Decreased Multiple Myeloma Cell Research Articles

Anti‐myeloma Effect of Xanthohumol on Multiple Myeloma Cells

Multiple myeloma (MM) is characterized as a type of blood cell cancer, specifically affecting plasma cells within bone marrow. This disease results in approximately 13,000 deaths each year in the United States. The over‐accumulation of malignant plasma cells crowds out normal, healthy blood and immune cells, leaving the host potentially immunocompromised and prone to weakness, fatigue, weight loss and broken bones. Xanthohumol (XN), a prenylated flavonoid derived from the common hop plant Humulus lupulus, has been shown to have profound anti‐obesity, anti‐inflammatory and anti‐cancer effects. However, its effect on MM was rarely evaluated. The purpose of our study was to examine cytotoxic and apoptogenic effects of XN on human MM cells. Our results show that XN decreased MM cell viability in a time (24, 48, 72 hours) and dose (1, 5, 10, 20 and 30 μM) ‐dependent manner. Apoptogenic effect of XN was confirmed by flow cytometry analysis and western blotting. Our data demonstrated that, as the concentration of XN administered increased, the number of apoptotic MM cells increased. Protein expression of apoptosis‐related factors including caspase 3, caspase 8, caspase 9 and poly‐(ADP‐ribose) polymerase (PARP) was examined and showed that the amounts of cleaved caspase 3 and PARP1 were increased corresponding to an increase of XN. In order to determine whether the AMPK pathway is involved in XN‐induced apoptosis of MM cells, we will pre‐treat MM cells with either an activator (AICAR) or an inhibitor (Compound C) of AMPK followed by XN treatment in MM cells to elucidate the role of AMPK on anti‐myeloma effect of XN.

Blockade of HSP70 by VER-155008 synergistically enhances bortezomib-induced cytotoxicity in multiple myeloma

Proteasome inhibitor bortezomib is one of the most effective drugs currently available for the treatment of multiple myeloma (MM). However, the intrinsic and acquired resistance to bortezomib can limit its effectiveness. The activation of heat shock response has been characterized as a potential resistance mechanism protecting MM cells from bortezomib-induced cell death. In this study, in response to bortezomib therapy, we discovered that HSP70 is one of the most substantially upregulated heat shock proteins. In order to further explore approaches to sensitizing bortezomib-based treatment for MM, we investigated whether targeting HSP70 using a specific inhibitor VER-155008 combined with bortezomib could overcome the acquired resistance in MM. We found that HSP70 inhibitor VER-155008 alone significantly decreased MM cell viability. Moreover, the combination of VER-155008 and bortezomib synergistically induced MM cell apoptosis markedly in vitro. Notably, the combined treatment was found to increase the cleavage of PARP, an early marker of chemotherapy-induced apoptosis. Importantly, the reduction of anti-apoptotic Bcl-2 family member Bcl-2, Bcl-xL, and Mcl-1 and the induction of pro-apoptotic Bcl-2 family member BH3-only protein NOXA and Bim were confirmed to be tightly associated with the synergism. Finally, the ER stress marker CHOP (CCAAT-enhancer binding protein homologous protein), which can cause transcriptional activation of genes involved in cell apoptosis, was markedly induced by both VER-155008 and bortezomib. Taken together, our finding of a strong synergistic interaction between VER-155008 and bortezomib may support for combination therapy in MM patients in the future.

Functional Characterization of E3 Ligases and Their Regulators: Therapeutic Implications for Development of New Proteolysis-Targeting Chimeric Degraders of Oncoproteins

Functional Characterization of E3 Ligases and Their Regulators: Therapeutic Implications for Development of New Proteolysis-Targeting Chimeric Degraders of Oncoproteins

Targeting histone deacetylase 3 (HDAC3) in the bone marrow microenvironment inhibits multiple myeloma proliferation by modulating exosomes and IL-6 trans-signaling

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.

Citron Rho-interacting kinase silencing causes cytokinesis failure and reduces tumor growth in multiple myeloma

AbstractCitron Rho-interacting serine/threonine kinase (CIT) is a serine/threonine kinase that acts as a key component of the midbody and is essential for cytokinesis. CIT has been reported to be highly expressed in some tumor tissues and to play a role in cancer proliferation; however, the significance of CIT has not been investigated in multiple myeloma (MM). Here, we identified, by protein microarray and immunohistochemistry, that CIT is 1 of the upregulated proteins in the plasma cells of MM patients compared with healthy controls. Analysis of a gene expression profile data set showed that MM patients with high CIT gene expression had significantly worse overall survival compared with MM patients with low CIT gene expression. CIT silencing in MM cell lines induced cytokinesis failure and resulted in decreased MM cell proliferation in vitro and in vivo. TP53 expression was found to be an independent predictor of CIT dependency, with low-TP53 cell lines exhibiting a strong dependency on CIT. This study provides the rationale for CIT being a potential therapeutic target in MM in future trials.

Blockade of Ubiquitin Receptor PSMD4/Rpn10 Triggers Cytotoxicity and Overcomes Bortezomib-Resistance in Multiple Myeloma

