day13 (p = 0.029) and significantly prolonged survival (p = 0.001). Finally, Triplatin reduced total tumor burden of Panc02­luc pancreatic cancer peritoneal carcinomatosis model to 35% of the control by Day 9 (p

= 0.337) and the survival was significantly prolonged (p = 0.025). Conclusion: Triplatin demonstrated significant suppression lung metastatic tumor of 4T1­luc2 breast cancer, and peritoneal carcinomatosis of CT26­luc colon cancer and Panc02­luc pancreatic cancer. Newer platinum compounds with less toxicity and favorable pharmacokinetics warrant further investigation for advanced metastatic cancer. #5118 A novel polyamine sulfonamide with anti­leukemic activity. Vindhya Vijay, Amy Meacham, Leylah Drusbosky, Elizabeth Wise, Christopher Cogle. University of Florida, Gainesville, FL. The greatest challenge in treating Acute Myeloid Leukemia (AML) is refractory disease. Although 60­80% of AML patients achieve complete remission after induction chemotherapy, majority of these patients relapse and die of progressive disease. New treatment options targeting the vulnerabilities of AML biology are highly critical for disease regression, in particular the chemo­resistant leukemia­initiating population, to ensure relapse­free survival in patients. Identifying novel druggable targets that are selective to AML despite their location in the vascular niche is thus highly warranted. We developed an in vitro co­culture model consisting of AML cells and Bone Marrow­ derived Endothelial Cells (BMECs), mimicking the protective effect of the vascular niche in the bone marrow microenvironment. Using this unique model, and taking advantage of combinatorial chemistry, we performed high­throughput chemical­ phenotypic screening of approximately 30 million novel compounds and identified a novel polyamine sulfonamide 2470­51 that can selectively kill AML cells by overcoming the protective effect of the BMECs. In addition to AML blasts, 2470­51 also exhibited highly selective activity against the leukemic stem and progenitor cell population while sparing normal hematopoietic stem and progenitor cells. In vivo studies using patient­derived xenograft models indicated significant regression in AML engraftment post 2470­51 treatment. Target identification experiments involving unbiased label­free shotgun proteomic analysis in combination with targeted Selected Ion Monitoring (SIM) revealed covalent drug binding targets of 2470­51. By performing differential protein expression analysis using ITRAQ analysis, we identified downstream mechanisms that led to mitotic cell cycle arrest and cell death. Collectively, our findings display the in vitro as well as in vivo efficacy of a novel polyamine sulfonamide in eliminating AML, including the leukemia

initiating compartment. We also uncover a novel mechanism in AML that can be taken advantage of for selective toxicity. Furthermore, we establish the role of 2470­51 as a potential therapeutic agent in treating AML. #5119 Evaluation of Minnelide as potential targeted therapy for triple negative breast cancer. Mahendra K. Singh,1 Soham Shah,2 Nikita Satish Sharma,1 Bhuwan Giri,1Sulagna Banerjee,1 Ashok Saluja1. 1University of Miami School of Medicine, Miami, FL; 2University of Minnesota School of Medicine, Minneapolis, MN. Recent advances in diagnostics and better understanding of molecular mechanism underlying breast cancer has let to the better therapeutic options and disease outcome for majority of breast cancer patients. However, ~10 ­ 20% of all breast cancers often referred to as “triple negative” as they lack expression of the estrogen (ER), progesterone (PR), and human epidermal growth factor 2 (HER2) receptors convey a poor prognosis due in part to a lack of targeted therapies. The aim of the current study is to evaluate whether triptolide and its water soluble analog Minnelide is effective against triple negative breast cancer cells. We have previously shown that triptolide/Minnelide not only reduces tumor growth in various cancer models but it also regulates epithelial ­ mesenchymal transition (EMT), an important mechanism underlying metastasis. In our preliminary findings using three triple negative breast cancer (TNBC) cell lines, MDA­MB­231, MDA­MB­468, and MDA­ MB­157, we demonstrate that triptolide not only inhibits the proliferation of TNBC cells but also regulates the protein levels of EMT markers including β­Catenin and Vimentin. In order to elucidate the mechanism underlying triptolide mediated inhibition of cellular proliferation and regulation of EMT markers in TNBC cells, we identified Src kinase and Aurora kinase A as two new targets for triptolide action in TNBC cells. By targeting Src and Aurora kinase, triptolide disrupts the integrity of focal adhesion structures and reduces cell spreading via regulating FAK activity. Our preliminary findings regarding potential use of triptolide/Minnelide in TNBC based on in vitro experiments are promising. However, considering the complex pathophysiology of breast cancer and other biological factors playing role in a disease setting, in­

vivo experiments to test the efficacy of Minnelide in relevant mouse models for mammary cancers are currently underway. #5120 Cryptolepine a plant alkaloid, inhibits the growth of nonmelanoma skin cancer cells through inhibition of topoisomerase and induction of DNA damage. Harish C. Pal, Santosh K. Katiyar. University of Alabama at Birmingham, Birmingham, AL. Topoisomerases are highly specialized nuclear enzymes that remove superhelical tension on chromosomal DNA that allows replication and transcription of DNA. Many cancer chemotherapeutic drugs used in the clinics inhibit tumor growth by targeting topoisomerase functions resulting in DNA damage and cancer cell death. Cryptolepine, a major alkaloid isolated from Cryptolepis sanguinolenta plant’s roots, has shown anti­malarial, anti­bacterial, anti­fungal, and anti­inflammatory activities. In the present study, we examined the therapeutic effect of cryptolepine on non­melanoma skin cancer cells (NMSCC), SCC­13 and A431 as an in vitro model, and underlying mechanism of action with special emphasis on topoisomerases and DNA damage check points. Western blot analysis and enzyme activity evaluation assays demonstrated that SCC­13 and A431 cells express comparatively higher levels of topoisomerases and higher enzymatic activities compared with normal human epidermal keratinocytes (NHEK). Topoisomerase expression and activity was greatly reduced after 24 h treatment with 2.5, 5.0 and 7.5 µM cryptolepine. Inhibition of topoisomerases expression and function by cryptolepine resulted in significant DNA damage and enhanced expression of DNA­PK expressions. Cryptolepine induced DNA damage activated DNA damage response proteins as demonstrated by increased phosphorylation of ATM/ATR, BRCA1, Chk1/Chk2 and γH2AX. Consequences of cryptolepine induced DNA damage were also associated with activation of p53 signaling cascade. Cryptolepine induced post­ translational modifications such as phosphorylation and acetylation of p53, and inhibition of mdm2 protein expression resulted in activation and accumulation of p53 in SCC­13 and A431 cells. In turn, activated p53 enhanced protein expression of cyclin­dependent kinase inhibitors p16 and p21. Activation of p16 and p21 proteins downregulated expression of cyclin­dependent kinase 2 (CDK2), cyclin A, cyclin D1 and cyclin E and thus induced S­phase cell cycle arrest in NMSC cells with reduced

expression of cell division proteins Cdc25a and Cdc25b. Cryptolepine treatment also enhanced release of cytochrome c from mitochondria due to increased disruption of mitochondrial membrane potential. Inhibition of topoisomerase activity and activation of DNA damage response signaling culminated in significantly reduced cell viability and enhanced cell death of SCC­13 and A431 cells. However, NHEK cells were least sensitive to cryptolepine induced cell death. Results of our study strongly suggest for detailed preclinical investigations in animal models to further assess its anti­skin cancer potential and its possible use in human patients. #5121 PEG(E)­TC11, a novel polyethylene glycol­linked phthalimide derivative, inhibited high­risk MM cell growth in vivo and in vitro via cell cycle G2/M arrest in a CRBN­independent manner. Shuji Aida,1 Daiju Ichikawa,1 Kazuki Iida,1 Masashi Hozumi,1 Misa Nakamura,1 Ryo Uozaki,1 Nahoko Hashimoto,1 Mikio Okayama,2 Yuko Yonemura,3 Noriko Tabata,3 Taketo Yamada,4 Maiko Matsushita,1 Takeshi Sugai,1 Hiroshi Yanagawa,3 Yutaka Hattori1. 1Keio University Faculty of Pharmacy, Tokyo, Japan; 2Keio University School of Medicine, Tokyo, Japan; 3IDAC Theranostics Inc., Tokyo, Japan; 4Saitama Medical University, Saitama, Japan. Multiple myeloma (MM) is one of the hematological malignancy that is characterized by proliferation of malignant plasma cells. Recent advance in the treatment of MM using newly developed drugs, prognosis of the MM patients have been significantly improved. For example, immunomodulatory drugs (IMiDs) such as thalidomide, lenalidomide and pomalidomide have been developed for treatment of MM. However, IMiDs have only limited effects against MM patients with high risk chromosomal abnormalities such as t(4;14) and del17p (high­risk MM). In 2010, it was reported that IMiDs directly bind to cereblon (CRBN), a component of ubiquitin ligase 3 complex, and induced teratogenicity as well as anti­tumor effects. We have previously reported that a novel phthalimide derivative, 2­(2,6­diisopropylphenyl)­5­amino­1H­isoindole­ 1,3­dione (TC11) induced apoptosis against high­risk MM cells in vivo and in vitro, and inhibited differentiation of osteoclasts. We also reported that TC11 directly bound to α­tubulin and nucleophosmin­1 (NPM1), but did not bind to CRBN. However, TC11 was not well dissolved in water

with only 0.02 mg/mL solubility. Therefore, TC11 showed poor absorption into blood and limited anti­tumor activity when it was intraperitoneally administered in tumor­bearing mice. To resolve these problems, we synthesized PEG(E)­TC11, in which TC11 is linked to polyethylene glycol through an ester bond, and consequently enhanced water solubility of PEG(E)­TC11 to 88.9 mg/mL. PEG(E)­ TC11 revealed as potent growth inhibitory effect on high­risk MM cells as TC11 in vitro. In pharmacokinetic study, PEG­modification of TC11 improved the peak blood concentration (Cmax) from 2.6 to 24.4 μM and extended elimination half­life (t1/2) from 1.4 to 2.2 hr when 186 μM/kg of these compounds were intraperitoneally injected. More importantly, these pharmacokinetic improvement led to more potent growth inhibition of MM cells in vivo than TC11. We also explored mechanisms of anti­ myeloma effect of PEG(E)­TC11 and found that PEG(E)­TC11 induced apoptosis via G2/M cell cycle arrest. However, unlike IMiDs family, BIACORE assay revealed that PEG(E)­TC11 didn’t directly bind to CRBN, indicating that growth inhibitory effect of PEG(E)­TC11 against MM cells was independent of binding to CRBN. In conclusion, PEGylation of TC11 significantly increased water solubility, resulted in potentiated anti­myeloma activity in vivo. Furthermore, PEG(E)­TC11 inhibited cell growth via G2/M arrest in a CRBN­independent manner. Thus, PEG(E)­TC11 is considered as a candidate compound for overcoming high­risk MM. #5122 Utilizing zebrafish to study the effect strigolactone on breast cancer cells. Kira Lin,1 Christopher Grivas,1 Elema Boru,1 Yaron Dayani,2 Eric Berens,1 Anna T. Riegel,1 Eric Glasgow,2 Ronit I. Yarden1. 1Georgetown University, Washington, DC; 2Georgetown University, Bethesda, MD. Strigolactones (SLs) are a novel class of phytohormones that regulates shoot branching patterns and above­ground plant architecture by inhibiting the outgrowth of axillary lateral buds or meristems. The SL analogues (SLAs) inhibit the growth and survival of a wide array of cancer­derived cell lines including: prostate, colon, lung, melanoma, osteosarcoma and leukemic cell lines, while minimally affecting normal cultured cells. Interestingly, cancer cells with high metastatic potential are more sensitive to the inhibitory effect of SLAs than less aggressive cells.

Treatment of cancer cells with SLAs leads to the activation of stress­ related signaling including p38MAPKs and JNK1/2, and disrupt the microtubule network. Furthermore, a reduction in acetylated­alpha­ tubulin was observed in SLA­treated MDA­MB­231 cells. Microtubules dynamics has been proposed as one of the mechanisms that regulate cell migration by acting on lamellipodia formation. The ECIS invasion assay data suggests that even at low concentration of SLA and as soon as 6 hours, SLAs suppress MDA­MB­231 cells’ ability to invade the HUVEC monolayer. To further examine the effect of SLAs on the invasive and metastatic behaviors of cancer cells in vivo, fluorescently labeled MDA­ MB­231 cells were used as xenograft models in zebrafish. Embryos injected with MDA­MB­231 cells showed 66.6% metastasis in vehicle treated control fishes, as compared to 22.85% in SLAs treated fishes. This suggests that SLAs are potent inhibitors of cancer dissemination and metastasis. #5123 Targeted thieno[2,3­d]pyrimidines with fluorinated phenyl side chains as antitumor agents. Nian Tong,1 Aleem Gangjee,1 Adrianne Wallace Povrik,2 Carrie O’Connor,2 Aamod Aamod Dekhneb,2 Zhanjun Hou,3 Larry H. Matherly2. 1Duquesne Univ., Pittsburgh, PA; 2Wayne State University, Detroit, MI; 3Barbara Ann Karmanos Cancer Institute, Detroit, MI. The reduced folate carrier (RFC) is ubiquitously expressed in tissues and tumors and is the major tissue transporter for folate cofactors. Folate receptor (FR) α and β, as well as the proton­coupled folate transporter (PCFT), exhibit narrower patterns of tissue expression and are likely to serve more specialized physiologic roles. FRs are expressed in a subset of cancer cells (e.g., ovarian and non­small cell lung cancer for FRα, acute myelogenous leukemia for FRβ), whereas PCFT is expressed in a wide range of human solid tumors but not leukemias. Earlier generations of glycinamide ribonucleotide formyltransferase (GARFTase) and de novo purine nucleotide biosynthesis (e.g., Lometrexol) have shown promise as antitumor drugs. However, these compounds are excellent substrates for RFC and thus are non­selective for tumors, leading to dose­limiting toxicities. We previously reported a novel class of 6­substituted thieno[2,3­d] pyrimidines with a phenyl side chain and 3­ and 4­carbon bridge lengths (AGF17 and AGF23, respectively) which selectively

targeted FRs but not RFC or PCFT. To increase antitumor efficacy, we synthesized fluorinated analogs of AGF17 and AGF23, designated as AGF309 and AGF304, respectively. AGF309 and AGF304 potently inhibited proliferation of Chinese hamster ovary (CHO) cell lines engineered to individually express human FRα (IC50s of 1.08 and 2.27 nM, respectively) or FRβ (IC50s 0.48 and 1.14 nM, respectively). AGF304 and AGF309 showed nominal activity toward RFC­ and PCFT­ expressing CHO cells. Both AGF309 and AGF304 were also potently inhibitory toward FRα­expressing human KB human tumor cells (IC50s of 7.19 and 5.27nM, respectively). By analogy with previous iterations of 6­substituted thieno [2,3­d] pyrimidine compounds, growth inhibitory activity by AGF309 and AGF304 likely involves inhibition of de novo purine biosynthesis at GARFTase and potentially a secondary intracellular target. Collectively, our in vitro findings of the potent and selective antitumor activity and FR selectivity suggest that further preclinical evaluation of AGF309 and AGF304 as potential antitumor agents is warranted. #5124 GNS561 a new quinoline derivative inhibits the growth of hepatocellular carcinoma in a cirrhotic rat and human PDX orthotopic mouse models. Firas BASSISSI,1 Zuzana Macek Jilkova,2 Sonia Brun,1 Jerome Courcambeck,1 Jennifer Tracz,1 Keerthi Kurma,2 Gaël S. Roth,3 Cindy Khaldi,1 Corinne Chaimbault,1 Benoit Quentin,1 Emilie Asseraf,1 Antoine Beret,1 Eric Raymond,1 Philippe Halfon,1 Thomas Decaens3. 1Genoscience Pharma, Marseille, France; 2Université Grenoble Alpes, Institute for Advanced Biosciences, Research Center UGA / Inserm U 1209/CNRS 5309, Grenoble, France; 3Université Grenoble Alpes, Institute for Advanced Biosciences, Research Center UGA / Inserm U 1209/CNRS 5309, Clinique Universitaire d’Hépato­gastroentérologie, Pôle Digidune, CHU Grenoble, Grenoble, France. Background: Hepatocellular carcinoma (HCC) remains a major health problem, often diagnosed at late stages with limited number of therapeutic options. New drugs with original mechanisms of action are urgently aimed to improve current armamentarium in HCC patients. Quinoline derivatives are novel class of oral small molecules inducing

multiple cellular effects such as inhibition of autophagy, induction of apoptosis, and cell cycle modulation. The aim of these studies was to assess tolerance and efficacy of a new quinolone derivative GNS561. Material and methods: In vitro experiments were realized with viability, apoptosis and migration in tumor cells in HCC cell lines and primary tumor. Drug tolerance and plasma and liver pharmacokinetic were evaluated after single and repeated dosing in mice and rat. GNS561 and sorafenib efficacy in vivo were evaluated in a PDX orthotopic BALB/c­ nu mouse model and in a diethylnitrosamine (DEN)­induced HCC cirrhotic rat model. AFP, cell proliferation and tumor weight and size were assessed in mice. In rat tumor progression was followed by MRI, pathological analysis (tumor size and number), immunohistochemistry and PCR analysis after 6weeks of treatment. Results: GNS 561 shows potent anti­proliferative activity when assayed against a panel of human tumor cell lines and notably against a panel of HCC patient primry tumors even in those with sorafenib resistance (Mean EC50 3µM vs 11µM for sorafenib). GNS561 is highly selectively trapped in the liver. Plasma and liver PK in mice and rats after single and repeated doses confirm this selectivity with good tolerance and oral bioavailability. In PDX mouse model, tumor growth was significantly reduced by GNS561 with a dose­response manner, this tumor regression was associated with AFP level decreases by 72% with GNS561 (p=0.002) and 54% with sorafenib (p=0.046) compared to control. In rat model, mean number of tumors was significantly lower in GNS561 at 15mg/kg group (n=50.6), in sorafanib at 10mg/kg (n=65.1) and in combination group (n=40.6), when compared to control (n=100.4; p=0.0024, p=0.029 and p=0.0002). Tumor decrease measured by MRI was associated with a significantly reduced proliferation of tumor cells particularly in GNS561 group (70%) and combination (84%) compared to control, whereas the effect of sorafenib alone on proliferation was modest (30%). Conclusions: GNS561 is a liver selective drug with good tolerance and promising efficacy in different HCC animal models. GNS 561 was more efficient than sorafenib to control tumor growth in preclinical models. Based on its safe toxicity profile and potent activity in rodent models, GNS 561 is now aimed to further reach clinical development in patients with HCC in 2017. #5125 Muscadine grape extract inhibits breast cancer brain metastatic cells by multiple mechanisms.

Wenhong Chen, Patricia Gallagher, Ann Tallant, Linda J. Metheny­ Barlow. Wake Forest School of Medicine, Winston­Salem, NC. Tumor metastasis to the brain is a common complication of cancer, affecting 500,000 patients each year. Due to poor penetration of most chemotherapeutics into the brain, brain metastasis may occur even while systemic disease in under control. Despite aggressive treatments including radiation therapy, survival at 12 months following diagnosis is only 20%, underscoring the need for better means to prevent and/or treat brain metastases. Natural products have historically been a very successful source of new drugs. We are investigating the anti­cancer potential of a proprietary muscadine grape extract (MGE; from Piedmont Research & Development Corp.) on breast cancer cell lines that are metastatic to the brain. In a colony formation assay, MGE (10 μg/mL) decreased clonogenic survival of 4T1.luc2.Br5 cells by 50% (n=3, p 1 standard deviation over the median considered high c­MYC expression). Similarly two of six of AT/RT cell lines expressed high levels of c­MYC protein by western blot. DON treatment slowed cell growth of high c­MYC expressing AT/RT cell lines BT12 and CHLA06 (MTS assay p95 and >99% respectively) with an excellent in vitro stability, i.e. 90% after 10 days in human serum at 37ºC. An MTT cytotoxicity study demonstrated MC1R specific antiproliferative effect in cutaneous, ocular and uveal melanoma cell lines. A maximum tolerated dose (MTD) study was completed, where a range of 0­148 kBq (0­4 µCi) of 225Ac­MC1RL­DOTA was administered to groups of BALB/c mice. The radiopharmaceutical was well tolerated at even the highest doses and animals did not reach any clinical endpoints, such as weight loss, loss of kidney function or abnormal pathology. Biodistribution studies on MC1R expressing tumor bearing mice revealed tumor selectivity and a combination of renal and hepatic clearance with minimal retention in other normal tissues. A blood PK study in rats showed rapid clearance of the agent from the blood in 60%, 225Ac­DOTA­J591 mAb was purified by gel filtration and sterilized by membrane filtration. In a pilot pre­clinical study, we studied the toxicity in non­tumor bearing BALB/c mice. Results: The radiochemical purity is >95% and the immunoreactivity was >80%. Twenty­five BALB/c mice received from 2.1 to 6.3 KBq of 225Ac­J591. Treatment was well tolerated, with all mice alive and healthy at Day 26.

