Kortuem, K. Martin

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 Introduction: Customized gene panel sequencing is an attractive approach to genomic tumor characterization in clinical care. Based on published MM exome data we developed a MM Mutation Panel (M3 P) that includes the most commonly mutated genes, actionable drug targets, genes targeted by current standard of care (SOC) therapies and which allows tracking of clonal evolution, copy number and sample purity. Methods and Material: M3 P (v3.0) covers 88 genes (1327 amplicons, 181kb). MM samples from 504 patients (pts) have been analyzed (corresponding germline in 81%) through collaborations between Mayo Clinic and Hospital-12-de-Octubre (Madrid, Spain), the DSMM and GMMG (Würzburg, Ulm, Freiburg and Heidelberg, Germany) and the HOVON trial groups (Rotterdam, The Netherlands). The investigated cohort includes 410 untreated pts (81%), which includes a high risk cohort of 72 pts with del17p, 25 paired samples with later follow up from the same cohort, and 94 relapsed patients, of which 50 were relapsed and refractory. Results: Overall coverage per mutation averaged >500x depth. We identified 945 variants (1.9 per pt) and in 83% of the pts a mutation was found. Clonal heterogeneity was assessed with mutations ranging from 3%-100% variant reads suggesting the presence of a significant number of subclones (e.g. 21% of mutations were in < 10% of reads). The mutation incidence was compared with and closely resembles the most recent MM comprehensive genomic data from the MMRF CoMMpass study: We compare here all pts sequenced by M3 P, untreated pts sequenced by M3 P and CoMMpass: KRAS (24%/23%/24%), NRAS (20%/20%/18%), DIS3 (13%/14%/10%), BRAF (9%/7%/6%), FAM46C (6%/6%/8%) and TRAF3 (6%/6%/7%). TP53 mutation incidence, however, was significantly increased in our cohort (14%/12%/4.2%), a difference explained by the inclusion of del17p (untreated) and relapsed refractory MM in panel sequenced pts, cohorts with elevated incidences of TP53 mutations (32% / 26% respectively). Potentially actionable targets include BRAF mutationsin 43 patients (9%), with druggable p.V600E in 19 or 5%, 8 pts (2%) with FGFR3 (p.R248C and p.G375C one patient each), p.R132 mutation in 4 out of 5 IDH1 (1%) and p.R172K IDH2 mutation in 1 of 3 (1%) pts. Mutations in the MAPK pathway (NRAS, KRAS, BRAF) were detected in 59% of pts, ranging from 36% untreated MM to 72% in refractory MM. Similarly, the CRBN/CUL4B/IKZF1/IKZF3/IRF4 pathway, important for IMiD function, harbored a significant enrichment of mutations in advanced disease (6% untreated vs 17% relapsed), including CRBN mutations (0.5% vs 7%). Nine of 17 IRF4 mutations were located at the p.K123R hotspot, with minor difference between early or late disease (1% vs 3%). Notably, in 8 of 9 pts with CRBN mutation and clinical information, all were unresponsive to IMiD therapy, supporting association of these mutations with resistance to IMiDs. Conversely, M3 P genes related to other SOC therapies, including NR3C1 (targeted by steroids) and 5 proteasome subunit genes (proteasome inhibitors), were rarely mutated across the cohorts not exceeding 1% mutation incidence for each gene. Significant differences in DIS3 and FAM46C mutation incidences were observed across cohorts: DIS3 mutations are more common in untreated pts with a 1.7 fold increased predominance (14% untreated and 8% treated). FAM46C has an expected incidence of 8% but was rarely mutated in untreated del17p high risk disease with only one of 100 patients harboring both mutations. The significance of this finding needs to be determined but implies a possible overlap in function. Finally we assessed impact on survival of the mutation variants identified in 142 untreated Mayo patients and found STAT3 mutations negatively impacting PFS (p=0.034) and OS (p=0.001). This gene is rarely mutated in MM (2% of the total cohort) thus the sample size was small and this finding needs further validation. Conclusion: We here describe 504 MM patients sequenced using the M3 P gene panel, which identified mutations in >80% of investigated patients, overlaps well with published whole exome sequence data and provides clinically relevant information. New findings were the high frequency of minor clones, the relative lack of overlap of del17 and FAM46C mutation, a higher frequency