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Purpose: BRD9 is a defining component of the noncanonical SWI/SNF complex, which regulates gene expression by controlling chromatin dynamics. Although recent studies have found an oncogenic role for BRD9 in multiple cancer types including multiple myeloma, its clinical significance and oncogenic mechanism have not yet been elucidated. Here, we sought to identify the clinical and biological impact of BRD9 in multiple myeloma, which may contribute to the development of novel therapeutic strategies. Experimental Design: We performed integrated analyses of BRD9 in vitro and in vivo using multiple myeloma cell lines and primary multiple myeloma cells in established preclinical models, which identified the molecular functions of BRD9 contributing to multiple myeloma cell survival. Results: We found that high BRD9 expression was a poor prognostic factor in multiple myeloma. Depleting BRD9 by genetic (shRNA) and pharmacologic (dBRD9-A; proteolysis-targeting chimera; BRD9 degrader) approaches downregulated ribosome biogenesis genes, decreased the expression of the master regulator MYC, and disrupted the protein-synthesis maintenance machinery, thereby inhibiting multiple myeloma cell growth in vitro and in vivo in preclinical models. Importantly, we identified that the expression of ribosome biogenesis genes was associated with the disease progression and prognosis of patients with multiple myeloma. Our results suggest that BRD9 promotes gene expression by predominantly occupying the promoter regions of ribosome biogenesis genes and cooperating with BRD4 to enhance the transcriptional function of MYC. Conclusions: Our study identifies and validates BRD9 as a novel therapeutic target in preclinical models of multiple myeloma, which provides the framework for the clinical evaluation of BRD9 degraders to improve patient outcome.

Supplementary Materials

BRD9 occupies promoter regions of target gene.

Purpose:BRD9 is a defining component of the noncanonical SWI/SNF complex, which regulates gene expression by controlling chromatin dynamics. Although recent studies have found an oncogenic role for BRD9 in multiple cancer types including multiple myeloma, its clinical significance and oncogenic mechanism have not yet been elucidated. Here, we sought to identify the clinical and biological impact of BRD9 in multiple myeloma, which may contribute to the development of novel therapeutic strategies.Experimental Design:We performed integrated analyses of BRD9 in vitro and in vivo using multiple myeloma cell lines and primary multiple myeloma cells in established preclinical models, which identified the molecular functions of BRD9 contributing to multiple myeloma cell survival.Results:We found that high BRD9 expression was a poor prognostic factor in multiple myeloma. Depleting BRD9 by genetic (shRNA) and pharmacologic (dBRD9-A; proteolysis-targeting chimera; BRD9 degrader) approaches downregulated ribosome biogenesis genes, decreased the expression of the master regulator MYC, and disrupted the protein-synthesis maintenance machinery, thereby inhibiting multiple myeloma cell growth in vitro and in vivo in preclinical models. Importantly, we identified that the expression of ribosome biogenesis genes was associated with the disease progression and prognosis of patients with multiple myeloma. Our results suggest that BRD9 promotes gene expression by predominantly occupying the promoter regions of ribosome biogenesis genes and cooperating with BRD4 to enhance the transcriptional function of MYC.Conclusions:Our study identifies and validates BRD9 as a novel therapeutic target in preclinical models of multiple myeloma, which provides the framework for the clinical evaluation of BRD9 degraders to improve patient outcome.

GSEA_RNAseq (GOBP)

The bone marrow (BM) microenvironment actively promotes multiple myeloma pathogenesis, and therapies targeting both cancer cells and the niche are highly effective. We were interested in identifying novel signaling pathways supporting multiple myeloma–BM cross-talk. Mutations in the transmembrane receptor Roundabout 1 (ROBO1) were recently identified in patients with multiple myeloma; however, their functional consequences are uncertain. Through protein structure–function studies, we discovered that ROBO1 is necessary for multiple myeloma adhesion to BM stromal and endothelial cells and that ROBO1 knockout (KO) compromises BM homing and engraftment in a disseminated mouse model. ROBO1 KO significantly decreases multiple myeloma proliferation in vitro and intra- and extramedullary tumor growth in vivo. Mechanistically, the ROBO1 C-terminus is cleaved in a ligand-independent fashion and is sufficient to promote multiple myeloma proliferation. Vice versa, mutants lacking the cytoplasmic domain, including the human-derived G674* truncation, act dominantly negative. Interactomic and RNA-sequencing studies suggest that ROBO1 may be involved in RNA processing, supporting further studies.Significance:ROBO1 is highly expressed in t(4;14) multiple myeloma and supports homing and dissemination to the BM niche. ROBO1 knockout causes reduced tumor growth in intramedullary and extramedullary myeloma animal models, while the ROBO1 C-terminus is cleaved in multiple fragments and it is necessary and sufficient to sustain myeloma proliferation.

