ALBERT

Description: High-grade serous ovarian carcinoma (HGSOC) is the most common and aggressive form of epithelial ovarian cancer, for which few targeted therapies exist. To search for new therapeutic target proteins, we performed an in vivo shRNA screen using an established human HGSOC cell line growing either subcutaneously or intraperitoneally in immunocompromised mice. We identified genes previously implicated in ovarian cancer such as AURKA1, ERBB3, CDK2, and mTOR, as well as several novel candidates including BRD4, VRK1, and GALK2. We confirmed, using both genetic and pharmacologic approaches, that the activity of BRD4, an epigenetic transcription modulator, is necessary for proliferation/survival of both an established human ovarian cancer cell line (OVCAR8) and a subset of primary serous ovarian cancer cell strains (DFs). Among the DFs tested, the strains sensitive to BRD4 inhibition revealed elevated expression of either MYCN or c-MYC, with MYCN expression correlating closely with JQ1 sensitivity. Accordingly, primary human xenografts derived from high-MYCN or c-MYC strains exhibited sensitivity to BRD4 inhibition. These data suggest that BRD4 inhibition represents a new therapeutic approach for MYC-overexpressing HGSOCs.

Description: Current therapies for sarcomas are often inadequate. This study sought to identify actionable gene targets by selective targeting of the molecular networks that support sarcoma cell proliferation. Silencing of asparagine synthetase (ASNS), an amidotransferase that converts aspartate into asparagine, produced the strongest inhibitory effect on sarcoma growth in a functional genomic screen of mouse sarcomas generated by oncogenic Kras and disruption of Cdkn2a. ASNS silencing in mouse and human sarcoma cell lines reduced the percentage of S phase cells and impeded new polypeptide synthesis. These effects of ASNS silencing were reversed by exogenous supplementation with asparagine. Also, asparagine depletion via the ASNS inhibitor amino sulfoximine 5 (AS5) or asparaginase inhibited mouse and human sarcoma growth in vitro, and genetic silencing of ASNS in mouse sarcoma cells combined with depletion of plasma asparagine inhibited tumor growth in vivo. Asparagine reliance of sarcoma cells may represent a metabolic vulnerability with potential anti-sarcoma therapeutic value.

Description: Abstract 832 We hypothesized that new therapeutic targets for multiple myeloma (MM) could be discovered through the integrative computational analysis of genomic data. Accordingly, we generated gene expression profiling and copy number data on 250 clinically-annotated MM patient samples. Utilizing an outlier statistical approach, we identified HOXA9 as the top candidate gene for further investigation. HOXA9 expression was particularly high in patients lacking canonical MM chromosomal translocations, and allele-specific expression analysis suggested that this overexpression was mono-allelic. Indeed, focal copy number amplifications at the HOXA locus were observed in some patients. Outlier HOXA9 expression was further validated in both a collection of 52 MM cell lines and 414 primary patient samples previously described. To further verify the aberrant expression of HOXA9 in MM, we performed quantitative RT-PCR, which confirmed expression in all MM patients and cell lines tested, with high-level expression in a subset. To further investigate the mechanism of aberrant HOXA9 expression, we interrogated the pattern of histone modification at the HOXA locus because HOXA gene expression is particularly regulated by such chromatin marks. Accordingly, immunoprecipitation studies showed an aberrantly low level of histone 3 lysine 27 trimethylation marks (H3K27me3) at the HOXA9 locus. H3K27me3 modification is normally associated with silencing of HOXA9 in normal B-cell development. As such, it appears likely that the aberrant expression of HOXA9 in MM is due at least in part to defects in histone modification at this locus. To determine the functional consequences of HOXA9 expression in MM, we performed RNAi-mediated knock-down experiments in MM cell lines. Seven independent HOXA9 shRNAs that diminished HOXA9 expression resulted in growth inhibition of 12/14 MM cell lines tested. Taken together, these experiments indicate that HOXA9 is essential for survival of MM cells, and that the mechanism of HOXA9 expression relates to aberrant histone modification at the HOXA9 locus. The data thus suggest that HOXA9 is an attractive new therapeutic target for MM. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 277 NK cells play an important role in innate immune responses directed against autologous cells that have undergone viral or malignant transformation. The ability of NK cells to lyse targets is primarily dependent on the expression of various inhibitory or activating receptors. However, transformed cells have also developed mechanisms to evade immune surveillance and the molecular basis for target resistance to immune-mediated lysis is not well understood. To address this issue we undertook a genetic screening approach to identify novel pathways that modulate tumor cell susceptibility to NK cell lysis. Our genetic screen utilized a subset of the TRC1 lentiviral shRNA library developed at the Broad Institute of Harvard and MIT. The library subset targeted 476 protein kinases and 180 phosphatases that represent 88% and 80%, respectively, of known NCBI sequences with these functions. The library also targeted 372 genes representing tumor suppressors, DNA binding proteins as well as irrelevant shRNAs as controls. Each gene was targeted by 5 or more independent shRNAs tested individually in a 384 well format using robotic manipulations. A total of 6,144 individual shRNAs were incubated with IM-9 myeloma cells and subsequently tested for their ability to modulate response by NKL effector cells (an IL-2 dependent human NK cell line). The top 10 percentile of shRNAs inducing increased secretion of interferon-gamma (INF-γ) from NKL cells was identified. To reduce the likelihood of off-target effects, only genes that were positive for 2 or more independent shRNAs were selected for further analysis. Among the genes that increased target cell susceptibility to NK activity we found 2 members of the Jak family (Jak1 and Jak2) with Jak1 being one of the strongest hits in our screen. IM-9 myeloma cells with stable expression of at least 2 independent shRNAs targeting Jak1 and Jak2 were established and tested for their sensitivity to NKL, NK92 or primary NK cells using INF-γrelease and 51Cr release cytotoxicity assays. Stable suppression of both Jak1 and Jak2 in IM-9 cells induced a significant increase of INF-γsecretion from NK cells and increased sensitivity in cytotoxicity assay when compared to parental IM-9 or cells expressing irrelevant shRNAs. Western blot analysis showed a selective decrease of Jak1 and Jak2 protein in IM-9 cells expressing specific shRNAs but not irrelevant shRNAs. While target cells with reduced expression of Jak1 and Jak2 were more susceptible to NK cell activity, no effect was observed when Jak3 and TYK2 were silenced. We then tested the NK susceptibility of different tumor cell lines with reduced expression of Jak1 and Jak2. Seven additional tumor cell lines representing other hematologic malignancies expressing Jak1 and Jak2 shRNAs were established: myeloma (KM12BM), chronic myeloid leukemia (K562), Burkitt's lymphoma (Daudi), acute myeloid leukemia (U937, ML2 and KG1) and acute T cell leukemia (Jurkat). These experiments confirmed that Jak1 silencing can induce increased susceptibility to NK cell activity. However, this effect was more pronounced in some cases (IM-9, KM12BM, U937, KG1) compared to other cell lines where this effect was limited (K562, ML-2, Jurkat) or absent (Daudi). To investigate the mechanism for modulation of target cell susceptibility to NK cells by Jak1, we compared gene expression profiles of IM-9-Jak1-KO with IM-9 parental cells and IM-9 cells infected with an irrelevant shRNA. No difference in expression was found for ligands of activating NKG2D receptors (MICA, MICB, ULPB1, 2, 3) or ligands for NK inhibitory receptors (HLA Class I genes A, B, C, E). One of the most up-regulated genes in IM-9-Jak1-KO cells was TNFSFR10A (TRAIL-R1), a gene that is known to induce apoptotic signals upon TRAIL engagement. In contrast, FAIM3, an inhibitor of FAS (CD95) signaling, was significantly down-regulated. IM-9-Jak1-KO cells also over-expressed several GALNT genes, recently shown to be markers of TRAIL sensitivity. These results suggest that Jak1 and possibly Jak2 can modulate susceptibility of some tumor cells to NK cell lysis. The mechanism for this effect appears to be at least partly through increased sensitivity to engagement of the TRAIL/FAS extrinsic apoptotic pathway. Disclosures: No relevant conflicts of interest to declare.

