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PSMD4/Rpn10 is a subunit of the 19S proteasome unit that is involved with feeding target proteins into the catalytic machinery of the 26S proteasome. Since proteasome inhibition is a common therapeutic strategy in multiple myeloma (MM), we investigated Rpn10 and found that it is highly expressed in MM cells versus normal plasma cells. Rpn10 levels inversely correlated with overall survival in MM patients. Inducible knockout or knockdown of Rpn10 decreased MM cell viability both in vitro and in vivo by triggering the accumulation of polyubiquitinated proteins, cell cycle arrest, and apoptosis associated with activation of caspases and unfolded protein response-related pathways. Proteomic analysis revealed that inhibiting Rpn10 increased autophagy, antigen presentation and the activation of CD4+ T and NK cells. We developed an in vitro AlphaScreen binding assay for high-throughput screening and identified a novel Rpn10 inhibitor, SB699551 (SB). Treating MM cell lines, leukemic cell lines, and primary MM patient cells with SB decreased cell viability without affecting the viability of normal PBMCs. SB inhibited the proliferation of MM cells even in the presence of the tumor-promoting bone marrow milieu and overcame proteasome inhibitor (PI)-resistance without blocking the 20S proteasome catalytic function or the 19S deubiquitinating activity. Rpn10 blockade by SB triggered MM cell death via similar pathways as the genetic strategy. In MM xenograft models, SB was well-tolerated, inhibited tumor growth, and prolonged survival. Our data suggests that inhibiting Rpn10 will enhance cytotoxicity and overcome PI-resistance in MM, providing the basis for further optimization studies of Rpn10 inhibitors for clinical application.

Long noncoding RNAs (lncRNAs) can drive tumorigenesis and are susceptible to therapeutic intervention. Here, we used a large-scale CRISPR interference viability screen to interrogate cell-growth dependency to lncRNA genes in multiple myeloma (MM) and identified a prominent role for the miR-17-92 cluster host gene (MIR17HG). We show that an MIR17HG-derived lncRNA, named lnc-17-92, is the main mediator of cell-growth dependency acting in a microRNA- and DROSHA-independent manner. Lnc-17-92 provides a chromatin scaffold for the functional interaction between c-MYC and WDR82, thus promoting the expression of ACACA, which encodes the rate-limiting enzyme of de novo lipogenesis acetyl-coA carboxylase 1. Targeting MIR17HG pre-RNA with clinically applicable antisense molecules disrupts the transcriptional and functional activities of lnc-17-92, causing potent antitumor effects both in vitro and in vivo in 3 preclinical animal models, including a clinically relevant patient-derived xenograft NSG mouse model. This study establishes a novel oncogenic function of MIR17HG and provides potent inhibitors for translation to clinical trials.

The multiple myeloma (MM) therapeutic landscape has evolved significantly with the approval of numerous novel agents, including next generation proteasome inhibitors (PIs), immunomodulatory agents (IMIDs), and monoclonal antibodies (MoABs) targeting CD38 and SLAMF7. While these discoveries have led to an unprecedented improval in patient outcomes, the disease still remains incurable. Immunotherapeutic approaches have shown substantial promise in recent studies of chimeric antigen receptor T-cell (CAR T-cell) therapy, bispecific antibodies, and antibody drug conjugates targeting B-cell maturation antigen (BCMA). This review will highlight these novel and targeted therapies in MM, with particular focus on PIs, IMIDs, MoAb and BCMA-directed immunotherapy.

The accessibility of cell surface proteins makes them tractable for targeting by cancer immunotherapy, but identifying suitable targets remains challenging. Here we describe plasma membrane profiling of primary human myeloma cells to identify an unprecedented number of cell surface proteins of a primary cancer. We used a novel approach to prioritize immunotherapy targets and identified a cell surface protein not previously implicated in myeloma, semaphorin-4A (SEMA4A). Using knock-down by short-hairpin RNA and CRISPR/nuclease-dead Cas9 (dCas9), we show that expression of SEMA4A is essential for normal myeloma cell growth in vitro, indicating that myeloma cells cannot downregulate the protein to avoid detection. We further show that SEMA4A would not be identified as a myeloma therapeutic target by standard CRISPR/Cas9 knockout screens because of exon skipping. Finally, we potently and selectively targeted SEMA4A with a novel antibody–drug conjugate in vitro and in vivo.

Purpose: Most data on the prognostic impact of cytogenetic abnormalities come from the setting of newly diagnosed multiple myeloma, it remains unclear whether the dynamic changes of cytogenetic aberrations affect the prognostic evaluation in multiple myeloma. Methods: We analyzed the prognostic impact of dynamic changes of cytogenetic abnormalities with a cohort consisting of 80 paired patients with consecutive cytogenetic data both at diagnosis and recurrence among 568 patients with newly diagnosed multiple myeloma. Results : Three patterns of recurrence were established from 80 paired patients: Pattern A (40%) consisted of 32 patients without new cytogenetic abnormalities at the time of progression. Pattern B (15%) consisted of 12 patients harboring new standard risk (SR) cytogenetic aberrations. Pattern C (45%) consisted of 36 patients with new high-risk (HR) cytogenetic abnormalities. The median overall survival (mOS; P Conclusions: Dynamic cytogenetic changes significantly affected prognostic evaluation in multiple myeloma. Patients harboring new HR cytogenetic abnormalities and escalated genetic risk stratification at relapse had worse outcomes. Figure 1 Disclosures Anderson: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.

Clonal evolution drives tumor progression, dissemination, and relapse in multiple myeloma (MM), with most patients dying of relapsed disease. This multistage process requires tumor cells to enter the circulation, extravasate, and colonize distant bone marrow (BM) sites. Here, we developed a fluorescent or DNA-barcode clone-tracking system on MM PrEDiCT (progression through evolution and dissemination of clonal tumor cells) xenograft mouse model to study clonal behavior within the BM microenvironment. We showed that only the few clones that successfully adapt to the BM microenvironment can enter the circulation and colonize distant BM sites. RNA sequencing of primary and distant-site MM tumor cells revealed a progression signature sequentially activated along human MM progression and significantly associated with overall survival when evaluated against patient data sets. A total of 28 genes were then computationally predicted to be master regulators (MRs) of MM progression. HMGA1 and PA2G4 were validated in vivo using CRISPR-Cas9 in the PrEDiCT model and were shown to be significantly depleted in distant BM sites, indicating their role in MM progression and dissemination. Loss of HMGA1 and PA2G4 also compromised the proliferation, migration, and adhesion abilities of MM cells in vitro. Overall, our model successfully recapitulates key characteristics of human MM disease progression and identified potential new therapeutic targets for MM.

Isatuximab is a monoclonal antibody targeting the transmembrane receptor and ectoenzyme CD38, a protein highly expressed on hematological malignant cells, including those in multiple myeloma (MM). Upon binding to CD38-expressing MM cells, isatuximab is thought to induce tumor cell killing via fragment crystallizable (Fc)-dependent mechanisms, including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), as well as via direct Fc-independent mechanisms. Here, these mechanisms of action were investigated in MM and diffuse large B-cell lymphoma (DLBCL) cell lines, as well as in peripheral blood mononuclear cells derived from healthy donors, and in MM patient-derived samples. Our findings show that isatuximab-mediated cytotoxicity occurred primarily via ADCC and ADCP in MM cell lines and via ADCC and apoptosis in DLBCL cell lines expressing high levels of CD38. We identified the programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) pathway and MM cell-secreted transforming growth factor-beta (TGF-β) as tumor cell-related features that could suppress CD38-mediated ADCC. Furthermore, we established that isatuximab can directly activate natural killer (NK) cells and promote NK cell-mediated cytotoxicity via crosslinking of CD38 and CD16. Finally, isatuximab-induced CDC was observed in cell lines with high CD38 receptor density (>250,000 molecules/cell) and limited expression of inhibitory complement regulatory proteins (CD46, CD55, and CD59

