Published Ahead of Print on April 10, 2015, as doi:10.3324/haematol.2014.122069. Copyright 2015 Ferrata Storti Foundation.
Targeting the spliceosome in chronic lymphocytic leukemia with the macrolides FD-895 and pladienolide B by Manoj K. Kashyap, Deepak Kumar, Reymundo Villa, James J. La Clair, Chris Benner, Roman Sasik, Harrison Jones, Emanuela M. Ghia, Laura Z. Rassenti, Thomas J. Kipps, Michael D. Burkart, and Januario E. Castro Haematologica 2015 [Epub ahead of print] Citation: Kashyap MK, Kumar D, Villa R, La Clair JJ, Benner C, Sasik R, Jones H, Ghia EM, Rassenti LZ, Kipps TJ, Burkart MD, and Castro JE. Targeting the spliceosome in chronic lymphocytic leukemia with the macrolides FD-895 and pladienolide B. Haematologica. 2015; 100:xxx doi:10.3324/haematol.2014.122069 Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid dissemination of science. Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts that have completed a regular peer review and have been accepted for publication. E-publishing of this PDF file has been approved by the authors. After having E-published Ahead of Print, manuscripts will then undergo technical and English editing, typesetting, proof correction and be presented for the authors' final approval; the final version of the manuscript will then appear in print on a regular issue of the journal. All legal disclaimers that apply to the journal also pertain to this production process.
Targeting the spliceosome in chronic lymphocytic leukemia with the macrolides FD-895 and pladienolide-B
Manoj K. Kashyap,1 Deepak Kumar,1 Reymundo Villa,2 James J. La Clair,2 Chris Benner,5 Roman Sasik,4 Harrison Jones,1 Emanuela M. Ghia,1 Laura Z. Rassenti,1,3 Thomas J. Kipps, 1,3 Michael D. Burkart,2 Januario E. Castro1,3
1
Moores Cancer Center, 2Department of Chemistry and Biochemistry, 3CLL Research
Consortium, and Department of Medicine, 4Center for Computational Biology, Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, 5
Integrative Genomics and Bioinformatics Core, Salk Institute for Biological Studies, La
Jolla, CA 92037, USA Statement of equal author’s contribution: M.K.K., D.K., R.V. and J.J.L. contributed equally to this study. Running heads: Targeting the spliceosome in leukemia and lymphoma
Correspondence Januario E. Castro, Moores Cancer Center, University of California San Diego, 3855 Health Science Drive, La Jolla, CA 92093-0820, e-mail:
[email protected] or Michael D. Burkart, Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 920930358, e-mail:
[email protected]
Key words: splicing, CLL, polyketide, pladienolide, FD-895, spliceosome modulator, alternative splicing, intron retention, SF3b, RNA-seq.
1
ABSTRACT
RNA splicing plays a fundamental role in human biology. Its relevance in cancer is rapidly emerging as demonstrated by spliceosome mutations that determine the prognosis of patients with hematological malignancies. We report studies using FD-895 and pladienolide-B in primary leukemia cells derived from chronic lymphocytic leukemia patients and leukemia-lymphoma cell lines. We found that FD-895 and pladienolide-B induce an early pattern of mRNA intron retention – spliceosome inhibition. This process was associated with apoptosis preferentially in cancer cells as compared to normal lymphocytes. The pro-apoptotic activity of these compounds was observed regardless of poor prognostic factors such as Del(17p), TP53 or SF3B1 mutations and was able to overcome the protective effect of culture conditions that resemble the tumor microenvironment. In addition, the activity of these compounds was observed not only in vitro but also in vivo using the A20 lymphoma murine model. Overall, these findings show evidence for the first time that spliceosome modulation is a valid target in chronic lymphocytic leukemia and provide additional rationale for the development of spliceosome modulators for cancer therapy.
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INTRODUCTION
Chronic lymphocytic leukemia (CLL) is the most common adult leukemia.1 Despite of improvement in survival of patients that are treated with chemoimmunotherapy,2 still there is no cure for this disease, except for those patients that undergo allogeneic bone marrow transplant. High-risk patients, such as those with deletions in chromosome 17 Del(17p) or TP53 mutations, generally fail to respond to chemotherapy and have a very poor prognosis.3 As such, there is a need for development of therapeutic agents that target novel pathways in CLL.4 Splicing, the removal of introns and joining of exons from nascent pre-mRNA, has gained attention as a target for cancer therapy given the distinct splicing patterns identified both in tumor cells and metastatic tumor populations.5,6 Recently, a series of studies identified heterozygous missense mutations in U2AF1 and SF3B1 genes associated with myelodysplastic syndrome (MDS), and have shown that splicing factor 3B subunit 1 (SF3B1) is frequently mutated in MDS,7,8 and CLL.9,10 This, combined with the identification of small molecules that target the spliceosome, motivated us to explore the application of these agents to CLL. Identified in 1994,
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FD-895 was the first member of a large family of polyketides
isolated from related strains of Streptomyces platensis, which includes pladienolide-B (PLAD-B) and pladienolide-D (PLAD-D) (Supplementary Figure S1).12 PLAD-B targets the splicing factor subunit SF3b and this interaction is postulated to be responsible for its mechanism of antitumor activity.13 Pladienolide-resistant clones from WiDr and DLD1 colorectal-cancer cell-lines shared an identical mutation at Arg1074 (R1074H) in the SF3B1 gene suggesting that that this mutation is critical for its anti-cancer activity via spliceosome modulation.14 15 Additional work has also identified other small molecules with splicing modulator activity including spliceostatin A, herboxidiene, isoginkgetin, and E7107 a compound that has been tested in phase I clinical studies showing clinical activity, albeit with unexpected visual toxicities.16 Additional data will be required to define the role of this and other related compounds as potential anti-cancer agents. 3
Here, we present studies using FD-895 and PLAD-B on primary leukemia cells derived from CLL patients and leukemia and lymphoma cell lines. We anticipate that these studies will provide the foundation for future development of pharmacologicallyoptimized spliceosome modulators.17
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METHODS For in vivo study, PLAD-B was purchased from Santa Cruz Biotechnology (Catalog # sc391691). Fludarabine (F-ara-A) (Catalog # F9813) and Bendamustine (Catalog # B5437) were obtained from Sigma-Aldrich. Peripheral blood mononuclear cells (PBMC) from CLL patients were obtained from the CLL Research Consortium tissue bank. After CLL diagnosis was confirmed,18 patients provided written informed consent for blood sample collection on a protocol approved by the Institutional Review Board of UCSD, in accordance with the Declaration of Helsinki.19 The animal study protocol was approved by the Medical Experimental Animal Care Committee - University of California, San Diego. Normal B cell isolation: Normal B cells (NBC) were purified from buffy coats of healthy volunteer donors. Positive isolation with Dynabeads CD19 pan B (Life Technologies) and DETACHaBEAD CD19 (Life Technologies) were used to achieve more than 95% purity by flow cytometry analysis. Additional techniques, methods and list of PCR primers (Supplementary Table-1) used in the study are provided in the supplementary information (SI).
