Shi, Chang-Xin

Abstract We have conducted systematic small interfering RNA (siRNA) lethality screening of the kinome and “druggable genome” in human myeloma cell lines (HMCL) to functionally generate a comprehensive map of critical genes and vulnerabilities in human myeloma tumors. KMS11 human myeloma cells were screened with both an 1800 oligo (639 gene) siRNA library targeting the kinome and with a 13,984 oligo (6,791 gene) library targeting the druggable genome in duplicate using optimized conditions that resulted in >95% transfection efficiency and <5% background cytotoxicity. siRNA were used at low concentration (13nM) to minimise off-target cellular effects. Each gene was initially screened with at least 2 distinct siRNA. Universally lethal and non-silencing siRNA were tested in parallel as controls. Viability was measured at 96 hours by ATP-dependent luminescence and was normalized to the mean and distribution of controls by the B-score method. Approximately 14% of kinome siRNA and 5.8% of druggable genome siRNA caused important reductions in HMCL viability (greater than three standard deviations from control samples treated with non-silencing siRNA), compared with an anticipated rate due to chance of only 0.135%. Short-listed kinome vulnerabilities were validated by repeat targeting in a second lethality screening experiment in KMS11 using a total of four independent siRNA per gene. Forty-two kinases were confirmed as critical for myeloma cell survival. Unsupervised pathway analysis indicates that approximately half are concentrated within established pathways involved in cytokine (VEGFR, FGFR3, IGFR/IL6R) signaling and regulation of cell cycle, apoptosis or metabolism. The role of other survival kinases in myeloma are novel. The selectivity of kinome vulnerabilities identified in KMS11 for myeloma cells versus other tissues was examined by parallel silencing of these targets in KMS11 and JJN3 myeloma cells and in non-myeloma 293 and A549 epithelial cells. The most lethal kinase targets in myeloma that simultaneously show experimental evidence of tumor selectivity include aurora kinase A (AURKA), G-protein receptor coupled kinase 6 (GRK6) and protein kinase N1 (PKN1). By gene expression analyses, PKN1 and GRK6 are both preferentially expressed in plasma cells, compared with an analysis of >50 somatic tissues in which they are absent or only weakly expressed. The extent to which the kinome vulnerabilities identified in KMS11 occur in other HMCL was assessed by tertiary siRNA screening and by lentiviral shRNA techniques. More than 75% of kinases vulnerable in KMS11 cells are recurrently vulnerable in other myeloma tumor lines. Secondary validation studies of all genomic vulnerabilities identified from the much larger siRNA lethality screen of the druggable genome in myeloma cells is ongoing, however, to date 134 genes have been validated as essential myeloma survival factors during primary screening by concordant lethality of 2/2 independent siRNA (compared with an expected rate due to chance of n<1 genes). Significantly, proteosome subunits and specific ubiquitin ligases number amongst the most potently lethal genomic vulnerabilities in KMS11. Overall, this attempt to systematically and functionally document all genetic vulnerabilities in myeloma provides a strong rational impetus for the development of novel therapies that optimally target susceptibilities inherent within myeloma tumors.

