Abstract: Aurora-A is a mitotic kinase that regulates mitotic spindle formation and segregation. In multiple myeloma (MM), high Aurora-A gene expression has been correlated with centrosome amplification and proliferation; thus, inhibition of Aurora-A in MM may prove to be therapeutically beneficial. Here we assess the in vitro and in vivo anti-MM activity of MLN8237, a small-molecule Aurora-A kinase inhibitor. Treatment of cultured MM cells with MLN8237 results in mitotic spindle abnormalities, mitotic accumulation, as well as inhibition of cell proliferation through apoptosis and senescence. In addition, MLN8237 up-regulates p53 and tumor suppressor genes p21 and p27. Combining MLN8237 with dexamethasone, doxorubicin, or bortezomib induces synergistic/additive anti-MM activity in vitro. In vivo anti-MM activity of MLN8237 was confirmed using a xenograft-murine model of human-MM. Tumor burden was significantly reduced (P = .007) and overall survival was significantly increased (P 〈 .005) in animals treated with 30 mg/kg MLN8237 for 21 days. Induction of apoptosis and cell death by MLN8237 were confirmed in tumor cells excised from treated animals by TdT-mediated dUTP nick end labeling assay. MLN8237 is currently in phase 1 and phase 2 clinical trials in patients with advanced malignancies, and our preclinical results suggest that MLN8237 may be a promising novel targeted therapy in MM.

Abstract: Arginine-specific methyltransferases critically regulate cellular homeostasis by dictating the biological outcome of target proteins. Among them, Protein Arginine Methyltransferase 5 (PRMT5) has attracted growing interest due to its role as an enzyme mediating epigenetic regulation of anti-cancer target genes, as well as in methylation of non-histone proteins involved in growth-regulating and survival pathways including p53. However, little is known about its biologic function in multiple myeloma (MM). To first evaluate the clinical significance of PRMT5 in MM pathogenesis, we analyzed RNA-seq data from newly-diagnosed MM patients and identified highly upregulated PRMT5 in 320 patients' CD138+ cells compared to 16 samples of normal bone marrow (BM) plasma cells. Additional analysis of PRMT5 expression in two independent datasets also showed further PRMT5 mRNA upregulation during progression of MM. Immunohistochemical staining also confirmed elevated expression of PRMT5 in BM biopsies from MM patients as compared to healthy individuals and monoclonal gammopathy of undetermined significance (MGUS). Moreover, analysis of the prognostic significance of PRMT5 expression in MM patients enrolled on IFM/DFCI 2009 clinical study showed that high PRMT5 expression was associated with poor prognosis in terms of both event free (p= 0.016) and overall survival (p=0.018). Consistently, we also found upregulated PRMT5 expression at both mRNA and protein levels in MM cell lines (N=11) and patients CD138+ MM cells (N=3) as compared to PBMCs from healthy volunteers, associated with a parallel increase of cellular symmetric arginine di-methylation (SDMA) substrates. Interestingly, genetic depletion of PRMT5 in H929 (p53wt) and KMS11 (p53null) MM cell lines by shRNA decreased SDMA levels, associated with cell growth inhibition in a p53-independent manner. Likewise, pharmacological inhibition of PRMT5 with the small molecule inhibitor EPZ015666 triggered decreased SDMA levels, cell growth, survival, and clonogenicity, as well as induction of caspase-dependent apoptosis in MM cell lines. Moreover, although PRMT5 and SDMA levels were increased in MM cells cultured in the presence of BM stromal cell supernatant, cytotoxic activity of EPZ015666 was maintained. Notably, drug treatment significantly impaired cell proliferation of patient MM cells (n=2) even in the presence of BM mononuclear or stromal cells, without toxicity on normal PBMCs. At the level of gene expression modulation, PRMT5 inhibition was associated with downregulation of NF-kB-dependent transcription, evidenced by both gene set enrichment analysis (GSEA) and Ingenuity Upstream Regulator Analysis. Moreover, analysis of protein levels confirmed reduction of both canonical and non-canonical NF-kB pathways, evidenced by significantly decreased NF-kB DNA binding activity by ELISA. Importantly, Mass Spectrometry analysis identified TRIM21 as a new PRMT5 interactor; and EPZ015666-treated cells showed that PRMT5 methylates TRIM21 evidenced by WB analysis. Since TRIM21 mediates monoubiquitination of IKKbeta, thereby triggering its selective autophagy-mediated degradation, we next analyzed EPZ015666 effects on IKKbeta. Treatment increased both monoubiquitination of IKKbeta and the formation of IKKbeta-TRIM21-pBECLIN1-pULK1 autophagic complexes. Conversely, inhibition of autophagosome formation by 3-methyladenine abrogated the anti-MM activity of EPZ015666 and IKKbeta degradation, indicating that selective autophagic degradation of IKKbeta and inhibition of NF-kB signaling mediates EPZ015666-triggered anti-MM activity. Consistent with this view, confocal microscopy analysis also confirmed co-localization of IKKbeta in the autophagosome after EPZ015666 treatment. Finally, stable silencing of TRIM21 in MM cell lines significantly abrogated the anti-proliferative effect of EPZ015666. Collectively, these data delineate arginine methylation as a new control mechanism of MM cell growth, and demonstrate that inhibiting PRMT5 decreases tumor cell survival via blockade of NF-kB signaling, even in the context of the BM milieu. These data demonstrate the biologic and prognostic significance of PRMT5 in MM pathogenesis, and provide the rationale for novel therapies targeting PRMT5 to improve patient outcome in MM. Disclosures Hideshima: Acetylon: Consultancy; C4 Therapeutics: Equity Ownership. Munshi:Takeda: Consultancy; Celgene Corporation: Consultancy; Merck: Consultancy; Pfizer: Consultancy; Oncopep: Consultancy, Equity Ownership. Anderson:Gilead: 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; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Oncoprep: Equity Ownership; Oncoprep: Equity Ownership; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: 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.