To identify therapeutically actionable genetic dependencies we have pursued various approaches to derive a deeper understanding of the oncogenic hallmarks of myelomagenesis. We have studied the long noncoding RNA (lncRNA) landscape in multiple myeloma (MM) and identified a large number of differentially expressed lncRNAs in MM versus normal plasma cells. These lncRNAs presumably drive the tumorigenesis and MM cell growth, and in turn be susceptible to therapeutic intervention. To this end, we have developed and utilized a CRISPR interference (CRISPRi)-based platform for decoding and targeting the lncRNA dependencies (LongDEPs) in MM. In this study, we have used RNA-seq of patient-derived CD138+ MM cells (n=360) and MM cell lines (n=70) to generate a priority list of 913 expressed intergenic lncRNAs. Then, to systematically interrogate the role of these lncRNAs in MM cell growth, we have performed a CRISPRi viability screen transducing 3 MM cell lines engineered to express a dCAS9-KRAB fusion protein, with a pooled library consisting of 7 sgRNAs against each of the 913 transcription start sites (TSS) and 576 negative control sgRNAs. Relative representation of sgRNAs was assessed by deep sequencing after 3 weeks and analyzed using the MAGeCK robust rank aggregation (RRA) algorithm. The most enriched or depleted sgRNAs were further tested in a secondary CRISPRi viability screen. Focusing on depleted sgRNAs, we have identified >30 unique LongDEPs; which were further validated via an antisense oligonucleotide (ASO)-based loss-of-function study in a panel of MM cell lines (n=11). A comparative transcritpomic analysis comparing data from 360 newly-diagnosed and clinically-annotated MM patients and 16 healthy donors showed significant upregulation of these LongDEPs in MM patient cells. Of note, specific longDEPs were found selectively upregulated in genetically-defined patient subsets, including high-risk MM carrying t(4;14), 1q gain or del17p. Moreover, at least 18 LongDEPs were identified as independent risk-predictors of clinical outcome in newly-diagnosed MM patients. The lncRNA RROL was identified as a leading LongDEP, with a dependency score on a par with positive controls such as IRF4 or MYC. This lncRNA is specifically overexpressed in MM patients after disease relapse, and its higher expression in newly diagnosed MM patients could predict a worse clinical outcome. We have validated the essential role of RROL in support of the proliferation and survival of MM cells both in vitro and in vivo in NOD SCID mice, using ASO-based loss-of-function studies. To explain this effect, we have characterized its role in the control of the pro-survival de novo lipogenesis (DNL) pathway via an unbiased lipid profiling and by measuring the incorporation of C 14-radiolabeled glucose into the lipid pool. Mechanistically, we have shown that RROL promotes the DNL pathway via transcriptional regulation of rate-limiting enzymes including ACC1. Using in vitro (RNA protein pull down) and in cellulo (RNA yeast-3-hibrid) assays, we have identified the transcription factor c-MYC as a relevant protein interactor of RROL. This interaction occurs at the chromatin level and is required for i) MYC occupancy at DNL gene loci (e.g. ACC1), as shown by both ChIP-qPCR and single molecule dual RNA FISH coupled with immunofluorescence; ii) MYC interaction with a number of transcriptional co-activators, including WDR82, as assessed in vitro in 3 MM cell lines using co-immunoprecipitation followed by Mass spectrometry (Co-IP/MS) and in cellulo using the proximity-dependent biotin identification assay (BioID) in Flp-In T-REx cells expressing a FLAG-BirA*-MYC fusion protein. Overall, our data indicate that RROL provides the chromatin scaffold to assemble a transcriptionally activated ribonucleoprotein complex - minimally composed by RROL, MYC and WDR82 - at gene regulatory loci of DNL rate-limiting enzymes. To develop therapeutic inhibitors of LongDEPs, starting with RROL, we have tested >70 ASOs following a multi-step screening approach. The anti-MM activity of 2 leading compounds was demonstrated in vitro and in vivo in 2 clinically relevant animal models, including a BLI-based orthotopic model. In conclusion, our work establish LongDEPs as an additional source of genetic dependencies in MM paving the way for their biologic, clinical and therapeutic characterization in this disease context. Disclosures Young: Dewpoint: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Camp4 Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Membership on an entity's Board of Directors or advisory committees; Omega Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Gryaznov: MAIA Therapeutics: Current Employment. Anderson: Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Munshi: Novartis: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Celgene: Consultancy; Adaptive Biotechnology: Consultancy; Takeda: Consultancy; Karyopharm: Consultancy; Legend: Consultancy; Abbvie: Consultancy; Pfizer: Consultancy; Bristol-Myers Squibb: Consultancy.

Introduction: Multiple Myeloma (MM) is an incurable plasma cell malignancy commonly preceded by the asymptomatic stage smoldering multiple myeloma (SMM). MM is characterized with significant genomic heterogeneity of chromosomal gains and losses (CNVs), translocations, and point mutations (SNVs); alterations that are also observed in SMM patients. However, current SMM risk models rely solely on clinical markers and do not accurately capture progression risk. While incorporating some genomic biomarkers improves prediction, using all MM genomic features to comprehensively stratify patients may increase risk stratification precision in SMM. Methods: We obtained a total of 214 patient samples at SMM diagnosis. We performed whole-exome sequencing on 166 tumors; of these, RNA sequencing was performed on 100. Targeted capture was done on 48 additional tumors. Upon binarization of DNA features, we performed consensus non-negative matrix factorization to identify distinct molecular clusters. We then trained a random forest classifier on translocations, SNVs, and CNVs. The predicted clinical outcomes for the molecular subtypes were further validated in an independent SMM cohort of 74 patients. Results: We identified six genomic subtypes, four with hyperdiploidy (>48 chromosomes, HMC, HKR, HNT, HNF) and two with IgH translocations (FMD, CND) (Table 1). In multivariate analysis accounting for IMWG (20-2-20) clinical risk stages, high-risk (HMC, FMD, HKR) and intermediate-risk (HNT, HNF) genetic subtypes were independent predictors of progression (Hazards ratio [HR]: 3.8 and 5.5, P = 0.016 and 0.001, respectively). The low-risk, CND subtype harboring translocation (11;14) was enriched for the previously defined CD-2 MM signature defined by the B cell markers CD20 and CD79A (FDR = 0.003 ), showed upregulation of CCND1, E2F1, and E2F7 (FDR = 0.01, 0.0004, 0.08), and was enriched for G2M checkpoint, heme metabolism, and monocyte cell signature (FDR = 0.003, 0.003, 0.003, respectively). The FMD subtype with IgH translocations (4;14) and (14;16) was enriched for P53, mTORC1, unfolded protein signaling pathways and plasmacytoid dendritic cell signatures (FDR = 0.01, 0.005, 0.008, respectively). The HKR tumors were enriched for inflammatory cytokine signaling, MYC target genes, T regulatory cell signature, and the MM proliferative (PR) signatures (FDR = 0.02, 0.03, 0.007, 0.02, respectively). The APOBEC mutational signature was enriched in HMC and FMD tumors (P = 0.005), while there was no statistical difference across subtypes in the AID signature. The median follow-up for the primary cohort is 7.1 years. Median TTP for patients in HMC, FMD, and HKR was 3.8, 2.6, and 2.2 years, respectively; TTP for HNT and HNF was 4.3 and 5.2, respectively, while it was 11 years in CND patients (P = 0.007). Moreover, by analyzing the changes in MM clinical biomarkers over time, we found that patients from high-risk subgroups had higher odds of developing evolving hemoglobin and monoclonal protein levels over time (P = 0.01 and 0.002, respectively); Moreover, the absolute increase in M-protein was significantly higher in patients from the high-risk genetic subtypes at one, two, and five years from diagnosis (P = 0.001, 0.03, and 0,01, respectively). Applying the classifier to the external cohort replicated our findings where intermediate and high-risk genetic subgroups conferred increased risk of progression to MM in multivariate analysis after accounting for IMWG staging (HR: 5.5 and 9.8, P = 0.04 and 0.005, respectively). Interestingly, within the intermediate-risk clinical group in the primary cohort, patients in the high-risk genetic subgroups had increased risk of progression (HR: 5.2, 95% CI 1.5 - 17.3, P = 0.007). In the validation cohort, these patients also had an increased risk of progression to MM (HR: 6.7, 95% CI 1.2 - 38.3, P = 0.03), indicating that molecular classification improves the clinical risk-stratification models. Conclusion: We identified and validated in an independent dataset six SMM molecular subgroups with distinct DNA alterations, transcriptional profiles, dysregulated pathways, and risks of progression to active MM. Our results underscore the importance of molecular classification in addition to clinical evaluation in better identifying high-risk SMM patients. Moreover, these subgroups may be used to identify tumor vulnerabilities and target them with precision medicine efforts. Figure 1 Figure 1. Disclosures Bustoros: Janssen, Bristol Myers Squibb: Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria. Casneuf: Janssen: Current Employment. Kastritis: Amgen: Consultancy, Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Genesis Pharma: Honoraria; Janssen: Consultancy, Honoraria, Research Funding. Walker: Bristol Myers Squibb: Research Funding; Sanofi: Speakers Bureau. Davies: Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Dimopoulos: Amgen: Honoraria; BMS: Honoraria; Takeda: Honoraria; Beigene: Honoraria; Janssen: Honoraria. Bergsagel: Genetech: Consultancy, Honoraria; Oncopeptides: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Patents & Royalties: human CRBN mouse; GSK: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Yong: BMS: Research Funding; Autolus: Research Funding; Takeda: Honoraria; Janssen: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; GSK: Honoraria; Amgen: Honoraria. Morgan: BMS: Membership on an entity's Board of Directors or advisory committees; Jansen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees. Getz: IBM, Pharmacyclics: Research Funding; Scorpion Therapeutics: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Ghobrial: AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, Curio Science, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, GNS, GSK: Consultancy.