Abstract Immunomodulatory drugs (IMiDs) are widely used in the treatment of patients with Multiple Myeloma (MM) however, only 30% of relapsed MM patients respond to single agent therapy and most patients eventually develop drug resistance. The molecular target of IMiDs in MM is cereblon but other parallel pathways or downstream events which enhance or preclude drug responsiveness are unknown. We therefore conducted a genome scale small interfering RNA (siRNA) lethality study in MM in the presence of increasing concentrations of lenalidomide. Primary screening was performed in a single-siRNA-per-well format with the human druggable genome siRNA set V4 comprising four siRNAs targeting each of 6,992 genes (total 27968 siRNAs). Lenalidomide was added 24 hours post transfection and cell viability was measured by ATP-dependent luminescence at 144 hours after transfection. Primary screen data was rigorously evaluated for multiple quality control metrics and found to exceed all expected performance parameters with >98% global transfection efficiency, <0.25 CV values, and minimal plate-to-plate and set-to-set variations observed. Hit selection was performed by analysis of IC50 value shift in the presence of each testing siRNA compared with three different control siRNA oligos. 160 candidate genes that enhance lenalidomide sensitivity upon silencing (sensitizers) were selected and re-screened with four siRNA oligos targeting each gene. 50 genes were identified as reproducible lenalidomide sensitizers including three Peroxisome (PEX) family proteins (PEX1, PEX10 and PEX7) and seven RAB family proteins (RAB17, RAB1A, RAB26, RAB30, RAB36, RAB4A and RAB8A). Four kinase genes were also identified in sensitizer hits and two of these, I-Kappa-B Kinase-Alpha (IKK1 or CHUK) and ribosomal protein S6 kinase (RPS6KA3 or RSK2), encode proteins that associate with significance together with a phosphorylation dependent transcription factor (CREB1) in Toll signaling pathways (p-value 0.0068). RSK2 is a serine/threonine-protein kinase that acts downstream of oncogenic FGFR3 mediated signaling and is phosphorylated by ERK (MAPK1/ERK2 and MAPK3/ERK1 signaling) during hematopoietic transformation. Phosphorylated RSK2 was previously reported to be frequently expressed in myeloma cell lines and primary myeloma cells. Using lentiviral shRNA expression, we demonstrated that knockdown of RSK2 in three genetically variable MM cell lines induced cyctocytoxiticy and consistently sensitized to lenalidomide. Two selective small molecular inhibitors of RSK2 (SL 0101-1 and BI-D1870) were then demonstrated to synergize with lenalidomide to induce myeloma cell cytotoxicixity. To further understand the mechanism underlying sensitization, immunoblotting analysis was performed to look at downstream changes after either RSK2 knockdown or RSK2 inhibition by BI-D1870. We found that both RSK2 knockdown and BI-D1870 treatment, mimicking lenalidomide treatment or cereblon inhibition, induced downregulation of both IRF4 and MYC in MM cells. The combination of lenalidomide and BI-D1870 not only produced a substantial synergistic effect inducing MM cytotoxicity, but also demonstrated a significant enhancement of downregulation of IRF4 and MYC. Forced overexpression of RSK2 attenuated the synergistic effects of lenalidomide and BI-D1870. In summary, our high throughput screen identified multiple gene targets that associate with increasing sensitivity to IMiDs in MM cells, of which, RSK2 was further validated by both shRNA silencing and specific inhibitors as an effective target to cooperate with IMiDs to induce myeloma cytotoxicity. Clinical studies of RSK2 inhibition in concert with IMiD (cereblon inhibitor) therapy would be appropriate. Disclosures: Stewart: Onyx: Consultancy, Research Funding; Millennium: Honoraria, Research Funding; Celgene: Honoraria; BMS: Honoraria.

Abstract Abstract 602 The molecular target(s) which co-operate with proteasome inhibition in inducing drug sensitivity or resistance in Multiple Myeloma (MM) remain unknown. We therefore conducted a genome scale small interfering RNA (siRNA) lethality study in KMS11 MM cells in the presence or absence of bortezomib without regard to pre-conceived mechanistic notions. Primary screening was performed in a single-siRNA-per-well format with the human druggable genome siRNA set V4 comprising 13,984 siRNA targeting 6,992 genes and comprising two RNAi per gene. siRNA were transfected at low concentration (13nM) to minimize off-target effects using conditions that resulted in transfection of >95% cells and <5% background cytotoxicity. Bortezomib was added (at the IC10, 25, 70 and 90) 24 hours post transfection. After 96 hours (72 hours after bortezomib), viability was measured by ATP-dependent luminescence. In primary screening 320 candidate bortezomib sensitizing genes were