Conclusions: A stable alpha emitting­antibody complex has been produced with 225Ac­J591. Initial mouse safety experiments have been successful. A RIT dose escalation study of PSMA­targeted alpha emitter in a LNCaP xenograft model is underway. #5202 Additive benefits of radium­223 dichloride and bortezomib combination in a syngeneic 5TGM1 multiple myeloma mouse model. Mari I. Suominen,1 Jukka P. Rissanen,1 Anniina Luostarinen,1 Katja M. Fagerlund,1 Birgitta Sjöholm,2 Esa Alhoniemi,3 Sanna­Maria Käkönen,4 Dominik Mumberg,5 Jussi M. Halleen,1 Karl Ziegelbauer,5 Arne Scholz5. 1Pharmatest Services Ltd, Turku, Finland; 2Aurexel Life Sciences Ltd, Askainen, Finland; 3Avoltus Oy, Turku, Finland; 4University of Turku/Aurexel Life Sciences Ltd, Turku, Finland; 5Bayer, Berlin, Germany. Radium­223 dichloride (radium­223, Xofigo®), a targeted alpha­therapy, improves overall survival in prostate cancer patients with bone metastases. It inhibits disease progression by reducing tumor growth and tumor­induced pathological bone reaction in breast and prostate cancer mouse models. Radium­223 is actively incorporated into the bone matrix by osteoblasts. Multiple myeloma (MM) is characterized by increased osteoclast and reduced or no osteoblast activity. Bortezomib (Velcade®), a treatment for MM, restores the impaired osteoblast activity in MM. Here, we report the effects of radium­223, bortezomib and their combination on myeloma cell proliferation in vitro and on myeloma bone disease model in mice. Proliferation assays were performed with human plasma cell leukemia (JJN­3, L­363), human MM (LP­1, MOLP­8, RPMI­8226 and OPM­2), and mouse MM (5TGM1) cells. Corresponding in vivo effects were studied in a syngeneic 5TGM1 mouse MM model. Female C57BL/KaLwRij mice (7 weeks old, n=15/group) were inoculated with 5TGM1 cells via tail vein and 26 days later, radium­223 (300 kBq/kg, single iv injection) and/or bortezomib (1 mg/kg ip, twice a week; total of 3 doses) or vehicle control were administered. The development of osteolytic lesions was detected by radiography. Hind limbs were used for histological analyses and total activity measurement was performed by a gamma­counter. TRAP­stained osteoclasts were counted at tumor­bone

interface. Bortezomib inhibited proliferation of all cancer cell lines tested at 25 nM (JJN3 and OPM­2 at 2.5 nM) and radium­223 at 0.8 kBq/ml (L­363 and MOLP­8 at 0.2 kBq/ml) concentrations. Additive effects were observed with combination treatment in vitro. The 5TGM1 in vivo model demonstrated that both bortezomib and radium­223 decreased osteolytic lesion area as monotherapy (p95% of the samples achieved the minimum concentrations required for the TruSight® Tumor 170 assay. As a surrogate for DNA quality, we measured the amplification potential of the nucleic acid by assessing a ΔCq value using quantitative PCR after

normalization to a fixed input mass. To assess RNA quality, we used the DV200 metric, which measures the percentage of RNA fragments >200 nucleotides in length. We examined ΔCq and DV200 values across different tissues and didn’t find a significant difference between tissues. Finally, we assessed the ability of samples to pass the sample quality control (QC) metrics in the TruSight® Tumor 170 assay. These QC metrics ensure accurate variant calling, with a sensitivity and specificity of ≥95%. We found that samples that had a ΔCq value of ≤5 and a DV200 value of ≥20 achieved a QC success rate above 95%. This data highlights the need for further investigation into the methods for extraction, quantification and quality assessment of nucleic acids for solid tumor profiling and underscores the robustness of TruSight® Tumor 170 with FFPE samples.1 For Research Use Only. Not for use in diagnostic procedures. #5355 PANCeq ­ Development and validation of a targeted sequencing assay for detection of mutations and copy number changes in most recurrently mutated driver genes. Tobias Meissner, Anu Amallraja, Brandon Michael Young, Adam Mark, Cayce Conolly, Amanda Andrews, Casey Williams, Brian Leyland­Jones. Avera Cancer Institute, La Jolla, CA. Next generation sequencing is becoming increasingly prevalent as a tool for the identification of genetic variation to aid diagnosis and support therapy decisions in routine clinical care. It has been shown that tumors are comprised of subpopulations of cells with distinct genomic alterations, and hence tumor heterogeneity is becoming an important factor in deciding treatment. Deep sequencing of tumor samples allows for the determination of number and proportions of low variant allele frequency tumor subclones. Currently, high costs for whole­exome deep sequencing (> 250X) limit its application in routine clinical care, and there is a need for more affordable targeted approaches. Here we present PAN Cancer Sequencing (PANCeq), a targeted deep sequencing panel for the detection of mutations and copy number variants (CNVs) in most recurrently mutated cancer driver genes. A platform­independent computational Docker­based pipeline for data analysis and automatic report generation was developed. PANCeq uses customized targeted enrichment, followed by next­generation sequencing of (FFPE) tumor and

matched germline samples. We included 12 patient samples (breast, ovarian, kidney and prostate) as well as triplicates of 3 reference standards (HD200, HD753, HD733 ­ Horizon). These were sequenced on Illumina NextSeq o an average depth of 1000X. Sequencing data was aligned using BWA to hg19 human reference. Somatic variants and CNVs were identified using VarDict and CNVkit respectively. We mined TCGA data across 26 tumor types, and selected known mutations from the most recurrently mutated genes, yielding 2849 mutations across 467 genes. In addition, we included 79 pharmacogenomic relevant and 218 sample tracking SNPs as well as 80 cancer relevant CNVs (whole exon), and designed a custom panel based on their genomic locations, resulting in a panel size of 708kb. To assess performance, we sequenced reference standards with known variants spiked­in at varying VAF (1 ­ 41.5%). Somatic variant calling was performed against a wildtype sample. For HD200, out of 21 covered mutations, 14 were correctly classified as germline, and 7 as somatic. For HD733, out of 21 mutations, 3 were correctly identified as germline, 18 as somatic. For HD753, all 6 germline and somatic mutations were correctly identified. The 2 CNVs spiked­in were also correctly identified. In all cases we found a good agreement between observed and expected VAF. Somatic variant results for 12 patient samples were compared with variants reported by a private vendor. While we found good agreement at VAF > 20%, our panel detected on average 7 additional clinically relevant mutations at < 20%. We demonstrate that PANCeq is an accurate and sensitive method for timely identification of clinically actionable mutations and copy number variations across multiple cancer types. #5356 Validation of a clinically actionable cancer core gene test for solid tumors facilitating targeted molecular therapy and immunotherapy. Lin Ma, Michael Hua, Steven Rivera, Anna Gerasimova, Quoclinh Nguyen, Sirisha Sunkara, Robert Lagier, Alla Smolgovsky, David Wolfson, Jared F. Taylor, Frederick Racke, Charles Strom, Andrew Grupe, Joseph Catanese, Feras Hantash. Quest Diagnostics, San Juan Capistrano, CA. Molecular profiling of tumor mutations has expedited molecular targeted therapy for cancer patients. Additionally, tumor mutation load and DNA

microsatellite instability (MSI) status can help predict patient’s response to immunotherapy. We analytically validated a gene test targeting clinically actionable cancer mutations of all 4 major types: single nucleotide variations (SNVs); insertion/deletions (INDELs); whole gene copy number variations (CNVs); and structural rearrangements (translocations) as well as the MSI status. This test interrogates all coding exons from 49 core cancer genes, introns for a subset of genes selected for detection of prevalent gene rearrangements, and the TERT (telomerase reverse transcriptase) promoter region. MSI status is determined from a set of 5 intronic mono­nucleotide repeats collectively associated with microsatellite instability. Targeted DNA regions are captured by in­solution hybridization with complementary biotinylated RNA baits and sequenced on the Illumina NextSeq®500 platform. Paired formalin­fixed, paraffin­embedded (FFPE) tumor samples and whole blood samples are analyzed simultaneously for the detection of SNV, INDEL, CNV, translocation and MSI status. FFPE samples can also be analyzed alone if the whole blood sample is not available, eliminating the reporting of MSI status. A minimum of 50 ng FFPE DNA and 100 ng of whole blood DNA are required for this test. A total of 123 FFPE, 2 FFPE FNA samples, 19 paired FFPE and whole blood paired samples covering lung cancer, colorectal cancer, melanoma and breast cancer were included in this validation study. Analytical validation of assay performance demonstrated that, on average, 700­fold read depth was achieved across all targeted regions with >95% of these regions covered by a minimum of 300 unique reads. Analytical sensitivity was ≥5% mutation frequency for SNV and INDEL and ≥20% for translocation and CNV. Thirty­eight unique variants were confirmed between this test and orthogonal methodologies: 22 SNVs, 6 INDELs, 5 translocations, and 5 CNV. Fifty­ eight out of 59 (98% concordance) paired FFPE/blood samples achieved the same MSI status result with this targeted sequencing approach compared to the reference National Comprehensive Cancer Network (NCCN) Bethesda PCR test. MSI­positive samples, on average, were determined to have 5­fold higher mutation count compared to MSI­ negative samples, consistent with the previously reported higher mutation burden. In conclusion, we have developed and analytically validated a core cancer gene test employing NGS technology with demonstrated high analytical sensitivity and specificity. Coupled with clinical interpretation, this test will facilitate clinical decision­making for molecular targeted therapy and immunotherapy.

#5357 OncoGxSelectTM ­ a beneficial supplement to comprehensive cancer genetic test. Yang Han, Pengfei Yu, Qingxuan Song, Min Wei, Guanghui Hu. Admera Health LLC, South Plainfield, NJ. Genetic profiling of tumor has been widely accepted both in oncology research and clinical field. However, clinical doctors are sometimes overwhelmed by tremendous and complicated information provided by comprehensive genetic tests, although they are welcomed by research­ oriented oncologists. In order to provide accurate key findings to aid diagnosis and treatment in clinical practice, a careful tailored NGS based test ­ OncoGxSelectTM was developed and validated. Targeting 7 popular cancer types (NSCLC, Colorectal cancer, Breast cancer, Melanoma, Thyroid cancer, Esophageal and GIST cancer), OncoGxSelectTM detects all 4 types of genomic alterations including single nucleotide mutation, insertion/deletion, copy number variation and gene rearrangement in tumor tissue for 12 well­characterized clinical actionable genes strictly following NCCN (National Comprehensive Cancer Network) guidelines. OncoGxSelectTM offers high sensitivity and specificity greater than 99%, low cost and fast turnaround time using both DNA and RNA as starting material from FFPE samples with as low as 10% tumor cell content. This CLIA certified and CAP accredited genetic test panel is a valuable supplement to clinical oncologists to further facilitate diagnosis and treatment choosing. #5358 Comprehensive evaluation of Illumina’s TruSight® Tumor 170 Panel to estimate tumor mutational burden. Shile Zhang, Alex S. So, Shannon Kaplan, Kristina M. Kruglyak. illumina, San Diego, CA. Tumor mutational burden (TMB), or the density of variants in a tumor, is typically assessed by whole exome sequencing (WES) and has been shown to correlate with efficacy of immunotherapy treatment. Targeted cancer gene panels are broadly used to assess mutational status in cancer related genes but have not historically been used to estimate TMB. Recently, two studies have demonstrated that TMB can be accurately estimated using these cancer gene panelsi,ii. Illumina’s TruSight® Tumor

170 (TST170, research use only, RUO) panel is a comprehensive next­ generation sequencing (NGS) assay that covers the coding regions of 170 genes associated with solid tumors. TST170 targets DNA and RNA variants from the same FFPE tumor sample in a single sequencing run. Here we evaluate the performance of TMB estimation with TST170. TST170 is an enrichment­based targeted panel designed to capture mutational changes, including single nucleotide variant, indels, amplifications, splice variants and fusions. The TST170 analysis pipeline is able to call variants with frequencies down to 5%. In the current study, TMB was calculated as the number of reported variants per megabase after germline polymorphism filtering. First, we evaluated the performance of TST170 for TMB estimation using 6,000 TCGA samples that had been analyzed by WES. TMB estimated from the TST170 targeted regions showed a high correlation to TMB estimated from WES (R2=0.91). Next, we evaluated the prognostic value of TMB estimated from TST170 by overlapping the TST170 targeted regions with WES data for 202 subjects treated with CTLA­4 or PD­1 from three clinical studies. Higher TMB estimated from the TST170 targeted regions was observed in subjects who responded to checkpoint inhibitors. Finally, we assessed the correlation of TMB estimation using matched samples profiled with both WES and TST170 and again saw high correlation between the two methods. In summary, our analysis indicates that the panel content of TST170 can be used to accurately estimate TMB from tumor samples. iGarofalo A, Sholl L, Reardon B, et al. The impact of tumor profiling approaches and genomic data strategies for cancer precision medicine. Genome Med. 2016;8(1):79. doi:10.1186/s13073­016­0333­9. iiCampesato, L., Barroso­Sousa, R., Jimenez, L., et al. Comprehensive cancer­gene panels can be used to estimate mutational load and predict clinical benefit to PD­1 blockade in clinical practice. Oncotarget, 2015;6(33), 34221­34227. #5359 An ultrasensitive and highly reproducible hybridization capture­ based Next­Generation Sequencing clinical assay to enable precision oncology in patients with solid tumors. Moises Hernandez, Sara King, Sharanya Raghunath, Christopher Johnson, David Loughmiller, Lincoln Nadauld, Pravin J. Mishra. Intermountain Precision Genomics, St. George, UT.

Sequencing whole cancer exome and whole genome provides useful information to study cancer evolution. However, cost and amount of data may become overwhelming. Enrichment capture­based methods to design custom targeted gene panel have rapidly evolved recently in cancer genomics arena. Custom targeted gene sequencing entails several advantages (i.e., better quality, ethical, affordable, technically suitable, personalized and reimbursable) for cancer patients over whole exome and whole genome sequencing. With this in mind, and to enable precision oncology in patients with solid tumors, we developed ICG100 2.0 panel, a hybridization capture­based next­generation sequencing assay for targeted sequencing of all exons and flanking introns of 162 commonly mutated cancer genes in formalin­fixed, paraffin­embedded (FFPE) tumors. To establish ICG100 2.0 panel, we compared three commercially available capture based technologies and evaluated reproducibility, sensitivity, specificity and the detection limit for low­frequency variants using internally developed bioinformatics pipeline. Cell lines, reference standards/synthetic DNA and solid tumor samples with the known genetic information was utilized in this analysis. Results were derived from MiSeq and NextSeq platforms and cross­compared on other platforms, including MassArray and ddPCR for establishing concordance and uniformity. Intra and interrun replicates were utilized to assess the quality, precision and reproducibility of variant calling. Mean depth of coverage was observed at >300X with >99% sensitivity and specificity. These findings and observations will guide other clinical laboratories to establish new assay that require less DNA input, enzyme based fragmentation and reduced preparation time. While we show that the three capture based methods (after comparison) had an overall accuracy in SNP and CNV detection similar to each other with minor differences, we describe an approach to assess and establish the best assay from the clinical standpoint to guide treatment decisions and match cancer patients to the most appropriate clinical trials. #5360 DNA repair increases sequencing accuracy without altering actual mutation frequency in clinical samples. Pingfang Liu, Lixin Chen, Laurence Ettwiller, Eileen Dimalanta, Theodore B. Davis, Thomas C. Evans. New England Biolabs, Ipswich, MA.

Targeted cancer therapy based on genomic alterations can be remarkably effective. Currently, cancer genome profiling using next generation sequencing (NGS) is routinely applied in cancer care to guide personalized treatment. The accuracy of this profiling directly impacts therapeutic choices and the outcomes of patient care. We previously showed that false positive variants are abundant and can account for a major fraction of identified somatic variations in publicly available datasets (doi: http://dx.doi.org/10.1101/070334). These false positive variants show signs of mutagenic DNA damage. We further demonstrated that enzymatic DNA repair increases sequencing quality by lowering damage­induced background noise. Therefore, enzymatic DNA repair has the potential to improve sequencing accuracy, avoiding incorrect somatic variant calls and consequently reducing incorrect diagnostic conclusions. In this study, we investigated whether enzymatic DNA repair introduces any bias to NGS libraries using analysis by droplet digital PCR (ddPCR) and deep sequencing. DNA Reference Standards containing multiple common cancer mutations (Horizon Discovery, Inc.) were spiked into formalin­fixed paraffin­embedded (FFPE) DNA isolated from tumor samples from different tissue types at defined frequencies (0.5­10% quantified by ddPCR). Genotyping of the FFPE DNA ensured that they were free of any of the spiked­in mutations. After DNA repair and library preparation, mutation frequencies were quantified by ddPCR, and compared to the mutation levels in input DNA and control libraries without repair. Deep sequencing of 151 cancer genes including these spike­ins showed no difference in mutation frequency for the spiked­in mutations between the control and repair groups. However, the number of false positive variant calls was reduced in the repair group. Our data demonstrates that DNA repair significantly increases sequencing accuracy without altering the frequency of actual mutations in tumor samples. #5361 Predesigned gene content for rapid deployment of custom oncology panels. Andrew J. Barry,1 Amy Emerman,2 Sarah Bowman,2 Kruti Patel,2 Eileen Dimalanta,1 Scott Adams,1 Noa Henig,1 Fiona Stewart,1 Cynthia Hendrickson,2 Theodore Davis,1 Charles Elfe1. 1New England Biolabs, Ipswich, MA; 2Directed Genomics, Ipswich, MA.

Efficient utilization of targeted gene panels for oncology research is challenged by the wide variation in gene constituents specific to a given study. While focused gene panels efficiently provide the necessary depth of coverage for low frequency variant detection, the high costs and design challenges associated with de novo panel design present challenges. The NEBNext DirectTM technology utilizes a novel approach to selectively enrich nucleic acid targets ranging from a single gene to several hundred genes, without sacrificing specificity. The approach rapidly hybridizes both strands of genomic DNA with biotinyated probes prior to strepdavidin bead capture, enzymatic removal of off­target sequence, and conversion of captured molecules into sequencer­ready libraries. This results in a unique read coverage profile that results in uniform coverage across a given target. Unlike alternative hybridization methods, the approach does not necessitate upfront library preparation, and instead converts the captured molecules into dual­indexed illumina sequencer compatible libraries containing an 8 basepair sample ID and a 12bp Unique Molecule Index (UMI). The UMI individually tags each molecule prior to the final PCR amplification of the library, enabling identification of PCR duplicate molecules. The result is a 1­day protocol that enables the preparation of sequence­ready libraries from purified genomic DNA specific to the gene content included in the panel. We have designed and developed baits specific to the full exonic content of 450 genes associated with cancer. These are designed, balanced, and pooled on a per gene basis, and can be combined into customized panels, allowing rapid turnaround of specific custom gene subsets. Here, we present the ability to rapidly deploy custom gene panels across a variety of panel sizes and content, while maintaining high specificity, uniformity of coverage across target content, and sensitivity to detect nucleic acid variants from tumor samples. #5362 Targeting BRCA1 and BRCA2 with NEBNext Direct™. Scott M. Adams,1 Kruti M. Patel,1 Amy B. Emerman,1 Sarah K. Bowman,1 Charles D. Elfe,1 Noa Henig,1 Salvatore Russello,2 Andrew Barry,2 Theodore Davis,2 Cynthia L. Hendrickson1. 1Directed Genomics, Ipswich, MA; 2New England Biolabs, Ipswich, MA. The screening and detection of germline BRCA1 and BRCA2 mutations are critical for the effective management of patients with breast or ovarian

cancer and for the identification of individuals with a high risk of developing these cancers. In addition, somatic detection of pathogenic variants in BRCA1/2 can influence treatment decisions due to the susceptibility of tumors with BRCA mutations to PARP inhibitors. Here we introduce the NEBNext DirectTM BRCA enrichment panel for the interrogation of BRCA1 and BRCA2 by Illumina sequencing. NEBNext DirectTM is a novel, hybridization­based method to selectively enrich nucleic acid targets ranging from a single gene to several hundred genes. This approach includes features such as the incorporation of unique molecule identifiers (UMIs) and the ability to capture degraded DNA, enabling accurate detection of low­frequency variants from formalin­compromised DNA and other challenging sample types. We applied the NEBNext Direct BRCA1/2 panel to frozen tissue and achieved a high specificity for the BRCA targets (96% of the sequenced reads mapped to BRCA1 and BRCA2 and 80% of the sequenced bases were within the targeted regions). In addition, the resulting libraries were highly uniform in coverage, with 91% of the targeted bases covered at a value of at least 50% of the mean depth of coverage and 100% of the bases covered at 20% of the mean or greater. Some variability in the specificity was observed with formalin­fixed, paraffin embedded (FFPE) samples, and this effect was dependent on the quality of the FFPE DNA. The UMIs were used to identify PCR duplicates prior to variant calling, as well as for error correction, enabling the accurate detection of low­ frequency variants. The library preparation for all samples was completed in one day, and the entire process of library preparation, sequencing on the Illumina Miseq, and data analysis was completed in a total of two days. In summary, we demonstrate that application of the NEBNext DirectTM method to the enrichment of BRCA1 and BRCA2 provides a tractable approach for the rapid and highly sensitive analysis of these cancer­ associated genes. #5363 Measuring gene expression at the tumor microenvironment: A comparison between nCounter PanCancer Immune Profiling Panel and Oncomine Immune Response Research Assay. Ann Mongan, Warren Tom, Janice Au­Young, Aleksandr Pankov, Gauri Ganpule, Fiona Hyland. Thermo Fisher Scientific, San Francisco, CA.