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that charge tRNAs with their cognate amino acids. Recent studies have revealed that the expression of aaRSs is dynamically altered in response to cellular stresses, and that specific isoforms play pivotal roles in carcinogenesis and tumor progression. Despite emerging interest in aaRSs in cancer, the biologic sequelae of aaRSs in multiple myeloma (MM) have not yet been elucidated. We first examined RNA expression levels of aaRSs in tumor cells from MM patients. Gene expression analyses revealed that most aaRSs were upregulated in MM patients compared to healthy donors. Importantly, expression of 4 aaRSs, including glutamyl-prolyl-tRNA synthetase (EPRS), correlated with worse clinical prognosis: high EPRS expression correlated with disease progression (GSE6477; P< 0.001) and shorter survival (CoMMpass; P = 0.007). Consistent with clinical data, ERPS knockdown in MM cells (AMO1, RPMI 8226, and MM.1S) triggered significant cell growth inhibition, suggesting that EPRS is a growth and survival factor in MM cells. Halofuginone (HF) is an inhibitor of the prolyl-tRNA-synthetase (PRS) activity of EPRS and potently inhibits MM growth. However, HF is proline (Pro) competitive and increased levels of Pro abrogates the effect of HF, including the Pro-rich tumor microenvironment. To overcome this limitation, we developed a novel ATP competitive and Pro non-competitive PRS inhibitor, NCP26. NCP26 potently decreased growth in MM cell lines, including drug (Dox, Dex, PI, IMiDs) resistant isogenic derivatives, and patient MM cells (EC50 ≈ 500 nM), without significant toxicity to peripheral blood mononuclear cells. Neither anti-apoptotic cytokines (i.e., IL-6) nor co-culture with bone marrow (BM) stromal cells abrogated NCP26-induced MM cell death. Importantly, unlike HF, NCP26-induced cell growth inhibition was not altered by an excess of exogenous Pro. Since aaRSs inhibition results in accumulation of uncharged tRNAs sensed by the amino acid response (AAR), we next examined whether NCP26 activates the AAR pathway in MM cells. As expected, NCP26 increased both p-GCN2 and p-eIF2α, associated with upregulation of ATF4 and CHOP expression. Moreover, NCP26 treatment induced G0/G1 cell cycle arrest followed by apoptotic cell death, evidenced by cleavage of caspases and PARP. We also evaluated in vivo efficacy of NCP26 in the AMO1 xenograft mouse model: NCP26 (10 mg/kg, q.d.) significantly inhibited tumor growth (P = 0.005) and prolonged host survival (P < 0.001) compared to control, without affecting body weight. To further characterize the biological impact of NCP26 in MM cells, we performed single-cell RNA-seq using BM samples from MM patients. While both HF and NCP26 treatment reduced MM, NCP26 triggered more sustained and robust activation of integrated stress response, evidenced by induction of GCN2, CHOP, and IRE1, compared to HF. Gene set enrichment analysis also revealed significant alteration in chromatin processes, besides proteostasis, such as translation, signal peptide, and stress response, after NCP26 treatment. Since previous studies suggested that translation of genes bearing high Pro-Pro (PP) motifs is more susceptible to EPRS inhibition than non-PP-bearing genes, we next performed a quantitative proteomic analysis in AMO1 cells treated with NCP26: of 7,366 proteins, 84 were upregulated and 363 were downregulated after 6-hour treatment. We also performed in silico bioinformatic analysis to uncover PP-bearing genes, and incorporated CRISPR-screening data to identify genetic dependencies in MM. By overlapping the three datasets, we identified a group of 55 PP motif-containing proteins which play essential roles in MM cell pathogenesis, including MYC, PIM2, and CCND1/2. We also confirmed that PP motif-containing proteins were significantly downregulated by NCP26 treatment in MM cells both in vitro and in vivo. In conclusion, our results identify EPRS as a biomarker of poor prognosis and a novel therapeutic target in MM. NCP26, a novel ATP-competitive and Pro non-competitive EPRS inhibitor, induces MM cytotoxicity both in vitro and in vivo by triggering AAR and EPRS-Pro-rich proteins pathways. Our data provide the preclinical rationale for development of NCP26-based clinical candidates to improve patient outcomes in MM. Disclosures Gooding: Bristol Myers Squibb: Research Funding. Hagner: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Ramasamy: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; Celgene (BMS): Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive biotech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Immune therapies including CAR T cells and bispecific T cell engagers are demonstrating remarkable efficacy in relapsed refractory myeloma (MM). In this context, we have recently shown that proteasome inhibitor bortezomib (BTZ) results in immunogenic cell death (ICD) and in a viral mimicry state in MM cells, allowing for immune recognition of tumor cells. Induction of a robust anti-MM immune response after BTZ was confirmed both in vitro and in vivo: treatment of 5TGM1 MM cells with BTZ induced tumor regression associated with memory immune response, confirmed by ELISPOT of mouse splenocytes. We have confirmed the obligate role of calreticulin (CALR) exposure in phagocytosis and the ICD process, since BTZ-induced ICD is impaired in CALR KO MM cells both in vitro and in vivo. We further showed that the therapeutic efficacy of BTZ in patients was correlated with ICD induction: BTZ-induced ICD signature was positively correlated with OS (p=0.01) in patients enrolled in the IFM/DFCI 2009 study. Together, these studies indicate that ICD is associated with long-term response after BTZ treatment. In this work, we reasoned that genomic or transcriptomic alterations associated with shorter survival of MM patients after BTZ treatment may impair activation of the ICD pathway. To this aim, we performed a transcriptomic analysis of purified CD138+ cells from 360 newly diagnosed, clinically-annotated MM patients enrolled in the IFM/DFCI 2009 study. By focusing on genes involved in the ICD process, we found that low levels of GABA Type A Receptor-Associated Protein (GABARAP) were associated with inferior clinical outcome (EFS, p=0.0055). GABARAP gene locus is located on chr17p13.1, a region deleted in high risk (HR) MM with unfavorable prognosis. Remarkably, we found that correlation of low GABARAP levels with shorter EFS was significant (p=0.018) even after excluding MM patients with del17p; and GABARAP is therefore an independent predictor of clinical outcome. GABARAP is a regulator of autophagy and vesicular trafficking, and a putative CALR binding partner. Interestingly, among a panel of MM cell lines (n=6), BTZ treatment failed to induce exposure of CALR and MM cell phagocytosis by DCs in KMS11 cells, which carry a monoallelic deletion of GABARAP. This effect was rescued by stable overexpression of GABARAP. Moreover, CRISPR/Cas9-mediated KO of GABARAP in 3 ICD-sensitive cell lines (AMO1, H929, 5TGM1) abrogated CALR exposure and ICD induction by BTZ. GABARAP add-back by stable overexpression in KO clones restored both CALR exposure and induction of ICD, confirming GABARAP on-target activity. Similarly, pre-treatment of GABARAP KO cells with recombinant CALR restored MM phagocytosis, further confirming that GABARAP impairs ICD via inhibition of CALR exposure. Based on these findings, we hypothesized that GABARAP loss may alter the ICD pathway via CALR trapping, resulting in the ICD resistant phenotype observed in GABARAP null and del17p cells. To this end, we explored the impact of GABARAP KO on the CALR protein interactome, in the presence or absence of BTZ. Importantly, GABARAP KO produced a significant increase of CALR binding to stanniocalcin 1 (STC1), a phagocytosis checkpoint that mediates the mitochondrial trapping of CALR, thereby minimizing its exposure upon ICD. Consistently, GABARAP KO also affected CALR interactome in BTZ-treated cells, which was significantly enriched in mitochondrial proteins. Importantly, co-IP experiments confirmed GABARAP interaction with STC1. These data indicate a molecular scenario whereby GABARAP interacts with STC1 to avoid STC1-mediated trapping of CALR, allowing for the induction of ICD after treatment with ICD inducers; on the other hand, this mechanism is compromised in GABARAP null or del17p cells, and the STC1-CALR complex remains trapped in the mitochondria, resulting in ICD resistance. To functionally validate our findings in the context of the immune microenvironment, we performed mass Cytometry after T cell co-culture with DCs primed by both WT and GABARAP KO AMO1 clones. And we confirmed that treatment of GABARAP KO clones with BTZ failed to activate an efficient T cell response. In conclusion, our work identifies a unique mechanism of immune escape which may contribute to the poor clinical outcome observed in del17p HR MM patients. It further suggests that novel therapies to restore GABARAP may allow for the induction of ICD and improved patient outcome in MM. Disclosures Bianchi: Jacob D. Fuchsberg Law Firm: Consultancy; MJH: Honoraria; Karyopharm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Richardson: AstraZeneca: Consultancy; Regeneron: Consultancy; Protocol Intelligence: Consultancy; Secura Bio: Consultancy; GlaxoSmithKline: Consultancy; Sanofi: Consultancy; Janssen: Consultancy; Takeda: Consultancy, Research Funding; AbbVie: Consultancy; Karyopharm: Consultancy, Research Funding; Celgene/BMS: Consultancy, Research Funding; Oncopeptides: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding. Chauhan: C4 Therapeutics: Current equity holder in publicly-traded company; Stemline Therapeutics, Inc: Consultancy. Munshi: Legend: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Abbvie: Consultancy; Takeda: Consultancy; Adaptive Biotechnology: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Bristol-Myers Squibb: Consultancy. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Background: RAS/CDK-dependent pathways play essential roles in multiple myeloma (MM) pathogenesis. Targeting these pathways represents a novel therapeutic strategy in MM. Our ongoing studies (>420 patients) demonstrate that aberrantly spliced transcript expressions can predict MM patient survival outcomes better than gene expression alone, indicating a significant role of splicing mechanism in MM pathophysiology. These studies also identified intron retentions as the predominant recurrent alterations (~32% of spliced genes were retained introns) in MM. We evaluated splicing alterations associated with pathway-level responses after RAS/CDK inhibition in order to identify and validate novel molecular targets. Methods/results: MM cells were treated with selected Erk1/2 and CDK4/6 inhibitors (Ei, Ci) to inhibit RAS and CDK pathways. Our studies demonstrated strong synergistic (IC We next evaluated aberrantly spliced transcript expression in MM cells, with/without Erk1/2 knockdown (KD) or with Ei+Ci treatment. Unsupervised clustering of deregulated genes showed dose-dependent treatment effects. This observation was further supported by principal component analyses: upregulation in response to Erk1/2 KD and downregulation due to treatment with Ei+Ci were considered spliced gene-signatures linked to RAS/CDK modulation. Gene/pathway enrichment analyses of these genes showed their involvement in cell proliferation and regulation of epigenetic networks in MM. Importantly, these analyses suggest that overexpression of RAVER1/SNRPB core splicing regulator genes are associated with RAS/CDK pathway regulation. These genes encode subunits of U1/2/4/5 spliceosome complex and are involved in intron retention processes, a marker of malignant transformation. We compared signature-gene expressions from 558 MM patient samples to the signature-genes in plasma cells from normal donors and observed significant (p Conclusions: Our studies 1) show an association between RNA processing and RAS-CDK pathways in MM, 2) identify a core splicing protein, SNRPB/RAVER1, as a novel target for modulating this cascade, and 3) suggest that targeting spliceosome complexes represents a promising therapy in MM. Disclosures Letai: Zentalis Pharmaceuticals: Other: equity holding member of the scientific advisory board; Dialectic Therapeutics: Other: equity holding member of the scientific advisory board; Flash Therapeutics: Other: equity holding member of the scientific advisory board. Anderson: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Purpose: We study CD38 levels in immunosuppressive CD4+CD25highFoxp3+ regulatory T cells (Treg) and further define immunomodulating effects of a therapeutic CD38 mAb isatuximab/SAR650984 in multiple myeloma. Experimental Design: We evaluated percentages of CD38-expressing subsets in Tregs from normal donors and multiple myeloma patients. Peripheral blood mononuclear cells (PBMC) were then treated with isatuximab with or without lenalidomide or pomalidomide to identify their impact on the percentage and immunosuppressive activity of Tregs on CD4+CD25− T cells (Tcons). We investigated the mechanism of increased Tregs in multiple myeloma patients in ex vivo cocultures of multiple myeloma cells with PBMCs or Tcons. Results: CD38 expression is higher on Tregs than Tcons from multiple myeloma patients versus normal donors. CD38 levels and the percentages of CD38high Tregs are increased by lenalidomide and pomalidomide. Isatuximab preferentially decreases Treg and increases Tcon frequencies, which is enhanced by pomalidomide/lenalidomide. Isatuximab reduces Foxp3 and IL10 in Tregs and restores proliferation and function of Tcons. It augments multiple myeloma cell lysis by CD8+ T and natural killer cells. Coculture of multiple myeloma cells with Tcons significantly induces Tregs (iTregs), which express even higher CD38, CD25, and FoxP3 than natural Tregs. This is associated with elevated circulating CD38+ Tregs in multiple myeloma patients versus normal donors. Conversely, isatuximab decreases multiple myeloma cell- and bone marrow stromal cell–induced iTreg by inhibiting both cell–cell contact and TGFβ/IL10. Finally, CD38 levels correlate with differential inhibition by isatuximab of Tregs from multiple myeloma versus normal donors. Conclusions: Targeting CD38 by isatuximab can preferentially block immunosuppressive Tregs and thereby restore immune effector function against multiple myeloma. Clin Cancer Res; 23(15); 4290–300. ©2017 AACR.

