Stewart, A. Keith

Abstract The oncogenic transcription factor c-Maf has been proposed as an ideal therapeutic target for multiple myeloma (MM), but how to achieve it is still elusive. In the present study, we found the Otub1/c-Maf axis could be a potential target. Otub1, an OTU family deubiquitinase, was found to interact with c-Maf by mass spectrometry. Otub1 abrogates c-Maf K48-linked polyubiquitination, thus preventing its degradation and enhancing its transcriptional activity. Specifically, this deubiquitinating activity depends on its Lys71 and the N terminus but is independent of UBE2O, a known E2 of c-Maf. Otub1 promotes MM cell survival and MM tumor growth. In contrast, silence of Otub1 leads to c-Maf degradation and c-Maf-expressing MM cell apoptosis. Therefore, the Otub1/c-Maf axis could be a therapeutic target of MM. In order to explore this concept, we performed a c-Maf recognition element–driven luciferase-based screen against US Food and Drug Administration–approved drugs and natural products, from which the generic cardiac glycoside lanatoside C (LanC) is found to prevent c-Maf deubiquitination and induces its degradation by disrupting the interaction of Otub1 and c-Maf. Consequently, LanC inhibits c-Maf transcriptional activity, induces c-Maf-expressing MM cell apoptosis, and suppresses MM growth and prolongs overall survival of model mice, but without apparent toxicity. Therefore, the present study identifies Otub1 as a novel deubiquitinase of c-Maf and establishes that the Otub1/c-Maf axis is a potential therapeutic target for MM.

Abstract GX15-070 is a small-molecule antagonist of the BH3-binding groove of the Bcl-2 family of proteins and can thus induce apoptosis by inhibiting the interaction between pro- and anti-apoptotic proteins. In addition, GX15-070 activates a cellular pathway, which leads to the activation of the pro-apoptotic BH3-only protein Bim, a natural cellular inhibitor of anti-apoptotic members. Together, the direct inhibition of anti-apoptotic Bcl-2 family proteins and the activation of pro-apoptotic Bim by GX15-070 can sensitize tumor cells to apoptosis. Bcl-2 family members, particularly Bcl-XL and Mcl-1 have been implicated in the survival of myeloma cells and may be responsible, in part, for drug resistance. Consequently, GX15-070 was tested against the MMRC 12 validation cell line panel, a panel of 12 standardized and annotated human myeloma cell lines (HMCLs). Western blots preformed on 8 HMCLs confirmed expression of Bcl-2, Mcl-1 and/or Bcl-XL. Basal levels of Bcl-2 and MCl-1 were relatively similar across all HMCLs studied whereas levels of Bcl-XL were variable GX15-070 inhibited the viability of all 12 myeloma cell lines as determined by MTT assay. The mean IC50 was 215 nM; range (6.4-678nM) and sensitivity did not correlate with level of Bcl-2, MCL-1 or Bcl-XL expression. On the other hand cytotoxicity to normal human blood lymphocytes, stroma and bone marrow CFUs was not observed at concentrations which were effectively cytotoxic to HMCL. Time course experiments demonstrate that apoptosis and cleavage of caspase 3 began by 12 hours and continued to increase over a 96 hour period. Co-culture with human bone marrow stroma cells (BMSCs) failed to protect HMCL from GX15-070 induced cytotoxicity. Similarly, potent MM growth factors, Interleukin 6 (IL-6) and Insulin like growth factor (IGF)-1 did not confer resistance to GX15-070. Of interest, overexpression of Bcl-2 has been shown to attenuate PS-341-induced cell death (Mitisades); however, the combination of PS-341 and GX15-070 did not reveal synergistic interaction in preliminary results. Studies looking at other drug combinations are planned. Finally, GX15-070 produced cytotoxic responses in the first primary myeloma sample tested with further testing ongoing. Mouse xenograft studies are also ongoing and will be presented. These studies indicate extensive pre-clinical activity of GX15-070 in multiple myeloma and set the stage for planned Phase II clinical trials.

