ALBERT

Description: Background We showed a tumor-promoting and immunosuppressive role of plasmacytoid dendritic cells (pDCs; CD123/IL-3Rα+) in multiple myeloma (MM) pathogenesis (Chauhan et al. Cancer Cell, 2009;Ray et al, Leukemia, 2018). Importantly, tagraxofusp, an FDA-approved (for patients with blastic plasmacytoid dendritic cell neoplasm) novel targeted therapy directed against CD123, triggers anti-MM activity by reducing the viability of MM-promoting pDCs. These observations led to a recently completed phase 1 clinical trial of tagraxofusp and pomalidomide/dexamethasone in relapsed and refractory MM patients (NCT02661022). The treatment regimen demonstrated preliminary safety and efficacy, with 5 of 9 heavily pretreated patients achieving durable partial response (PR) (ASH 2019). Here, we report the initial results of our correlative science studies using bone marrow (BM), peripheral blood (PB), and serum from the study cohort. Materials and Methods Tagraxofusp is a bioengineered therapeutic protein developed by fusing human IL-3 to the catalytic translocation domain of truncated diphtheria toxin (DT) via a Met-His linker (Stemline Therapeutics, NY). pDCs and patient MM cells were purified from BM/PB samples after informed consent, and quantified using FACS, as described (Ray et al, Leukemia, 2018). A novel high-throughput seroproteomics platform SOMAscan was utilized to analyze 1,310 protein analytes in serum samples from MM patients (n = 9). SOMAscan data were subjected to meta-analysis to generate heatmaps, followed by hierarchical cluster analysis. SOMAscan results were validated with ELISA using supernatants from MM patient pDCs cultured with or without tagraxofusp. Results Analysis of BM/PB samples from MM patients receiving tagraxofusp therapy showed a marked reduction in the frequency of viable pDCs [average 2% at screening vs 0.75% post-tagraxofusp; n = 6; p = 0.036]. pDCs isolated from tagraxofusp-treated patients showed decreased ability to trigger MM cell growth. Seroproteomics analysis of MM patient serum before and after tagraxofusp therapy showed alterations in the levels of 100 proteins [Median Fold Change in expression: 0.39 to 4.5; n = 6; 3 each; p 〈 0.05]. Importantly, tagraxofusp treatment reduced pDC-related soluble proteins including IFN-α (fold change: 0.8, treated vs untreated; p 〈 0.05). Our earlier study showed that pDC-MM interactions triggered secretion of IL-3, which in turn promotes both pDC survival and MM cell growth. Importantly, tagraxofusp decreased serum IL-3 (fold change 0.75, treated vs untreated; p 〈 0.05), consistent with tagraxofusp decreasing survival of tumor-promoting pDCs. Conclusions Our current correlative science studies validate target specificity of tagraxofusp against MM pDCs in relapsed and refractory MM patients enrolled in a phase 1 clinical trial and support further evaluation for this novel therapeutic to improve the clinical outcome of patients with MM. Further combination studies are planned. Disclosures Mo: Celgene/BMS: Membership on an entity's Board of Directors or advisory committees. Olguin:Stemline Therapeutics: Current Employment. Chen:Stemline Therapeutics: Current Employment. Brooks:Stemline: Current Employment. Mughal:Stemline: Current Employment. Richardson:Celgene/BMS, Oncopeptides, Takeda, Karyopharm: Research Funding. Chauhan:consultant to Stemline Therapeutics, Inc., and Equity owner in C4 Therapeutics.: Consultancy, Other: Equity owner in C4 Therapeutics.; Oncopeptide AB: Consultancy. Anderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; 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.

