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Clonal proliferation of plasma cells within the bone marrow (BM) affects local cells, such as mesenchymal stromal cells (MSCs), leading to osteolysis and fatality in multiple myeloma (MM). Consequently, there is an urgent need to find better mechanisms of inhibiting myeloma growth and osteolytic lesion development. To meet this need and accelerate clinical translation, better models of myeloma within the BM are required. Herein we have developed a clinically relevant, three-dimensional (3D) myeloma BM coculture model that mimics bone cell/cancer cell interactions within the bone microenvironment. The coculture model and clinical samples were used to investigate myeloma growth, osteogenesis inhibition, and myeloma-induced abnormalities in MM-MSCs. This platform demonstrated myeloma support of capillary-like assembly of endothelial cells and cell adhesion-mediated drug resistance (CAM-DR). Also, distinct normal donor (ND)- and MM-MSC miRNA (miR) signatures were identified and used to uncover osteogenic miRs of interest for osteoblast differentiation. More broadly, our 3D platform provides a simple, clinically relevant tool to model cancer growth within the bone-useful for investigating skeletal cancer biology, screening compounds, and exploring osteogenesis. Our identification and efficacy validation of novel bone anabolic miRs in MM opens more opportunities for novel approaches to cancer therapy via stromal miR modulation.

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family that has been recently linked to tumor development. However, its role in modulating multiple myeloma (MM) biology and disease progression remains unexplored. We first demonstrated that patients with MM present with higher expression of Pyk2 compared with healthy individuals. By using loss-of-function approaches, we found that Pyk2 inhibition led to reduction of MM tumor growth in vivo as well as decreased cell proliferation, cell-cycle progression, and adhesion ability in vitro. In turn, overexpression of Pyk2 promoted the malignant phenotype, substantiated by enhanced tumor growth and reduced survival. Mechanistically, inhibition of Pyk2 reduced activation of Wnt/β-catenin signaling by destabilizing β-catenin, leading to downregulation of c-Myc and Cyclin D1. Furthermore, treatment of MM cells with the FAK/Pyk2 inhibitor VS-4718 effectively inhibited MM cell growth both in vitro and in vivo. Collectively, our findings describe the tumor-promoting role of Pyk2 in MM, thus providing molecular evidence for a novel tyrosine kinase inhibitor as a new therapeutic option in MM.

The PI3K/Akt pathway plays a crucial role in the pathogenesis of multiple myeloma (MM) in the bone marrow (BM) milieu. However, efficacy of selective and potent Akt inhibition has not yet been fully elucidated. In this study, we, therefore, examined the biologic impact of selective and potent Akt inhibition by a novel allosteric inhibitor TAS-117. TAS-117 induced significant growth inhibition, associated with downregulation of phosphorylated Akt (p-Akt), selectively in MM cell lines with high baseline p-Akt. Cytotoxicity of TAS-117 was also observed in patient MM cells, but not in normal peripheral blood mononuclear cells. Importantly, TAS-117 induced significant cytotoxicity in MM cells even in the presence of BM stromal cells, associated with inhibition of IL6 secretion. Oral administration of TAS-117 significantly inhibited human MM cell growth in murine xenograft models. TAS-117 triggered apoptosis and autophagy, as well as induction of endoplasmic reticulum (ER) stress response with minimal expression of C/EBP homologous protein (CHOP), a fatal ER stress marker. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity, associated with increased CHOP and PARP cleavage and blockade of bortezomib-induced p-Akt, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. Carfilzomib-induced cytotoxicity was similarly enhanced by TAS-117. Importantly, TAS-117 enhanced bortezomib-induced cytotoxicity in vivo, associated with prolonged host survival. Our results show that selective and potent Akt inhibition by TAS-117 triggers anti-MM activities in vitro and in vivo, as well as enhances cytotoxicity of proteasome inhibition, providing the preclinical framework for clinical evaluation of selective Akt inhibitors, alone and in combination with proteasome inhibitors in MM. Cancer Res; 74(16); 4458–69. ©2014 AACR.

Wnt/β-catenin signaling underlies the pathogenesis of a broad range of human cancers, including the deadly plasma cell cancer multiple myeloma. In this study, we report that downregulation of the tumor suppressor microRNA miR-30-5p is a frequent pathogenetic event in multiple myeloma. Evidence was developed that miR-30-5p downregulation occurs as a result of interaction between multiple myeloma cells and bone marrow stromal cells, which in turn enhances expression of BCL9, a transcriptional coactivator of the Wnt signaling pathway known to promote multiple myeloma cell proliferation, survival, migration, drug resistance, and formation of multiple myeloma cancer stem cells. The potential for clinical translation of strategies to re-express miR-30-5p as a therapeutic approach was further encouraged by the capacity of miR-30c and miR-30 mix to reduce tumor burden and metastatic potential in vivo in three murine xenograft models of human multiple myeloma without adversely affecting associated bone disease. Together, our findings offer a preclinical rationale to explore miR-30-5p delivery as an effective therapeutic strategy to eradicate multiple myeloma cells in vivo. Cancer Res; 74(6); 1801–13. ©2014 AACR.

Proteasome inhibitors have demonstrated that targeting protein degradation is effective therapy in multiple myeloma (MM). Here we show that deubiquitylating enzymes (DUBs) USP14 and UCHL5 are more highly expressed in MM cells than in normal plasma cells. USP14 and UCHL5 short interfering RNA knockdown decreases MM cell viability. A novel 19S regulatory particle inhibitor b-AP15 selectively blocks deubiquitylating activity of USP14 and UCHL5 without inhibiting proteasome activity. b-AP15 decreases viability in MM cell lines and patient MM cells, inhibits proliferation of MM cells even in the presence of bone marrow stroma cells, and overcomes bortezomib resistance. Anti-MM activity of b-AP15 is associated with growth arrest via downregulation of CDC25C, CDC2, and cyclin B1 as well as induction of caspase-dependent apoptosis and activation of unfolded protein response. In vivo studies using distinct human MM xenograft models show that b-AP15 is well tolerated, inhibits tumor growth, and prolongs survival. Combining b-AP15 with suberoylanilide hydroxamic acid, lenalidomide, or dexamethasone induces synergistic anti-MM activity. Our preclinical data showing efficacy of b-AP15 in MM disease models validates targeting DUBs in the ubiquitin proteasomal cascade to overcome proteasome inhibitor resistance and provides the framework for clinical evaluation of USP14/UCHL5 inhibitors to improve patient outcome in MM.

Introduction Multiple myeloma (MM) is characterized by dissemination and accumulation of plasma cells in the bone marrow (BM), which promotes tumor cell growth and therapy resistance. ROBO1 is a conserved transmembrane receptor of the Ig superfamily with no intrinsic catalytic activity, and its role in MM pathogenesis is unknown. Material and Methods We first analyzed ROBO1 expression via western blot and/or immunohistochemistry (IHC). Gene expression profiling in a cohort of 170 newly diagnosed MM patients (IFM170) was used to compare ROBO1 expression across primary MM and BM stroma cells (BMSC), and normal BM plasma cells (PC). We used short hairpin RNA (shRNA) for stable ROBO1 knock down (KD) and CRISPR-Cas9 for ROBO1 knock out (KO). For protein structure-function and rescue studies, ROBO1 KO MM cells were transduced with a lentiviral vector expressing either full-length (FL) or truncated ROBO1 mutants devoid of extracellular (Cyt) or intracellular domain (DeltaCyt), including patient-derived truncating mutations, with a C-terminus triple FLAG tag. FLAG immunoprecipitation (IP) followed by mass spectrometry or western blotting and immunofluorescence (IF) were used to identify ROBO1 interacting partners and ROBO1 cellular localization. We used a hydrogel encapsulation system to study proliferation in a 3D system. To study extramedullary and intramedullary MM growth in vivo, WT and ROBO1 KO OPM2 were injected either subcutaneously (plasmacytoma model) or intra-medullary in femoral bones of donor mice which were then implanted subcutaneously in recipient SCID mice (µ-SCID model). PET-CT was used to assess tumor volume. Mouse tumors were retrieved for IHC and RNA extraction followed by RNA sequencing. To study dissemination and homing, KO and FL addback OPM2 cells were injected intravenously in SCID mice. Femurs and plasmacytoma were retrieved at endpoint for IHC. Results ROBO1 is highly expressed in human MM cell lines and primary MM cells with highest expression in cells carrying the high risk t(4;14) cytogenetic and low/absent expression in normal PC. Of human cancer cell lines, ROBO1 expression was limited to late B cell lineage; and ROBO1 KD was selectively cytotoxic against MM, but not other hematologic cancers. ROBO1 KO significantly decreases proliferation in a 3D culture system and tumor growth in extramedullary (mean tumor volume KO versus WT plasmacytoma: 457 versus 1323 mm3, p value= 0.02) and intramedullary (mean tumor volume KO versus WT: 823 versus 2684 mm3, p value= 0.001) murine models of human MM. ROBO1 KO MM cells show decreased adhesion to BM endothelial and BMSC, which is fully rescued by FL ROBO1 addback. To address whether ROBO1 loss alters dissemination/homing of MM cells in vivo, we injected mice intravenously with ROBO KO or FL addback OPM2 cells. While ROBO1 KO resulted in a modest, non-statistically significant prolongation in mouse OS (90 versus 75 days, respectively, p value 0.2), the pattern of disease was strikingly different. As expected, ROBO1 FL mice developed hindlimb paralysis with extensive BM infiltration with MM. Importantly, ROBO1 KO mice demonstrated reduced BM infiltration and developed solitary plasmacytoma. We next showed that ROBO1 C-terminus domain is necessary and sufficient to rescue ROBO1 KO proliferative defect while expression of ROBO1 truncations, including patient-derived frameshift mutations, acted as dominant negative. IP showed avid interaction of ROBO1 with ABL1. Interestingly, we showed that the cytosolic domain of ROBO1 undergoes cleavage and translocates to the nucleus, where its function is now being studied. Conclusions We show that ROBO1 is necessary for MM homing to the BM niche and for MM growth within and outside the BM space. ROBO1 cytosolic domain undergoes proteolytic cleavage and translocates to the nucleus and is necessary and sufficient to rescue ROBO1 KO defective proliferation. Based on our data, we propose a dual model for ROBO1 in MM: the full transmembrane receptor is involved in regulating adhesion, dissemination and homing of MM cells within the BM niche; the cleaved intracellular C-terminus domain participates in transcriptional regulation, promoting MM proliferation. These data suggest that ROBO1 C-terminus may be a novel molecular target in MM. Disclosures Roccaro: Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; European Hematology Association: Research Funding; Transcan2-ERANET: Research Funding; European Hematology Association: Research Funding; AstraZeneca: Research Funding; Transcan2-ERANET: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding. Ghobrial:Takeda: Consultancy; Sanofi: Consultancy; Amgen: Consultancy; BMS: Consultancy; Celgene: Consultancy; Janssen: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

Long noncoding RNAs (lncRNA) are major regulators of chromatin dynamics and gene expression. We have recently performed deep RNA sequencing of CD138+ cells from 360 uniformly-treated, newly-diagnosed multiple myeloma (MM) patients (IFM/DFCI 2009) and described the lncRNA landscape and their role as independent risk predictors for clinical outcome in MM. Moreover, we have identified one of these lncRNAs - lnc-17-92 - as an independent risk predictor highly correlating with EFS and OS in newly-diagnosed MM providing rationale to define its molecular role in MM. Lnc-17-92 is generated at MIR17HG gene locus and is known for being involved in the biogenesis of miR-17-92 cluster of microRNAs. We here establish, for the first time, role of this transcript as a lncRNA with microRNA-independent function and molecular and biological implications in MM. Having confirmed its expression in MM cell lines and primary MM cells, we have utilized antisense oligonucleotides (n=3) to suppress lnc-17-92 expression in large panel of human MM cell lines (HMCLs) (n=12) and primary patient MM cells (n=13). Lnc-17-92 inhibition impaired MM cell proliferation leading to apoptotic cell death. This inhibitory effect was not rescued by ectopic expression of miR-17-92 microRNAs, confirming independent activity of lnc-17-92 on MM cell growth and viability. The microRNA-independent role of lnc-17-92 in transcriptional control was further confirmed using DROSHAKOcells. Analysis of transcriptomic changes after lnc-17-92 modulation in HMCLs and primary MM cells identified bona fide transcriptional targets of lnc-17-92. Using two independent MM RNA-seq datasets, we observed high correlation (R> 0.4) between lnc-17-92 expression and the expression of 12 of the transcriptional targets identified above. Interestingly, these genes were significantly enriched within metabolic pathways, suggesting an unexplored role for lnc-17-92 in MM cell metabolism. Further analysis using an RNAi-based loss-of-function screening in 3 HMCLs revealed Acetyl-CoA Carboxylase Alpha (ACC1) as a novel myeloma vulnerability. ACC1 encodes the limiting enzyme in the de novo lipogenesis pathway. Analysis of incorporation of C14-radiolabeled glucose into lipids in MM cells revealed that inhibition of ACC1 or lnc-17-92 strongly inhibited de novo lipogenesis in HMCLs and in primary MM cells. We have used ACC1 conditional KD MM cells expressing IPTG-inducible ACC1 shRNAs and confirmed significant role of ACC1 in MM cell growth and survival, both in vitro and in vivo in SCID mice model. Importantly, supplementation of palmitate, the main downstream product of ACC1 activity, significantly reverses the growth inhibitory effect of either ACC1 or lnc-17-92 suppression in MM cells. These data suggest an important role for lipogenesis pathway on lnc-17-92-promoted MM cell growth. We have further investigated mechanism by which lnc-17-92 may exert its transcriptional control. Protein-RNA pulldown assay established MYC as interacting partner of lnc-17-92. This interaction was confirmed by immunoprecipitation of MYC-bound RNA followed by qRT-PCR with specific primers for detection of lnc-17-92. ChIP-seq analysis revealed a direct binding of MYC at regulatory regions of ACC1 in MM.1S cells; these data were corroborated by the decreased ACC1 expression observed in MYC KD MM cells. Taken together, these data suggest that lnc-17-92 may function as a scaffold between MYC and the E-box motifs present on ACC1 intronic sequences, facilitating MYC binding and its transcriptional activity on ACC1. Finally, for translational application, we have pre-clinically investigated ND-646, a clinically applicable small molecule inhibitor of ACC1. Analysis of incorporation of C14-radiolabeled glucose into lipids confirmed its effect on lipogenesis in MM, which was associated with a significant in vitro growth inhibitory activity in large panel of HMCLs and primary patient MM cells. In vivo studies in murine model of human MM, using this oral agent, are ongoing and will be presented. In conclusion, we here report for the first time the microRNA-independent role of lnc-17-92 in MM pathobiology with direct impact on transcriptional control of lipogenesis. The availability of oral inhibitor of this pathway may allow the clinical application of this unique targeted therapy in MM. Disclosures Anderson: Janssen: Other: Advisory Board; Gilead Sciences: Other: Advisory Board; OncoPep: Other: Scientific founder ; Sanofi-Aventis: Other: Advisory Board; C4 Therapeutics: Other: Scientific founder . Munshi:Abbvie: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Adaptive: Consultancy; Oncopep: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Oncopep: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Abbvie: Consultancy.

