Citations

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 Abstract 3513 To identify targets for rational combination therapies with 5-Azacytidine (5-Aza) in myeloid malignancies, we utilized high-throughput RNA-interference (RNAi) viability screening. A siRNA library targeting 572 kinases and a custom collection of 289 putative cancer targets, including cell cycle and apoptosis regulatory genes, were screened alone and in combination with 5-Aza in TF-1 and ML-2 myeloid leukemia cell lines to identify synergistic interactions in reducing cell viability. Of the 572 kinases that were individually silenced, less than 1% significantly increased sensitivity to 5-Aza. The kinase library was also screened in combination with 5-Aza in a third myeloid cell line, THP-1, confirming that few kinases sensitize to 5-Aza when inhibited. While few kinases sensitized to 5-Aza, the anti-apoptotic Bcl-2 family of genes emerged as potent sensitizers to 5-Aza from RNAi screens. Therefore, silencing by siRNA of BCL-XL, BCL-2, BCL-W, MCL-1 and BFL-1 was evaluated in combination with 5-Aza treatment in an expanded panel of myeloid cell lines including TF-1, HEL, THP-1, ML-2 and MDS-L. BCL-XL validated as a vulnerability and potent sensitizer to 5-Aza in erythroid leukemia cell lines TF-1 and HEL, whereas MCL-1 was a strong vulnerability in the monocytic leukemia cell line THP-1 and also a moderate sensitizer to 5-Aza in ML-2, THP-1, TF-1 and HEL. Published proteomics data from our group indicate that M6 and M7 leukemias exhibit higher levels of BCL-XL, while additional unpublished data suggest elevated levels of MCL-1 in M4 and M5 leukemias, supporting our functional observations. Additionally, data from the public database Oncomine suggest that BCL-XL expression is elevated in M6 and M7 leukemias while MCL-1 shows a trend towards elevation in M4 and M5 leukemias. Based on RNAi screening results, siRNA validation experiments and proteomic/mRNA expression data, we evaluated the BCL-2/BCL-XL/BCL-W inhibitor ABT-737 in combination with 5-Aza. ABT-737 resulted in dose-dependent sensitization to 5-Aza in all AML-derived cell lines examined (including M7, M6, M5, M4 and M2 FAB subtypes) and in the MDS cell line MDS-L; however, no sensitization was observed in the CML cell line K562. In extensive ex vivo experiments with 17 primary specimens, potent synergy between 5-Aza and ABT-737 was observed in AML, MDS and MPN samples, but not in most CML samples examined. Calculations with CalcuSyn software demonstrate synergy, with combination index values as low as 0.2, between 5-Aza and ABT-737 both ex vivo and in vitro. The combination of 5-Aza with ABT-737 resulted in substantial induction of apoptosis, measured by the induction of cleaved caspase 3 in TF-1 and HL-60 cells, as compared to either compound alone. Interestingly, although siRNA silencing of MCL-1 in combination with 5-Aza was potent across several cell lines, and silencing of BCL-XL preferentially in an erythroid differentiation background, ABT-737 with 5-Aza sensitized across a variety of cell lines and all myeloid primary specimens ex vivo. We suggest that inhibition of anti-apoptotic Bcl-2 family members is a most promising rational combination strategy with 5-Aza for the treatment of leukemias. Our results also highlight the potential utility of more specific anti-apoptotic Bcl-2 family inhibitors in the lineage-specific treatment of myeloid malignancies. Disclosures: Off Label Use: AraC in AML. Experimental Agent MK1775. Mesa:Incyte: Research Funding; Lilly: Research Funding; SBio: Research Funding; Astra Zeneca: Research Funding; NS Pharma: Research Funding; Celgene: 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 Introduction: We employed a customized Multiple Myeloma (MM) specific Mutation-Panel (M3 P) to screen a homogenous cohort of refractory MM (rMM) patients in a timely and cost-effective manner. rMM was defined as progressive