The tumor microenvironment, especially infiltrating T lymphocytes and inflammatory molecules, is believed to be highly relevant to the tumor’s sensitivity to cancer checkpoint blockade therapy. At the same time, the exact markers that are predictive of response for each therapeutic agent are still the subject of active investigations. To address the need for better understanding of the effect of different T cell subsets, antigen presentation, and tumor killing, gene expression profiling presents an attractive means to simultaneously evaluate the tumor microenvironment and cancer cells. In this study we compare the results and performance of the nCounter PanCancer Immune Profiling Panel and the Oncomine Immune Response Research Assay, both of which are designed to measure the expression of genes indicative of an immune response and potential immune­editing activities by tumor cells. The nCounter panel detects gene expression by counting unique probes that hybridize target mRNA, while the Oncomine panel employs NGS to sequence and count reads derived from the targets. While both panels are designed to work with FFPE samples, The nCounter panel expects 100 ng of unamplified RNA, while the Oncomine panel requires only 10 ng total RNA with its AmpliSeq technology. The two panels share 254 common genes, which constitute the basis for this comparison. Across 12 cancer samples (breast, H&N, melanoma, NSCLC, and RCC), results show that the Oncomine panel offers 20% higher dynamic range, thereby providing more robust readouts about the differences among samples. More importantly, with virtually no background noise, the underlying NGS technology provides an absolute zero readout for non­expressing genes which significantly improves the sensitivity for detecting low expressing genes, as their presence can be confirmed by as few as two reads. The two technologies show moderate correlation (R ~ 0.7), with the Oncomine panel more strongly correlating with qPCR (R ~ 0.9). Finally, when clustered using all genes on the panel, only the Oncomine panel provides clear stratification of cancer types, thus allowing the panel to be used for tissue type confirmation in addition to evaluating the immune response. #5364 A targeted NGS solution to evaluate gene expression signature of the tumor microenvironment from 40 NSCLC FFPE and matched fresh frozen samples. Yuan­Chieh Ku, Warren Tom, Yongming Sun, Alex Pankov, Tim Looney, Fiona Hyland, Janice Au­Young, Ann Mongan. Thermo Fisher

Scientific, South San Francisco, CA. Cancer cells and their surrounding non­malignant cells, including immune cells, signaling molecules, stromal and extracellular matrix, create the tumor microenvironment (TME). The composition of this TME plays important roles in tumor progression, evading growth suppressors and activating metastasis. However, the regulatory mechanism and function of each constituent remains poorly understood. With several checkpoint blockade therapy studies, the presence of PD­L1 has been reported to be a promising marker to predict positive response. Current IHC methods to measure PD­L1 are subjective and highly variable. A higher­throughput and standardized solution that can systematically measure gene expression of cells present in the TME has emerged to be a more desirable alternative. Here, we applied the OncomineTM Immune Response Research Assay to measure the expression of 395 genes in non­ small cell lung cancer (NSCLC) samples from 40 matched FFPE and fresh frozen sample types. This assay leverages NGS technology to sequence and count reads derived from the original transcript. With an input requirement of 10 ng of total RNA, libraries were generated, templated on the Ion ChefTM and sequenced on the Ion S5TM System. Results showed that, despite small input amount, the expression profiles of FFPE and fresh frozen samples are highly correlated with an average correlation greater than 0.9. We selected 22 genes out of the panel to validate expression with qPCR using FFPE samples. These genes were selected to cover a range of low, medium, and high expressors per our NGS data. Again, we observed a strong correlation (R ~ 0.9) between NGS and qPCR data. Approximately 80% of the 40 samples show moderate to high expression of CD8+ T cell cytokines, IFNG and TNFa. We further found that the expression of CD8A and CD8B are highly correlated with CD4, suggesting the co­presence of both cytotoxic and helper T cells. High correlation among CD4, FOXP3, TGFB1, and IL2RA (CD25) also suggests that their expression can be used as markers for the presence of Treg cells. We conducted a differential expression analysis between a group of samples (n=8) with high percentage of surrounding and infiltrating lymphocytes and another group (n=5) with low stromal content but devoid of infiltrating lymphocytes. Interestingly, we found a large number of genes which annotated as markers for infiltrating lymphocytes (CTSS, CXCR4, CD37, SRGN, FCER1G, SAMHD1, and GZMA) are significantly up­regulated in samples with high percentage of surrounding and infiltrating lymphocytes. In summary,

this study highlights the robustness of using a targeted panel to understand the composition and regulatory mechanism of the TME and tumor immune response. #5365 Combining enzymatic DNA fragmentation with NGS library construction results in high quality, high yield libraries. Fiona Stewart, Lynne Apone, Vaishnavi Panchapakesa, Karen Duggan, Timur Shtatland, Bradley Langhorst, John Murdoch, Christine Sumner, Christine Rozzi, Pingfang Liu, Keerthana Krishnan, Deyra Rodriguez, Joanna Bybee, Danielle Rivizzigno, Laurie Mazzola, Eileen Dimalanta, Theodore Davis. New England Biolabs, Inc., Ipswich, MA. The use of Next Generation Sequencing (NGS) data has been instrumental in advancing our understanding of human genetics, identifying the molecular events that contribute to human disease, and supporting drug development targeted towards precision medicine. Continued advancement relies on overcoming the limitations and bottlenecks associated with NGS. In this work, we have focused on NGS library preparation, where the requirement for expensive equipment and numerous steps can lead to sample loss, errors, and limited throughput. Specifically, we have developed a library construction method that integrates enzymatic DNA fragmentation into the workflow and combines fragmentation with end repair and dA­tailing in a single step. Integrating these reactions eliminates the need for costly equipment to shear DNA and reduces the number of sample transfers and losses. Adaptor ligation is also carried out in the same tube, after which a single cleanup step is performed. For low input samples, PCR amplification is performed prior to sequencing. This method is compatible with a broad range of DNA inputs and insert sizes. Libraries generated using this streamlined method with inputs ranging from 500 pg to 500 ng of intact DNA show no significant difference in coverage uniformity or sequence quality metrics, compared to libraries generated with mechanically sheared DNA. Similarly, libraries generated to contain insert sizes that range from 150bp to 1kb display no significant difference in sequence quality from each other or from those generated with mechanically sheared DNA. Finally, this streamlined method generates libraries of substantially higher yields than those generated using mechanically fragmented DNA, allowing the use of lower DNA inputs and fewer PCR cycles. The ability to generate high quality NGS libraries from intact

DNA without the need for costly equipment and numerous cleanup or liquid transfer steps substantially reduces the time, cost and errors associated with library construction. In addition, these advances will enable greater use and adoption of NGS technologies in clinical and diagnostic settings. #5366 Detection of low­frequency somatic variants using single­molecule, real­time sequencing. Primo Baybayan, Laura Nolden. Pacific Biosciences, Menlo Park, CA. Detection of somatic mutations, especially in heterogeneous tumor samples where variants may be present at a low level, is challenging. Single Molecule, Real­Time (SMRT®) Sequencing is ideal for minor variant detection because of its ability to sequence single molecules with very high accuracy (>QV40) using the circular consensus sequencing (CCS) approach. Here, we characterize the Sequel System for the detection of low­ frequency somatic variants using constructs containing mutations in coding regions in EGFR, NPM1, AKT1 and JAK2 representing deletion, insertion, substitution and homopolymer variants. Wild type and mutant amplicons, provided by SeraCare, were mixed and serially diluted from 10% down to 0.1% allelic frequency. Independent SMRTbell libraries were constructed for each dilution point, sequenced and analyzed using SMRT Sequencing to identify the variants and determine the observed frequency. The random error profile and high­accuracy CCS reads make it possible to accurately detect low­frequency somatic variants. We will demonstrate sensitivity of the PacBio Systems to detect mutations down to 0.1%. #5367 Ngago: no evidence of targeted endonuclease activity. Sabine Topka, Sara Kazzaz, Kenneth Offit, Vijai Joseph. Memorial Sloan Kettering Cancer Center, New York, NY. The development of a versatile and easy to use genome editing tool based on the bacterial CRISPR/Cas9 system has led to a vast increase in applications of genome editing in laboratories around the world. As an extremely powerful and versatile tool it has facilitated important discoveries in both basic and translational research as well as in a

potentially wide range of therapeutic applications. Natronobacterium gregoryi Argonaute (NgAgo) protein was described as a favorable alternative to Cas9 due to its simplicity and higher endonuclease specificity. Thus far, there are no formal reports documenting the success of NgAgo in other laboratory settings and cellular systems. Utilizing the identical cellular background and protocol that was made publicly available by the authors of the initial study, we were unable to reproduce the endonuclease function of NgAgo. These results do not support the role of NgAgo in gene editing experiments. #5368 Comparative analysis of multiple copy number alteration tools in the detection of amplifications and deletions on both whole­exome and targeted NGS panel platforms. Selene M. Virk, Sean Michael Boyle, Ravi Alla, Jennifer Yen, Richard Chen. Personalis Inc., Menlo Park, CA. Somatic copy number alterations, or CNAs, are frequent occurrences in the tumor landscape and can present in the form of focal alterations or as chromosome level events. Reliable detection of CNAs is crucial to understanding the impact of these events on a wide range of factors including tumor progression and treatment outcomes. The clinical use of NGS panels, and more recently exomes, continues to expand in cancer but the reliable detection of CNAs remains a challenge. A major factor contributing to the challenge is the availability of reliable tools that have been well tested and validated. We sought to characterize the sensitivity and specificity of multiple CNA detection tools with a focus on clinically relevant genes in NGS panel and whole­exome. Our approach utilized a variety of publicly available CNA tools to identify whole gene amplifications and deletions in a set clinically relevant genes identified in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. The tested CNA tools included two that supported both whole exome and panel NGS data, allowing cross platform in addition to cross tool comparisons. This work involved more than 20 well characterized cell lines derived from a broad spectrum of tumor types, including breast, lung, melanoma, and prostate cancers. The cell lines were analyzed in both tumor/normal and tumor only (which utilizes a proxy normal) modes. Analyses of patient­matched and proxy­ normal data were performed with both assays. Using our approach, we were able to identify clinically relevant CNAs on

both platforms. The performance of the tools tested varied, with some tools performing best on either amplification or deletion events. These results represent a comprehensive comparison of recent copy number alteration tools and provides data that can be utilized to make the tools even more robust and reliable.

Epigenetics 5 #5369 Epigenetic, transciptomic and ubiquitomic changes associated with BAP1 loss in uveal melanoma. Stefan Kurtenbach, Jeffim N. Kuznetsov, Matthew G. Field, Rohit Reddy, Margaret I. Sanchez, Christina L. Decatur, J William Harbour. University of Miami Miller School of Medicine, Miami, FL. Uveal melanoma (UM) is the most common primary and aggressive ocular cancer. Up to 50% of the patients develop metastasis, which are notoriously resistant to all forms of therapy and despite medical treatment leads to death within a mean time of 5­7 months, with a mortality rate of over 90%. We previously described that gene expression profiling can be used to classy UM tumors into two basic categories, class 1 (low metastatic risk) and class 2 (high metastatic risk). We further described that inactivating mutations in the ubiquitin hydrolase BAP1 are found in over 85% of class 2 tumors. BAP1 is involved in removing ubiquitin from specific proteins to regulate their function, like histone H2A, thereby regulating gene expression. Building on these findings, we have generated uveal melanoma and melanocyte cell lines that allow for the inducible knockdown of BAP1. First, we performed RNAseq on multiple cell lines before and after BAP1 knockdown and compare the results to gene expression of class 1 and class 2 tumors. We identified significant overlap in key genes linked to metastasis between the primary tumors, uveal melanoma and melanocyte cell lines. Using ChIP­seq to interrogate uveal melanoma cells depleted of BAP1, we identified changes in genome wide histone marks and RNA polymerase localization associated with BAP1. To gain further insight into non­histone deubiquitination targets of BAP1, we performed ubiquitination proteomic profiling using the UbiScan® technology and identified a list of high probability BAP1 substrates. Taken together, these complementary genome­wide investigations provide a global picture of the cellular functions of BAP1 and they provide novel insights into the metastasis­promoting effect of BAP1 loss in UM. #5370 Examination of DNA looping near oncogenes reveals variable patterns of epigenetic landscapes in cancer.

Monika Perez, Ty Johannes. University of Tulsa, Tulsa, OK. INTRODUCTION: The compaction of chromatin in the nucleus into hierarchical three­ dimensional (3D) structures plays a key role in the regulation of gene expression. Advancements in high­throughput chromatin conformation capture assays have enabled the determination of this hierarchical structure in a variety of cell states, including cancer. Of note, recent studies have implicated pathogenic alterations in 3D topology of the genome with the activation of proto­oncogenes in a multitude of cancers. METHODS: To characterize the topological changes associated with different cancer phenotypes, we examine publically available epigenetic profiles from the ENCODE Project, including 3D data from Chromatin Interaction Analysis by Paired­End Tag Sequencing (ChIA­PET) from breast cancer and leukemia cell lines. Integrating the chromatin topology data with transcription factor binding, DNase accessibility, and histone modification data, we uncover variable epigenetic landscapes associated with differentially active oncogenes in these cancer cell lines. RESULTS: We provide evidence of topological variability influencing differentially expressed oncogenes and tie these observations to other epigenetic changes. In particular, we show insulated­neighborhoods mediated by CTCF looping partition segments of the genome containing active oncogenes away from repressive markings like H3K4me3. Additionally, we show that variation in topology differentially localizes H3K27ac in distal regulatory regions to promoters of oncogenes, providing a means for gene activation in non­coding regions. CONCLUSIONS: Variable patterns of chromatin topology provide a unique signature and mechanism of oncogenes for cancerous phenotypes. We use publically available data to show mechanisms of activity associated with chromatin topology variation. #5371 Effect of DNA methyltransferase inhibitor 5­azacitidine on 3D chromatin structure measured by Hi­C. Yuchen Vincent Bai,1 John Whitaker,1 Emanuele Palescandolo,2 Vinod Krishna,1 Vipul Bhargava,1 Satya Saxena,1 Xiang Yao,1 David

Pocalyko,1 Kurt Bachman1. 1J&J, Spring House, PA; 2J&J, Beerse, Belgium. Therapies targeting epigenetic modifiers such as the DNA methyltransferase (DNMT) inhibitor, 5­azacytidine (Aza), are used in treating hematologic malignancies and also show promising results in subsets of solid tumors. The molecular mechanisms for these results, however, are not fully understood. Nonetheless, a number of clinical studies using combinations of epigenetic therapies plus chemo or immunotherapy to enhance the tumor response are ongoing. HCT­116, a colon cancer cell line genetically defined by microsatellite instability (MSI), resulting in a highly mutated, yet primarily diploid genome, has been routinely used to study epigenetic mechanisms and response to DNMT inhibition. In a previous study, we integrated a combination of data types including DNA methylation, DNA accessibility, histone modifications and transcriptomic data that revealed strong correlations between epigenomic changes and gene expression following Aza treatment. However, for some genes, we could not predict expression changes using these unidimensional, region­specific analysis methods. As others have demonstrated, genes do not work as single, isolated units, but rather interact with distal regulatory elements. These regulatory elements, often several Mb away can control gene expression through physical interactions such as bending and looping. We reason that in addition to direct demethylation of the immediate regulatory elements within the gene, Aza treatment can affect long­range chromosomal communications. In order to further understand the chromatin remodeling effect of Aza treatment, we performed high resolution genome­wide chromosome conformation capture (Hi­C) followed by NGS. HCT­116 cells were treated with DMSO or 1 μM Aza for 42 and 96 hours. Hi­C and RNA­ Seq data was analyzed and integrated with other datatypes. The results showed that AZA treatment alters topologically associating domains (TAD), with the formation of new TAD boundaries and disappearance of others. The genome was then separated into 25K base pair consecutive bins, with each bin marked as type A (active) or type B (inactive). We observed bin switching following AZA treatment and differential loop formations (e.g. promoter­enhancer). Bins that switched from B to A at both time points were found enriched in genes that are related to acute phase response, interferon pathway, and cancer including PI3KCB, DDX58, CD274 and CDKN2A. Our findings using Hi­C were found to be in agreement with previous results using alternative experimental

methods, which identified Aza and CTCF as the top upstream regulators of these genes. Our data also suggests that overall changes in 3D chromatin activities measured by Hi­C could be a better predictor of transcriptional regulation compared to H2K27me3 and H3K4me3 alone, particularly in situations where there are no significant changes in those marks but where changes in gene expression exist. #5372 Alterations in the chromatin accessibility in nonalcoholic steatohepatitis­associated hepatocellular carcinoma. Mekonnen Lemma Dechassa. NCTR/FDA, Jefferson, AR. Hepatocellular carcinoma (HCC) is one of the leading causes of cancer­ related deaths worldwide. Accumulated evidence in the past decade has stablished that NASH, in addition to other well­identified risk factors, is becoming a major cause of HCC in United States. In the present study, we investigated cancer­associated chromatin alterations in the Stelic Animal Model (STAM) of NASH­related HCC. Using Assay for Transposase­Accessible Chromatin with high throughput sequencing (ATAC­seq), we identified 1666 differentially opened chromatin regions in HCC tumor tissue as compared to normal livers. Annotation of these regions showed that they were associated with 1773 differentially expressed genes, which were enriched in lipid and steroid metabolism, obesity, and cancer. Among the differentially opened chromatin regions, 10% resided within 5 kb from the transcription start site (TSS), while the remaining 90% were located distal to the TSS (> 5 kb). By combining ATAC­seq and transcriptomic profiles, we demonstrated that 182 of the up­regulated genes were associated with open chromatin regions in tumor tissue. Furthermore, 24 of these up­regulated genes, including Apoa4, Nupr1, Anxa2, and Igfbp1, had accessible chromatin regions located proximal to the TSS. Importantly, analysis of histone modifications at the open chromatin regions of the over­expressed cancer­related Apoa4 and Anxa2 genes demonstrated enrichment by H3K4 mono­methylation (H3H4me1) and H3K27 acetylation (H3K27ac), histone marks that associated with the active gene promoters (H3K27ac) and enhancers (H3Kme1 and H3K27ac). In conclusion, these data provide evidence for alterations in the chromatin accessibility in tumor tissue and potential role of open chromatin regions in the over­expression of critical cancer­related genes in HCC.

#5373 3D genome architecture changes during cancer cell migration and metastasis. Rachel Patton McCord, Rosela Golloshi. University of Tennessee, Knoxville, TN. Metastasis, the most deadly part of cancer, results from cancer cell migration and invasion from a tumor into surrounding tissues. During this migration, the cell often has to squeeze and deform its nucleus through tight spaces. This nuclear squeezing is a rate­limiting step in migration, and the deformations can even rupture the nucleus. The organization of the genome inside the nucleus into loops, domains, and territories is important for proper gene regulation, DNA replication, and genome repair. However, little is known about the characteristics of nuclear organization that might allow for the nuclear deformation of metastatic cancer cells. We are using Chromatin Conformation Capture (Hi­C) and microscopy techniques to study genome organization during cancer cell migration. Using photoconvertible fluorophore Dendra2­H4 we draw and monitor patterns on nuclei of cells migrating through dense collagen matrices, Transwell filters, or microfluidic channels. Our data show changes in these patterns, indicating spatial reorganization of the genome during migration. Previous work has suggested that cancer cell migration requires global condensation of the genome. Some cancer chemotherapeutics act on epigenetic marks to decondense chromatin. We are testing whether such chromatin decondensation affects the physical deformability of the nucleus and thus effective migration. We have used Transwell and wound healing migration assays to monitor migration of cells treated with histone deacetylase inhibitors and histone methyltransferase inhibitors. Our results show that the drugs inhibit migration in a Transwell assay but not in the wound healing assay. This suggests that genome decondensation prevents the passage of the nucleus through narrow 3D spaces. But, a condensed genome organization is not necessary for 2D cell motility, where nuclei are unconfined. Hi­C experiments on migrating cells with deformed nuclei and drug­treated cells will characterize the specific rearrangements in genome structure that occur during cancer cell migration and whether these specific changes are counteracted by epigenetically active drugs. #5374 Polyunsaturated fatty acids (PUFAs) alter the epigenetic landscape in

MDA­MB­231 triple negative breast carcinoma (TNBC). Amy M. Chattin, Mykelti O'Brien, Ronald S. Pardini. Univ. of Nevada, Reno, NV. Breast cancer is the second leading cause of cancer­related deaths in US women; 10­20% of which are triple­negative breast cancer (TNBC) subtypes and have the poorest short­term prognosis. This is probably due to an epithelial to mesenchymal transition (EMT) that tumors undergo, de­differentiating into cancer stem cells. Epigenetic reprogramming is proposed to be required for malignant transformation and invasion, particularly in cells undergoing EMT transitions such as TNBC. Therefore, altering the epigenetic landscape in metastatic TNBC lines would provide researchers with a unique opportunity to sensitize tumors to treatment and induce apoptosis. Epigenetic research chiefly focuses on inhibitors of co­repressors histone deacetylase (HDACi) and DNA methyltransferases (DNMTi). Short chain fatty acids, have proven to be effective HDACi’s, suggesting a possible role for other fats in epigenetic regulation. Research has shown that several omega­3 polyunsaturated fatty acids (PUFAs), specifically docosahexaenoic acid (DHA; C22:6, n­3) and eicosapentaenoic acid (EPA; C20:5, n­3), inhibit tumorigenesis whereas the omega­6 PUFA linoleic acid (LA; 1C8:2, n­6) promotes neoplastic growth; supporting the association of elevated cancer incidence with n­6 PUFA dietary intake. We initiated studies to evaluate the impact of omega­3 fatty acids on tumor epigenetic pathways. Therefore we monitored epigenetic alterations in MDA­MB­231 TNBC cells in response to both omega­3 and omega­6 PUFAs in a time and dose­dependent manner. DHA, EPA, and arachidonic acid (AA; 20:5, n­6) all elicit apoptosis in a similar manner, indicating that initiating apoptosis is not omega­6 or omega­3 specific. We now show that PUFA incorporation and the resulting attenuated metabolism are not sufficient to induce apoptosis in MDA­ MB­231 TNBC at 3 and 12 hour timepoints. Additionally, plasma membrane integrity was not appreciably altered at 3 or 12 hour time points. These data, combined with a lack apoptosis at 12 hours, indicate that the level of intact PUFA in cell membranes does not initiate apoptosis. Moreover, mitochondrial membrane potential was universally diminished early and throughout the time course and, although not limiting, decreased ATP levels were observed at 12 hours (relative to vehicle control). Furthermore, activity of HDAC classes (I, IIa and IIb) were altered early at 3 and 48 hours, but typically punctuated with a

recovery to basal levels noted at the 12 hour time point. Chromatin condensation was observable primarily in 12 and 48 hour time points, although it was noted in DHA and EPA treatments at 3 hours. Collectively, these data demonstrate that PUFA­mediated epigenetic changes occur on a time scale proceeding apoptotic induction, indicating that PUFAs may be useful as an epigenetically­targeted cancer therapeutic agent or adjuvant therapy. #5375 Oncogenic BRAF mutation induces widespread DNA hypermethylation in a murine model for human serrated colorectal neoplasia. Vicki Whitehall,1 Catherine Bond,1 Cheng Liu,1 Futoshi Kawamata,1 Diane McKeone,1 Saara Jamieson,1 Sally Pearson,1 Susan Woods,2 Tamsin Lannagan,1 Lochlan Fennell,1 Winnie Fernando,1 Mark Bettington,1 Daniel Worthley,3 Barbara Leggett1. 1QIMR Berghofer Medical Research Institute, Herston, Australia; 2University of Adelaide, Herston, Australia; 3University of Adelaide, Adelaide, Australia. BACKGROUND: The serrated colorectal neoplasia pathway describes the progression of morphologically serrated polyps to cancer and accounts for approximately one third of all colorectal cancer cases. Human serrated polyps are characterised by activating mutation of the BRAF oncogene and widespread DNA methylation changes termed the CpG Island Methylator Phenotype. A causative versus synergistic relationship between BRAF mutation and this methylator phenotype has not been determined. We aimed to address this by developing a murine model for serrated neoplasia driven by BRAF mutation. METHODS: BrafV637E conditionally active mice were crossed with intestine­specific, inducible Villin­CreERT2 mice to direct the BRAF mutation to the intestine at 2 weeks of age. The proximal ilieum or proximal colon were sampled at defined time points including 10 days, 10 weeks, 5 months, 8 months, 10 months, 12 months and 14 months. Macroscopic lesions larger than 10mm were bisected for molecular and histological assessment. The entire remaining intestine was fixed and examined histologically. DNA methylation was investigated for 94 genes known to by methylated in colorectal cancer using Epitect MethylII Complete PCR Arrays (Qiagen).