Background and Rationale Proteasome inhibitors (PIs) are standard of care therapy for patients diagnosed with multiple myeloma (MM). However, prolonged treatment can be associated with toxicity and development of drug resistance. Our research efforts have therefore focused on designing therapeutic strategies that can overcome PI-resistance. In this context, our RNA-interference-based loss-of-function studies have identified 19S proteasome-associated Ubiquitin Receptor (UbRs) Rpn10 and Rpn13 as potential therapeutic targets. Both Rpn10 and Rpn13 UbRs are associated with the 19S regulatory lid of the proteasome that recognizes ubiquitylated proteins marked for degradation by 20S core particle. We have previously reported the role of Rpn13 in MM (Song et., al Luekemia 2016 Sep;30(9):1877-86). Here we functionally characterized the role of Rpn10 in MM.Materials and Methods Cytotoxicity assays were performed using a panel of MM cell lines, primary patient cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors. Cell viability was assessed using WST-1, MTT, and Cell Titer-Glo assay.Signal transduction pathways were evaluated using immunoblotting. Proteasome activity was measured as previously described (Chauhan et al., Cancer Cell 2005, 8:407-419; Chauhan et al., Cancer Cell 2012, 22(3):345-358). Statistical significance of data was determined using a Student's t test.Results Functional characterization of Rpn10 in MM:Gene expression (GEP) analysis showed inverse correlation between Rpn10 and overall patient survival (n=175) (p= 0.00064). Immunoblot analysis showed high Rpn10 protein levels in primary patient cells and MM cell lines versus normal plasma cells or PBMCs. Rpn10 knockdown by siRNA significantly decreased MM cell viability in both bortezomib-sensitive and -resistant MM cell lines. To assess the Rpn10 function, we generated a doxycycline-inducible Rpn10-shRNA knockout stable MM cell line. Rpn10-shRNA knockdown decreased MM cell proliferation. Mechanistic studies show that Rpn10 knockdown-triggered MM cell death is associated with 1) accumulation of cells in early and late apoptotic phase; 2) increase in polyubiquinated proteins; 3) arrest of cell cycle; 4) induction of ER stress; and 5) activation of caspases mediating apoptotic pathways. In order to determine if blockade of Rpn10 affects cellular proteasome function, we next utilized a reporter cell line expressing ubiquitin-tagged GFP that is constitutively targeted for proteasomal degradation. Interestingly, Rpn10-siRNA increased accumulation of the Ub-GFP reporter, reflecting impaired proteasome-mediated protein degradation. Finally, treatment of MM cells with peptides targeting UIM2 domain of Rpn10 significantly decreased MM cell viability, suggesting a potential therapeutic approach in MM.ConclusionOur preclinical data validates targeting 19S proteasome ubiquitin receptor Rpn10 upstream of the proteasome in the ubiquitin proteasomal cascade, and provides the framework for clinical evaluation of Rpn10 inhibitors to overcome PI resistance and improve patient outcome in MM. DisclosuresAnderson:C4 Therapeutics: Equity Ownership, Other: Scientific founder; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Millennium Takeda: Consultancy; OncoPep: Equity Ownership, Other: Scientific founder.

Abstract 1066: Imipridone ONC201 efficacy in refractory multiple myeloma via Erk1/2 inhibition and pro-apoptotic Bim upregulation: single agent and combinatorial approaches

Abstract Disease relapse and drug resistance occurs in the majority of multiple myeloma (MM) patients despite improvements offered by new treatments. There is an urgent unmet need for new therapies that can overcome drug resistance and prolong patient survival after failure of standard-of-care. ONC201, founding member of the impridone class of compounds, has robust preclinical efficacy in a variety of tumor types. A first-in-human ONC201 clinical study has demonstrated exceptional safety, therapeutic pharmacokinetics and evidence of tumor engagement that has led to several Phase I/II advanced cancer studies. Given the pronounced sensitivity of B-cell lymphomas to ONC201, we assessed the efficacy of ONC201 in MM preclinical models. We treated human MM cell lines and patient-derived cells isolated from bone marrow aspirates with ONC201 for 72 hours. ONC201 treatment decreased MM cell viability in CellTiter-Glo assays, with IC50 values that were 1 μM to 1.5 μM, even in high risk MM cell line RPMI8226. Consistent with prior reports, the efficacy of ONC201 was independent of TP53 status, as MM.1S or NCI-H929 cells with wild-type TP53 and OPM-2 or RPMI8226 with mutated TP53 had a similar sensitivity towards ONC201. Additionally, ONC201 was equally effective in paired MM cell lines with TP53 wild type or knockout. Western blot analysis showed increased apoptosis, cleavage of caspase-9, caspase-3, and PARP. We also found that ONC201 induced expression of the pro-apoptotic protein Bim in MM cells, which can occur downstream of ERK inactivation. shRNA knockdown of Bim expression in MM cells abrogated ONC201-induced apoptosis in annexin-V binding assays. Phosphorylation of Bim at Ser69 by Erk1/2 has been shown to promote proteasomal degradation of Bim. In accordance with this mechanism, we observed that ONC201 treatment reduced levels of phosphorylated Erk1/2, an indicator of Erk1/2 kinase activity, and downregulated Bim pSer69. In addition, ONC201 induced apoptosis in dexamethasome-, bortezomib-, and carfilzomib-resistant MM cell lines with the same efficacy as in wild-type cells. As a rational strategy to increase the efficacy of ONC201 by enhancing its inhibition of proteasome-mediated Bim degradation, we tested combinations of ONC201 with proteasome inhibitors. Synergistic reduction in cell viability and enhanced Bim expression and PARP cleavage was observed with ONC201 in combination with bortezomib or carfilzomib in MM cells. The combination of ONC201 and bortezomib enhanced the levels of Bim and cleaved PARP in MM cells. Overall, these findings demonstrate that ONC201 inhibits the Erk1/2 signaling pathway and induces Bim expression to induce apoptosis in MM regardless of p53 status and resistance to standard-of-care therapies. Our studies provide a strong rationale for clinical trials of ONC201 as a single agent or in combination with approved drugs in relapsed/refractory MM. Citation Format: Yongsheng Tu, Jin He, Huan Liu, Richard Eric Davis, Robert Z. Orlowski, Varun Vijay Prabhu, Joshua Allen, Jing Yang. Imipridone ONC201 efficacy in refractory multiple myeloma via Erk1/2 inhibition and pro-apoptotic Bim upregulation: single agent and combinatorial approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1066. doi:10.1158/1538-7445.AM2017-1066

Blockade of deubiquitylating enzyme Rpn11 triggers apoptosis in multiple myeloma cells and overcomes bortezomib resistance.