disease (PD) during therapy or within 60 days after discontinuation of therapy. The M3 P provides a practical alternative to whole genome/exome sequencing approaches and includes 88 genes selected for being either actionable targets, potentially related to drug response or part of known key pathways in MM biology. Patients and Methods: Tumor and germline samples of 50 rMM patients were screened using M3 P. Patients received a median of five lines of treatment at the time of tumor sampling (range 2-15 lines). All patients received at least one immunomodulatory agent (IMiD) and proteasome inhibitor (PI), and 88%, 76% and 66% were refractory to either an IMiD, PI or both. All patients had received lenalidomide and bortezomib, 48% and 34% were additionally exposed to pomalidomide and thalidomide and 18% to carfilzomib at any time prior to sampling. The majority of patients had a progressive and refractory disease immediately prior to sampling (82% and 78%), with 43% of them being IMiD-refractory and 46% being PI-refractory in the most recent line of therapy. Cytogenetic data at time of sampling were available from 78% of patients. Adverse cytogenetics were detectable in 82% of patients with gain 1q21 > 2 copies (62%), deletion 17p (33%), t(4;14) (13%) and t(14;16) (8%) being the most common. Results: An average sequencing depth of 750x was obtained across the samples. Our screening revealed an increased prevalence of mutations in the MAPK pathway (72%), including 34% of KRAS, 26% of NRAS, 18% of BRAF (with one patient carrying two BRAF mutations and 8% in actionable p.V600E). Additionally, two mutations (4%) were found in RASA2, a RAS inhibitor. Other potentially actionable mutations were seen in p.R132H IDH1 and p.R248C FGFR3 (2% each). Most prominently, the CRBN pathway was found mutated in 22% of cases, including CRBN (12%, one case with concomitant CUL4B mutations), CUL4B (4%), IRF4 (4%) and IKZF1 (2%). In CRBN we found four cases with mutations leading to a truncated protein (frameshift, nonsense and splicing) and two cases with missense mutations located within the thalidomide-binding domain, suggesting a causal effect on IMiD resistance. All CRBN mutated patients and 91% of the CRBN pathway mutated patients were at least once unresponsive to IMiD based treatment. In one patient with IRF4 mutation sensitivity to IMiDs remained unknown. A majority of patients were also resistant to PI treatment (76%), however mutations in proteasome subunits (PSMD1 and PSMB8) and XBP1 wererare (4%), but only a limited number of this gene family were represented in our panel. Other recurrently mutated genes were TP53 (26%), FAM46C (12%), ATM (10%) and TRAF3 (8%). Notably, DIS3 mutations were identified in only 6% of cases. As previously reported, TP53 mutations were associated with deletion 17p (8/13 cases, 62%). A correlation between the absolute number of mutations and previous lines of treatment was not observed (r=0.05, p=0.73). Conclusion: Targeted sequencing on a homogenous cohort of heavy pretreated rMM patients gives key insights in the landscape of the rMM genome, uncovering an increasing frequency of MAPK, TP53 and CRBN mutations. Importantly, this is the first study showing recurrent mutations in CRBN in patients unresponsive to IMiD treatment, supporting a potential association with resistance to IMiD based therapies. Disclosures Mai: Onyx: Other: Travel Grant; Janssen-Cilag: Other: Travel Grant; Celgene: Other: Travel Grant; Mundipharma: Other: Travel Grant. Merz:Janssen: Other: Travel grants; Celgene: Other: Travel grants. Hillengass:Takeda: Honoraria, Other: Travel support; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding; Janssen-Cilag: Honoraria, Other: Travel support; Celgene: Honoraria, Other: Travel support. Goldschmidt:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Chugai: Research Funding, Speakers Bureau. Stewart:Oncospire Genomics: Research Funding.