RESULTS: Braf mutant mice displayed histologic changes analogous to the human serrated neoplasia pathway. Extensive intestinal hyperplasia developed by 10 days post induction of the BRAF mutation. By 10 weeks, 50% mice had developed areas of crypt dilation reminiscent of human sessile serrated adenomas. By 8 months, the majority of mice had murine serrated adenomas with dysplasia and invasive cancer developed in 40% of mice by 14 months, one of which metastasised to the liver. Compared to age­matched control mice, Braf mutant mice showed significant, gene­specific increases in DNA methylation from 5 months (p0.5. Furthermore, we observed 44 genes with a correlation coefficient of >0.7 in at least two tissue types. The majority of genes with the strongest correlations are transcription factors known to play roles in differentiation and development, with an enrichment of zinc finger and homeobox­containing genes. Further analysis of these genes exhibit divergent 3’ methylation when comparing normal and tumor tissues. Importantly, the extent of 3’ methylation of these genes is associated with patient overall survival in 5 of the 10 tumor types analyzed, strongly suggesting that this process plays a role in cancer pathogenesis. Conclusion: DNA methylation of the 3’ region is a functionally and clinically relevant epigenetic modification, and may serve as a novel target for inhibiting tumorigenesis and tumor progression. #5378 Genes involved in development and differentiation are commonly methylated in cancers derived from multiple organs: A single­ institutional methylome analysis using 1007 tissue specimens. Kentaro Ohara,1 Eri Arai,1 Yoriko Takahashi,2 Nanako Ito,1 Ayako Shibuya,3 Koji Tsuta,4 Ryoji Kushima,4 Hitoshi Tsuda,4 Hidenori Ojima,1 Hiroyuki Fujimoto,4 Shun­ichi Watanabe,4 Hitoshi Katai,4 Takayuki Kinoshita,4 Tatsuhiro Shibata,3 Takashi Kohno,3 Yae Kanai1. 1Keio University School of Medicine, Tokyo, Japan; 2Mitsui Knowledge Industry Co., Ltd., Tokyo, Japan; 3National Cancer Center Research Institute, Tokyo, Japan; 4National Cancer Center Hospital, Tokyo, Japan. Aim: The aim of this study was to clarify the significance of DNA methylation alterations shared by cancers derived from multiple organs. Background: Little is known about DNA methylation alterations during carcinogenesis shared by various organs. In this single­institutional study, consistency of sample quality, diagnostic criteria and technical platforms

may be advantageous for providing an overall view of DNA methylation profiles of cancers arising in multiple organs. Methods: We analyzed single­institutional methylome data by Infinium HumanMethylation27 or HumanMethylation450 BeadChip (Illumina) for 1,007 samples of non­cancerous tissue (N) and corresponding cancerous tissue (T) obtained from the lung, stomach, kidney, breast and liver. Results: Principal component analysis revealed that N samples of each organ showed distinct DNA methylation profiles, DNA methylation profiles of N samples of each organ being inherited by the corresponding T samples and DNA methylation profiles of T samples being more similar to those of N samples in the same organ than those of T samples in other organs. We identified 2,636, 2,209, 1,915, 2,914 and 5,665 probes that were aberrantly methylated in lung adenocarcinomas, gastric adenocarcinomas, clear cell renal cell carcinomas, breast cancers (e.g. invasive ductal carcinomas, ductal carcinomas in situ and invasive lobular carcinomas) and hepatocellular carcinomas, respectively, in comparison with the corresponding N samples. When we examined pairs of organs, only 6.9% (between stomach cancers and kidney cancers) to 35.4% (between lung cancers and breast cancers) of aberrantly methylated probes were shared between cancers of any two organs, indicating that the majority of DNA methylation alterations were diverse among multiple organs. In contrast to such organ and/or carcinogenetic factor­specificity of DNA methylation profiles, when compared to the corresponding N samples, 231 genes commonly showed DNA hypermethylation in T samples in four or more organs. Gene ontology enrichment analysis showed that such commonly methylated genes were enriched among “transcriptional factors” participating in development and/or differentiation, which reportedly show bivalent histone modification in embryonic stem cells. Pyrosequencing and quantitative reverse transcription­PCR revealed an inverse correlation between DNA methylation levels and mRNA expression levels of representative commonly methylated genes, such as ALX1, ATP8A2, CR1 and EFCAB1, in tissue samples. Conclusion: These data suggest that disruption of the differentiated state of precancerous cells via alterations of expression, independent of differences in organs and/or carcinogenetic factors, may be a common feature of DNA methylation alterations during carcinogenesis in multiple organs. #5379

TET2 loss and the lymphoma­associated RHOA mutation cooperate to disrupt CD4+ T cell function. Shengbing Zang,1 Jia Li,1 Haiyan Yang,2 Wei Han,1 Jixiang Zhang,1 Minjung Lee,1 Yubin Zhou,1 Deqiang Sun,1 Yun Huang1. 1Texas A&M University, Houston, TX; 2Zhejiang Cancer Department, Houston, TX. Peripheral T cell lymphomas (PTCLs) are a group of rare but aggressive lymphomas derived from mature T cells or natural killer cells with a dismal prognosis. Current treatments for PTCL are ineffective with high relapse rates, largely owing to the lack of targeted therapeutics and a deep mechanistic understanding of the molecular etiology. Recent exome sequencing has unveiled the frequent co­existing somatic mutations in RHOA (G17V) and TET2 in PTCLs, but not in other types of hematological malignancies. Most notably, mutations in both TET2 and RHOA are frequently observed (~60­70%) in angioimmunoblastic T­cell lymphoma (AITL), which is among the most common subtypes of PTCLs with a median overall survival of approximately 1.5 years. Patients with AITL are often associated with autoimmune manifestations, but the mechanistic underpinnings remain unresolved. To examine whether TET2 loss and RhoAG17V alter mature T cell function, we performed adoptive T cell transfer experiments with WT or Tet2 knockout transgenic mice. Using this strategy, we generated four groups of recipient mice transferred with T cells as follows: WT, RhoAG17V, Tet2­ /­, or Tet2­/­RhoAG17V. Among these four groups, only recipient mice transferred with double mutant T cells displayed severe inflammatory­ like phenotypes that ultimately led to early lethality in mice. Moribund mice transferred with Tet2­/­RhoAG17V T cells showed substantial weight loss, severe skin ulcer on tail/paw/ear accompanied with pruritus, and lymphomegaly as typically seen in AITL patients. Histopathological analysis on major organs derived from the Tet2­/­RhoAG17V group indicated severe infiltration of both T and B lymphocytes in major organs, as well as a pronounced increase in the numbers of follicles and activated germinal centers in lymph nodes. Further analysis indicates that Tet2 loss and RhoAG17V mutation cooperated together to exclusively promote CD4+ T cell proliferation and survival, and exert no adverse effects on CD8+ T cells, which recapitulates the mutation spectrum

detected in AITL patients, i.e., RhoAG17V is primarily detected in CD4+ T cells but not other T cell subsets. The functional consequence of Tet2 loss and RhoAG17V expression in CD4+ T cells is characterized by an imbalance between the effector and regulatory T cells, which may account for the overt immunoinflammatory phenotypes seen in mouse models. The transcriptome analysis further revealed a pronounced remodeling of the immune signaling network that points to the aberrant expression of several key transcriptional factors. In summary, our study demonstrated a previously­unappreciated cooperativity between Tet2 loss and RhoAG17V mutation in disrupting mature CD4+ T cells function, which is made through the synergy between epigenetic and GTPase signaling pathways. The results obtained from this study provide an additional basis for future development of new diagnosis, prognosis and therapy for PTCL patients. #5380 Inactivation of endogenous genes in cancer cells using targeted promoter DNA methylation via CRISPR­DNMT3a fusion protein. Simon D. Spivack. Albert Einstein College of Medicine, Bronx, NY. Background: Developing technologies for precise manipulation of individual DNA methylation loci is an attractive challenge in cancer and lung biology, because aberrant expression via hyper­ or hypo­methylation is so common. Projects such as ENCODE and the Roadmap Epigenetics Project, have identified thousands of epigenetic marks from human genome. However, the functional evaluation of these marks has been largely limited to determining their associations with gene expression. Technologies for targeting manipulation of epigenetic marks would allow direct experimental testing of the impact of DNA methylation at specific residues, mechanisms of gene regulation, and potential use as interventions, for example to silence constitutively active oncogenes. Methods: Here, we developed a CRISPR Cas9­based tool for specific DNA methylation in which the catalytic domain of DNMT3a (DNMT3a­ CD) is fused to the carboxy­terminus of Cas9 D10A­H840A mutant (dCas9). Both construct promoter strength and transfection strategies were optimized. Results: We demonstrated targeted and consistent CpG methylation in 30~100bp regions downstream of the PAM (binding) site of the gRNA guided fusion protein in HEK293 cells. The multiple guide RNAs could

target the dCas9­DNMT3A to multiple sites consistently. DNA methylation activity was specific for the targeted region and was heritable across cell divisions. We also found directed promoter DNA methylation of DAL1, GATA5 and C­MYC could decrease the corresponding mRNA expression by up to 80%; different DNA methylation positions had consequently different effects on gene expression. We have now performed C­MYC methylation­mediated knockdown in A549 lung cancer cells and are evaluating resulting cell cycle and apoptosis phenotypes. Conclusion: We believe this new targeted epigenetic technology can be directed to cancer cells for the purpose of reprogramming lung cancer, and unregulated stromal and microenvironment cells, for preventive and therapeutic uses. #5381 A highly sensitive method for noninvasive cancer profiling through targeted methylation sequencing of circulating cell­free DNA. Li Liu,1 Jonathan M. Toung,1 Raakhee Vijayaraghavan,1 Ruoyu Zhang,1 Helen J. Huang,2 Toshinori Hinoue,3 Hui Shen,3 Neeraj Salathia,1 Marina Bibikova,1 Richard Shen,1 Karen Gutekunst,1 Peter W. Laird,3 Filip Janku,2 Jian­Bing Fan1. 1Illumina, San Diego, CA; 2MD Anderson Cancer Center, Houston, TX; 3Van Andel Research Institute, Grand Rapids, MI. Liquid biopsies, by analyzing circulating cell­free DNA (cfDNA), have emerged as a promising tool for noninvasive cancer diagnostics and monitoring. Compared to the limited number of scattered DNA mutations in cancer patients, the genome­wide distribution of numerous densely clustered DNA methylation alterations may enable more robust cancer signal detection and higher sensitivity in cancer diagnostics. Furthermore, the cancer type specific methylation signatures potentially can be used for the identification of cancer tissue origin. To enable multivariate analysis of genome­wide methylation markers, we have developed a targeted methylation sequencing assay that can measure the methylation status of thousands of affected CpG (5'­C­phosphate­G­3') sites simultaneously. This assay allows low cfDNA input as little as 3ng while retaining good efficiency. The targeted sites were particularly selected for hypermethylated markers in more than 20 major cancer types based on The Cancer Genome Atlas (TCGA) database. Following bisulfite

sequencing of cfDNA, methylation signals from targeted CpG sites were aggregated into a single score that measures deviation of a sample’s methylation profile from a reference baseline. Cancer/normal classification was performed by comparing the methylation score of a test sample to a cutoff established by a group of plasma samples from healthy individuals. Subsequently, methylation signals from cancer type specific markers were used to perform cancer type classification. In a study to evaluate the potential clinical applications of this method, we processed 25 blood samples collected from healthy individuals and 70 blood samples collected from 63 advanced cancer patients. The assay demonstrated a specificity of 100% (n=25), and a sensitivity of 94.3% in colorectal cancer (n=36), 72.7% in breast cancer (n=11), and 52.6% in lung cancer (n=19) detection, respectively. We further performed cancer type classification on the samples that were correctly classified as true positives in the cancer/normal classification. Our results showed a classification accuracy of 82.4% for colorectal cancer (n=34), 87.5% for breast cancer (n=8), and 70% for lung cancer (n=10). In summary, we have developed a comprehensive method that allows us to perform noninvasive cancer profiling through targeted methylation sequencing assay and novel analysis algorithms. This method can potentially be used in multiple validated clinical applications, such as early cancer detection, cancer diagnostics, and monitoring of minimal residual disease. #5382 A DNMT3A­independent hypomethylator phenotype is a unifying epigenetic signature of AML with good risk cytogenetics. Andrew D. Kelly, Jozef Madzo, Priyanka Madireddi, Patricia Kropf, Charly R. Good, Jaroslav Jelinek, Jean­Pierre J. Issa. Lewis Katz School of Medicine at Temple University, Philadelphia, PA. Background: Acute myeloid leukemia (AML) causes the most leukemia­ related deaths in the United States, and has frequent mutations in epigenetic regulators, including DNMT3A, IDH, and TET2. Such aberrations have been proposed to transform the epigenetic state in cancer, often involving DNA hypomethylation, however, the genomic specificity, causes, and clinical consequences of such methylation changes in AML remain unclear. Methods: We queried genome­wide CpG methylation using The Cancer Genome Atlas (TCGA) AML samples (n=194) run on Illumina 450k arrays. We used RNA­seq data to study gene expression changes

associated with hypomethylator phenotypes (HP). Analysis was done using R. Results: Genome­wide analysis of CpG sites that are highly methylated in normal blood, and variably methylated in AML (β­value standard deviation in AML > 0.2; average β­value in normal blood > 0.8) revealed two distinct HPs by hierarchical clustering: Good­risk (GR) HP which included favorable cytogenetics, and DNMT­HP, which was enriched for DNMT3A mutations. We refined DNA methylation signatures of each HP cluster by differential methylation analysis and re­classified patients accordingly. Strikingly, all patients with t(8;21), inv(16), or t(15;17) belonged to the GR­HP+ group, suggesting that a common epigenetic thread connects these otherwise disparate genetic aberrations. From a clinical perspective GR­HP+ patients were younger than GR­HP­ patients, and had significantly longer overall survival (median OS, years: GR­HP+ = Not reached; GR­HP­ = 1.00; P < 0.001). In contrast, DNMT­ HP+ cases were statistically equivalent to DNMT­HP­ except for an enrichment for higher WBC counts, including no difference in survival (median OS, years: DNMT­HP+ = 0.92; DNMT­HP­ = 1.34; P = 0.27). From an epigenetic perspective the two HP clusters harbored distinct DNA methylation changes; although both favored hypomethylation within non­CpG islands relative to CpG islands, the enrichment was more pronounced for DNMT­HP (Odds ratio: hypomethylated CpG islands/hypomethylated non­CpG islands, GR­HP = 0.64; DNMT­HP = 0.18). Genetic analysis revealed that GR­HP+ leukemia had wild­type IDH, DNMT3A, and NPM1 genes. In contrast, DNMT­HP+ AML had significantly more FLT3, NPM1, and DNMT3A mutations compared to DNMT­HP­ patients. RNA­seq revealed significant up­regulation of genes in both HP phenotypes (216, and 150 genes for GR­HP and DNMT­HP, respectively at FDR < 0.01 and FC > 2). Pathway analysis of these genes revealed enrichments for ion channels and the complement pathway in DNMT­HP, and for nervous system and developmental genes in GR­HP. Conclusions: Our data suggest that two HPs exist in AML with unique epigenetic and transcriptomic signatures. The striking association between GR­HP and different favorable cytogenetic changes suggests that a common set of epigenetic features may contribute to improved survival in these patients. #5383 Non­invasive diagnosis of early­stage lung cancer via targeted high­

throughput DNA methylation sequencing of circulating tumor DNA (ctDNA). Xuyu Cai,1 Yangbin Gao,1 Hui Shen,2 Peter Laird,2 Jian­bing Fan,1 Weihong Xu,1 Wenhua Liang,3 Jianxing He3. 1AnchorDx Medical Co. Ltd., Guangzhou, China; 2Van Andel Research Institute (VARI), Grand Rapids, MI; 3First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China. Current state­of­the­art lung cancer early screening involves using low­ dose CT scan to identify lung nodules smaller than 3cm in diameter. However, it’s still a clinical dilemma to differentiate between malignant and benign nodules. We took the approach of methylation profiling by high­throughput bisulfite DNA sequencing in tissue samples to identify specific methylation signatures. We learned methylation patterns that differentiate malignant vs. benign lesions from tissue samples by in­depth data mining, and then used pattern matching to classify plasma samples. Given the usual low amount of ctDNA in plasma, we also developed an ultra­ sensitive library preparation method to perform targeted bisulfite DNA sequencing from as low as 1ng of cfDNA or 1mL of plasma. From a training set of 88 tissue samples, which includes 60 malignant specimens with different subtypes (invasive adenomas, IA; minimally invasive adenomas, MIA; atypical adenomatous hyperplasia, AAH; adenocarcinoma in situ, AIS; carcinoid; lymphoepithelioma­like carcinoma of the lung, LELC; squamous carcinoma, SC; as well as 28 benign specimens in different categories including hematomas, granuloma, tuberculosis, inflammatory pseudotumor (IPT), sclerosing hemangiomas (SHL) and infections, we were able to achieve a sensitivity of 95% for identification of malignant lesions, with a specificity of 78.6%. From an independent validation set of 45 plasma samples, we achieved a sensitivity of 94.7% and a specificity of 85.7% for differentiating patients with malignant tumor from patients with benign lesions. Specifically, our assay is demonstrated to be highly sensitive towards early­stage lung cancer detection, with a sensitivity of 93.3% in a total of 15 patients with stage IA/B lung cancer. In summary, we have developed a highly sensitive blood­based non­ invasive diagnostic assay for identification of early stage lung cancer, which can aid clinical decisions for patients with a CT scan positive for lung nodules. This approach can also be extended to non­invasive early

screening for various cancer types. #5384 Aberrant methylation of ANK1, the host gene for miR­486, distinguishes lung tumors by histology and smoking status. Mathewos Tessema,1 Christin M. Yingling,1 Maria A. Picchi,1 Guodong Wu,1 Tyron Ryba,2 Young Lin,1 Steven A. Belinsky1. 1Lovelace Respiratory Research Institute, Albuquerque, NM; 2New College of Florida, Sarasota, FL. MicroRNAs (miRs) regulate many fundamental biological processes primarily through inhibiting the transcription and/or translation of their target genes. MiR­486 is an intragenic miR located within ANK1, a gene that encodes for the adapter protein ankyrin­1. Although the tumor suppressor role of miR­486­5p and its down­regulation in non­small cell lung cancer (NSCLC) is well established, the mechanism(s) leading to its repression is unclear. In this study, we investigated epigenetic regulation of ANK1 and its impact on miR­486­5p expression in lung cancer. DNA methylation and expression levels of ANK1 and miR­486­5p in lung tumor­normal pairs, NSCLC cell lines, as well as various normal blood and lung epithelial cell controls were evaluated using qualitative and quantitative assays. The genome­wide impact of epigenetic repression of ANK1 was evaluated using siRNA. The results show that miR­486­5p expression in lung tumors and NSCLC cell lines was significantly reduced compared to distant normal lung tissue pairs (p = 0.0006) and strongly correlated with ANK1 expression. In NSCLC cell lines with strongly reduced expression of miR­486­5p and ANK1, treatment with the DNA methylation inhibitor 5­aza­2­deoxycytidine significantly increased the expression of both the miR and its host gene. Human ANK1 is a large (~230kb) gene with three promoter regions regulating tissue specific expression of three (ANK1B, ANK1E, and ANK1A) transcript variants. ANK1B (the longest of the three variants) is the primary transcript expressed in lung tissue, while ANK1E and ANK1A are weakly and rarely expressed in lung epithelial cells. ANK1B promoter contains a large CpG island that is methylated in 45% (118/262) of lung tumors but not in normal lung or blood samples. The prevalence for ANK1B methylation was significantly higher (p < 0.001) in lung adenocarcinoma (101/200, 51%) compared to lung squamous cell carcinomas (17/62, 27%) after adjustment for smoking. Comparison of

methylation by patients’ smoking history also revealed that ANK1B methylation in lung adenocarcinoma was significantly more prevalent in smokers (57%, 73/128) than in never smokers (39%, 28/72), p = 0.014; HR = 2.086 (CI: 1.157 ­ 3.759). These findings were confirmed by quantitative methylation assays and independently validated using publicly available methylation data for large (n = 809) NSCLC cases from the Cancer Genome Atlas database. Finally, siRNA mediated knockdown of ANK1 in lung cancer cell lines followed by transcriptome­ wide expression and pathway analysis revealed that ANK1 repression primarily alters cancer development and progression pathways. Taken together, our data indicates that aberrant methylation of ANK1B promoter is highly prevalent in lung cancer, discriminates lung tumors by histology and smoking history, and represses the expression of ANK1 along with the tumor­suppressor miR­486­5p it hosts within its last intron. #5385 Colorectal cancer methylome and laterality. Sho Hirabayashi, Masamichi Hayashi, Goro Nakayama, Keisuke Kurimoto, Hiroshi Tanabe, Mitsuro Kanda, Hideki Takami, Yukiko Niwa, Naoki Iwata, Daisuke Kobayashi, Chie Tanaka, Suguru Yamada, Hiroyuki Sugimoto, Masahiko Koike, Tsutomu Fujii, Michitaka Fujiwara, Yasuhiro Kodera. Nagoya University Graduate School of Medicine, Nagoya, Japan. Background: Colorectal cancers are anatomically derived from Midgut area (from cecum to mid transverse colon) and Hindgut area (from mid transverse to anal canal). The laterality of colorectal cancers was reported as one of the prognosis markers of these cancers. Midgut originated cancers had statistically poorer overall survivals than Hind gut (Yahagi et al, J Gastrointest Surg, 2016). In addition to clinical reasons, for example its symptomless nature of the bowel obstruction on the right side, the difference of genetic and/or epigenetic profiles between both side colorectal cancers may exists. Methods: Surgically resected 30 colorectal cancers (Midgut n=9, Hidgut n=21) were included. Any clinicopathological factors except for tumor site was not statistically different between 2 groups. We decided to focus on colon cancer methylome and laterality in this study. Two novel methylation markers of colon cancers (PAX5 and VGF) which were extracted from microarray analysis were used, and quantitative

methylation­specific PCR (QMSP) assay was performed for each marker.Results: Both PAX5 and VGF methylations were extremely tumor­specific markers (P500X) targeted DNA sequencing using the NimbleGen SeqCap EZ Choice system and Illumina technology. Our custom panel included key tumor suppressors (e.g. TP53, RB1, CDKN2A), cell cycle drivers (e.g. CCND1, CCNE1), DNA repair genes (e.g. ATM, BAP1, ERCC2), PI3K pathway genes (e.g. PIK3CA, PTEN, AKT1), and other oncogenes (e.g. RAS/RAF, EGFR, ERBB2, PPARG, FGFR3). Results: The majority of patients with metastatic BCa (18/31 patients, 24/43 samples) had robust evidence of somatic alterations and therefore ctDNA. Our findings were consistent with the known landscape of BCa, including mutations in TP53 and ARID1A (and other chromatin modifiers), hotspot activating mutations in PIK3CA, as well as typical copy number changes such as focal amplifications of ERBB2, KRAS, and CCNE1. In addition, we identified complex gene rearrangements including in one case an activating FGFR3 gene fusion. Some samples had evidence of very high mutation rates, indicative of somatic ‘hypermutation’. Interestingly, in contrast to the metastatic setting, 85% of patients with localized BCa had no evidence of ctDNA, despite elevated cfDNA yields in some patients. Conclusion: The majority of metastatic BCa patients have high levels of ctDNA suitable for standard targeted sequencing approaches, while patients with localized muscle­invasive tumors appear not to harbor ctDNA at fractions greater than 1­2%. The robust detection of so­called ‘actionable’ copy number alterations, mutations and rearrangements in ctDNA provides an unparalleled opportunity for practical molecular stratification of patients in clinical trials of novel targeted agents. #5398 High­throughput clonal analysis of AML tumors with droplet microfluidics. Dennis Eastburn,1 Maurizio Pellegrino,1 Sebastian Treusch,1 Adam Sciambi,1 Bill Hyun,2 Jamie Yates1. 1Mission Bio, Inc., San Francisco, CA; 2University of California, San Francisco, San Francisco, CA. Single cell analysis tools are crucial to better understand the role that rare or heterogeneous cancer cells play in the evolution of tumor progression.