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RESULTS
FD-895 and PLAD-B induce intron retention – spliceosome modulation Analysis of RNA-seq data between CLL and NBC showed differences in splicing efficiency between CLL and NBC. In order to quantitatively assess splicing efficiency, the intron retention (IR) ratios were calculated for each gene by comparing the RNA-seq read density in exons, measured in fragments per kilobase per million fragments mapped (FPKM), relative to the RNA-seq density of introns (Supplementary Table-2). Comparison of intron retention ratios showed that several genes have widespread changes in splicing efficiency, resulting in a basal increase of intron / exon RNA ratios in untreated CLL compared to NBC (1.4 fold average increase, Figure 1A and Supplementary Figure 2A, 2B). On an average 71% of sequenced genes showed increased IR in CLL compared to NBC (Supplementary Table-3). More over, after incubation of CLL cells with FD-895 or PLAD-B there was an additional increase in IR ratios (2.2 fold average increase, Figure 1B, Supplementary Figure 2C-2D, and 3A), but this was not observed after F-ara-A treatment (Figure 1C-1D). We observed that treatment with FD-895 or PLAD-B induced IR more significantly in genes with the lowest basal IR suggesting a “de novo” spliceosome inhibition induced by this compounds rather than exacerbation of an existing abnormal splicing process (Supplementary Figure 4). DNAJB1, a member of the spliceosome system, was one the genes that showed increase in the IR ratio after treatment with FD-895 or PLAD-B (7 fold increase - Figure 1 A-C) and because of that, this gene was selected for validation studies described below. We performed an unsupervised cluster analysis of 3,500 highly expressed genes using IR ratios of CLL and NBC samples treated with FD-895, PLAD-B, F-ara-A or untreated controls. We observed that IR ratios from untreated samples clustered together with Fara-A samples, indicating minimal IR changes induced by F-ara-A regardless of the cell type (CLL or NBC). Distinctively, FD-895 and PLAD-B treated samples showed higher IR ratios compared to untreated controls or F-ara-A treated samples (Figure 1D, Supplementary Figure 5). Additionally, we analyzed gene expression profiles of the same 6
samples and we found that unlike IR ratios, gene expression profiles were segregated by cell type forming NBC and CLL clusters. Untreated cells and F-ara-A treated samples clustered together while samples treated with FD-895 or PLAD-B showed similar gene expression profiles (Figure 1E). We selected the top 50 genes that were affected by intron retention induced by FD-895 and found that the majority of those belong to pathways representing RNA splicing and gene regulation, signal transduction, ER stress and apoptosis (representative genes from those pathways include: DNAJA1, DNAJB1, ABT1, NFKB1, GPX1, SLC2A3, HERPUD1, RBM4-RBM14, RBM8A, and WTAP) – (Figure 1F, Supplementary Figure 5). There was a similar pathway distribution when the gene sampling was expanded to the top 900 targets affected by IR (Supplementary Figure 3B). Using Gene ontology (GO) functional enrichment analysis we found that incubation with FD-895 and PLAD-B induced IR mainly in pathways that are critical for RNA editing / processing, cell survival and cell cycle regulation (Figure 1G, Supplementary Figure 3B). Splicing abnormalities in these pathways were present as a baseline finding in untreated CLL cells compared with NBC, although with higher p values (Supplementary Figure 3C). We also analyzed the GO enrichment of differential gene expression induced by these compounds and found different pathways that were enhanced, including immune regulation and cell survival, while gene expression of the RNA processing and editing pathway was significantly downregulated (Figure 1G). Validation of FD-895 and PLAD-B induced IR / spliceosome modulation in CLL Because IR is a process considered to be a surrogate marker of spliceosome modulation 20
, and because of the RNA-seq data suggested a process of IR, we decided to perform a
series of validation studies in cells treated with FD-895 and PLAD-B under different conditions. For these experiments, we selected DNAJB1, a gene that has been used in previously studies9, and one of the highest ranked genes showing IR pattern (Figure 1B1C, 1F). CLL samples were incubated with a concentration gradient of FD-895, PLAD-B, bendamustine, or F-ara-A for 4 h (Figure 2A). After treatment, the levels of spliced and unspliced gene expression were evaluated by RT-PCR and qRT-PCR. While CLL cells 7
treated with nanomolar concentrations of FD-895 or PLAD-B showed IR, this was not observed in samples treated with bendamustine or F-ara-A, even at supra-physiological concentrations (Figure 2A). FD-895 or PLAD-B did not induce IR in the housekeeping gene GAPDH (Figure 2A and Supplementary Figure 6A). IR was observed in cells treated with FD-895 or PLAD-B for all three genes examined, including: DNAJB1, RIOK3 (Figure 2B, Supplementary Figure 6B), and BRD2 (data not shown). The process of IR was time-dependent and occurred within 15 min of treatment (Figure 2B). We quantified IR using real-time qRT-PCR and observed a time-dependent increase of unspliced DNAJB1 in samples treated with FD-895 or PLAD-B, but not with F-ara-A (Figure 2C). In addition, we found that FD-895 and PLAD-B induced ~4 fold increased IR for both DNAJB1 and RIOK3 in CLL cells when compared to NBC (p < 0.0001, Figure 2D-2E). The activity of FD-895 and PLAD-B in CLL cells is associated with regulation of alternative splicing The RNA-seq data showed IR in MCL-1 gene after treatment with FD-895 (Supplementary Figure 6C). Thus, we investigated whether or not spliceosome modulation / IR by FD-895 and PLAD-B was associated with regulation of alternative splicing (AS) using MCL-1 and BCL-X, two genes involved in apoptosis, which are known to depend on AS for their function - pro-apoptotic (short isoform) and antiapoptotic (long isoform).21 We observed that treatment of CLL cells with FD-895 or PLAD-B induced expression of the short / pro-apoptotic alternatively spliced isoforms for both MCL-1 (Figure 3A) and BCL-X (Figure 3B). This process was time dependent, occurring within 30 min of treatment. Contrary to that, there was no evidence of AS in NBC (Figure 3A-3B). In addition, we did not observe IR or AS in GAPDH (Figure 3C and Supplementary Figure 6A). Using gel electrophoresis and densitometric analysis we calculated the ratio of small to large (S/L) isoforms for MCL-1 and BCL-X, which is used as a marker for apoptosis.21 We found a higher S/L ratio (p < 0.0001) for MCL-1 (Figure 3D) and BCL-X (Figure 3E) in CLL cells treated with FD-895 or PLAD-B, but there were no changes in NBC. S/L ratios remained unchanged in F-ara-A treated cells. 8
FD-895 and PLAD-B induce early irreversible commitment to apoptosis in CLL cells within 2 h of treatment We cultured CLL cells in 100 nM FD-895 or 100 nM PLAD-B, and examined apoptosis over time. The minimum time for the irreversible development of apoptosis was determined by exposing the CLL cells to FD-895 or PLAD-B over increasing incubation times up to 48 h, followed by washing twice with media, and continuing to culture for a total of 48 h. We found that the effect of both FD-895 and PLAD-B is irreversible and required incubation for at least 2 h to induce apoptosis (Figure 4A). Determination of FD-895 and PLAD-B IC50 values and effect of co-culture conditions that resemble the tumor microenvironment FD-895 and PLAD-B induced apoptosis in CLL at nanomolar concentrations with an IC50 in the range of 5.1-138.7 nM (Figure 4B-4C). In contrast, NBC and normal T cells were resistant to the activity of both compounds, and the IC50 value was not achieved even when concentrations > 1 µM were used (Figure 4D). In order to assess the activity of FD-895 and PLAD-B under stringent conditions that resemble the anti-apoptotic tumor microenvironment,16 we incubated CLL cells with increasing concentrations of FD-895, PLAD-B (0.01 -1 µM) and F-ara-A (1-10 µM) alone or with stroma-NK-tert cells. Stroma-NK-tert cells increased the viability of CLL cells and inhibited the activity of F-ara-A by more than 50%. However, FD-895 and PLAD-B were able to overcome the anti-apoptotic effect of the stroma cell support without causing direct cytopathic effect on NK-tert cells (Figure 4E). FD-895 and PLAD-B selectively induce apoptosis in CLL cells but not in normal lymphocytes in a TP53 independent manner We evaluated the anti-leukemia activity of FD-895 or PLAD-B in cells derived from CLL patients with Del(17p) and /or inactivating mutations in TP53. The samples were cultured alone or with stroma-NK-tert cells and harvested post-treatment at 48 h and apoptosis was measured by flow cytometry. Cells derived from CLL patients with Del(17p) and/or TP53 mutations (% Del(17p) by FISH 66-99.5%) displayed resistance to F-ara-A, with IC50 values > 10 µM (Figure 4C), while wild type CLL samples underwent
