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Blocking of MEK-ERK pathway overcomes myeloma cells resistance to immunomodulatory drugs in the bone marrow microenvironment., Immunomodulatory drugs (IMiDs) have markedly improved patient outcome in multiple myeloma (MM); however, resistance to IMiDs commonly underlies relapse of disease. Here, we identify that tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) knockdown (KD)/knockout (KO) in MM cells mediates IMiD resistance via activation of noncanonical nuclear factor κB (NF-κB) and extracellular signal–regulated kinase (ERK) signaling. Within MM bone marrow (BM) stromal cell supernatants, TNF-α induces proteasomal degradation of TRAF2, noncanonical NF-κB, and downstream ERK signaling in MM cells, whereas interleukin-6 directly triggers ERK activation. RNA sequencing of MM patient samples shows nearly universal ERK pathway activation at relapse on lenalidomide maintenance therapy, confirming its clinical relevance. Combination MEK inhibitor treatment restores IMiD sensitivity of TRAF2 KO cells both in vitro and in vivo. Our studies provide the framework for clinical trials of MEK inhibitors to overcome IMiD resistance in the BM microenvironment and improve patient outcome in MM.

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.

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..

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.

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.

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.

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.

Dysregulated translational control and high protein turnover is a feature of multiple myeloma (MM) cells; as a result, proteasome inhibitors (PI), triggering a misfolded protein stress and death response, have been extremely successfully in MM therapy. But despite success of PIs and other novel therapeutic approaches, majority of patients progress; thus, investigating the mechanisms driving the drug sensitivity and/or resistance is important. We here report a novel chaperone protein 14-3-3ε with significant impact on MM cell pathobiology and patient outcome. 14-3-3 proteins are a family of master regulators of intracellular signaling that influence several cellular processes through binding to specific serine/threonine-phosphorylated residues on a diverse array of cellular proteins. With known interaction of 14-3-3 proteins with several functionally diverse molecules and well recognized chaperon function, we sought to evaluate their role in impacting proteasome activity. We compared the sensitivity of 14 MM cell lines to two proteasome inhibitors, Bortezomib (BTZ) and Carfilzomib (CFZ) with expression of all 14-3-3 proteins at both mRNA and protein levels. We compared the measured drug activity and individual 14-3-3 gene expression across all cell lines by Pearson correlation coefficients and observed that the expression level of only YWHAE (coding gene for the isoform 14-3-3ε) showed a significant negative correlation with both BTZ and CFZ response. We therefore evaluated its expression in primary MM cells and found its lower expression to be associated with poor outcome in MM patients receiving a bortezomib (Btz)-based therapy. To elucidate the underlying molecular mechanism responsible for 14-3-3ε contribution to PIs sensitivity, we performed integrated analysis of protein interactome and transcriptomic changes following 14-3-3ε gene modulation. These studies showed that 14-3-3ε impacts mTORC1 signaling in MM cells by binding to serine-phosphorylated residues on mTOR and its upstream negative regulator TSC2, resulting in mTORC1 activation. Conversely, depletion of 14-3-3ε inhibits TSC2 phosphorylation, causing a subsequent inhibition of mTORC1 signaling. One major conserved function of mTORC1 is to promote mRNA translation and therefore protein synthesis via activation of S6 kinase 1 (S6K1) as well as inhibition of the eukaryotic initiation factor 4E (eIF4E) binding protein (4E-BP). We have therefore assessed the impact of 14-3-3ε on translational efficiency in MM cells. In the viable MM cell population, 14-3-3ε depletion caused up to 50% reduction of protein synthesis in three MM cell lines, whereas 14-3-3ε overexpression in the KMS20 cell line or addback in YWHAE-KO cells (H929 and KMS11) resulted in a marked upregulation of protein synthesis. As MM is characterized by excess production of monoclonal immunoglobulins, we evaluated impact of 14-3-3ε perturbation on intracellular and secretion light chains production. We observed a significant decrease in the intracellular abundance and secretion of the light chains with 14-3-3ε KD in all MM cell lines tested. These observations were corroborated by gain-of-function studies where ectopic overexpression of 14-3-3ε in MM cells was associated with increased protein load (and an enhanced sensitivity to PIs) in vitro as well as in vivo. Importantly, we confirmed a significant correlation between 14-3-3ε expression, PIs sensitivity and protein load (evaluated as M protein production) in primary MM cells from two independent datasets. Moreover, MM patients with del17p, where 14-3-3ε is located, have lower 14-3-3ε expression and decreased monoclonal protein level, providing an explanation for inability of BTZ to overcome high-risk feature associated with del17p. In conclusion, we here report for the first time a unique function for 14-3-3ε in modulating the sensitivity to PIs through regulation of protein synthesis and M protein load in MM cells. Altogether these observations suggest that 14-3-3ε is a predictor of clinical outcome and may serve as a potential target to modulate PIs sensitivity in MM. Disclosures Anderson: Sanofi-Aventis: Other: Advisory Board; OncoPep: Other: Scientific founder ; C4 Therapeutics: Other: Scientific founder ; Janssen: Other: Advisory Board; Gilead Sciences: Other: Advisory Board. Munshi:Takeda: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Abbvie: Consultancy; Abbvie: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Oncopep: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Janssen: Consultancy.