of DIS3 mutation at diagnosis compared to relapse, the prognostic significance of STAT3 mutation and the frequent presence of CRBN pathway mutation in drug resistant relapsed patients. Disclosures Sonneveld: Janssen-Cilag, Celgene, Onyx, Karyopharm: Honoraria, Research Funding; novartis: Honoraria. Mai:Janssen-Cilag: Other: Travel Grant; Onyx: Other: Travel Grant; Mundipharma: Other: Travel Grant; Celgene: Other: Travel Grant. Goldschmidt:Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millenium: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Chugai: Honoraria, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Knop:Celgene Corporation: Consultancy. Kull:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Martinez-Lopez:Novartis: Honoraria, Research Funding; Bristol-Meyer Squibb: Honoraria; Celgene: Honoraria; Janssen: Honoraria. Einsele:Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Amgen/Onyx: Consultancy, Honoraria, Speakers Bureau. Raab:Novartis: Research Funding. Stewart:Oncospire Inc.: Equity Ownership; Celgene: Consultancy; Novartis: Consultancy; BMS: Membership on an entity's Board of Directors or advisory committees.

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 Background:A goal of “post-genomic” cancer care is development of specific, affordable, reliable and rapid next-generation sequencing-based assays determining prognosis, therapeutic selection and minimal disease monitoring, replacing older technologies in the diagnostic and monitoring workup. In this regard, we designed and deployed a custom gene panel for identification of mutations, copy-number changes and clonal frequency in multiple myeloma (MM) samples. Methods: The MM Mutation Panel Version 2.0 (M3P 2.0)1 includes 1271 amplicons from 77 genes that are either mutated in MM (genes with a published mutation incidence ≥3%), represent critical pathophysiological pathways, are targetable with small molecule-based therapies and/or are associated with drug resistance (eg. XBP-1, CRBN, IZKF, IRF4). The amplicons are spread across 21 chromosomes, allowing the identification of copy-number changes in these regions. Results: We have sequenced 38 MM cases (150 will be presented) with paired tumor-germline DNA samples, using only 20ng of DNA for library preparation. Patients (Pts) included 29 untreated and 9 MM pts with multi drug refractory disease. Coverage depth averaged 964X. Hyperdiploid MM was successfully identified in 45% of cases, del(13q) in 34%, del(1p) in 18%, del(17p) in 8% and gain of 1q in 13%. Array CGH in representative cases confirmed concordance with semiconductor sequencing. Overall, 71 exonic missense and 3 nonsense mutations were detected, and 7 frameshift indels. In 89% of the pts a mutation in at least one M3P2.0 gene was identified (range 1-8). 92% of mutations were predicted damaging (Provean, Polyphene-2 or SIFT). Most commonly mutated were KRAS (37%), NRAS (21%), BRAF (13%), TP53, CDKN1B, DIS3 (each 11%), FAM46C, STAT3, and IRF4 (each 8%). The MEK-ERK pathway was mutated in 61% of cases and in 3 cases more than one gene in this pathway (NRAS, KRAS, BRAF) was simultaneously mutated. Six mutations in BRAF were identified in 5 untreated pts, 3 of them known to be activating and targetable (Val600E). Other commonly mutated pathways were the NFKB pathway in 24% of pts and the Cyclin D pathway, mutated in 16% of cases. Genes of the cereblon pathway (CRBN, IRF4, IKZF3), essential for the action of lenalidomide (len) were mutated in 5 Pts. One newly diagnosed and len refractory Pt harbored a subclonal CRBN mutation (Asn316Lys, allelic fraction [AF] 20%) as well as 4 clonal IRF4 mutations within the DNA binding domain of the gene (Lys89Asn (AF 78%), Lys77Glu, Ser48Arg and Cys49Ser (AF 80% each). Another IRF4 mutated (Lys123Arg, AF 90%) hemodialysis dependent Pt was responsive to dose adapted treatment of len and dexamethasone (dex). Mutations in the C2H2-type 3 region of IKZF3 in a len resistant (Gly191Arg, AF 48%) pt occured as well as in a len and dex responsive (Arg242Gly, AF 52%) Pt. Furthermore, we identified a truncating XBP1 mutation (p.Leu232*, 97% AF) in a Pt refractory to bortezomib. One patient harbored a mutation in the steroid receptor NR3C1 (Pro255Leu, 22% variant reads). Finally, integrated mutation and copy-number data identified biallelic deletions of CDKN2C and FAM46C