Although, it is now feasible to perform single­cell RNA­Seq on thousands of, several challenges remain for high­throughput single­cell DNA sequencing. To address these challenges and enable the characterization of genetic diversity in cancer cell populations, we developed a novel approach that barcodes amplified genomic DNA of individual cells confined to microfluidic droplets. The barcodes are used to reassemble the genetic profiles of individual cells from next generation sequencing data. A key feature of our approach is the “two­step” microfluidic workflow that releases genomic DNA from cellular proteins prior to amplification. The microfluidic workflow first encapsulates individual cells in droplets, lyses the cells and prepares the lysate for genomic DNA amplification using proteases. Following this lysate preparation step, the proteases are inactivated and droplets containing the genomes of individual cells are then paired with molecular barcodes and PCR reagents. We demonstrate that the two­step microfluidic approach is vastly superior to workflows without the two­step process for efficient DNA amplification on tens of thousands of individual cells per run with high coverage uniformity and low allelic dropout of targeted genomic regions. To apply our single­cell sequencing technology to the study of acute myeloid leukemia (AML), we developed a targeted panel to sequence genes frequently mutated in AML including TP53, DNMT3A, FLT3, NPM1, NRAS, KRAS, JAK2, IDH1 and IDH2. Using this panel, we were able to identify clonal populations from AML research samples; moreover, the single­cell nature of our approach enabled the correlation of multiple mutations within subclones and determination of whether the mutations existed as as a homozygote or heterozygote. Collectively, our results show a greater degree of heterogeneity in AML tumor samples than is commonly appreciated with bulk sequencing methods. #5399 Investigating phenotypic plasticity in breast cancer with high­ throughput nanogrid single­nucleus RNA sequencing. Ruli Gao,1 Charissa Kim,1 Emi Sei,1 Jie Yang,1 Leo Chan,2 Maithreyan Srinivasan,2 Hong Zhang,1 Funda Meric­Bernstam,1 Nicholas E. Navin1. 1University of Texas MD Anderson Cancer Center, Houston, TX; 2WaferGen Inc., CA. Single­cell RNA sequencing (RNA­seq) is a powerful tool for investigating rare tumor subpopulations and resolving intra­tumor

heterogeneity, but is low throughput, expensive, and requires fresh tissue samples. To address these limitations, we developed a 5’ high­throughput single­nucleus RNA sequencing (SNRS) approach that uses nanogrid technology to perform single­cell imaging and sequencing of 500­2500 nuclei in parallel. The automated image scanning procedure allowed us to exclude doublets and select live cells with DAPI/PI staining. This approach allows the transcriptomic profiling of frozen tissue samples, in which the cytoplasmic membrane is ruptured in cells, but leaves the nuclear membrane intact. We validated SNRS in a breast cancer cell line (SK­BR­3) and compared the transcriptomes of 500 nuclei to 500 whole cells, which revealed a high concordance in the number of genes expressed as well as their expression levels. We also performed bulk RNA­seq of isolated nuclear and cellular fractions from 5 breast cancer cell lines, which showed a high concordance in genes and expression levels. Differentially expressed genes in the nucleus mainly included lincRNAs, pseudogenes and mitochondria genes, but did not affect most cancer genes and pathway analysis. We further applied SNRS to sequence 500 nuclei from a triple­negative breast cancer patient and identified diverse phenotypes in tumor cells, including variation in cell proliferation, migration, invasion, and epithelial­to­mesenchymal transition. These studies demonstrated the technical feasibility of using a nanogrid platform to perform high­throughput single­cell RNA sequencing and showed that nuclei from cell lines and tumors can be used to study signaling pathways and gene networks that play an important role in tumor progression. #5400 Accurate identification of single nucleotide variants in whole genome amplified single cells. Xiao Dong, Lei Zhang, Brandon Milholland, Moonsook Lee, Alexander Y. Maslov, Tao Wang, Jan Vijg. Albert Einstein College of Medicine, Bronx, NY. Single cell sequencing for analyzing DNA mutations across the genome in somatic tissues is critically important for studying development, cancer and aging. However, current procedures are prone to artifacts and to date a reliable protocol for single­cell somatic mutation analysis remains to be developed. Here we address the two largest sources of artifacts, i.e., DNA denaturation­related cytosine deamination and allelic bias­driven whole genome amplification errors. We first reconfigured multiple displacement

amplification (MDA) into an efficient protocol for whole genome amplification of single cells without cytosine deamination artifacts, i.e., Single Cell MDA (SCMDA). We then developed a new single­cell SNV caller (SCcaller) that distinguishes real somatic mutations and amplification errors by utilizing a SNP­based localized estimate of allelic amplification bias. The procedure was validated by comparing SCMDA­ amplified single cells with unamplified clones derived from single cells from the same population. Together with SCcaller, SCMDA provides a firm foundation for analyzing cellular heterogeneity in somatic mutational landscapes in tumor and surrounding, normal tissues. #5401 Assessment of whole genome amplification for whole exome sequencing in detecting genetic mutation. Crystal Xue, Laura Gardner, Guanglong Jiang, Fei Shen, Bryan Schneider. Indiana Univ. Cancer Ctr., Indianapolis, IN. Whole exome sequencing (WES) has been widely used for studying genetic mutations in DNA coding regions to elucidate cancer etiology and for identifying biomarkers to optimize chemotherapy. However, these studies can be limited when only small amount of DNA is available. Whole genome amplification (WGA) technology can be combined with WES to make these studies technically possible. Here, we evaluated WGA using a phi 29 polymerase prior to library preparation for WES in samples with various DNA concentrations. WES was performed by targeted exon amplification followed by massively parallel sequencing. We compared the base calls of single nucleotide variants for individual same samples with or without WGA prior to library construction to determine the concordance rate of variant calls. We also assessed genetic variant call rate in the same samples with or without WGA. The number of variants obtained ranged from 159,851,999 to 390,784 in the non­ amplified samples and from 353,215 to 384,118 in the same amplified samples. The average concordance rate of identical variants in the samples with or without WGA was 96.5%. Our results indicate that WGA in prior to WES can increase detection efficiency of single nucleotide variants in samples with relatively small amount of DNA and/or low DNA concentration. #5402 Single cell sequencing of high grade serous ovarian cancer.

Timothy K. Starr,1 Boris Winterhoff,1 Makayla Maile,2 Kenneth Beckman,2 Jerry Daniel,2 Melissa Geller,2 Martina Bazzaro,2 Molly Klein,2 Raffaele Hellweg,2 Juan Abrahante,2 Amit K. Mitra,2 Atilla Sebe,3 Sally A. Mullany1. 1Univ. of Minnesota Medical School, Minneapolis, MN; 2Univ. of Minnesota, Minneapolis, MN; 3Paul Ehrlich Institute, Langen, Germany. We performed RNA sequencing of single cells derived from a high grade serous ovarian cancer (HGSOC) specimen to determine the extent of heterogeneity and to determine if it was feasible to identify cancer stem cells or gene expression signatures of chemo resistance. To perform RNA sequencing we enzymatically digested a fresh specimen from an HGSOC derived from the ovary. Immune cells were depleted by flow cytometry and single cell sequencing was performed using the Fluidigm C1 chip in tandem with Illumina HiSeq 2500 sequencing. Multiple bioinformatics tools were used to identify subgroups and activated pathways. Immunohistochemistry was performed on an adjacent tumor section to analyze markers of epithelium, stroma and stem cells. We found that gene expression patterns in single cells could be used to separate cells into stroma­like and epithelial­like groups. Gene set enrichment analysis identified proliferative genesets (oxidative phosphorylation and MYC targets) associated with the epithelial­like cells while epithelial­to­ mesenchymal­transition (EMT) genes associated with the stroma­like cells. Neither group was significantly associated with genesets derived from chemo­resistant cells. Using known marker analysis, we could identify a small percentage of cells that expressed ovarian cancer stem cell markers and we could group cells into functional categories. Using four molecular subtypes established from large­scale bulk sequencing studies we show that single cells from a single patient are heterogeneous and each molecular subtype is represented. In conclusion, we show the feasibility of performing single cell sequencing on an epithelial ovarian cancer and reveal a heterogeneous population of cells. Expanding these findings to a larger cohort of patients could allow for identification of targetable sub­populations of cells that were previously undetectable in studies that use bulk samples to interrogate the transcriptome and genome of ovarian cancer patients. #5403 Reproducible elevation of RNA versus DNA mutation signal in low

purity breast tumors. Jerez Te,1 Coralie Viollet,1 Xijun Zhang,1Jatinder Singh,1 Jeffrey A. Hooke,2 Harvey B. Pollard,1 Hai Hu,3 Craig D. Shriver,2 Clifton L. Dalgard,1 Matthew D. Wilkerson1. 1Uniformed Services University; The American Genome Center, Bethesda, MD; 2John P. Murtha Cancer Center; Walter Reed National Military Medical Center, Bethesda, MD; 3Chan Soon­Shiong Institute of Molecular Medicine at Windber, Windber, PA. Background: Accurate detection of somatic mutations is critical for informing targeted therapy options. Prevalent non­cancer cell admixture complicates this detection in breast cancer. Conventional mutation detection relies on DNA sequencing; however in prior work, we demonstrated that combining RNA and DNA sequencing increases mutation signal strength, or mutant allele fraction (MAF). The ratio of RNA MAF versus DNA MAF (RNA:DNA MAF) was greatest in low purity breast tumors. We hypothesized that this elevation is biologically driven and would be conserved in a second, distinct tissue specimen of the same tumors. Here, we compare mutation characteristics between two tissue blocks in a cohort of breast tumors (n = 8) to evaluate possible preservation of RNA versus DNA mutation signal throughout the tumor. Methods: We selected four high purity and four low purity breast tumors (“Block1”) from The Cancer Genome Atlas (TCGA) cohort and associated ABSOLUTE purity analysis. For these tumors, we acquired a second tissue block (“Block2”) not analyzed by TCGA, cut sections, analyzed sections by H&E stains, and extracted nucleic acids. Whole genome DNA sequencing and mRNA sequencing was performed for Block2 specimens using Illumina X and NextSeq 500 sequencers, respectively. Somatic mutations in Block2 were detected using UNCeqR and compared to published UNCeqR somatic mutations from TCGA. We then evaluate MAF characteristics in the entire TCGA breast tumor cohort (n = 695). Results: Tumor purity estimates, determined by histology and by sequencing, were reduced in Block2 of the low purity tumor set versus the high purity tumor set, consistent with Block1 analysis. Molecular properties of genome­wide gene expression and somatic DNA copy number were highly similar between block­mated specimens (p < 0.01). We then identified expressed mutations present in Block1 and Block2 of the same tumor and compared the MAFs on these common mutations.

DNA MAF and RNA MAF were each significantly correlated between Block1 and Block2 (p < 1e­12 in both cases). The average RNA:DNA MAF was 2.5 for the cohort, indicating that RNA mutation signal is greater than DNA in general. In Block2 specimens, the RNA:DNA MAFs were significantly greater in the low purity tumor set than the high purity tumor set (mean 2.7 versus 2.1, p < 6e­5), reflecting the same trend observed in Block1 specimens. Analyzing the entire TCGA cohort, RNA:DNA MAF was positively correlated with proliferation pathway gene expression (p < 3e­16) and was greatest in the Basal subtype versus other subtypes (p < 2e­9). Conclusion: Mutant allele fraction both of DNA and of RNA was conserved across breast tumor subsections. Low purity and basal subtype breast tumors had elevated RNA:DNA MAF supporting a relationship to underlying biology and identifying classes of tumors with pronounced benefit for DNA and RNA integrated mutation analysis. #5404 Functional analysis of lncRNAs in pancreatic ductal adenocarcinoma. Luis Arnes, Jiguang Wang, Zhaoqi Liu, Carlo Maurer, Lori Sussel, Kenneth P. Olive, Raul Rabadan. Columbia University, New York, NY. Pancreatic ductal adenocarcinoma (PDA) is anticipated to be the second leading cause of cancer related death in western countries by 2030 with a 5­year survival rate of 6%. Recently, genome wide sequencing and transcriptome analysis have been applied to identify cancer­driving mutations and mRNA expression profiles of pancreatic tumors. Despite a better understanding of the molecular drivers and the signaling pathways dysregulated in PDA, this knowledge has not been able to identify novel therapeutic targets. A common limitation for the aforementioned studies is that they have been performed in bulk samples, and they have been focused on mutations and gene expression analysis of protein­coding genes, which represents less than 2% of the genome. Long non­coding RNAs are emerging as essential players in the biology and progression of a variety of tumors. However, this concept has not been extensively explored to the study of PDA. We have developed computational methods and performed experimental validation to identify functional lncRNAs drivers of tumor progression in pancreatic cancer. Specifically, we have developed computational tools to identify lncRNAs associated with genomic traits of PDA using genome

wide high throughput data. The analysis uncovered the existence of 1741 lncRNAs that we termed ncPDAs (non­coding PDA). Using a unique RNA­seq dataset from >200 laser capture microdissected pancreatic tumors, we selected those ncPDAs that are expressed specifically in the neoplastic epithelium of human pancreatic tumors. Within the top 100 ncPDAs enriched in neoplastic epithelial cells, we identified known lncRNAs drivers in other tumor types such as CRNDE (ncPDA4), Pvt1 (ncPDA 9), HOTAIR (ncPDA 39), Malat1 (ncPDA 68) and Neat1 (ncPDA 71). We are currently exploring the role of several ncPDAs in pancreatic cancer cell lines. Loss of function studies suggest that several ncPDAs are required for tumor progression. Our investigation will continue to unravel the role of ncPDAs in the initiation and progression of pancreatic cancer. #5405 Comprehensive analysis of long non­coding RNAs in human stomach adenocarcinoma molecular subtypes. Jian Chen, Jiun­Sheng Chen, Keping Xie, John R Stroehlein, Marta Davila, Xiaoping Su. UT MD Anderson Cancer Ctr., Houston, TX. Objective: Long non­coding RNA (lncRNA) plays key role in cell biology including epigenetic remodeling and post­transcriptional regulation. The potential roles of lncRNAs as biomarkers and therapeutic targets have been proven in solid tumors. Molecular profiling in stomach adenocarcinoma (STAD) has been characterized using The Cancer Genome Atlas (TCGA) database. However, there are no currentlncRNA comprehensive analyses and the relationships between patterns of molecular subtypes and the signature of lncRNA in in human stomach adenocarcinoma are unclear. Methods: (1) The Cancer Genome Atlas (TCGA) 312 primary STAD RNA­Seq data (bam files) and their related clinical data were obtained from the Cancer Genomics Hub and TCGA Data Portal. (2) We filtered the dataset to remove lncRNAs with low expression, defined as having an RPKM value 100 compounds show improved ability to recapitulate known biomarkers and drug targets compared to other state of the art methods. For the MEK1/2 inhibitor selumetinib, we successfully recapitulated BRAF mutation as the top­scored biomarker of sensitivity. In addition, we identified four genes that carry additional information that can be used to stratify samples into sensitive or resistant groups: all were wild type in the sensitive cell lines and mutated in some of the resistant lines. We hypothesize that these genes could be used in conjunction with BRAF

mutation status to refine the stratification to selumetinib therapy. We also identified several highly expressed genes in the resistant cell lines, and we hypothesize that these genes may mediate drug resistance. Similar analysis are currently ongoing for olaparib and palbociclib, comparing our findings to the subtypes currently proposed in the literature. Conclusions: Our integrated approach identifies drug­specific gene signatures in PDAC cell lines. We derived genomic signatures for two clinically relevant to PDAC compounds in the context of the established in the literature PDAC subtypes, and hypothesize that these signatures could be used to identify patients most likely to respond to these therapies. #5549 Novel network predictor for drug sensitivity in cell line response data. Ana Brandusa Pavel,1 Bin Li,2 Andrew Krueger2. 1Graduate Program in Bioinformatics, Boston University, Boston, MA; 2Takeda Oncology, Cambridge, MA. Sequencing patient tumors has enabled the design of treatment regimens that exploit sensitizing genomic alterations. Associating drugs with mutations that enhance their effect is a key component of the American Society of Clinical Oncology vision for the next two decades of cancer treatment. Methods that distinguish drug sensitizing molecular events from the millions of extraneous alterations common within a tumor will help researchers implement precision medicine strategies. Here we use molecular data from the cancer cell line encyclopedia; including mutations, copy number alterations and gene expression changes along with drug response data, to identify subnetworks of interacting proteins that contain drug sensitizing alterations. Building upon the ability of fuzzy logic models to capture gene activity from different molecular data types, we create ‘Network­FLM’, a method to identify drug sensitizing molecular markers using a subnetwork model that distinguishes a drug sensitive sample from a drug insensitive one. Because the subnetwork model incorporates the sign and direction of network edges and the magnitude of gene activity changes, biologically meaningful features are captured. We integrate protein interaction information from Metacore database with somatic mutation, copy number and gene expression measurements, profiled by CCLE. Using cross validation, we evaluate the mean AUC of the predictor for 24 anti­cancer

compounds (Barretina et al., 2012). We build subnetwork classifiers for each compound using mutation, copy number and expression data separately and in all possible combinations. We find that the Network­FLM approach performs well for targeted agents such as Sorafenib, and also for drugs with pleitropic mechanisms of action. Gene expression data alone creates effective predictive subnetworks for 8 compounds (Nilotinib, Sorafenib, Irinotecan, PLX4720, Paclitaxel, Topotecan, TAE684, and Erlotinib). Adding copy number changes to gene expression data created better predictive networks for 8 compounds (PF2341066, AZD6244, L685458, RAF265, PD0325901, ZD6474, PHA665752, and 17­AAG). Likewise, three compounds benefit from adding mutation data to expression data (Panobinostat, PD0332991, and Lapatinib). Combining all datatypes created the best predictors for AEW541 and AZD0530. For three compounds, mutation data alone and together with copy number alterations is the best input for building predictive models. We identified hyper­active subnetworks in cancer cell lines and used them to predict drug sensitivity. We plan to further explore the potential of these networks to improve patient response to anti­cancer drugs. #5550 Collaborative analyses for delineating mutation variations among different ethnic patients of prostate cancer based on genomic data integration. Qingyu Xiao,1 Yidi Sun,1 Hong Li,1 Yixue Li,1 Guo­Ping Zhao,1 Wendy Wang,2 Sudhir Srivastava2. 1CAS­MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China; 2National Cancer Institute, National Institutes of Health, Bethesda, USA, Bethesda, MD. Emerging data support the hypothesis that both inherited and somatic alterations of genomes of African American (AA) prostate cancer (CaP) patients are significantly different from that of Caucasian American (CA) men. Recent studies from our and other groups have established that frequencies of the most common genomic alteration, TMPRSS2­ERG fusion, which apparently causes the high expression of ERG oncoprotein, is markedly lower in AA than in CA patients. Some works indicated that the frequency of CaP ERG fusion is the lowest in Asian patients, but this frequency changed from 11% to 78% in different Asian cohorts. Further

analysis in Asian men is necessary. In order to systematically and comprehensively address this and related problems, Uniformed Services University of the Health Sciences­Center for Prostate Disease Research (CPDR), the US National Cancer Institute (NCI) and CAS­MPG Partner Institute for Computational Biology (PICB), Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS), China, launched an international collaboration project. The overall goal of the project is to evaluate difference of driver gene mutations of prostate cancer in various ethnic groups by integrating genomic and transcriptomic data from CPDR, TCGA and the Chinese Prostate Cancer Consortium (CPCC), specially focusing on somatic alterations (ERG and others), microbiome, mitochondrial sequences and microRNAs. We integrated gene fusion, copy number, and gene expression data to detect potential driver genes and, as anticipated ERG alteration was one of the most commonly detected. The frequency of ERG fusion is the highest for CA, intermediate for AA and the lowest for Asian men, while its expression is significantly higher in ERG­fusion tumors than other tumors cross over all of the three ethnic groups. We did find some genes altered more frequently in Asian patients than in American patients, subject to further validations. We explored the methodology for microbiota profiling using prostate cancer WGS sequencing data. To this date, we have developed an analysis pipeline for the detection of microbiome from prostate tumor tissues and the corresponding blood samples with high confidence but inadequate sensitivity likely due to the low availability of both microbial sequences and bacterial reference genomes. Our preliminary analyses highlight the potential of international collaboration. CPDR­PICB­NCI will work on a seamless approach for data integration, particularly of the raw data to truly enhance CaP diagnosis and treatment by providing more informative biomarker and therapy targets relevant to ethnically diverse patient populations. #5551 Systematic analysis of novel noncoding genes associated with poor prognostic pathways in lung adenocarcinoma. Jou­Ho Shih, Yuh­Shan Jou. Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan. Non­coding genes are shown to play important roles in cancer progression with potential for serving as theranostic biomarkers. However, there is still a lack of studies to predict the biological roles of

novel non­coding genes in tumorigenic pathways and associations with patient survival. Lung adenocarcinoma (LUAD) is the predominant histological subtype of lung cancer, which is the leading cause of cancer death. Nowadays, target therapies have improved LUAD patients’ survival about 6­12 months, but limited improvement of long­term survival, suggesting that discovering new therapeutic targets are important. Therefore, here we applied weighted correlation network analysis (WGCNA) to cluster highly correlated genes of transcriptomic data to accurately investigate the functions of non­coding genes with poor prognosis in lung adenocarcinoma (LUAD). We discovered a total of 627 differential expressed coding and non­ coding genes (DEGs) from 6 transcriptomic datasets derived from LUAD patients with survival information. By using WGCNA, we discovered 6 non­coding candidates (PTTG3P, MIR497HG, HSP078, TBX5­AS1, LOC100506990, and C14orf64) and their core networks of DEGs, which were highly associated with patient survival and clustered in modules functioning in cell cycle and migration. We noticed that a previously known processed pseudogene, PTTG3P (Pituitary Tumor­transforming Gene 3), is highly correlated with poor survival and associated with mitosis of the cell cycle. Up­regulated PTTG3P expression in LUAD was in silico validated of RNA­seq data from TCGA compared to normal lung tissues (fold­change= 2, p­value< 0.0001) and associated with poor 5­year survival rates (HR= 1.83, p­ value= 0.012). We further confirmed PTTG3P is highly expressed and associated with poor survival outcome of LUAD patients in Taiwan (HR= 1.75, p­value= 0.037) with RNA­ISH experiments. To examine the coding potential of pseudogene PTTG3P, we found that PTTG3P might encode a consensus 21 kDa novel polypeptide from a single exon by using various prediction programs of coding potential. Ectopic expression of PTTG3P in lung cancer cells indicated that PTTG3P protein can be detected resulted in shortening the process of metaphase to anaphase in cell cycle progression and promotion of cell proliferation. Knockdown experiments of PTTG3P further reversed aforementioned experiments. Together, we established a powerful and systematic strategy for functional classification of non­coding genes in association with poor prognosis of LUAD patients. We revealed that commonly annotated and processed pseudogenes defined as non­coding genes for lacking of introns and promoter regions could still be