9
apoptosis when treated with F-ara-A at IC50 values of ~1 µM (Figure 4B). Contrary to the differential sensitivity to F-ara-A based on TP53 status, we observed that CLL samples underwent apoptosis with FD-895 or PLAD-B at IC50 values of 10-50 nM, regardless of the presence of Del(17p) or TP53 mutations (Figure 4B-4C, Figure 5). Moreover, B and T lymphocytes from healthy volunteers were resistant to the pro-apoptotic activity of FD895 or PLAD-B, and show significantly lower levels of apoptosis after 48 h in culture compared with CLL cells (Figure 4D, Figure 5). Evaluation of the activity of FD-895 and PLAD-B across different human leukemia and lymphoma cell lines In order to validate our observations in CLL, we studied the pro-apoptotic activity of FD895 and PLAD-B in different cancer cell lines. Both FD-895 and PLAD-B induced apoptosis in TP53 mutant cell lines including Raji, Jurkat, and Ramos (Figure 4F-H) with IC50 values in the nanomolar range (1-70 nM for FD-895 and 5.1-73.1 nM for PLAD-B). FD-895 and PLAD-B induce apoptosis in CLL cells independently of TP53 and SF3B1 mutational status We also tested whether treatment with FD-895 or PLAD-B could induce apoptosis in cells from CLL patients with wild type or mutant SF3B1 (Supplementary Table 4). We found that treatment with 100 nM FD-895 (Figure 5A) or 100 nM PLAD-B (Figure 5B) induced cell death in CLL cells regardless of their SF3B1 mutational status with levels of apoptosis that were significantly higher compared to normal lymphocytes (Figure 5). FD-895 and PLAD-B induce apoptosis via caspase-dependent pathway We performed a colorimetric-proteolytic assay to determine if FD-895 or PLAD-B induced apoptosis through a caspase-dependent mechanism. Treatment of CLL cells with FD-895 or PLAD-B induced activation of caspases 3, 6, 8, and 9 (Figure 6A). There was no caspase activation in NBC treated with either FD-895 or PLAD-B (Figure 6A). Caspase dependency was corroborated by co-culturing the cells with different compounds (F-ara-A, FD-895 or PLAD-B) in the presence of increasing concentrations of Z-VAD, a pan-caspase inhibitor. We observed that Z-VAD inhibited apoptosis induced by FD-895
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and PLAD-B suggesting that caspase activation is necessary for FD-895 and PLAD-B induced apoptosis (Figure 6B). Apoptosis induced by FD-895 or PLAD-B in CLL is associated with regulation of PARP and Mcl-1 We analyzed the levels of PARP and Mcl-1 by western blot analysis in CLL cells after treatment with 100 nM FD-895, 100 nM PLAD-B or 10 µM F-ara-A. CLL cells were treated for 6 h and 24 h for Mcl-1 and PARP, respectively. We observed increased levels of cleaved PARP (89 kDa) in CLL cells treated with FD-895, PLAD-B or the control Fara-A. However, as observed in the Mcl-1 AS experiments, only samples treated with FD-895 or PLAD-B showed downregulation of the anti-apoptotic isoform of Mcl-1 (48 kDa) (Figure 6C). Induction of intron retention and apoptosis correlates with tumor regression in vivo FD-895 and PLAD-B both induce apoptosis and IR at nanomolar concentrations in a wide range of cell lines including A20 murine lymphoma (Figure 6D). To assess if the in vitro activity of these compounds correlates with antitumoral activity in vivo, we treated BALB/c mice bearing subcutaneous A20 lymphoma tumors with intraperiotoneal injections of PLAD-B for 5 consecutive days. We observed tumor regression and improved survival (p < 0.001) in the group of mice treated with PLAD-B. There was a dose dependent effect that favored higher doses of this compound (3mg/Kg/Day vs. 10mg/Kg/Day). In contrast, mice injected with vehicle or dexamethasone control continued to show tumor progression (Figure 6E). After 35 days of follow up, none of the vehicle or dexamethasone control treated mice survived while 33% (low dose) and 83% (high dose) of PLAD-B treated mice were still alive (Figure 6F). None of the mice treated with PLAD-B showed evidence of toxicity, behavioral changes, diarrhea, rough coat, withdrawal, weigh loss or gross visual impairment.22
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DISCUSSION Gene expression consists of several steps, including transcription, pre-mRNA processing (capping, splicing, and poly-adenylation), mRNA surveillance, and mRNA export. These steps are extensively coupled to form ‘gene expression factories,23 whose modulation, if selective, offers new avenues into cancer therapy. Among those events, the regulation of mRNA maturation by the spliceosome has gained significant recognition as potential therapeutic target due to the recent discovery of mutations in genes associated with the spliceosome. These include mutations in U2AF35, ZRSR2, SRSF2 and SF3B1 that have been reported in solid tumors as well as hematological malignancies including CLL and MDS.9,10,24 In CLL, SF3B1 mutations are found in ~10-15% of cases and constitute an independent prognostic factor associated with rapid progression and short survival with a frequency that increases after exposure to chemotherapy, suggesting clonal selection of SF3B1 mutant cells after treatment.9,10,25 SF3B1 is upregulated in CLL compared with NBC, possibly due to epigenetic regulation through hypomethylation.26 In addition, in silico data strongly suggest that hot spot mutations of SF3B1 in CLL are in fact oncogenic gainof-function mutations conferring SF3B1 attributes of a proto-oncogene.27 Therefore, it is plausible that the expression of mutated SF3B1 could result in “hyper-activation” of the spliceosome system leading to the generation of oncogenic / leukemogeneic alternative spliced mRNA isoforms that participate in cell survival, proliferation and possibly chemoresistance. Although the causative link between SF3B1 mutation and CLL pathogenesis remains unclear, recent findings suggest that SF3B1 mutations might be linked not only to deregulation of the spliceosome but also to genomic instability and epigenetic alterations.28 In this project, we studied FD-895 and PLAD-B, two polyketides, whose biological activity has been associated with the ability to modulate splicing by targeting SF3b.15 We evaluated the effects of these two compounds in primary leukemia cells from CLL patients as well as normal lymphocytes and leukemia-lymphoma cells lines. RNA-seq transcriptome analysis allowed us to analyze the effect of FD-895 and PLAD-B from a global perspective. We use FPKM intron / exon ratios as a surrogate marker of IR 12