Supplementary Figure 1 showing ROBO1 expression in primary MM cells versus normal donor bone marrow plasma cells via gene expression profiling and immunohistochemistry. Supplementary Figure 2 showing distribution of ROBO1 expression in the CoMMpass database; ROBO1 expression in CoMMpass database according to cytogenetics; ROBO1 expression in IFM170 database according to cytogenetics; ROBO1 expression in t(4;14) cell lines WT or KO for MMSET. Supplementary Figure 3 showing ROBO1 protein down regulation after ROBO1 knock down via shRNA. Supplementary Figure 4 showing effects of shRNA4- and shRNA-5 mediated ROBO1 KD on cell viability across a panel of MM cell lines with western confirming proper protein down regulation. Supplementary Figure 5 showing successful ROBO1 KO across several KMS11 monoclones, but not H929 monoclones. Supplementary Figure 6 showing expression of ROBO1 FL construct in polyclonally and monoclonally-transduced OPM2 cells. Supplementary Figure 7 showing expression of FL ROBO1 addback in distinct KMS11 ROBO1 KO monoclones. Supplementary Figure 8 showing radiographic images of WT versus KO ROBO1 mu-SCID mice. Supplementary Figure 9 showing adhesion defect in ROBO1 KO KMS11 cells that is fully rescued by FL ROBO1 addback. Supplementary Figure 10 showing Kaplan-Meier survival curve of disseminated mouse model injected with ROBO1 KO versus FL OPM2 cells. Supplementary Figure 11 showing cleaved cytosolic ROBO1 fragments in 293T cells transduced with FL ROBO1 tagged with C-LAP tag and in WT MM.1S cells. Supplementary Figure 12 showing interacting partners of ROBO1 cytosolic domain as deconvoluted via STRING database. Supplementary Figure 13 showing differentially expressed genes in ROBO1 WT versus KO plasmacytoma represented via Volcano plot. Supplementary Figure 14 showing gene set enrichment analysis of differentially expressed genes in ROBO1 WT versus KO plasmacytoma. Supplementary Figure 15 showing schema of patient-derived ROBO1 mutation and expression of G674 truncation in WT and KO ROBO1 OPM2 cells. Supplementary Table 1 showing clinical annotation of newly diagnosed MM parents used in Supplementary figure 1.

Supplementary Methods

The proteasome inhibitor bortezomib induces apoptosis in multiple myeloma cells and has transformed patient outcome. Using in vitro as well as in vivo immunodeficient and immunocompetent murine multiple myeloma models, we here show that bortezomib also triggers immunogenic cell death (ICD), characterized by exposure of calreticulin on dying multiple myeloma cells, phagocytosis of tumor cells by dendritic cells, and induction of multiple myeloma–specific immunity. We identify a bortezomib-triggered specific ICD gene signature associated with better outcome in two independent cohorts of patients with multiple myeloma. Importantly, bortezomib stimulates multiple myeloma cell immunogenicity via activation of the cGAS/STING pathway and production of type I IFNs, and STING agonists significantly potentiate bortezomib-induced ICD. Our study therefore delineates mechanisms whereby bortezomib exerts immunotherapeutic activity and provides the framework for clinical trials of STING agonists with bortezomib to induce potent tumor-specific immunity and improve patient outcome in multiple myeloma.Significance:Our study demonstrates that cGAS/STING-dependent immunostimulatory activity mediates bortezomib anti-myeloma activity in experimental models and associates with clinical response to bortezomib in patients with multiple myeloma. These findings provide the rationale for clinical evaluation of STING agonists to further potentiate anti–multiple myeloma immune response.See related commentary by Zitvogel and Kroemer, p. 405.This article is highlighted in the In This Issue feature, p. 403

Supp. table S1 shows immune genes regulated after treatment with bortezomib in vivo.