in a multi drug refractory Pt. Mutation and/or copy-number losses in both alleles were found in DIS3 in 4 Pts, in CYLD, TP53 and TRAF3 in 2 Pts and in CARD11, CDKN2A, CDKN1B, RB1and STAT3 in one Pt each. Conclusion: In summary, we report the initial results from 38 MM Pts using a custom MM sequencing panel which employs small amounts of DNA requiring very few cells and providing deep coverage in clinically meaningful timeframes. Our findings frequently include prognostically significant information, actionable targets and mutations in genes related to drug resistance. Targeted mutation profiling will likely become part of the clinical workup in MM and our M3P2.0 mutation panel is a suitable tool to provide information needed to guide precision therapy and to set the basis for individually tailored treatment decisions. 1. Kortuem KM. et al. Development Of a 47 Gene Sequencing Panel For Common Mutations Present In Multiple Myeloma: Preliminary Results In 77 p53 Deleted Patients, ASH 2013. Disclosures Stewart: Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Array BioPharma: Consultancy; Sanofi: Consultancy; Takeda Pharmaceuticals International Co.: Research Funding; Novartis: Consultancy. Bruins:OncoSpire: Salary support Other. Vasmatzis:OncoSpire: Salary Support Other. Kumar:Celgene: Consultancy, Research Funding; Millennium: The Takeda Oncology Co.: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding; Sanofi-Aventis: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Skyline Diagnostics: Membership on an entity's Board of Directors or advisory committees. Fonseca:Medtronic, Otsuka, Celgene, Genzyme, BMS, Lilly, Onyx, Binding Site, Millennium, AMGEN: Consultancy, patent for the prognostication of MM based on genetic categorization of the disease. He also has sponsored research from Cylene and Onyx Other, Research Funding. Bergsagel:MundiPharma: Research Funding; OncoEthix: Research Funding; Constellation Pharmaceutical: Research Funding; Novartis: Research Funding.

Abstract Abstract 3968 To better understand the genetic events associated with extra-medullary multiple myeloma (MM) we completed whole exome, long-insert whole genome and RNA sequencing on a patient with t(14;16) and neck adenopathy, as well as whole genome sequencing on two additional patients with respectively: t(4;14) and plasma cell leukemia, and del 17 with chest wall mass. All patients had multi-drug resistant disease and del 13. The frequency of somatic non-synonymous single nucleotide variants (SNV) ranged from 119 to 498, including an average of 15 truncating SNV, as well an average of 75 structural variants (range 2–147). Importantly, in one patient a truncating mutation, Q99*, on one allele and minor clone point mutation, R283K, on the second allele was observed in CRBN, a gene whose expression is required for effective lenalidomide and pomalidomide anti-myeloma activity. In the same patient a non-synonymous mutation, E171K, was also observed in PSMG2, a proteasome assembly protein, and a non-synonymous mutation, G369A, in NR3C1, a glucocorticoid receptor. Subsequently we have sequenced CRBN in 27 patients and 7 cell lines and have found no further examples of mutation in CRBN but have previously reported a bi-alleleic focal deletion in the MM.1Sres cell line. Mutations in NR3C1 have not previously been described in other MM genomes. Other, potentially clinically relevant mutations in the three patients were observed in FGFR3, PRDM1, STAT5B, NRAS, KRAS, PIK3CA, NF1, ATM, NFKB2, CKS1B and ERBB4. Although the three patients did not share a unifying event, a number of mutated genes observed were shared with other published myeloma genomes including RB1, TP53, MLL3, JAG2, VAV3 and SETD2. We also looked at pathway activation across the three genomes with the most statistically mutated pathways including VEGF signaling and activation, cell adhesion and chemokines, cytoskeleton remodeling, integrins in carcinoma progression, TP53 signaling and NOTCH signaling. In conclusion, we have identified, for the first time, the presence of a mutation in CRBN in a myeloma patient as well as mutations in PSMG2 and NR3C1, likely associated with drug resistance. Disclosures: Schmidt: Karyopharm: Research Funding. Carpten:Life Technologies: Research Funding. Stewart:Millenium: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy.