/ spliceosome modulation.13,29 By calculating IR ratios, we can estimate the relative efficiency of splicing for each gene and indirectly assess the functional status of the spliceosome system. Our initial experiments showed an increased pattern of IR in untreated CLL compared with NBC. This was somehow unexpected, however, amplified IR has been recently reported in solid tumors including lung and breast cancer.30,31 To our knowledge, this is the fist time that a basal increase in IR has been reported in CLL or hematological malignancies. This suggests that base line abnormalities which involve RNA processing / spliceosome system could be implicated in CLL leukemogenesis. We observed that FD-895 and PLAD-B induced very rapidly a generalized process of IR where the majority of genes (>82%) showed intronic sequences after incubation with this agent but no intron retention in housekeeping gene e.g. GAPDH and intronless genes. The majority of genes (25%) with the highest levels of IR belonged to the Gene Regulation / RNA splicing pathway. This strongly suggests that the IR effect mediated by FD-895 and PLAD-B involves a broad process that is not stochastic in nature and that certain pathways, mainly RNA processing / editing, are highly sensitive to the effect of these compounds and may provide an explanation for their mechanism of action. In addition, genes with a high basal IR ratio showed the lowest rates of IR increase after treatment. Contrary to that, genes that initially had low levels of IR were the ones that showed the highest post-treatment increase in IR (Supplementary Figure 4). Overall, this suggests that the activity of the compounds used in our experiments induced a “de novo” pattern of spliceosome inhibition more than exacerbation of an existing abnormal splicing process. Our data show that structurally similar macrolides (PLAD-B & FD-895) demonstrate common potent pro-apoptotic activity at nanomolar concentrations in vitro and in vivo. FD-895 and PLAD-B induced an early and irreversible commitment to apoptosis within 2 hours of treatment (Figure-4A). These nanomolar concentrations compare favorably with pharmacokinetic data of E7107, a compound used in clinical trials.16 In addition, these polyketides showed not only to be active in vitro but also demonstrated encouraging
13
clinical activity in vivo using the A20 lymphoma mouse model. Overall, this suggests the potential applicability of these compounds in cancer therapy. We found a striking difference between the activity of FD-895 and PLAD-B compared with chemotherapy agents such as bendamustine and F-ara-A. Even though CLL cells ultimately die after chemotherapy incubation, we never observed evidence of IR induced by these agents. Contrary to that, a hallmark of apoptosis induced by FD-895 and PLADB was an early IR induction. IR after incubation with FD-895 and PLAD-B was not only very rapid, within minutes of incubation, but also occurred at nanomolar concentrations and was at least 50% to 75% higher in CLL cells compared with NBC. Overall, these findings strongly suggest that the mechanism(s) of action of FD-895 and PLAD-B is mediated, at least in part, by IR / spliceosome modulation. The pro-apoptotic activity of FD-895 and PLAD-B was observed in all malignant cells tested (CLL, Ramos, Jurkat, Raji, A20 mouse lymphoma cell line), but it was much lower in normal T and B-lymphocytes suggesting that malignant cells may be more dependent on the spliceosome system for their survival. This finding shows a therapeutic window that could facilitate future clinical development of this class of compounds. Not only we observed IR induced by FD-895 and PLAD-B but also we found that these compounds have the ability to regulate AS. We demonstrated this by using apoptosis related genes (MCL-1, BCL-X), which are known to be dependent on AS for their function.32 After incubation with FD-895 and PLAD-B we observed a change of isoform ratios that favor the expression of pro-apoptotic isoforms. This was not observed with chemotherapy agents such as F-ara-A or bendamustine. It is very likely that modulation of AS induced by these compounds is broader and may involve other genes critical for cancer survival, proliferation etc. Notoriously, we did not observe AS induced by FD895 or PLAD-B in normal lymphocytes suggesting that this is probably a very important pro-apoptotic mechanism of action that explains the differential sensitivity observed in CLL compared to NBC. In our experiments we used an in vitro model of stromal cell support that mimics conditions present in the tumor microenvironment. This is important, as the tumor microenvironment has been shown to provide anti-apoptotic protection to cancer cells 14
including CLL and could be responsible for chemoresistance as well as persistence of minimal residual disease (MRD).33 Under these conditions, we showed that the proapoptotic activity of FD-895 and PLAD-B overcomes the protective effect of the microenvironment, suggesting that spliceosome modulators may have potential applications in refractory cancer and MRD eradication. The pro-apoptotic activity of FD-895 and PLAD-B was independent of Del(17p) - TP53 or SF3B1 mutations found in CLL. Both of these compounds displayed comparable activity in cells from CLL patients as well as cell lines with these genetic abnormalities and resistance to F-ara-A (known for its p53 dependent cytotoxicity). The fact that the activity of FD-895 and PLAD-B is independent of p53 opens significant opportunities for development of therapies for refractory malignancies where the prevalence of p53 dysfunction is significantly high.34 In addition, finding that the pro-apoptotic activity of these agents was independent of SF3B1 mutational status was somehow unexpected, mainly because in vitro data have shown that SF3B1 mutations may confer resistance to this kind of compounds.15 Potential explanations for this observation include the following: First, all the mutations described in CLL (including the patients tested at UCSD) cluster in the heat repeat region 2-6 of the SF3b1 protein.24,35 On the other hand, the R1074H mutation that confers resistance to PLAD-B is located in a distant domain (9th heat repeat region, Supplementary Figure 7). This highlights functional differences of these mutations and their impact on FD-895 and PLAD-B activity. Second, the activity of PLAD-B and FD-895 may be more complex than simply binding to SF3B1 as these compounds can also target other members of this protein family including SF3B1, SF3B3, SF3B4, and SF3B2 and potentially other proteins of the spliceosome system. Third, the fact that all CLL samples tested were sensitive to FD-895 and PLAD-B regardless of SF3B1 mutation status, highlights the importance of this pathway in CLL and the potential relevance of a more global spliceosome deregulation in cancer. We found that the pro-apoptotic activity of FD-895 and PLAD-B in CLL cells was Caspase dependent (with activation of the intrinsic and extrinsic pathways) and correlated with changes in apoptosis related proteins including poly (ADP-ribose) polymerase (PARP) cleavage and Mcl-1 (long isoform) down regulation. It is possible that IR and AS induced by FD-895 and PLAD-B, tips over the strong anti-apoptotic protein balance that 15
favors survival in CLL and other malignancies.36 This could have potential implications in terms of synergistic activity of these compounds in combination with other anti-cancer agents. Together, our studies provide a solid foundation for further therapeutic development of this class of compounds. We have shown for the first time that FD-895 and PLAD-B induce a global pattern of IR / spliceosome modulation and regulation of alternative RNA splicing at low nanomolar concentrations, and that the functional consequence of this process leads to apoptosis in vitro and in vivo preferentially in cancer cells.
This
constitutes evidence that the spliceosome system is a relevant target in leukemia and lymphoma and potentially other cancers. Our current efforts using key structure−activity relationships are focused on adapting this knowledge to further guide analog discovery and synthesis,17 with the ultimate goal to develop novel spliceosome modulators for cancer therapy.
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ACKNOWLEDGEMENTS The authors would like to thank the following organizations for their grant support: Lymphoma Research Foundation (LRF, grant #285871) to J.E.C. and M.D.B., the American Cancer Society (RSG–06–011–01–CDD) to M.D.B., the National Institutes of Health (R01-GM086225) to M.D.B, the National Institutes of Health (PO1-CA081534)CLL Research Consortium Grant to T.J.K, J.E.C. UC San Diego Foundation Blood Cancer Research Fund (T.J.K.) and the Bennett Family Foundation (J.E.C.). AUTHORSHIP CONTRIBUTIONS M.K.K., D.K., R.V. and J.J.L. contributed equally to this study. J.E.C., M.D.B., and J.J.L conceived and guided the research. J.E.C., M.D.B., J.J.L., M.K.K., and D.K. were involved in designing of the experiments, analysis of the data, and interpretation of the results, and participated in writing the manuscript. J.J.L and R.V. and M.D.B. prepared the samples of FD-895 used within this program. M.K.K, D.K, and H.J performed experiments; L.R., E.G., T.J.K. and J.E.C. provided patient samples, and patient clinical and laboratory data.
