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A Systematic Approach to the Development of Novel Therapeutics for Lung Cancer Using Genomic Analyses Y Daigo1, A Takano1, K Teramoto1, S Chung2 and Y Nakamura2,3 Molecularly targeted drugs for cancer therapy represent a therapeutic advance, but the proportion of patients who receive clinical benefit is still very limited. We present here the rationale and initial results of our program to define molecules involved in lung carcinogenesis with the goal of identifying new therapeutic targets and/or predictive biomarkers for drug response. We have used gene expression analysis of 120 lung cancers followed by RNA interference, tumor-tissue microarray analysis, and functional analyses to systematically distinguish potential target molecules specifically expressed in cancer cells. Through this approach, we have identified oncoproteins that provide the starting point for the development of therapeutic antibodies, dominant negative peptides, small-molecule inhibitors, and therapeutic cancer vaccines. We believe that the approach we describe should result in new molecularly targeted therapies with minimal risk of adverse events. Lung cancer is one of the most common malignant tumors in the world, and non–small cell lung cancer (NSCLC) accounts for nearly 80% of those cases.1 Because the majority of lung cancers are diagnosed at an advanced stage, patients are unlikely to be cured of their disease.2 Over the past decade, cytotoxic agents including paclitaxel, docetaxel, gemcitabine, and vinorelbine have emerged to offer multiple therapeutic choices for patients with advanced NSCLC; however, these regimens provide only modest survival benefits as compared with cisplatin-based ­therapies.3 In addition to these cytotoxic drugs, several molecularly targeted agents such as monoclonal antibodies against vascular endothelial growth factor (i.e., bevacizumab) and epidermal growth factor receptor EGFR (i.e., cetuximab), as well as inhibitors of epidermal growth factor receptor tyrosine kinase (i.e., gefitinib and erlotinib) and anaplastic lymphoma receptor kinase tyrosine kinase (i.e., crizotinib) have been developed and applied in clinical practice.4–7 However, each of these new regimens can provide clinical benefits to only a small subset of lung cancer patients. In addition, these regimens have been reported to cause very serious adverse events such as interstitial pneumonia (epidermal growth factor receptor inhibitors) or hemorrhage (anti–vascular endothelial growth factor antibody).4–6 Therefore, it is imperative that we continue to develop new molecularly targeted therapies involving antibody-based

drugs, small molecules, and cancer-vaccine immunotherapies, along with their predictive biomarkers, in order to provide the appropriate treatment for each individual patient. The increasingly well-accepted concept that an individual patient’s response to anticancer drugs may depend on genetic and/or epigenetic changes in the tumor forms the rationale for much of new cancer drug development, as well as the ratio­ nale for the program we present here. The information obtained through human genomic and proteomic analyses has been accelerating the discovery of new molecules associated with various types of cancer and is expected to contribute to the development of many new molecular therapies (i.e., immunotherapy and novel drugs targeting these cancer-causing molecules).8 Molecularly targeted drugs are expected to be more specific to cancer cells and cause few or no adverse events due to their welldefined mechanisms of action. One way to achieve this goal is to combine a variety of scientific approaches. To this end, we have combined the following: (i) genome-wide analysis of genes to screen molecules that are expressed in the majority of cancer cells but are scarcely expressed in normal tissues, (ii) highthroughput screening of protein expression related to prognosis of patients by means of tissue microarray, and (iii) examination of loss-of-function phenotypes by RNA interference systems.8–14 Through this systematic approach, we have identified

1Department of Medical Oncology and Cancer Center, Shiga University of Medical Science, Otsu, Japan; 2Department of Medicine, University of Chicago, Chicago,

Illinois, USA; 3Department of Surgery, University of Chicago, Chicago, Illinois, USA. Correspondence: Y Nakamura ([email protected])

Received 21 December 2012; accepted 30 April 2013; advance online publication 19 June 2013. doi:10.1038/clpt.2013.90 218

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state a set of molecules that fall into the category of oncoproteins, whose upregulation is an important feature of the malignant nature of lung cancers, including disease progression and drug sensitivity.15–51 Here, we summarize our progress using this systematic genomics approach to help to identify new therapeutic targets and to develop new molecularly targeted therapies in lung cancer via the use of a cohort of patients from Hokkaido University, Kanagawa Cancer Center, Saitama Cancer Center, Keiyukai Sapporo Hospital, and Niigata Cancer Center. STRATEGY FOR NOVEL THERAPEUTIC TARGET DISCOVERY