Long noncoding RNA (lncRNAs) are key epigenetic factors that drive the origin and progression of human cancers via mechanisms that are largely unknown. We have previuosly reported the clinical significance of a lncRNA signature in multiple myeloma (MM) as independent risk predictor for clinical outcome; and recently identified a lncRNA RROL (RNA Regulator of Lipogenesis) with impact on MM cell proliferation. Here we describe a unique regulatory function for RROL in the control of gene networks involved in the de novo lipogenesis (DNL) pathway, ultimately impacting MM cell growth and survival. Based on growth and survival impact of RROL depletion, we performed integrated transcriptomic analysis of RNA-seq data after RROL depletion in MM cell lines and CD138+ patient MM cells, and identified a set of significantly modulated metabolic genes including the acetyl Co-A Carboxylase 1 (ACC1) gene, encoding a rate-limiting enzyme of the DNL pathway. This metabolic pathway converts nutrients into fatty acids serving for energy storage or biosynthesis of membranes and signaling molecules. Consistent with the transcriptional control of ACC1, we have observed that RROL inhibition in cell lines and primary MM cells significantly decreased the incorporation of C14-radiolabeled glucose into de novo synthesized lipids. Importantly, supplementation with exogenous palmitate, the main downstream product of DNL pathway, rescued the growth inhibitory effect of RROL depletion on MM cells, further confirming the importance of the DNL pathway in the oncogenic activity of RROL in MM. To understand the molecular mechanism through which RROL regulates ACC1 expression and its metabolic axis, we evaluated the RROL interactome in MM cells. RNA-Protein Pull Down (RPPD) and in vivo RNA yeast three-hybrid (Y3H) assays led to the identification of MYC as relevant direct partner of RROL. These results were further validated by qRT-PCR analysis of MYC-bound RNA obtained through RNA immunoprecipitation (RIP) assay. Using experimental model of conditional MYC KD (P493-6), we found that RROL exerts regulatory activity on ACC1 only in the presence of MYC. Mapping of MYC genomic occupancy by ChIP-seq and gene expression after MYC KD in MM cells revealed that ACC1 is a direct transcriptional target of MYC in cells expressing RROL. These data indicate that RROLand MYC cooperate in the transcriptional control of ACC1. Moreover, we found that RROL itself is transcriptionally regulated by MYC, suggesting the existence of a feed-forward regulatory loop in which MYC enhances the expression of RROL that, in turn, drives MYC transcriptional activity to ACC1. We hypothesized that RROL may shape the protein interacting network of MYC to confer specificity for ACC1 promoter. To this end, we performed mass spectrometry analysis of MYC interactome in three MM cell lines in the presence or in the absence of RROL and identified the transcriptional modulator WDR82 as RROL-dependent MYC partner. Interestingly, WDR82 directly interacts with RROL as assessed in the RPPD and RNA Y3H assays, and transcriptionally regulates ACC1 in RROL-dependent manner. These data indicate that RROL catalyzes the interaction of MYC with the transcriptional modulator WDR82 to form a transcriptional ternary complex regulating ACC1 expression. To therapeutically antagonize the RROL lipogenic signaling we have pre-clinically tested small molecule inhibitors of ACC1 (ACC1i). We have observed significant anti-MM activity of ACC1i in vitro in a large panel of MM cell lines and primary MM cells from patients; and in vivo in mouse models of human MM including the localized subcutaneous model and the disseminated model that establish a more aggressive systemic disease. Importantly, we have now developed clinically applicable ASOs and small molecule-like compounds to directly target RROL in MM cells. These studies are ongoing and will be presented. In conclusion, we here report a unique regulatory function of a novel lncRNA supporting MM cell growth via its control of the lipogenic metabolic axis. The availability of oral inhibitors of ACC1 as well as the ongoing development of RROL inhibitors may allow clinical application of this unique targeted therapy in MM. Disclosures Fulciniti: NIH: Research Funding. Chauhan:Oncopeptide AB: Consultancy; consultant to Stemline Therapeutics, Inc., and Equity owner in C4 Therapeutics.: Consultancy, Other: Equity owner in C4 Therapeutics.. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy.

Immunotherapeutically targeting CD138 has not been successfully translated into substantial clinical benefit in multiple myeloma (MM) patients. We here developed and determined in preclinical models of human MM the efficacy of VIS832, a novel humanized monoclonal antibody (MoAb) with differentiated CD138 target binding to the anti-CD138 MoAb BB4 within indatuximab ravtansine (BT082). Compared with BB4, VIS832 has significantly improved binding (P8). In the reporter-based antibody-dependent cellular cytotoxicity (ADCC) bioassay, EC50 of VIS832 was approximately 99.31 ng/ml (0.66 nM), whereas the BB4 possessing an isotype matched human IgG1 minimally induced ADCC against U266 MM cells. Evaluation of VIS832-induced ADCC in NK-MM cell co-cultures determined its EC50 values ranged from 2.22 + 0.37 to 15.3 + 2.71 ng/ml, with % maximal lysis of 37.06 + 1.45 % to 97.3 + 3.34 %, across tested MM cell lines (n=12), both sensitive or resistant to current therapies including dexamethasone, IMiDs, and bortezomib. While VIS832 effectively induced ADCC against parental cells with high and low CD138 levels, no lysis was detected in paired CD138-knockout MM cells. In the presence of BM stromal cells or osteoclasts, two key MM-supporting accessory cells in the BM milieu, VIS832-induced MM cytolysis was minimally affected. Importantly, VIS832 eliminated autologous MM cells of bone marrow (BM) samples from relapsed/refractory (RRMM) patients, with > 60% maximal lysis and > 90% killing in 4 out of 7 patient BM samples. EC50 values of VIS832 were 8.58-86.04 ng/ml in this patient cohort, comparable to those (17.28-179.3 ng/ml) of its non-humanized predecessor mAb 2810 against another RRMM patient cohort (n=6). In contrast to its potent autologous CD138+ patient cell lysis, VIS832 did not affect CD138-negative patient BM cells. VIS832 induced higher maximal lysis (~3-fold) of all target MM cell lines than CD38-targeting daratumumab (dara), regardless of resistance to lenalidomide and pomalidomide. These results also confirmed significantly higher CD138 vs CD38 levels in all MM cell lines and patient MM cells (P60 days (P Disclosures Chaganty: Visterra Inc: Current Employment. Ramakrishnan:Visterra Inc: Current Employment. Wollacott:Visterra Inc: Current Employment. Viswanathan:Visterra Inc: Current Employment. Adari:Visterra Inc: Current Employment. Munshi:Karyopharm: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy; Takeda: Consultancy; AbbVie: Consultancy. Babcock:Visterra Inc: Current Employment. Shriver:Visterra Inc: Current Employment. Myette:Visterra Inc: Current Employment. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