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

Description: Background and Rationale Patients with multiple myeloma (MM) are at a higher risk of viral infection due to immune deficiency. Importantly, recent studies highlighted the severity of COVID-19 in MM. To date, however, the mechanism(s) underlying the lack of anti-viral immune response in MM are unclear. Plasmacytoid dendritic cells (pDCs) play a key role in both recognition of viral nucleic acid and initiating anti-viral activity via type 1 interferon (IFN) response signaling. We showed that interactions of dysfunctional pDCs with MM cells, T cells, and NK cells confer immune suppression in the host-MM bone marrow (BM) microenvironment (Chauhan et al, Cancer Cell 2009; 16: p309;Ray et al, Leukemia 2015; 29: p1441). Here, we analyzed the immune-pathway proteins implicated in Covid-19-host interactions [Gordon et al, Nature 2020; 583, p459] to assess whether pDC-MM interactions modulate these pathways to confer immune suppression and susceptibility to COVID-19. We identified the TLR pathway serine/threonine kinase TBK1 (TANK-binding kinase 1), involved in type I IFN induction, as a potential immunotherapeutic target in MM. Moreover, Covid-19 relies on host-Ubiquitin-proteasome-system for propagation, and we found that targeting ubiquitin receptor Rpn13 with a specific inhibitor RA190 restores TBK1 expression in MM. Methods For our studies, we examined SARS-CoV-2-human protein-protein interactions (PPIs) maps (Gordon et al, 2020). pDC-MM co-cultures, and RNAseq using next- generation sequencing (NGS): Purified MM patient pDCs were cocultured with autologous MM cells (1pDC/5MM) for 48h, followed by separation of MM cells from pDCs using FACS. Total RNA from MM cells was subjected to RNAseq analysis using Illumina NGS. Raw sequence data were analyzed to generate differential expression (DEseq2). Linear model (Limma) and its GUI (Glimma) were also used. Statistical significance: log2FC (fold change) values in coculture vs control, with a False Discovery Rate value of 95). The heatmap analysis was done using Morpheus software (Broad Institute, MIT). MM patient and pDCs data used for bioinformatics were from the NCBI GEO. Results A total of 41 genes involved in the Covid-19 host-pathogen immune interactions are also differentially expressed in MM after coculture with pDCs. (log2Fold change: ± 3.5-fold; p 〈 0.00001; pDC-MM vs MM alone). The gene expression widely varies in pDC-MM vs MM [Median (log10): -0.13 to 4.5; adj p 〈 0.001]. We identified 3 specific innate immunity-linked genes TBK1, IRF3 (Interferon regulatory factor 3), and RAE1 (interferon-inducible mRNA nuclear export factor) which are essential for IFN production in MM. Importantly, pDCs decrease TBK1 (Log2FC: -0.5) and RAE1 (Log2FC: -0.3) as well as induce IRF3 (Log2FC: 1.5) in MM (p 〈 0.0001). Analysis of publicly available gene profiling datasets on relapsed MM patient showed low levels of TBK1 and RAE1 and higher IRF3 (n = 50) [Log2FC: TBK1: -0.208; RAE1: -0.286; IRF3: 0.273; vs normal; p 〈 0.05). Of note, low TBK1 expression correlates with poor survival in MM patients (n = 350) and elevated TBK1 correlates with a better prognosis (p = 0.026). Similar analysis showed that most of 41 genes involved in the Covid-19 host-pathogen immune interactions are expressed in pDCs. In unstimulated pDCs, TBK1 expression is significantly lower than IRF3 and RAE1 (2-3-fold, p 〈 0.05). In functional studies, treatment of pDCs with IFN-α activating CpG-ODN type-A increases both TBK1 (adj P = 0.004) and RAE1 (adj P = 0.043), without significantly altering IRF3 expression. Taken together, we show that TBK1 is downregulated in pDCs, and pDCs-MM interactions further decreases TBK1 in MM, thereby attenuating IFN-mediated anti-viral immune response signaling in MM. Finally, we found that blockade of proteasome-mediated protein degradation via inhibition of Ubiquitin receptor Rpn13 upregulates TBK-1 expression, indicating potential clinical use of targeting Rpn13 in restoring anti-viral immune responses in MM. Conclusion We found that pDC-MM interactions downregulate TBK1, which in turn reduces IFN response signaling essential to generate an effective anti-viral immune response in the MM BM microenvironment. Our findings suggest that: 1) low levels of TBK1 may confer increased susceptibility of MM patients to COVID-19; and 2) targeting TBK1 may restore anti-viral immune response and reduce COVID-19 severity in MM. Disclosures Chauhan: consultant to Stemline Therapeutics, Inc., and Equity owner in C4 Therapeutics.: Consultancy, Other: Equity owner in C4 Therapeutics.; Oncopeptide AB: Consultancy. Anderson:Janssen: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: 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.; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees.