Syndecan-1 (SDC1), also known as CD138, is a member of integral membrane heparin sulfate proteoglycans constantly expressed in plasma cells (PCs) and a primary diagnostic marker for human multiple myeloma (MM). We here further define new functions of SDC1 in the MM pathobiology. Firstly, flow cytometry and qRT-PCR analysis showed that SDC1 is expressed at relatively higher levels in AMO-1, U266, OPM2, H929, MM1S, and MM1R MM cells when compared with JJN3, RPMI 8226, and ANBL6 MM cells. SDC1 levels are comparable in paired MM cell lines sensitive or resistant to current anti-MM therapies including lenalidomide, pomalidomide, and bortezomib. Significantly increased SDC1 mRNA levels in advanced MM stages (p Disclosures Munshi: Celgene: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Oncopep: Consultancy; Abbvie: Consultancy; Abbvie: Consultancy; Amgen: Consultancy; Adaptive: Consultancy. Anderson:Celgene: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau.

MEDI2228, an antibody-drug conjugate (ADC) comprised of an anti-BCMA antibody site-specifically conjugated to a DNA cross-linking pyrrolobenzodiazepine dimer, is currently under clinical development for the treatment of human multiple myeloma (MM) (NCT03489525). MEDI2228 induces DNA damage responses (DDR) prior to apoptosis, as demonstrated by phosphorylation of ATM/ATR, CHK1/2, and gH2AX in MM cells regardless of p53 status and responsiveness to current MM therapies including bortezomib and IMiDs. Since activation of DDR alters expression of ligands for NKG2D receptors critical for NK-mediated immune surveillance, we here examined whether the ATM/ATR-CHK1/2 signaling cascades activated by MEDI2228 treatment would increase NKG2D ligands in MM cells. Using real-time quantitative RT-PCR and flow cytometry analysis, we found that treatment with MEDI2228 increased the expression of major histocompatibility complex (MHC) class I chain-related proteins A and B (MICA/B) in MM cell lines (n>5) and CD138+ MM cells from patients with relapsed and refractory disease (n=4). In addition, expression of the MHC class I molecules/NKG2D ligands ULBP-1, -3, -2/5/6 increased following MEDI2228 treatment. Next, we evaluated NK cell-mediated lysis of MM target cells (n>3) with or without pretreatment with MEDI2228 and found increased NK cell-mediated lysis of MEDI2228-pretreated vs control MM cells in an effector-target ratio-dependent manner. In parallel, we examined whether MEDI2228 stimulates STAT1- and IFN-related signaling pathways since they are activated by DDR and play a crucial role in innate and adaptive immunity. We found that MEDI2228 treatment significantly increases phosphorylation of STAT1 in H929 and its derived IMiD-resistant cells, and further augments expression of IFN-induced genes (IFITs), IFIT1, 2, 3, and 5, which have been shown to inhibit proliferation and promote apoptosis in cancer cells. Significantly, CD38 is upregulated by MEDI2228 treatment, with increased mRNA expression as well as membrane expression detected by flow cytometry in MM cell lines and MM cells from newly diagnosed and refractory patients (n=5). Consequently, MEDI2228-pretreated MM cells (n>3) are more susceptible to NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) by daratumumab, which targets CD38. Taken together, our data show that MEDI2228-induced DDR primes MM cells to NK cell-mediated cytotoxicity by increasing expression of MICA/B in MM cells to enhance binding and activating NK cytolytic activity. Simultaneously, MEDI2228 induces IFN-stimulated genes, including CD38, resulting in enhanced MM cell lysis by daratumumab. These results indicate additional mechanisms of anti-MM activity for MEDI2228 and suggest that a combination of MEDI2228 and anti-CD38 mAbs may further improve outcome for MM patients. Disclosures Kinneer: AstraZeneca: Employment. Munshi:Janssen: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Adaptive: Consultancy. Anderson:Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau.

AMG 701 is a half-life extended BiTE® (bispecific T-cell engager) targeting the B cell maturation antigen (BCMA). Here, we confirmed AMG 701-mediated T cell-redirected lysis of MM cells, defined immunomodulatory effects of AMG 701, and investigated combination potential of AMG 701 with immunomodulatory drugs (IMiDs) in human MM. Firstly, AMG 701 induced specific and efficacious T cell-dependent cytotoxicity (TDCC) against all MM cell lines tested, regardless of sensitivity to current anti-MM agents and expression levels of BCMA. AMG 701-induced TDCC was minimally affected in the presence of myeloma-supporting cells and cytokines in the bone marrow (BM) microenvironment, including osteoclasts (OCs), BM stromal cells (BMSCs), and a proliferation-inducing ligand (100 ng/ml). Importantly, AMG 701 induced lysis of autologous patient cells from the relapse and refractory stage of MM (RRMM). AMG 701 rapidly upregulated cell surface expression of CD107a and the production of IFNγ and TNFα, more so in CD8 than CD4 T subsets. It stimulated the proliferation and activation of T cells, to a greater extent in CD8 vs CD4 T cells, leading to significantly increased ratios of CD8/CD4 T cells. Significantly, AMG 701 induced differentiation of naive T cells (CD4 and CD8) to T cells with memory phenotype. This includes central memory (CM), effector memory (EM) T cells, and stem cell like memory cells. Time-course immunophenotyping studies showed that AMG 701 transiently upregulated the expression of key immune checkpoint and costimulatory markers on both CD4 and CD8 T cells. The induced T cells purified from ex vivo co-cultures still effectively lysed MM cells with lower BCMA levels. This may suggest an increased T cell clonality. Furthermore, IMiDs (len or pom) enhanced AMG 701-mediated TDCC against MM cells at earlier time points, lower E/T ratios, lower concentrations, or in the presence of immunosuppressive OCs or BMSCs. The combination AMG 701 and IMiDs maximized MM cell lysis accompanied with a decreased EC50 value. Combined treatments induce a more pronounced immunomodulation than AMG 701 alone in the presence of OCs, as evidenced by higher percentage of CM+EM and CD8/CD4 ratio at d8. AMG 701 with IMiDs combination significantly enhance AMG 701-mediated autologous patient MM cell lysis in a synergistic manner (combination index < 1). In the human NCI-H929 xenograft model reconstituted with human T effector cells, AMG 701 effectively blocked tumor growth 5d after the first injection, regardless of doses (0.02-2 mg/kg). Tumors were completely eradicated following 3 separate injections in the host without weight loss. Next, sub-optimal doses and treatment schedules for AMG 701 and len were then used to investigate in vivo anti-MM effects by the combination vs monotherapy. Mice receiving MM cells were treated, from d15 until the end of the study, with len once daily, AMG 701 once weekly, or combination of AMG 701 and len. Two days after the first drug administration, all three treatments significantly inhibited MM tumor growth in mice (p Disclosures Munshi: Oncopep: Consultancy; Janssen: Consultancy; Abbvie: Consultancy; Takeda: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Celgene: Consultancy. Wahl:Amgen Research GmbH: Employment. Matthes:Amgen Research GmbH: Employment. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Chapman-Arvedson:Amgen Research: Employment.

The development of novel agents including immunomodulatory drugs (IMiDs) lenalidomide (Len) and pomalidomide (Pom) has led to improved patient outcome in multiple myeloma (MM); however, acquired resistance to IMiDs commonly underlies relapse during the course of treatment. Previous studies show that IMiDs bind to the CRL4CRBN ubiquitin ligase cereblon (CRBN) and promote proteasomal degradation of IKZF1 and IKZF3 followed by downregulation of c-Myc and IRF4, resulting in MM cell growth inhibition. Therefore, CRBN is the primary binding target and master regulator of IMiDs sensitivity; however, the molecular mechanisms regulating resistance to IMiDs have not been fully defined. Importantly, some MM cells show resistance to IMiDs despite harboring high CRBN expression levels. To delineate the molecular mechanisms underlying IMiDs resistance, we first performed genome-wide knockout screening in IMiDs-sensitive MM.1S cells using a CRISPR-Cas9 GeCKOv2 library containing 6 unique sgRNAs against each of 19,050 genes and 4 sgRNAs against each of 1,864 miRNAs. We observed that knockout (KO) of twenty-eight genes and one miRNA were associated with resistance to IMiDs. Of note, all six sgRNAs targeting CRBN were identified as a positive regulator of IMiDs sensitivity, consistent with previous studies. Among these genes, we found that three different sgRNAs targeting TRAF2 were enriched after IMiDs treatment. Therefore, we next individually cloned the sgRNAs of TRAF2 into the Cas9 lentiviral vector, and then re-introduced them into MM.1S cells. Importantly, TRAF2-KO MM.1S cells acquire significant resistance to Pom and Len treatment. To examine whether TRAF2 KO induced IMiDs resistance was CRBN-pathway dependent, we assessed CRBN and its downstream interacting protein levels. TRAF2 KO showed no effect on CRBN expression; moreover, IMiDs can still trigger IKZF1 and IKZF3 degradation, associated with downregulation of IRF4. Taken together, these data suggest that TRAF2 mediates sensitivity to IMiDs in a mechanism independent of CRBN-IKZF1/3 axis. Since TRAF2 is a member of the TNF receptor associated factor (TRAF) protein family required for activation of several signaling pathways including NF-ĸB and JNK, we next examined the biologic impact of TRAF2 KO. Importantly, TRAF2 KO cells show significantly increased processing of precursor p100 to p52 (NF-ĸB2), resulting in hyperactivation of the non-canonical NF-κB pathway; conversely, p52 KO re-sensitizes the cells to IMiDs treatment. The activity of the canonical NF-κB pathway was not similarly altered in TRAF2 KO cells. These results suggest that TRAF2 predominantly activates non-canonical NF-κB pathway, associated with resistance to IMiDs in MM cells. We next examined the relevance of TRAF2 downregulation in the context of the bone marrow (BM) microenvironment. Co-culture of MM cells with either bone marrow stromal cells (BMSCs) or culture supernatants (BM-CS) confers resistance to IMiDs. Importantly, TRAF2 is downregulated in BMSCs and BM-CS, suggesting that IMiDs resistance in the BM microenvironment may be mediated by TRAF2 downregulation induced by soluble factors. Cytokine array assay confirmed detectable TNF-α in BM-CS. Indeed, MM cells treated with TNF-α showed decreased TRAF2 expression, associated with resistance to IMiDs. These data demonstrate that TNF-α secreted by BMSCs induces TRAF2 downregulation, thereby inducing IMiDs resistance. We further analyzed the mechanism of TNF-α-mediated TRAF2 downregulation. Since previous studies have shown that TRAF2 is a substrate of the proteasome, we treated MM cells with TNF-α in the presence and absence of proteasome inhibitor. TRAF2 downregulation by TNF-α was partially abrogated by proteasome inhibitor, associated with accumulation of ubiquitinated TRAF2. These data confirm that TNF-α induces TRAF2 downregulation is due, at least in part to proteasomal degradation. In conclusion, our data show that TRAF2 is a CRBN-independent regulator of IMiDs sensitivity and provide the preclinical rationale for combining IMiDs with inhibitors of non-canonical NF-κB or TNF-α signaling to overcome IMiDs resistance and improve patient outcome. Disclosures Anderson: Sanofi-Aventis: Other: Advisory Board; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder.