identified and rescreened with four siRNA oligos each gene. 57 of the top sensitizer hits were selected for which at least two distinct siRNA decreased the EC50 by 2 standard deviations from cells treated with control siRNAs. By cross referencing gene expression profile (GEP) data of KMS11, we furthered pared the list to 37 plausibly expressed targets (0.5% of those genes originally screened) as bortezomib sensitizers. After silencing, 50% of these 37 genes also sensitized the lung cancer cell line A549 to bortezomib. After further parsing of genes which also modulated the sensitivity of MM to Melphalan (non specific chemosensitizers), 34 genes remained: The strongest sensitizers to bortezomib were the proteasome subunits PSMA5, PSMB2, PSMB3, PSMB7 but included less obvious targets such as BAZ1B, CDK5, CDC42SE2, MDM4, NME7, TFE3, TNFAIP3, TNK1, TOP1, VAMP2 and YY1 were also identified. Of these, the most potent synergetic effects were observed with siRNAs against the proteasome subunits and against cyclin dependent kinase 5 (CDK5), which caused the greatest shift in EC50. CDK5 is of particular interest as a therapeutic target as it is expressed at high levels in MM and neural tissues but has low expression in other organs. Using viral shRNA expression, silencing CDK5 consistently increased the sensitivity of genetically variable MM cell lines (n=5) to all of the proteasome inhibitors tested: bortezomib, carfilzomib and PR047, and the effect could be at least partially rescued by overexpression of an RNAi resistant CDK5. To explore therapeutic relevance the small molecule CDK5 inhibitor, Roscovitin, was shown to be synergistic or additive with bortezomib in both MM cell lines and primary patient samples. Gene expression profiling was then performed to seek an explanation for the CDK5 sensitization effect and regulation of a proteasome subunit PSMB5 by CDK5 was identified as a probable route to sensitization. In summary inhibition of the existing proteasome either directly by suppression of proteasome subunits, or indirectly by suppression of modulators such as CDK5 appears to confer the greatest sensitization effect suggesting that combinations of bortezomib with other unique proteasome inhibitor drugs or combinations with inhibitors of CDK5 is a logical avenue for clinical exploration. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 127 The precise molecular mechanism of action and targets through which thalidomide and related immunomodulatory drugs (IMiDs) exert their anti-tumor effects remains unclear. A landmark paper has recently identified cereblon (CRBN) as a primary target of thalidomide teratogenicity. We hypothesized that this protein would also be required for myeloma (MM) cytotoxicity. To examine this issue we first tested 12 MM cell lines: 4 of which (OCIMY5, OPM1, SKMM2 and KMS12 PE) were very resistant to treatment with both lenalidomide and pomalidomide. Of these, OCIMY5 and OPM1 showed CRBN gene expression levels at the bottom 10% across the human MM cell lines. We next used CRBN shRNA lentiviral expression constructs to knock down CRBN in 5 human-derived myeloma cell lines (HMCLs) (KMS18, MM1.S, H929, OPM2 and JJN3). HMCLs infected with CRBN shRNAs have a significant reduction of CRBN expression and subsequent diminished cell viability (reduced 65–78%). Surviving MM cells with CRBN knock down demonstrate an acquired (essentially complete) resistance to lenalidomide when compared with controls but retained sensitivity to melphalan, dexamethasone and bortezomib. Gene expression changes induced by lenalidomide were dramatically suppressed in those CRBN depleted cells, e.g., OPM2 cells only showed 30 down regulated (3% of control) and 150 up-regulated genes (24% of control) after lenalidomide treatment, further emphasizing that CRBN is required for lenalidomide activity. We next compared by array comparative genomic hybridization the differences between MM1.S (lenalidomide sensitive) and its isogenic lenalidomide resistant line (MM1.S res), generated by culturing MM1.S in gradually increasing concentrations of lenalidomide. Only three structural abnormalities were noted differentiating the two lines one of which was deletion of CRBN. In fact the baseline MM1.S cell line has a minor population of 9% of cells with CRBN depletion which becomes dominant after lenalidomide treatment. However, further scrutiny of CRBN status in additional HMCLs and MM patients (irrespective of therapy and stage) suggested that copy-number abnormalities affecting this gene are uncommon events in MM, with 12% of HMCLs (7 out of 60) and only 1.2% of MM patients (3 out of 238) showing CRBN monoallelic deletion. Next, we examined by quantitative polymerase chain reaction (Q-PCR) ten MM patients who had become resistant to lenalidomide therapy. CRBN expression level by Q-PCR in 8 of 10 patient