Abstract: 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.

Abstract: Introduction: The bone marrow (BM) niche promotes multiple myeloma (MM) growth, survival and drug resistance. Therapies targeting both cancer cells and the microenvironment are highly effective. We were interested in identifying novel signaling pathways supporting MM pathogenesis through MM-BM crosstalk. The transmembrane receptor Roundabout 1 (ROBO1) plays a role in growth and dissemination of solid tumors, however its function in MM is unknown. Material and Methods: We analyzed ROBO1 expression in cell lines and primary samples via western blot, immunohistochemistry (IHC) and gene expression profiling. We used short hairpin RNA and CRISPR-Cas9 for ROBO1 knock down (KD) and knock out (KO), respectively. For protein structure-function and rescue studies, we stably expressed full-length (FL) or mutant ROBO1 devoid of extracellular (Cyt) or intracellular domain (DeltaCyt), including patient-derived G674* truncation, with a C-terminus FLAG tag. We used a 3D hydrogel encapsulation system to study proliferation; FLAG immunoprecipitation (IP) followed by mass spectrometry to identify ROBO1 interacting partners; and immunofluorescence to detect ROBO1 localization. To study tumor growth in vivo, we performed PET-CT of mice inoculated subcutaneously or intramedullary with WT or ROBO1 KO MM cells and retrieved tumors for RNA sequencing. To study dissemination and homing, KO and FL addback MM cells were injected intravenously in SCID mice. Mice were monitored for development of tumors or hindlimb paralysis and femora/tumors harvested once mice reached endpoint. Results: ROBO1 is highly expressed in MM cell lines and primary cells but low/absent in normal plasma cells and other hematologic cancer cell lines. ROBO1 KD is specifically cytotoxic for MM cells and ROBO1 KO decreases proliferation, a phenotype fully rescued by FL ROBO1. Compared to WT, ROBO1 KO significantly decreases intramedullary (mean tumor volume (MTV): 1323 vs 457 mm3, p value= 0.02) and extramedullary (MTV: 2684 vs 823 mm3, p value= 0.001) tumor growth in vivo. We further discovered that ROBO1 KO decreases adhesion of MM to BM endothelial and BMSC, which is fully rescued by FL ROBO1. In a disseminated mouse model, ROBO1 KO cells generate bone plasmacytoma with reduced BM invasion, as compared to the extensive BM infiltration observed with ROBO1 FL cells. Consistently, in primary samples from patients, we detected ROBO1 expression only in 1 out of 10 solitary plasmacytoma (dim staining) as compared to 14 out of 14 MM bone marrow biopsy samples tested (11 strong, 3 dim, p value= 0.0001). Mechanistically, we show for the first time that ROBO1 C-terminus is cleaved in a ligand-independent fashion; translocates to the nucleus; and is necessary and sufficient to rescue ROBO1 KO proliferative defect. Viceversa, mutants lacking the cytoplasmic domain, including the G674* truncation, act dominantly negative. Interactomic and RNA sequencing studies point to a previously unknown function of ROBO1 in RNA processing, setting the bases for future studies. Conclusions: We show for the first time that ROBO1 is necessary for MM growth and homing to the BM. Cleaved ROBO1 cytosolic domain translocates to the nucleus and is necessary and sufficient to rescue ROBO1 KO proliferative defect, possibly by participating in RNA processing. These data suggest that ROBO1 C-terminus may be a novel molecular target in MM. Citation Format: Giada Bianchi, Peter G. Czarnecki, Matthew Ho, Aldo M. Roccaro, Antonio Sacco, Yawara Kawano, Annamaria Gulla, Anil Aktas Samur, Tianzeng Cheng, Kenneth Wen, Yu-Tzu Tai, Maria Moscvin, Xinchen Wu, Gulden Camci-Unal, Matteo Claudio Da VIa, Niccolo Bolli, Ruben D. Carrasco, Irene M. Ghobrial, Kenneth C. Anderson. ROBO1 promotes homing, dissemination, and survival of multiple myeloma within the bone marrow microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB217.