Description: Background and Rationale 20S proteasome-based therapies are the mainstay of treatment of patients with multiple myeloma (MM); however, resistance to proteasome inhibitor (PI) therapies commonly develops underlying relapse of disease. We showed that inhibition of 19S-associated ubiquitin receptor (UbR) ADRM1/Rpn13, upstream of 20S proteasome, inhibits MM cell growth and overcomes PI-resistance in MM. hRpn13 recognizes polyubiquitinated proteins and facilitates their disassembly via 19S-associated deubiquitinating enzyme UCHL5, allowing for protein degradation via 20S proteasomal catalytic activities. To date, however, hRpn13-modulated protein substrates and downstream signaling remains unclear. Here, we utilized multiplexed proteomics with tandem mass spectrometry; GeneOntology (GO) enrichment; as well as pathway database KEGG and Reactome to identify hRpn13-associated signaling molecules and delineate functionally significant biological pathways. Materials and Methods CRISPR-Cas9 genome editing was performed to generate hRpn13-knockout (hRpn13-KO) HCT116 cells which were subjected to multiplexed proteomics. Functionally relevant proteins were categorized using GO, as well as KEGG- and Reactome-pathway analysis. The prognostic relevance of identified proteins was derived using Gene Expression Profiling (GEP) databases on uniformly treated MM patients. Protein expression level was determined by western blotting. CRISPR-Cas9-mediated hRpn13 deletion was confirmed using PCR and sequencing, as well as immunoblot analysis. Statistical significance was assessed with Student's t test Results Among 8766 proteins analyzed, 206 proteins were down-regulated, whereas 65 proteins were significantly up-regulated in hRpn13-KO cells compared to hRpn13-WT cells. Targeted hRpn13-deletion upregulated physiological pathways related to amino acid biosynthesis and transport, metabolism, proliferation, and apoptosis. We validated 5 such upregulated proteins (SLC1A3, THBS1, GLYR1, GCLM, and TIMM8A) using immunoblotting. As for hRpn13 deletion, treatment of MM.1S cells with Rpn13 inhibitor RA190 triggered a similar increase in SLC1A3, THBS1, GLYR1, GCLM, and TIMM8A levels. These data indicate that SLC1A3, THBS1, GLYR1, GCLM, and TIMM8A proteins are direct substrates of UbR hRpn13. On the other hand, hRpn13-deletion decreased levels of proteins involved in cell adhesion, biological regulation, antigen recognition, and extracellular matrix interactions. Clinical relevance in MM: We next screened 271 proteins against GEP datasets on MM patients (GSE6477; GSE13591; GSE6691). Among the 65 molecules upregulated in hRpn13-KO cells, 18 were expressed at significantly lower levels in MM patients vs normal plasma cells; and importantly, 2 (SLC1A3, THBS1) of these 18 proteins correlated with poor survival. In a functional validation study, blockade of hRpn13 with RA190 induced increased SLC1A3 and THBS1 levels, suggesting that targeting SLC1A3/THBS1 may restore abnormal amino acid metabolism in MM. We next screened 206 of 271 proteins that were downregulated in Rpn13-KO cells in GEP database of MM patients. Results show that 32 of these 206 molecules were highly expressed in MM patient samples vsnormal plasma cells, and that 2 (SLC16A7 and DBF4) of 32 correlated with poor survival. SLC16A7 encodes for monocarboxylate transporter 2 (MCT2), a key component of glycolysis, and DBF4 regulates DNA replication/cell proliferation. Importantly, RA190 decreased SLC16A and DB4 levels. Together, these data suggest that: 1) SLC16A, DBF4, SLC1A3, and THBS1 are downstream signaling targets of hRpn13; and 2) hRpn13-inhibition triggered MM cell death involves blockade of elevated glycolysis and DNA replication/cell growth via SLC16A and DBF4, respectively, as well as restoration of normal amino acid synthesis via SLC1A3. Conclusion Collectively, our study utilized CRISPR gene-editing, biochemical, and molecular strategies to identify UbR hRpn13-mediated proteomic alterations. We identified novel targets including SLC16A7, DBF4, SLC1A3, and Thrombospondin-1 which may serve as prognostic biomarkers in MM. Importantly, our findings further support hRpn13-directed therapeutics, as well as preclinical evaluation of novel strategies targeting SLC16A, DBF4, SLC1A3, and Thrombospondin-1, to enhance cytotoxicity and improve patient outcome in MM. Disclosures Chauhan: Oncopeptide AB: Consultancy; consultant to Stemline Therapeutics, Inc., and Equity owner in C4 Therapeutics.: Consultancy, Other: Equity owner in C4 Therapeutics.. Anderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: 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; 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; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees.