Chimeric antigen receptor (CAR) T cells targeting BCMA are positioned to transform treatment of multiple myeloma (MM), and virally-generated anti-BCMA CAR T cells have shown impressive early stage clinical results. However, the safety risk/benefit, manufacturing constraints, and relevant patient populations of viral anti-BCMA CAR T have yet to be fully defined. Here we present preclinical characterization of an autologous mRNA-generated anti-BCMA CAR T cell product (Descartes-08) designed to reduce safety risk and broaden the fitness-for-use of anti-BCMA CAR T cell therapy. Descartes-08 are autologous CD8+ T cells that express anti-BCMA CAR on up to 90% of cells with duration of CAR expression for several days with subsequent reduction in expression to background approximately 1 week after their generation. The manufacturing process is clinically scalable with high purity and viability of Descartes-08 following cryopreservation. Descartes-08 undergo cytotoxic degranulation and produce cytokines IFNg, TNFα, IL-2, in response to multiple BCMA-expressing multiple myeloma target cell lines in an effector-to-target-ratio-dependent manner. Furthermore, Descartes-08 kills MM lines that are both resistant and sensitive to lenalidomide and pomalidomide, and/or MM cells that are grown in the presence of bone marrow stromal cells that clinically support MM survival. Moreover, Descartes-08 are highly cytotoxic against MM cells from both newly-diagnosed and relapsed patients. The magnitude of cytolytic and cytokine responses correlates with duration of anti-BCMA CAR expression and declines after 4 days, indicating a temporal limit in activity that is predicted to dramatically decrease the risk of severe cytokine release syndrome. In a mouse model of disseminated human MM, Descartes-08 shows CAR-specific suppression of myeloma that is maintained throughout the duration of treatment. Taken together, these results illustrate features of RNA-generated anti-BCMA CAR T cells that promise key clinical advantages, thereby supporting ongoing clinical development of Descartes-08 for treatment of MM (NCT03448978). Disclosures Kurtoglu: Cartesian Therapeutics: Employment. Zhang:Cartesian Therapeutics: Employment. Stewart:Cartesian Therapeutics: Employment. Anderson:Sanofi-Aventis: Other: Advisory Board; OncoPep: Other: Scientific founder ; C4 Therapeutics: Other: Scientific founder ; Gilead Sciences: Other: Advisory Board; Janssen: Other: Advisory Board.

A novel anti-BCMA antibody drug conjugate (ADC) MEDI2228 preferentially binds to membrane-bound vs soluble BCMA (sBCMA) to effectively deliver the pyrrolobenzodiazepine (PBD) payload tesirine to multiple myeloma (MM) cells (Leukemia. 2019;33: 766). In preclinical models, this ADC targets both MM cells in bulk and CD19+CD138- patient MM progenitor cells. We here study the potency of MEDI2228 in MM cells in the bone marrow (BM) microenvironment and examine molecular mechanisms whereby MEDI2228 overcomes drug resistance. First, MEDI2228, more effectively than its microtubule-binding monomethyl auristatin-F (MMAF) ADC homolog, inhibits proliferation (>1-2-log) and survival of all MM cell lines and MM cells from patients with multiple relapsed and refractory diseases, regardless of BCMA levels, p53 status, and the protection conferred by BM stromal cells and IL-6. Significantly, MM cells with lower BCMA expression and resistance to bortezomib or immunomodulatory drugs (IMiDs, i.e., lenalidomide, pomalidomide) are more susceptible to MEDI2228 vs its MMAF ADC homolog. MEDI2228, but not its MMAF ADC homolog, activates critical DNA damage responses (DDR) via phosphorylation of ATM/ATR kinases, checkpoint kinases (CHK)1/2, and H2AX, associated with induction of multiple DDR pathway-associated genes. Low doses of MEDI2228 and bortezomib (btz) synergistically induce apoptosis of drug-sensitive and -resistant MM cells, at least in part, through modulation of RAD51, a DNA damage and repair protein. Importantly, MEDI2228 further triggers the ATM/ATR-CHK1/2 signaling cascade, associated with increased gH2AX, p21, and apoptosis molecules in MM1S-xenografted tumors in mice. In vivo, a single sub-optimal dose of 0.4 mg/kg MEDI2228 induces superior anti-MM activity than btz, indicating that MEDI2228 is significantly more effective and selective than btz as single agent therapy in vivo. Furthermore, combined treatments with MEDI2228 and btz result in potent tumor depletion and significantly prolonged host survival via increased nuclear gH2AX-expressing microfoci, DNA damage-induced growth arrest and cell death. Significant tumor necrosis is observed earlier in mice receiving both drugs than either agent alone. At 177d, 15% mice in the combination treatment group remain alive and without any tumor. Importantly, no weight loss is noted in all groups, indicating a favorable safety profile of MEDI2228, alone or with btz, in vivo. Moreover, DDR checkpoint inhibitors, i.e., AZD0156 (ATMi), AZD6738 (ATRi), AZD1775 (WEE1i), synergize with MEDI2228 to enhance MM cell cytotoxicity (combination index < 1). This study therefore further supports clinical development of MEDI2228 (NCT03489525) as an important next-generation immunotherapy to improve outcome of MM patients. Disclosures Kinneer: AstraZeneca: Employment. Munshi:Amgen: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Adaptive: Consultancy; Celgene: Consultancy. Anderson:Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Sanofi-Aventis: Other: Advisory Board.

Apolipoprotein B mRNA editing catalytic polypeptide-like 3B (APOBEC3B, A3B) is one of 7-membered DNA cytosine deaminase family, causing cytosine-to-uracil (C-to-U) deamination in single-stranded DNA and promoting mutations in multiple human cancers including multiple myeloma (MM). High APOBEC3B expression is found in a significant portion of MM patients with MAF overexpression among t(14;16) and t(14;20). A3B upregulation is further associated with poor prognosis in MM, suggesting its role in the MM pathophysiology. However, approximately 23% MM patients with high APOBEC3 activity are associated with MAF/MAFA/MAFB translocations, the remainder of patients with high APOBEC3 carry neither translocations nor overexpression of these genes. Besides, studies are lacking on how A3B is regulated and the role of A3B in drug responses in MM. We here defined new mechanisms controlling A3B expression and further characterized its impact on treatment responses to current anti-MM therapies. Using qRT-PCR, A3B transcript is significantly higher than other members of the APOBEC3 gene family in MM cell lines (n=19) and MM patients, indicating that A3B may play a major role in MM. Using immunoblotting analysis, A3B protein expression was further confirmed in MM cell lines with various levels (n=10). Importantly, A3B mRNA upregulation by 1.34-42.64 folds was observed in CD138-purified cells from majorities of MM patients (83.3%) when compared to PBMC from the same individual (n=12). In MM cell lines without MAF/MAFA/MAFB translocation as a study model, higher A3B protein expression is associated with higher DNA damage levels as evidenced by higher γ-H2AX. These results suggest that A3B expression might be influenced by DNA damage levels in MM cells. Following a short time treatment of gamma-irradiation to cause DNA damages, A3B expression in viable MM cells was enhanced in a dose-dependent manner. We next treated MM cells (n=5) with common anti-MM drugs such as Melphalan (Mel) and Bortizomib (btz), both of which induce DNA damages, followed by examination of changes in A3B and γ-H2AX. Under sublethal treatment conditions of Mel or btz, A3B was consistently induced at both mRNA and protein levels in multiple MM cell lines regardless of the baseline A3B expression. Significantly, A3B was upregulated and associated with increased γ-H2AX in patient MM cells treated with Mel or btz under sub-lethal doses. Since DNA damages activate the ATR/ATM pathway, we next investigated whether these kinases mediate A3B induction following treatments with these compounds in MM cells. The presence of ATM or ATR inhibitors blocked A3B upregulated by these DNA damage-inducing treatments in MM cell lines (n=3), indicating an ATM/ATR-dependent pathway for A3B changes. Next, gene-specific CRISPR knock out (KO) and inducible-shRNA knockdown (KD) were used to determine the functional impact of perturbation of A3B in proliferation and survival of MM cells. Both KO and KD of A3B decreased growth and viability of MM cell lines regardless of sensitive or resistant to dexamethasone or lenalidomide. Using LIVE/DEAD fixable Aqua Stain and annexin V-based flow cytometric analysis, A3B inhibition enhanced growth arrest followed by apoptosis in MM cells. Significantly, A3B KD by its shRNA in RPMI8226 MM cells enhanced sensitivity to pomalidomide. Taken together, these data indicate that increased A3B level plays a critical role in MM cell survival and drug responses. DNA damages triggered by IR, Mel, or btz further enhance A3B expression via ATM/ATR pathway, which in turn increases subclonal diversity leading to drug resistance. The role of A3B in disease pathophysiology and progression, coupled with its function in mediating treatment response, suggest potential utility of targeting A3B in MM. Disclosures Munshi: Celgene: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Takeda: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

A proliferation inducing ligand (APRIL) is a natural ligand for B cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI), two receptors overexpressed in human multiple myeloma (MM) patient cells. Specifically, BCMA is highly expressed in plasma cells of all MM patients and BCMA-based immunotherapies has recently shown impressive response rates in patients with relapsed and refractory diseases. APRIL, mainly secreted by myeloma-supporting bone marrow (BM) accessory cells, i.e., macrophages, osteoclasts (OC), promotes MM cell progression in vitro and in vivo. It further induces survival and function of regulatory T cells (Treg) via TACI, but not BCMA, to support an immunosuppressive MM BM microenvironment (Leukemia. 2019;33:426). Here, we study effects of APRIL in current immunotherapies in MM and determine whether APRIL influences antibody-dependent cellular cytotoxicity (ADCC) induced by therapeutic anti-BCMA (J6M0) or anti-CD38 (daratumumab) mAbs via FcR-expressing immune effector cell-dependent mechanisms. Using anti-human IgG1 to detect J6M0 binding to the cell membrane BCMA, we first showed that APRIL, in a dose-dependent manner (31-500 ng/ml), competed with J6M0 for binding to BCMA. Such effects were inhibited by the blocking anti-APRIL monoclonal antibody (mAb) (Apry-1-1), as confirmed by flow cytometry and confocal microscopy. APRIL still inhibited J6M0 binding to BCMA at 4°C, arguing against that APRIL induces shedding of BCMA receptor. Using PE labeled anti-FLAG to detect APRIL-FLAG bindings to MM cell surface BCMA, J6M0 (0.25-4 µg/ml) did not alter APRIL binding to BCMA following 2h or 1d incubation. High concentrations of J6M0 (>10 µg/ml) only blocked ~50% of APRIL (0.2 µg/ml)-induced NFκB activity as determined by specific DNA binding assays, indicating that APRIL-induced signaling cascade via BCMA or TACI in MM cells is not completely blocked by J6M0. In parallel, data analysis using mRNA-seq identified 594 or 355 differentially expressed genes (Log2-Fold-change > 1.5 and adjusted p < 0.05) in APRIL- and BCMA-overexpressed RPMI8226 MM cell transfectants, respectively, when compared with control parental cells. KEGG and Reactome pathway enrichment analysis further defined that these differentially expressed genes are enriched in cell adhesion, migration, chemokine signaling pathways, and JAK/STAT signaling pathways, in addition to proliferation and survival in MM cells. We next asked whether overnight treatment with APRIL in MM cell lines decreased their baseline lysis by FcR-expressing effector cells, i.e., NK, monocytes. In a dose-dependent manner, APRIL (10-200 ng/ml) downregulated baseline MM cell lysis mediated by these effector cells. Importantly, in a similar fashion, ADCC was decreased against all APRIL-treated vs control MM cell lines induced by J6M0 or daratumumab. Conversely, blocking anti-APRIL mAbs reverted APRIL-suppressed cytotoxicity against MM cells induced by J6M0 or daratuzumab. These results were validated by decreased J6M0-induced NK cell degranulation following co-incubation with APRIL-treated vs control MM cells. In contrast, anti-APRIL neutralizing mAbs specifically blocked APRIL-inhibited NK cell membrane CD107a expression. Furthermore, co-cultures with MM-supporting OCs or macrophages decreased ADCC against MM cells by NK cells; conversely the neutralizing anti-APRIL mAb significantly blocked APRIL-reduced MM cell lysis by J6M0- or Daratumumab. Finally, APRIL reduced J6M0-induced patient MM cell lysis when freshly isolated BM mononuclear cells from MM patients (n=10) were incubated with NK cells from the same individual. Anti-APRIL mAbs still blocked APRIL blockade in J6M0-induced autologous patient MM cell lysis. Taken together, our data further indicate that therapies directed at the APRIL/BCMA and APRIL/TACI axes may simultaneously target MM cells and counteract APRIL-reduced MM cell lysis induced by therapeutic mAbs targeting MM cells. These results thus support combination strategies of blocking APRIL mAbs with BCMA- or CD38-directed immunotherapies to further overcome MM cell-induced immunosuppressive BM microenvironment, thereby enhance Disclosures Munshi: Abbvie: Consultancy; Abbvie: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Janssen: Consultancy; Janssen: Consultancy; Oncopep: Consultancy; Amgen: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Adaptive: Consultancy; Celgene: Consultancy. Anderson:Gilead Sciences: Other: Advisory Board; Janssen: Other: Advisory Board; Sanofi-Aventis: Other: Advisory Board; OncoPep: Other: Scientific founder ; C4 Therapeutics: Other: Scientific founder .

