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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.

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.

Purpose: Accumulating evidence indicates that intratumor heterogeneity is prevalent in multiple myeloma and that a collection of multiple, genetically distinct subclones are present within the myeloma cell population. It is not clear whether the size of clonal myeloma populations harboring unique cytogenetic abnormalities carry any additional prognostic value. Experimental Design: We analyzed the prognostic impact of cytogenetic aberrations by fluorescence in situ hybridization at different cutoff values in a cohort of 333 patients with newly diagnosed myeloma and 92 patients with relapsed myeloma. Results: We found that nearly all IgH-related arrangements were observed in a large majority of the purified plasma cells; however, 13q deletion, 17p deletion, and 1q21 amplification appeared in different percentages within the malignant plasma cell population. Based on the size of subclones carrying these cytogenetic aberrations, the patients were divided into four groups: 0%–10%, 10.5%–20%, 20.5%–50%, and >50%. Receiver-operating characteristics analysis was applied to determine the optimal cutoff value with the greatest differential survival and showed that the most powerful clone sizes were 10% for 13q deletion, 50% for 17p deletion, and 20% for 1q21 gains, which provided the best possible cutoffs for predicting poor outcomes. Conclusions: Our study indicated that the impact of clone size on prognostic value varies between specific genetic abnormalities. Prognostic value was observed for even a subgroup of plasma cells harboring the cytogenetic aberration of 13q deletion and 1q21 gains; however, 17p deletion displayed the most powerful cutoff for predicting survival only if the predominant clones harbored the abnormality. Clin Cancer Res; 21(9); 2148–56. ©2015 AACR.

Purpose: The efficacy of peptide vaccines may be enhanced by stimulating immune cells with multiple peptides derived from distinct tumor-associated antigens. We have evaluated the heteroclitic XBP1-US184–192 (YISPWILAV), heteroclitic XBP1-SP367–375 (YLFPQLISV), native CD138260–268 (GLVGLIFAV), and native CS1239–247 (SLFVLGLFL) peptides, which have strong HLA-A2 affinity and immunogenicity in combination, for their ability to elicit multiple myeloma antigen–specific responses. Experimental Design: Multipeptide-specific cytotoxic T lymphocytes (MP-CTL) were generated by the stimulation of CD3+ T lymphocytes from HLA-A2+ individuals with either autologous mature dendritic cells or T2 cells pulsed with a cocktail of these four peptides. Results: The peptide cocktail did not compromise tumor antigen–specific activity of CTLs. MP-CTLs displayed increased total, effector memory (CCR7−CD45RO+), and activated (CD69+) CD3+CD8+ T lymphocytes. In addition, MP-CTL showed IFN-γ production, cell proliferation, and cytotoxicity against HLA-A2+ multiple myeloma cells, including cells of HLA-A2+ patients with multiple myeloma. Importantly, MP-CTLs showed specific responses in functional assays to each relevant peptide but not to an irrelevant HLA-A2–specific CMV pp65 (NLVPMVATV) peptide. Conclusions: These results highlight the potential therapeutic application of vaccination with a cocktail of HLA-A2–specific peptides to induce CTLs with a broad spectrum of immune responses against multiple myeloma antigens. Clin Cancer Res; 18(17); 4850–60. ©2012 AACR.

Purpose: The transcription factor specificity protein 1 (Sp1) controls number of cellular processes by regulating the expression of critical cell cycle, differentiation, and apoptosis-related genes containing proximal GC/GT-rich promoter elements. We here provide experimental and clinical evidence that Sp1 plays an important regulatory role in multiple myeloma (MM) cell growth and survival. Experimental Design: We have investigated the functional Sp1 activity in MM cells using a plasmid with Firefly luciferase reporter gene driven by Sp1-responsive promoter. We have also used both siRNA- and short hairpin RNA–mediated Sp1 knockdown to investigate the growth and survival effects of Sp1 on MM cells and further investigated the anti-MM activity of terameprocol (TMP), a small molecule that specifically competes with Sp1-DNA binding in vitro and in vivo. Results: We have confirmed high Sp1 activity in MM cells that is further induced by adhesion to bone marrow stromal cells (BMSC). Sp1 knockdown decreases MM cell proliferation and induces apoptosis. Sp1-DNA binding inhibition by TMP inhibits MM cell growth both in vitro and in vivo, inducing caspase-9–dependent apoptosis and overcoming the protective effects of BMSCs. Conclusions: Our results show Sp1 as an important transcription factor in myeloma that can be therapeutically targeted for clinical application by TMP. Clin Cancer Res; 17(20); 6500–9. ©2011 AACR.