Immune escape underlies progression of disease and resistance to therapy in MM, and dysfunction of both innate and adaptive immunity highlight the urgent need for scientifically-informed strategies to restore anti-MM immunity and improve patient outcome. Here we demonstrate that proteasome inhibitor bortezomib (BTZ) modulates the immunosuppressive bone marrow milieu by inducing immunogenic cell death (ICD) and a type-I interferon (IFN) response which contribute to its clinical efficacy. Moreover, we identified STING as a mediator of its anti-MM immune response, and provide the preclinical rationale for clinical trials of BTZ-STING agonist combination therapy. We first show that BTZ induces hallmarks of ICD in both human and murine MM cell lines, including exposure of endoplasmic reticulum protein calreticulin (CALR), that functions as an "eat me signal". Specifically, co-culture with BTZ-treated MM cells induced functional maturation of dendritic cells (DCs) and enhanced uptake of BTZ-treated MM cells, assessed by flow cytometry and confocal microscopy. Notably, these functional sequelae were abrogated when DCs were co-cultured with CALRKO MM cells; and add-back experiments by stable overexpression of CALR in KO clones confirmed the specific role of CALRin BTZ-induced immunogenicity. We next validated these findings in 2 in vivo syngeneic models. First, we observed that anti-MM activity of BTZ resulted in more potent murine 5TGM1 tumor cell shrinkage in immunocompetent than immunodeficient hosts, an effect mediated by ICD induction since it was abrogated in immunocompetent mice bearing CALRKO tumors. Second, in vitro BTZ-treated 5TGM1 cells were used as a vaccine to induce a protective anti-MM immune response: vaccination protected against tumor growth upon rechallenge with 5TGM1 cells; conversely, palpable tumors were detected in non-vaccinated mice by 1 week. Moreover, vaccination with BTZ-treated CALRKO 5TGM1 cells abrogated the protective effect of BTZ since only 50% mice were tumor free by 30 days. Consistently, ELISPOT assay on mice splenocytes confirmed that vaccination with BTZ-treated 5TGM1 cells triggered a MM-specific T cell response. Next, we performed RNAseq analysis of BTZ-treated vs untreated tumors from both CALRWT and CALRKO cells growing in immunocompetent mice; and then carried out an integrative analysis of RNAseq data from clinically-annotated MM patients (n=327) uniformly treated with BTZ-based regimens (IFM/DFCI 2009). We identified a specific ICD signature induced by BTZ only in CALRWT tumors in mice; and importantly, we found that increased expression of the human orthologs of this ICD signature was strongly and positively correlated with clinical outcome (overall survival (OS), p value=0.01). Moreover, the predictive value of this signature was confirmed in an independent dataset of BTZ-treated patients (GSE9782) (OS p value=0.024). Gene ontology analysis of the ICD signature genes revealed enrichment in inflammatory response pathways, including IFN stimulated genes (ISGs). Using RNAseq analysis and qRT-PCR validation, we showed that MM cells demonstrate a type-I IFN response after BTZ treatment which contributes to its in vivo efficacy, since neutralization of type-I IFNs signaling in both MM and host cells with a type-I IFNs receptor 1 (IFNAR) blocking antibody significantly reduced BTZ anti-MM activity. Finally, we showed that BTZ increases genomic instability/micronuclei formation in MM cells and activates innate cGAS/STING immune pathway, thereby stimulating a type-I IFN response. Conversely, STINGKO in MM cells abrogated this effect and decreased BTZ-triggered anti-MM T cell response. Moreover, expression of the ISGs included in ICD signature was positively correlated with STING expression in MM patients. Importantly, pharmacological activation of STING with STING agonist induced potent anti-MM activity in vivo; and combination of STING agonist with BTZ further potentiated this in vivo anti-MM response, with increased T cell infiltration into retrieved tumors, evidenced by IHC analysis. In conclusion, our studies delineate a novel mechanism whereby BTZ triggers anti-MM immune responses, and show that STING agonists can enhance this response. These findings provide the framework for clinical evaluation of STING agonists in combination with BTZ to induce potent anti-MM immune responses and thereby improve patient outcome. Disclosures Fulciniti: NIH: Research Funding. Richardson:Celgene/BMS, Oncopeptides, Takeda, Karyopharm: Research Funding. Chauhan:Oncopeptide AB: Consultancy; consultant to Stemline Therapeutics, Inc., and Equity owner in C4 Therapeutics.: Consultancy, Other: Equity owner in C4 Therapeutics.. Munshi:Karyopharm: Consultancy; Takeda: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy. Anderson:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Celgene: Membership on an entity's Board of Directors or advisory committees.

Whole-genome sequencing analysis of newly diagnosed and relapsed multiple myeloma (MM) samples identified recurrent mutations in genes involved in the MAPK pathway, highlighting the potential of RAS/RAF/MEK/ERK signaling as a therapeutic target. Genomic studies identified translocations that involve IGH and set of partner genes MMSET, FGFR3, and CCND1 as primary events in MM. CDK4/CDK6 is overexpressed in MM, and CDK6 overexpression correlates with poor OS, suggesting that CDK4/6 are promising targets for MM therapy. Recent studies demonstrated synergistic activity of combined novel ERK1/2i inhibitor LY3214996 and CDK4/6i LY2835219 in solid tumors, but analogous studies have not been done in MM. Here we used preclinical models of MM to investigate inhibiting Erk1/2, CDK4/6, or both using ERK1/2i, CDK4/6i, or combination therapy. MM cell lines, RAS mutated or wild type (WT), were sensitive to ERK1/2i at IC50 To address the underlying mechanism of the synergism between Erk1/2i and CDK4/6i, we evaluated their cellular and transcriptional activity in MM cells. Gene expression profiling showed significant downregulation of RAS and CDK4/6 signaling pathway genes in MM cells as a result of ERK1/2i and CDK4/6i treatment at specific concentration ratios (3:1/1:3). Further evaluation of functional effects of ERK1/2i and CDK4/6i, alone or in combination, demonstrated that the synergistic effect of these inhibitors in MM cells is achieved through inhibition of p-S6, downregulation of c-myc, and correlate with ERK1/2i+CDK4/6i induced cell arrest in the G1 cell cycle phase. We noted increased ERK1/2 phosphorylation, which generally results in compensatory activation of parallel signaling pathways or in the loss of negative feedback. Regardless, ERK1/2i+CDK4/6i retained the inhibitory activity of the downstream signaling network, as demonstrated by the inhibition of cytoplasmic (p-RSK1) and nuclear (c-myc) targets of ERK at protein and mRNA levels. Treatment with ERK1/2i+CDK4/6i significantly decreased the levels of p-Rb and E2F1, downstream targets of CDK4/6. Recent studies shown that, in addition to cell cycle regulation, CDK4 and CDK6 induce tumorigenesis through regulation of inflammatory cytokines that are induced via NFκB pathway activation. CDK4/6i functional effects on MM cells cannot be limited to cell cycle arrest, CDK4/6i might also inhibit cytokines, which are produced in MM cells by NFκB activation. Overall, we shown that ERK1/2i+CDK4/6i induced cell proliferation and led to the key target molecule (p-c-myc, p-RSK, p-S6, p-RB, and E2F1) downregulations suggesting on-target activity of these inhibitors in MM cells. Importantly, our studies demonstrate strong synergistic anti-MM activity with ERK1/2+CDK4/6 therapy, providing a preclinical framework for clinical trials to improve patient outcome in MM. Disclosures Letai: Novartis: Research Funding; AbbVie: Consultancy; AstraZeneca: Consultancy; Zentalis: Membership on an entity's Board of Directors or advisory committees; Flash Therapeutics: Membership on an entity's Board of Directors or advisory committees; Dialectic: Membership on an entity's Board of Directors or advisory committees; Chugai: Other: Lecture Fees. Anderson:Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics..