Immune escape underlies progression of disease and resistance to therapy in multiple myeloma (MM). Conversely, restoration of anti-tumor immunity primed by immunogenic cancer cells and intratumoral dendritic cells (DCs) may be exploited to convert the immunologically "cold" MM into a "hot" MM; and may lead to long-term clinical benefit, even in patient subgroups with high-risk (HR) cytogenetics and poor survival. Here, we investigated the mechanisms, biologic sequelae, and clinical benefits of bortezomib (BTZ)-induced immunogenic cell death (ICD), the immunogenic consequence of apoptosis resulting in specific anti-MM immunity via T-cell priming by DCs. We first show that BTZ can induce hallmarks of ICD in human and murine MM cell lines (n=5), including exposure of endoplasmic reticulum protein calreticulin (CALR) that functions as an "eat me signal". Specifically, our data show that co-culture with BTZ-treated MM cells can induce phenotypic and functional changes in immature DCs including higher expression of CD86/CD83 on cell surface and enhanced uptake of BTZ-treated MM cells, as assessed by flow cytometry- and confocal-based phagocytosis assay, respectively. Notably, we show that CALR has a key role in BTZ-induced immunogenicity, since these functional sequelae were abrogated in vitro when DCs were co-cultured with CALRKO MM cells. We then validated these findings in 2 different in vivo syngeneic models. First, we observed that anti-MM activity of BTZ resulted in more potent 5TGM1 tumor cell shrinkage in immunocompetent hosts; and that this effect was directly linked to ICD induction, since it was abrogated in mice bearing CALRKO tumors. Second, in vitro BTZ-treated 5TGM1 cells were used as a vaccine to enhance an anti-MM immune response: injection of live tumor cells resulted in palpable tumors in non-vaccinated mice by 1 week; conversely, injection of live tumor cells in vaccinated mice did not result in detectable tumor after 30 days. In contrast, in mice similarly vaccinated with BTZ-treated CALRKO 5TGM1 cells and challenged with injection of live WT cells, only 50% of vaccinated mice were tumor-free at day 30. Next, we performed RNAseq analysis of BTZ-treated vs untreated tumors from both immunodeficient or immunocompetent mice; and also carried out an integrative analysis of RNAseq data from newly diagnosed and clinically annotated MM patients (n=360) uniformly treated with BTZ-based regimes (IFM/DFCI 2009). Importantly, increased expression of the human orthologs of the immune genes induced in mice by BTZ was strongly and positively correlated with patient clinical outcome (OS p value=0.00089). The predictive value of this signature was confirmed in an independent dataset (GSE9782) (OS p value=0.024). Moreover, by interrogating the IFM/DFCI patient dataset, we identified the gamma-aminobutyric acid receptor-associated protein (GABARAP) as a top differentially expressed gene among patients with longer survival rate (>5 years) as compared to those with poor survival ( In conclusion, our studies demonstrate the clinical benefits of BTZ-induced ICD in MM; and that loss-of-function of GABARAP, particularly in HR patients with 17p deletion, abrogates induction of antitumor immunity after drug exposure. These studies provide the framework for novel combination treatments to trigger anti-MM immunity and improve patient outcome in MM. Disclosures Chauhan: Stemline Therapeutics: Consultancy; C4 Therapeutics.: Equity Ownership. Munshi:Celgene: Consultancy; Adaptive: Consultancy; Oncopep: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Abbvie: Consultancy; Amgen: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

Constitutive genomic complexity, ongoing DNA damage, and accumulating mutations are observed with progression from monoclonal gammopathy of undetermined significance (MGUS) to active multiple myeloma (MM) to relapsed/refractory disease. Apolipoprotein B mRNA editing catalytic polypeptide-like 3B (APOBEC3B), a DNA cytosine deaminase in cancer mutagenesis, is recently linked to sub-clonal diversification, intra-tumor heterogeneity, and tumor evolution in MM. We here studied the upstream mechanisms of APOBEC3B dysregulation and further defined functional consequences of molecular manipulation of APOBEC3B in MM cells. We first asked whether APOBEC3B levels are altered in MM cells upon treatments with Melphalan (Mel), an alkylating agent used to treat MM which is known to induce replication stress, or with ionizing radiation (IR). Sub-lethal doses of Mel or IR induce APOBEC3B transcript and protein expression in a dose- and time-dependent manner in MM cell lines (n=7), associated with phosphorylation of γH2AX. Significantly, Bortezomib (btz), even at sub-lethal doses triggering DNA damage signaling, induced APOBEC3B expression in multiple MM cell lines. Next, inhibition of ATR or ATM activation pathway significantly decreased Mel- or IR or btz-induced APOBEC3B, suggesting that replication stress induced by Mel, IR, or btz activates ATM/ATR-dependent APOBEC3B induction. We used gene-specific CRISPR knock out (KO), shRNA knockdown (KD), and inducible-shRNA KD to study the functional impact of perturbation of APOBEC3B in MM cells. Both KO and KD of APOBEC3B decreased growth and survival in multiple MM cell lines sensitive or resistant to dexamethasone or lenalidomide. APOBEC3B inhibition significantly enhanced growth arrest followed by apoptosis in MM cells, suggesting that increased APOBEC3B levels contribute to MM cell survival. We next analyzed available data sources for MM cell lines from Cancer Cell Line Encyclopedia and the Genomics of Drug Sensitivity in Cancer, which include microarray gene expression and drug sensitivity information. APOBEC3B expression negatively correlates with MM cell sensitivity to JQ1, a BET inhibitor which inhibit MM cell growth and survival in vitroand in vivo. Importantly, in MM cell lines which are relatively resistant to pomalidomide and JQ1 than other cell lines, APOBEC3B KD by its shRNA enhances sensitivity to both drugs. Taken together, our findings provide new insights into the role of APOBEC3B in triggering cytidine deaminase-induced mutagenesis associated with progression of disease. Furthermore, DNA replication stress triggered by Mel, IR, or btz upregulates APOBEC3B expression, which in turn confers drug resistance. The role of APOBEC in disease pathogenesis and progression, coupled with its role mediating drug resistance, suggest potential utility of targeting APOBEC3B in novel MM therapies. Citation Format: Lijie Xing, Jiye Liu, Liang Lin, Shih-Feng Cho, Kenneth Wen, Tengteng Yu, Gang An, Ligui Qiu, Kenneth Anderson, Yu-Tzu Tai. APOBEC3B is induced by activation of DNA repair pathway and modulates the survival and treatment response in human multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 383.

e14302 Background: We investigated the ability of CD138 antigen-specific CD8+ cytotoxic T lymphocytes (CD138-CTL), generated using a novel HLA-A2-specific CD138260-268 (GLVGLIFAV) peptide, in combination with various immune checkpoint inhibitors, to target breast, colon, and pancreatic cancer. Methods: This study was designed to evaluate anti-tumor activities of CD138-CTL, alone or in combination with Pembrolizumab (anti-PD1), against solid tumors. Results: Upregulation of CD138 protein expression was detected on breast, colon, and pancreatic cancer cells. CD138-CTL induced ex vivo by repeated stimulations with HLA-A2-specific CD138260-268 (GLVGLIFAV) peptide were enriched for CD45RO+ memory CTL and demonstrated antigen-specific and HLA-A2-restricted anti-tumor activities. Treatment of CD138-CTL with anti-PD1, anti-TIM3, or anti-LAG3 or tumor cells with anti-PDL1, anti-Gal9 or anti-LAG3 resulted in an increased proliferation of CD138-specific CD8+ CTL, which express costimulatory and activation markers (CD28, CD40L, 41BB, CD69, CD38). However, expression of immune checkpoints (PD1, LAG3, CTLA4) was also induced on these CD138-CTL upon stimulation. Importantly, anti-tumor activities of CD138 peptide-CTL was significantly enhanced in combination with Pembrolizumab, especially within the antigen-specific memory CD8+ T cell subsets (central memory > effector memory), directed against HLA-A2+ breast cancer, colon cancer and pancreatic cancer. Conclusions: These data provide the framework for an immunotherapeutic strategy encompassing a CD138260-268 (GLVGLIFAV) peptide-based cancer vaccine in combination with Pembrolizumab to enhance antigen-specific central memory cells with more potent and long-lasting CD8+ CTL immune responses in patients with solid tumors. Based on these result, clinical trials evaluating CD138260-268 (GLVGLIFAV) in multi-peptide cancer vaccine are on-going in patients with triple negative breast cancer in combination with anti-PD1 (Pembrolizumab) in metastatic setting (NCT 03362060) or anti-PD-L1 (Durvalumab) in stage II/III, adjuvant setting (NCT 02826434).

Background: Multiple myeloma (MM), the second most common hematologic malignancy in the US, is characterized by the proliferation and accumulation of clonal malignant plasma cells in the bone marrow, associated with hypercalcemia, renal dysfunction and bone disease. Despite recent advances in treatment including novel therapeutics in combination with transplantation, MM still remains incurable. We have recently developd a highly immunogenic HLA-A2-specific multipeptide cancer vaccine targeting XBP1 (X-box binding protein 1), CD138 (Syndecan-1) and CS1 (SLAMF7) antigens. In both pre-clinical studies and Phase 1/2a clinical trials in patients with smoldering multiple myeloma and triple negative breast cancer, vaccination with this multi-peptide based cancer vaccine induced antigen-specific and Th1-type anti-tumor immune responses, which were maintained for long-term within tetramer+/memory (CD45RO+) CD8 cytotoxic T lymphocytes (CTL). The immune responses were further enhanced in patients who received vaccination in combination with lenalidomide or checkpoint inhibitor. To allow for vaccination against additional tumor-associated antigens, we have recently investigated B Cell Maturation Antigen (BCMA), a cell surface antigen restricted to MM and normal plasma cells, as well as accessory dendritic cells. As the receptor for binding of B cell activating factor (BAFF) and of a proliferation-inducing ligand (APRIL), BCMA promotes MM cell growth, drug resistance, as well as survival of long lived plasma cells. Due to its restricted expression pattern, targeting of this antigen with antibodies, immunotoxins, and CAR T-cells is already ongoing in MM at present. Objective: We aimed to identify immunogenic BCMA peptides in order to generate antigen-specific CD8+ effector cytotoxic T lymphocytes against MM cells. Findings: We identified novel immunogenic HLA-A2 native and engineered heteroclitic BCMA peptides that induce tumor-specific CTL against MM. The engineered heteroclitic BCMA72-80 and BCMA54-62 peptides have improved HLA-A2 binding affinity and stability compared to their native BCMA72-80 and BCMA54-62 peptides. In preclinical studies, each of the heteroclitic BCMA peptide induced antigen specific CTL, which expressed increased T-cell activation (CD38, CD69) and co-stimulatory (CD40L, OX40, GITR) molecules. The engineered heteroclitic BCMA72-80 triggered a more robust immune response than BCMA54-62 peptide, associated with increased HLA-A2 binding affinity and stability. Importantly, the heteroclitic BCMA72-80 peptide-specific CTL induced selective and robust proliferative and cytolytic activities in response to MM patients’ tumor cells. Specifically, these tetramer+ CD8+ CTL demonstrated HLA-A2-restricted immune responses against MM, associated with CD107a degranulation, proliferation, Th-1 cytokine (IFN-γ/IL-2/TNF-α) cytokine production, perforin/granzyme B up-regulation, and increased 41BB expression. Furthermore, the heteroclitic BCMA72-80 peptide has demonstrated its immunogenicity through development central memory and effector memory CTL, which showed polyfunctional antitumor activities against MM including patients’ primary tumor cells and cell lines. This novel engineered heteroclitic BCMA72-80 peptide may therefore be useful for generation of antigen-specific and tumor-selective CTL in MM. Significance: We have identified BCMA-specific native and engineered heteroclitic peptides which induce BCMA antigen specific CD8+ CTL with robust anti-MM activity. This immunogenic BCMA peptide will be utlized in clinical protocols of vaccination and/or adoptive T-cells immunotherapy to trigger antigen-specific central and effector memory CTL anti-MM immunity. Citation Format: Jooeun Bae, Teru Hideshima, Yu-Tzu Tai, Nikhil Munshi, Kenneth Anderson. Development of fargeted multiple myeloma cancer vaccine and antigen-specific T-cell immunotherapy using novel Immunogenic-engineered heteroclitic BCMA peptides [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B100.

Homologous recombination (HR), a mechanism to accurately repair DNA in normal cells, is deregulated in cancer. Elevated/deregulated HR is implicated in genomic instability and telomere maintenance, which are critical lifelines of cancer cells. We have previously shown that HR activity is elevated and significantly contributes to genomic instability in Barrett's esophageal adenocarcinoma (BAC). The purpose of this study was to evaluate therapeutic potential of HR inhibition, alone and in combination with telomerase inhibition, in BAC. We demonstrate that telomerase inhibition in BAC cells increases HR activity, RAD51 expression, and association of RAD51 to telomeres. Suppression of HR leads to shorter telomeres as well as markedly reduced genomic instability in BAC cells over time. Combination of HR suppression (whether transgenic or chemical) with telomerase inhibition, causes a significant increase in telomere attrition and apoptotic death in all BAC cell lines tested, relative to either treatment alone. A subset of treated cells also stain positive for β-galactosidase, indicating senescence. The combined treatment is also associated with decline in S-phase and a strong G2/M arrest, indicating massive telomere attrition. In a subcutaneous tumor model, the combined treatment resulted in the smallest tumors, which were even smaller (P=0.001) than those that resulted from either treatment alone. Even the tumors removed from these mice had significantly reduced telomeres and evidence of apoptosis. We therefore conclude that although telomeres are elongated by telomerase, elevated RAD51/HR assist in their maintenance/stabilization in BAC cells. Telomerase inhibitor prevents telomere elongation but induces RAD51/HR, which contributes to telomere maintenance/stabilization and prevention of apoptosis, reducing the efficacy of treatment. Combining HR inhibition with telomerase renders telomeres more vulnerable to degradation and significantly increases/expedites their attrition, leading to apoptosis. We therefore demonstrate that a therapy targeting HR and telomerase has the potential to prevent both tumor growth and genomic evolution in BAC.

Multiple myeloma (MM) is a plasma cell neoplasm that proceeds through a premalignant state of monoclonal gammopathy of unknown significance; however, the molecular events responsible for myelomagenesis remain uncharacterized. To identify cellular pathways deregulated in MM, we addressed that sumoylation is homologous to ubiquitination and results in the attachment of the ubiquitin-like protein Sumo onto target proteins. Sumoylation was markedly enhanced in MM patient lysates compared with normal plasma cells and expression profiling indicated a relative induction of sumoylation pathway genes. The Sumo-conjugating enzyme Ube2I, the Sumo-ligase PIAS1, and the Sumo-inducer ARF were elevated in MM patient samples and cell lines. Survival correlated with expression because 80% of patients with low UBE2I and PIAS1 were living 6 years after transplantation, whereas only 45% of patients with high expression survived 6 years. UBE2I encodes the sole Sumo-conjugating enzyme in mammalian cells and cells transfected with a dominant-negative sumoylation-deficient UBE2I mutant exhibited decreased survival after radiation exposure, impaired adhesion to bone marrow stroma cell and decreased bone marrow stroma cell–induced proliferation. UBE2I confers cells with multiple advantages to promote tumorigenesis and predicts decreased survival when combined with PIAS1. The sumoylation pathway is a novel therapeutic target with implications for existing proteasomal-based treatment strategies.