Supp. Fig. 1 shows that bortezomib induces ICD in vitro. Suppl. Fig.2 shows that bortezomib treatment of co-culture of dendritic and T cells does not induce T cell maturation. Suppl. Fig. 3 shows that bortezomib-induced MM cell death promotes proliferation of CD4+ and CD8+ naïve T cells. Suppl. Fig.4 shows that bortezomib treatment generates a robust immune response against MM cells. Suppl. Fig. 5 shows pathways enriched by bortezomib treatment in vivo. Suppl. Fig. 6 shows Gene Ontology analysis of ICD signature genes. Suppl. Fig. 7 shows that MM cells engage in a type-I IFN response after treatment with bortezomib. Suppl. Fig. 8 shows type-I IFN response after bortezomib in MM is STING dependent. Suppl. Fig. 9 shows STING agonists potentiate bortezomib-induced anti-tumor immunity.

Univariate and multivariate analysis of BRD9 expression

Supplementary materials

Purpose:Efforts are required to improve the potency and durability of CD38- and BCMA-based immunotherapies in human multiple myeloma. We here delineated the molecular and cellular mechanisms underlying novel immunomodulatory effects triggered by BCMA pyrrolobenzodiazepine (PBD) antibody drug conjugate (ADC) MEDI2228 which can augment efficacy of these immunotherapies.Experimental Design:MEDI2228-induced transcriptional and protein changes were investigated to define significantly impacted genes and signaling cascades in multiple myeloma cells. Mechanisms whereby MEDI2228 combination therapies can enhance cytotoxicity or overcome drug resistance in multiple myeloma cell lines and patient multiple myeloma cells were defined using in vitro models of tumor in the bone marrow (BM) microenvironment, as well as in human natural killer (NK)-reconstituted NOD/SCID gamma (NSG) mice bearing MM1S tumors.Results:MEDI2228 enriched IFN I signaling and enhanced expression of IFN-stimulated genes in multiple myeloma cell lines following the induction of DNA damage–ATM/ATR-CHK1/2 pathways. It activated cGAS-STING-TBK1-IRF3 and STAT1-IRF1–signaling cascades and increased CD38 expression in multiple myeloma cells but did not increase CD38 expression in BCMA-negative NK effector cells. It overcame CD38 downregulation on multiple myeloma cells triggered by IL6 and patient BM stromal cell-culture supernatant via activation of STAT1-IRF1, even in immunomodulatory drug (IMiD)- and bortezomib-resistant multiple myeloma cells. In vitro and in vivo upregulation of NKG2D ligands and CD38 in MEDI2228-treated multiple myeloma cells was further associated with synergistic daratumumab (Dara) CD38 MoAb-triggered NK-mediated cytotoxicity of both cell lines and autologous drug-resistant patient multiple myeloma cells.Conclusions:These results provide the basis for clinical evaluation of combination MEDI2228 with Dara to further improve patient outcome in multiple myeloma.

Immune profiling in patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and multiple myeloma (MM) provides the framework for developing novel immunotherapeutic strategies. Here, we demonstrate decreased CD4+ Th cells, increased Treg and G-type MDSC, and upregulation of immune checkpoints on effector/regulatory and CD138+ cells in MM patients, compared MGUS/SMM patients or healthy individuals. Among the checkpoints profiled, LAG3 was most highly expressed on proliferating CD4+ Th and CD8+ Tc cells in MM patients BMMC and PBMC. Treatment with antibody targeting LAG3 significantly enhanced T cells proliferation and activities against MM. XBP1/CD138/CS1-specific CTL generated in vitro displayed anti-MM activity, which was further enhanced following anti-LAG3 treatment, within the antigen-specific memory T cells. Treg and G-type MDSC weakly express LAG3 and were minimally impacted by anti-LAG3. CD138+ MM cells express GAL-3, a ligand for LAG3, and anti-GAL-3 treatment increased MM-specific responses, as observed for anti-LAG3. Finally, we demonstrate checkpoint inhibitor treatment evokes non-targeted checkpoints as a cause of resistance and propose combination therapeutic strategies to overcome this resistance. These studies identify and validate blockade of LAG3/GAL-3, alone or in combination with immune strategies including XBP1/CD138/CS1 multipeptide vaccination, to enhance anti-tumor responses and improve patient outcome in MM.