Abstract In approximately 50% of Multiple Myeloma (MM), translocation of non random genes into the IgH locus is believed to be the seminal event in the pathogenesis of the disease. Another 50% of cases are hyperdiploid and trisomic to certain autosomes, but do not harbor any translocation and thus are believed to have genetic alterations in unidentified loci. These observations, together with the finding of somatic mutations in FGFR3, N- and K-RAS, MYC, TP53 and CDKN2C/p18INK4C during the later stages of MM indicate that defective signaling pathways likely play a role in the progression of this malignancy. Of relevance then, in recent years recurrent mutations in kinases have frequently been implicated in malignancies including notably colon cancer and melanoma. We have therefore begun a comprehensive effort to sequence the tyrosine kinome for mutations and genetic polymorphisms in MM. Of particular interest are 90 receptor tyrosine kinases, 43 receptor tyrosine kinase-like, 5 receptor guanylate cyclase, and a lipid kinase. We report here results from our pilot high throughput exon scanning in 32 human MM cell lines which initially focused on 13 kinases known to be somatically mutated in human cancers. To date we have expanded this effort to assess 30 genes with sequence obtained which currently spans 80% coverage of the kinase domains of these genes. A total of 1.9 million bp have been sequenced across 235 exons. No recurrent mutations have been identified in the kinase domains of the cancer-associated genes: SRC, ILK1, KIT, GUCY2F, PDGFRA; in the genetic disorder-associated kinases: BTK, EPHA4, LAMA2, EPHB6, ACVR2; and in the mutation hot spots of frequently mutated cancer gene PIK3CA. A novel missense mutation is however identified upstream of the kinase domain of FGFR3 changing a Ser residue to Arg at codon 433. This residue, which is conserved across species and in FGFR1 and FGFR2, has not been reported in myeloma and in thanatophoric dysplasia, but the biological significance of this mutation is unknown. Several single nucleotide polymorphisms were identified in the coding regions of some of these kinases. Notably, synonymous polymorphisms in the kinase domains of EPHA4, PDGFRA3, KIT, MLK1, ILK1, NTRK3, FLT3, ABL1, FES, MLK4, and EGFR1 were identified that changed a codon but not the amino acid. More importantly, we identified non-synonymous amino acid variations in the kinase domains of EPHA4, GUCY2F, PTK2, and PIK3CA genes that are more likely to effect variability in the activity of these kinases. In summary, no recurrent kinase mutations of significance in Myeloma development or progression have yet been identified. Sequencing of the known cancer associated kinases in MGUS and hyperdiploid MM patients is now underway and our data set is being expanded to include all 139 kinases.

Abstract Dexamethasone is a powerful anti-inflammatory, immunosuppressive agent widely used in the treatment of MM with pleiotropic effects on both the tumor, and host immune cells. We report on a completely novel approach to immune modulation using an agent with almost directly opposite effects to glucocorticoids on innate immune cells. LCL161 is an IAP antagonist targeting the cellular inhibitor of apoptosis proteins cIAP1 and -2 (cIAP1/2) that in a phase I study in solid tumors was well tolerated with a cytokine release syndrome as the dose limiting toxicity. We previously reported that loss of cIAP1/2 in MM cells results in the stabilization of NIK and activation of the non-canonical NFkB pathway, a pathway we found frequently activated by a promiscuous array of mutations in multiple myeloma (MM). As expected, we found that LCL161 has no direct anti-tumor activity against MM cells in vitro and ex vivo. However, the non-canonical NFkB pathway is also a key activator of the innate immune response. Remarkably, we found that in vivo LCL161 has dramatic, apoptosis-independent activity associated with marked phagocytosisagainst MM that develops spontaneously in an immunocompetent genetically engineered mouse model of MM (Vk*MYC). Cyclophosphamide (Cy), which has been shown to induce an acute secretory activating phenotype in tumor-associated macrophages, markedly potentiated the anti-tumor effects of LCL161. We show that phagocytes (depleted by liposomal clodronate), but not adaptive immune cells, are required for the anti-tumor effect. Consistently we show that LCL161 activates dendritic cells and macrophages in vitro, and in vivo, and upregulates production of inflammatory cytokines. A phase 2 clinical trial of LCL161 was conducted in 24 patients with relapsed MM previously exposed to both IMiDs and PIs, but less than 5 prior lines of therapy. Patients received LCL161 1200mg po weekly, with Cy 500mg po weekly added for progressive disease or lack of response. Single agent LCL161 was well-tolerated, with grade 3 or 4 non-hematologic toxicity in four patients, and following the addition of cyclophosphamide in 9 patients. No patients responded to single agent LCL161, and the best response was stable disease in 6/24. In 21 patients, Cy was added after a median of 2 cycles, in whom the best response was stable disease or better in 18/21 (p<0.001 compared to LCL161 alone), and a PR or better (including 1 VGPR and 1 CR) in 5/21 (p<0.001 compared to LCL161 alone). Six months following the initiation of Cy, 35% of patients remain event-free. Two patients had stable disease for more than nine months after stopping treatment. One, relapsing after VDT-PACE, HDM200, RVd x 3 years with t(11;14) and del17p, received only three months of LCL161, the last month in combination with Cy. He then achieved a PR, reconstitution of his uninvolved IgG to normal levels for the first time in more than 3 years and still has not progressed one year after stopping therapy. Similar changes in serum and bone marrow inflammatory cytokines and gene expression were observed in patients following treatment with LCL161 to those observed in the mice. We conclude that LCL161 is able to modulate the tumor microenvironment to exert long-term disease control in combination with Cy, and deserves further study in combination with agents that more directly depend on phagocytic cell activity (e.g., monoclonal antibodies, inhibitors of "Don't eat me" signals). Disclosures Bergsagel: Novartis: Research Funding; Constellation Pharmaceuticals: Research Funding; Incyte: Consultancy; Jannsen: Consultancy; Mundipharma: Consultancy. Off Label Use: Use of LCL161 to treat multiple myeloma. Kumar:Celgene: Research Funding; Millenium/Takeda: Research Funding; Onyx: Research Funding; AbbVie: Research Funding; Janssen: Research Funding; Sanofi: Research Funding; Celgene, Millenium, Sanofi, Skyline, BMS, Onyx, Noxxon,: Other: Consultant, no compensation,; Skyline, Noxxon: Honoraria. Stewart:Oncospire Genomics: Research Funding.