Purpose: Nicotinamide phosphoribosyltransferase (Nampt) regulates intracellular NAD+ pool and is highly expressed in a number of malignancies. FK866, a selective inhibitor of Nampt, depletes intracellular NAD+ levels, thereby blocking cellular metabolism and triggering sensitization to other drugs and cell death. Here we characterized the antitumor effects of Nampt inhibition in Waldenström macroglobulinemia. Experimental Design: We investigated Nampt role in MW cells using both mRNA and protein expression analyses. We have also used loss-of-function approaches to investigate the growth and survival effects of Nampt on MW cells and further tested the anti-MW activity of dual Nampt and BTK inhibition in vitro and in vivo. Results: We found that Waldenström macroglobulinemia cells exhibit high levels of Nampt compared with normal B cells. Loss of function studies suggested a potential oncogenic role of Nampt in Waldenström macroglobulinemia cells, and BTK-inhibitor ibrutinib and FK866 resulted in a significant and synergistic anti-Waldenström macroglobulinemia cell death, regardless of MYD88 and CXCR4 mutational status. Cell death was associated with: (i) activation of caspase-3, PARP and downregulation of Mcl-1, (ii) enhanced intracellular ATP and NAD+ depletion, (iii) inhibition of NF-κB signaling, and (iv) inhibition of multiple prosurvival signaling pathways. In a murine xenograft Waldenström macroglobulinemia model, low-dose combination FK866 and ibrutinib is well tolerated, significantly inhibits tumor growth, and prolongs host survival. Conclusions: Our results show intracellular NAD+ level as crucial for proliferation and survival of Waldenström macroglobulinemia cells, and provides the mechanistic preclinical rationale for targeting Nampt, either alone or with Ibrutinib, to overcome drug resistance and improve patient outcome in Waldenström macroglobulinemia. Clin Cancer Res; 22(24); 6099–109. ©2016 AACR.

Here we show that overexpression or activation of B-cell maturation antigen (BCMA) by its ligand, a proliferation-inducing ligand (APRIL), promotes human multiple myeloma (MM) progression in vivo. BCMA downregulation strongly decreases viability and MM colony formation; conversely, BCMA overexpression augments MM cell growth and survival via induction of protein kinase B (AKT), MAPK, and nuclear factor (NF)-κB signaling cascades. Importantly, BCMA promotes in vivo growth of xenografted MM cells harboring p53 mutation in mice. BCMA-overexpressing tumors exhibit significantly increased CD31/microvessel density and vascular endothelial growth factor compared with paired control tumors. These tumors also express increased transcripts crucial for osteoclast activation, adhesion, and angiogenesis/metastasis, as well as genes mediating immune inhibition including programmed death ligand 1, transforming growth factor β, and interleukin 10. These target genes are consistently induced by paracrine APRIL binding to BCMA on MM cells, which is blocked by an antagonistic anti-APRIL monoclonal antibody hAPRIL01A (01A). 01A is cytotoxic against MM cells even in the presence of protective bone marrow (BM) myeloid cells including osteoclasts, macrophages, and plasmacytoid dendritic cells. 01A further decreases APRIL-induced adhesion and migration of MM cells via blockade of canonical and noncanonical NF-κB pathways. Moreover, 01A prevents in vivo MM cell growth within implanted human bone chips in SCID mice. Finally, the effect of 01A on MM cell viability is enhanced by lenalidomide and bortezomib. Taken together, these data delineate new molecular mechanisms of in vivo MM growth and immunosuppression critically dependent on BCMA and APRIL in the BM microenvironment, further supporting targeting this prominent pathway in MM.

miRs play a critical role in tumor pathogenesis as either oncogenes or tumor-suppressor genes. However, the role of miRs and their regulation in response to proteasome inhibitors in multiple myeloma (MM) is unclear. In the current study, miR profiling in proteasome inhibitor MLN2238-treated MM.1S MM cells shows up-regulation of miR33b. Mechanistic studies indicate that the induction of miR33b is predominantly via transcriptional regulation. Examination of miR33b in patient MM cells showed a constitutively low expression. Overexpression of miR33b decreased MM cell viability, migration, colony formation, and increased apoptosis and sensitivity of MM cells to MLN2238 treatment. In addition, overexpression of miR33b or MLN2238 exposure negatively regulated oncogene PIM-1 and blocked PIM-1 wild-type, but not PIM-1 mutant, luciferase activity. Moreover, PIM-1 overexpression led to significant abrogation of miR33b- or MLN2238-induced cell death. SGI-1776, a biochemical inhibitor of PIM-1, triggered apoptosis in MM. Finally, overexpression of miR33b inhibited tumor growth and prolonged survival in both subcutaneous and disseminated human MM xenograft models. Our results show that miR33b is a tumor suppressor that plays a role during MLN2238-induced apoptotic signaling in MM cells, and these data provide the basis for novel therapeutic strategies targeting miR33b in MM.

Purpose: The efficacy of peptide vaccines may be enhanced by stimulating immune cells with multiple peptides derived from distinct tumor-associated antigens. We have evaluated the heteroclitic XBP1-US184–192 (YISPWILAV), heteroclitic XBP1-SP367–375 (YLFPQLISV), native CD138260–268 (GLVGLIFAV), and native CS1239–247 (SLFVLGLFL) peptides, which have strong HLA-A2 affinity and immunogenicity in combination, for their ability to elicit multiple myeloma antigen–specific responses. Experimental Design: Multipeptide-specific cytotoxic T lymphocytes (MP-CTL) were generated by the stimulation of CD3+ T lymphocytes from HLA-A2+ individuals with either autologous mature dendritic cells or T2 cells pulsed with a cocktail of these four peptides. Results: The peptide cocktail did not compromise tumor antigen–specific activity of CTLs. MP-CTLs displayed increased total, effector memory (CCR7−CD45RO+), and activated (CD69+) CD3+CD8+ T lymphocytes. In addition, MP-CTL showed IFN-γ production, cell proliferation, and cytotoxicity against HLA-A2+ multiple myeloma cells, including cells of HLA-A2+ patients with multiple myeloma. Importantly, MP-CTLs showed specific responses in functional assays to each relevant peptide but not to an irrelevant HLA-A2–specific CMV pp65 (NLVPMVATV) peptide. Conclusions: These results highlight the potential therapeutic application of vaccination with a cocktail of HLA-A2–specific peptides to induce CTLs with a broad spectrum of immune responses against multiple myeloma antigens. Clin Cancer Res; 18(17); 4850–60. ©2012 AACR.

The CS1 antigen provides a unique target for the development of an immunotherapeutic strategy to treat patients with multiple myeloma (MM). This study aimed to identify HLA-A2(+) immunogenic peptides from the CS1 antigen, which induce peptide-specific cytotoxic T lymphocytes (CTL) against HLA-A2(+) MM cells. We identified a novel immunogenic HLA-A2-specific CS1(239-247) (SLFVLGLFL) peptide, which induced CS1-specific CTL (CS1-CTL) to MM cells. The CS1-CTL showed a distinct phenotype, with an increased percentage of effector memory and activated CTL and a decreased percentage of naïve CTL. CS1(239-247) peptide-specific CD8(+) T cells were detected by DimerX analyses and demonstrated functional activities specific to the peptide. The CTL displayed HLA-A2-restricted and antigen-specific cytotoxicity, proliferation, degranulation and γ-interferon (IFN-γ) production against both primary MM cells and MM cell lines. In addition, the effector memory cells subset (CD45RO(+) CCR7(-) /CD3(+) CD8(+) ) within CS1-CTL showed a higher level of CD107a degranulation and IFN-γ production as compared to effector cells (CD45RO(-) CCR7(-) /CD3(+) CD8(+) ) against HLA-A2(+) primary MM cells or MM cell lines. In conclusion, this study introduced a novel immunogenic HLA-A2-specific CS1(239-247) peptide capable of inducing antigen-specific CTL against MM cells that will provide a framework for its application as a novel MM immunotherapy.

HM1.24, an immunologic target for multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). In this study, we investigated in vitro and in vivo anti-MM activities of XmAb5592, a humanized anti-HM1.24 mAb with Fc-domain engineered to significantly enhance FcγR binding and associated immune effector functions. XmAb5592 increased antibody-dependent cellular cytotoxicity (ADCC) several fold relative to the anti-HM1.24 IgG1 analog against both MM cell lines and primary patient myeloma cells. XmAb5592 also augmented antibody dependent cellular phagocytosis (ADCP) by macrophages. Natural killer (NK) cells became more activated by XmAb5592 than the IgG1 analog, evidenced by increased cell surface expression of granzyme B–dependent CD107a and MM cell lysis, even in the presence of bone marrow stromal cells. XmAb5592 potently inhibited tumor growth in mice bearing human MM xenografts via FcγR-dependent mechanisms, and was significantly more effective than the IgG1 analog. Lenalidomide synergistically enhanced in vitro ADCC against MM cells and in vivo tumor inhibition induced by XmAb5592. A single dose of 20 mg/kg XmAb5592 effectively depleted both blood and bone marrow plasma cells in cynomolgus monkeys. These results support clinical development of XmAb5592, both as a monotherapy and in combination with lenalidomide, to improve patient outcome of MM.

Purpose: The transcription factor specificity protein 1 (Sp1) controls number of cellular processes by regulating the expression of critical cell cycle, differentiation, and apoptosis-related genes containing proximal GC/GT-rich promoter elements. We here provide experimental and clinical evidence that Sp1 plays an important regulatory role in multiple myeloma (MM) cell growth and survival. Experimental Design: We have investigated the functional Sp1 activity in MM cells using a plasmid with Firefly luciferase reporter gene driven by Sp1-responsive promoter. We have also used both siRNA- and short hairpin RNA–mediated Sp1 knockdown to investigate the growth and survival effects of Sp1 on MM cells and further investigated the anti-MM activity of terameprocol (TMP), a small molecule that specifically competes with Sp1-DNA binding in vitro and in vivo. Results: We have confirmed high Sp1 activity in MM cells that is further induced by adhesion to bone marrow stromal cells (BMSC). Sp1 knockdown decreases MM cell proliferation and induces apoptosis. Sp1-DNA binding inhibition by TMP inhibits MM cell growth both in vitro and in vivo, inducing caspase-9–dependent apoptosis and overcoming the protective effects of BMSCs. Conclusions: Our results show Sp1 as an important transcription factor in myeloma that can be therapeutically targeted for clinical application by TMP. Clin Cancer Res; 17(20); 6500–9. ©2011 AACR.

Recent advances regarding the introduction of anti-adhesion strategies as a novel therapeutic concept in oncology hold great promise. Here we evaluated the therapeutic potential of the new-in-class-molecule selective-adhesion-molecule (SAM) inhibitor Natalizumab, a recombinant humanized IgG4 monoclonal antibody, which binds integrin-α4, in multiple myeloma (MM). Natalizumab, but not a control antibody, inhibited adhesion of MM cells to non-cellular and cellular components of the microenvironment as well as disrupted the binding of already adherent MM cells. Consequently, Natalizumab blocked both the proliferative effect of MM-bone marrow (BM) stromal cell interaction on tumour cells, and vascular endothelial growth factor (VEGF)-induced angiogenesis in the BM milieu. Moreover, Natalizumab also blocked VEGF- and insulin-like growth factor 1 (IGF-1)-induced signalling sequelae triggering MM cell migration. In agreement with our in vitro results, Natalizumab inhibited tumour growth, VEGF secretion, and angiogenesis in a human severe combined immunodeficiency murine model of human MM in the human BM microenvironment. Importantly, Natalizumab not only blocked tumour cell adhesion, but also chemosensitized MM cells to bortezomib, in an in vitro therapeutically representative human MM-stroma cell co-culture system model. Our data therefore provide the rationale for the clinical evaluation of Natalizumab, preferably in combination with novel agents (e.g. bortezomib) to enhance MM cytotoxicity and improve patient outcome.

The development of an immunotherapeutic strategy targeting CD138 antigen could potentially represent a new treatment option for multiple myeloma (MM). This study evaluated the immune function of CD138 peptide-specific cytotoxic T lymphocytes (CTL), generated ex vivo using an HLA-A2-specific CD138 epitope against MM cells. A novel immunogenic HLA-A2-specific CD138(260-268) (GLVGLIFAV) peptide was identified from the full-length protein sequence of the CD138 antigen, which induced CTL specific to primary CD138(+) MM cells. The peptide-induced CD138-CTL contained a high percentage of CD8(+) activated/memory T cells with a low percentage of CD4(+) T cell and naive CD8(+) T cell subsets. The CTL displayed HLA-A2-restricted and CD138 antigen-specific cytotoxicity against MM cell lines. In addition, CD138-CTL demonstrated increased degranulation, proliferation and γ-interferon secretion to HLA-A2(+) /CD138(+) myeloma cells, but not HLA-A2(-) /CD138(+) or HLA-A2(+) /CD138(-) cells. The immune functional properties of the CD138-CTL were also demonstrated using primary HLA-A2(+) /CD138(+) cells isolated from myeloma patients. In conclusion, a novel immunogenic CD138(260-268) (GLVGLIFAV) peptide can induce antigen-specific CTL, which might be useful for the treatment of MM patients with peptide-based vaccine or cellular immunotherapy strategies.