To identify molecules involved in pulmonary carcinogenesis and select those that could lead to the development of new molecular therapies and/or predictive biomarkers, we have established a systematic screening system (Figure 1). We collected 120 frozen lung cancer tissue samples from 2002 to 2005, including 86 NSCLCs (71 adenocarcinomas, 14 squamous-cell carcinomas, and 1 adenosquamous carcinoma) and 34 small-cell lung cancers. The screening strategy for molecular targets discovery was as follows: (i) identification of highly transactivated genes in a large proportion of 120 lung cancers by genome-wide screening using the complementary DNA microarray representing 27,648 genes or expressed sequence tags from enriched tumorcell populations (cancer cells dissected by laser microdissection); (ii) verification of little or no expression of each of the candidate genes in normal tissues by complementary DNA microarray and northern blot analyses; (iii) validation of the clinicopathological significance of high levels of expression of the gene with tissue microarray containing approximately 300 archived lung cancer samples; (iv) verification of the critical role of each target gene in the growth or invasiveness of cancer cells by RNA interference and cell growth/invasion assays (i–iv representative results of two genes, CDCA1 and KNTC2, are shown in Figure 2a–c); (v) evaluation of their usefulness as targets for passive immunotherapy and/or as a serum biomarker for lung cancer by high-throughput enzyme-linked immunosorbent Selection of genes overexpressed in the majority of lung cancers (cDNA microarray – 120 clinical lung cancers)

Selection of genes upregulated in lung cancer, but not expressed in normal tissues (northern blotting) Verification of the clinicopathological significance of the gene products (tissue microarray) Measurement of the serum protein levels (ELISA) Selection of genes that were essential for cell growth and invasion (RNA interference and/or antibody-based assays)

New therapies Therapeutic antibodies Small molecular compounds Interacting peptides Cancer peptide vaccines

New biomarkers Early cancer detection Molecular staging of disease Chemosensitivity prediction Companion diagnostics

Figure 1  Genomics-based approach toward new medicine development. cDNA, complementary DNA. Clinical pharmacology & Therapeutics | VOLUME 94 NUMBER 2 | August 2013

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assay and proteomics analysis; and (vi) screening of the epitope peptides recognized by human histocompatibility leukocyte (HLA)-A*0201- or HLA-A*2402-restricted cytotoxic T lymphocyte (CTL).8–14 Using this systematic approach, we have identified 30 molecules that appear to fall into the category of oncoantigens whose overexpression is an important feature of the malignant nature of cancer cells and that have very high immunogenicity to induce antigen-specific CTLs in cancer patients.15–51 We went on to further validate the molecules identified as potential targets for the development of antibodies, small-molecular compounds, growth-suppressive cell-permeable peptides, and cancer vaccines. THERAPEUTIC ANTIBODIES

Cell-surface and/or secretory proteins unique to lung cancer cells are considered more accessible to immune mechanisms and drug-delivery systems, and therefore identification of such proteins is an effective strategy for the development of novel therapeutics. We have therefore been screening genes encoding such proteins and have identified overexpression of ADAM8 (a disintegrin and metalloproteinase domain-8), DKK1 (dickkopf-1), LY6K (lymphocyte antigen 6 complex locus K), NECTIN4, and EBI3 (Epstein–Barr virus–induced gene 3) in the majority of lung cancer cases.38–44 We subsequently established enzyme-linked immunosorbent assays to measure serum levels of these proteins and found that serum ADAM8, DKK1, LY6K, NECTIN4, and EBI3 levels were higher in lung cancer patients than in healthy controls, suggesting that these tumor markers individually or in combination could significantly improve the screening sensitivity of lung cancer.38–44 Validation of these results using a different set(s) of samples will be required before widespread applicability can be envisioned. Our data revealed that some of the secreted proteins we identified, such as NECTIN4 and DKK1, appeared to play an important role in cell growth in an autocrine/paracrine ­manner.42,43 Therefore, we examined whether antibodies to these proteins could be used as therapeutic neutralizing antibodies. We found that both anti-DKK1 antibody and anti-NECTIN4 antibody inhibited the growth of lung cancer cells in vitro and suppressed the growth of engrafted lung tumors in vivo. Tumor tissues treated with anti-DKK1 or anti-NECTIN4 antibodies displayed significant fibrotic changes and a decrease in viable cancer cells without apparent toxicity in mice, suggesting antibodies to NECTIN4 or DKK1 as potential theranostic tools for diagnosis and treatment of lung cancer.42,43 ANTICANCER CELL-PERMEABLE PEPTIDE THERAPY