17
REFERENCES 1. Rai KR. Pathophysiologic mechanisms of chronic lymphocytic leukemia and their application to therapy. Exp Hematol. 2007;35(4 Suppl 1):134-136. 2. Hallek M, Fischer K, Fingerle-Rowson G, et al. Addition of rituximab to fludarabine and cyclophosphamide in patients with chronic lymphocytic leukaemia: a randomised, open-label, phase 3 trial. Lancet. 2010;376(9747):1164-1174. 3. Castro JE. Treatment of patients with chronic lymphocytic leukemia with 17p deletion: the saga continues. Leuk Lymphoma. 2012;53(2):179-180. 4. Hallek M. Signaling the end of chronic lymphocytic leukemia: new frontline treatment strategies. Blood. 2013;122(23):3723-3734. 5. Kornblihtt AR, Schor IE, Alló M, Dujardin G, Petrillo E, Muñoz MJ. Alternative splicing: a pivotal step between eukaryotic transcription and translation. Nat Rev Mol Cell Biol. 2013;14(3):153-165. 6. Kim E, Goren A, Ast G. Insights into the connection between cancer and alternative splicing. Trends Genet. 2008;24(1):7-10. 7. Visconte V, Tabarroki A, Rogers HJ, et al. SF3B1 mutations are infrequently found in non-myelodysplastic bone marrow failure syndromes and mast cell diseases but, if present, are associated with the ring sideroblast phenotype. Haematologica. 2013;98(9):e105-107. 8. Visconte V, Rogers HJ, Singh J, et al. SF3B1 haploinsufficiency leads to formation of ring sideroblasts in myelodysplastic syndromes. Blood. 2012;120(16):3173-3186. 9. Wang L, Lawrence MS, Wan Y, et al. SF3B1 and other novel cancer genes in chronic lymphocytic leukemia. N Engl J Med. 2011;365(26):2497-2506. 10. Landau DA, Carter SL, Stojanov P, et al. Evolution and impact of subclonal mutations in chronic lymphocytic leukemia. Cell. 2013;152(4):714-726. 11. Seki-Asano M, Okazaki T, Yamagishi M, et al. Isolation and characterization of a new 12-membered macrolide FD-895. J Antibiot (Tokyo). 1994;47(12):1395-1401. 12. Asai N, Kotake Y, Niijima J, Fukuda Y, Uehara T, Sakai T. Stereochemistry of pladienolide B. J Antibiot (Tokyo). 2007;60(6):364-369. 13. Kotake Y, Sagane K, Owa T, et al. Splicing factor SF3b as a target of the antitumor natural product pladienolide. Nat Chem Biol. 2007;3(9):570-575. 14. Webb TR, Joyner AS, Potter PM. The development and application of small molecule modulators of SF3b as therapeutic agents for cancer. Drug Discov Today. 2013;18(12):43-49. 15. Yokoi A, Kotake Y, Takahashi K, et al. Biological validation that SF3b is a target of the antitumor macrolide pladienolide. FEBS J. 2011;278(24):4870-4880. 16. Eskens FA, Ramos FJ, Burger H, et al. Phase I pharmacokinetic and pharmacodynamic study of the first-in-class spliceosome inhibitor E7107 in patients with advanced solid tumors. Clin Cancer Res. 2013;19(22):6296-6304. 17. Villa R, Kashyap MK, Kumar D, et al. Stabilized Cyclopropane Analogs of the Splicing Inhibitor FD-895. J Med Chem. 2013;56(17):6576-6582. 18. Matutes E, Owusu-Ankomah K, Morilla R, et al. The immunological profile of B-cell disorders and proposal of a scoring system for the diagnosis of CLL. Leukemia. 1994;8(10):1640-1645.
18
19. WMA Declaration of Helsinki - Ethical Principles for Medical Research Involving Human Subjects. [http://www.wma.net/en/30publications/10policies/b3/index.html%5D. 20. Landau DA, Wu CJ. Chronic lymphocytic leukemia: molecular heterogeneity revealed by high-throughput genomics. Genome Med. 2013;5(5):47. 21. Miura K, Fujibuchi W, Unno M. Splice variants in apoptotic pathway. Exp Oncol. 2012;34(3):212-217. 22. Fox MW. The visual cliff test for the study of visual depth perception in the mouse. Anim Behav. 1965;13(2):232-233. 23. Proudfoot NJ, Furger A, Dye MJ. Integrating mRNA processing with transcription. Cell. 2002;108(4):501-512. 24. Schwaederlé M, Ghia E, Rassenti LZ, et al. Subclonal evolution involving SF3B1 mutations in chronic lymphocytic leukemia. Leukemia. 2013;27(5):1214-1217. 25. Quesada V, Ramsay AJ, Lopez-Otin C. Chronic lymphocytic leukemia with SF3B1 mutation. N Engl J Med. 2012;366(26):2530. 26. Rossi D, Bruscaggin A, Spina V, et al. Mutations of the SF3B1 splicing factor in chronic lymphocytic leukemia: association with progression and fludarabinerefractoriness. Blood. 2011;118(26):6904-6908. 27. Wu X, Tschumper RC, Jelinek DF. Genetic characterization of SF3B1 mutations in single chronic lymphocytic leukemia cells. Leukemia. 2013;27(11):2264-2267. 28. Ramsay AJ, Rodríguez D, Villamor N, et al. Frequent somatic mutations in components of the RNA processing machinery in chronic lymphocytic leukemia. Leukemia. 2013;27(7):1600-1603. 29. Cvitkovic I, Jurica MS. Spliceosome database: a tool for tracking components of the spliceosome. Nucleic Acids Res. 2013;41(Database issue):D132-141. 30. Zhang Q, Li H, Jin H, Tan H, Zhang J, Sheng S. The global landscape of intron retentions in lung adenocarcinoma. BMC Med Genomics. 2014;7:15. 31. Eswaran J, Horvath A, Godbole S, et al. RNA sequencing of cancer reveals novel splicing alterations. Sci Rep. 2013;3:1689. 32. Gao Y, Koide K. Chemical perturbation of Mcl-1 pre-mRNA splicing to induce apoptosis in cancer cells. ACS Chem Biol. 2013;8(5):895-900. 33. Burger JA, Tsukada N, Burger M, Zvaifler NJ, Dell'Aquila M, Kipps TJ. Bloodderived nurse-like cells protect chronic lymphocytic leukemia B cells from spontaneous apoptosis through stromal cell-derived factor-1. Blood. 2000;96(8):2655-2663. 34. te Raa GD, Malcikova J, Pospisilova S, et al. Overview of available p53 function tests in relation to TP53 and ATM gene alterations and chemoresistance in chronic lymphocytic leukemia. Leuk Lymphoma. 2013;54(8):1849-1853. 35. Oscier DG, Rose-Zerilli MJ, Winkelmann N, et al. The clinical significance of NOTCH1 and SF3B1 mutations in the UK LRF CLL4 trial. Blood. 2013;121(3):468475. 36. Chu P, Deforce D, Pedersen IM, et al. Latent sensitivity to Fas-mediated apoptosis after CD40 ligation may explain activity of CD154 gene therapy in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A. 2002;99(6):3854-3859.