Anti-CD38 monoclonal antibody (MoAb) treatments including daratumumab (DARA) are effective therapies for both newly diagnosed and relapsed multiple myeloma (MM). In this study, we examined the soluble factors that modulate CD38 expression and are associated with sensitivity to DARA-mediated antibody-dependent cellular cytotoxicity (ADCC) in the bone marrow (BM) microenvironment. Importantly, primary BM stromal cell (BMSC) culture supernatant (BMSC-sup) and interleukin-6 (IL-6) downregulated CD38 expression and reduced DARA-mediated ADCC. Both cytokine profiling of the BMSC-sup and genome-scale clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) knockout screening in MM cell lines identified and validated the JAK-STAT3 signaling pathway mediating CD38 downregulation, whereas the JAK-STAT1 pathway mediated CD38 upregulation. STAT3 knockdown abrogated BMSC-sup– and IL-6–induced CD38 downregulation on MM cell lines. We also confirmed that STAT3 and CD38 is negatively correlated in primary MM cells. To assess potential clinical relevance, pharmacological inhibition of the JAK-STAT pathway on BMSC-sup–induced CD38 downregulation was further examined. JAK inhibitor ruxolitinib inhibited STAT3 phosphorylation in MM cell lines, upregulated CD38 expression in MM cell lines and primary patient MM cells, and augmented DARA-mediated ADCC against MM cell lines. Taken together, our results suggest that CD38 expression on MM cells in the BM microenvironment is regulated by both STAT1 (positively) and STAT3 (negatively), and that inhibition of the JAK-STAT3 pathway represents a novel therapeutic option to enhance CD38 expression and anti-CD38 MoAb-mediated MM cytotoxicity.

High protein load is a feature of multiple myeloma (MM), making the disease exquisitely sensitive to proteasome inhibitor (PIs). Despite the success of PIs in improving patient outcome, the majority of patients develop resistance leading to progressive disease; thus, the need to investigate the mechanisms driving the drug sensitivity vs resistance. With the well-recognized chaperone function of 14-3-3 proteins, we evaluated their role in affecting proteasome activity and sensitivity to PIs by correlating expression of individual 14-3-3 gene and their sensitivity to PIs (bortezomib and carfilzomib) across a large panel of MM cell lines. We observed a significant positive correlation between 14-3-3ε expression and PI response in addition to a role for 14-3-3ε in promoting translation initiation and protein synthesis in MM cells through binding and inhibition of the TSC1/TSC2 complex, as well as directly interacting with and promoting phosphorylation of mTORC1. 14-3-3ε depletion caused up to a 50% reduction in protein synthesis, including a decrease in the intracellular abundance and secretion of the light chains in MM cells, whereas 14-3-3ε overexpression or addback in knockout cells resulted in a marked upregulation of protein synthesis and protein load. Importantly, the correlation among 14-3-3ε expression, PI sensitivity, and protein load was observed in primary MM cells from 2 independent data sets, and its lower expression was associated with poor outcome in patients with MM receiving a bortezomib-based therapy. Altogether, these observations suggest that 14-3-3ε is a predictor of clinical outcome and may serve as a potential target to modulate PI sensitivity in MM.

Purpose: The number and activity of osteoclasts (OCs) are strongly enhanced by myeloma cells, leading to significant bone lesion in patients with multiple myeloma (MM). Mechanisms remain elusive whether myeloma-supporting OCs also induce suppressive immune bone marrow (BM) microenvironment. Methods: We examined if OCs could modulate T cell-mediated anti-MM immunity; Inhibitory immune molecules expression during osteoclastogenesis; The impact of APRIL on PDL1 expression in MM and the pathway; The impact of Anti-CD38 mAb on osteoclastogenesis and T cell response; Results: Here, we first show that OCs significantly protect MM cells against T cell-mediated cytotoxicity via direct inhibition of proliferating CD4+and CD8+ T cells. The immune checkpoint molecules PDL1, Galectin-9, HVEM, and CD200, as well as T cell metabolism regulators indoleamine 2, 3-dioxygenase (IDO) and CD38 are significantly up-regulated during osteoclastogenesis. Importantly, levels of these molecules, except CD38, are higher in OCs than MM cells. Anti-PDL1 monoclonal antibody and IDO inhibitor partly overcome OC-inhibited T cell responses against MM cells, confirming their roles in OC-suppressed MM cell lysis by cytotoxic T cells. In addition, Galectin-9 and a proliferation-induced ligand (APRIL), mainly secreted by OCs, are significantly up-regulated during osteoclastogenesis. Galectin-9 specifically induces apoptosis of T cells while sparing monocytes and MM cells. APRIL induces PDL1 expression in MM cells, providing additional immune inhibition by OCs. Moreover, CD38 is significantly up-regulated during osteoclastogenesis. When targeted by an anti-CD38 monoclonal antibody, suppressive T cell function by OCs is alleviated, associated with down-regulation of HVEM and IDO. Conclusion: Taken together, these results define expression of multiple immune proteins and cytokines in OCs essential for suppressive MM BM milieu. These results further support combination of targeting these molecules to improve anti-MM immunity. Disclosures Anderson: Celgene: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Oncoprep: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

Patient-derived multiple myeloma (MM) cells are difficult to establish in culture or propagate in vivo in murine model. Here, we describe a zebrafish xenograft model that permits rapid, reliable growth of human MM cells injected into the perivitelline space of albino zebrafish (Casper) embryos 48 hours postfertilization. MM1S and MM1R MM cell lines and primary CD138(+) MM cells were stained with CM-Dil red fluorescent dye and suspended in Matrigel prior to their injection. The cells grew at the site of injection and disseminated throughout the developing embryos and larvae. Tumor size was quantified by fluorescent microscopy, and cell fate was followed for 4 days. All of the cell line xenografts showed responses similar to those previously observed with in vitro assays. CD138(+) plasma cell xenografts derived from MM patients also grew and were inhibited by the same drugs patients had responded to clinically. Using this technique, we can assess drug sensitivity or resistance with a small number of MM cells in a short period. This raises the possibility that one might be able to assess drug sensitivity in real time with readily obtainable clinical samples.

Multiple myeloma (MM) is characterized by a highly unstable genome, with aneuploidy observed in nearly all patients. The mechanism causing this karyotypic instability is largely unknown, but recent observations have correlated these abnormalities with dysfunctional DNA damage response. Here, we show that the NAD(+)-dependent deacetylase SIRT6 is highly expressed in MM cells, as an adaptive response to genomic stability, and that high SIRT6 levels are associated with adverse prognosis. Mechanistically, SIRT6 interacts with the transcription factor ELK1 and with the ERK signaling-related gene. By binding to their promoters and deacetylating H3K9 at these sites, SIRT6 downregulates the expression of mitogen-activated protein kinase (MAPK) pathway genes, MAPK signaling, and proliferation. In addition, inactivation of ERK2/p90RSK signaling triggered by high SIRT6 levels increases DNA repair via Chk1 and confers resistance to DNA damage. Using genetic and biochemical studies in vitro and in human MM xenograft models, we show that SIRT6 depletion both enhances proliferation and confers sensitization to DNA-damaging agents. Our findings therefore provide insights into the functional interplay between SIRT6 and DNA repair mechanisms, with implications for both tumorigenesis and the treatment of MM.

Dysregulated homologous recombination (HR) contributes to the acquisition of genomic changes and development of drug resistance over time in multiple myeloma (MM). We have now further investigated the molecular intermediates that may play an important role in HR process. We observe that ABL kinase, which regulates RAD51 through its phosphorylation, contributes to increased HR activity and genomic instability in myeloma. Moreover, Cellular response after DSBs involves nuclear re-localization of ABL1 upon DNA damage. A cellular localization of ABL1 has been confirmed in MM. Consistently, we observe that ABL kinase inhibitor reduces HR activity and genomic instability (as assessed by micronucleus assay) in MM cells. Based on our observation that melphalan increases genomic instability (as assessed by micronuclei assay and by whole genome sequencing), we sought to investigate impact of melpahalan on HR and role of ABL1 kinase in this process. We show that treatment with melphalan leads to increase in RAD51 expression and HR activity in MM cells in a dose-dependent manner. Evaluation by RNA sequencing showed that treatment of MM1S cells with melphalan is associated with upregulation of p53 signaling (containing multiple genes involved in DNA damage, detection of damaged sites, recombination/repair and genomic instability) as the topmost pathway. These findings suggest that melphalan-induced DNA damage leads to a concerted overexpression of genes involved in DNA damage response, recombination, genomic instability and chemoresistance. With the role of ABL1-kinase following DSB, we investigated and report that treatment with melphalan induces micronuclei formation, whereas nilotinib significantly reduces both the basal and melphlalan-induced micronuclei in all MM cell lines tested. We have now confirmed these observations by evaluating copy number alterations using single nucleotide polymorphism (SNP) arrays. To evaluate the impact of nilotinib, melphalan and their combination on genomic instability, we cultured MM cells in the presence of nilotinib (2.5 µM), melphalan (1 µM) and combination of both drugs for three weeks and investigated the acquisition of new copy number events, relative to "day 0" cells (serving as baseline genome), using SNP arrays. Treatment with melphalan led to massive increase in the acquisition of amplification and deletion events, whereas nilotinib not only reduced the acquisition of copy number events under spontaneous condition but also almost completely reversed/prevented those induced by melphlalan. Importantly, the treatment with nilotinib could also significantly sensitized MM cell lines and bone marrow plasma cells from relapsed MM patients to melphalan treatment. These data confirm that ABL1 inhibition reduces spontaneous and melphalan-induced genomic instability in MM cells. Since components of cell cycle play critical role in the maintenance of genome stability and growth, we investigated the impact of ABL1-inhibitor nilotinib, alone and in combination with melphalan, on cell cycle. Treatment of MM1S cells with melphalan for 48hrs led to 2.5-fold increase in the accumulation of cells in S-phase, suggesting an increase in replication stress by melphalan. Nilotinib reduced both the spontaneous and melphalan-induced fraction of S-phase cells by 27% (±2%) and 30% (±1%), respectively. Combined treatment with nilotinib and melphalan also increased sub G1 fraction of cells by 2.3-fold compared to those treated with melphalan alone, suggesting increased apoptosis in these cells. These data are consistent with our observation that treatment of MM cells with melphalan increase the phosphorylation of RPA32, a marker of replication stress while nilotinib reduces both the endogenous and melphalan-induced phosphorylated RPA32 level, suggesting that nilotinib might also be helpful in combating replication stress-mediated genomic instability. Taken together, these data demonstrate the critical role of ABL1-kinase in both spontaneous and drug (melphalan)-induced genomic instability and its inhibition could reduce/delay genomic evolution while enhancing cytotoxicity in multiple myeloma. Disclosures Fulciniti: NIH: Research Funding. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy.