samples had a significant reduction (20% to 90% reduction compared to baseline) at time of drug resistance, suggesting CRBN expression as a potential biomarker to predict IMiD response. Notably however some clearly resistant patients have normal CRBN, demonstrating that while CRBN is absolutely required for response, multiple resistance mechanisms are likely present. By gene and protein expression downstream targets of CRBN were examined and included interferon regulatory factor 4 (IRF4) previously reported to also be a target of lenalidomide. In summary, we demonstrate here that CRBN is essential for IMiD activity and preliminary data support that low levels of CRBN predict for poor drug response. In addition, our data suggest that CRBN is a critical molecule but not the unique source of IMiD resistance. We suggest that the “IMiDs” be renamed Cereblon binding small molecules to more accurately reflect their mechanism of action. Disclosures: Orlowski: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract Introduction: Partial loss of chromosome arm 1p frequently occurs in multiple myeloma (MM), and is associated with a poor prognosis. Several minimally altered regions on 1p have been identified, including 1p32.3, 1p31.3, 1p22.1-1p21.3, and 1p12. Cytoband 1p12 was deleted in 19% of cases, and this deletion was associated with shorter overall survival (OS) in univariate analysis. The target of homozygous deletion 1p12 was FAM46C. In addition, mutations of FAM46C were identified in 3.4% to 13% of primary MM tumors and 25% of 16 human myeloma cell lines (HMCLs), implying its potential pathogenic relevance. In other work we have suggested that FAM46C mutation is a progression event and have shown that it is rarely seen in newly diagnosed del17 patients, inferring some overlap in function. However, there is no published functional annotation of FAM46C and its role in MM remains unknown. In the present study, we aimed to identify the biological role of FAM46C in myeloma cells. Materials/Method: The expression of FAM46C in HMCLs was analyzed by western blot. Lentiviral constructs expressing wild type and mutated FAM46C were generated and transduced into HMCLs, followed by cell viability assay and cell cycle analysis. Cells were harvested and processed to measure gene expression and cell signaling changes after introduction of FAM46C by mRNAseq, pathway analysis and immunoblotting assay. Results: The expression of FAM46C protein is generally low in most HMCLs. Introduction of wild type FAM46C in HMCLs induced a substantial cell growth inhibition and apoptosis, especially in two HMCLs including MM1.S and KMS11. Cell viability of KMS11 and MM1.S was reduced by 50% to 80% at day 6 after introduction of FAM46C, compared to 0-30% growth retardation detected in HMCLs and non-myeloma cell lines that do not carry FAM46C deletion. We identified 88 genes whose mRNA expression was significantly altered after enforced expression of FAM46C in MM1.S cells. Pathway analysis revealed that FAM46C-regulated genes are enriched in the canonical pathways associated with unfolded protein response, cell cycle control and DNA damage repair. Critical MM genes that are downregulated by FAM46C expression include IRF4 and MYC, which are also downstream targets of immunomodulatory drugs (IMiDs). Consistently, some HMCLs such as KMS11 and OPM2 show an enhanced sensitivity to lenalidomide after introduction of FAM46C. Next, lentiviral constructs expressing various FAM46C mutants were generated in order to understand the consequence of FAM46C mutation. The mutant constructs mimic mutations identified in MM patients or HMCLs. Those mutants and wild type FAM46C were transduced and tested together in MM1.S cells. We found that three published misssense mutations, one frame-shift mutation and deletion of the sequence between aa172 and aa186 of FAM46C (which has been found in previous studies as a hot spot of mutation) all abolished FAM46C-mediated anti-myeloma activity, thus would be expected to confer a MM cell survival advantage. Conclusion: Our data demonstrated that enforced FAM46C expression in myeloma cells induced myeloma growth inhibition and apoptosis. Mutations in FAM46C and TP53 in newly diagnosed patients seem mutually exclusive but not in relapsed patients from our patients sequencing studies, suggesting it may associate with disease progression. Together, these studies suggest that FAM46C may function as a tumor suppressor in myeloma. We also found that published mutations of FAM46C confer a survival advantage to MM cells, and that FAM46C overexpression downregulates IRF4 and MYC and is thus associated with loss of myeloma cell survival. Disclosures Stewart: Novartis: Consultancy; Oncospire Inc.: Equity Ownership; Celgene: Consultancy; BMS: Membership on an entity's Board of Directors or advisory committees.