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.

Targeting CD38 Suppresses Induction and Function of T Regulatory Cells to Reverse Immunosuppression in Multiple Myeloma

We here study targeting CD38 to overcome immunosuppression by CD4+CD25highFoxp3+ T regulatory cells (Tregs) in multiple myeloma (MM). CD38 is differentially expressed on T cell subsets with higher levels on Tregs than CD4+CD25- conventional T cells (Tcons) from MM patients vs. normal donors. CD38 levels and the percentages of CD38high Tregs are further increased by low doses of Pomalidomide (Pom) or Lenalidomide (Len), which could confer further sensitivity to CD38 targeting. This result further support combined targeting CD38 with immunomodulatory drugs (IMiDs) to mitigate tumor-related immunosuppression. Importantly, anti-CD38 mAb SAR650984 (SAR) preferentially decreases Treg while increases Tcon frequencies, which is enhanced by Pom/Len. SAR induces apoptosis and inhibits proliferation of Tregs in Fc-independent manner. It further reduces Foxp3 and IL10 in Tregs, blocks migration of Tregs, and restores proliferation and function of Tcons. Importantly, SAR augments MM cell lysis by CD8+ T and natural killer cells, as seen by enhanced cell surface CD107a for degranulation and IFNγ production. Pom/Len further enhances these effector functions induced by SAR. Ex vivo cocultures of MM cells with peripheral blood mononuclear cells (PBMCs) or Tcons significantly induce Tregs (iTregs) which express even higher CD38 than natural occurring Tregs (nTregs) in a time-dependent manner. CD38 is increased at even higher extent on iTregs induced from Tcons than PBMCs when cocultured with MM cells, indicating the conversion of Tcons into iTregs. This is associated with elevated circulating CD38+ Tregs in MM patients vs. normal donors. Besides upregulated CD38, iTregs, when compared with Tcons alone, express higher levels of CD25, Foxp3, CD44, ICOS, and PD1, while low CD127. PDL1 is concurrently increased on MM cell membrane in these cocultures. Since anti-TGFb, -PD1, or -PDL1 mAb, when added alone, partially blocks iTreg induction from Tcon, cell-cell contact via PD1/PDL1 interaction and TGFb are attributed to induction of iTregs. SAR decreases MM cell- and bone marrow stromal cell-induced iTregs and production of inhibitory cytokines TGFb and IL10, further indicating that SAR targets immunosuppressive function in CD38high iTregs. Finally, CD38 levels correlate with differential inhibition by SAR on Tregs from MM vs normal donors. Taken together, these results show that targeting CD38 can preferentially block potent immunosuppressive Tregs while restore effector function to further against MM. DisclosuresAnderson:Oncoprep: Equity Ownership; Acetylon: Equity Ownership; C4 Therapeutics: Equity Ownership; Oncoprep: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Gilead: 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; 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; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

Critical Analysis on the Mechanism of Action (MoA) of the Anti-CD38 Monoclonal Antibody Isatuximab in Multiple Myeloma (MM)

Monoclonal antibodies (mAbs) targeting CD38 are demonstrating remarkable efficacy, particularly when combined with anti-MM agents. Thus, in-depth understanding of the MoA of anti-CD38 mAbs is of utmost importance to design rational treatment combinations. Notably, while there is considerable data about the MoA of Daratumumab, there is virtually no data about the MoA of Isatuximab.Here, we started by analyzing in a selected panel of MM cell lines (RPMI8226, H929, MM1S and OPM2) the effect of Isatuximab on 1) proliferation, 2) direct cell death, 3) complement dependent cytotoxicity (CDC), 4) antibody dependent cell mediated cytotoxicity (ADCC), and 5) antibody dependent cellular phagocytosis (ADPC). Our results show that Isatuximab had no effect on proliferation, and did not directly killed any of the MM cell lines tested. Interestingly, while Isatuximab (similar to drugs such as Cetuximab) binds to C1q, CD38 receptor density in MM cell lines was insufficient to trigger CDC based on the absence of C3 deposition and impact on cell survival. Upon co-culture with human blood leukocytes, Isatuximab induced ADCC particularly on cells showing higher expression of CD38 (P=0.02). Upon co-culture with human-derived M2-like macrophages, we noted a trend (P=.1) towards decreased MM cell expansion; thus, in order to establish the exact contribution of ADCC and ADCP on the efficacy of Isatuximab, we co-cultured RPMI8226 MM cells with human leukocytes and prior to treatment (10µg/mL), used sensitive FACS sorting to remove key immune cell populations such as NK cells, macrophages and T cells from the culture. Our results show that the presence of NK cells was critical for the efficacy of Isatuximab, whereas macrophages and T cell depletion had no effect on MM cell viability in the presence of Isatuximab.We next tested Isatuximab (10µg/mL) in 10 primary patient samples. In the absence of C3 deposition in the majority of samples (ie. no CDC), we observed Isatuximab-induced cell death in all patients tested (P=0.002), along with increased proliferation and activation of NK cells upon Fc recognition. Due to broad expression of CD38 in hematopoietic cells, we also investigated presence of off-target effects of Isatuximab. Importantly, with the exception of CD38bright B cell precursors (P=.0006), there was no toxicity observed in other progenitor and mature hematopoietic cells. We also investigated the potential synergism of Isatuximab in combination with Bortezomib-Dexamethasone; interestingly, the addition of Isatuximab resulted in a (P=0.06) 2.7-fold increment in MM cell death.In order to further understand the direct effect of Isatuximab in both MM and (CD38bright) NK cells, we determined the molecular signature of both subsets in co-culture and upon treatment with Isatuximab. Interestingly (though consistent with the lack of impact in proliferation and direct apoptosis), MM cells showed no gene dysregulation upon treatment with Isatuximab and in the absence of NK cells. By contrast, in the presence of NK cells and respective ADCC, MM cells chemoresistant to Isatuximab showed 184 dysregulated genes, including significant down-regulation of CD38. Noteworthy, Isatuximab binding induced in NK cells up-regulation of genes related to antigen binding, chemokine receptor activity, G-protein coupled peptide receptor or hydrolase activity. One such gene was CD137, which up-regulation was subsequently confirmed at the protein (surface antigen) level.In summary, this critical analysis on Isatuximab's MoA using a comprehensive set of assays, MM cell lines and primary patient samples, suggests that ADCC is the major efficacy contributor for this particular mAb. Thus, drugs that can further stimulate NK cells might be optimal candidates for rational combinations with Isatuximab; in this regard, we showed that Isatuximab induces up-regulation of CD137 which warrants further investigations on the value of combined Isatuximab and anti-CD137 immune therapies. [Display omitted] DisclosuresPaiva:Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding.