Abstract Abstract 2810 Poster Board II-786 We wished to explore the genetic events associated with disease progression and development of drug resistance in multiple myeloma (MM). To do so 11 patients were studied in whom at least two (range 2-3) temporally distinct samples of tumor DNA and RNA were available. The baseline genetic initiating event was defined for all patients (3 were genetic high risk; one with t(14;16) two with t(4;14)) as well as the gene expression profile (GEP) defined risk score using the Little Rock 70 or 17 gene panel (only one, the t(14;16) was GEP defined high risk). High resolution array CGH and gene expression were then performed on each sample. Of the 8 patients with a “low risk” tumor initiating event and low risk GEP score, 6 patients had no, or only one, copy number abnormality (CNA) change between the two temporally distinct MM samples. In stark contrast the 3 genetic high risk at baseline had between 17 and 40 distinct CNA changes at the time of progression. For all 11 patients 89 CNA were acquired with progression whereas 19 previously abnormal regions disappeared suggesting clones with these abnormalities were extinguished by the therapy received. In total we detected 0-40 CNA changes between the various timepoints, median 1, mean 10.7. The acquisition of new CNA was much more common than the loss of CNA. We then focused more specifically on the t(4;14) patient with the highest number of CNA changes. This patient has a well documented clinical course of having a sustained two year VGPR to Len/dex and then progressing while still taking Len/dex. Comparison of the pre and post-Len/dex samples identified 40 CNA changes(the most of any pair studied to date). Only six CNA were shared between the two samples, which included deletions of chr4, 9, 12, 13, and X plus a t(4;14) translocation. These likely represent the initiating “driver” tumor events. The new CNA we identified originated from both remodeled genomic changes and the emergence of unique changes, indicating a new tumor clone had emerged while the previously dominant clone had regressed (e.g. a deletion of a large segment of chromosome 8 at diagnosis was no longer observed in the relapse sample). The newly acquired CNA encompassed 3968 genes (13.7% of the genes in the genome), however, only 1235 of these genes (4%) were expressed in this patient at diagnosis (1188 in the typical myeloma patient). Since 1235 genes is still a large number we hypothesized that whole genome sequencing (WGS) would help elucidate the mechanism of lenalidomide resistance. We isolated DNA from germline tissue and CD138 purified tumor cells including: diagnostic, first relapse and second relapse samples. Utilizing SOLiD (Applied Biosystems, Foster City, CA) sequencing technology, we have completed fragment library WGS on both the germline and the final tumor samples. Quality control measures report the average number of sequence reads per start point to be less than 1.2, indicating the library is primarily composed of unique molecules. In addition, approximately 40% of the sequence reads map uniquely to the genome. Together these quality measures indicate our sample libraries are complex and provide good representation of the genome. Data on the whole genome sequence of myeloma at diagnosis and at the time of progression will be presented. Disclosures: Bergsagel: Celgene: Consultancy.