Chimeric antigen receptor (CAR) T-cell therapy targeting B cell maturation antigen (BCMA) has provided deep (73% - 100%) responses in relapsed/refractory multiple myeloma (MM). However, median PFS has been less than 12 months, and amongst the small number of patients retreated at the time of progression with the same CAR T product, responses have been infrequent. This highlights development of resistance that may preclude effectiveness of the 2ndinfusion, and may also underly relapse following response to the initial CAR-T cell therapy. Here, we have investigated one of the resistance mechanisms using longitudinal single cell transcriptomic and bulk genomic analysis. This patient had relapsed/refractory IgG lambda MM with hypodiploidy and a complex karyotype with t(8;12) (q24;q14), clonal t(11;14) (q13;q32), and clonal deletion 13. Patient received 150x106CAR+ T cells (ide-cel) and achieved partial response, with duration of response of 8 months. The patient was retreated with 450 x106CAR+ T cells at relapse, but with no response. To delineate the resistance mechanism, we evaluated the bone marrow (BM) niche using 37658 cells from eight time points from before 1st CAR T cell infusion to 2 months after 2nd CAR T cell infusion, and identified 13 clusters consisting of hematopoietic cells and MM/plasma cells. Using RT-PCR based detection, we observed engineered CAR T cells only at 2 weeks after first infusion, when maximal CAR T cell expansion was observed. We did not observe infused CAR T cells with single cell RNAseq after 2ndinfusion, but a limited expansion was confirmed using RT-PCR.Re-clustering of the T cell cluster showed an increased proportion of CD4+ helper and T regulatory cells (Treg) 2 weeks after 1st infusion. In contrast, TREG proportion remained constant at the 2nd infusion, suggesting other causes for lack of expansion of CAR-T cells. We also did not identify any significant increase in the proportion of cells expressing immune check point inhibitory markers or in accessory cell types with immune inhibitory function in MM BM. Since we did not delinate a role of the BM milieu mediating suppression of CAR-T cell expansion and function following 2ndinfusion, we next explored tumor intrinsic factors. Soluble BCMA level (produced predominantly by MM cells) was high before the first CAR T cell infusion and dropped significantly to a very low level coinciding with the clinical response; however, it remained low even at the time of relapse with increase burden of MM, indicating a lack of BCMA production by MM cells. We therefore investigated genomic changes in MM cells at the time of relapse. Our single cell analysis of BM samples identified 3 samples (at the time of relapse and post 2ndCAR T cell infusion) with significant numbers of MM cells, evidenced by expression of CD138 and XBP1 (marker of plasma cells), CCND1 (upregulated in this patient with t(11;14)) and lack of RB1 (downregulated in this patient with del13). Imputation of copy number alterations scRNAseq showed that the majority of MM cells had a deletion of 16p, including the BCMA locus located on 16p13.13. We further validated these findings using deep whole exome sequencing (WES) of purified CD138+ cells collected after the second CAR T infusion. Before first CAR T cell infusion, 4% MM cells showed deletion 17p, while after second infusion both WES and scRNAseq prediction showed that del17p and del16p were clonal, and longitudinal scRNAseq analysis indicated that del17p and del16p co-occurred in the same clone. Moreover, WES identified a subclonal nonsense mutation (p.Q38*) in BCMA that creates an early stop codon in the BCMA gene. This biallelic BCMA deletion, acquired with one copy loss and a 2ndloss-of-function mutation, provides the molecular basis for lack of BCMA expression in MM cells at the time of relapse. Our data showed that both TP53 and BCMA had deletion in one allele and mutation in the second allele. These results identify biallelic loss of BCMA locus as a potential resistance mechanism to BCMA targeting therapy. Our results highlight the need to investigate sBCMA as a potential indicator of BCMA loss at relapse, and to carry out detailed transcriptomic or genomic analysis to confirm mutations. Moreover, these data also demonstrate the ability of MM cells to survive without BCMA expression. With the growing number of BCMA targeting therapeutic modalities under development, we would expect to see such occurrences more commonly in the future. Disclosures Fulciniti: NIH: Research Funding. Campbell:BMS: Current Employment, Current equity holder in publicly-traded company. Petrocca:bluebird, bio: Current Employment, Current equity holder in publicly-traded company. Hege:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Patents & Royalties: numerous, Research Funding; Celgene (acquired by Bristol Myers Squibb): Ended employment in the past 24 months; Mersana Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Arcus Biosciences (Former Board of Directors): Divested equity in a private or publicly-traded company in the past 24 months. Kaiser:BMS: Current Employment, Current equity holder in publicly-traded company. Anderson:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.. Munshi:C4: Current equity holder in private company; Legend: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy; Janssen: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy.

The development of novel agents including immunomodulatory drugs (IMiDs) lenalidomide (Len) and pomalidomide (Pom) has improved patient outcome in multiple myeloma (MM). Previous studies have shown that IMiDs directly bind cereblon (CRBN), a substrate adaptor of Cullin4 Ring Ligase (CRL4), and activate CRL4CRBN ligase, thereby selectively targeting two B cell transcription factors IKZF1 and IKZF3 for ubiquitylation and proteasomal degradation. We have shown that IMiDs also directly bind and inhibit TP53-regulating kinase activity, resulting in MM cell growth inhibition. Importantly, IMiDs have achieved additive and synergistic anti-MM activity when combined with proteasome inhibitors and monoclonal antibodies in vitro, and are now utilized in combinations to treat newly diagnosed MM. However, development of resistance to IMiDs commonly underlies relapse of disease. To delineate mechanisms of IMiDs resistance, the majority of previous studies have focused on CRBN. Although downregulation or mutations in CRBN can be associated with IMiDs resistance, MM cells can manifest resistance without any CRBN dysfunction, indicating alternative mechanisms of IMiDs resistance. We and others have shown a critical role of the BM microenvironment in MM pathogenesis: secretion of soluble factors from cellular components activates intracellular signaling pathways that promote MM cell migration, proliferation, survival, and drug resistance. To date, however, the mechanisms whether tumor microenvironment mediates IMiDs resistance have not been fully delineated. In our genome-wide CRISPR-Cas9 knockout screening, we have validated that CSN9 signalosome complex regulates sensitivity to IMiDs by modulating CRBN expression. Our screen also identified TRAF2, a member of the TNF receptor associated factor protein family, to regulate IMiDs sensitivity. To confirm modulation of IMiDs sensitivity by TRAF2, we here individually cloned TRAF2 sgRNAs into LentiCRISPRv2 vector, and then re-introduced them into MM cells. As expected, TRAF2-KO MM cells acquired significant resistance to Pom and Len treatment. Importantly, TRAF2 KO showed no effect on CRBN expression; moreover, IMiDs treatment of TRAF2 KO MM cells still triggered IKZF1 and IKZF3 degradation associated with downregulation of IRF4, the main effector of MM cell survival. Importantly, we observed that TRAF2 KO MM cells expressed high levels of p-ERK. We and others have shown that the bone marrow (BM) microenvironment activates ERK signaling pathway and plays a crucial role in drug resistance, and here showed that co-culture of MM cells with BM stromal cells (BMSCs) or culture supernatants (SC-sup) confers resistance to IMiDs, associated with downregulation of TRAF2. These results suggest that IMiDs resistance in the BM microenvironment may be mediated by ERK signaling pathway induced by soluble factors. AZD6244 is a potent and highly selective MEK inhibitor, and we next investigated whether AZD6244 inhibits ERK1/2 activation and overcome IMiDs resistance in TRAF2 KO MM cells. Importantly, addition of AZD6244 overcame resistance to IMiDs in both TRAF2 KO MM cells and SC-sup treated TRAF2 wild-type (WT) cells. In our human MM murine xenograft model, we observed that TRAF2 WT MM cells were sensitive to Pom treatment in vivo, while TRAF2 inducible KD MM cells demonstrated resistance. Importantly, the combination of AZD6244 and Pom significantly reduced growth even of TRAF2 KD MM cells, indicating that MEK inhibitor can inhibit activation of ERK1/2 and overcome IMiDs resistance in vivo. Finally, we confirmed that the number of patient samples with activated ERK pathway was significantly enriched at relapse while on lenalidomide maintenance therapy compared to diagnosis (BIOCARTA ERK pathway analysis 67% vs 39%, respectively, FDR In summary, we have identified and validated TRAF2 as a CRBN-IKZF1/3 axis-independent regulator of BM microenvironment-mediated sensitivity to IMiDs. These studies not only identify a novel mechanism of IMiDs resistance in the tumor microenvironment, but also provide the preclinical rationale for combining inhibitors of MEK/ERK signaling with IMiDs to overcome IMiDs resistance and improve patient outcome. Disclosures Munshi: Legend: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; BMS: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; C4: Current equity holder in private company. Richardson:Celgene/BMS, Oncopeptides, Takeda, Karyopharm: Research Funding. Anderson:Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