Abstract Abstract 2801 Poster Board II-777 To provide a rationale basis for targeted drug development for multiple myeloma patients, we have conducted high-throughput genome-scale RNA interference (RNAi) synthetic lethality studies in human myeloma cell lines (HMCL) to generate a comprehensive map of critical genes and molecular vulnerabilities in human myeloma tumor cells. KMS11 human myeloma cells were screened with a 13,982-oligo library targeting the ‘druggable' genome (6,991 genes) using optimized conditions that resulted in >95% transfection efficiency. Each gene was screened with 2 or more distinct oligos, in duplicate, using a single-siRNA-per-well format, testing >34,000 wells. Replicate high throughput experiments yielded highly reproducible results (R2=0.82). Viability was measured at 96h by ATP-dependent luminescence. Universally lethal siRNA and non-targeted siRNA were employed as controls. The specificity (FDR) of lethal RNAi was evaluated by custom-developed statistical methods based on RNAi result concordancy. From screening, 5.8% of druggable genome siRNA caused statistically relevant 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%). Of these, two hundred and nineteen genes, targeted by the most lethal siRNA, were forwarded to validation studies. Validation high-throughput RNAi studies, using 4 oligos per gene, were conducted in order to verify target gene vulnerability. Ultimately, seventy two genes were validated as highly critical for myeloma cell survival (each with multiple concordant siRNA hits plus high reproducibility of R2 =0.94). Among top-ranked lethal molecular vulnerabilities in KMS11 myeloma cells we recurrently identified the proteasome (8 PSM subunits were independently identified as highly vulnerable); and BCL2 family member MCL1; both known therapeutic targets, validating a functional genomics approach to target discovery. In addition, a number of novel, equally lethal, molecular vulnerabilities were identified in KMS11 including WEE1, kinetochore complex component KNTC2, the aurora kinases, polo-like kinase 1, a previously uncharacterized DNA methyl transferase, ribonucleotide reductase, ribosomal protein L38, leptin receptor overlapping transcript LEPROT and SLC25A23 (a carrier responsible for mitochondrial adenine nucleotide flux). Various ubiquitous cellular proteins involved in RNA or protein processing (e.g. SF3a, SNW1, SNRPA1, EIF3s8, amongst others) were also identified as non redundant and critical for cellular viability. Thirty nine top-ranked molecular vulnerabilities in KMS11 were evaluated for comparative vulnerability in a second myeloma cell line, JJN3, and in A549 and 293 epithelial cells. A majority were vulnerable in JJN3. While 25 genes proved equally lethal on silencing in A549 and 293 epithelial cells, several, notably including MCL1 and three proteasome subunits, appear differentially susceptible in myeloma cells versus epithelial cells. From this genome scale study, MCL1 ranks third as a target capable of inducing cell death in myeloma cells, highlighting the potent vulnerability of myeloma cell viability to alterations in this anti-apoptosis regulator. Moreover, from active comparative studies of molecular vulnerability in myeloma versus epithelial cell lines, MCL1 as an RNAi target demonstrates perhaps the greatest cytotoxic selectivity between myeloma and epithelial cell lines of all targets examined. Gene expression studies indicate that MCL1 is highly expressed in myeloma tumor cells but is absent or only weakly expressed in human primary somatic tissues. Overall, therefore, from an unsupervised genome-scale screening approach, our data support MCL1 as an optimal Achilles heel molecular target in multiple myeloma, potentially offering greater specificity and deeper cytotoxic effect than proteasome inhibition. Disclosures: No relevant conflicts of interest to declare.

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 Abstract 1807 We have recently demonstrated that cereblon (CRBN) mediates the direct anti-myeloma activity of immunomodulatory drugs (IMiDs). However, the genes/pathways downstream of CRBN associated with anti-myeloma activity remain unclear. We, and others, identified interferon regulatory factor 4 (IRF4) as one of the downstream targets of CRBN-associated signaling. Both lenalidomide treatment and CRBN knockdown downregulate IRF4. IRF4 levels return to baseline in IMiD resistant cells surviving CRBN silencing. To determine whether IMiD-induced IRF4 downregulation is critical to anti-MM activity, we overexpressed IRF4 in two IMiD-sensitive human MM cell lines (HMCLs), KMS11 and MM1.S, followed by lenalidomide treatment. Lenalidomide-induced cytotoxicity was greatly impaired in both HMCLs overexpressing IRF4 compared with the control virus infected cells. Further analysis indicated that IRF4 over-expression does not completely prevent lenalidomide-induced growth arrest, but reduces cell death by 70% after