The purpose of these studies was to identify human leukocyte antigen (HLA)-A2(+) immunogenic peptides derived from XBP1 antigens to induce a multiple myeloma (MM)-specific immune response. Six native peptides from non-spliced XBP1 antigen and three native peptides from spliced XBP1 antigen were selected and evaluated for their HLA-A2 specificity. Among them, XBP1(184-192), XBP1 SP(196-204) and XBP1 SP(367-375) peptides showed the highest level of binding affinity, but not stability to HLA-A2 molecules. Novel heteroclitic XBP1 peptides, YISPWILAV or YLFPQLISV, demonstrated a significant improvement in HLA-A2 stability from their native XBP1(184-192) or XBP1 SP(367-375) peptide, respectively. Cytotoxic T lymphocytes generated by repeated stimulation of CD3(+) T cells with each HLA-A2-specific heteroclitic peptide showed an increased percentage of CD8(+) (cytotoxic) and CD69(+)/CD45RO(+) (activated memory) T cells and a lower percentage of CD4(+) (helper) and CD45RA(+)/CCR7(+) (naïve) T cells, which were distinct from the control T cells. Functionally, the cytotoxic T lymphocytes (CTL) demonstrated MM-specific and HLA-A2-restricted proliferation, interferon-γ secretion and cytotoxic activity in response to MM cell lines and importantly, cytotoxicity against primary MM cells. These data demonstrate the distinct immunogenic characteristics of unique heteroclitic XBP1 peptides, which induce MM-specific CTLs and highlights their potential application for immunotherapy to treat the patients with MM or its pre-malignant condition.

In the recent years, the development of immunotherapy has revolutionized the cancer treatment including multiple myeloma (MM). Therapeutic agents killing MM cells and activating immune cell function to overcome immunocompromised microenvironment further improved clinical outcome. Importantly, adoptive cellular therapy using chimeric antigen receptor (CAR) T cell targeting specifically expressed antigen on tumor cells has become one of the exciting breakthrough in MM immunotherapy.

14-3-3 proteins are chaperone and scaffold proteins that exert a widespread influence on cellular processes through binding to serine/threonine-phosphorylated residues on target proteins, forcing conformational changes or influencing their interactions with other molecules. Altered 14-3-3 expression is associated with development and progression of cancer. We therefore evaluated the status of all 14-3-3 isoforms in plasma cells disorders in publically available gene expression profiling (GEP) data. Using independent patient datasets, we observed a consistent higher expression of YWHAE (coding gene for the isoform 14-3-3ε) in MM and plasma cell leukemia (PCL) patients, while no consistent differences were observed with the other isoforms. Moreover, we also confirmed higher expression of YWHAE in our RNA-seq data from 420 newly-diagnosed MM patients, with relatively low expression in normal plasma cells. Finally, 14-3-3ε was also found to be constitutively expressed at protein level in primary patient MM cells and in a large panel of MM cell lines, with significantly lower expression in healthy donor B cells. To evaluate if 14-3-3ε represents a functional dependency in MM, we performed genetic perturbation of YWHAE in a panel of MM cell lines. Depletion of YWHAE using 3 different shRNA inhibited cell proliferation and induced cell apoptosis across 5 different cell lines, independently of their genetic background. We next performed CRISPR-Cas9-mediated YWHAE knock out (KO) in H929 and JJN3 cells and observed a significant decrease in cell viability and a robust apoptotic response. H929 YWHAE KO cells infected with FLAG-YWHAE addback lentiviral construct completely rescued this phenotype, confirming that loss of YWHAE is responsible for the defective cell viability and apoptotic phenotype. These observations were corroborated by ectopic overexpression of YWHAE in H929 WT cells that significantly promoted MM cell viability. To elucidate the underlying molecular mechanisms, proteins immunocomplexed co-precipitated with FLAG in H929 KO cells with 14-3-3ε-FLAG addback were analyzed by mass spectrometry. Protein analysis revealed interaction of 14-3-3ε with a large number of proteins, enriched in mTORC1, PI3K-AKT-mTOR and unfolded protein response (UPR) pathway-related genes. Among these, TSC2 and mTORC1 proteins were further studied. WB analysis confirmed interaction of 14-3-3ε with p-mTOR (S2448) and its upstream negative regulator p-TSC2 (S939), while mTORC1 downstream targets, p-p70 S6k and p-4E-BP1, did not interact with 14-3-3ε. WB analysis also revealed activation of TSC2 and consequent inhibition of mTORC1 (via decrease of p-mTOR S2448 levels) in YWHAE KD cells. YWHAE-FLAG addback reversed these effects. Additionally, GEP data in KD cells confirmed a significant impact on mTORC1 pathways. Importantly, YWHAE expression highly correlated (R> 0.8) with genes involved in the mTORC1 pathway, including PSMC4, COPS5, EIF2S2, in our RNA-seq dataset, demonstrating a clinical significance of 14-3-3ε and mTORC1 cooperation in the context of myeloma. One of the most conserved functions of mTORC1 is to promote translation. We therefore assessed the impact of YWHAE on global translational efficiencies in MM cells, and observed significant impact on nascent protein synthesis by YWHAE modulation. 14-3-3ε KD induced 4EBP1 de-phosphorylation through inhibited mTORC1, and concomitantly induced EIF2α phosphorylation. Both effects inhibited translation initiation complex formation, mechanistically supporting a strong protein synthesis arrest. These data show the modulation of several hubs of the signaling apparatus controlling translation initiation in response to YWHAE modulation, ultimately producing a marked protein synthesis inhibition. Deregulated translational control is a central feature of MM. Our findings highlight a unique function for YWHAE as promoter of MM cell survival through regulation of mTOR-dependent protein synthesis and apoptosis. Pharmacological inhibition of YWHAE/14-3-3ε is therefore a possibility to specifically target malignancies with deregulated translational control such as MM. Disclosures Anderson: C4 Therapeutics: Equity Ownership, Other: Scientific founder; Millennium Takeda: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; OncoPep: Equity Ownership, Other: Scientific founder; Celgene: Consultancy. Munshi:OncoPep: Other: Board of director.

CD38 is highly expressed on malignant plasma cells from patients with multiple myeloma (MM), and anti-CD38 monoclonal antibodies (i.e. daratumumab, SAR650984) trigger cytotoxicity through antibody-dependent cellular cytotoxicity (ADCC) and activation of complement-dependent cytotoxicity (CDC). Although remarkable response has been observed in relapsed and refractory MM, relapse of disease is common. To improve the clinical efficacy of CD38-targeting immunotherapies, novel combination treatment strategies have been evaluated in preclinical studies and clinical trials. For example, all-trans retinoic acid or histone deacetylase inhibitors enhance daratumumab-induced cytotoxicity via upregulation of CD38 expression. To date, however, the biologic impact of the bone marrow microenvironment on CD38-targeting immunotherapies has not been characterized. In this study, we evaluated the impact of bone marrow stromal cells (BMSCs) from MM patients on CD38 expression and anti-CD38 Antibody-induced ADCC. We first cultured MM cells (RPMI8226, MM.1S, MOLP8) with culture supernatant from BMSCs and measured CD38 expression by flow cytometry. A significant reduction of CD38 expression on all MM cell lines was noted in a time-dependent fashion. For example, CD38 expression (mean fluorescence intensity) was reduced 44%, 32%, and 42% on RPMI8226, MM.1S and MOLP8 cells, respectively, after 48 h culture with BMSC supernatants. To identify mediators of this effect, we next examined the effect of MM-relevant cytokines (IL-6, IGF-1, IL-1β) on CD38 expression. Importantly, IL-6 significantly downregulated CD38 expression on MM cells whereas IGF-1 or IL-1β had no significant impact. We further performed quantitative real-time PCR to evaluate CD38 mRNA level in RPMI8226 and MOLP8 cells cultured with BMSC supernatant or IL-6 and confirmed that CD38 transcript was downregulated by both treatments in these cells. Since IL-6, but not IGF-1, downregulated CD38, we hypothesized that JAK-STAT3 pathway might mediate this IL-6 triggered downregulation of CD38 expression. As with IL-6 treatment, transfection of MM.1S cells with constitutively active STAT3 significantly reduced CD38 expression. Conversely, knockdown of STAT3 by siRNA partially restored CD38 expression on RPMI8226 cells in the presence of IL-6. Moreover, we cultured RPMI8226 and MM.1S cells with JAK inhibitor ruxolitinib (1, 3, 10 μM) in the presence of BMSC supernatant or IL-6. We observed significant recovery of CD38 expression on MM cells. We next evaluated the impact of these modulations of CD38 expression on ADCC. Natural killer cell-resistant RPMI8226 cells were pre-treated with BMSC supernatant for 48 h and then labeled with calcein-AM for 30 min, followed by co-culture with peripheral blood mononuclear cells from healthy donors (effector/target ratio of 30:1) in the presence of anti-CD38 antibody (1 μg/ml of SAR650984). As expected, ADCC was reduced by BMSC supernatant. Our studies, therefore, show that BMSC supernatant downregulates CD38 expression through IL-6-STAT3 signaling pathway in MM cells, and thereby reduces sensitivity to CD38-targeted ADCC in MM cells. They provide the rationale for novel treatment strategies of CD38-targeting immunotherapy in combination with JAK-STAT pathway inhibitors to enhance sensitivity or overcome resistant to CD38-targeted ADCC in the context of the MM BM microenvironment. Disclosures Anderson: Celgene: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Millennium Takeda: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Bristol Myers Squibb: Consultancy; OncoPep: Equity Ownership, Other: Scientific founder.

The first Bispecific T-cell Engager (BiTE®) targeting B-cell maturation antigen (BCMA) on multiple myeloma (MM) cells and CD3 on T cells is currently in clinical development (NCT02514239). This first-generation BiTE® has a short serum half-life and is delivered by continuous IV infusion. We here investigated the T cell-redirected cytotoxicity and immunomodulatory effects of AMG 701, a BCMA-targeting BiTE® with extended half-life, alone and in combination with lenalidomide (len), in MM cell lines and patient-derived samples. AMG 701 has a plasma half-life of 112 hr. in non-human primates (Cancer Res 2018;78(13 Suppl):Abstract nr LB-299) and is currently being evaluated clinically (NCT03287908). We here show that AMG 701 significantly induced T cell-mediated lysis of BCMA-positive MM cells resistant or sensitive to current anti-MM agents including bortezomib and lenalidomide (len). EC50 values ranged from 0.64-2.54 ng/ml following overnight treatment with AMG 701 at effector to target (E/T) ratio of 10 to 1. Moreover, following overnight treatment AMG 701-induced MM cell lysis remained robust even at low concentrations (< 2 ng/ml) and low E/T ratios (2/1, 1/2). Importantly, the presence of myeloma-supporting osteoclasts or bone marrow stromal cells did not significantly alter the ability of AMG 701 to lyse MM cells. In the presence of BCMA-positive target cells, AMG 701 rapidly induced degranulation of CD4+ and CD8+ T cells in a dose-dependent manner, evidenced by upregulated surface CD107a expression. AMG 701 triggered lysis also induced secretion of IFNγ and TNFα, to a greater extent in CD8+ than CD4+ T cell subsets. Importantly, AMG 701 (1d treatment) induced lysis of autologous patient tumor cells from relapsed and refractory MM. Combined effects of AMG 701 with len were next investigated using effector cells (PBMC or CD3+ T) pretreated with len. Len enhanced AMG 701-mediated T cell lysis of MM cells even in the presence of osteoclasts. In the presence of BCMA-positive target cells, len pretreatment also further enhanced AMG 701-induced secretion of IFNγ and TNFα from T cells, to a greater extent in CD8+ than CD4+ T cell subsets. In the absence of AMG 701, len-specific lysis of MM cells was observed, confirming len-enhanced cytotoxic potential of T cells against MM cells. We next studied the potential immunomodulatory effects of AMG 701 by flow cytometry analysis. AMG 701 was added to co-cultures of MM cells and CellTraceTM violet-labeled effector cells and T cells were analyzed at various time points. After 4d-co-incubation, AMG 701 induced more proliferation of CD8+ than CD4+ T cells (9.94% vs 0.8% at 1 ng/ml; and 47.5% vs 16.7% at 10 ng/ml, respectively). Higher levels of CD25 and CD69 were also observed in CD8+ than CD4+ T cells. Transient but not persistent upregulation of immune checkpoint molecules PD-1, TIM-3, and LAG-3 were seen on CD4+ and CD8+ T cells after coincubation with AMG 701 and BCMA-positive target cells. In parallel, the CD8+/CD4+ T cell ratios were increased (1.21 to 3.48-fold at d1 to d5, p Disclosures Munshi: OncoPep: Other: Board of director. Anderson:Bristol Myers Squibb: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Gilead: Membership on an entity's Board of Directors or advisory committees; OncoPep: Equity Ownership, Other: Scientific founder; Millennium Takeda: Consultancy; Celgene: Consultancy.

