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The anti-CD38 monoclonal antibody SAR650984 (SAR) is showing promising clinical activity in treatment of relapsed and refractory multiple myeloma (MM). Besides effector-mediated antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity, we here define molecular mechanisms of SAR-directed MM cell death and enhanced anti-MM activity triggered by SAR with Pomalidomide (Pom). Without Fc-cross-linking agents or effector cells, SAR specifically induces homotypic aggregation (HA)-associated cell death in MM cells dependent on the level of cell surface CD38 expression, actin cytoskeleton and membrane lipid raft. SAR and its F(ab)'2 fragments trigger caspase 3/7-dependent apoptosis in MM cells highly expressing CD38, even with p53 mutation. Importantly, SAR specifically induces lysosome-dependent cell death (LCD) by enlarging lysosomes and increasing lysosomal membrane permeabilization associated with leakage of cathepsin B and LAMP-1, regardless of the presence of interleukin-6 or bone marrow stromal cells. Conversely, the lysosomal vacuolar H+-ATPase inhibitor blocks SAR-induced LCD. SAR further upregulates reactive oxygen species. Pom enhances SAR-induced direct and indirect killing even in MM cells resistant to Pom/Len. Taken together, SAR is the first therapeutic monoclonal antibody mediating direct cytotoxicity against MM cells via multiple mechanisms of action. Our data show that Pom augments both direct and effector cell-mediated MM cytotoxicity of SAR, providing the framework for combination clinical trials.

The key nuclear export protein CRM1/XPO1 may represent a promising novel therapeutic target in human multiple myeloma (MM). Here we showed that chromosome region maintenance 1 (CRM1) is highly expressed in patients with MM, plasma cell leukemia cells and increased in patient cells resistant to bortezomib treatment. CRM1 expression also correlates with increased lytic bone and shorter survival. Importantly, CRM1 knockdown inhibits MM cell viability. Novel, oral, irreversible selective inhibitors of nuclear export (SINEs) targeting CRM1 (KPT-185, KPT-330) induce cytotoxicity against MM cells (ED 50 <200 n M ), alone and cocultured with bone marrow stromal cells (BMSCs) or osteoclasts (OC). SINEs trigger nuclear accumulation of multiple CRM1 cargo tumor suppressor proteins followed by growth arrest and apoptosis in MM cells. They further block c-myc, Mcl-1, and nuclear factor κB (NF-κB) activity. SINEs induce proteasome-dependent CRM1 protein degradation; concurrently, they upregulate CRM1, p53-targeted, apoptosis-related, anti-inflammatory and stress-related gene transcripts in MM cells. In SCID mice with diffuse human MM bone lesions, SINEs show strong anti-MM activity, inhibit MM-induced bone lysis and prolong survival. Moreover, SINEs directly impair osteoclastogenesis and bone resorption via blockade of RANKL-induced NF-κB and NFATc1, with minimal impact on osteoblasts and BMSCs. These results support clinical development of SINE CRM1 antagonists to improve patient outcome in MM.

Recent studies have delineated cancer-type-specific roles of histone 3 lysine 27 (H3K27) demethylase KDM6B/JMJD3 depending on its H3K27 demethylase activity. Here we show that KDM6B is expressed in multiple myeloma (MM) cells; and that shRNA-mediated knockdown and CRISPR-mediated knockout of KDM6B abrogate MM cell growth and survival. Tumor necrosis factor-α or bone marrow stromal cell culture supernatants induce KDM6B , which is blocked by IKKβ inhibitor MLN120B, suggesting that KDM6B is regulated by NF-κB signaling in MM cells. RNA-seq and subsequent ChIP-qPCR analyses reveal that KDM6B is recruited to the loci of genes encoding components of MAPK signaling pathway including ELK1 and FOS , and upregulates expression of these genes without affecting H3K27 methylation level. Overexpression of catalytically inactive KDM6B activates expression of MAPK pathway-related genes, confirming its function independent of demethylase activity. We further demonstrate that downstream targets of KDM6B, ELK1 and FOS, confer MM cell growth. Our study therefore delineates KDM6B function that links NF-κB and MAPK signaling pathway mediating MM cell growth and survival, and validates KDM6B as a novel therapeutic target in MM.

Histone deacetylase (HDAC) inhibitors have been extensively investigated as therapeutic agents in cancer. However, the biological role of class IIa HDACs (HDAC4, 5, 7 and 9) in cancer cells, including multiple myeloma (MM), remains unclear. Recent studies show HDAC4 interacts with activating transcription factor 4 (ATF4) and inhibits activation of endoplasmic reticulum (ER) stress-associated proapoptotic transcription factor C/EBP homologous protein (CHOP). In this study, we hypothesized that HDAC4 knockdown and/or inhibition could enhance apoptosis in MM cells under ER stress condition by upregulating ATF4, followed by CHOP. HDAC4 knockdown showed modest cell growth inhibition; however, it markedly enhanced cytotoxicity induced by either tunicamycin or carfilzomib (CFZ), associated with upregulating ATF4 and CHOP. For pharmacological inhibition of HDAC4, we employed a novel and selective class IIa HDAC inhibitor TMP269, alone and in combination with CFZ. As with HDAC4 knockdown, TMP269 significantly enhanced cytotoxicity induced by CFZ in MM cell lines, upregulating ATF4 and CHOP and inducing apoptosis. Conversely, enhanced cytotoxicity was abrogated by ATF4 knockdown, confirming that ATF4 has a pivotal role mediating cytotoxicity in this setting. These results provide the rationale for novel treatment strategies combining class IIa HDAC inhibitors with ER stressors, including proteasome inhibitors, to improve patient outcome in MM.

Epigenetic signaling pathways are implicated in tumorigenesis and therefore histone deacetylases (HDACs) represent novel therapeutic targets for cancers, including multiple myeloma (MM). Although non-selective HDAC inhibitors show anti-MM activities, unfavorable side effects limit their clinical efficacy. Isoform- and/or class-selective HDAC inhibition offers the possibility to maintain clinical activity while avoiding adverse events attendant to broad non-selective HDAC inhibition. We have previously reported that HDAC3 inhibition, either by genetic knockdown or selective inhibitor BG45, abrogates MM cell proliferation. Here we show that knockdown of HDAC3 , but not HDAC1 or HDAC2 , as well as BG45, downregulate expression of DNA methyltransferase 1 (DNMT1) mediating MM cell proliferation. DNMT1 expression is regulated by c-Myc, and HDAC3 inhibition triggers degradation of c-Myc protein. Moreover, HDAC3 inhibition results in hyperacetylation of DNMT1, thereby reducing the stability of DNMT1 protein. Combined inhibition of HDAC3 and DNMT1 with BG45 and DNMT1 inhibitor 5-azacytidine (AZA), respectively, triggers synergistic downregulation of DNMT1, growth inhibition and apoptosis in both MM cell lines and patient MM cells. Efficacy of this combination treatment is confirmed in a murine xenograft MM model. Our results therefore provide the rationale for combination treatment using HDAC3 inhibitor with DNMT1 inhibitor to improve patient outcome in MM.