lenalidomide treatment. Immunoblotting analysis of KMS11 cells indicated that IRF4 over-expression blocks lenalidomide-induced activation of caspase 8, reduces up-regulation of p21waf and increases CDK6 expression but does not significantly affect lenalidomide-induced MYC down-regulation. Although cereblon and IRF4 are broadly expressed in MM, baseline levels of expression are only weakly correlated (r=0.22) in primary MM patient gene expression analysis. Gene expression studies revealed statistical changes in 1,368 genes when comparing high versus low CRBN expression in primary myeloma samples. Interestingly genes associated with high CRBN expression included cyclin D2, SOCS3 and IL4 while genes associated with low cereblon expression included cyclin D1, FRZB and CD200. In order to understand how CRBN is connected with downstream anti-myeloma signaling, a structure-function study was performed to determine which CRBN domain is required for lenalidomide-induced IRF4 down-regulation and cytotoxicity. Lentiviral constructs expressing wild-type CRBN and a series of mutated CRBN were generated, including mutations at thalidomide binding site (Y384A/W386A), deletion of DDB1 binding region (ΔMid) and truncations at N-terminal and C-terminal. Lentiviruses from these constructs were used to infect IMiDs resistant HMCLs, OCI-MY5 and MM1.S res. Both of these cell lines have very low endogenous CRBN expression and they became sensitive to lenalidomide after introduction of wild-type CRBN. Conversely, introduction of CRBN with mutated thalidomide-binding site or with DDB1 binding region depletion failed to mediate lenalidomide toxicity and down-regulation of IRF4. OCI-MY5 cells expressing either N-terminal or C-terminal truncated CRBN showed substantial reduced responses (more than 50%) to lenalidomide compared with wild-type CRBN expressing cells. Deletion of only 20–30 amino acids at either ends of CRBN greatly impaired the protein function, suggesting that protein folding might be important for CRBN-mediated IMiD response. Our data indicate that IMiD induced myeloma cytotoxicity is largely mediated by modifying CRBN associated E3 ubiqutin ligase and subsequent IRF4 downregulation, suggesting the CRBN-IRF4 axis is a potential target for development of new anti-myeloma drugs. Disclosures: Schmidt: Karyopharm: Research Funding. Stewart:Millenium: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy.

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 Previous work in our laboratory has identified the importance of the NFB signaling pathway for plasma cell (PC) survival and accumulation in Multiple Myeloma (MM). Although up to 43% of patient PCs appear to have activated NFkB by gene expression profiling (GEP), the remainder must rely on an alternate signaling pathway for their survival. We examined our MM GEP datasets and supervised the clustering of patients according to an index of NFkB activity. The majority of patients not activating NFkB were associated with a high IL-6/Stat3 signature, which included the protein phosphatase of regenerating liver-3 (PRL-3). Interestingly, analysis of the PRL-3 promoter revealed competing binding sites for both NFkB and STAT3 within the first intron, suggesting that these play a role in the transcriptional regulation of this gene. PRL-3 has been previously demonstrated to be absent in most normal tissues while its expression in MM cells is quite elevated. As normal PCs have low levels of PRL-3 expression, we determined the upper limit of normal to be 3SD above the mean of these cells. Using this cutoff we report that elevated PRL-3 expression is observed in plasma cells from 1/15 normal individuals, 4/22 MGUS patients and 47/126 patients with either smoldering or overt MM (6.7%, 18.2% and 45.6% respectively). Less than 10% of patients were seen to have both an elevated NFkB index and high PRL-3 expression. Patients with higher expression of PRL-3 were found to have statistically significant higher mean values of creatinine, C-reactive protein, lower albumin levels and a lower overall survival in our Mayo Clinic MM dataset. To examine the role PRL-3 in MM biology we first stimulated MM cell lines with IL-6 and observed PRL-3 up-regulation in two cell lines (INA6, OPM1) but not in JK6L (an NFkB activated cell line). Using a luciferase reporter we have determined that STAT3 induction of PRL3 expression requires the first intron of the PRL3 gene which contains the putative STAT3 binding site. Finally we show that PRL-3 inhibition by shRNA leads to decreased cell growth and loss of IL-6 responsiveness in vitro in at least one cell line. Based on these results, we propose that the majority of MM patients depend on either the NFkB or STAT3 signaling pathways for their survival and that these are to a large degree mutually exclusive. Our experimental results suggest that PRL-3 plays a role in IL-6/STAT3 signaling and that it is a promising target for drug development.