Chimeric antigen receptor (CAR) T-cell therapy remains limited to select centers that can carefully monitor adverse events. To broaden use of CAR T cells in community clinics and in a frontline setting, we developed a novel CD8+ CAR T-cell product, Descartes-08, with predictable pharmacokinetics for treatment of multiple myeloma. Descartes-08 is engineered by mRNA transfection to express anti-BCMA CAR for a defined length of time. Descartes-08 expresses anti-BCMA CAR for 1 week, limiting risk of uncontrolled proliferation; produce inflammatory cytokines in response to myeloma target cells; and are highly cytolytic against myeloma cells regardless of the presence of myeloma-protecting bone marrow stromal cells, exogenous a proliferation-inducing ligand, or drug resistance including IMiDs. The magnitude of cytolysis correlates with anti-BCMA CAR expression duration, indicating a temporal limit in activity. In the mouse model of aggressive disseminated human myeloma, Descartes-08 induces BCMA CAR-specific myeloma growth inhibition and significantly prolongs host survival (p

The bone marrow (BM) microenvironment actively promotes multiple myeloma pathogenesis, and therapies targeting both cancer cells and the niche are highly effective. We were interested in identifying novel signaling pathways supporting multiple myeloma–BM cross-talk. Mutations in the transmembrane receptor Roundabout 1 (ROBO1) were recently identified in patients with multiple myeloma; however, their functional consequences are uncertain. Through protein structure–function studies, we discovered that ROBO1 is necessary for multiple myeloma adhesion to BM stromal and endothelial cells and that ROBO1 knockout (KO) compromises BM homing and engraftment in a disseminated mouse model. ROBO1 KO significantly decreases multiple myeloma proliferation in vitro and intra- and extramedullary tumor growth in vivo. Mechanistically, the ROBO1 C-terminus is cleaved in a ligand-independent fashion and is sufficient to promote multiple myeloma proliferation. Vice versa, mutants lacking the cytoplasmic domain, including the human-derived G674* truncation, act dominantly negative. Interactomic and RNA-sequencing studies suggest that ROBO1 may be involved in RNA processing, supporting further studies. Significance: ROBO1 is highly expressed in t(4;14) multiple myeloma and supports homing and dissemination to the BM niche. ROBO1 knockout causes reduced tumor growth in intramedullary and extramedullary myeloma animal models, while the ROBO1 C-terminus is cleaved in multiple fragments and it is necessary and sufficient to sustain myeloma proliferation.

Oncogenic activated RAS mutations have been detected in 50% of de novo and 70% of relapsed multiple myeloma (MM) patients. Translocation t(11;14) involving IgH/CCDN1 and overexpression of cyclin-Ds are early events in MM pathogenesis, enhancing uncontrolled MM cell growth. We hypothesized that targeting both RAS/MAPK pathway molecules including Erk1/2 along with cyclin-Ds enhances MM cytotoxicity and minimizes side effects. Recent studies have demonstrated the high potency of Erk1/2 and CDK4/6 inhibitors in metastatic relapsed cancers, and here we tested anti-MM effects of the Erk1/2 + CDK4/6 inhibitor combination. Our studies showed strong synergistic (IC