Targeting proteasome ubiquitin receptor Rpn13 in multiple myeloma.

Proteasome inhibitor bortezomib is an effective therapy for relapsed and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance can limit its long-term utility. Recent research has focused on targeting ubiquitin receptors upstream of 20S proteasome, with the aim of generating less toxic therapies. Here we show that 19S proteasome-associated ubiquitin receptor Rpn13 is more highly expressed in MM cells than in normal plasma cells. Rpn13-siRNA (small interfering RNA) decreases MM cell viability. A novel agent RA190 targets Rpn13 and inhibits proteasome function, without blocking the proteasome activity or the 19S deubiquitylating activity. CRISPR/Cas9 Rpn13-knockout demonstrates that RA190-induced activity is dependent on Rpn13. RA190 decreases viability in MM cell lines and patient MM cells, inhibits proliferation of MM cells even in the presence of bone marrow stroma and overcomes bortezomib resistance. Anti-MM activity of RA190 is associated with induction of caspase-dependent apoptosis and unfolded protein response-related apoptosis. MM xenograft model studies show that RA190 is well tolerated, inhibits tumor growth and prolongs survival. Combining RA190 with bortezomib, lenalidomide or pomalidomide induces synergistic anti-MM activity. Our preclinical data validates targeting Rpn13 to overcome bortezomib resistance, and provides the framework for clinical evaluation of Rpn13 inhibitors, alone or in combination, to improve patient outcome in MM.

A novel hypoxia-selective epigenetic agent RRx-001 triggers apoptosis and overcomes drug resistance in multiple myeloma cells.

The hypoxic bone-marrow (BM) microenvironment confers growth/survival and drug-resistance in multiple myeloma (MM) cells. Novel therapies targeting the MM cell in its hypoxic-BM milieu may overcome drug resistance. Recent studies led to the development of a novel molecule RRx-001 with hypoxia-selective epigenetic and Nitric Oxide-donating properties. Here we demonstrate that RRx-001 decreases the viability of MM cell lines and primary patient cells, as well as overcomes drug-resistance. RRx-001 inhibits MM cell growth in the presence of BM stromal cells. RRx-001 induced apoptosis is associated with: 1) activation of caspases; 2) release of ROS and nitrogen-species; 3) induction of DNA damage via ATM/γ-H2AX; and 4) decrease in DNA methytransferase (DNMT) and global methylation. RNA interference study shows a predominant role of DNMT1 in MM cell survival versus DNMT3a or DNMT3b. Deubiquitylating enzyme USP7 stimulates DNMT1 activity; and conversely, USP7-siRNA reduced DNMT1 activity and decreased MM cell viability. RRx-001 plus USP7 inhibitor P5091 triggered synergistic anti-MM activity. MM xenograft studies show that RRx-001 is well tolerated, inhibits tumor growth, and enhances survival. Combining RRx-001 with pomalidomide, bortezomib or SAHA induces synergistic anti-MM activity. Our results provide the rationale for translation of RRx-001, either alone or in combination, to clinical evaluation in MM.

The KDM3A-KLF2-IRF4 axis maintains myeloma cell survival.

KDM3A is implicated in tumorigenesis; however, its biological role in multiple myeloma (MM) has not been elucidated. Here we identify KDM3A–KLF2–IRF4 axis dependence in MM. Knockdown of KDM3A is toxic to MM cells in vitro and in vivo. KDM3A maintains expression of KLF2 and IRF4 through H3K9 demethylation, and knockdown of KLF2 triggers apoptosis. Moreover, KLF2 directly activates IRF4 and IRF4 reciprocally upregulates KLF2, forming a positive autoregulatory circuit. The interaction of MM cells with bone marrow milieu mediates survival of MM cells. Importantly, silencing of KDM3A, KLF2 or IRF4 both decreases MM cell adhesion to bone marrow stromal cells and reduces MM cell homing to the bone marrow, in association with decreased ITGB7 expression in MAF-translocated MM cell lines. Our results indicate that the KDM3A–KLF2–IRF4 pathway plays an essential role in MM cell survival and homing to the bone marrow, and therefore represents a therapeutic target.