translated into functional polypeptides. We found processed pseudogene PTTG3P can be translated to a protein, which might play an important role in predicted function of mitosis and impact on poor survival in LUAD patients. #5552 Differential proteomic responses of luminal­A and basal­like breast cancer cell lines during growth inhibition induced by co­culture with agarose encapsulated murine renal adenocarcinoma (RENCA) cells. Melissa A. Laramore,1 Peter James,2 Prithy C. Martis,1 Atira Dudley,1 Lawrence S. Gazda,1 Carl A. Borrebaeck,2 Barry H. Smith3. 1The Rogosin Institute ­ Xenia Division, Xenia, OH; 2Lund University, Lund, Sweden; 3The Rogosin Institute, New York, NY. The existence of a tumor growth regulatory network that is conserved across tumor types and species has been hypothesized. Our evidence for this regulatory network is derived from studies demonstrating tumor growth inhibition by agarose encapsulated cancer cells (cancer macrobeads). The ability of encapsulated, thus growth restricted cells, to inhibit freely growing cancer cells has been shown when the encapsulated cells are murine (RENCA) or human (e.g., J82). Clinical trials are underway (NCT01053013, NCT02046174). We have shown that RENCA macrobeads release >10 known tumor inhibitory proteins targeting several signaling pathways including Akt/PI3. To better understand the mechanism(s) of growth inhibition, we used a systems biology approach to identify protein profiles and interactions from macrobead­treated human breast carcinoma cell lines that are either mildly aggressive (MCF7) or highly aggressive (MDA­ MB231 [MDA]). Target cells were co­cultured with RENCA macrobeads or left untreated for 5 days. Lysates of target cells were prepared in Laemmli buffer, frozen, and sent for MS/MS. Samples were run in duplicate, protein intensities normalized, and the treated/untreated ratio log10 transformed to get a normal distribution. Protein profiles were analyzed using Key Pathway Analysis (KPA) and Metacore software. Growth inhibition by RENCA macrobeads was confirmed (MCF725%; MDA 57%). KPA of MCF7 proteins showed upregulation of ubiquitin pathways involved in degradation of misfolded proteins. Stress induced apoptosis and DNA damage (which correlates with the growth inhibition) were the

top 2 pathways upregulated using Pathway and Process Enrichment analysis. This is in line with a strong epigenetic gene deregulation (Metacore). Transcription Factor and Network analyses show upregulation of CREB, A2MR and ATF3 networks. Also, Regulated Network analysis suggests a role for A2MR or RAGE in the inhibition of proliferation and increased apoptosis. Macrobead­treated MDA cells showed no significant changes using KPA. Pathway, Network and Process enrichment (Metacore) showed 5 of the top 10 upregulated processes were associated with cytoskeleton remodeling. Cell cycle and protein folding processes were also upregulated. Transcription factors associated with this response were similar to that of MCF7. RAGE again appears in many of the pathways as indicated by Network and Regulated Network analysis. The protein response to RENCA macrobeads differs for these 2 cell lines. Proteins related to apoptosis are upregulated in the MCF7 cells whereas MDA have a preference for cytoskeleton remodeling. These data support the hypothesis that distinctive tumors may respond differently to RENCA macrobead exposure at both an epigenetic and protein level, nonetheless resulting in growth inhibition. #5553 A computational model for integrating genomic data with public datasets for molecular tumor board recommendations. R Joseph Bender,1 Edik Blais,1 Apoorva Kulkarni,1 Michael J. Pishvaian,2 David Halverson,1 Jonathan R. Brody,3 Emanuel Petricoin,1 Subha Madhavan2. 1Perthera, Inc, McLean, VA; 2Georgetown University Lombardi Comprehensive Cancer Center, Washington, DC; 3Thomas Jefferson University Pancreas, Biliary and Related Cancer Center, Philadelphia, PA. Recent genomic profiling studies in pancreatic adenocarcinoma (PDA) have revealed actionable mutations affecting multiple signaling pathways, but in spite of these mutations, targeted inhibitors of these pathways have low success rates. A possible reason for these failures is that single­gene biomarkers (e.g. a KRAS mutation as an indicator of MEK inhibitor sensitivity) fail to account for crosstalk within and between dysregulated pathways. We have previously curated a knowledgebase of published studies as evidence to support molecular tumor board recommendations to cancer patients after multi­omic profiling. Here we present a

computational framework for integrating this knowledgebase with drug response data from cancer cell lines to propose “actionable” biomarkers based on a panel of pathways instead of targeting a single gene mutation. We constructed a computational model encompassing a broad range of cancer­related pathways, including RAS/RAF/MEK/ERK, PI3K/AKT, cell cycle regulation, and DNA repair. The model consisted of a set of ordinary differential equations (ODE) with protein interactions following Hill­type kinetics and the rate of cell division and apoptosis modeled dependent on key signaling nodes, including the level of phosphorylated ERK and AKT. We integrated two sources of publicly available data: 1) published studies correlating phosphoprotein measurements and resistance pathways to targeted inhibitors in clinical development; and 2) mutation data correlated with drug­specific response metrics (e.g. IC50 values), such as CCLE and NCI­60. We systematically screened frequently observed overlapping disrupted signaling pathways (i.e., combinations of mutations) by simulating predicted IC50 values for targeted inhibitors. Based on these simulations, we then simulated the effect of pairs of drugs to explore which drug combinations may be best suited for inhibiting tumor growth when tumors harbor multiple mutations. We present two applications of this computational approach: a comparison of CDK4/6 inhibition in CDKN2A­mutated PDA vs. hormone receptor­positive breast cancer and a comparison of PARP inhibition in BRCA1/2­mutated PDA and ovarian cancer. The predictions generated by our simulations were consistent with clinical observations in that fewer combinations of mutations in PDA were sensitive to these inhibitors than in breast and ovarian cancer, suggesting ways to refine biomarkers for sensitivity to these drugs in PDA. The computational approach presented here takes into account multiple datasets from a knowledgebase to provide a prioritized list of treatments that match a patient’s molecular profile while also providing the rationale for the recommendation. This represents a step toward incorporation of systems biology in precision oncology. #5554 Metabolic reprogramming in non­small cell lung cancer: a precision oncology approach. Iman Tavassoly, Ravi Iyengar. Icahn School of Medicine at Mount Sinai, New York, NY. Lung cancers are among the most common invasive cancers worldwide

and annually lead to high mortality and morbidity. Genomic alterations have been known to control the evolution of hallmarks of cancer in a dynamic way. These molecular alterations combined with epigenomic and post­genomic modifications contribute to formation of these neoplasms. Multiplicity of these changes has made development of personalized therapeutic regimens for these cancers a complex problem. Metabolic reprogramming is one of the main mechanisms in progression of cancers. There have been efforts to model the metabolic reprogramming in cancer using metabolic networks of cancer cells, but there has been no computational framework to model these metabolic transitions in cancer for precision and personalized medicine. We have combined computational, mathematical and experimental methodologies to develop a platform for precision oncology in non­small cell lung cancer (NSCLC) by in silico models of metabolic switches. Our integrative analysis of genomic data from NSCLC has led to discovery of genomic signatures controlling metabolic reprogramming in NSCLC with KRAS mutations. This discovery was proved in vivo and in vitro using drugs blocking different metabolic pathways. We have shown that NSCLC cells and tumors which carry KRAS mutations and have these genomic signatures are addicted to the pentose phosphate pathway (PPP). We have verified and proved the predictive value of these genomic signatures using Patient Derived Xenograft (PDX) tumor models of NSCLC. We are developing a mathematical and computational framework to model these metabolic switches. Our platform is capable of using genomic data from a cell line or tumor to determine the metabolic dependency of them quantitatively and predict the optimized personalized treatments for modulating metabolic pathways aiming to control cancer progression. #5555 Integrative network variomics reveals complex genotype to phenotype relationships in cancer. Nidhi Sahni, Song Yi. UTMD Anderson Cancer Ctr., Houston, TX. Proteins interact with other macromolecules in complex cellular networks for signal transduction and biological functions. Recently a deluge of genomic information becomes available from patients stricken by a variety of cancer types. In cancer, genetic aberrations have been traditionally thought to abolish the entire gene function. It has been increasing appreciated that each genomic mutation of a gene could have a

subtle but unique effect on protein function or network rewiring, contributing to diverse phenotypic consequences across cancer patient populations. In this study, we designed an integrative multi­omics platform using systems biology approaches to functionally assess the effects of cancer genetic variants. We compared a broad spectrum of mutation classes for a wide range of mechanistic effects on gene function in the context of signaling networks. Our results reveal a functional landscape of context­specific signaling network perturbations underlying cancer genetic heterogeneity at the base­pair resolution. Such information is critical for understanding the complex pleiotropic effect of cancer genes, and provides a possible link between genotype and phenotype in cancer. Furthermore, this study suggests new perspectives in targeted therapy and precision medicine. #5556 Widespread protein interactome network rewiring in human cancer. Song Yi, Nidhi Sahni. UTMD Anderson Cancer Ctr., Houston, TX. In the past decade, genome and exome sequencing projects have identified thousands of genetic variants in patients across a large number of cancer types. However, the explosion of genomic information has left many fundamental questions regarding genotype­phenotype relationships unresolved. One critical challenge is to distinguish causal disease mutations from non­pathogenic polymorphisms. Even when causal mutations are identified, the functional consequence of such mutations is often elusive. Classical “one gene, one function, one disease” models can not reconcile with the complexity that different mutations of the same gene often lead to different phenotypes. The extent to which network perturbations are involved in disease malfunction and how distinct interaction perturbation patterns can distinguish cancer mutations are largely unknown. Here we report a systematic approach to investigate genetic variant­specific effects on molecular interactions at large scale across diverse human cancers. Remarkably, in comparison to non­disease polymorphisms, disease mutations are more likely to associate with interaction perturbations. A large fraction of missense disease mutations are found to cause protein interaction alterations. While some result in loss of all their interactions, many exhibit selective elimination of specific interactions (“edgetic”). Different mutations of the same gene give rise to different interaction profiles, accounting for distinct disease outcomes. Edgetic mutations perturb interactions through disrupting specific

interaction interfaces, and the perturbed partners are more likely expressed in relevant disease tissue. Together, our approach is insightful in prioritizing disease­causing variants, and uncovering patient mutation­ specific disease mechanisms at a base­pair resolution, a critical step towards personalized precision medicine. Furthermore, our results suggest distinct interaction perturbations as a widespread mechanism underlying genetic heterogeneity, providing a fundamental link between genotype and phenotype in cancer. #5558 Deconstruction of a human tumor microenvironment. Frances R. Balkwill,1 Oliver M. Pearce,1 Robin Delaine­Smith,1 Eleni Maniati,1 Sam Nichols,1 Jun Wang,1 Conrad Bessant,2 Martin Knight2. 1Barts Cancer Inst., London, United Kingdom; 2Queen Mary University of London, London, United Kingdom. The purpose of this study was to understand the relationships between the molecular mechanisms of disease progression and higher­order features such as tissue stiffness, extent of disease and cellularity in the tumor microenvironment, TME. Using samples of human high­grade serous ovarian cancer metastases, ranging from normal to heavily diseased, we identified molecular components of the TME using transcriptomic and proteomic analysis. We integrated these data against higher­order features of the tissue, namely the biomechanics, cellularity, and disease score. We then used bioinformatics and multivariate statistics to identifying components of the TME that best model the higher­order features. For the first time, we revealed the complexity of extracellular matrix remodeling during metastases development, defining patterns of extracellular­matrix associated genes and proteins that predicted both extent of disease and tissue modulus. This allowed us to identify a core group of twenty­two matrix­associated molecules that modeled the dynamic process of tissue remodeling during tumor progression. We used these data to generate a ‘matrix index’, a quantitative measure of the gene expression of the twenty­two molecules. In cancer transcriptomic databases, this matrix index had prognostic significance in thirteen solid cancers including high­ grade serous ovarian cancer, even after multivariate analysis. We conclude that there may be a common host matrix response to human solid cancers.

#5559 Using cancer dependency data to discover tumor suppressive and oncogenic functional modules. Joshua Pan,1 Robin M. Meyers,2 Brittany C. Michel,1 Ann E. Sizemore,2 Francisca Vazquez,2 Barbara A. Weir,2 William C. Hahn,1 Aviad Tsherniak,2 Cigall Kadoch1. 1Dana­Farber Cancer Institute, Boston, MA; 2Broad Institute, Boston, MA. Efforts to define protein complexes and their functional networks are critical for systems­level understanding of the pathways involved in human cancer. Current methods to catalog human protein complexes via physical interaction are often unable to resolve functional differences between complex members or infer relationships governed by sub­ stoichiometric interactions. While functional wiring maps in yeast have been generated by measuring epistatic interactions between pairs of genes, efforts to scale this concept in individual human cell lines have been met with challenges and have only been able to characterize limited numbers of genes at a time. We have developed a scalable approach that can measure functional similarity without the constraints of pairwise genetic interaction experiments. Using data from genome­wide RNAi and CRISPR dropout screens performed in hundreds of cancer cell lines, we leveraged the heterogeneity of gene dependencies across cancer types to measure functional similarity between thousands of genes at once, which in turn allowed us to recreate known inter­ and intra­complex functional relationships and to uncover tumor suppressive and oncogenic functional modules in cancer­relevant pathways such as proteolysis, metabolism and transcription. Applying these approaches to the mammalian SWI/SNF (BAF) chromatin remodeling complex, which is mutated in over 20% of human cancer, revealed three functional modules that arose separately during metazoan evolution, one of which is entirely novel and uncharacterized. We then performed biochemical experiments that fully support three specialized complex configurations, each with distinct size, subunit composition, and function. These data reorganize the BAF complex into previously unrecognized modules that better explain mutational burden in human cancer. Notably, we observe that that all known BAF­driven, highly penetrant rare cancers and neurodevelopmental disorders involve disruption within a single functional module we defined, underscoring the

value of evaluating disease genomics through the lens of functional modularity. #5560 Systemic map of protein phosphatase 2A (PP2A)­regulated phosphotargets and drug responses in cancer cells. Otto Kauko,1 Susumu Imanishi,2 Evgeny Kulesskiy,3 Teemu D. Laajala,1 Laxmana Yetukuri,1 Artur Padzik,1 Mikael Jumppanen,1 Pekka Haapaniemi,1 Bhagwan Yadaw,3 Veronika Suni,1 Taru Varila,1 Garry Corthals,1 Wennerberg Krister,3 Tero Aittokallio,1 Jukka Westermarck1. 1University of Turku, Turku, Finland; 2Meijo University, Nagoya, Japan; 3University of Helsinki, Helsinki, Finland. Despite the pivotal role of phosphatases in cancer cell signalling, systemic understanding of phosphatase targets is still at infancy. Protein phosphatase 2A (PP2A) is a human tumor suppressor complex. PP2A inhibition is a requirement for human cell transformation and PP2A regulates many cancer critical signalling pathways. Importantly, emerging data indicates that reactivation of PP2A tumor suppressor activity could provide entirely novel approach for cancer therapy. Here, we present first systemic analysis of phosphoprotein targets (dephosphorylome) regulated by PP2A in cancer cells. Based on data, PP2A regulates cancer critical signalling pathways, including entire EGFR­RAS­RAF­MEK­ERK cascade, and functions as a master regulator of MYC function. At network level, PP2A targets critical cellular processes such as chromosome organization, RNA splicing, and nuclear envelope assembly. Surprisingly, soft clustering of PP2A dephosphorylome revealed that most phospho­target residues are subject to only unidirectional regulation in cancer cells. Moreover, targets show intracellular gradient where phosphatase inhibition dominates nuclear phosphorylation balance. Since phosphoregulation is critical for cancer drug responses, dephosphorylome was correlated with cancer cell responses to over 300 drugs. Importantly, cancer therapies could be broadly classified based on their dephosphorylome, both at quantitative and qualitative manner. Finally, we demonstrate the utility of this large dataset by validating the role of PP2A in MEK inhibitor resistance in KRAS mutant cancer cells via regulation of RAF, mTor and MYC. The study presents first systemic resource to understand potential of PP2A manipulation in cancer cell

signalling and drug responses. The study also uncovers generally important insights to phosphoregulation. #5561 Single­cell analysis reveals an adaptive, transiently heritable, slowly­ dividing, drug­resistant state inhibitable by drug combinations. Mohammad Fallahi­Sichani,1 Verena Becker,1 Benjamin Izar,2 Gregory J. Baker,1 Jia­Ren Lin,1 Sarah A. Boswell,1 Levi A. Garraway,2 Peter K. Sorger1. 1Harvard Medical School, Boston, MA; 2Dana Farber Cancer Institute, Boston, MA. Adaptation and fractional response of tumor cells to targeted inhibitors of oncogenic pathways creates a population of viable tumor cells from which fully resistant clones can ultimately arise. Thus, understanding transient drug adaptation is key for both improving the effectiveness of treatment and delaying/controlling acquired resistance. Despite the wealth of information available about feedback mechanisms associated with adaptive resistance, most of our knowledge in this area comes from studying drug response in bulk tumor cell populations. Furthermore, the phenotypic consequences of drug adaptation have been often studied at a few fixed time­points, when drug­adapted cells exhibit a high population­ average activity in multiple pro­growth signaling cascades. It therefore remains unclear how the initial responses to drug relate to subsequent phenotypes such as cell death or adaptation. This is likely a key point for designing novel approaches to overcome fractional drug response in tumor cells and to achieve durable therapy. We use real­time live­cell imaging, single­cell analysis and molecular profiling to show that exposure of BRAFV600E melanoma cells to RAF/MEK inhibitors elicits a time­variable and heterogeneous response in which some cells die, some arrest and the remainder adapt to drug. Drug­adapted cells up­regulate markers of the neural crest (e.g. NGFR), a melanocyte precursor, and grow slowly. The drug­induced slowly­cycling NFGRHigh state is only transiently stable, reverting to the drug­naïve state within two weeks of drug withdrawal as measured by the restoration of RAF/MEK inhibitor sensitivity, accelerated rate of cell division and reduced expression of NGFR. Transcriptional and biochemical profiling of cell lines and human tumors implicates a role for the c­ Jun/ECM/FAK/Src cascade in driving the de­differentiated resistance program. We identify multiple drugs targeting this cascade as well as

BET bromodomain inhibitors that block this resistance program in cell lines and in a BRAFV600E melanoma xenograft model and increase sensitivity and maximal effect (Emax) of RAF/MEK inhibitors. Our study reveals directly how drug adaptation happens in individual tumor cells leading to emergence of heterogeneous cell sub­populations with reduced drug­sensitivity that may be targeted by drug combinations. #5562 Integrating evolutionary dynamics into treatment of metastatic castrate­resistant prostate cancer: a pilot multidisciplinary study. Jingsong Zhang,1 Jessica J. Cunningham,1 Joel S. Brown,2 Robert A. Gatenby1. 1H Lee Moffitt Cancer Center, Tampa, FL; 2University of Illinois at Chicago, Chicago, IL. Background: Prior studies have investigated “intermittent therapy” to delay hormone resistance in prostate cancer but did not explicitly include quantitative analysis of the evolutionary dynamics. We hypothesize that evolution­informed strategies may prolong time to progression but require a multidisciplinary effort to simulate intratumoral Darwinian dynamics and design a clinically feasible treatment protocol. Methods: We investigate intratumoral evolutionary dynamics during treatment of metastatic castrate resistant prostate cancer (mCRPC) with abiraterone, which blocks CYP17A1 autosynthesis of testosterone from endocrine precursors. We build a mathematical model assuming three competing phenotypes: (i) TP cells express CYP17A1 and produce testosterone; (ii) T+ cells require exogenous androgen; and (iii) T­cells are androgen­independent and resistant to abiraterone. Model predictions were tested in a pilot clinical trial. Results: Mathematical Model: Computer simulations demonstrate continuous maximum dose abiraterone treatment produces competitive release of resistant T­cells. However, limited treatment designed to maintain residual TP and T+ populations and suppress proliferation of T­ cells was predicted to prolong response while lowering the required drug dose. Clinical Trial: In a pilot clinical trial, 11 men with asymptomatic mCRPC were treated with abirateone according to an evolution­informed, patient­specific algorithm based on the modeling results. Pre­treatment biopsies (available for 3 patients) demonstrated the predicted tumor subpopulations. Cycles of response and regrowth similar to model simulations were observed with cycle lengths varying from 3 months to >

1 year. Over a median follow up period of 18 months, 10 subjects remain responsive to Abiraterone without PSA or radiographic progression. Median time to PSA progression significantly (P 35­fold), followed by OC (< 13­fold), MBC (< 4­fold), and PC (< 2­fold). PDL1 expression of 46/57 (80.7%) LC, 17/30 (56.7%) MBC, 6/19 (31.5%) PC, and 20/72 (27.8%) OC was greater than the population median of all tumors combined. Within the LC samples with PDL1 expression equal to the top quartile of the population, 10 (71.4%) were KRAS mutant lesions and 4 (28.6%) were WT tumors. Finally, amongst LC and PC harboring a KRAS mutation, PC showed an overall lower expression of PDL1 with only 2/19 (10.5%) cases been above the population median and none within the top quartile of the population. Conclusions: The LCM­RPPA workflow has the unique ability to capture immune checkpoint expression on a continuous quantitative scale as well as capture its broad dynamic range. Because RPPA is unconstrained by antigen retrieval issues as well as subjectivity of IHC interpretation, this approach may generate more accurate cut­point of therapeutic response

prediction. Overall the dynamic range of PDL1 was broader in LC compared to other solid tumors, and LC had a much higher proportion of patients with tumors expressing high levels of PDL1. These quantitative differences may explain therapeutic efficacy of PDL1 inhibition across tumor types. Such speculative hypothesis should be further validated in prospective clinical trials. Finally, these preliminary data suggest that organ specific microenvironments more than specific driving mutations (e.g. KRAS) may strongly influence PDL1 expression in malignant lesions. #5657 Objective measurement and significance of VISTA (PD­1H) expression in non­small cell lung cancer (NSCLC). Franz Villarroel­Espindola,1 Ila J. Datar,1 Vamsidhar Velcheti,2 David L. Rimm,1 Roy S. Herbst,3 Kurt A. Schalper1. 1Yale School of Medicine, New Haven, CT; 2Cleveland Clinic, Cleveland, OH; 3Medical Oncology Yale Cancer Center, New Haven, CT. Introduction: VISTA (PD­1H) is a member of the B7 family of immune co­regulatory molecules and has been proposed as a novel anti­cancer immunotherapy target. The intra­ and extracellular domains of VISTA show homology to PD­1 and PD­L1, respectively, suggesting a role in anti­tumor immune evasion. The expression of VISTA, its association with PD­1 axis components and biological role in human NSCLC are unknown. Methods: Using multiplex quantitative immunofluorescence (QIF), we simultaneously measured the levels of VISTA (clone D1L2G, CST), PD­ L1 (clone 405.9A11, CST) and PD­1 (clone EH33, CST) protein in 732 stage I­IV NSCLCs from 3 retrospective collections represented in tissue microarray format (cohort #1 [n=297, Yale], cohort #2 [n=329, Greece]; and cohort #3 [n=106, Yale]). To evaluate the tumor tissue distribution, VISTA was also selectively measured in cytokeratin+ tumor cells, CD3+ T­cells, CD4+ T­helper cells, CD8+ cytotoxic T­cells and CD20+ B­ lymphocytes. Associations between the marker levels, clinico­ pathological­molecular variables and survival were studied. Results: VISTA protein was detected in all NSCLCs, showed a membranous staining pattern and was localized predominantly in the tumor cells in 27.4% of cases; and in the stromal compartment in 98.5%. Although VISTA was detected in all major tumor infiltrating lymphocyte