Purpose: Efforts are required to improve the potency and durability of CD38- and BCMA-based immunotherapies in human multiple myeloma. We here delineated the molecular and cellular mechanisms underlying novel immunomodulatory effects triggered by BCMA pyrrolobenzodiazepine (PBD) antibody drug conjugate (ADC) MEDI2228 which can augment efficacy of these immunotherapies. Experimental Design: MEDI2228-induced transcriptional and protein changes were investigated to define significantly impacted genes and signaling cascades in multiple myeloma cells. Mechanisms whereby MEDI2228 combination therapies can enhance cytotoxicity or overcome drug resistance in multiple myeloma cell lines and patient multiple myeloma cells were defined using in vitro models of tumor in the bone marrow (BM) microenvironment, as well as in human natural killer (NK)-reconstituted NOD/SCID gamma (NSG) mice bearing MM1S tumors. Results: MEDI2228 enriched IFN I signaling and enhanced expression of IFN-stimulated genes in multiple myeloma cell lines following the induction of DNA damage–ATM/ATR-CHK1/2 pathways. It activated cGAS-STING-TBK1-IRF3 and STAT1-IRF1–signaling cascades and increased CD38 expression in multiple myeloma cells but did not increase CD38 expression in BCMA-negative NK effector cells. It overcame CD38 downregulation on multiple myeloma cells triggered by IL6 and patient BM stromal cell-culture supernatant via activation of STAT1-IRF1, even in immunomodulatory drug (IMiD)- and bortezomib-resistant multiple myeloma cells. In vitro and in vivo upregulation of NKG2D ligands and CD38 in MEDI2228-treated multiple myeloma cells was further associated with synergistic daratumumab (Dara) CD38 MoAb-triggered NK-mediated cytotoxicity of both cell lines and autologous drug-resistant patient multiple myeloma cells. Conclusions: These results provide the basis for clinical evaluation of combination MEDI2228 with Dara to further improve patient outcome in multiple myeloma.

Background Efforts are required to improve potency and durability of CD38- and BCMA-based immunotherapies in human multiple myeloma (MM). Methods We here delineated the molecular and cellular mechanisms underlying novel immunomodulatory effects triggered by BCMA pyrrolobenzodiazepine (PBD) antibody drug conjugate MEDI2228 which may augment efficacy of these immunotherapies. RNA sequencing followed by gene set enrichment analysis showed that MEDI2228 significantly enriched IFN I-signaling and induced type I interferon (IFN I)-stimulated genes (ISGs) in MM cell lines. The most MEDI2228-enhanced IFN-driven genes include chemokines/cytokines and receptors (i.e., CXCL9/10, CCL4L1/2, CCL22, CCL1/3/4/5, CCL3L1/3, TNFSF9/10, CSF2 (GM-CSF), CCR7), RSAD2, CASP1, ISGs (i.e., XAF1, TRIMs, IFITs, ISG15, GBP2/3, OAS1/2/L, RNASEL, MX1/2, FAS), RUNX3, GZMB, IKBKE, IRF1/6/7/9, STAT1/2/4/6, MB21D1(CGAS), TMEM173 (STING), IFIT1/2/3/5, and SOCS1/2/3. Results Regardless of genetic heterogeneity and resistance to current anti-MM therapies, MEDI2228 induced dose- and time-dependent DNA damage-ATM/ATR-CHK1/2 pathways, activation of cGAS-STING-TBK1-IRF3 and STAT1-IRF1-signaling cascades, as well as increased CD38 expression. It overcame CD38 downregulation triggered by IL6 and bone marrow stromal cell culture supernatant (BMSC-sup), via activation of STAT1-IRF1 without phosphorylation of STAT3 in immunomodulatory drugs (IMiDs)- and bortezomib-resistant MM cell lines. In contrast, MEDI2228 did not change CD38 expression and survival in BCMA-negative NK effector cells. Significantly, MEDI2228 with anti-CD38 monoclonal antibody Daratumumab (Dara) synergistically induced NK-mediated lysis of MM cell lines and autologous resistant patient MM cells, even in the presence of BMSC-sup and BMSCs. In parallel, MEDI2228 increased membrane expression of NKG2D ligands (NKG2DLs), i.e., MICA/B and ULBPs, in all MM cells tested, including Dara-resistant patient cells, correlating with enhanced MM cell susceptibility to NK cell killing. Since MEDI2228, but not its MMAF-ADC homolog M3 even used at >1-log higher concentrations enhanced surface expression of CD38 and NKG2DLs on MM cells, the potent DDR-mediated immunomodulation triggered by MEDI2228 vs M3 is critical in rendering MM cells more susceptible to Dara-induced NK cell killing. Importantly, M2 still activated STAT1/IRF1 signaling to induce CD38 and MICA/B expression in MM tumors grown in mice. All MM1S tumor-bearing NSG mice reconsituted with human NK cells became tumor-free following a single low dose M2 with Dara treatment, with 100% host survival. Conclusions Taken together, our data showed that MEDI2228 restored MM sensitivity to CD38 targeting by Dara without depleting NK cells and potentiated immunogenic cell death of MM cells. These results therefore provide the mechanistic rationale for clinical evaluation of combination CD38- and BCMA-directed immunotherapies to further improve patient outcome in MM.