The Hypoxia-Selective Epigenetic Agent, Rrx-001, Triggers Apoptosis and Overcomes Drug-Resistance in Multiple Myeloma Cells

Introduction The bone marrow (BM) microenvironment confers growth, survival, and drug resistance in multiple myeloma (MM) cells (Chauhan et al, Cancer Cell 2009, 16:309-323) BM hypoxia (low oxygenation) plays a role in promoting MM cell survival, drug resistance, migration, and metastasis. Novel therapies that selectively target the MM cell in its hypoxic BM milieu may therefore overcome conventional drug resistance. Recent studies led to the development of a novel aerospace industry-derived Phase 2 molecule RRx-001 with hypoxia-selective epigenetic and NO-donating properties. A Phase I clinical trial demonstrated promising evidence of anti-tumor activity in a heavily pretreated population with no dose-limiting toxicities (Reid et al. J Clin Oncol 32:5s, 2014 suppl; abstr 2578, Reid et al, Lancet Oncology, in press). RRx-001 is currently under investigation in multiple Phase II clinical trials. Here we examined both the mechanism of action and anti-MM activity of RRx-001 using in vitro and in vivo models of MM.Methods Cell viability, apoptosis, and migration assays were performed using MTT, Annexin V staining, and transwell Inserts, respectively. ROS and NO generation was measured as previously described (Chauhan et al., Blood, 2004, 104:2458). Synergistic anti-MM activity was assessed by isobologram analysisusing "CalcuSyn" software program. In vitro angiogenesis was assessed using matrigel capillary-like tube formation assays. DNMT1 activity was measured using DNMT1 assay kit. USP7 siRNA was purchased from Dharmacon. CB-17 SCID-mice were subcutaneously inoculated with MM.1S cells as previously described (Chauhan et al., Cancer Cell 2012, 11:345-358). Statistical significance of data was determined using a Student's t test. RRx-001 was obtained from EpicentRx, CA, USA; USP7 inhibitor P5091, bortezomib, SAHA, and pomalidomide were purchased from Selleck chemicals, USA.Results Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, OPM2, H929, Dox-40 ARP-1, KMS-11, ANBL6.WT, ANBL6.BR, and LR5) and primary patient cells for 48h significantly decreased their viability (IC50 range 1.25nM to 2.5nM) (p < 0.05; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for RRx-001. Tumor cells from 3 of 5 patients were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Moreover, RRx-001 inhibits proliferation of MM cells, even in the presence of BM stromal cells. Washout experiments showed that a short time (3h) exposure of MM cells to RRx-001 triggered irreversible cell death. RRx-001-triggered apoptosis is associated with: 1) induction of DNA damage response signaling via ATM/p53/gH2AX axis; 2) activation of caspases mediating both intrinsic and extrinsic apoptotic pathways; 3) increase in oxidative stress through release of ROS and generation of NO; and 4) decrease in DNMT1 activity and global methylation levels. Furthermore, RRx-001 blocked migration of MM cells and angiogenesis. Deubiqyitylating enzyme USP7 stimulates DNMT1 enzymatic activity. USP7-siRNA reduced DNMT1 activity and decreased MM cell viability. Importantly, the combination of USP7 inhibitor P5091 and RRx-001 triggered synergistic anti-MM activity associated with a robust decrease in DNMT1 activity, as well as increased degradation of USP7 substrate MDM2 and induction of downstream p21/p53 signaling axis. In vivo studies using a subcutaneous human MM xenograft model shows that RRx-001 is well tolerated, inhibits tumor growth, and enhances survival. Finally, combining RRx-001 with pomalidomide, bortezomib or SAHA induces synergistic anti-MM activity in p53-WT and p53-null MM cells, and overcomes drug resistance.Conclusion Our preclinical studies demonstrate that RRx-001, a ROS-mediated epigenetic inhibitor with anti-angiogenic properties selectively targets MM cells in vivo and synergizes with existing anti-MM agents to overcome therapeutic resistance. Our data also suggest a potential mechanism of action for RRx-001-induced epigenetic changes via USP7-DNMT1 complex and downstream p53/p21 signaling cascade. Collectively, these results provide a rationale for rapid translation of RRx-001, either alone or in combination, in a clinical trial of relapsed refractory MM. DisclosuresOronsky:epicentrx: Employment. Scicinski:epicentrx: Employment. Chauhan:Stemline Therapeutics: Consultancy.

Targeting 19S-Proteasome Deubiquitinase Rpn11/POH1/PSMD14 in Multiple Myeloma

Introduction Deregulation of the ubiquitin-proteasome system (UPS) is linked to pathogenesis of various human diseases, including cancer. Targeting the proteasome is an effective therapy in multiple myeloma (MM) patients. Recent research efforts led to the discovery of newer agents that target enzymes modulating protein ubiquitin-conjugation/deconjugation rather than the proteasome itself, with the goal of generating more specific and less toxic antitumor therapies. Ubiquitylation is a dynamic reversible process coordinated by many enzymes: ubiquitin ligases attach ubiquitin to proteins allowing for their degradation, whereas deubiquitylating (DUB) enzymes deconjugate ubiquitin from target proteins, thereby preventing their proteasome-mediated degradation. Rpn11 is a DUB enzyme associated with the 19S regulatory particle lid of the proteasome that removes ubiquitin from target proteins to facilitate protein degradation by 20S proteasome core particle. Here we examined the role of Rpn11 in MM using both biochemical and RNA interference strategies.Materials and Methods Cell viability and apoptosis were assessed using WST and Annexin V staining, respectively. MM.1S MM cells were transiently transfected with control short interfering RNA (siRNA), RPN11 ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. Isobologram analysisand CalcuSyn software program were utilized to assesssynergistic/additive anti-MM activity. Ub-AMC assay Proteasome activity was measured, as in our prior study (Chauhan et al., Cancer Cell 2005, 8:407-419). Signal transduction pathways were evaluated using immunoblotting. Statistical significance of data was determined using a Student's t test. O-phenanthroline (OPA) was purchased from EMD Millipore, USA; and bortezomib, lenalidomide, and pomalidomide were purchased from Selleck chemicals, USA.Results Gene expression (GEP) analysis of Rpn11 showed a significantly higher level in primary patient MM cells (n=73) versus normal plasma cells (n=15) (p < 0.05). We found a statistically significant inverse correlation between Rpn11 levels and overall patient survival (p =0.035). Western blot analyses show higher Rpn11 levels in MM cell lines and patient cells compared to normal cells. Rpn11-siRNA significantly decreased MM cell viability (p < 0.001; n=3). To further validate our siRNA data, we utilized Rpn11 inhibitor O-phenanthroline (OPA) (Verma et al., Science 2002, 298:611-5). Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, ARP-1, Dox40, LR5, INA6, ANBL6.WT, and ANBL6.BR) and primary patient cells for 48h significantly decreased their viability (IC50 range 8µM to 60µM; p < 0.001 for all cell lines; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for OPA. Tumor cells obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies remained sensitive to OPA. Moreover, the cytotoxicity of OPA was observed in MM cell lines sensitive and resistant to conventional (dex) and novel (bortezomib) therapies. Furthermore, OPA inhibits proliferation of MM cells even in the presence of BM stromal cells or pDCs. OPA inhibits Rpn11 DUB activity without blocking 20S proteasome activities. Mechanistic studies show that OPA-triggered MM cell death is associated with 1) accumulation of cells in early and late apoptotic phase; 2) increase in polyubiquinated proteins; and 3) activation of caspases mediating both intrinsic and extrinsic apoptotic pathways. Importantly, OPA-induced apoptosis in MM cells occurs in a p53-independent manner, since OPA triggered significant apoptosis in both p53-null (ARP-1) and p53-mutant (RPMI-8226) MM cells (p < 0.004). Finally, combining OPA with lenalidomide, pomalidomide, or bortezomib induces synergistic/additive anti-MM activity, and overcomes drug resistance.Conclusion Our preclinical data showing efficacy of OPA in MM disease models validates targeting 19S proteasome-associated DUB Rpn11 upstream of the proteasome in the ubiquitin proteasomal cascade to overcome proteasome inhibitor resistance, and provides the framework for clinical evaluation of Rpn11 inhibitors to improve patient outcome in MM. DisclosuresChauhan:Stemline Therapeutics: Consultancy.