Description: Lenalidomide (Len) and pomalidomide (Pom) are immunomodulatory drugs (IMiDs) effective in hematologic malignancies, in combination therapies for multiple myeloma (MM) in particular. Cereblon (CRBN), a component of the CRL4CRBN E3 ligase, is required for IMiD's anti-myeloma activity. Emerging evidence suggests that IMiDs bind CRBN and block an endogenous substrate MEIS2 from binding to CRBN, thereby facilitating the recruitment of transcription factors IKZF1 and IKZF3 to CRL4CRBN and their degradation. This then leads to loss of IRF4 necessary for myeloma survival. The clinical relevance of these novel findings, however, has not been defined. To address this question, we've investigated the mechanism of IMiD action and the functional consequences in freshly isolated primary bone marrow myeloma cells (BMMCs) (n=31) in stromal co-culture ex vivo in the context of the clinical response to Len or Pom in vivo before or after biopsy. We showed by whole transcriptome sequencing, protein analysis and functional assays that 1) BMMCs are addicted to IKZF3-IRF4 for survival; 2) Len-mediated IRF4 loss leads to de-repression of IRF7, induction of interferon (IFN) response genes and TRAIL-mediated apoptosis; and 3) the magnitude of IFN induction is tightly associated with killing of BMMCs by Len. Importantly, the IMiD sensitivity in BMMCs ex vivo correlated with the prior or subsequent clinical response to IMiD-based therapies in individual myeloma patients, suggesting that the clinical response to IMiDs in myeloma is largely intrinsic to myeloma cells. IMiDs have been reported to cause cell cycle arrest. We found that before evidence of killing, Len and Pom induced late G1 arrest by both repressing CCNA2 (encoding cyclin A) mRNA synthesis and elevating p21 and p27 proteins independent of Rb and p53. This result suggests that IMiDs preferentially kill cells in G1 arrest, and that induction of p rolonged early G1 arrest (pG1) beyond the normal G1 transit time by selective inhibition of CDK4/CDK6 with palbociclib (PD 0332991, Ibrance) may sensitize MM cells to IMiD killing, as it does to killing by other agents. Indeed, induction of pG1 by palbociclib overrides cell cycle regulation by Len, and sensitizes BMMCs to Len-mediated apoptosis by augmenting the loss of IRF4 protein and the induction of IRF7, IFNb and TRAIL. Further investigation revealed that induction of pG1 by CDK4/CDK6 inhibition sensitizes primary myeloma cells to IMiD killing by rapid acceleration of Len-mediated loss of IKZF1 and IKZF3 proteins, within one hour of IMiD addition. Loss and gain of function studies demonstrates that MEIS2 opposes pG1 sensitization to Len killing; however, MEIS2 itself is regulated by the cell cycle. Induction of pG1 reduces the ratio of MEIS2 to CRBN by both reducing the MEIS2 protein rapidly and increasing the CRBN protein at a later time in cooperation with Len. In summary, our data provide the first evidence that induction of prolonged early G1 arrest by selective inhibition of CDK4/CDK6 amplifies IMiD killing of primary myeloma cells by both repressing MEIS2 and increasing CRBN protein in cooperation with Len. This leads to a profound reduction in the ratio of MEIS2 to CRBN that accelerates the loss of IKZF1, IKZF3 and IRF4, and enhances IFN and TRAIL induction. Reducing the MEIS2/CRBN ratio thus represents a novel mechanism by which CDK4/CDK6 inhibition sensitizes myeloma to IMiDs, and a means for developing mechanism-based IMiD therapy through cell cycle control. Disclosures Huang: Celgene: Research Funding. Off Label Use: Palbociclib (PD 0332991) is a specific CDK4/CDK6 inhibitor used to stop the cell cycle.. Rossi:Calgene: Speakers Bureau. Pearse:Celegen: Consultancy. Mark:Calgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Niesvizky:Celgene: Consultancy, Speakers Bureau. Chen-Kiang:Celgene: Consultancy.