Abstract Introduction Lenalidomide is an immunomodulatory drug (IMID) used to treat Multiple Myeloma (MM). Although a role for cereblon (CRBN)-mediated degradation of Ikaros proteins (IKZF1 and IKZF3) has been shown, the complete molecular and biochemical mechanisms responsible for lenalidomide-mediated anti-MM activity and/or resistance are undiscovered. Therefore, we aimed to analyze whether IMIDs (thalidomide, lenalidomide, and pomalidomide) are inducing oxidative stress in MM and what determines these drugs varying sensitivity and/or resistance. Methodology Amplex Red Assay has been performed to analyze IMIDs-mediated inhibition of H2O2 decomposition in both, in-vitro and in-vivo assays. Lentiviruses were prepared in 293T cells for CRBN, IgL-λ & IgL-k, and Bim knockdown experiment. Quantification of MM cellular anti-oxidative capacity for determining IMID sensitivity was standardized with H2O2-mediated oxidation of FADH2 and NAD(P)H. To measure apoptosis and gene expression analysis 106 cells were incubated with lenalidomide for 24 to 96 hours before they were examined by annexin-PI and FACS analysis. Gene and protein expression were measured by RT-PCR, western blot, and immunohistochemistry. Results We discovered that IMIDs inhibit peroxidase-mediated decomposition of H2O2 in both, in vitro horseradish peroxidase (HRP) assays and in human MM cell lines (HMCLs). Of the IMIDs analyzed, pomalidomide was the more potent inhibitor. H2O2 treatment effectively degraded IKZF1 and IKZF3 in HMCLs. To confirm the central role of CRBN in IKZF1 and IKZF3 degradation by H2O2-induced oxidative stress, we used CRBN knockdown OPM2 isogeneic cells and the CRBN-overexpressing OCIMY-5 cell line. We treated both sets of isogenic cell lines with lenalidomide and H2O2 for 3 hours, and we showed that H2O2 similarly mediates IKZF1 and IKZF3 degradation in a CRBN-dependent fashion. Next, we tested viability of CRBN present and absent cell lines with increasing concentrations of lenalidomide and H2O2 for 3 days. Lenalidomide-induced cytotoxicity was CRBN dependent, but H2O2 was not after 3 days, as shown by MTT assays. The capacity of MM cells to decompose H2O2 was measured via a biochemical test that quantitatively measured cellular anti-oxidative capacity. IMID sensitivity was well correlated with cellular anti-oxidative capacity, likely, cells more efficiently decompose H2O2was resistant and cells were not sensitive to IMID. This result shows that antioxidant capacity determines lenalidomide sensitivity among HMCLs with similar CRBN protein expression. We discovered that lenalidomide-mediated cytotoxicity in MM was attributable to oxidative damage of intracellular immunoglobulin proteins. By using several sets of isogenic cells lines with and without CRBN expression, we confirmed that lenalidomide treatment caused accumulation of IgL dimers only in CRBN-positive cells. Lenalidomide-induced IgL dimerization lead to decreased secretion and consequent intracellular accumulation of IgL, as evidenced by unchanged IgL mRNA expression, increased total intracellular IgL protein, and decreased secretion of IgL. After 72 hours of lenalidomide treatment we found decreased XBP-1u, increased XBP-1s, and over-expressed GRP78/BiP endoplasmic reticulum stress (ERS) marker proteins in CRBN positive cells but not in CRBN knock-down cells. We observed Bim requirement, especially BimEL, after lenalidomide treatment in CRBN-positive lenalidomide-sensitive cells. Our data reveals that lenalidomide-mediated; progressive ERS can positively enhance bortezomib-induced apoptosis in an in-vitro MM model. We pretreated MM cells with lenalidomide and then treated them with bortezomib. OPM2 cells pretreated with lenalidomide for 2 days clearly showed increased sensitivity to bortezomib-induced apoptosis compared with cells that were not pretreated. Conclusion IMIDs inhibit H2O2 decomposition. Ikaros protein degradation is a consequence of H2O2 mediated oxidative stress. Therefore, cells producing high H2O2 and with less antioxidative capacity are more sensitive to IMIDs. On the basis of this discovery, we would be able to predict which patients will benefit from IMIDs-mediated therapy and develop new drugs other than IMIDs that can inhibit intracellular H2O2 decomposition in MM. At present, CRBN may be required for IMIDs to effectively inhibit H2O2decomposition. Disclosures Chirackal: Mayo Clinic: Patents & Royalties: Filed a professional US patent for quantifying cellular anti-oxidative capacity. Fonseca:Mayo Clinic: Patents & Royalties: Filed a professional US patent for quantifying cellular anti-oxidative capacity.