A proliferation inducing ligand (APRIL) is a natural ligand with higher affinity than BAFF for both B cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI), which are overexpressed on multiple myeloma (MM) cells. APRIL, which is abundantly secreted by myeloma-supporting osteoclasts and macrophages, promotes MM cell progression in vivo and further induces regulatory T cells (Treg) via TACI, but not BCMA, to promote an immunosuppressive MM bone marrow (BM) microenvironment (Blood 2017;130:3066). In preclinical studies, an antagonistic APRIL monoclonal antibody (mAb) significantly inhibited human MM cell growth in SCID-hu mice and abrogated APRIL-induced immunosuppression mediated by Tregs. Here we characterized the ability of BION-1301, a neutralizing APRIL mAb, currently under clinical development in MM (NCT03340883) to overcome protection conferred by APRIL against MM cell lysis induced by the anti-BCMA J6M0 mAb (Blood 2017 130:499). Our studies show that APRIL protects against MM cell lysis induced by J6M0 in the presence of FcR-expressing effector cells (PBMC, monocytes or NK), an effect which is blocked by neutralizing anti-APRIL mAb. APRIL also downregulates J6M0-induced cell membrane CD107a expression on NK cells co-cultured with BCMA-expressing MM cells, which is similarly abrogated by anti-APRIL mAb. Importantly, anti-APRIL mAb also significantly decreases protection conferred by osteoclasts against MM cell lysis induced by J6M0. These data indicate that blocking APRIL with anti-APRIL mAb may enhance BCMA mAb targeted (J6M0)-induced MM cell lysis. Next, using anti-human IgG1 to detect J6M0 binding to MM cell surface BCMA, we found that APRIL in a dose-dependent manner directly competes with J6M0 for binding to BCMA, which was confirmed by ELISA. In addition, APRIL effectively inhibits J6M0 binding to BCMA at 4°C, which argues against APRIL-induced BCMA receptor shedding and/or internalization. In contrast, BAFF affects J6M0 binding to BCMA only at higher concentrations (>1 µg/mL), consistent with > 2-log higher affinity of APRIL vs. BAFF for BCMA. We further assessed APRIL, BAFF, BCMA, and TACI levels in the serum of patients with MM at various stages of disease. Specifically, we used ELISA to measure free APRIL and the ELLA automated immunoassay platform to determine the levels of soluble BAFF, soluble BCMA, and soluble TACI in serum samples from patients with MM (n=193) as well as serum samples from healthy volunteers (HV, n=100). Patient samples included monoclonal gammopathy of undetermined significance (MGUS, n=12), smoldering MM (SMM, n=20), newly diagnosed MM (ND, n=39), post induction pre-autologous stem cell transplant (ASCT, n=55), post-ASCT (n=6), and relapsed refractory MM (RR, n=61). We found that free APRIL levels are significantly increased in serum samples from patients with MM at all stages of disease, when change from baseline levels were compared to those from HVs (post-ASCT: p=0.0003; other groups (MGUS, SMM, ND, pre-ASCT, and RR): p Our studies therefore indicate that therapies directed at the APRIL/BCMA and APRIL/TACI axes may simultaneously target MM cells and counteract APRIL-induced immunosuppression, and that combination strategies targeting APRIL with BCMA directed therapy may augment anti-MM activity. Moreover, elevated free APRIL serum levels in MGUS and all stages of MM suggest a role for APRIL in mediating immunosuppression during the development and in the pathogenesis of MM. Disclosures Dulos: Aduro Biotech: Employment. Guelen:Aduro Biotech Europe: Employment. van Zandvoort:Aduro Biotech Europe: Employment. van de Wiel:Aduro Biotech Europe: Employment. van de Crommert:Aduro Biotech Europe: Employment. Lejeune:Aduro Biotech Europe: Employment. Namini:Aduro Biotech: Employment. Eenennaam:Aduro Biotech Europe: Employment, Equity Ownership. van Elsas:Aduro Biotech Europe: Employment. Richardson:Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees. Munshi:OncoPep: Other: Board of director. Anderson:Takeda Millennium: Consultancy; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Oncopep: Equity Ownership.

Constitutive genomic complexity, ongoing DNA damage, and accumulating mutations are observed with progression from monoclonal gammopathy of undetermined significance (MGUS) to active multiple myeloma (MM) to relapsed/refractory disease. Apolipoprotein B mRNA editing catalytic polypeptide-like 3B (APOBEC3B), a DNA cytosine deaminase, plays a prominent role in inducing mutations in multiple human cancers. In MM, APOBEC3B is linked to sub-clonal diversification, intra-tumor heterogeneity, and tumor evolution. Moreover, upregulation of APOBEC3B is associated with poor MM prognosis, suggesting that targeting MM cells with high APOBEC3B may represent a novel therapeutic approach. We here studied the upstream mechanisms of APOBEC3B dysregulation and further defined functional consequences of molecular manipulation of APOBEC3B in MM cells. We characterized its sequelae to identify novel strategies for cancer prevention or treatment by targeting this key driver gene of cancer mutagenesis. Since the expression of APOBEC3B is associated with replication stress in breast cancer, we first asked whether APOBEC3B levels are altered in MM cell lines upon treatments with Melphalan (Mel), an alkylating agent used to treat MM which is known to induce replication stress; or with ionizing radiation (IR). Using quantitative RT-PCR and Western blotting, we found that sub-lethal doses of Mel or IR induce APOBEC3B expression in a dose- and time-dependent manner in MM cell lines (n=7) associated with the phosphorylation of γH2AX. Interestingly bortezomib (btz), even at sub-lethal doses which triggers DNA damage signaling, also induced APOBEC3B expression in H929, MM1S, and U266 MM cells. Since DNA replication stress activates the ATR/ATM pathway, we next investigated whether these kinases mediate APOBEC3B induction following Mel- or IR- or btz-induced DNA replication stress. H929 and MM1S cells were treated with Mel or IR in the presence or absence of ATM or ATR inhibitors, and these cells were then lysed and assayed for APOBEC3B expression. Importantly, inhibition of ATR or ATM activation pathway significantly decreased Mel- or IR or btz-induced APOBEC3B, suggesting that replication stress induced by Mel, IR, or btz, activates transcription of APOBEC3B via an ATM/ATR dependent pathway in vitro. To test the effect of APOBEC3B on cell growth and survival, we used gene-specific CRISPR knock out (KO), shRNA knockdown (KD), and inducible-shRNA KD to study the functional impact of perturbation of APOBEC3B in MM cells. Both KO and KD of APOBEC3B decreased growth and survival in multiple MM cell lines sensitive or resistant to dexamethasone or lenalidomide. Using zombie aqua and annexin V-based flow cytometric analysis, we showed that APOBEC3B inhibition enhanced growth arrest, followed by apoptosis, in these MM cells. These data suggest an important role of increased APOBEC3B levels in MM cell survival. We next analyzed available data sources for MM cell lines from Cancer Cell Line Encyclopedia (CCLE) and the Genomics of Drug Sensitivity in Cancer (GDSC), which include microarray gene expression and drug sensitivity information. APOBEC3B expression negatively correlates with MM cell sensitivity to JQ1, a BET inhibitor which has been reported to inhibit MM cell growth and survival in vitro and in vivo. Importantly, in MM cell lines which are relatively resistant to pomalidomide and JQ1 than other cell lines, APOBEC3B KD by its shRNA enhances sensitivity to both drugs. Taken together, our findings provide new insights into the role of APOBEC3B in triggering cytidine deaminase-induced mutagenesis associated with progression of disease. Furthermore, we show that DNA replication stress triggered by Mel, IR, or btz upregulates APOBEC3B expression, which in turn confers drug resistance. The role of APOBEC in disease pathogenesis and progression, coupled with its role mediating drug resistance, suggest potential utility of targeting APOBEC in novel MM therapies. Disclosures Munshi: OncoPep: Other: Board of director. Anderson:Bristol Myers Squibb: Consultancy; Millennium Takeda: Consultancy; Celgene: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; OncoPep: Equity Ownership, Other: Scientific founder; Gilead: Membership on an entity's Board of Directors or advisory committees.

A proliferation-inducing ligand (APRIL) is produced by multiple accessory and myeloid cells in the bone marrow niche. Through binding to its receptors B-cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI), APRIL plays an important role in the development and maintenance of cells derived from the B cell lineage. Both APRIL and its receptors have been identified as promotors of multiple myeloma (MM), a disease that is characterized by malignant proliferation of plasma cells in the bone marrow. We have shown that BION-1301, a first-in-class APRIL-targeting humanized antibody, blocks APRIL-induced effects on MM cell survival and the tumor microenvironment (TME). Despite recent approval of new therapies for MM, this type of cancer remains incurable as patients relapse and eventually become refractory to the current therapeutic regimens. We hypothesize that the presence of high levels of APRIL in the bone marrow of patients with MM establishes a protected environment favoring tumor cell survival and proliferation. The effects of APRIL were studied on tumor cell lines alone or in co-culture with osteoclasts, which are thought to produce APRIL in the bone marrow niche. We further hypothesize that engaging APRIL with BION-1301 sensitizes MM cells for other MM-targeting therapeutic agents. First, stimulation of MM cells with APRIL led to rapid phosphorylation of proteins in the NFκB pathway, that was completely blocked by the BION-1301 parental antibody hAPRIL.01A. In addition, by validating expression of known NFκB target genes, APRIL appears to protect MM cells from drug-induced toxicity by upregulation of anti-apoptotic genes (e.g. MCL-1), since APRIL enhanced survival of MM cells that were treated with anti-MM drugs, such as dexamethasone. Importantly, addition of hAPRIL.01A blocked this pro-survival effect, highlighting the potential of BION-1301 to enhance MM sensitivity towards MM-targeting agents. We confirmed the immune suppressive potential of APRIL by showing that APRIL upregulates known immunosuppressive proteins such as IL-10 and PD-L1, indicating that BION-1301 could increase sensitivity to therapies such as daratumumab that depend on the immune system. Finally, in vitro APRIL increased secretion of the chemokines MIP-1α and MIP-1β by MM cell lines, which are known to stimulate osteoclast differentiation and activation. This suggests a positive feedback loop in which APRIL, produced mainly by osteoclasts in the bone marrow, induces chemokine production by MM cells, and these chemokines in turn stimulate osteoclasts. Importantly, when hAPRIL.01A is added to block APRIL, chemokine production drops, indicating that in addition to sensitizing MM tumor cells for drug treatment, BION-1301 also suppresses osteoclast activity. Altogether, we demonstrate in vitro that APRIL induces anti-apoptotic signaling, triggers expression of an immune suppressive phenotype, and could potentially modulate the tumor microenvironment through the production of key chemokines. Blocking APRIL with BION-1301 may dampen these effects, illustrating the potential of BION-1301, as a therapeutic agent for MM, particularly in combination therapies. BION-1301 is currently in a phase 1 clinical trial for MM. Disclosures Bakker: Aduro Biotech: Employment. Habraken:Aduro Biotech: Employment. Dulos:Aduro Biotech: Employment. Van Elsas:Aduro Biotech Europe: Employment. Vink:Aduro Biotech Europe: Employment. Eenennaam:Aduro Biotech Europe: Employment, Equity Ownership.

Major improvements in patient outcome have resulted from the development of novel agents targeting multiple myeloma (MM) cells in the bone marrow (BM) microenvironment. Immunomodulatory drugs (IMiDs) including lenalidomide and pomalidomide bind to the CRL4CRBN ubiquitin ligase and promote proteasomal degradation of IKZF1/3 followed by induction of growth inhibition in MM cells. Although IMiDs-based treatments have achieved significant responses and improved outcomes in MM patients, acquired resistance to IMiDs commonly underlies relapse. Although the molecular mechanisms regulating sensitivity to IMiDs have not been fully defined, cereblon (CRBN) is considered to be the primary binding target of IMiDs. However, some MM cells show resistance to IMiDs despite harboring high CRBN expression levels. In this study, we show that TRAF2, a member of TNF receptor associated factor protein family, represents a novel regulator of IMiDs sensitivity in MM cells. To study the molecular mechanisms underlying IMiDs resistance, we first performed genome-wide knockout screening in IMiDs-sensitive MM.1S cells using a CRISPR-Cas9 GeCKOv2 library containing 6 unique sgRNAs against each of 19,050 genes and 4 sgRNAs against each of 1,864 miRNAs Twenty-eight genes and one miRNA were identified which were associated with resistance to IMiDs. Of note, all six sgRNAs targeting CRBN were identified, consistent with previous studies. Among these novel genes, we found that three different sgRNAs targeting TRAF2 were enriched after IMiDs selection. Therefore, we next individually cloned the sgRNAs of TRAF2 into the Cas9 lentiviral vector, and then re-introduced them into MM.1S cells. Importantly, TRAF2 knockout (KO) MM.1S cells acquire significant resistance to pomalidomide and lenalidomide treatments. To examine whether TRAF2 KO-induced IMiDs resistance was CRBN-pathway dependent, we assessed CRBN and its downstream protein levels. TRAF2 KO showed no effect on CRBN expression; IMiDs can still induce IKZF1/3 degradation, associated with downregulation of IRF4 in TRAF2 KO cells. Taken together, these data suggest that TRAF2 mediates sensitivity of IMiDs, independent of CRBN-IKZF1/3 axis. Since TRAF2 is a member of the TNF receptor associated factor (TRAF) protein family required for activation of several signal pathways including NF-ĸB and JNK, we next examined signaling cascades modulated by TRAF2 KO. Importantly, TRAF2-KO MM cells revealed increased processing of p52 (NF-ĸB2) from its precursor p100, resulting in hyperactivation of the non-canonical NF-κB pathway. The soluble factors (ie, cytokines) secreted by MM cells have important paracrine role on MM cell growth and drug resistance. We therefore performed cytokine analysis of proteins secreted by TRAF2 KO cells, and found that chemokine (C-C motif) ligand 5 (CCL5), a target gene of NF-kB pathway, was upregulated in TRAF2 KO cells. Moreover, upregulation of CCL5 was also observed in acquired IMiDs resistant MM1.S cells, which was confirmed by quantitative real-time PCR. In addition, exogenous CCL5 in the culture medium partially downregulated sensitivity to IMiDs treatment. In conclusion, we here identify and validate TRAF2 as a novel regulator of IMiDs sensitivity, independent of CRBN. Our mechanistic studies provide the preclinical rationale for combination treatment strategies with non-canonical NF-kB inhibitors to overcome IMiDs resistance in MM. Disclosures Zhou: WuXi App Tec Inc: Employment. Anderson:Oncopep: Equity Ownership; C4 Therapeutics: Equity Ownership; Celgene: Consultancy; Takeda Millennium: Consultancy; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees.