The development of small-molecular compounds to inhibit protein–protein interaction has been known to be very difficult. However, several groups recently reported that inhibition of the protein–protein interaction of cancer-related proteins could effectively block the function of the complex in vivo and in vitro.52,53 For example, the cell-permeable peptide derived from AMP1 specifically blocked the AMP1/cortactin binding and effectively inhibited breast cancer invasion and metastasis.52 219

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Figure 2  Two representative proteins identified as drug target molecules through our systematic approach. (a) Expression of CDCA1 (left) and KNTC2 (right) in 23 normal human tissues by northern blot analysis. Among 23 normal organs we examined, these two genes were exclusively and abundantly expressed in testis. (b) Immunohistochemical analysis of clinical samples from surgically resected NSCLC tissues using anti-CDCA1 (left) and anti-KNTC2 (right) antibodies. We classified patterns of CDCA1/KNTC2 expression as absent/weak (scored as 0 or 1+) or strong (scored as 2+). Of the 282 NSCLC cases examined, 95 (33.7%) revealed strong CDCA1 staining (score 2+), 113 (40.1%) were stained weakly (score 1+), and no staining (score 0) was observed in 74 cases (26.2%). For KNTC2, strong staining (score 2+) was observed in 112 cases (39.7%), weak staining (score 1+) in 122 cases (43.3%), and no staining (score 0) in 48 cases (17%). All of these tumors were surgically resected NSCLC cases, and no staining was observed in any of their adjacent normal lung tissues. (c) Growth-suppressive effect by RNAi for CDCA1 or KNTC2. The cells that were transfected either si-CDCA1 or si-KNTC2 showed reduction of their transcripts (RT-PCR) and suppression of the growth of A549 lung cancer cells (colony formation assay). (d) Kaplan–Meier analysis of tumor-specific survival times according to CDCA1 (top) and KNTC2 (bottom) expression based on the results of tissue microarrays (n = 282). (e) Inhibition of cell growth of LC319 NSCLC cells by the peptide corresponding to a part of CDCA1. The cell-permeable peptide (11R-CDCA1398–416) inhibited the binding of CDCA1–KNTC2. MTT assay shows the growth-suppressive effect of the 11R-CDCA1398–416 peptide that was introduced into LC319 cells overexpressing both CDCA1 and KNTC2. CDCA1, cell division–associated 1; KNTC2, kinetochoreassociated 2; NSCLC, non–small cell lung cancer; RNAi, RNA interference; RT-PCR, reverse-transcription polymerase chain reaction. Reprinted from ref. 34.

The anti-MDM2 peptide blocking TP53–MDM2 interaction was also reported to induce rapid accumulation of TP53, activation of apoptosis-inducing genes, preferential killing of retinoblastoma cells, and minimal retinal damage after intravitreal 220

injection.53 The concept of selective killing of tumor cells with no or minimum toxic effect on normal cells by targeting functional interaction of particular molecules is something we decided to pursue in our data set. VOLUME 94 NUMBER 2 | August 2013 | www.nature.com/cpt