Inhibition of P-Selectin and PSGL-1 Using Humanized Monoclonal Antibodies Increases the Sensitivity of Multiple Myeloma Cells to Proteasome Inhibitors

Introduction: Multiple myeloma (MM) is a plasma cell malignancy localized in the bone marrow (BM). Despite the introduction of novel therapies more than 70% of MM patients relapse. We have previously shown that inhibition of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) play a key role on proliferation of MM and that its inhibition with small-molecule inhibitors sensitized MM cells to therapy. However, these small molecule inhibitors had low specificity to P-selectin and showed poor pharmacokinetics. In this study, we tested inhibition of P-selectin and PSGL-1 using functionally blocking monoclonal antibodies to sensitize MM cells to therapy.Methods: The humanized monoclonal antibodies anti-P-selectin (SelG1) and anti-PSGL-1 (SelK2) were obtained from Selexys Pharmaceuticals. Endothelial cells, stromal cells derived from MM patients, and MM cell lines (MM1s, H929, OPM1 and RPMI8226) were used for in vitro expression, proliferation, apoptosis and immuno-blotting assays. The expression of P-selectin and PSGL-1 were tested by the interaction of SelG1 (5-20µg/mL) and SelK2 (5-20µg/mL) antibodies with endothelial cells, stromal cells and MM cells for 1hr, followed by addition of a secondary-FITC antibody and flow cytometry analysis. For adhesion assay, cells were treated with increasing concentrations of SelG1 and SelK2 for 1hr; pre-labeled MM cells were then applied to unlabeled endothelial or stromal cells for 1hr, non-adherent cells were washed, and adherent cells were analyzed by a fluorescent reader. For proliferation, MM1s-GFP+ cells cultured alone, with stroma, or with endothelial cells; and were treated with, or without, bortezomib and carfilzomib, in the presence, or absence, of SelG1 and SelK2 antibodies, and proliferation was determined by flow cytometry. Protein expression associated with survival, apoptosis and cell cycle signaling was analyzed by western blotting.For in vivo, MM1s-Luc-GFP cells were injected intravenously into SCID mice and tumor progression followed for 4 weeks. Anti-mouse-P-selectin antibody was used to inhibit P-selectin in the mouse endothelial cells and stroma. Likewise, SelK2 and anti-mouse PSGL-1 were used to inhibit PSGL-1 on human MM cells in the mouse microenvironment, respectively. Mice were divided into 6 groups (1) vehicle treated control, (2) anti-mouse-P-selectin (5mg/kg) alone, (3) SelK2 (5mg/kg) and anti-mouse-PSGL-1 (5mg/kg) alone, (4) bortezomib (0.5mg/kg) alone, (5) a combination of anti-mouse-P-selectin and bortezomib, and (6) a combination of SelK2 (5mg/kg), anti-mouse-PSGL-1 (5mg/kg) and bortezomib. Anti-mouse-P-selectin, anti-mouse–PSGL-1, SelK2 and bortezomib were administered intraperitoneally twice a week.Results:The half-life of SelG1 in a Phase I clinical study was previously shown to be 363 hours while the half-life of SelK2 in a primate study was approximately 100 hours. P-selectin expression was detected on endothelial and stromal cells using the SelG1 antibody, while no expression was found on MM cells. PSGL-1 was highly expressed on MM cells as well as on endothelial cells and stromal cells as detected by SelK2 monoclonal antibody. Inhibition of P-selectin and PSGL-1 with SelG1 or SelK2, respectively, decreased MM cell adhesion to endothelial and stromal cells, and decreased the proliferation of MM cells induced by stromal and endothelial cells. Similarly, inhibition of the interaction between P-selectin and PSGL-1 sensitized MM cells to bortezomib and carfilzomib in vitro. In vivo results demonstrated that inhibition of the P-selectin or PSGL-1 as single agents delay tumor growth compared to non-treated mice and that it enhances the effect of bortezomib.Conclusions: These results demonstrate that inhibition of P-selectin and PSGL-1 by SelG1 and SelK2 antibodies, respectively, disrupts the interaction between MM cells and BM microenvironment and decreases adhesion and proliferation of MM cells. Moreover, inhibition of P-selectin and PSGL-1 increased the sensitivity of MM cells to bortezomib and carfilzomib in vitro, and to bortezomib in vivo. These data provides a basis for future clinical trials for sensitization of refractory MM patients to therapy by inhibition of P-selectin and PSGL-1 using SelG1 and SelK2 monoclonal antibodies. DisclosuresRollins:Selexys: Employment. Alvarez:Selexys: Employment. Kawar:Selexys: Employment. Azab:Selexys: Research Funding.