Abstract In an attempt to identify novel genetic abnormalities in multiple myeloma (MM) we analyzed 68 MM patient samples and 42 human myeloma cell lines (HMCL) with high-resolution (60kb) array-based comparative genomic hybridization (aCGH). We concentrated on bi-allelic genomic deletions with the expectation that these regions might contain novel tumor suppressor genes and identified a common abnormality at 14q32. Fine mapping of the 14q32 microdeletion by PCR demonstrated a 45 kb minimal deletion encompassing two genes; TRAF3, a negative regulator of the non-canonical NF-kB pathway, and AMN. To determine the frequency of copy number abnormalities of this region a cohort of 161 patients were screened with a FISH probe mapping to the minimally deleted region. This identified 7 (4.4%) patients with bi-allelic deletion and 19 (11.8%) patients with a single copy of the target region. Sequencing of all TRAF3 coding exons in 62 patients and 42 HMCL identified inactivating mutations in 9 patient samples and 4 HMCL. Overall, the incidence of TRAF3 inactivation, bi-allelic deletion or mutation, is 7/42 (16.7%) in HMCL and 12/62 (19.4%) in patients, making it one of the most common genetic abnormalities identified in MM. Importantly, dysregulation of NF-kB pathways is a common event in MM, but the mechanism for this dysregulation is largely unknown. Although TRAF3 is a negative regulator of the non-canonical NF-kB pathway it has not previously been implicated in cancer. Therefore, we tested the tumor suppressor function of TRAF3 by reintroducing TRAF3 into HMCL harboring TRAF3 abnormalities using an adenovirus gene transfer system. Expression of TRAF3 in HMCL with TRAF3 abnormalities rapidly reversed cellular processing of NFKB2 from p100 (an inactive isoform) to p52 (active) and additionally led to a 60% reduction in cellular proliferation (range, 10–89%) associated with Go/G1 cell cycle arrest and induction of cell death by apoptosis. At 48 hrs post infection the proportion of cells entering S-phase or G2/M declined by 31% (range, 8.1–56.1) and the proportion undergoing apoptosis increased by 28% (range 11.7–47.4%). In contrast, over-expression of TRAF3 in control HMCL without TRAF3 abnormalities had no effect. Overall, TRAF3 is identified as a novel tumor suppressor that regulates the non-canonical NF-kB pathway and is inactivated in ~ 20% of MM patients. Given the involvement of the NF-kB pathway in multiple human malignancies and the near constitutive expression of TRAF3 we propose that inactivation of TRAF3, a novel tumor suppressor identified for the first time in this study, may occur in other cancers. In this regard, we have recently identified a Burkitt lymphoma cell line that does not express TRAF3 and thus has constitutive processing of p100 to p52. The clinical and therapeutic consequences of this finding and its frequency in other tumor types are currently the focus of further investigation. In conclusion, the abnormalities identified in this study represent the first mutations identified that cause constitutive NF-kB activation in MM.