Description: Introduction Proteasome inhibitors (PIs) are a backbone standard of care in the treatment of relapsed/refractory and newly diagnosed multiple myeloma (MM). Although PIs represent a major advance, not all MM responds, and relapse develops due to the development of drug-resistance. Besides the 26S proteasome, the ubiquitin proteasome pathway harbors many other sites for therapeutic intervention, with the potential to overcome PI-resistance. We showed that inhibition of deubiquitylating enzyme USP7, upstream of the 20S proteasome, induces cell death even in MM cells resistant to PI therapies. To date, however, the effect of USP7 inhibition on the immunosuppressive and tumor-promoting MM-host bone marrow (BM) accessory cells such as plasmacytoid dendritic cells (pDC) remains unclear. Here, we utilized our co-culture models of pDCs, T cells, and NK cells with autologous patient MM cells to examine whether USP7 inhibition also stimulates anti-MM immunity. Methods MM cell lines were validated by fingerprinting, and MM patient BM/PB samples (n = 9) were obtained after informed consent. We used minimally cytotoxic concentrations of a novel USP7 inhibitor XL177A (0.1-0.5 µM). Activation/maturation assays MM-pDCs were treated with XL177A (0.2 uM; 24h), followed by evaluation of CD86/HLA-DR markers by FACS. USP7-HA-UbVME Labeling: pDC representative Cal-1 cells were treated with DMSO or XL177A for 6h; proteins were labeled with ubiquitin (Ub) active site probe Ub-vinyl methyl ester (HAUbVME) for 30 mins, followed by immunoblot analysis using anti-USP7 antibodies. CTL/NK activity assays MM CD8+ T- or NK-cells cells were cultured with autologous pDCs (1:10 pDC:T/NK ratio) with XL177A for 3 days; after washing to remove drug, cells were cultured with pre-stained (CellTrace Violet) target MM cells (10:1 E/T ratio; T/NK:MM) for 24h, followed by FACS quantification of viable MM cells. Statistical parameters were calculated using GraphPad Prism. Results 1) Treatment of MM cell lines and patient MM cells with XL177A for 24h significantly decreases their viability (IC50 range 0.6 uM to 5 uM) (mean ± SD; p 〈 0.05; n=4). 2) Blockade of USP7 by XL177A activates MM patient BM-pDCs, evidenced by an increase in pDC maturation markers CD86 and HLA-DR. As a negative control, treatment of pDCs with XL177B, an inactive (R) enantiomer of XL177A, had no effect on CD86 or HLA-DR expression on pDCs. 3) XL177A specificity against USP7 DUB activity was assessed using competition assays between XL177A and Ub-active site labeling probe HAUbVME: treatment of pDC-Cal-1 cells with XL177A blocked the labeling HAUbME probe, confirming its specificity. Similar results were noted using another USP7 inhibitor P5091. 4) Functional studies showed that XL177A stimulates MM-specific CD8+ CTL activity, evidenced by a decrease in the viability of patient MM cells (p = 0.0004; n= 9). 5) XL177A triggered pDC-induced NK cell-mediated MM-specific cytolytic activity (p

Description: 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.