Abstract Abstract 4034 Through literature review we identified 376 pre-clinical studies in which a small molecule or antibody has been demonstrated to be cytotoxic to multiple myeloma (MM) cells either as cell lines, primary tumor samples or in mouse models. Indeed it seems there is no limit to the number of compounds that are cytotoxic to MM cell lines at high enough concentration. Since the pipeline to clinical trials is then large the utility of such assays in predicting future clinical value is debatable. We therefore sought to examine the utility of applying very early Phase I or Phase II trial experiences to predict for future clinical adoption. Using Pubmed, ASCO, ASH, EHA and IMWG abstracts we identified 117 drugs explored as single agents in 211 early stage clinical MM trials between 1961 and 2012 involving 7,166 patients. As definitions have changed over time, our analysis used partial responses (PR) or better as a response. When multiple trials were reported with a single drug, the average and the highest response rate for that agent was identified. Results were considered regardless of whether single or multi center trial, elderly or younger patients, stage of disease, targeted or non targeted therapy. High dose therapies requiring autologous stem cell support (eg. melphalan and etoposide), trials with less then 5 patients (e.g. siltuximab) or trials with concomitant steroid application which obscure single agent activity (e.g. pomalidomide, marizomib or daratumumab) were excluded in the analysis. Results: 17 drugs (14.5%) showed a best result of PR in more than 20% of patients, but when average response across all reported trials is considered, doxorubicin, arsenic trioxide and idarubicin drop out leaving 14 active drugs. Twelve drugs (10.3%) report as a best result PR in 10–20% of patients. 88 drugs (75.2%) reported a PR response in less than 10% of patients, 68 of which (58.1%) had no reported activity. A striking finding of our study is that all drugs approved for, or commonly used in clinical practice (prednisone, dexamethasone, cyclophosphamide, melphalan, bendamustine, thalidomide, lenalidomide, bortezomib, carfilzomib) have an average single agent PR response of at least 20%. Pomalidomide, although not approved, meets this threshold in Phase I testing. Older agents including teniposide, fotemustine, paclitaxel, and interferon also appear active by this criteria but have fallen from favor or were never adopted due to perceived toxicity. Of note, a number of newer agents which are in, or which recently completed Phase 3 testing, such MLN9708, elotuzumab, panobinostat, vorinostat or perifosine show little single agent activity. A weakness in this approach is that changes in measurement of response over time might bias the results, especially in favor of older studies. Furthermore, the chance of responding to a new drug has likely slowly declined as therapies have improved and patients have entered such trials at increasingly late stages of disease. It also does not rule out the possibility of synergistic activity or clinical benefit from a cytostatic drug. Nevertheless, our analysis suggests that a cut off of 20% single agent partial response activity averaged across all trials with that agent is highly predictive of future clinical success. If best reported response over 20% is considered no drug except vincristine has yet reached the clinic and has subsequently fallen from favor. Thus only drugs with 20% PR activity are in widespread use and thus this benchmark provides a framework for guiding choice of drugs for late stage clinical testing. Disclosures: Jimenez-Zepeda: MMRF: Research Funding; Jansenn Ortho: Honoraria. Fonseca:Onyx: Consultancy, Research Funding; Cylene: Research Funding; Medtronic: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Genzyme: Consultancy; BMS: Consultancy; Lilly: Consultancy; Binding Site: Consultancy; Millenium: Consultancy; AMGEN: Consultancy; Mayo Clinic: Patents & Royalties. Stewart:Millenium: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy.