Background RAS/CDK-dependent pathways play essential roles in multiple myeloma (MM) pathogenesis; both pathways are undruggable. We evaluated molecular changes associated with pathway-level responses after RAS/CDK inhibition to identify novel molecular targets. Method in our previous studies MM cells were treated with selected Erk1/2 and CDK4/6 inhibitors (Ei, Ci) to target RAS/CDK pathways. Our studies indicate strong synergistic (IC Results We evaluated mRNA splicing changes in MM cells, with and without Erk1/2 knockdown or with Ei+Ci treatment. Unsupervised clustering of deregulated genes showed dose-dependent treatment effects. Upregulation in response to Erk1/2 knockdown 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 complexes and are involved in intron retention processes, a marker of malignant transformation. We evaluated expressions of RAVER1 and SNRPB in 558 MM patent samples and 10 normal donor BM PCs and observed significant (p Conclusions Our studies show an association between RNA processing and RAS-CDK pathways in MM, identify a core splicing protein, SNRPB, as a novel target for modulating this undruggable cascade, and suggest that targeting spliceosome complexes is a promising therapy.

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.

We investigated here the novel immunomodulation and anti–multiple myeloma (MM) function of T cells engaged by the bispecific T-cell engager molecule AMG 701, and further examined the impact of AMG 701 in combination with immunomodulatory drugs (IMiDs; lenalidomide and pomalidomide). AMG 701 potently induced T-cell–dependent cellular cytotoxicity (TDCC) against MM cells expressing B-cell maturation antigen, including autologous cells from patients with relapsed and refractory MM (RRMM) (half maximal effective concentration

Therapeutically targeting CD138, a define multiple myeloma (MM) antigen, is not yet approved for patients. We here developed and determined the preclinical efficacy of VIS832, a novel therapeutic monoclonal antibody (MoAb) with differentiated CD138 target binding to BB4 that is anti-CD138 MoAb scaffold for indatuximab ravtansine (BT062). VIS832 demonstrated enhanced CD138-binding avidity and significantly improved potency to kill MM cell lines and autologous patient MM cells regardless of resistance to current standard-of-care therapies, via robust antibody-dependent cellular cytotoxicity and phagocytosis mediated by NK and macrophage effector cells, respectively. Specifically, CD38-targeting daratumumab-resistant MM cells were highly susceptible to VIS832 which, unlike daratumumab, spares NK cells. Superior maximal cytolysis of VIS832 vs. daratumumab corresponded to higher CD138 vs. CD38 levels in MM cells. Furthermore, VIS832 acted synergistically with lenalidomide or bortezomib to deplete MM cells. Importantly, VIS832 at a sub-optimal dose inhibited disseminated MM1S tumors in vivo as monotherapy (P

Multiple myeloma (MM) is an incurable cancer that derives pro-survival/proliferative signals from the bone marrow (BM) niche. Novel agents targeting not only cancer cells, but also the BM-niche have shown the greatest activity in MM. Histone deacetylases (HDACs) are therapeutic targets in MM and we previously showed that HDAC3 inhibition decreases MM proliferation both alone and in co-culture with bone marrow stromal cells (BMSC). In this study, we investigate the effects of HDAC3 targeting in BMSCs. Using both BMSC lines as well as patient-derived BMSCs, we show that HDAC3 expression in BMSCs can be induced by co-culture with MM cells. Knock-out (KO), knock-down (KD), and pharmacologic inhibition of HDAC3 in BMSCs results in decreased MM cell proliferation; including in autologous cultures of patient MM cells with BMSCs. We identified both quantitative and qualitative changes in exosomes and exosomal miRNA, as well as inhibition of IL-6 trans-signaling, as molecular mechanisms mediating anti-MM activity. Furthermore, we show that HDAC3-KD in BM endothelial cells decreases neoangiogenesis, consistent with a broad effect of HDAC3 targeting in the BM-niche. Our results therefore support the clinical development of HDAC3 inhibitors based not only on their direct anti- MM effects, but also their modulation of the BM microenvironment.