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 .

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

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

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

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

Dysregulation of transcriptional control is a common phenomenon associated with oncogenesis. Inhibitors of DNA binding (ID) proteins are critical actors in lymphopoiesis, acting as regulators of transcription through a helix-loop-helix (HLH) domain which enables heterodimerization with basic HLH (bHLH) proteins inhibiting their binding to DNA. ID proteins have been implicated in malignant transformation, but their role in multiple myeloma (MM) is unknown. Here, we evaluated the role of ID proteins in biology and transcriptional dysregulation in MM. We first evaluated the expression of the four ID proteins in normal and malignant plasma cells using RNA sequencing data from a cohort of 360 newly diagnosed MM patients and 16 normal plasma cells. We observed significant downregulation of ID2 in primary patient MM cells in comparison to normal plasma cells (p 0.0013). To study ID2 function in MM cells, we next overexpressed ID2 in 2 MM cell lines (MM1S and NCIH929) and observed a significant decrease in proliferation rate, together with G0/G1 phase cell cycle arrest. We performed RNA-sequencing to evaluate the transcriptomic changes following ID2 overexpression. Gene set enrichment analysis (GSEA) revealed significant downregulation of genes involved in E2F pathway and significant changes in pathways related to immune response, regulation of cell death and cell proliferation. In addition, analysis of upstream cis-regulatory motifs of genes significantly dysregulated in both cell lines (>1.5 fold change) showed a highly significant enrichment for bHLH class I transcription factors (E proteins) binding motifs. Conversely, stable ID2 knockdown in 4 MM cell lines (MM1S, NCIH929, RPMI8226 and KMS11) expressing intermediate levels of ID2, showed an increased proliferation rate, assessed by cell counting, H3-thymidine incorporation and ATP production. RNA-sequencing after ID2 knockdown in MM1S and NCIH929 cells showed 600 common genes upregulated in both cell lines (>1.5 fold change). GSEA revealed upregulation of pathways involved in inflammatory response and epithelial-to-mesenchymal transition, while upstream cis regulatory motifs analysis showed a highly significant enrichment for binding motifs of bHLH class I transcription factors E proteins, in particular Tcf3 (p Next, we sought to investigate the mechanisms involved in ID2 downregulation in MM. Since the role of the microenvironment is critical in myelomagenesis, we evaluated the impact of BM microenvironment on ID2 expression in a co-culture system. Using bone marrow stromal cells (BMSC) derived from MM patients and stromal cell line (HS5) in co-culture with various MM cell lines, we observed that both cell-cell interactions and soluble factors secreted by BMSC or HS5 were able to significantly downregulate ID2 expression at the RNA and protein level. Furthermore, ID2 overexpression in MM cell lines (MM1S and NCIH929) abrogated the impact of BMSC on MM cell proliferation. Next, we evaluated ID2 promoter methylation profile and binding motifs using Sequenom mass array and the assay for transposase-accessible chromatin sequencing (ATAC-seq), respectively. While we didn't observe any increase in methylation of CpG islands located in ID2 promoter in co-culture, explaining ID2 downregulation, we identified several binding motifs corresponding to known driver transcription factors in MM. Especially, we identified SP1 binding motif and we confirmed SP1 binding to ID2 promoter by ChIP-sequencing in MM1S, NCIH929 and U266. These data demonstrate that in MM, ID2 acts as a tumor suppressor by promoting major transcriptomic changes and cell cycle arrest. Bone marrow stromal cells further induce significant downregulation of ID2 in myeloma cells suggesting that ID2/bHLH axis and other ID2 related pathways represent a potential new therapeutic target in myeloma. Disclosures Anderson: Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; Celgene: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; OncoPep: Equity Ownership, Other: Scientific founder; Millennium Takeda: Consultancy. Munshi:OncoPep: Other: Board of director.

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

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

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

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