(TIL) subsets, the signal was higher in CD20+ B­cells than in CD3+ T­ lymphocytes (P 1) with 351 lincRNAs differentially expressed between MM and normal plasma cells. Using only the expressed lincRNAs, we applied log rank tests for quartile 1 (Q1) versus Q2 through Q4 and Q4 versus Q1 through Q3 in order to identify under­ and overexpressed prognostic genes, respectively. Four under and seven overexpressed genes were selected for final model. We used Más­o­ menos for final predictive model, which simply calculates the risk score, by using expression values. The Kaplan­Meier estimates of EFS at 4 years were 53.3% (95% CI, 45.1% to 63.1%) and 32.6% (95% CI, 25.1% to 42.2%), and OS at 4 years were 93.2% (95% CI, 88.9% to 97.6%) and 71.1% (95% CI, 62.9% to 80.3%) in our patients having a low or high risk score. When applied to patient cohort separated by other risk categorization including minimal residual disease status (MRD), cytogenetic risk status (del17p, t(4;14) and t(14;16)) and International Staging System (ISS), lincRNA signature was able to further identify patients with significant differential survival outcomes. In summary, we report that lincRNAs have an independent effect on survival outcome in MM and provides rational for its use in risk stratification as well as to understand biological impact. #5720 Serum interleukin­18 and metalloproteinase­1 predict long­term prognosis of breast cancer. Ji­Yeon Kim,1 Kyunghee Park,2 Eunjin Lee,2 Hae Hyun Jung,3 Seok Jin Nam,1 Jeong Eon Lee,1 Seok Won Kim,1 Jong Han Yoo,1 Se Kyung Lee,1 Soo Youn Bae,1 Jin Seok Ahn,1 Young­Hyuck Im,1 Yeon Hee Park1. 1Samsung Medical Center, Seoul, Republic of Korea; 2Samsung Genome Institute, Seoul, Republic of Korea; 3Samsung Advanced Institute for Health Sciences and Technology, Seoul, Republic of Korea. Introduction: Intrinsic subtyping and twenty­one gene expression assay using tumor tissue indicates gene expression profile had advantage to predict breast cancer(BC) patients’ prognosis. However, these tumor biomarker assays had many limitations as like as high cost, demand for large amount of tumor sample and limited use for specific subtype of BC. Therefore, we conducted this study to find serum biomarker predicting BC prognosis. Materials and Methods: This study was conducted by retrospective analysis of patients with stage I to IIIC invasive breast cancer who

received curative surgery at Samsung Medical Center between 2003 and 2004. We performed multiplex immunoassay using 45 cytokine and 8 MMP multiplex immunoassay panels (Bio­Rad Laboratories Inc, Irvine, CA, USA). Among clinical factors, operational stage was re­categorized into stage I & II vs. stage III and Hormone receptor(HR) status was grouped into HR positive vs. negative. For each protein, proteins significantly associated with overall survival(OS) in univariate analyses were candidates for the multivariate analysis. Results: In total, 246 patients who received curative surgery for breast cancer were enrolled. Of 246 patients, serum cytokine analyses were performed in 229 patients. Of clinical characteristics, ten year OS of BC with stage III was 76.2% compared to 92.7% of stage I and 88.6% of stage II (hazard ratio (Stage III vs. stage I/II): 2.62 (95% CI: 1.26­5.48), p­value =0.016) and HR expressed BC had 91.1% of 10year OS, compared to 81.9%of HR negative BC (hazard ratio (HR positive): 0.47 (95% CI: 0.23­0.95), p­value =0.035). In terms of serum biomarker, high expression of IL­18 was associated with good prognosis (hazard ratio: 0.45 (95% CI: 0.21­0.96), p­value =0.033) in contrast with poor prognosis of MMP­1 expression (hazard ratio: 1.96 (95% CI: 0.94­4.08), p­value =0.067). Multivariate analysis showed that BC stage, HR positivity, serum IL­18 and MMP­1 affected patients’ OS. Stage I/II HR positive BC with low MMP­1 and high IL­18 increased OS about 5 times compared to stage III HR negative BC with high MMP­1 and low IL­18 (hazard ratio:0.21 (95% CI:0.1­0.48), c­index:0.731, p­value G in DTX1 promoter region, we conducted dual­luciferase reporter assays. The Gallele showed significantly higher luciferase activity than the A allele (PG polymorphism may affect DTX1 expression and may be a useful marker for predicting the prognosis of patients with early stage NSCLC. #5728 Metabolomic pathway alterations in endometrial cancer. Tatiana Altadill. Vall d'Hebron Research Institute, Barcelona, Spain. Introduction: Endometrial cancer is the most frequent gynecological cancer diagnosed in United States, accounting for 76,000 deaths every year. Up to now, it has been broadly described the proteomic and the

transcriptomic profiles of endometrial cancer (EC). However, the metabolomic content of EC tissues is largely unknown. In this work, we aim to characterize the pathways dysregulated in EC tumorigenesis and the changes that the metabolites experience along the progression of the disease. Methods: We performed an untargeted discovery using liquid chromatography coupled with electro­spray quadrupole time of flight mass spectrometry (UPLC­ESI­QTOF­MS). We analyzed the metabolomic and lipidomic profile of a set of 56 tumor tissues, corresponding to 39 different stages of endometrioid EC tumors (10 FIGO stages IA, 9 stages IB, 10 stages II and 10 stages III), and 17 benign endometrium tissues. Verification of initiation and progression markers was performed by MS/MS. Furthermore the dysregulated pathways were validated by Real Time quantitative PCR (RTqPCR) and Immunohistochemistry (IHQ) in an independent cohort of 60 and 109 patients, respectively. Results: A total of 8,146 features were detected in the positive and 7,558 in the negative electrospray ionization mode. Among those, a list of 40 significant differentially expressed metabolites (FC>2) was verified. The lipid metabolism appeared highly upregulated; with an important number of PCs, PSs, PEs, PIs and PGs. Linoleic acid, 3­Deoxyvitamin D3, and UDP­N­acetyl­D­galactosamine were also upregulated in EC. Differently, the peptide Glu Phe Arg Trp, some amides (palmitamide, stearamide and oleamide), PA (18:0/18:1), PE (20:1/22:6), PE (22:6/P­18:1), PG (19:0/22:4), inosine and picolinic acid were downregulated in the tumor tissue. Regarding tumoral progression, we showed a significant and non­ gradual dysregulation of PC (16:0/20:5), the PC (16:0/22:6), PE (16:0/22:6), PE (22:6/P­18:1) and PE (18:1/22:6) at different stages of cancer progression. A significant decrease of the levels of UDP­N­acetyl­ D­galactosamine and Arachidonic acid at advanced stages of the tumor was detected. We confirmed by RTqPCR and by IHQ the downregulation of the 3 adenosine­to­inosine editing enzymes (ADAR1, ADAR2 and ADARB3) in EC patients compared to the control group. We also observed a higher expression of the enzymes in the most aggressive (serous EC tumors) compared to the less aggressive tumors (endometrioid EC tumors). We are currently performing studies to understand the role of the RNA editing pathway in EC. Conclusions: In this study we described the metabolome and lipidome of EC initiation and progression. We verified and validated several metabolites that are significantly differentially found in control subjects

compared to EC patients and along tumoral progression. These results also depictured the metabolism of EC and deciphered, for the first time, an alteration in the adenosine­to­inosine editing pathway in EC. #5729 Loss of USP10 and p14ARF protein expression is associated with poor prognosis patients with small intestinal adenocarcinoma. Joon Seon Song,1 Hanbyoul Cho,2 Joon­Yong Chung,3 Ilseon Hwang,4 Hee Jin Lee,1 Seung­Mo Hong,1 Stephen M. Hewitt5. 1University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea; 2Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Republic of Korea; 3Experimental Pathology Laboratory, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD; 4Keimyung University Dongsan Medical Center, Daegu, Republic of Korea; 5Experimental Pathology Laboratory, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Bethesda, MD. Ubiquitin Specific Peptidase 10 (USP10) is a member of the ubiquitin­ specific protease family and its related pathways are apoptosis, autophagy and DNA damage. P14ARF is encoded by an alternative reading frame within the INK4a/ARF locus at chromosome 9p21, which is related with cell cycle arrest and apoptosis. Recently, ablation of USP10 prevented c­ myc induced cellular senescence by destabilization of p14ARF and p53, and accelerated hyper­proliferation and transformation of normal cells, suggesting USP10 have a crucial role in oncogenes induced senescence by keeping the stability of p14ARF and p53, key regulator of cellular senescence. However, knowledge on the clinical and prognostic significance of USP10 and p14ARF expression in patients with small intestine adenocarcinoma is limited. In the present study, we investigated the prognostic significance of USP10 and p14ARF by immunohistochemistry. 195 of resected small intestine adenocarcinomas were retrieved from 22 South Korean institutions by Korean Small Intestinal Cancer Study Group. Tissue microarrays were constructed using formalin­fixed paraffin­embedded specimen. 70 (35.9%) out of 195 patients showed immunoreactivity for USP10 and 124 (63.6%) showed loss of immunoreactivity for USP10. 120 (61.5%) of 195

patients were immunopositive for p14ARF and 75 (38.5%) patients showed loss of immunoreactivity for p14ARF. Loss of USP 10 was correlated with male predominancy (p=0.014), higher pT stage (p=0.044), presence of lymphatic invasion (p=0.033), absences of sporadic adenoma (p=0.024) and absences of peritumoral dyplasia (p=0.019). On multivariate survival analysis, loss of USP10 expression was independent poor prognostic factor (HR=3.04, 95% CI= 1.79­5.18, p < 0.001) and also both loss of USP10 and p14ARF expressions showed similar result ( HR=3.76, 95% CI= 1.93­7.23, p < 0.001) while loss of p14ARF was not an independent prognostic factor (p=0.587). Our findings indicate that loss of USP10, associated with loss of p14ARF protein expression, could be poor prognostic factors in small intestine adenocarcinoma. #5730 Increased FGFRL1 expression is associated with prostate cancer progression. Lan Yu,1 Mervi Toriseva,1 Andrew Erickson,2 Heikki Seikkula,3 Martti Nurmi,3 Pekka Taimen,3 Peter Boström,3 Tuomas Mirtti,4 Kalle Alanen,3 Markku Kallajoki,3 Johanna Tuomela,1 Mattias Nees,1 Pirkko Härkönen1. 1Institute of Biomedicine, Cell Biology and Anatomy, University of Turku, Turku, Finland; 2Institute for Molecular Medicine Finland, Helsinki, Finland; 3University of Turku, Turku, Finland; 4Institute for Molecular Medicine Finland and Helsinki University Hospital (HUSLAB), Helsinki, Finland. Prostate cancer (PCa) is the most diagnosed cancer in men in developed countries. Fibroblast growth factor receptors (FGFRs) have been demonstrated to play an important role in PCa initiation and progression. Fibroblast growth factor receptor like 1 (FGFRL1) is the most recently identified member of the FGFR family. Its extracellular domain shares high similarity to FGFR1­4 but it lacks the cytoplasmic domain with tyrosine kinase activity. Thus, FGFRL1 is able to bind FGF ligands but the subsequent intracellular signaling cascade is defective. We observed up­regulation of FGFRL1 mRNA expression in PCa in a public genome­ wide cancer transcriptome data base (cBioPortal). To validate the expression data and to investigate the putative role of upregulated FGFRL1 protein in normal prostate and PCa, tissue microarrays containing different types of benign and malignant prostate tissue were

used. Altered FGFRL1 protein expression was correlated with clinical parameters; and both in vitro and in vivo experiments were applied to study the biological functions FGFRL1 in PCa cell lines. Our results confirmed that FGFRL1 expression is upregulated in PCa compared to normal prostate. More specifically, increased cytoplasmic and nuclear FGFRL1 expression was associated with high Gleason score and Ki67 expression, while membrane­associated FGFRL1 showed the opposite correlation. To investigate the effects of androgens on FGFRL1 in vitro, PCa cells were treated with dihydrotestosterone and/or MDV3100, an androgen receptor inhibitor. Dihydrotestosterone increased FGFRL1 expression and MDV3100 inhibited endogenous FGFRL1 expression, but not when stimulated by dihydrotestosterone, suggesting indirect androgen­mediated regulation. The in vitro studies also showed that FGFRL1 is able to attenuate the phosphorylation of FRS2α and ERK1/2 by FGF ligands, providing evidence for the assumed decoy function of membranous FGFRL1. However, knock­down of FGFRL1 in PC3M cells resulted in reduced cell growth in tumor xenografts. Additionally, mRNA sequencing on PC3M cells with FGFRL1 knock­down revealed differential expression of about 250 molecules, including several metalloproteinases and FGFR1, compared to control cells (FDRlogFC>1). In conclusion, we suggest that upregulation and altered subcellular localization of FGFRL1, in PCa, directly or indirectly, promotes tumor growth and progression. The results suggest that FGFRL1 is a potent regulator of gene expression and may be involved in diverse functions depending on its cellular localization. #5731 The prognostic significance of EGFR, HER­2, HER­4, EGFRvIII, c­ MET, Ki67 and CD44 in patients with FIGO stages III and IV ovarian cancer. Soozana Puvanenthiran,1 Sharadah Essapen,2 Ben Haagsma,2 Izhar Bagwan,2 Alan Seddon,1 Helmout Modjtahedi1. 1Kingston University, Kingston­upon­Thames, United Kingdom; 2Royal Surrey County Hospital, Guildford, United Kingdom. Overexpression and activation of HER (human epidermal growth factor receptor) family members have been reported in a wide range of epithelial tumours. Although several monoclonal antibodies (mAbs) and small molecule tyrosine kinase inhibitors (TKIs) specific for the HER members

have been approved for the treatment of patients with a wide range of tumours, none has yet been approved for the treatment of patients with ovarian cancer. In some studies, the co­expression of other growth factor receptors (e.g. c­MET), the presence of cancer stem cells (CSCs) have been associated with resistance to therapy with the HER inhibitors. The aim of the present study was to determine the expression level and prognostic significance of the EGFR, HER­2, HER­4 and EGFRvIII, as well as c­MET, CD44 and cell proliferation marker Ki67 in 60 patients with FIGO stage III and IV ovarian cancer. The expression levels of these markers were determined, at different cut off values, using immunohistochemistry (IHC), and their associations with the overall survival and disease free survival were evaluated using Chi­squared and Kaplan­Meier survival curves and log­rank test as well as the univariate Cox­regression analysis. At cut off values of >5% of tumour cells with positive immunostaining, 62%, 93%, 45%, 3%, 21%, 48%, and 95%, of the cases were positive for EGFR, HER­2, HER­4, EGFRvIII, c­MET, CD44, Ki67 respectively and 28% were positive for the co­expression of EGFR/HER­2/HER­4. The cellular location of immunostaining was membranous for EGFR, HER­2, c­MET and CD44 and was present in 33%, 10%, 3% and 50% of the cases examined respectively. In univariate analysis, the expression of EGFR at cut­off values of >50% (HR, 3.57; 95% CI, 1.07 to 11.85; P= 5% (HR, 8.20; 95% CI, 2.02 to 33.2; P=5% of tumour cells, was associated with better overall survival (HR, 0.13; 95% CI, 0.02 to 0.73; P= 0.021 respectively). In addition, at cut­off value of >5% of tumour cells with positive immunostaining, EGFR expression (HR, 2.83; 95% CI 1.18 to 6.77; P = 0.019) and >10% (HR, 2.40; 95% CI 1.07 to 5.37; P= 0.032), as well as the co­expression of EGFR/HER­2 (HR, 2.83; 95% CI 1.18 to 6.77; P=0.019), EGFR/c­MET (HR, 3.05; 95% CI 1.2 to 7.75; P= 0.019) and EGFR/Ki67 (HR, 2.83; 95% CI 1.18 to 6.77; P=0.019) were all associated with poorer disease free survival in these patients. Our results suggest that co­expression of EGFR\HER­2\HER4 is common in patients with Stages III and IV ovarian cancers and support the need for investigations on the therapeutic potential of various forms of pan­HER inhibitors in such patients. #5732 PI3K/Akt activity regulates androgen receptor expression and

predicts poor clinical outcome in non­metastatic hormone­naïve prostate cancer. Sami Blom,1 Petra Mäki­Teeri,1 Andrew Erickson,1 Lassi Paavolainen,1 Tuomas Mirtti,2 Antti Rannikko,2 Swapnil Potdar,1 Päivi Östling,1 Wytske van Weerden,3 Olli Kallioniemi,1 Teijo Pellinen1. 1Institute for Molecular Medicine Finland FIMM, Helsinki, Finland; 2University of Helsinki, Helsinki, Finland; 3Erasmus MC, Helsinki, Netherlands. Activation of PI3K/Akt pathway is associated with adverse outcome and aggressive disease in many cancers. In prostate cancer (PCa), the activity of this pathway has been shown to promote disease progression and metastasis. However, it is still controversial how PI3K/Akt regulates androgen receptor (AR), a central signaling molecule in prostate pathophysiology, and whether it has an active role in hormone naïve non­ metastatic PCa. Here, we show using immunohistochemistry (IHC) and advanced quantitative multiplexed IHC that the expression of phosphorylated­Akt(S473) and AR are highly correlated in clinical PCa, even at the cellular level. Furthermore, we found that high expression of p­Akt(S473) predicts poor clinical outcome in two independent hormone­ naïve non­metastatic PCa cohorts. To study whether PI3K/Akt regulates AR expression, we performed an in vitro drug screen with 32 PI3K/Akt/mTOR inhibitors in PC346C, an AR expressing cell line derived from a hormone­naïve primary tumor of prostate. We observed a strong correlation between p­Akt(S473) and AR also in vitro in individual cells independent of the inhibitor used. Although both PI3K and Akt specific inhibition reduced cell viability, the response in nuclear expression of AR was highly dependent on the target of inhibition: Akt specific inhibition reduced AR nuclear expression and resulted in large, spindle­shaped cells, whereas PI3K specific inhibition increased AR nuclear expression and resulted in smaller, round­shaped cells. These data suggest that PI3K and Akt have different roles in sustaining AR activity in PCa as perturbations of the two components leads to differential responses in terms of AR nuclear expression and cell morphology. In conclusion, activated Akt associates with AR expression and predicts poor clinical outcome in hormone­naïve non­metastatic PCa. Furthermore, the differing roles of PI3K and Akt in AR regulation warrants for further studies as it may have implications in the design of PCatherapy targeting PI3K/Akt, especially when the inhibitors are administered in combination with anti­androgens.

#5733 Expression levels of UL16 binding protein 1 and natural killer group 2 member D in patients with gastric cancer. Kiyoshi Yoshimura,1 Moeko Inoue,1 Tetsuhiko Asao,1 Masanori Fuse,1 Satoshi Wada,2 Atsuo Kuramasu3. 1National Cancer Center, Chuo­ku, Tokyo, Japan; 2Kanagawa Cancer Center, Yokohama, Kanagawa, Japan; 3Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan. UL16 binding protein 1 (ULBP1) expressed on the tumor cell surface binds to the natural killer group 2 member D (NKG2D) receptor presenting on natural killer (NK), CD8+ T, and gamma delta T cells. However, the roles of ULBP1 and NKG2D expression and associated immune responses in gastric cancer are unclear. Here, we investigated the relationships between ULBP1 and NKG2D expression and clinical outcomes in patients with gastric cancer. The expression levels of ULBP1 and NKG2D were examined in human gastric cancer cell lines and gastric cancer tissues from 98 patients who underwent surgery from 2004 to 2008. MKN­72 cells expressed ULBP1 with ULBP2, ­5, or ­6. NKG2D was expressed at a higher level after activation on T cells and NK cells. Among tissue sections positive for NKG2D expression, six cases were positive for CD8 and CD56. In all tissues, NKG2D­expressing cells were typically aCD8+ T cells. Patients with NKG2D expression in tumors had significantly longer overall survival (OS) than patients without NKG2D expression in tumors (p = 0.0217). The longest OS was observed in patients positive for both ULBP1 and NKG2D, whereas the shortest OS was observed in patients negative for both ULBP1 and NKG2D. The interaction between ULBP1 and NKG2D may improve OS in patients with gastric cancer and may have applications in immunotherapy for induction of adaptive immunity in patients with cancer. Moreover, ULBP1 and NKG2D may be useful as prognostic biomarker in gastric cancer. #5734 The influence of body mass index on overall survival following surgical resection of non­small cell lung cancer. Xi Liu,1 Boris Sepesi,1 Kathryn A. Gold,2 Arlene M. Correa,1 John V.