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FIGURE LEGENDS Figure 1. RNA Sequencing analysis in samples treated with FD-895 and PLAD-B. RNA transcriptome analysis was conducted on RNA obtained from two separate CLL samples (wild type for TP53 and SF3B1) and normal B cells from healthy controls. CLL and normal B cells were incubated with 100 nM FD-895, 100 nM PLAD-B or 10 µM Fara-A for 2 h prior to harvesting. (A) Comparison of IR log2 ratios (FPKM intron / FPKM exon) in untreated CLL and untreated normal B cells. The black line represents the diagonal where IR ratios are equal in both samples. (B) Comparison of IR log2 ratios in CLL control and FD-895 treated samples. (C) RNA-seq read densities at the DNAJB1 locus, a sample gene with high IR pattern. FD-895 and PLAD-B treated samples but not F-ara-A or untreated controls showed a pattern of IR with accumulation of read densities in the regions expanding introns 1 and 2. (D) Hierarchical clustering for approximately 3,500 genes shown in the heat-map depicting the relative IR log2 ratios, and (E) Gene expression across normal B cells and CLL2 samples treated with FD-895, PLAD-B and F-ara-A shown in the heat-map for approximately 4,000 genes. A similar clustering was observed with additional CLL and normal B cells samples. (F) RNA-seq heat map showing 50 genes with high level of IR after treatment with either FD-895 or PLAD-B. (G) Heat-map showing Gene Ontology pathway enrichment (p-values) using genes with greater than 3-fold increases in either their IR or mRNA expression ratios in CLL or normal B cells in untreated control samples compared with samples incubated with FD895.
20
Figure 2. Induction of intron retention / spliceosome modulation mediated by FD895 and PLAD-B. (A) RT-PCR was used to access spliced (S) and unspliced (U) isoforms of DNAJB1 and GAPDH in CLL cells. (B) IR was evaluated by RT-PCR for DNAJB1, RIOK3 and GAPDH (used as loading control) in CLL cells after treatment (C) qRT-PCR analysis of CLL cells after treatment to evaluate levels of unspliced mRNA for DNAJB1. GAPDH was used for normalization. (D) Intron retention for DNAJB1 and (E) RIOK3 was evaluated by qRT-PCR in CLL and normal B cells using specific primers that allowed detection of intron containing regions (Supplementary Table 1). The untreated controls (CLL or NBCs) for DNAJB1 and RIOK3 were set to value 1. GAPDH was used as a control for normalization.
21
Figure 3. Regulation of alternative splicing after treatment with FD-895 or PLAD-B. (A-C). Normal B cells and CLL cells were treated with 100 nM FD-895, 100 nM PLAD-B or 10
μM F-ara-A for the indicated times. RT-PCR for (A) MCL-1, (B) BCL-X and (C) GAPDH was conducted. The RT-PCR generated two different products; long or antiapoptotic isoform (L) and short or pro-apoptotic splice isoform (S). (D-E) The levels of the L and S isoforms were evaluated after incubation 100 nM FD-895, 100 nM PLAD-B or 10 μM F-ara-A by densitometry of RT-PCR bands stained with ethidium bromide using Quantity One software (Bio-Rad). The S/L ratio was plotted as a mean of two separate experiments as shown in panel (D) for MCL-1 and (E) for BCL-X. Error bars represent SD and *** = p 4,000 mm3 (4 cm3). The treatment phase initiated when the tumors reached 5 to 7 mm in diameter, and it was administered by intraperitoneal (IP) injection daily for 5 consecutive days. Treatment cohorts included the following: (1) DMSO vehicle, (2) Dexamethasone (5mg/Kg/Day), (3) PLAD-‐B, low dose (3mg/Kg/day) or high dose (10mg/Kg/day). Statistical analysis: Data was analyzed using Prism (GraphPad Software Inc.). IC50 values were calculated by fitting sigmoid dose-‐response curves with GraphPad Prism 5.0 (GraphPad Software, Inc.). The error bars represent standard deviation (SD). Statistical differences for the
4
mean values are indicated as follows: *, **, and *** denote ples#(Q)# Quartiles (Q)
Quar>les#based#on#IR#baseline#pre6treatment#(CLL/NBC):# Q1 Q2 Q3
Q4#
Q4
Quar>le#
IR#fold#change# CLL/NBC#
%#of#genes#with#>2#fold#change#aFer# treatment#with#FD6895#
Q1#
0%0.957#
46.94%#
Q2#
0.958%1.224#
37.56%#
Q3#
1.225%1.611#
29.84%#
Q4#
1.612%16.315#
14.38%#
12
Supplementary Figure S4. Quartile analysis for assessment of change IR after FD-‐895 treatment in CLL Using the RNA sequence data presented in Figure 1A, we calculated a fold change of IR for genes found in CLL compared to a control sample from NBC. These fold change of IR (CLL vs. NBC) data was divided in quartiles using the ranges shown in this table. Then, we examined the percentage of genes from each quartile that showed a ≥ 2 fold increase in IR after treatment with FD-‐895. We found that the % of genes with ≥ 2 fold IR increase after treatment with FD-‐ 895 was inversely proportional to the base line IR ratio, with more genes that fell in this category in Q1 (lowest IR base line) compared with the other quartiles. The figure represents the Mean+/S.D. value drawn from both two independent CLL samples (CLL1 and CLL2). Y-‐axis represents the % genes showing > 2-‐fold IR increase and the x-‐axis shows each one of the quartiles (Q) -‐ Q1-‐4.
13
CLL1-Control CLL1-F-ara-A CLL2-Control CLL2-F-ara-A NBC-Control NBC-F-ara-A CLL1-FD-895 CLL1-PLAD-B CLL2-FD-895 CLL2-PLAD-B NBC-FD-895 NBC-PLAD-B
Top 50 Intron Retained Genes with FD treatement
Relative Intron Retention Ratio
GPX1 ABT1 MED4-AS1 SYS1 IL1RN CREM TMEM115 TRAPPC2P1 SLC2A3 SYAP1 HERPUD1 MRPS18A UQCRFS1 PNRC2 MRPL44 REL GNG2 B4GALT1 BIN3 EIF2S1 FTH1 TRMT10C NFKB1 ISCA1 RNF6 TXLNA SNX9 WTAP USP36 BCL10 COX6A1 DNAJA1 MRPL14 MOB4 MRPL50 IL10RA PRKAR1A MFAP1 RPN1 DNTTIP2 MT2A DNAJB1 STK17B BUD31 TNFRSF1B IL1B LLPH IFIT2 COPB2 TXNDC5
3x 2x 1.5x 1x -1.5x -2x -3x
14
Supplementary Figure S5. Hierarchical clustering for 50 genes in the heat map depicting the relative IR log2 ratios The heat map shows 50 genes with intron retention after FD-‐895 treatment. These genes belong to pathway representing RNA splicing, gene regulation, signal transduction, ER stress and apoptosis.