state Through the use of immunoprecipitation and subsequent mass-spectrometric analysis, we have identified several combinations of oncogenic proteins whose interaction plays key roles in the proliferation and/or survival of lung cancer cells. The interactions we found are between cell division–associated 1 (CDCA1) and kinetochore-associated 2 (KNTC2); cell division cycle–associated 8 (CDCA8) and aurora kinase B (AURKB); TBC1 domain family, member 7 (TBC1D7) and tuberous sclerosis complex (TSC1); and cell division–associated 5 (CDCA5) and extracellular signal–regulated kinase (ERK). We believe that these interactions are important features of lung cancer and are therefore exploring the possibility of designing new anticancer peptides as well as small molecules to specifically target the activity of CDCA1/KNTC2, CDCA8/AURKB, TBC1D7/TSC1, and CDCA5/ERK1/2.34–37 For example, we showed that oncoprotein TBC1D7 interacts with the TSC1 protein in lung cancer cells.36 The introduction of TSC1 into cells increased the level of the TBC1D7 protein, whereas knockdown of TSC1 expression decreased its level, suggesting that TBC1D7 is probably stabilized through interaction with TSC1. Inhibition of the binding complex by a TBC1D7-derived 20-amino-acid cell-permeable peptide (11R-TBC1D7152–171), which corresponded to the binding domain to TSC1, effectively suppressed cancer cell growth.36 Similarly, as shown in Figure 2d,e, CDCA1 interacts with KNTC2, and higher levels of expression of these two proteins were associated with poor prognosis.34 We found that CDCA1 binds to a C-terminal region of KNTC2, and this interaction could be blocked by treatment with an 11R-CDCA1398–416 cellpermeable peptide and result in the suppression of cancer cell growth.34 These two results suggest that selective inhibition of the protein–protein interaction by peptides, or possibly by small molecules that mimic the peptide, could be a promising therapeutic strategy for development of novel lung cancer therapeutics. In addition, we found that ERK interacted with and phosphorylated oncoprotein CDCA5 at serine 209 in vivo.37 Functional inhibition of the interaction between CDCA5 and ERK by a cellpermeable peptide corresponding to a 20-amino-acid sequence in CDCA5, which included the serine 209 phosphorylation site by ERK, significantly reduced phosphorylation of CDCA5 and resulted in growth suppression of lung cancer cells.37 Selective suppression of the ERK-CDCA5 pathway by this cell-permeable peptide or a small molecule that mimics the peptide could be a

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an effective strategy. Our data indicate that inhibition of the interaction of oncoproteins and their binding partners that are highly expressed in lung cancers may be a potential strategy for development of new types of molecular therapies. SMALL-MOLECULAR COMPOUNDS

On the basis of the gene-expression profiles of lung cancer cases using complementary DNA microarray, we identified TTK (therine threonine kinase; alias monopolar spindle 1), whose expression was more than five times higher in the majority of lung cancer tissues than in their corresponding normal tissues. The expression of TTK was hardly detectable in normal tissues, except for testis.9–13,45 We believe TTK kinase is an attractive cancer drug target owing to the important role in the centrosome duplication, the spindle assembly checkpoint, and the maintenance of chromosomal stability. Therefore, we performed high-throughput screening and found lead compounds that inhibited the TTK kinase activity.51 A design based on JNK inhibitors with aminopyridine scaffold and subsequent modifications identified diaminopyridine (compound no. 9) with a half-maximal inhibitory concentration of 37 nmol/l. An X-ray structure of compound no. 9 revealed that the Cys604 carbonyl group of the hinge region flips to form a hydrogen bond with the aniline NH group in compound no. 9. In addition, optimization of compound no. 9 led to compound no. 12, which had improved cellular activity, suitable pharmacokinetic profiles, and good in vivo efficacy in a mouse A549 xenograft model, suggesting that TTK can serve as a promising target for the development of anticancer therapeutics. Moreover, compound no. 12 displayed excellent selectivity over 95 kinases we tested, indicating that the unusual flipped-peptide conformation of the kinase probably contributed to its selectivity.51 CANCER VACCINES

Oncoantigens are proteins that are very specifically expressed in cancer cells and have oncogenic activity and high immunogenicity.54,55 Oncoantigens are considered promising targets for immunotherapy such as therapeutic cancer vaccines. Using the systematic screening system for oncoantigens described above, we searched for targets for cancer vaccines and identified cancer-testis or oncofetal antigens that were transactivated in the majority of lung cancers and are essential for the growth and/or survival of cancer cells. For the development of c

Figure 3  Comparison of diagnostic imaging in lung adenocarcinoma patient who showed a complete response to cancer-vaccine monotherapy. (a) Chest CT scan at diagnosis. (b) Chest CT scan before vaccination monotherapy. (c) Chest CT scan after 5 months of peptide vaccination monotherapy. CT, computed tomography. Clinical pharmacology & Therapeutics | VOLUME 94 NUMBER 2 | August 2013