The approval of the first two monoclonal antibodies targeting CD38 (daratumumab) and SLAMF7 (elotuzumab) in late 2015 for treating relapsed and refractory multiple myeloma (RRMM) was a critical advance for immunotherapies for multiple myeloma (MM). Importantly, the outcome of patients continues to improve with the incorporation of this new class of agents with current MM therapies. However, both antigens are also expressed on other normal tissues including hematopoietic lineages and immune effector cells, which may limit their long-term clinical use. B cell maturation antigen (BCMA), a transmembrane glycoprotein in the tumor necrosis factor receptor superfamily 17 (TNFRSF17), is expressed at significantly higher levels in all patient MM cells but not on other normal tissues except normal plasma cells. Importantly, it is an antigen targeted by chimeric antigen receptor (CAR) T-cells, which have already shown significant clinical activities in patients with RRMM who have undergone at least three prior treatments, including a proteasome inhibitor and an immunomodulatory agent. Moreover, the first anti-BCMA antibody-drug conjugate also has achieved significant clinical responses in patients who failed at least three prior lines of therapy, including an anti-CD38 antibody, a proteasome inhibitor, and an immunomodulatory agent. Both BCMA targeting immunotherapies were granted breakthrough status for patients with RRMM by FDA in Nov 2017. Other promising BCMA-based immunotherapeutic macromolecules including bispecific T-cell engagers, bispecific molecules, bispecific or trispecific antibodies, as well as improved forms of next generation CAR T cells, also demonstrate high anti-MM activity in preclinical and even early clinical studies. Here, we focus on the biology of this promising MM target antigen and then highlight preclinical and clinical data of current BCMA-targeted immunotherapies with various mechanisms of action. These crucial studies will enhance selective anti-MM response, transform the treatment paradigm, and extend disease-free survival in MM.

Immunomodulatory drugs and monoclonal antibody-based immunotherapies have significantly improved the prognosis of the patients with multiple myeloma (MM) in the recent years. These new classes of reagents target malignant plasma cells (PCs) and further modulate the immune microenvironment, which prolongs anti-MM responses and may prevent tumor occurrence. Since MM remains an incurable cancer for most patients, there continues to be a need to identify new tumor target molecules and investigate alternative cellular approaches using gene therapeutic strategies and novel treatment mechanisms. Osteoclasts (OCs), as critical multi-nucleated large cells responsible for bone destruction in >80% MM patients, have become an attractive cellular target for the development of novel MM immunotherapies. In MM, OCs are induced and activated by malignant PCs in a reciprocal manner, leading to osteolytic bone disease commonly associated with this malignancy. Significantly, bidirectional interactions between OCs and MM cells create a positive feedback loop to promote MM cell progression, increase angiogenesis, and inhibit immune surveillance via both cell–cell contact and abnormal production of multiple cytokines/chemokines. Most recently, hyper-activated OCs have been associated with activation of programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway, which impairs T cell proliferation and cytotoxicity against MM cells. Importantly, therapeutic anti-CD38 monoclonal antibodies and checkpoint inhibitors can alleviate OC-induced immune suppression. Furthermore, a proliferation-inducing ligand, abundantly secreted by OCs and OC precursors, significantly upregulates PD-L1 expression on MM cells, in addition to directly promoting MM cell proliferation and survival. Coupled with increased PD-L1 expression in other immune-suppressive cells, i.e., myeloid-derived suppressor cells and tumor-associated macrophages, these results strongly suggest that OCs contribute to the immunosuppressive MM BM microenvironment. Based on these findings and ongoing osteoimmunology studies, therapeutic interventions targeting OC number and function are under development to diminish both MM bone disease and related immune suppression. In this review, we discuss the classical and novel roles of OCs in the patho-immunology of MM. We also describe novel therapeutic strategies simultaneously targeting OCs and MM interactions, including PD-1/PD-L1 axis, to overcome the immune-suppressive microenvironment and improve patient outcome.

Extra-medullary disease (EMD) in Multiple Myeloma (MM) is associated with poor prognosis and resistance to chemotherapy. However, molecular alterations that lead to EMD have not been well defined. We developed bone marrow (BM)- and EMD-prone MM syngeneic cell lines and identified that epithelial-to-mesenchymal-tranistion (EMT) transcriptional patterns were significantly enriched in the both clones compared to parental cells, together with higher levels of CXCR4 protein; and demonstrated that CXCR4 enhanced the acquisition of an EMT-like phenotype in MM cells with a phenotypic conversion for invasion, leading to higher bone metastasis and EMD dissemination in vivo. In contrast, CXCR4-silencing led to inhibited tumor growth and reduced survival. Ulocuplumab, a monoclonal anti-CXCR4 antibody, inhibited MM cell dissemination, supported by suppression of the CXCR4-driven EMT-like phenotype. These studies suggest that targeting CXCR4 may act as a regulator of EMD through EMT-like transcriptional modulation, thus representing a potential therapeutic strategy to prevent MM disease progression.

Combination of a novel HDAC6 inhibitor ACY-241 and anti-PD-L1 antibody enhances anti-tumor immunity and cytotoxicity in multiple myeloma

Regulatory B cell-myeloma cell interaction confers immunosuppression and promotes their survival in the bone marrow milieu

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.

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.

Background: Recent attempts have focused on identifying fewer magnitude of minimal residual disease (MRD) rather than exploring the biological and genetic features of the residual plasma cells (PCs). Interphase fluorescence in situ hybridization (iFISH) analyses in sequential samples provide a simple and reliable method to longitudinal track the dynamic changes in clonal architecture and speculate the possible evolutionary pattern. Here, we report for the first time the incidence and prognostic significance of cytogenetic abnormalities (CA) existed in the PCs of patients achieving at least partial remission. Methods: A cohort of 193 patients with at least one CA at diagnosis were analyzed using data from the prospective, non-randomized clinical trial (BDH 2008/02), and iFISH analyses were performed in patient-paired diagnostic and post-therapy samples. Results: Persistent CA in residual tumor cells were observed for the majority of patients (63%), even detectable in 28/63 (44%) patients with MRD negativity ( According to the change of the clonal size of specific CA, patients were clustered into five groups, reflecting five patterns of clone selection under therapy pressure. 1) Pattern A were observed in 36 (19%) patients where a minor subpopulation or undetectable subclone in the pre-treatment sample became dominant after therapy. 2) Pattern B was identified in 29 (15%) patients whose fractions of PCs harboring different CA were decreased with inconsistent extent. 3) Identical CA fraction in residual PCs were found in 22 (11%) patients as Pattern C. 4) Pattern D. The fractions of PCs harboring specific CA in 35 patients (18%) were uniformly declined. 5) 71 patients lost their abnormal cytogenetic clone after therapy (less than cut-off level) were classified as Pattern E. The cytogenetic dynamics of pattern A and B can be interpreted as a therapy-induced selection process with comparable inferior survival (m-TTP 1.2 vs. 1.6 years, respectively). Patients with pattern E experienced the most favorable outcome (m-TTP 5.0 years), following those with pattern D and C (m-TTP 3.5 and 2.5 years). Among the 65 patients with clonal selection, 24 underwent upfront auto-transplantation that experienced significantly improved survival. However, upfront transplant failed to completely reverse the inferior outcome caused by therapy-induced clonal selection. Longitudinal cytogenetic studies at relapse were available in 43 patients. The results suggested that sequential cytogenetic dynamics were observed in most patients, and the cytogenetic architecture of residual cells could to some extent predict the evolutional pattern at relapse. Conclusions: The repeat cytogenetic evaluation in residual cells could not only serves as a good complementary tool for MRD detection, but also provides a better understanding of clinical response and clonal evolution. Therapy-induced clonal selection was associated with inferior outcome regardless of the baseline cytogenetic profiles. The early identification of resistant clone may contribute to guide better tailored therapy strategies based on the feature of the residual tumor cells. Figure Disclosures Munshi: Celgene: Consultancy; Adaptive: Consultancy; Oncopep: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Abbvie: Consultancy.

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.