15
A
B hg19
5 kb
CLL1-Control
hg19
CLL1-Control
CLL2-Control
CLL2-Control Relative RNA-Seq Read Density
Relative RNA-Seq Read Density
20 kb
NBC-Control
CLL1-FD895
CLL2-FD895
NBC-Control
CLL1-FD895
CLL2-FD895
NBC-FD895 NBC-FD895
GAPDH
C 5 kb
RIOK3
hg19
CLL1-Control
Relative RNA-Seq Read Density
CLL2-Control
NBC-Control
CLL1-FD895
CLL2-FD895
NBC-FD895
MCL1
16
Supplementary Figure S6. Screenshot of GAPDH, RIOK3, and MCL1 genes showing relative RNA-‐seq read density mapping to the UCSC reference genome (hg19) to show effect on intron retention after two hrs of treatment with FD-‐895 (100nM). Panel A: The RNA-‐seq reads mapping of house keeping gene GAPDH to show effect of spliceosome modulator on intron retention between different treatments. Panel B: The RNA-‐ seq reads mapping of RIOK kinase 3 encoding gene (RIOK3) to show effect of spliceosome modulator on intron retention between different treatments. Panel C: The RNA-‐seq reads mapping of anti-‐apoptotic gene MCL1 to show effect of spliceosome modulator on intron retention between different treatments.
17
SF3B1 mutational analysis in the CLL-B patient sample in this and other study
HR2
HR3
HR4
R1074H
G740A
K666N/T
T663I
E622D
K700E
HR5
HR !"6 Exon14
HR !"7 16
HR !"8
HR!" 9
HR !"11 !"10 HR
Exon 22
SF3B1 Green: Wild Purple: Mutated Red Box: Resistance to Pladienolide B but there is no report of presence of this mutation in CLL-B. References: 1. Yokoi A et al, FEBS J. 2011;278 (24):4870-80 2. Webb RT et al. Drug Discovery Today. 2013;18 (1-2):43-9 3. Wan Y and Wu CJ, Blood. 2013;121 (23):4627-34 4. Schwaederle M et al. Leukemia. 2013;27:1214-1217 5. Oscier DG et al. Blood. 2012; 121 (3): 468-75 6. Landau DA et al. Cell. 2013; 152 (4):714-726
18
Supplementary Figure S7. Schema showing distribution of mutations in different Heat repeats regions (HR) of SF3B1 gene. The figure showing distribution of mutations identified in our and studies by other groups in SF3B1 gene. For the position of the mutations, the purple letter indicates the original amino acid followed by position of amino acid and the purple color indicates the amino acid after the mutation. The R1074H mutations shown in red box in HR9 has never been reported in any of the study on CLL but in colorectal cancer cell lines after maintaining those in media containing PLAD-‐B for prolonged period.
19
Supplementary Tables Supplementary Table 1: Sequences of primers used in the RT-‐PCR and qRT-‐PCR Table&1.&Sequences&of&primers&used&in&the&RT9PCR&and&qRT9PCR&experiments&
experiments ! for&testing&DNAJB1,&RIOK3,&BCL.X,&and&MCL.11
RT#PCR#primers# Primer#
Location#
Sequence#
DNAJB1"!FW'
Exon!2!
5’!GAACCAAAATCACTTTCCCCAAGGAAGG!3’!
DNAJB1!"!RV'
Exon!3!
5’!AATGAGGTCCCCACGTTTCTCGGGTGT!3’!
RIOK3'"!FW'
Exon!3!
5’!CCAGTGACCTTATGCTGGCTCAGAT!3’!
RIOK3'"!RV'
Exon!4!
5’!GGTCTGTAGGGATCATCACGAGTA!3!
BCL/X'"!FW'
Exon!2!
5’!GAGGCAGGCGACGAGTTTGAA!3’!
BCL/X'"!RV'
Exon!3!
5’!TGGGAGGGTAGAGTGGATGGT!3’!
MCL/1'"!FW'
Exon!1!
5’!CTCGGTACCTTCGGGAGCAGGC!3’!
MCL/1'"!RV'
Exon!3!
5’!CCAGCAGCACATTCCTGATGCC!3’!
GAPDH'"!FW'
Exon!3!
5’!TGGTCACCAGGGCTGCTT!3’!
GAPDH'"!RV'
Exon!4!
5’!AGCTTCCCGTTCTCAGCCTT!3'!
qRT#PCR#primers# DNAJB1"!FW!
Intron!2!
5’!GGCCTGATGGGTCTTATCTATGG!3’!
DNAJB1"!RV!
Intron!2!
5’!TTAGATGGAAGCTGGCTCAAGAG!3’!
RIOK3!"!FW!
Intron!3!
5’!TCAATGGAGATAGCAAAGGTATTATAAC!3’!
RIOK3!"!RV!
Intron!3!
5’!AGATTTACTTAGGAGCACATTATGAGTG!3’!
GAPDH!"!FW!
Exon!3!
5’!TGGTCACCAGGGCTGCTT!3’!
GAPDH!"!RV'
Exon!4!
5’!AGCTTCCCGTTCTCAGCCTT!3'!
FW!and!RV!denote!forward!and!reverse!primers!
20
Table-4: detail of the parameters from RNA-seq data Supplementary Table derived 2: Details of the parameters from RNA-‐seq data Directory/Experiment File
Genome
Total reads
% Aligned
% Unique Match
% Multimappers
% unmapped
Aligner
Genome
Total reads in analysis
Total positions in analysis
Est. Genome Size
Reads per bp
Avg. Reads per position
GCcontent
NBC-PLAD-B
hg19
32437585
51.10%
45.30%
5.90%
48.90%
STAR
hg19
14693711
7497476
3092662209
0.004751
1.413
0.483
NBC-F-ara-A
hg19
33181747
33.80%
30.80%
3.00%
66.20%
STAR
hg19
10234242
7731582
3093307713
0.003309
0.882
0.469
NBC-FD895
hg19
23057170
90.40%
75.00%
15.40%
9.60%
STAR
hg19
17296312
4784233
3093561832
0.005591
2.772
0.496
NBC-control
hg19
23540499
84.20%
73.70%
10.50%
15.80%
STAR
hg19
17347130
7583077
3061420103
0.005666
1.748
0.478
CLL2-PLAD-B
hg19
43358539
84.30%
76.60%
7.70%
15.70%
STAR
hg19
33206561
10793915
3093242291
0.010735
2.105
0.482
CLL2-F-ara-A
hg19
49132267
18.70%
17.50%
1.10%
81.30%
STAR
hg19
8620084
6677622
3092153001
0.002788
0.823
0.472
CLL2-FD895
hg19
27607036
23.30%
21.60%
1.70%
76.70%
STAR
hg19
5965561
3940652
3092691589
0.001929
1.026
0.477
CLL2-control
hg19
43670385
23.80%
22.30%
1.40%
76.20%
STAR
hg19
9747419
7348961
3092779879
0.003152
0.856
0.474
CLL1-PLAD-B
hg19
25885854
88.60%
74.90%
13.70%
11.40%
STAR
hg19
19381741
6101538
3093181770
0.006266
2.311
0.479
CLL1-F-ara-A
hg19
27481703
75.30%
65.80%
9.60%
24.60%
STAR
hg19