Abstract: The bone marrow (BM) microenvironment confers growth, survival, and drug resistance in multiple myeloma (MM) cells. Here we have characterized the role of plasmacytoid dendritic cells (pDCs) in the MM BM milieu. Immunochemistry (IHC) analysis of tissue microarrays on MM patient BM biopsies with Abs specific against pDCs (CD123) and MM cells (CD138) shows pDCs in proximity of MM cells. Quantification of pDCs obtained by direct isolation from MM patient BM aspirates or peripheral blood (PB) showed increased numbers of pDCs in MM-BM compared to PB. Freshly isolated pDCs from normal healthy donors stimulate significant growth of MM cells: 4.1 ± 0.8 fold increase {3H}-thymidine uptake in MM cells co-cultured with pDCs versus control MM cells alone, (P 〈 0.005). Irradiated pDCs retain their ability to trigger proliferation of MM cells; furthermore, pDC-depleted PBMCs did not trigger significant growth of MM cells, confirming a specific MM cell growth-promoting activity of pDCs. Co-culture of patient MM cells with pDCs triggered a significant growth of tumor cells, but not normal BM-derived plasma cells. Importantly, both allogeneic and autologous MM-derived pDCs induced tumor cell growth. To determine whether pDCs enhance MM cell growth in vivo, mice were implanted subcutaneously with pDCs alone, MM cells alone or pDCs + MM cells, and tumor growth was monitored over 3 weeks. A robust growth of tumor in mice receiving pDC + MM occurred within 12 days, whereas mice injected with MM cell alone showed a similar tumor growth only at day 21. We further examined the ability of pDCs to prolong ex-vivo survival of patient MM cells. Co-culture of pDCs with patient MM cells significantly increased the survival of patient tumor cells (59%, n=5 P 〈 0.05), and IHC analysis of pDCs-MM cells co-cultures at 4 weeks confirmed that MM cells are clonal. We next examined the effect of anti-MM agents bortezomib and dexamethasone on the viability of pDCs and pDC-induced MM cell growth. Treatment of pDCs with bortezomib (20 nM) or dexamethasone (500 nM) does not significantly decrease viability of these cells (P = 0.25), higher concentrations of bortezomib (50 and 100 nM) decrease the viability of pDCs by less than 10%. Importantly, proliferation assays confirmed that pDCs triggered MM cell growth even in the presence of bortezomib, albeit to a lesser extent than without bortezomib(P 〈 0.05). Microarray analysis showed that the pDCs-MM cells interaction triggered significant changes in transcriptional activity of genes related to growth, survival, anti-apoptosis, and migration in MM cells. Cytokine bead array analysis of supernatants from pDCs-MM cells co-cultures showed a marked increase in the secretion of MM cell growth, survival and chemotactic factors, such as IL-10, IL-6, IL-8, TNF-α, IL-1Rα, IL-1α, IL-13, IL-15, CD40L, MCP-1, MIP-1β, IP10 and VEGF. Overall, our data therefore show that pDCs predominantly localize in the MM BM and functionally interact with MM cells via cell-cell contact and subsequent cytokine secretion, allowing for MM cell growth and survival even in the presence of conventional and novel drugs. These studies will provide the basis for novel therapeutic approaches targeting pDC-MM interaction to improve patient outcome in MM.

Abstract: 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.