We investigate here how APRIL impacts immune regulatory T cells and directly contributes to the immunosuppressive multiple myeloma (MM) bone marrow (BM) microenvironment. First, APRIL receptor TACI expression is significantly higher in regulatory T cells (Tregs) than conventional T cells (Tcons) from the same patient, confirmed by upregulated Treg markers, i.e., Foxp3, CTLA-4. APRIL significantly stimulates proliferation and survival of Tregs, whereas neutralizing anti-APRIL monoclonal antibodies (mAbs) inhibit these effects. Besides TACI-dependent induction of cell cycle progression and anti-apoptosis genes, APRIL specifically augments Foxp3, IL-10, TGFβ1, and PD-L1 in Tregs to further enhance Treg-inhibited Tcon proliferation. APRIL further increases MM cell-driven Treg (iTreg) via TACI-dependent proliferation associated with upregulated IL-10, TGFβ1, and CD15s in iTreg, which further inhibits Tcons. Osteoclasts producing APRIL and PD-L1 significantly block Tcon expansion by iTreg generation, which is overcome by combined treatment with anti-APRIL and anti-PD1/PD-L1 mAbs. Finally, APRIL increases IL-10-producing B regulatory cells (Bregs) via TACI on BM Bregs of MM patients. Taken together, these results define novel APRIL actions via TACI on Tregs and Bregs to promote MM cell survival, providing the rationale for targeting APRIL/TACI system to alleviate the immunosuppressive BM milieu 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.

Background Resistance to immune approaches poses a major challenge to effective immunotherapy and long-term clinical outcome in multiple myeloma (MM). Here we identified loss-of-function of gamma-aminobutyric acid receptor-associated protein (GABARAP) as a tumor-intrinsic mechanism of resistance to bortezomib (BTZ)-induced immunogenic cell death (ICD), the immunogenic consequence of apoptosis resulting in specific anti-MM immunity via T-cell priming by dendritic cells (DCs). Methods We found that BTZ induces ICD in human and murine MM cell lines that is dependent on the exposure of calreticulin (CALR), which drives DCs-mediated phagocytosis of MM cells. DCs-phagocytosis is followed by a specific T cell activation, with a significant increase of CD4+ effector memory (EM), total CD8+, CD8+ EM, and CD8+ terminally differentiated EM cells. Isolated T cells after co-cultures showed the presence of MM specific CTLs that were able to efficiently induce lysis of MM cells. Our results were validated using primary cells from MM patients. Moreover, induction of a protective immune response was confirmed in vivo. Specifically, treatment of 5TGM1 tumors with BTZ induced a tumor regression in a syngeneic model; and injection of live 5TGM1WT two weeks after regression did not result in tumor development, consistent with induction of immunological memory as confirmed by ELISPOT of mouse splenocytes. We identified a specific ICD signature induced by BTZ in mice; and we found that increased expression of the human orthologs of this signature was positively correlated with OS (p=0.01) in patients enrolled in the IFM/DFCI 2009 study. Notably, these functional immunologic sequelae were abrogated after BTZ treatment of CALRKO MM cells both in vitro and in vivo, confirming the obligate role of CALR exposure in the ICD process. Results By interrogating the IFM/DFCI dataset and focusing on genes involved in ICD processes that were correlated with MM patients clinical outcome, we found that low levels of GABARAP (chr17p13.1), an autophagy regulator and putative CALR binding partner, negatively impact MM patient clinical outcome (EFS, p=0.0032); even excluding HR patients with 17p deletion (EFS, p=0.018). Interestingly, KMS11 cells carrying monoallelic deletion of GABARAP were resistant to induction of ICD by BTZ; and sensitivity was restored after overexpression of the gene. Moreover, GABARAPKO in 3 ICD-sensitive cell lines abrogated the induction of ICD by BTZ; and add-back experiments by pre-treatment with recombinant CALR or GABARAP overexpression in KO clones restored ICD. Finally, CyTOF confirmed that treatment of GABARAPKO cells with BTZ failed to activate an efficient T cell response. Conclusions our study demonstrates that loss-of-function of GABARAP, particularly in HR patients with 17p deletion, contributes to tumor immune evasion and ICD resistance. These studies provide the framework for novel combination treatments to restore anti-MM immunity and improve patient outcome in HR MM.

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

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.