Abstract Introduction A unique biological characteristic of MM plasma cells (PCs) is the substantial production of immunoglobulin proteins. This process requires disulfide bond formations that in turn generate large amounts of H2O2. The Trx-TrxR reductase system exists to combat this oxidative stress; however, cytotoxicity can occur when the cells' antioxidative capacity is overwhelmed. Recently, we demonstrated that IMiDs inhibit peroxidase-mediated decomposition of H2O2, and that lenalidomide sensitivity in MM correlates with the degree of antioxidative capacity (Sebastian et al. 2015 ASH). In this study we sought to better understand this inhibitory process. Methods We used various human MM cell lines with different sensitivity to lenalidomide. Cell viability was assessed by the MTT assay. Antioxidative capacity was measured via the total cellular oxidation of reduced flavin adenine dinucleotide (FADH2) and reduced nicotinamide adenine dinucleotide (NAD(P)H) after H2O2treatment. A CRBN CRISPR/Cas9 KO plasmid was used to generate CRBN knockout cells and lentivirus system was used to over-express CRBN in MM cells. Results We found that IMiDs inhibit TrxR mediated intracellular H₂O₂ decomposition in a CRBN dependent fashion. Unlike lenalidomide, the direct TrxR inhibitor auranofin was very effective in inducing cell death in MM cell lines, in a CRBN independent fashion. By using MM.1R isogeneic cells with and without CRBN, and the CRBN-overexpressing OCIMY-5 cell line (transfected with wt-CRBN) we showed that auranofin treatment caused cytotoxicity irrespective of CRBN level of expression, while lenalidomide was only effective in CRBN expressing cells. Clinically achievable concentrations of auranofin treatment induced cell signals similar to lenalidomide, including degradation of IKZF1, IKZF3. As with lenalidomide, the sensitivity of CRBN expressing cell lines to auranofin correlated with their basal antioxidative capacity (MM.1S cells are sensitive and have with lower anti-oxidative capacity while RPMI-8226 is resistant due to its high anti-oxidative capacity). We also found that targeting Trx (downstream effector of TrxR) with the direct inhibitor PX12 overcame this cellular anti-oxidative capacity and inhibited cell proliferation in both RPMI-8226 and MM.1S in a CRBN independent fashion. Combination of auranofin with bortezomib showed a synergistic effect, but only in cells with lower anti-oxidative capacity. In contrast, PX-12, showed a synergistic effect with bortezomib in all analyzed MM cells lines, irrespective of their anti-oxidative capacity and CRBN expression. Conclusions IMiDs inhibit TrxR-mediated intracellular H2O2decomposition in CRBN expressing MM. While the direct TrxR inhibitor, auranofin, bypassed the CRBN requirement for inducing cytotoxicity in MM, its cytotoxic effect could be overcome by the cells antioxidative capacity. Direct Trx inhibition (such as PX-12) seems like a potent strategy for MM therapy to overcome cellular CRBN requirements and higher anti-oxidative capacity cells. Disclosures Bergsagel: Novartis: Research Funding; Amgen, BMS, Novartis, Incyte: Consultancy. Stewart:celgene: Consultancy. Fonseca:Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Janssen: Consultancy; Bayer: Consultancy; Millennium, a Takeda Company: Consultancy; Millennium, a Takeda Company: Consultancy; BMS: Consultancy; Millennium, a Takeda Company: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Celgene: Consultancy; AMGEN: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Millennium, a Takeda Company: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Janssen: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Bayer: Consultancy; AMGEN: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Novartis: Consultancy; AMGEN: Consultancy; Sanofi: Consultancy; AMGEN: Consultancy; Sanofi: Consultancy; BMS: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms.