11571 Background: Relapsed/refractory (R/R) non-Hodgkin lymphomas (NHL) have a poor prognosis with limited treatment options. Our expanding knowledge of molecular alterations seen in R/R NHL allows identification of patients that potentially may benefit from a precision medicine approach. However, experience in routine clinical implementation of precision medicine has been limited. Here, we summarize our clinical experience in molecular characterization of RR NHL targeted therapy (TT) using next-generation sequencing (NGS), and selection of targeted therapy (TT) based on molecular profile. Methods: We conducted a prospective study in RR NHL through the Center for Individualized Medicine at Mayo Clinic. Consenting patients underwent NGS using FoundationOne Heme panel from biopsies done at time of relapse. Results of NGS were discussed at the Genomic Tumor Board and recommendations for TT were given based on matching specific molecular alteration(s) with potential agent(s) predicted to be active based on NGS. The agents could include FDA-approved, off-label use and clinical trial therapies. Results: 28 cases were enrolled: 18 aggressive NHL, 10 follicular lymphoma (FL). Molecular alterations were present in all cases. In aggressive B-cell NHL, CDKN2A/B gene cluster alterations were seen in 73% (8/11), while seen in only 1/7 T-cell lymphomas (TCL), and 1/10 FL. TP53 deletions were second most common genomic alterations in DLBCL (57%) and seen in 40% FL. JAK-STAT and ERBB pathways were altered in TCL (2/7 each). IGH-BCl-2 gene rearrangement were common in FL (70%), followed by MLL gene alterations (50%). Targetable mutations were present in 86% (24/28) of cases. A TT was recommended in all 24 cases, but received by 2 patients only. Remaining patients did not due to benefit from current therapy (10/24), ineligibility or lack of clinical trial (7/24) or interim clinical deterioration (5/24). Conclusions: Targetable mutations were identified in most cases of RR NHL with TT recommended for all cases. However, access to TT limits potential clinical benefit of molecular-based matching strategy. More studies are needed to assess impact on clinical outcomes.

Abstract The attrition rate for anticancer drugs entering clinical trials is unacceptably high. For multiple myeloma (MM), we postulate that this is because of preclinical models that overemphasize the antiproliferative activity of drugs, and clinical trials performed in refractory end-stage patients. We validate the Vk*MYC transgenic mouse as a faithful model to predict single-agent drug activity in MM with a positive predictive value of 67% (4 of 6) for clinical activity, and a negative predictive value of 86% (6 of 7) for clinical inactivity. We identify 4 novel agents that should be prioritized for evaluation in clinical trials. Transplantation of Vk*MYC tumor cells into congenic mice selected for a more aggressive disease that models end-stage drug-resistant MM and responds only to combinations of drugs with single-agent activity in untreated Vk*MYC MM. We predict that combinations of standard agents, histone deacetylase inhibitors, bromodomain inhibitors, and hypoxia-activated prodrugs will demonstrate efficacy in the treatment of relapsed MM.

Purpose To assess the impact of age on outcome and to analyze the projected years of life lost in patients with multiple myeloma. Patients and Methods Ten thousand five hundred forty-nine patients were evaluated; 6,996 patients were treated with conventional chemotherapy, and 3,553 patients were treated with high-dose therapy with autologous stem-cell transplantation. Results Mean observed and relative overall survival times in the entire cohort were 3.7 and 3.9 years, respectively. Observed survival decreased steadily from 6.4 years in patients younger than age 50 years to 2.5 years in patients ≥ age 80 years. A similar decrease was noted for relative survival. Higher age correlated significantly with higher International Staging System (ISS) stage. Relative excess risk of death differed significantly between 10-year age cohorts beginning from age 40 years (P < .001 for age 50 to 59 v age 40 to 49, P < .001 for age 60 to 69 v age 50 to 59, P < .001 for age 70 to 79 v age 60 to 69, and P = .009 for age ≥ 80 v 70 to 79). The average years of life lost per patient was 16.8 years in the entire patient cohort and decreased steadily from 36.1 years in patients younger than 40 years old to 4.6 years in patients ≥ age 80 years. Conclusion Age is associated with higher ISS stage and is an important risk factor for early mortality. Survival declined continuously by each decade from age 50 to age ≥ 80 from more than 6 to less than 3 years. The average of years of life lost in patients with myeloma is higher than in many other cancers and amounts to more than 30 years in patients younger than 40 years old but decreases to less than 5 years in patients age 80 years or older.

AbstractA paucity of validated kinase targets in human multiple myeloma has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in myeloma tumor lines bearing common t(4;14), t(14;16), and t(11;14) translocations to identify critically vulnerable kinases in myeloma tumor cells without regard to preconceived mechanistic notions. Fifteen kinases were repeatedly vulnerable in myeloma cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. Whereas several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly G protein–coupled receptor kinase, GRK6, appeared selectively vulnerable in myeloma. GRK6 inhibition was lethal to 6 of 7 myeloma tumor lines but was tolerated in 7 of 7 human cell lines. GRK6 exhibits lymphoid-restricted expression, and from coimmunoprecipitation studies we demonstrate that expression in myeloma cells is regulated via direct association with the heat shock protein 90 (HSP90) chaperone. GRK6 silencing causes suppression of signal transducer and activator of transcription 3 (STAT3) phosphorylation associated with reduction in MCL1 levels and phosphorylation, illustrating a potent mechanism for the cytotoxicity of GRK6 inhibition in multiple myeloma (MM) tumor cells. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma.