Citations

Abstract Abstract 852 Adult acute lymphoblastic leukemia (ALL) is an aggressive disease with <30% long-term survival. This relatively poor outcome can be explained in part by an increased frequency of high-risk molecular subtypes compared to childhood ALL, such as BCR/ABL (20%-40% in adults vs 2%-5% in children). We hypothesized that aberrant epigenetic gene regulation contributes to the pathogenesis and clinical features of adult ALL. We therefore performed genome-wide DNA methylation and gene expression microarray studies of 215 adult patients with B-lineage ALL enrolled in the ECOG E2993 phase III trial. Patients had a median follow-up 4.75 years (3.5 months to 13 years) and median age 39 years (17 to 63 years). BCR/ABL(+) cases (n=83) had a worse overall survival (OS, p=0.08) than BCR/ABL(-) cases (n=132). The smaller difference in survival in this series between BCR/ABL(+) and (-) cases is likely due to the use of imatinib in some of these patients. The HELP microarray assay was used to measure DNA methylation at 50,000 CpGs annotated to ∼22,000 RefSeq promoters. The accuracy of HELP was confirmed by extensive quantitative single locus validation studies. Supervised analysis revealed the presence of a markedly aberrant DNA methylation signature (166 genes) in BCR/ABL(+) ALL with the cutoff values of p<0.001 (FDR<0.002) and log fold change>1, and a differential gene expression profile of 416 genes at p<0.001 (FDR<0.004) and log fold change>1. Integrative analysis of expression and DNA methylation indicated that many of these genes are functionally connected within a gene network centered around IL8, which was over expressed and hypomethylated in BCR/ABL(+) cases, along with IL2RA(CD25), CEBPB, ABL1, ID1, IL15, BCL2L13, CD69, NOV, S100A8 and S100A9. KEGG and BioCarta pathway analysis also showed enrichment for IL8 signaling, NF-kB Activation, B Cell Development and Antigen Presentation pathways, suggesting these cytokine networks might play central and distinct roles in BCR/ABL(+) ALL. To identify a core set of functionally relevant genes, we explored the overlap of the DNA methylation and gene expression signatures. The overlap consisted of 13 genes, among which 11 showed inverse correlation, including CD200, GAB1, HLA-DQA1, HLA-DQB1, IL2RA (CD25), LST1, LTB, NOV, ROB04, S100A9, and CD38. Consistent with previous ECOG findings, CD25 positivity was a more dominant predictor than BCR/ABL, and could further stratify BCR/ABL(+) patients into a favorable (CD25-) and a poor (CD25+) outcome group (OS, p=0.07). When comparing CD25(+) and CD25(-) groups among BCR/ABL(+) cases, we found RhoH and UBE2J1 as the top differentially methylated and IL2RA(CD25) as the top differentially expressed genes, and all three genes showed concordant corresponding changes in expression and methylation. The aberrant DNA methylation signature of MLL/AF4 cases was even more dramatic, with 469 identified as differentially methylated (p<0.001 (FDR<0.015), log fold change>1) and 1108 genes differentially expressed (p<0.001 (FDR<0.009), log fold change>1). Integrated analysis of DNA methylation and expression implicated gene pathways centered around TNFA and MYC, respectively. A core set of 44 genes were identified overlapping between the methylation and gene expression signatures, among which 34 showed inverse correlation, including ANXA5, BRE, CAPG, CEBPA, FAIM, FLT3, FUT4, IGFBP7, IL1R2, ITGA7, ITGAE, MAP1A, MAP7, MRPL33, PARP8, RBKS, SLITRK4 and TFR2 with overexpression and hypomethylation, and BTBD3, CCR6, FYN, GAB1, GYPC, HPS4, IL2RA, KCNK3, LCK, LST1, LTB, PRKCH, QPCT, S100A13, SGPL1 and ZAP70 with underexpression and hypermethylation in MLL/AF4(+) ALL. All signatures were independent of B-ALL differentiation stage. Using univariate Cox Hazard Regression model adjusted by age, WBC, CD25 and BCR/ABL status, we furthermore identified 259 expression and 115 methylation markers which were significantly correlated with patient OS risk (p<0.01). Molecularly or immunophenotypically defined poor prognosis adult B- ALLs thus feature specific aberrant DNA methylation profiles with associated inversely correlated gene expression. These gene sets delineate specific biological functions that may contribute to disease phenotype and offer an opportunity for development of targeted therapy. Aberrantly methylated genes in adult B-ALL correlate with clinical risk independent of other clinical or molecular risk factors. Disclosures: No relevant conflicts of interest to declare.

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 This study was coordinated by the ECOG-ACRIN Cancer Research Group (Robert L. Comis, MD and Mitchell D. Schnall, MD, PhD, Group Co-Chairs) and the Medical Research Counsel, United Kingdom, and supported in part by Public Health Service Grants CA180820, CA180794, CA180790, CA189859, CA180853, CA180791, and from the National Cancer Institute, National Institutes of Health and the Department of Health and Human Services. Its content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute. Background: Late relapse in acute leukemia is considered a relatively rare event. Patients with acute myeloid leukemia (AML) are often considered cured of the disease at 3 years, but information regarding adult acute lymphoblastic leukemia (ALL) patients is scarce. Data are presented from one of the largest prospective adult ALL studies, the MRC UKALLXII/ECOG E2993, to evaluate the rate and characteristics of late relapse in ALL. For this purpose, late relapse was defined, arbitrarily, as relapse 3 years post achievement of complete remission (CR) and very late relapse was defined as relapse > 5 years from CR. Methods: The UKALLXII/ECOG E2993 was an international ALL trial conducted jointly by the MRC in the United Kingdom and ECOG in the United States. All patients received identical induction therapy, followed by central nervous system prophylaxis. Patients with a sibling donor (or a matched unrelated donor in Philadelophia-chromosome-positive ALL) were assigned to receive an allogeneic hematopoietic stem cell transplant (HSCT); all others were randomized to undergo an autologous transplant or protracted standard consolidation/ maintenance therapy. The study accrued 2109 patients from 1993 to 2008. Following relapse, patients were followed for survival. For this report only patients registered before the tyrosine kinase inhibitors era are included in the analysis. Results: 1518 study patients were eligible for this analysis, 1208 (79.6%) Philadelphia-chromosome negative (Ph-neg) and 267 (17.5%) Philadelphia-chromosome positive (Ph-pos). 1381 (91%) of the patients achieved CR; 93% of the Ph-neg and 82% of the Ph-pos. 572 patients (37.7%) underwent allogeneic HSCT. The median duration of follow-up of patients who achieved CR was 10 years. Among the 1381 patients who achieved CR, 626 (45.3%) had a documented relapse; 566 (90.4%) relapsed within 3 years of CR and 60 (9.6%) relapsed beyond 3 years ('late relapse') (Figure 1). Among these 60 patients, 18(2.9%) relapsed after 5 years ('very late relapse'). Table. Patients n CR All relapses Relapses< 3 years Relapses≥ 3 years Relapses≥ 5 years All patients 1518 1381 (91%) 626 (45.3%) 566 (90.4%) 60 (9.6%) 18 (2.9%) Ph-neg 1208 (79.6%) 1123 (93%) 485 (40.1%) 429 (88.5%) 56 (11.5%) 17 (3.5%) Ph-pos 268 (17.5%) 219 (82%) 124 (56.6%) 122 (98.4%) 2 (1.6%) 1 (0.8%) Relapse beyond 3 years occurred in 4.3% of all who achieved CR, in 5% of Ph-neg and 0.01% of Ph-pos patients. Among the 60 late relapses, the median time to relapse was 46 months. 61.7% of the late-relapse patients were males, median age was 32 years, 88.3% were B-lineage ALL and the median white cell count at diagnosis was 6000/ul. 56.7% were in cytogenetic standard risk, 8.3% at high risk and the data of 35% are unknown. The median survival for the late relapse patients was longer than for those who relapsed within 3 years. The overall survival (OS) of the 56 Ph-neg patients who relapsed beyond 3 years is shown in Fig 2. Table.Relapse > 3 yearsRelapse > 3 yearsMedian survival from relapse (months)5.411.23-year OS from relapse6.5%29%5-year OS from relapse5.6%19% Conclusions: Late relapses in adults with Ph-neg ALL are not uncommon. About 10% of relapses occur beyond 3 years and 4.3% of all ALL patients who achieved a CR can expect to have a late relapse. These data are in contrast to AML where only 1% of patients relapse beyond 3 years (Watts JM et al, 2014). Most of the late relapse patients were at standard risk and appeared to have a relatively favorable outcome post relapse. Patients with ALL, particularly those who are Ph-neg, cannot be considered as cured at 3 years and need to be closely followed. Figure 1. Time to relapse of Ph-pos and Ph-neg ALL Figure 1. Time to relapse of Ph-pos and Ph-neg ALL Figure 2. Survival from relapse for Ph-neg patients who relapsed after 3 years from CR. Figure 2. Survival from relapse for Ph-neg patients who relapsed after 3 years from CR. Disclosures Rowe: Amgen: Consultancy; BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx Ltd.: Consultancy. Douer:Gilead: Consultancy.

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 Both genome wide association studies (GWAS) of common variation and exome wide association studies (EXWAS) of rare variation have successfully identified disease susceptibility variants for a variety of diseases. One GWAS of inherited susceptibility to Acute Myeloid Leukemia (AML) has been conducted, but no EXWAS have been performed to measure risk of AML attributable to low-frequency constitutional genetic variation. We performed the first EXWAS of risk of AML as a nested case-control study in the DISCOVeRY-BMT (Determining the Influence of Susceptibility Conveying Variants Related to one-Year mortality after BMT) cohorts. The DISCOVeRY-BMT parent study examined transplant-related mortality in leukemia patients undergoing unrelated donor allogeneic BMT. To identify low frequency variants and genes contributing to increased susceptibility to AML we used genotype data from the Illumina HumanExome BeadChip typed in the DISCOVeRY-BMT cohorts; the HumanExome BeadChip contains 242,901 variants, which are mainly protein-coding variants. The optimal sequence kernel association test (SKAT-O) was used to analyze gene-level associations with risk of AML. These gene-based tests evaluate the cumulative effects of multiple single gene variants on risk of AML. Analyses were performed in all European American AML cases and two subtypes: 1) de novo AML, 2) de novo AML with normal cytogenetics. Models were adjusted for age at transplant and principal components to control for population stratification. For gene-based tests at least 2 variants with minor allele frequency (MAF) ≤ 5%, were required to be present in the gene. This yielded a total of 13,687 genes tested, and a Bonferroni corrected significance level of P<3.65 x 10-6. Association tests were performed in 1,189 AML cases reported to CIBMTR 2000-08 (Cohort 1) and 327 AML cases reported to CIBMTR from 2009-11 (Cohort 2). Controls in Cohorts 1 (n=1,986) and 2 (n= 515) were 10/10 HLA-matched unrelated donors who passed a comprehensive medical exam and deemed healthy. We used metaSKAT to combine Cohorts 1 and 2 and obtain p-values of association with AML. We present the results of gene-level tests significant in both cohorts. The likely pathogenicity of these variants was determined in silico using SIFT, PolyPhen and MutationTaster. Patient characteristics are in Table 1. DNMT3A, on chromosome 2, was associated in the gene-based test with risk of AML (Pmeta=1.70x10-9, Table 2). Three missense variants at MAF <1% comprise both overall AML and de novo AML gene-based association: exm177559 (Asn->Ser), exm177507 (Arg->His), and exm177543 (Arg->Trp). Normal cytogenetics de novo AML gene-based assocations consisted of only 2 of these variants: exm177559 and exm177507 (Table 2). While prevalence of exm177507 is <1% for all AML cases, in de novo AML with normal cytogenetics the MAF was higher at 3%. The other 2 variants had a MAF<1% irrespective of subtype. Somatically, DNMT3A is most frequently mutated in hematologic malignancies, with >30% of de novo AML cases with a normal karyotype and >10% of MDS patients having DNMT3A mutations. Although these are germline gene associations all three of the variants found have been reported somatically in hematologic malignancies. In 200 AML cases from The Cancer Genome Atlas (TCGA) p.R882H (represented as exm177507 on the exome chip) was a frequent somatic mutation (25%). Exm177543 (p.R635W) and exm177559 (p.N501S) are reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) as somatic mutations involved in hematopoietic and lymphoid tissue in both cell lines and humans. Exm177507 and exm177543 show evidence of pathogenicity in all three in silico tools, while exm177559 was reported as deleterious and disease causing by Sift and MutationTaster, respectively. Our results show that multiple potentially pathogenic missense germline variants in DNMT3A comprise the gene-based association with AML, specifically de novo AML with normal cytogenetics. Given the functional nature of these variants it is possible germline risk stratification could be informative in determining AML risk, and subsequently development of AML harboring DNMT3A mutations. Confirmation of these findings in additional cohorts could have implications for individualized risk screening, prediction and prognosis. Additional cytogenetic subgroup analyses, including treatment-related AML, are underway. Disclosures Hahn: Novartis: Equity Ownership; NIH: Research Funding. McCarthy:Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; The Binding Site: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gamida Cell: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sucheston-Campbell:NIH/NCI: Research Funding.

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 Multiple myeloma tumors universally target one of the three human cyclin D genes (CCND1, CCND2 or CCND3) for dysregulation (Bergsagel et al., 2005, Blood, 106:296). Using a lentivirus expressing CCND2 RNAi we first tested the effects of selective cyclin D2 knock down on My5 and H929 myeloma cell lines and found G0/G1 phase arrest, increased apoptosis and significant selective disadvantage in transfected cells. By comparison, knockout mouse models indicate that most somatic tissues can develop in the total absence of cyclin D1, D2 and D3 (Kozar et al., 2004, Cell,118:477). Targeted inhibition of specific cyclin D expression is therefore a rational therapeutic strategy in myeloma. To identify novel pharmaceutical inhibitors of CCND2 transactivation we developed an assay employing NIH 3T3 cells stably co-expressing the CCND2 transactivator c-Maf and the cyclin D2 promoter driving firefly luciferase (luc) and screened the Lopac (n=1280), Prestwick (n=1120) and Spectrum (n=2000) libraries of drugs and natural compounds. In a parallel MTS assay, the effect of each compound on 3T3 viability was determined, allowing exclusion of compounds that caused secondary suppression of CCND2 due to non-specific cytotoxicity. From the screen we identified 10 c-Maf independent putative CCND2 inhibitors. These included monensin, patulin, β-lapachone, camptothecin, dihydrogambogic acid, gentian violet, thapsigargin, brefeldin A, pristimerin and kinetin riboside. Three of the 10 compounds (gentian violet, thapsigargin and patulin) were not studied further due to toxicity cited in the literature. Subsequent validation studies using selected compounds in human myeloma cell lines (HMCL) confirmed successful suppression of both cyclin D2 and D1 proteins. Each of these compounds was then shown to be cytotoxic to a genetically diverse and standardized panel of 14 HMCL in MTT assays: monensin (10–760 nM), camptothecin (5–700nM), dihydrogambogic acid (250–800 nM), pristimerin (150–500 nM) and kinetin riboside (2.5–20μM). Cell cycle analysis confirmed induction of G0/G1 phase arrest for most compounds, consistent with cyclin D inhibition. However, camptothecin and b-lapachone induced S-phase arrest, suggesting secondary suppression of cyclin D by virtue of S-phase activity. Unsorted myeloma patient bone marrow samples demonstrated selective activity for pristimerin, dihydrogambogic acid and kinetin riboside against CD138+ myeloma cells compared with non malignant hematopoietic cells; by contrast monensin showed almost equal toxicity for normal cells. The triterpenoid, pristimerin, showed potent anti-myeloma activity and was examined in greater detail. Studies confirm that pristimerin rapidly inhibits cyclin D1, D2 and D3 expression (<6 hours) at nanomolar concentrations and induces apoptosis of primary myeloma cells characterized by caspase 9 cleavage and Annexin V binding. While pristimerin is cytotoxic to HMCL and patient myeloma cells at 0.1–0.15 mg/L, toxicity studies in vivo indicate that the drug is tolerated in mice at 2.5 mg/kg i.p. daily. In vivo activity against a xenograft model is currently being determined. Overall this targeted chemical biology screen has identified several compounds, including the triterpenoid, pristimerin, that are being further characterized for promising preclinical anti-myeloma activity.

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.