Description: Karyotypic complexity is one of the hallmarks of multiple myeloma, complicating efforts to identify the specific genetic alterations involved in the development of multiple myeloma. To identify genes essential for myeloma proliferation, we used a high-throughput loss-of-function approach using lentivirally delivered short hairpin RNA (shRNA) library targeting human kinases as part of a Multiple Myeloma Research Foundation sponsored pilot project. Three myeloma cell lines, MM.1S, RPMI8226 and INA6, were infected in an arrayed 384-well format. All infections were performed in quadruplicate; two replicates were treated with puromycin to select for infected cells and two were left untreated. Wells in which more than 25% of the cells survived puromycin treatment, as determined by a cell viability assay, were included in future analysis. Genes in which 2 or more hairpins scored below a defined threshold were considered as ‘hits’. To identify physiologically relevant hits, we have integrated these findings with whole genome analysis of copy number and gene expression of multiple myeloma. We have identified a number of genes that are both amplified in and required for multiple myeloma cell viability. These observations suggest potential new targets for therapeutic targeting in myeloma.

Description: CDK4 and CDK6 are rarely mutated but are overexpressed or hyperactivated at a very high frequency in human cancers. By inhibiting CDK4/CDK6 with an exceptionally selective and reversible inhibitor, palbociclib (PD 0332991), we have developed a novel strategy to reprogram cancer cells for cytotoxic killing through induction of prolonged early G1 arrest (pG1). We have demonstrated that pG1 sensitizes cancer cells expressing Rb, the substrate of CDK4 and CDK6, to cytotoxic killing by forcing an imbalance in gene expression because only genes scheduled for early G1 are expressed. This sensitization is exacerbated after palbociclib withdrawal due to incomplete restoration of gene expression despite S phase synchronization (pG1-S). This study aims to identify genes that mediate pG1-S sensitization to two clinically-relevant agents for myeloma, the proteasome inhibitors carfilzomib and bortezomib, in model cell lines by a sensitizing pool genome-wide shRNA screen, and by validating the hits in a clinical trial of palbociclib in combination with bortezomib and dexamethasone. We ranked the hits based on the enrichment of target shRNAs, and representation in replica of each cell lines and among different human myeloma cell lines (HMCLs) as well as functional analyses. In myeloma cells, cell cycle control by palbociclib was intact in all hits, demonstrating that CDK4 and CDK6 are indispensable for myeloma replication. Among the top ranking 20 candidates, we found that NEDD4L was essential for proteasome inhibitor killing, FTH1 modulated the threshold of killing by diverse agents especially in pG1-S, and IL10RAappeared to be required for pG1-S sensitization to proteasome inhibitors. Moreover, RNA-sequencing analysis of primary myeloma cells from a phase II clinical trial targeting CDK4/CDK6 with palbociclib in combination with bortezomib in myeloma revealed that a higher level of FTH1 expression in myeloma cells in vivo correlated with sensitivity to this therapy, suggesting a role for FTH1 in differential sensitivity to this CDK4/CDK6-based therapy in myeloma. Selective inhibition of CDK4/CDK6 with palbociclib, or another specific inhibitor such as LY2835219 or LEE011, in combination therapy has now achieved unprecedented clinical efficacy in diverse human cancers. Most notably, palbociclib more than doubled the progression free survival of metastatic breast cancer patients when it was combined with letrozole, and has been designated a “breakthrough therapy” by the FDA for breast cancer. Our work provides the first insight into genes that mediate cell cycle sensitization to cytotoxic killing through selective CDK4/CDK6 inhibition. It provides an exciting potential for further investigation in a clinical context, such as the ongoing phase I clinical trial combining palbociclib with the immunomodulatory drug lenalidomide in patients with relapsed/refractory myeloma. Disclosures Huang: Celgene: Research Funding. Off Label Use: PD 0332991 (palbociclib) is a specific CDK4/CDK6 inhibitor used to stop the cell cycle.. Niesvizky:Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Chen-Kiang:Celgene: Consultancy, Research Funding.