18068764
7152187
3093136971
0.005842
1.948
0.474
CLL1-FD895
hg19
48559602
25.20%
22.10%
3.10%
74.80%
STAR
hg19
10739956
6610220
3093379330
0.003472
1.153
0.479
CLL1-control
hg19
36518872
34.60%
31.50%
3.10%
65.40%
STAR
hg19
11501147
8003113
3093323972
0.003718
0.977
0.468
Pladienolide-B
PLAD-B
Fludarabine
F-ara-A
21
Supplementary Table 3: A partial list showing top 50 genes with intron retention in untreated CLL as compared to control normal B cells
Rank%Order%Based% on%ratios%of%IR% ratios%between% CLL/NBC
Refseq%ID
Chromosme% Location
1
NM_019037
chr8
2 3 4 5 6 7
NM_024081 NM_001004304 NR_040662 NM_001099694 NM_000137 NM_001145347
chr11 chr12 chr6 chr19 chr15 chr19
8
NM_016042
chr9
9 10 11
NM_018019 NM_005101 NM_013375
chr17 chr1 chr6
12
NM_002575
chr18
13 14
NM_001160154 NM_172211
chr2 chr1
15
NM_001135191
chr2
16 17 18
NM_000882 NM_181708 NM_001285549
chr3 chr12 chr2
19
NM_001093771
chr12
20 21 22 23 24 25
NM_002657 NM_001134655 NM_018132 NM_004209 NM_152266 NM_003131
chr20 chr16 chr6 chr16 chr19 chr6
26
NM_020745
chr6
27 28
NM_001164721 NR_038943
chr1 chr10
29
NM_001370
chr2
30
NM_145294
chr16
31 32 33 34
NM_032043 NM_023007 NR_027131 NM_181553
chr17 chr1 chrX chr16
35
NM_003830
chr19
36
NM_005456
chr11
37
NM_006963
chr10
38
NM_001256508
chr11
39
NM_001127596
chr1
40 41 42 43
NM_006779 NM_014818 NM_032429 NM_144706
chr11 chr11 chr10 chr2
44
NM_004673
chr1
45 46 47 48 49
NR_037861 NM_032387 NM_001097579 NM_001136482 NR_109990
chr6 chr17 chrX chr19 chr20
50
NM_001135768
chr19
Gene%Description
EXOSC4|RRP41|RRP41A|Rrp41p|SKI6|Ski6p|hRrp41p|p12A|> |8q24.3|protein>coding PRRG4|PRGP4|TMG4|>|11p13|protein>coding ZNF740|Zfp740|>|12q13.13|protein>coding HCP5|6S2650E|D6S2650E|P5>1|>|6p21.3|ncRNA ZNF578|>|>|19q13.41|protein>coding FAH|>|>|15q25.1|protein>coding ZNF576|>|>|19q13.31|protein>coding EXOSC3|PCH1B|RP11>3J10.8|RRP40|Rrp40p|bA3J10.7|hRrp> 40|p10|CGI>102|9p11|protein>coding MED9|MED25|>|17p11.2|protein>coding ISG15|G1P2|IFI15|IP17|UCRP|hUCRP|>|1p36.33|protein>coding ABT1|hABT1|>|6p22.2|protein>coding SERPINB2|HsT1201|PAI|PAI>2|PAI2|PLANH2|>|18q21.3|protein> coding MGAT4A|GNT>IV|GNT>IVA|GnT>4a|>|2q12|protein>coding CSF1|CSF>1|MCSF|RP11>195M16.2|1p13.3|protein>coding ASAP2|AMAP2|CENTB3|DDEF2|PAG3|PAP|Pap>alpha|SHAG1|> |2p25|2p24|protein>coding IL12A|CLMF|IL>12A|NFSK|NKSF1|P35|>|3q25.33|protein>coding BCDIN3D|>|>|12q13.12|protein>coding ZDBF2|>|>|2q33.3|protein>coding TXNRD1|GRIM>12|TR|TR1|TRXR1|TXNR|>|12q23>q24.1|protein> coding PLAGL2|ZNF900|>|20q11.21|protein>coding ZNF213|CR53|ZKSCAN21|ZSCAN53|>|16p13.3|protein>coding CENPQ|C6orf139|CENP>Q|>|6p12.3|protein>coding SYNGR3|>|>|16p13|protein>coding C19orf40|FAAP24|>|19q13.11|protein>coding SRF|MCM1|>|6p21.1|protein>coding AARS2|AARSL|COXPD8|MT>ALARS|MTALARS|RP11> 444E17.1|6p21.1|protein>coding PTAFR|PAFR|>|1p35>p34.3|protein>coding ADD3>AS1|>|>|>|ncRNA DNAH6|DNHL1|Dnahc6|HL>2|HL2|hCG_1789665|2p11.2|protein> coding WDR90|C16orf15|C16orf16|C16orf17|C16orf18|C16orf19|> |16p13.3|protein>coding BRIP1|BACH1|FANCJ|OF|>|17q22.2|protein>coding JMJD4|>|>|1q42.13|protein>coding NKAPP1|CXorf42|>|Xq24|pseudo CMTM3|BNAS2|CKLFSF3|>|16q21|protein>coding SIGLEC5|CD170|CD33L2|OB>BP2|OBBP2|SIGLEC>5|>|19q13.3|protein> coding MAPK8IP1|IB1|JIP>1|JIP1|PRKM8IP|>|11p11.2|protein>coding ZNF22|HKR>T1|KOX15|ZNF422|Zfp422|RP11>285G1.13> 001|10q11|protein>coding TBC1D10C|CARABIN|EPI64C|>|11q13.2|protein>coding FCGR3A|CD16|CD16A|FCG3|FCGR3|FCGRIII|FCR> 10|FCRIII|FCRIIIA|IGFR3|RP11>5K23.1|1q23|protein>coding CDC42EP2|BORG1|CEP2|>|11q13|protein>coding TRIM66|C11orf29|TIF1D|TIF1DELTA|>|11p15.4|protein>coding LZTS2|LAPSER1|RP11>108L7.8|10q24|protein>coding C2orf15|>|>|2q11.2|protein>coding ANGPTL1|ANG3|ANGPT3|ARP1|AngY|UNQ162|dJ595C2.2|PSEC0154| 1q25.2|protein>coding PPT2>EGFL8|>|>|6p|ncRNA WNK4|PHA2B|PRKWNK4|>|17q21>q22|protein>coding GPR34|>|RP11>204C16.6|Xp11.4|protein>coding C19orf38|HIDE1|>|19p13.2|protein>coding RP5>1103G7.4|>|>|>|ncRNA PVR|CD155|HVED|NECL5|Necl>5|PVS|TAGE4|>|19q13.2|protein> coding
22
%%CLL/NBC
454 308 153 125 112 107 100 93 79 70 55 49 44 43 32 30 30 26 26 25 25 22 22 22 16 16 14 14 14 13 12 11 11 11 11 10 9 9 9 8 8 8 8 8 7 7 6 6 4 3
Supplementary,Table,2.,SF3B1,mutations,observed,within,the,examined,
!!
Supplementary Table 4: SF3B1 mutations observed within the examined patient-‐derived patient9derived,CLL,cells, CLL cells
Patient, CLL001! CLL002!
Mutation, ND! ND!
Exon, N/A! N/A!
CLL003! CLL004! CLL005! CLL006! CLL007! CLL008! CLL009! CLL010! CLL011!
E622D! K700E! K700E! T663I! E622D! G740E! K700E! K700E! K666T!
14! 15! 15! 14! 14! 15! 15! 15! 14!
Nucleotide, Change, N/A! N/A!
COSMIC, ID, N/A! N/A!
c.1866G>C! 132938! c.2098A>G! 84677! c.2098A>G! 84677! c.1988C>T! 145921! c.1866G>C! 132938! c.2219G>A! 133120! c.2098A>G! 84677! c.2098A>G! 84677! c.1997A>C! 131556! c.1996G>C! CLL012! K666N! 14! 132937! CLL001!and!CLL002!were!used!for!RNAEseq!analysis.!! ND:!No!mutation!detected;!N/A:!Not!applicable! ! ! !
23