Heymach,1 Ara A. Vaporciyan,1 Jason Roszik,1 Ethan Dmitrovsky1. 1UT MD Anderson Cancer Ctr., Houston, TX; 2UC San Diego Health, San Diego, CA. Prior work implicated an association between increased body mass index (BMI) and lower risk of mortality from lung cancer. The aim of our study was to comprehensively evaluate the influence of BMI on long­term overall survival in surgical patients with non­small cell lung cancer. This study investigated 1935 patients who underwent surgical resection for lung cancer at MD Anderson Cancer Center (from 2000 ­ 2014). Study variables included both patient and treatment related characteristics. Univariate and multivariate Cox regression analyses were performed to identify variables associated with overall survival. By univariate analysis, significant predictors of better survival were higher BMI, pathologic tumor stage (stage I versus stages II, III, or IV), type of surgery (lobectomy/pneumonectomy versus wedge resection/segmentectomy), younger age, female gender, and adenocarcinoma histology (versus squamous) (all p < 0.05). Patients considered morbidly obese (BMI ≥35) had a trend towards better outcomes than those classified as obese (BMI ≥ 30 and 1.3 fold. We also found that the frequency of ALDH+ PDAC CSCs increased by 12­19 fold compared to control cells. CSCs are also highly migratory and express factors suggestive of the epithelial to mesenchymal transition (EMT) suggesting that they play an important role

in metastatic disease. Following co­culture, the migration of PDAC cells increased by 1.2­2.1 fold (p≤ 0.05) with increased expression of the EMT associated genes SNAIL1, SNAIL2, ZEB1, ZEB2, N­cadherin, and Vimentin. PDAC cells induced CAF activation as evidenced by enhanced αSMA expression. Since activated CAFs are known to secret extracellular matrix proteins, we examined the activation of FAK downstream of integrin signaling and found increased phosphorylation of FAK at Y397. To determine the importance of FAK activation on the interaction between CAFs and PDAC cells, cells were treated with the FAK inhibitor PF573228 that significantly inhibited tumor colony formation. In contrast, the overexpression of FAK­FL further increased CAF­mediated PDAC clonogenic growth. Conclusion: CAFs enhance PDAC CSCs including clonogenic growth, migration, and self­renewal. These effects are partially mediated by FAK, and FAK inhibitors may represent a novel strategy to modulate the interaction of PDAC cells and the tumor microenviroment. #5890 Osteoblasts are educated into a tumor­associated stromal cell by disseminated breast cancer cells and mediate breast cancer cell proliferation in the bone microenvironment. Frank Marini,1 Julia Chifman,2 Janet Tooze,1 Candelaria Gomez­ Manzano,3 Karen M. Bussard4. 1Wake Forest Comprehensive Cancer Center, Winston­Salem, NC; 2American University, Washington, DC; 3The University of Texas MD Anderson Cancer Center, Houston, TX; 4Thomas Jefferon Univ. Kimmel Cancer Ctr., Philadelphia, PA. Breast cancer has a predilection for bone metastasis, where the five­year survival rate is bleak. Disseminated breast cancer cells invade bone and can remain undetectable and untreatable for decades during a period of reduced proliferation. Our work reveals that osteoblasts are educated into a tumor­ associated stromal cell by disseminated breast cancer cells and alter their production of exosomal microRNAs that regulate cancer cell cycle. Osteoblasts were grown to various stages of maturity and incubated with the conditioned medium (CM) from human breast cancer cells to produce tumor­associated osteoblasts (TAOs). TAOs and their CM were characterized, then TAO cells admixed with human breast cancer cells in­ vitro or in­vivo to elucidate the impact of TAOs on the tumor microenvironment. Osteoblasts undergo an inflammatory stress response and gain tumor­associated stromal cell­like properties when either co­

cultured or treated with the CM of human breast cancer cells. These changes were mediated by crosstalk via gap junction intercellular communication (connexin 43) and exosome exchange that occurred between TAO cells and human breast cancer cells. TAO­derived exosomes were found to contain increased amounts of microRNAs 320a and 193b compared to normal osteoblasts or breast cancer cells. microRNA 320a is associated with decreased cellular proliferation and induction of G0 phase of the cell cycle. miR 193b has been shown to regulate cyclin D1 expression and repress cellular proliferation. Knock­down of miRs 320a and 193b in breast cancer cells resulted in a reduced number of cancer cells in G0 and increased numbers of cancer cells in G1/S/G2/M phases of the cell cycle. Furthermore, TAO cell CM led to decreased activation of TRAP+ osteoclasts. When TAO cells were admixed with human breast cancer cells and inoculated in­vivo in mice, tumors grew more slowly and were at least 50% smaller than tumors composed of normal osteoblasts plus breast cancer cells, or breast cancer cells inoculated alone. Mice inoculated with an admix of TAO cells plus human metastatic breast cancer cells lived ~20 days longer than mice inoculated with an admix of normal osteoblasts plus metastatic breast cancer cells, or breast cancer cells alone. These data also suggest that TAO cells regulate the proliferation of metastatic breast cancer cells in the tumor microenvironment. Overall, these date suggest that osteoblasts are an important source of factors, specifically exosomal microRNAs, in breast cancer bone metastasis. The nature of these factors suggest their importance for facilitating disseminated breast cancer cell proliferation, as well as osteoclast activation in the bone microenvironment. Supported by NIH 1RC1 CA146381, 1R01NS06994, P50 CA083639 for FCM; (NRSA) T32 CA079448, NIH 3 R00 CA178177 for KMB. #5891 DCIS to invasive progression in breast cancer is delayed by restoring CCN5. Sandipto Sarkar, Arnab Ghosh, Gargi Maity, Snigdha Banerjee, Sushanta Banerjee. University of Kansas Medical Center/ VA Medical Center, Kansas City, KS. Malignant progression of breast cancer from pre­invasive to invasive lesions remains a mechanistically unknown event and a major challenge in medical research. By revealing the mechanism of action, our new and substantially different approach aims to demonstrate that CCN5/WISP2 might play a role in negative regulation of progression of pre­invasive lesion ductal carcinoma in­situ (DCIS) to invasive carcinoma (IC). DCIS to

IC transition results primarily from the loss of the myoepithelial cell (MEC) layer surrounding the breast ducts & lobules and basement membrane (BM) degradation followed by invasion of cancer cells into the surrounding stromal tissue and vasculature. It has been recently discovered that CCN5, a matricellular protein, is highly expressed in DCIS patient specimens and facilitates regression of aggressive phenotypes. Our in­vitro studies with myoepithelial cell lines (MECs) indicate that CCN5 may prevent the DCIS to IC transition through the protection of the MEC layer. CCN5 performs it’s protective role by regulating sonic hedgehog (SHh) expression in MECs. It has been previously shown in separate studies that Neuropilin1 (Nrp1) positively regulates expression of SHh and Nrp1 is exclusively expressed in MEC layer in breast tissues. An extension of our studies indicate that CCN5 might regulate the integrity of the mammary ductal architecture by protecting the MEC layer through a novel Nrp1­SHh signaling axis. Collectively, our studies indicate that regulating CCN5 expression level in breast cancer tissues might help us controlling the rate of progression of the disease from DCIS to an invasive stage. #5892 Induction of stromal fibrosis accelerates tumorigenesis in NeuT mice. Robert I. Glazer, Hongyan Yuan, Lu Jin. Georgetown University Medical Center, Washington, DC. Background: One of the central challenges in cancer prevention is the identification of factors in the tumor microenvironment (TME) that increase breast cancer susceptibility. The stromal composition of the breast is largely adipose and fibroblast tissue, and thus it is important to understand how alterations in these constituents affect in the onset of stromal fibrosis (SF) and cancer progression. To address these questions, MMTV­NeuT mice were crossed into the FAT­ATTAC (fat apoptosis through targeted activation of caspase 8) background (NeuT/ATTAC), wherein conditional dimerization of the FKBP­caspase 8 fusion transgene in female mice results in mammary fat ablation and its replacement with fibrotic tissue. Results: Induction of mammary fat ablation in NeuT/ATTAC mice over 4 weeks resulted in increased ductal proliferation and SF as denoted by increased collagen deposition and expression of Fibroblast Activation Protein and α­smooth muscle actin. Maintaining SF over 3­5 months resulted in a 40% reduction in tumor onset and a 2.5­fold increase in tumor multiplicity, which was associated with increased infiltration by myeloid­ derived suppressor cells. Gene expression profiling of tumors from mice with and without SF identified increased expression of the immune­related

genes Cxcl1, Ly6d and CD14, as well as acute phase proteins Saa1 and S100a6 in NeuT/ATTAC mice with SF. Conclusion: Induction of SF in an erbB2­dependent breast cancer model resulted in a tumor microenvironment more conducive to tumor progression and immune tolerance. These results suggest alternative therapeutic approaches that could be taken, eg. chemokine receptor antagonists, to increase the effectiveness of immunotherapy and chemotherapy. Supported by a grant from the Avon Foundation for Women and Award 1P30CA051008 from the National Cancer Institute, NIH, to the Lombardi Comprehensive Cancer Center. #5893 Bioenergetic signature from cocultures of pancreatic tumor cell lines and fibroblasts. Mª Teresa Agulló­Ortuño,1 Elena Prieto­García,1 C. Vanesa Díaz­García,1 Irene Otero Blas,2 Inmaculada García­Ruíz,1 José A. López­Martín2. 1Instituto de Investigación Sanitaria Hospital 12 de Octubre, Madrid, Spain; 2Hospital Universitario 12 de Octubre, Madrid, Spain. Pancreatic ductal adenocarcinoma (PDAC) is one of the tumors with greater invasiveness and metastasis. PDAC is associated with a large desmoplastic reaction, characterized by fibroblastic proliferation and extracellular matrix secretion. Besides, cellular bioenergetics has become a central issue of investigation in cancer biology, because the altered energy metabolism of cancer cells has been proposed as a potential target for cancer treatment. We have established co­cultures between PDAC cell lines (Capan­1 or PL­ 45) and fibroblasts (LC5). A reverse phase protein microarray (RPMA) approach has been applied to quantify proteins of energy metabolism in theses co­cultures, with the aim of identifying potential biomarkers in PDACs. The fifteen proteins of energy metabolism studied included members of the mitochondrial oxidation of pyruvate, the tricarboxylic acid cycle, β­oxidation of fatty acids, electron transport and oxidative phosphorylation, glycogen metabolism, glycolysis and oxidative stress using highly specific antibodies. Co­cultures modified proteins expression of energy metabolism, respect to monocultures. Capan­1 in co­culture increased several proteins belonging to OXPHOS (NDUFS3, SDHB, CORE2 and COXII), the antioxidant SOD2, the mitochondrial HSP60, and G6PDH. The expression of the glycolitic PKM2 decreased. Their fibroblasts partners increased the expression of SDHB and also decreased PKM2. PL­45 in co­culture, experimented an increase in HADHA. Their

fibroblasts partners showed a decrease in PKM2, LDHA, IF1, PDHe and HSP60.Capann­1 and PL­45 in co­culture with fibroblast, differ en their energy metabolism phenotype. Overall, the results indicate that the quantification of bioenergetic signature offers potential biomarkers that could be implemented to refine the understanding of the biological principles of PDAC and, eventually, the management of patients with these tumors. #5894 Stromal fibroblasts from metastatic breast cancer promote proliferation and migration of breast cancer cell and regulate its stemness. Yirui Gui, Adriana Aguilar­Mahecha, Marguerite Buchanan, Mark Basik. Ladi Davis Institute for Medical Research, Jewish General Hospital, McGill University, Montreal, Quebec, Canada. Breast cancer is one of the most common causes of cancer­related death in women in the world. Although most research is focused on the tumor cells, it is important to elucidate the molecular nature of the tumor­stromal relationship to truly understand the biology of breast tumor growth. Cancer­ associated fibroblasts (CAFs) are the major cellular constituents in the tumor microenvironment. CAFs are in direct contact with the adjacent cancer cells and crosstalk with them through soluble cytokines, growth factors and exosomes during carcinogenesis in human breast cancer. To determine for the first time the function of CAFs in metastatic sites of breast cancer, we collected CAFs from human patients of metastatic sites (m­CAFs) and compared them with CAFs from primary tumor (p­CAFs) and normal fibroblasts (NFs). We found that m­CAFs expressed higher levels of α­smooth muscle actin (SMA) compared with p­CAFs and NFs. The proliferation of MDA­MB­436 breast cancer cells was significantly increased by treatment with conditioned medium (CM) from m­CAFs compared with p­CAFs in vitro. Also, CM by m­CAFs induced paclitaxel resistance in MDA­MB­436 cells, suggesting that soluble factors produced by m­CAFs promote chemo­resistance, to a greater extent than CM from p­ CAFs. The migration and invasion ability of MDA­MB­436 cells co­ cultured with m­CAFs CM was significantly greater than that of the p­ CAFs CM. m­CAFs outperformed the p­CAFs and NFs in terms of their ability to promote sphere­forming activity in a 3D in vitro culture model. Furthermore, m­CAFs protected MDA­MB­436 cells from the cytotoxic effects of doxorubicin more than p­CAFs in this spheroid 3­D culture. When MDA­MB­436 cells were sorted from these spheroids and grew in

the respective CMs, they showed higher expression of the stemness markers of CD44+CD24­ compared with cells obtained from p­CAFs/MDA­MB­436 spheroids. Also these cells from the co­culture with m­CAFs experienced with the cadherin switch. Nanog has been demonstrated to promote chemoresistant and epithelial­mesenchymal transition (EMT). The results displayed that MDA­MB­436 cells incubated with the CM from the m­ CAFs had a higher expression of Nanog compared with that of p­CAFs. RNA­seq results showed that IGF2 is one of the most different genes between m­CAFs and p­CAFs, its expression was more significant in m­ CAFs co­cultured with MDA­MB­436 cells than p­CAFs. The signaling pathway in MDA­MB­436 cells were comparable between co­cultured with m­CAFsCM and the IGF2 stimulation at 2 hours. All these data illustrates that fibroblasts isolated from metastatic site differed in terms of their ability to promote breast cancer cells progression compared to p­CAFs. Our data suggests that m­CAFs are more potent than p­CAFs in inducing the proliferation, migration, drug resistance and cancer stemness of breast cancer cells in an in vitro model. #5895 Circulating CAF and cCAF circulating tumor cell co clusters are associated with metastatic breast cancer. Utsav Sharma, Philip Miller, Kelsie Medina­Saenz, Pedro Ferrer, Svetlana Speransky, Toni Yeasky, Dorraya El­Ashry. University of Miami, Miami, FL. Background: Breast cancer metastasis is the cause of breast cancer­related mortality. The tumor microenvironment (TME) plays a critical role in governing tumor initiation, progression and metastasis. In breast cancer, cancer associated fibroblasts (CAFs) are master regulators of the TME. Cancer metastasis occurs in part by transport of circulating tumor cells (CTCs) in the circulation. CTCs in clusters, rather than individual CTCs, have a greater capacity to establish metastases. We recently demonstrated that CAFs also circulate (cCAFs), both in clusters with CTCs and individually, in the blood of the majority of breast cancer patients with metastases, some patients without overt metastases, and in no patients with no evidence of disease. Our lab has also established primary breast cancer and primary CAF cell lines from dissociated breast tumors of different molecular subtypes; these are ideal models to dissect tumor­stromal interactions, both in vitro and in vivo. We hypothesize that cCAFs originate from the primary tumor, and further, that cCAFs cluster with CTCs to facilitate metastatic seeding.

Methods: We used separately labeled CAFs and primary breast cancer cells (DT28) co­injected into NSG mice and followed by a novel 2­color IVIS. We also modeled the ability of CAFs and breast cancer cells to form clusters in culture and used our microfluidic filter technology to evaluate the composition of clusters resulting from admixed CAFs and BC cells of differing metastatic capacity – metastatic DT28 and non­metastatic MCF­7. Results: Through sequential dual­color IVIS we demonstrate that we can monitor the presence of CAFs and breast cancer cells at orthotopic tumor injection sites and to sites of distant metastasis, indicating that cCAFs originate from the primary tumor. Evaluation of cCAFs and CTCs in tumor bearing mice indicates that breast cancer cells with high metastatic potential mobilize greater numbers of cCAFs. CAFs formed robust clusters with metastatic DT28 cells, while non­metastatic MCF­7 cells clustered with each other but not with CAFs suggesting that the ability of breast cancer cells to form clusters with CAFs, both in vitro and in vivo, is reflective of the metastatic capability of the breast cancer cell line. Conclusions: The intrinsic metastatic capabilities of breast cancer cells are augmented by contact and clustering with cCAFs. In turn, the ability of CAFs to mobilize into circulation is in part conferred by properties of aggressive breast cancer cells. These data corroborate observations from our pilot clinical study that indicated the presence of cCAFs is overwhelmingly associated with the presence of metastatic breast cancer. #5896 IL­8 signaling enhances TNBC growth and metastasis via crosstalk with stromal components. Kideok Jin, Niranjan B. Pandey, Aleksander S. Popel. Johns Hopkins Univ. School of Medicine, Baltimore, MD. Triple negative breast cancer (TNBC) as a metastatic disease is currently incurable. Reliable and reproducible methods for testing drugs against metastasis are not available. We have previously developed a robust metastatic model in which mice are pretreated with tumor cell­conditioned media (TCM) from human TNBC cells (MDA­MB­231 and SUM149) for 2 weeks prior to tumor cell inoculation. In this model we found reproducible spontaneous metastases in lymph nodes (LN) and lungs within 4­5 weeks after orthotropic tumor inoculation. We have discovered that the TNBC tumor cells secrete large amounts of interleukin­6 (IL­6) that “educates” lymphatic endothelial cells (LEC) in the LN and lungs. Stat3, a transcription factor, gets activated and induces the synthesis of CCL5 and VEGF among other factors. CCL5 recruits the tumor cells to the LN and

lungs; VEGF helps build blood vessels in the LN to facilitate tumor cell survival; VEGF produced in the lung helps the tumor cells extravasate into the lung. We have confirmed the importance of these factors by showing that inhibitors of these factors significantly inhibit metastasis. In this report, using a human antibody array, we identified factors secreted by fibroblasts and macrophages upon induction by MDA­MB­231 TCM. We ranked the expression level of each factor by real time qRT­PCR and determined that interleukin 8 (IL­8) was the top candidate. We confirmed by ELISA that IL­8 secreted from either fibroblasts or macrophages treated with MDA­MB­231 TCM was upregulated compared to treatment with serum free media (SFM). Our data showed that the proliferation of MDA­ MB­231 cells co­cultured with fibroblasts or macrophages was enhanced compared to monoculture. Furthermore, MDA­MB­231 cell migration, a key step in tumor metastasis, was promoted by CM from TCM­induced fibroblasts or macrophages. Knockdown of CXCR2, IL­8 receptor, expression by CRISPR­Cas9 system reduces MB231 cell proliferation and migration compared to wild type. In a mouse xenograft tumor model, the tumor growth of MB231­CXCR2­/­ cell was significantly decreased. In addition, the incidence of thoracic metastasis of MB231­CXCR2­/­ tumor was reduced compared to WT. We found that the auto­ and paracrine loop of IL­8 exists between TNBC cells and stroma, which results in enhanced IL­8 secretion from the stromal components. Significantly, inhibition of the IL­8 signaling pathway by Reparixin, an inhibitor of the IL­8 receptor CXCR1/2, abrogated MDA­ MB­231 tumor growth and metastasis. These findings implicate IL­8 signaling as a critical event in TNBC tumor growth and metastasis via crosstalk with stromal components. Further, these studies suggest that IL­8 acts as a key regulator orchestrating TNBC metastatic breast cancer. Therefore, we have provided evidence that supports the hypothesis that functional antagonism of the IL­8 signaling pathway has the potential to circumvent TNBC breast cancer growth and metastasis. #5897 Single cell­derived analysis of desmoid tumors for studying tumor­ stroma interactions. Mushriq Al­Jazrawe,1 Steven Xu,2 Qingxia Wei,2 Raymond Poon,2 Benjamin Alman3. 1University of Toronto, Toronto, Ontario, Canada; 2Hospital for Sick Children, Toronto, Ontario, Canada; 3Duke University, Durham, NC.

Cancer associated fibroblasts play an important role in the maintenance and remodeling of the tumor microenvironment, providing the appropriate conditions for neoplastic cell growth and invasion. Desmoid tumors (DT), also called aggressive fibromatosis, are rare, locally invasive soft tissue tumors that consist of fibroblastic cells embedded in extracellular matrix. Identification of the stromal cells to study tumor­stroma interactions is difficult due to both populations displaying a fibroblastic phenotype, and no cell marker exists that reliably differentiates between the two populations. Majority of DT arise sporadically due to somatic activating mutations in beta­catenin (CTNNB1), a major effector molecule of canonical Wnt signaling. We established single cell derived colonies from multiple DT samples and characterized the beta­catenin mutation status of each clone by Sanger sequencing. Indeed, we were able to establish both mutant and non­ mutant colonies from DT samples. Quantitative PCR for beta­catenin targets AXIN2 and LEF1 confirmed differential activity between the mutant and non­mutant colonies. The specific CTNNB1 codon mutation had no difference on beta­catenin transcriptional activity. We next performed a high throughput surface antigen screen to identify cell markers that can distinguish between the two subpopulations. Our screen found CD142 to be uniquely expressed by the mutant colonies, while the non­mutant colonies uniquely expressed Podoplanin. Quantitative PCR confirmed the differential expression of these markers. Furthermore, the CD142­positive population in heterogeneous DT samples correlated with their mutation frequency. Importantly, CD142­based cell sorting allowed the isolation of the mutant subpopulation even in samples that appeared as wild­type by Sanger sequencing. We also studied the expression of secreted factors in our mutant and non­ mutant populations. We observed that CTHRC1, a ligand related to the Wnt/PCP pathway, is highly elevated exclusively in the mutant subpopulations. Recombinant CTHRC1 increased the proliferation rate of DT primary cultures, as measured by BrdU incorporation, while neutralizing antibodies against CTHRC1 decreased cell proliferation. The importance of tumor­stroma interactions cannot be studied without first identifying and characterizing the two populations. This has been especially difficult in soft tissue sarcomas where both the neoplastic and stromal cells exhibit a mesenchymal phenotype. Our study offers a novel method for identifying the mutant and non­mutant subpopulations within desmoid tumors to study how they may interact. Rapidly quantifying tumor composition will also support efforts to understand the natural progression of disease and how it responds to therapy.

#5898 The consomic xenograft model identifies genetic changes in the tumor microenvironment that alter the growth and metastasis of head and neck cancers. Michael W. Straza,1 Amy Rymaszewski,1 Kwangok P. Nickel,2 Anne Frei,1 Anirban Chatterjee,2 Rachel Schlaak,1 Amit Joshi,1 Michael Flister,1 Randy J. Kimple,2 Carmen Bergom1. 1Medical College of Wisconsin, Milwaukee, WI; 2University of Wisconsin, Madison, WI. Background: The tumor microenvironment (TME) is known to impact tumor growth, metastatic potential, and treatment response. Nearly all studies of head and neck cancer (HNC) have focused on somatic mutations in the malignant cells. We hypothesized that genetic determinants limited to the microenvironment would influence HNC growth and metastatic potential. Approach: To demonstrate the impact of genetic differences in the TME on HNC cell line in vivo growth we utilized a novel tool, the consomic xenograft model (CXM). A consomic rat has an entire chromosome substituted into the isogenic background of another inbred strain by selective breeding. Use of immunodeficient (IL2Rγ­/­) consomic rats allows one to study the influence of stromal genetics on tumor biology without the confounding effect of differences in the immune system through the orthotopic implantation of cancer cells into different consomic rat strains. In this system, any differences in tumor growth or metastases are due to differences in the TME rather than cancer cells or immune response. We utilized SS and SS.BN3 consomic rat strains, previously shown to affect the growth of breast tumors, to study the effects of the TME on HNC tumor growth using two well­characterized HPV negative HNC cell lines, SCC­6 (base of tongue derived) and SCC­22b (derived from a hypopharyngeal cancer that had metastasized to lymph nodes). Both cell lines were modified to stably express luciferase. HNC cells were inoculated into the tongue of SS and SS.BN3 animals and tumor growth was monitored by biophotonic imaging after luciferin injection. Results: A significant difference in the tumor growth was seen between rat strains for both cell lines, with the SS.BN3 rats exhibiting less tumor growth and metastasis. Median luciferase activity from baseline increased by 4.1­fold vs. 1.1 fold in SCC­6 tumors in SS vs SS.BN3 rats, respectively (p