Description: The Multiple Myeloma Research Consortium (MMRC) Genomics Initiative was instigated to harness the power of multiple genomic approaches to further the understanding of multiple myeloma. To date, 137 samples from patients with newly diagnosed and relapsed myeloma (out of an estimated final total of 250 by the end of the year) have been subjected to expression profiling and array comparative genomic hybridization. To identify regions of recurrent copy number alteration with a high degree of confidence, we have used the Genomic Identification of Significant Targets In Cancer (GISTIC) algorithm, which detects such regions and assigns a probability to each. Application of GISTIC to the MMRC collection identified 14 significant regions of amplification and 15 significant regions of deletion. The algorithm further detects peaks of copy number change that contribute to each region’s significance. Of genes that were expressed in our dataset, a total of 64 across the genome were found to lie within the boundaries of significant amplification peaks and 30 were found to lie within significant deletion peaks. Given the likelihood that this gene collection is highly enriched for genes important in the pathogenesis of myeloma and potential therapeutic targets, it has been prioritized for further validation with the amplified genes submitted for arrayed RNAi and the deleted genes expedited for re-sequencing. In order to define a poor prognosis group, we developed and applied a model based on the gene expression dataset of Shaughnessy and colleagues. To identify pathways activated in poor prognosis disease, we applied Gene Set Enrichment Analysis with the Molecular Signatures Database. This demonstrated enrichment of multiple canonical pathways within the poor prognosis group, including those associated with proliferation, cell cycle progression and DNA repair. A search for recurrent copy number events associated with poor prognosis disease revealed that by far the most significant event, and the only one surviving correction for multiple hypothesis testing, was mono- or bi-allelic deletion of CDKN2C (p18). Interestingly, the poor prognosis group also demonstrated higher expression of p18 in those samples without bi-allelic deletion and of CDKN2A (p16). Deletion and increased expression of p18 in high proliferation index myeloma samples has previously been described. However, our analyses demonstrate the pre-eminence of p18 loss for defining poor prognosis disease compared to other recurrent copy number changes. Furthermore, it expands on work in other tumors demonstrating feedback mechanisms between cyclin dependent kinase inhibitor pathways, suggesting a more complex model of interplay between the pathways than previously described. In conclusion, the MMRC reference collection is proving an important tool in the fight to understand key genetic events in multiple myeloma and would ultimately be anticipated to contribute towards development of improved therapy for the disease.

Description: Abstract 1409 MYC is among the most prolific oncogenes in cancer, yet pharmacologic strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have undertaken to target c-Myc transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative co-activator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain-containing proteins as transcriptional regulatory factors for c-Myc. BET inhibition with JQ1 rapidly downregulates c-Myc transcription, followed by depletion of chromatin-bound c-Myc and genome-wide downregulation of Myc-dependent target genes. In translational model systems of multiple myeloma and Burkitt's lymphoma, both canonical MYC -dependent hematologic malignancies, JQ1 treatment produces a potent antiproliferative effect associated with cell cycle arrest. In multiple myeloma, adhesion to bone marrow stroma is associated with upregulation of BRD4, a BET bromomdomain coactivator protein. Inhibition of Myc function with JQ1 leads to impaired adhesion to stroma and cellular senescence, a classical Myc-specific phenotype. Mechanistically, JQ1 treatment depletes BRD4 from IgH enhancers, leading to prompt and robust downregulation of MYC transcription. In vivo efficacy of JQ1 in two disseminated models of multiple myeloma and in a Burkitt's lymphoma human xenograft establishes the therapeutic rationale for BET bromodomain inhibition in these diseases. Together, these studies identify a mechanistic rationale for targeting c-Myc in human cancer, and potentially other undruggable oncogenes driven by immunoglobulin rearrangement. Note: G.C.I. and J.E.D. have made equal contributions to this research; C.S.M. and J.E.B. are jointly senior authors Disclosures: Richardson: Millennium: Advisory Board; Celgene: Advisory Board; Johnson & Johnson: Advisory Board; Novartis: Advisory Board; Bristol Myers Squibb: Advisory Board. Ghobrial:Bristol-Myers Squibb: Research Funding; Millennium: Research Funding; Noxxon:; Millennium:; Celgene:; Novartis:. Anderson:Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Kung:Novartis Pharmaceuticals: Consultancy, Research Funding. Mitsiades:Millennium Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Kosan: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centocor: Consultancy, Honoraria; Amnis Therapeutics: Consultancy, Honoraria; PharmaMar:; OSI Pharmaceuticals: Research Funding; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding; Genzyme: Research Funding; Johnson & Johnson: Research Funding. Bradner:Acetylon Pharmaceuticals: Scientific Founder; SHAPE Pharmaceuticals: Scientific Founder; Tensha Therapeutics: Scientific Founder.