Multiple Myeloma (MM) initiation and progression is driven by recurrent cytogenetic events, i.e. multiple trisomies or translocations within the immunoglobulin locus. Gene mutations have been extensively studied, and they are generally involved in late phases of disease development. On the contrary, very little is known about non-recurrent structural variations (SV), which are increasingly emerging as critical driver in several cancers. To determine the extent to which the MM genome is shaped by such events, we performed whole genome sequencing (WGS) on 67 CD138+ purified bone marrow MM samples from 30 patients (median of 2 samples per patient; range 1-4), to which we added 22 previously published cases (Chapman et al, Nature 2011) for a total of 52 patients and 89 tumor samples. We defined SVs as inversions, translocations, internal tandem duplications and deletions, which we analysed using publicly available tools developed at the Wellcome Sanger Institute. We found a stunning 1887 unique SVs in the whole cohort, with a variable distribution across the entire series (median 29 per patient, range 0-156). To derive a homogeneous catalogue of SV across the different MM patients and biological subgroups, we annotated events according to the recently proposed classification on >2500 cancer genomes (Li Y. et al BioRxiv 2018). IGH and MYC translocations were the most frequent recurrent events and accounted for just 5.3% of the entire SV catalogue. We defined as complex events the following SV classes: chromothripsis, chromoplexy, multiple inversions (distinct between Local_n_Jumps and Local_and_distant_n_jumps in case of translocation involvement), templated insertion between more than 2 chromosomes. According to this classification, in 93% of patients a single, private complex SV was responsible for multiple and simultaneous CNAs across different chromosomes, thus providing a novel pathogenetic explanation for many recurrent CNAs in MM. Overall, 136 complex events were observed in 43/52 patients (83%). We found 34 instances of chromotripsis (Korbel J.O. et al., Cell 2013) in 18/52 (34%) patients. The vast majority (30/34) were clonal and conserved during evolution, suggesting an early role in MM pathogenesis. In addition, we observed 5 chromoplexy events (Korbel J.O. et al., Cell 2013) acquired in 5 patients. More interestingly, evidence of templated insertion on more than 2 chromosomes was observed in 13 patients (25%). This event is composed by multiple concatenated translocations causing small CNAs (mostly gains) and in 77% it resulted in a translocation involving an important MM oncogene (8 MYC and 2 CCND1), suggesting that this is a novel relevant driver mechanism in MM. Given the variety of the landscape of SV between patients, we investigated the presence of SV patterns (SV signatures) by the hierarchical dirichlet process (hdp) (https://github.com/nicolaroberts/hdp). Six main SV signatures were extracted: SV signature #1 was characterized by multiple isolated deletions; SV signature #2 was associated with chromotripsis; SV signature #3 was characterized by reciprocal translocation, local_and_distant_n_jumps and templete insertion between 2 chromosomes. Signature #4 was mostly characterized by local_n_jumps; Signature #5 and #6 were associated with temple insertions on multiple chromosomes with or without large tandem duplication, respectively. Different patients showed differential contribution from different signatures, and based on this we observed 5 distinct clusters. Interestingly some of these clusters were associated with distinct and known MM drivers. For example t(4;14)(MMSET;IGH) cases were enriched for SV signature #1. A significant fraction of patients without any recurrent IGH translocation were characterized by high prevalence of chromothripsis. SV signatures #5 and #6 were mostly associated with hyperdiploid patients with MYC translocation and low genomic impairment. In this study, we describe the landscape of SVs and complex events in MM, suggesting that this notation may represent an important step forward in disentangling the genomic complexity and heterogeneity of MM. Disclosures Moreau: Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: 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; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Corradini:Roche: Honoraria, Other: Advisory Board & Lecturer; Janssen: Honoraria, Other: Lecturer; Sandoz: Other: Advisory Board; Novartis: Honoraria, Other: Advisory Board & Lecturer; Abbvie: Honoraria, Other: Advisory Board & Lecturer; Celgene: Honoraria, Other: Advisory Board & Lecturer; Gilead: Honoraria, Other: Advisory Board & Lecturer; Takeda: Honoraria, Other: Advisory Board & Lecturer; Sanofi: Honoraria, Other: Advisory Board & Lecturer; Amgen: Honoraria, Other: Advisory Board & Lecturer. Anderson:Celgene: Consultancy; Oncopep: Equity Ownership; C4 Therapeutics: Equity Ownership; Takeda Millennium: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy. Avet-Loiseau:Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees. Munshi:OncoPep: Other: Board of director. Bolli:Celgene: Honoraria.

CD138 (Syndecan-1), a member of integral membrane family of heparan sulfate proteoglycans (HSPGS), is highly expressed on differentiated plasma cells (PC) and is both a primary diagnostic biomarker of multiple myeloma (MM) as well as an indicator of poor clinical prognosis. This surface antigen is an attractive candidate for targeted immunotherapy for MM, given its constitutive expression during disease progression, including smoldering myeloma, a relatively early asymptomatic phase of disease that is potentially amenable to early treatment. We here investigated the targeted use of chimeric anti-CD138 monoclonal antibody (mAb) 1610 and confirm its in vitro anti-tumor potency based on an immune directed cellular cytotoxicity against a diverse panel of CD138 positive MM cell lines, both resistant or sensitive to conventional and current MM therapies and varying levels of CD138 expression as measured by cell immunostaining and quantitative RT-PCR. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated using a calcein-AM based release assay in the presence of human natural killer (NK) effector cells purified from four different healthy donors. MAb 1610 lysed CD138-expressing MM cell lines in a dose dependent manner. This ADCC activity was mAb 1610 specific (in comparison to isotype control), CD138 target dependent, and mediated in the presence of human NK effector cells (co-cultured at an effector:target cell ratio of 20:1). MAb 1610 dependent-cytotoxicity was observed at concentrations as low as 0.01 µg/ml with maximal lysis occurring at approximately 1 µg/ml and extrapolated sub-nanomolar ED50 potencies (Table 1) based on these data. All MM cell lines were subject to mAb 1610-mediated lysis, albeit with slightly different sensitivities that modestly correlated with their relative CD138 cell surface expression levels. This anti CD138 mAb-dependent cellular toxicity included MM1SR and H929R cell lines, both of which are resistant to lenalidomide. MAb 1610 induced specific cell lysis of JJN3 cells, but not of CD138 knock out JJN3 cells or CD138-negative B lymphocytes, further confirming that mAb 1610 specifically induced ADCC against-CD138 expressing MM cells in a target specific manner. Using an orthogonal cytometric based assay, the ability of mAb 1610, in a dose-dependent manner, to activate NK cells was also shown in the presence of CD138 target cells, as evidenced by increased expression of CD107 (a marker for NK cell degranulation) and cytokine production in NK cells. Importantly, the CD138 targeting cytotoxic activities of mAb 1610 translationally extend to MM cells autologously derived directly from MM patients with newly diagnosed and relapsed/refractory diseases. The concomitant use of autologously derived effector cells from these patients to mediate antibody dependent myeloma cell killing further suggests the relevance of anti-CD138 directed immune-based therapeutic strategy in humans. In further replication of human disease, we also co-cultured MM1.S or MM1.R cells with human bone marrow stromal cells (BMSCs) which support myeloma cell growth by promoting an immunosuppressive microenvironment within the BM. Importantly, mAb 1610-dependent cytotoxicity against MM1.S or MM1.R cells was not attenuated by the co-presence of BMSCs. Similarly, IL-6 (10 ng/ml) did not significantly affect mAb 1610-induced ADCC activity, indicating a mechanism of action that can overcome growth promotion, immune suppression, and drug resistance conferred by the tumor promoting BM microenvironment. Taken together, these in vitro studies further demonstrate as a proof-of-concept the use of an antibody CD138 targeting strategy mediated through an immune based mechanism of myeloma plasma cell killing. Based on these results, optimization and further biological characterization of chimeric mAb 1610 in advance of pre-clinical studies is anticipated. Disclosures Myette: Visterra Inc.: Employment. Chaganty:Visterra Inc.: Employment. Adari:Visterra Inc.: Employment. Tissire:Visterra Inc.: Employment. Deotale:Visterra Inc.: Employment. Shriver:Visterra Inc.: Employment. Munshi:OncoPep: Other: Board of director. Anderson:Millennium Takeda: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; OncoPep: Equity Ownership, Other: Scientific founder; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Celgene: Consultancy; Bristol Myers Squibb: Consultancy.

Multiple myeloma (MM) is a plasma cell malignancy characterized by clinical and genomic heterogeneity. Recurrent IgH translocations, copy number abnormalities and somatic mutations have been reported to participate in myelomagenesis; however no universal driver of the disease has been identified. Here, we hypothesize that transcriptional deregulation is critical for MM pathogenesis and the maintenance of the MM cell state. In order to capture signatures of transcription factor engagement with the myeloma epigenome, we performed the assay for transposase-accessible chromatin sequencing (ATAC sequencing), deep RNA sequencing in 23 primary myeloma samples and 5 normal plasma cell samples (NPC) from healthy donors along with whole genome sequencing and H3K27ac ChIP-seq in a cohort of these primary MM samples. We identified 22,603 variable accessible loci between MM and NPC and correlated impact of these on expression of associated genes using RNA-seq data. Together with robust differential analysis of open chromatin regions and nuclease-accessibility footprints to identify discrete transcription factor binding events, we have discerned the myeloma-specific open chromatin landscape, identified transcription factor dependencies and potential new myeloma drivers. In our dataset we observe a vast number of loci with heterogeneous chromatin states across the sample cohort, and the majority of the open chromatin sites identified are unique to a single sample. However, distinct variable chromatin accessibility signatures indicative of the MM chromatin state when compared to normal plasma cells were observed. Remarkably, we observed more frequent recurrent loss of variable accessible loci compared to gains. In addition, specific open chromatin profiles evident in hyperdiploid and non-hyperdiploid MM were also identified. Accessibility footprinting revealed MM-specific enrichment for transcription factors known to be essential for MM cell survival including Interferon Regulatory Factors (IRFs), Nuclear Factor Kappa B (NFkB), Ikaros, and Sp1. Interestingly, we also identify the myocyte enhancer factor 2 (MEF2) family of transcription factors as being specifically enriched in open chromatin regions in MM cells. Using a CRISPR-Cas9 knockout system, we identify the MEF2 family member MEF2C as essential for MM cell proliferation and survival. MEF2C is significantly overexpressed at the RNA level in our study as well as in several independent cohorts and is a central enhancer-localized transcription factor in MM core regulatory circuitry as determined by H3K27ac ChIP-sequencing profiles of primary MM samples. In order to evaluate MEF2C as a therapeutic target, we used small molecule inhibitors targeting MEF2C activity via inhibition of MEF2C phosphorylation using inhibitors of salt-induced kinases (SIK) and microtubule-associated protein/microtubule affinity regulating kinases (MARK). SIK/MARK have been described to specifically activate MEF2C. SIK and MARK inhibition resulted in both dose- and time-dependent inhibition of MM cell growth and survival in a panel of 12 MM cell lines with various genotypic and phenotypic characteristics, revealing a potential approach to targeting the dysregulated gene regulatory state of myeloma. To conclude, here we identify here an altered chromatin accessibility landscape in multiple myeloma that likely contributes to oncogenic transcription states through the activity of transcription factors such as MEF2C, representing a new MM dependency and potential therapeutic target. Disclosures Anderson: Millennium Takeda: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; OncoPep: Equity Ownership, Other: Scientific founder. Young:Camp4 Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Omega Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Munshi:OncoPep: Other: Board of director.

The bone marrow (BM) microenvironment consists of extracellular-matrix and the cellular compartment including immune cells. Multiple myeloma (MM) cell and BM accessory cell interaction promotes MM survival via both cell-cell contact and cytokines. Immunomodulatory agents (IMiDs) target not only MM cells, but also MM cell-immune cell interactions and cytokine signaling. Here we examined the in vitro effects of IMiDs on cytokine signaling triggered by interaction of effector cells with MM cells and BM stroma cells. IMiDs diminished interleukin-2, interferonγ, and IL-6 regulator suppressor of cytokine signaling (SOCS)1 expression in immune (CD4T, CD8T, natural-killer T, natural-killer) cells from both BM and PB of MM patients. In addition, coculture of MM cells with healthy PBMCs induced SOCS1 expression in effector cells; conversely, treatment with IMiDs down-regulated the SOCS1 expression. SOCS1 negatively regulates IL-6 signaling and is silenced by hypermethylation in MM cells. To define the mechanism of inhibitory-cytokine signaling in effector cells and MM cells, we next analyzed the interaction of immune cells with MM cells that were epigenetically modified to re-express SOCS1; IMiDs induced more potent CTL responses against SOCS1 re-expressing–MM cells than unmodified MM cells. These data therefore demonstrate that modulation of SOCS1 may enhance immune response and efficacy of IMiDs in MM.

A proliferation inducing ligand (APRIL), abundant in serum samples of multiple myeloma (MM) patients, promotes MM cell progression in vivo predominantly via MM-specific receptor B cell maturation antigen (BCMA) (Blood. 2016; 127:3225). Here, we study whether APRIL directly regulate non-MM subpopulations to influence immunosuppressive bone marrow (BM) milieu and further define molecular mechanisms regulating these processes. First, transmembrane activator and calcium modulator (TACI) protein, the other APRIL receptor expressed at lower levels than BCMA on MM cell membrane, is consistently expressed at higher levels on CD4+CD25highFoxP3high T regulatory (Treg) vs autologous CD4+CD25- conventional T cells (Tcon) from MM patients (n=10, p10-fold in purified Tregs vs autologous Tcon (n=12, p Citation Format: Yu-Tzu Tai, Liang Lin, Liji Xing, Liji Xing, Kenneth Wen, Nikhil Munshi, Paul Richardson, Kenneth Anderson. A proliferation-inducing ligand (APRIL) directly impacts immune regulatory subsets to regulate suppressive multiple myeloma bone marrow microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3832.