SAR 650984, a Therapeutic Anti-CD38 Monoclonal Antibody, Blocks CD38-CD31 Interaction in Multiple Myeloma

CD38, a type II transmembrane glycoprotein with both ecto-enzyme activity and receptor function, is highly expressed at the surface of many hematological cancer cells. Recently, SAR 650984 (SAR), a novel humanized anti-CD38 monoclonal antibody in early clinical development, has shown an impressive and durable single agent activity in patients with multiple myeloma (MM). To further characterize the biological function of CD38 in MM cells, we generated RPMI8226 MM cells overexpressing CD38 (R-CD38) by lentiviral infection followed by blascitidin selection. R-CD38, maintained in 10 mg/ml of blascitidin-containing media, expresses 6-fold greater CD38 mRNA and protein levels than control RPMI8226 cells, as confirmed by RNAseq and quantitative flow cytometry. To study whether CD38 levels affect cell signaling cascade in MM cells, the phosphorylated forms of various signaling proteins were analyzed using Kinexä Antibody Microarray. At least 20 and 8 phospho-signaling molecules were significantly increased and reduced, respectively, in R-38 vs control RPMI8226 cells. Upregulated kinases include HO1, CREB1, IRAK1, and ERK1/2, whereas calreticulin and RSK1/2 are downregulated. RNAseq profiling on Illumina HiSeq was next done to identify CD38-targeted genes in MM cells. A volcano plot shows that 123 genes are induced and 32 repressed with FC>= 4 (positive or negative) and p value <=0.01. Enrichment in functional categories in these 155 gene differentially regulated genes further revealed predominant changes in plasma membrane, cytoskeleton, cellular adhesion, synaptic plasticity, neuronal transmission, transport, and Ca2+ regulation. Gene set enrichment analysis on log2(R-CD38/control RPMI8226) profile also indicates downregulated genes involved in interferon response and MHC class I, whereas myogenic and astrocytic marks are upregulated. Ongoing studies include NextBio/Meta analysis in SAR-treated R-38 vs RPMI8226 cells. Importantly, we found that R-CD38 cells are more sensitive than control RPMI8226 to lysis induced by SAR-mediated antibody-dependent cellular cytotoxicity (ADCC) using at least 3 donor effector cells, confirming CD38 overexpression in R-CD38 vs control cells. In addition, SAR directly induces apoptosis of R-CD38 cells but not control cells, with neither effector cells nor FcgR cross-linking (Abstract #72699). Although overexpression of CD38 in R-CD38 cells did not alter cell proliferation, R-CD38 showed increased adherence to bone marrow stromal cells (BMSCs) derived from MM patients (n=3) relative to controls. Importantly, SAR (0-10 mg/ml) inhibited MM cell adherence in a dose-dependent manner. Similar SAR effect in MM cell adhesion was seen when HS-5 stromal cells were used. In order to investigate the role of CD31, a physiological ligand for CD38, in MM cell adhesion to bone marrow (BM) microenvironment, we next performed MM cell adhesion assays using CD14+ monocytes and HUVEC cells which express significantly higher CD31 levels than BMSCs and HS-5 cells. At the same concentrations of SAR, % of inhibition in adhesion of MM cell lines (R-CD38, MOLP8, MM1S, H929) to monocytes was approximately 10-fold higher than to BMSC. Furthermore, SAR blocked MM cell adhesion to HUVEC more potently than adhesion to BMSC. These results indicate that SAR decreases MM cell adhesion to BM accessory cells via blockage of CD31-CD38 interaction, thus suggesting a potential new mechanism of action of SAR to elicit its anti-MM activity. DisclosuresCai:Sanofi: Employment. Yang:Sanofi: Employment. Song:Sanofi: Employment. Theilhaber:Sanofi: Employment. Adrian:Sanofi: Employment. Anderson:Celgene: Consultancy; Onyx: Consultancy; Gilead Sciences: Consultancy; Sanofi-Aventis US: Consultancy; Acetylon: Scientific Founder Other; Oncoprep: Scientific Founder Other.

Weighted Gene Co-Expression Network Analysis (WGCNA) Identifies Highly Proliferative Myeloma Subgroup Responsive to CDK4/ARK5 Inhibition