Purpose: We investigated the in vitro and in vivo anti-multiple myeloma activity of monoclonal antibody (mAb) 1339, a high-affinity fully humanized anti-interleukin 6 mAb (immunoglobulin G1), alone and in combination with conventional and novel anti-multiple myeloma agents, as well as its effect on bone turnover. Experimental Design: We examined the growth inhibitory effect of 1339 against multiple myeloma cell lines in the absence and in the presence of bone marrow stromal cells, alone or in combination with dexamethasone, bortezomib, perifosine, and Revlimid. Using the severe combined immunodeficient (SCID)–hu murine model of multiple myeloma, we also examined the effect of 1339 on multiple myeloma cell growth and multiple myeloma bone disease. Results: mAb 1339 significantly inhibited growth of multiple myeloma cell in the presence of bone marrow stromal cell in vitro, associated with inhibition of phosphorylation of signal transducer and activator of transcription 3, extracellular signal-regulated kinase 1/2, and Akt. In addition, mAb 1339 enhanced cytotoxicity induced by dexamethasone, as well as bortezomib, lenalidomide, and perifosine, in a synergistic fashion. Importantly mAb 1339 significantly enhanced growth inhibitory effects of dexamethasone in vivo in SCID-hu mouse model of multiple myeloma. mAb 1339 treatment also resulted in inhibition of osteoclastogenesis in vitro and bone remodeling in SCID-hu model. Conclusions: Our data confirm in vitro and in vivo anti-multiple myeloma activity of, as well as inhibition of bone turnover by, fully humanized mAb 1339, as a single agent and in combination with conventional and novel agents, providing a rationale for its clinical evaluation in multiple myeloma. (Clin Cancer Res 2009;15(23):7144–52)

Purpose: We investigated the antitumor effect of murine/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives against multiple myeloma (MM) cells in vitro and in vivo. Experimental Design: We examined the growth inhibitory effect of BT062-SPDB-DM4, BT062-SMCC-DM1, and BT062-SPP-DM1 against MM cell lines and primary tumor cells from MM patients. We also examined in vivo activity of these agents in murine MM cell xenograft model of human and severe combined immunodeficient (SCID) mice bearing implant bone chips injected with human MM cells (SCID-hu model). Results: Anti-CD138 immunoconjugates significantly inhibited growth of MM cell lines and primary tumor cells from MM patients without cytotoxicity against peripheral blood mononuclear cells from healthy volunteers. In MM cells, they induced G2-M cell cycle arrest, followed by apoptosis associated with cleavage of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase. Nonconjugated nBT062 completely blocked cytotoxicity induced by nBT062-maytansinoid conjugate, confirming that specific binding is required for inducing cytotoxicity. Moreover, nBT062-maytansinoid conjugates blocked adhesion of MM cells to bone marrow stromal cells. The coculture of MM cells with bone marrow stromal cells protects against dexamethasone-induced death but had no effect on the cytotoxicity of immunoconjugates. Importantly, nBT062-SPDB-DM4 and nBT062-SPP-DM1 significantly inhibited MM tumor growth in vivo and prolonged host survival in both the xenograft mouse models of human MM and SCID-hu mouse model. Conclusion: These results provide the preclinical framework supporting evaluation of nBT062-maytansinoid derivatives in clinical trials to improve patient outcome in MM.

Purpose: CD1d-restricted invariant natural killer T (iNKT) cells are important immunoregulatory cells in antitumor immune responses. However, the quantitative and qualitative defects of iNKT cells in advanced multiple myeloma hamper their antitumor effects. Therefore, the development of functional iNKT cells may provide a novel strategy for the immunotherapy in multiple myeloma. Experimental Design: We activated and expanded iNKT cells from multiple myeloma patients with α-galactosylceramide (α-GalCer)-pulsed dendritic cells, characterized their antitumor effects by the cytokine production profile and cytotoxicity against multiple myeloma cells, and explored the effects of immunomodulatory drug lenalidomide on these iNKT cells. We also investigated the expression of CD1d by primary multiple myeloma cells and its function to activate iNKT cells. Results: We established highly purified functional iNKT cell lines from newly diagnosed and advanced multiple myeloma patients. These CD1d-restricted iNKT cell lines produced high level of antitumor Th1 cytokine in response to α-GalCer-pulsed primary multiple myeloma cells, CD1d-transfected MM1S cell line, and dendritic cells. Moreover, iNKT cell lines displayed strong cytotoxicity against α-GalCer-pulsed primary multiple myeloma cells. Importantly, lenalidomide further augmented the Th1 polarization by iNKT cell lines via increased Th1 cytokine production and reduced Th2 cytokine production. We also showed that CD1d was expressed in primary multiple myeloma cells at mRNA and protein levels from the majority of multiple myeloma patients, but not in normal plasma cells and multiple myeloma cell lines, and CD1d+ primary multiple myeloma cells presented antigens to activate iNKT cell lines. Conclusions: Taken together, our results provide the preclinical evidence for the iNKT cell-mediated immunotherapy and a rationale for their use in combination with lenalidomide in multiple myeloma treatment.