Proteasome inhibition is an effective therapy for multiple myeloma (MM) patients; however, the emergence of drug resistance is common. Novel therapeutic strategies to overcome proteasome inhibitor resistance are needed. In this study, we examined whether targeting deubiquitylating (DUB) enzymes upstream of 20S proteasome overcomes proteasome inhibitor resistance. Gene expression analysis, immunohistochemical studies of MM patient bone marrow, reverse transcription–PCR and protein analysis show that Rpn11/POH1, a DUB enzyme upstream of 20S proteasome, is more highly expressed in patient MM cells than in normal plasma cells. Importantly, Rpn11 expression directly correlates with poor patient survival. Loss-of-function studies show that Rpn11-siRNA knockdown decreases MM cell viability. Pharmacological inhibition of Rpn11 with O -phenanthroline (OPA) blocks cellular proteasome function, induces apoptosis in MM cells and overcomes resistance to proteasome inhibitor bortezomib. Mechanistically, Rpn11 inhibition in MM cells activates caspase cascade and endoplasmic stress response signaling. Human MM xenograft model studies demonstrate that OPA treatment reduces progression of tumor growth and prolongs survival in mice. Finally, blockade of Rpn11 increases the cytotoxic activity of anti-MM agents lenalidomide, pomalidomide or dexamethasone. Overall, our preclinical data provide the rationale for targeting DUB enzyme Rpn11 upstream of 20S proteasome to enhance cytotoxicity and overcome proteasome inhibitor resistance in MM.

Arginine methyltransferases critically regulate cellular homeostasis by modulating the functional outcome of their substrates. The protein arginine methyltransferase 5 (PRMT5) is an enzyme involved in growth and survival pathways promoting tumorigenesis. However, little is known about the biologic function of PRMT5 and its therapeutic potential in multiple myeloma (MM). In the present study, we identified and validated PRMT5 as a new therapeutic target in MM. PRMT5 is overexpressed in patient MM cells and associated with decreased progression-free survival and overall survival. Either genetic knockdown or pharmacological inhibition of PRMT5 with the inhibitor EPZ015666 significantly inhibited growth of both cell lines and patient MM cells. Furthermore, PRMT5 inhibition abrogated NF-κB signaling. Interestingly, mass spectrometry identified a tripartite motif-containing protein 21 TRIM21 as a new PRMT5-partner, and we delineated a TRIM21-dependent mechanism of NF-κB inhibition. Importantly, oral administration of EPZ015666 significantly decreased MM growth in a humanized murine model of MM. These data both demonstrate the oncogenic role and prognostic relevance of PRMT5 in MM pathogenesis, and provide the rationale for novel therapies targeting PRMT5 to improve patient outcome.

Homologous recombination (HR), a mechanism to accurately repair DNA in normal cells, is deregulated in cancer. Elevated/deregulated HR is implicated in genomic instability and telomere maintenance, which are critical lifelines of cancer cells. We have previously shown that HR activity is elevated and significantly contributes to genomic instability in Barrett’s esophageal adenocarcinoma (BAC). The purpose of this study was to evaluate therapeutic potential of HR inhibition, alone and in combination with telomerase inhibition, in BAC. We demonstrate that telomerase inhibition in BAC cells increases HR activity, RAD51 expression, and association of RAD51 to telomeres. Suppression of HR leads to shorter telomeres as well as markedly reduced genomic instability in BAC cells over time. Combination of HR suppression (whether transgenic or chemical) with telomerase inhibition, causes a significant increase in telomere attrition and apoptotic death in all BAC cell lines tested, relative to either treatment alone. A subset of treated cells also stain positive for β-galactosidase, indicating senescence. The combined treatment is also associated with decline in S-phase and a strong G2/M arrest, indicating massive telomere attrition. In a subcutaneous tumor model, the combined treatment resulted in the smallest tumors, which were even smaller ( P =0.001) than those that resulted from either treatment alone. Even the tumors removed from these mice had significantly reduced telomeres and evidence of apoptosis. We therefore conclude that although telomeres are elongated by telomerase, elevated RAD51/HR assist in their maintenance/stabilization in BAC cells. Telomerase inhibitor prevents telomere elongation but induces RAD51/HR, which contributes to telomere maintenance/stabilization and prevention of apoptosis, reducing the efficacy of treatment. Combining HR inhibition with telomerase renders telomeres more vulnerable to degradation and significantly increases/expedites their attrition, leading to apoptosis. We therefore demonstrate that a therapy targeting HR and telomerase has the potential to prevent both tumor growth and genomic evolution in BAC.