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therapeutic cancer vaccines, we searched for the epitope peptides recognized by human HLA-A*0201 and/or HLA-A*2402restricted CTL by ELISPOT assay,45–50 that is, epitope peptides that can elicit CTL to the TTK, lymphocyte antigen 6 complex locus K (LY6K), insulin-like growth factor (IGF)-II mRNAbinding protein 3 (IMP-3), CDCA1, kinesin family member 20A (KIF20A), cell division cycle 45-like (CDC45L), and forkhead box M1 (FOXM1). We screened dozens of 9- or 10-amino-acid peptides, each of which corresponded to a part of TTK, LY6K, IMP-3, CDCA1, KIF20A, CDC45L, or FOXM1, and found that five epitope peptides could strongly induce CTL activity in cancer patients.45–50 Because the data strongly imply that LY6K, CDCA1, and KIF20A are novel tumor-associated antigens recognized by CTL, and the HLA-A*24-restricted epitope peptides can induce potent and specific immune responses against lung cancer cells, which express LY6K, CDCA1, and KIF20A, we moved on to conduct a phase I clinical trial (NCT01069575) for advanced, HLA-A*2402-positive NSCLC patients who failed standard therapy. In this study, we used the combination of a 10-mer LY6K peptide, a 9-mer CDCA1 peptide, and a 10-mer KIF20A peptide that correspond to epitopes recognized by CTLs. NSCLC patients were immunized with 1, 2, or 3 mg/body of each peptide mixed with 1-ml Montanide ISA-51 once a week. This study investigated both the feasibility and the toxicity of cancer-vaccine monotherapy, and the primary end point was safety. There are currently 18 patients enrolled in the study, and the peptide vaccine has been well tolerated in all immunized patients. The clinical response of the vaccination has been evaluated according to RECIST (version 1.0; Response Evaluation Criteria in Solid Tumors), and to date there has been one complete response (CR) (Figure 3), eight patients who have stable disease (SD), and nine who had progressive disease (PD), indicating that the diseasecontrol rate [(CR+SD/CR+SD+PD) × 100] is 50%. The mean overall survival time and the mean progression-free survival to date are 617 and 109 days, respectively. As for the patient who showed CR with no sign of recurrence at May 2013 to cancervaccine monotherapy, this patient was treated with the cocktail of 1 mg each of the three-peptide vaccine after standard therapies including chemoradiotherapy (60Gy irradiation to left lobe primary tumor + two cycles of carboplatin and taxol) and additional systemic chemotherapy including two cycles of cisplatin and gemcitabine. The patient showing CR has been stable for 5 months after the administration of the peptide monotherapy and is receiving the therapy without any signs of recurrence of the tumor (progression-free survival period is 994 days). The cancervaccine therapy using the cocktail of three peptides demonstrated good tolerability as well as a promising disease control rate, and it therefore warrants further clinical studies. On the basis of evidence obtained in several clinical studies using epitope peptides derived from oncoantigens identified by our group, we are conducting an International Conference on Harmonisation—Good Clinical Practice–based clinical study using the combination of three peptides derived from LY6K, CDCA1, and IMP-3 proteins in patients with advanced NSCLC refractory to standard therapy (NCT01592617). 222

CONCLUSION

Through the availability of human genome and proteome information and new technologies for high-throughput genetic/ genomic analysis, we have been able to characterize “individual cancer profiles” and identify molecules involved in critical pathways of pulmonary carcinogenesis. Although the applicability of our data with respect to tumor heterogeneity and interpatient variability will need to be examined in various independent sample sets, a comprehensive approach of characterization of cancer profiles, biomarker identification, validation, revalidation, and ultimately application using hundreds of archived tumor tissue samples is an effective strategy for selecting a defined set of genes that may be good target molecules for the development of novel therapies. These markers can also serve as companion diagnostics that may be able to predict responsiveness of cancer patients to drugs. Using this unique approach, we have identified more than 80 epitope peptides, from 38 cancerspecific proteins of many cancer types, that are able to induce CTLs very effectively. Twenty-five of these have already been tested in clinical trials for various types of human solid tumors (i.e., esophageal cancer). ACKNOWLEDGMENTS The clinical samples were provided as part of collaborative studies with Hokkaido University, Kanagawa Cancer Center, Saitama Cancer Center, Keiyukai Sapporo Hospital, and Niigata Cancer Center. We acknowledge all our collaborators and their colleagues. CONFLICT OF INTEREST Y.N. holds stock in and is a scientific adviser to OncoTherapy Science. The other authors declared no conflict of interest. © 2013 American Society for Clinical Pharmacology and Therapeutics

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