Abstract Abstract 573 Using high throughput RNA interference screening on 6,722 druggable genes we previously identified XPO1/CRM1 as one of the 50 most vulnerable targets in Multiple Myeloma (MM)1. XPO1 knockdown proved lethal in MM cell lines, but had no effect on human embryonic kidney (293) cells or lung cancer (A549) cells, showing that XPO1 is a specific myeloma vulnerability, and that myeloma cell survival is dependent upon XPO1 expression. XPO1 encodes the protein exportin 1, a nuclear transport protein that exports tumor suppressor proteins from the nucleus, where they are active, to the cytoplasm, where they become inactive. We next analyzed XPO1 in MM via gene expression profiling (GEP). XPO1 expression is up-regulated as the disease progresses: patients with active MM have a higher level of XPO1 compared to normal plasma cells (p<0.04) and to patients with monoclonal gammopathy of undetermined significance or smoldering MM (p<0.0001). The highest levels were in human MM cell lines. TC classification revealed highest levels in t(11;14) and lowest levels in t(4;14) disease. Selective inhibitors of nuclear export (SINE) compounds have recently been developed that irreversibly inhibit XPO1/CRM1 and its nuclear export function. One such inhibitor, KPT-276, decreased the viability of all 12 MM cell lines tested in vitro, as shown by MTT assay. After 72 hours of drug treatment, a median IC50 value of approximately 175 nM (range 30–1000 nM) was observed. No synergy with other commonly used anti-MM therapeutics was observed in vitro. In contrast, the drug had little effect in 8 solid tumor cell lines with the exception of the B cell lymphoma line Ramos. KPT-276 was also consistently active in inducing apoptosis against MM primary patient samples. Using an IC80 dose of KPT-276, drug-treated samples had a reduced population of cells in S phase (8%) compared to cells treated with DMSO (21%). Using the vkappa*myc transgenic MM model, KPT-276 reduced monoclonal spikes (by a mean of 56%) in all mice treated orally with 150 mg/kg dose three times per week for 4 weeks. Furthermore, KPT-276 significantly reduced tumor growth in a xenograft MM1.S mouse model. GEP was performed in the presence or absence of drug in two different MM cell lines. Two genes of probable relevance, cell division cycle 25 homolog A (CDC25A) and Bromodomain-containing protein 4 (BRD4), were dysregulated by SINE treatment. Both are involved in cell cycle control and have been linked to MYC. RT-PCR and western blotting confirm that MYC, CDC25A and BRD4 are down-regulated, as soon as six hours, after treatment with KPT-276. KPT-276 has shown marked anticancer activities against B cell malignancies in vitro and is active and tolerated in Phase I canine studies. KPT-330, a close analog of KPT-276, is currently in Phase 1 studies in human with advanced hematological and solid tumors. Disclosures: Schmidt: Karyopharm: Research Funding. McCauley:Karyopharm Therapeutics Inc: Employment. Shacham:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment. Stewart:Millenium: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy.

Abstract Human chromosome 13q14 is one of the hotspots of chromosomal deletion in prostate cancer, multiple myeloma, and chronic lymphocytic leukemia. Chromosome Condensation 1-like (CHC1L) is an uncharacterized gene in this region. CHC1L is found within the smallest common region of loss of heterozygosity in prostate cancer, and its decreased expression is linked to pathogenesis and progression of both prostate cancer and multiple myeloma. In the present study, we describe the generation and characterization of Chc1L gene knockout mice. Knockout mice develop normally and display an exaggerated proliferative response to LPS stimulation of cultured bone marrow and spleen cells at ten weeks of age. At approximately two years of age, both homozygous and heterozygous mice have markedly increased incidence of tumorigenesis, 80% and 56% respectively, compared to wild-type mice, 26%. The tumors are distributed most commonly to the spleen, mesenteric lymph nodes, liver and small intestine. The morphologic and immunophenotypic features of the tumor cells are consistent with those of tissue histiocytes, and most Chc1L knockout and heterozygote mice succumb to either Histiocytic Sarcoma or Histiocyte-Associated Lymphoma. These data provide the first direct evidence that Chc1L is a tumor suppressor gene involved in suppression of histiocyte-rich neoplasms. Citation Format: David Spillane, Ding Yan Wang, Changxin Shi, Youdong Wang, Anthony Gramolini, Keith Stewart, Susan Newbigging, Mingyao Liu, Xiao-Yan Wen. Chc1L is a tumor suppressor involved in development of histiocyte-rich neoplasms. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C41.