Abstract Abstract 194 Background: Immunomodulatory drugs (IMiDs) are key components of treatment for hematologic malignancies, especially multiple myeloma (MM). Recently, we demonstrated that CRBN expression is the major mediator of IMiD action operating at least in part via interferon regulatory factor 4 (IRF4), however the clinical significance of this finding remains unclear. Methods: To better understand clinical impact, we retrospectively reviewed 148 MM patients whose tumor cells were tested for CRBN expression by Gene Expression Profiling (GEP) prior to treatment with an IMiD containing therapy. Results: GEP studies show no significant differences in CRBN expression between MGUS, smoldering MM (SMM), untreated symptomatic MM and normal plasma cells. However, differences were observed between MM cell lines of varying drug sensitivity to IMiDs and between other B-cell Neoplasia (eg low grade lymphoma has higher levels). Within the genetic subtypes of MM, levels were significantly higher for hyperdiploid patients (median 1.26) as opposed to significantly lower levels for TC class D2 (median 0.76) and average for the 4p16 (median 0.97). We next screened the University of Arkansas Medical School (UAMS) GEP database of MM patients treated with combination therapies and did not find significant correlation between CRBN expression and survival in this multi-agent regimen. We then focused on patients that received only single agent IMiD therapy with low dose dexamethasone. Review of the twelve lowest expressing patients demonstrated a lack of response to IMiDs in any patient. Finally we analyzed 53 relapsed or relapsed/refractory patients treated homogeneously in a study with pomalidomide 2–4mg daily and weekly dexamethasone 40mg. CRBN levels were assessed prior to therapy initiation. Response rates varied significantly based on gene expression level. The percentage of patients that demonstrated at least a partial response was 0% for CRBN <0.81, 19% for CRBN expression of 0.81–0.90, and 33% for CBN expression >0.9. Significant differences in PFS (3.0 months vs. 8.9 months, p = 0.0006) and in OS (9.1 months vs. 27.2 months, p = 0.01) were observed when the lowest quartile of CRBN expression was compared to the top three quartiles. Values used for the cut offs are as follows: (25%: 0.889687; 50%: 1.026542, 75%: 1.211133). While this study demonstrates a positive correlation between CRBN expression and response to pomalidomide and dexamethasone, an important caveat with respect to PFS and OS is that CRBN mRNA level is primarily a reflection of the number of copies of CRBN. Since focal deletions are uncommon, this mostly reflects chromosome 3p copy number. Since trisomy 3 is common in hyperdiploid, good prognosis disease, CRBN not only is required for IMiD function but higher levels also serve as a surrogate for low risk disease. Optimal gene expression cut offs for survival were determined using the Contal and O'Quigley method. The optimal cut point for PFS was 1.18443 and for OS was 1.17816. Conclusion: In summary, this study demonstrates, for the first time, a clinical correlation between CRBN expression and response to IMiD and dexamethasone therapy. CRBN expression is also predictive of survival outcomes and when quantified prior to therapy could potentially be utilized as a predictive biomarker for response to an IMiD based treatment. Disclosures: Stewart: Celgene: Consultancy, Honoraria.

Key Points High-throughput RNAi screening identified lenalidomide sensitizer genes, including RSK2, RAB, peroxisome, and potassium channel family members. Knockdown or inhibition of RSK2 synergized with lenalidomide to induce myeloma cytotoxicity and downregulation of interferon regulatory factor 4 and MYC.

Key PointsWe identified CRBN-binding proteins and confirmed recent work implicating the Ikaros transcription factors as important members. Low IZKF1 levels predict lack of IMiD responsiveness and shorter OS; IZKF1 expression is a potential predictive biomarker for clinic application.