Multiple myeloma (MM) is an incurable plasma cell malignancy accounting for more than 10,000 deaths in the US each year. Hence the pursuit for novel therapeutic agents remains critically important. Myeloma pathogenesis is associated in part with aberrant cell cycle progression. Inhibition of cyclin dependent kinases CDK4/6 results in cell cycle arrest and sensitization to Bortezomib and other active agents in MM (Huang, Blood 2012). Here, we show that ARK5, a novel member of the human AMPKfamily, is overexpressed in 70% of MM and helps promote proliferation and cell cycle progression via G1/S phase activation in an mTOR dependent manner.We examined the role of ARK5 using loss of function studies by ARK5 siRNA transfection in MM1.S, NCI-H929 cells as well as treatment with ON 123300, a dual CDK4/ARK5 kinase inhibitor. ARK5 siRNA knockdown decreased MM cell viability and cell proliferation via G1/S arrest compared to control siRNA. ARK5 siRNA treatment significantly (~70%) induced apoptosis in MM cells as detected by Annexin V/PI staining. We observed that phosphorylation of Rb, a critical cell cycle protein was significantly reduced in ARK5 depleted cells. Moreover, mTOR pathway inhibition was confirmed by reduction of pS6K in ARK5depleted cells as compared to control siRNA treated cells.ON 123300 decreased viability in MM cell lines and patient cells but was not lethal to normal PBMCs. A single treatment of 50nM drug stratified MM cell lines into 2 groups, 5 resistant (MM.1R, KMS11, U266, RPMI-8226 and ARP1) and 4 sensitive cell lines (>80% cell kill-MM.1S, EJM, JJN3, NCI-H929). ARK5 protein expression by western blot analysis was much higher in sensitive cell lines. ON 123300 triggered G0/G1 cell cycle arrest and induced apoptosis similar to the effect of ARK5 siRNA (80% vs 70%). ON 123300 treatment also reduced phosphorylation of pRb and pS6K downstream of mTOR pathway. These results confirm that cell inhibitory effects of ON 123300 in MM are mediated in a large part via inhibition of ARK5. Co-culture experiments with BMSCs showed that ON 123300 not only targets MM cells but also overcomes the cytoprotective effects of the MM-host BM microenvironment. 4/5 ARK5 positive primary samples with adverse cytogenetics including 1q amplification and CyclinD1 translocation were sensitive to ON 123300 (>80% cytotoxicity) at 50nM. Further, IP injection of ON 123300 (100mg/kg) in tumor xenograft models (MM1.S, NCI-H929) showed that ON 123300 is well tolerated and significantly inhibits tumor growth in vivo(p<0.001).To study the mechanism of action for ON 123300, we performed geneset enrichment analysis (GSEA) on drug induced gene expression signature of RNA-Seq data from pre-post treated cell lines. We interrogated a wide array of geneset libraries, including MSigDB (Subramanian, PNAS 2005), drug induced transcriptional modules (Iskar, Mol Sys Bio. 2013) and disease signatures (Sirota, Sci Transl Med 2011). GSEA showed significant representation of genes that are enriched in normal plasma cells and rapamycin sensitive geneset.Next, we developed a weighted co-expression network (WGCNA, Langfelder BMC Bioinformatics 2008) based classifier using 304 MM samples from MMRC collection and RNA-Seq from 28 MM patients from Mt. Sinai Hospital. WGCNA defines a network that continuously links all genes and then clusters the most highly co-expressed genes in defined modules. These network modules were associated with clinical traits and UAMS GEP classification (Zhan, Blood 2006) of each sample. There was significant overlap between highly proliferative “PR” and “Cyclin D1/2” patient subsets based on classification and sensitivity to CDK4/ARK5 inhibition. Our classifier accurately discriminated 4 sensitive primary samples from one resistant sample, all tested in vitro. All sensitive samples were either Cyclin D1/2 or PR as per UAMS classification.Our preclinical studies provide the basis for clinical evaluation of CDK4/ARK5 inhibitor ON 123300 due to its selective cytotoxicity on MM cells in vitro and in vivo. Using WGCNA we establish a systematic framework by constructing for the first time, MM-associated gene co-expression networks contributing to tumorigenesis and progression. Thus, WGCNA modeling is a novel approach for identification of MM patient subgroups that have a higher likelihood of response in clinical trials with CDK4/ARK5 inhibitors. DisclosuresChari:Array BioPharma: Scientific Advisory Board Other; Celgene Corporation: Scientific Advisory Board, Scientific Advisory Board Other; Millennium Pharmaceuticals, Inc: Scientific Advisory Board, Scientific Advisory Board Other; Onyx Pharmaceuticals, Inc.: Scientific Advisory Board, Scientific Advisory Board Other. Reddy:Onconova Therapeutics, Inc.: Consultancy, Patents & Royalties. Reddy:Onconova Therapeutics, Inc.: Consultancy, Patents & Royalties. Dudley:Ecoeos, Inc: Consultancy, Equity Ownership; GNS Healthcare: Consultancy; GlaxoSmithKline: Consultancy; Ayasdi, Inc: Equity Ownership; NuMedii, Inc: Equity Ownership; Ubalo, Inc.: Equity Ownership. Jagannath:Onyx Pharmaceuticals, Inc.: Consultancy; Celgene Corporation: Scientific Advisory Board, Scientific Advisory Board Other; Millennium Pharmaceuticals, Inc. : Scientific Advisory Board, Scientific Advisory Board Other.

Cytoskeleton Regulator PAK4 Plays a Role in Growth and Survival of Myeloma with a Potential Therapeutic Intervention Using PAK4 Allosteric Modulators (PAMs)

P21-activated serine/threonine kinase 4 (PAK4) is a major effector of Cdc42 and is essential for cytoskeleton reorganization. PAK4 is activated in cancer cells, promotes cell migration and anchorage-independent cell growth, and protects against apoptosis induction. With cellular migration playing a significant role in multiple myeloma (MM) cell growth and survival, we investigated the expression and subcellular localization of PAK4 in MM cells. We observed a high level of un-phosphorylated PAK4 in the cytoplasm and high levels of phosphorylated PAK4 in the nucleus. In a gain-of-function study, over-expression of PAK4-eGFP in PAK4-deficient MM cells (RPMI8226) significantly increased cell proliferation and survival. Conversely, in a loss-of-function study, conditional knock-down of PAK4 expression with TRIPZ-lentiviral vectors decreased MM cell proliferation and survival proportionally to the reduction in PAK4. With a significant impact of PAK4 on MM cell growth, we identified a class of orally bioavailable PAK4 allosteric modulators (PAMs; e.g. KPT-6604, -7189, -7657, -8752). We observed inhibition of MM cell growth and survival after treatment with PAMs even in the presence of bone marrow microenvironment. In addition, there is a significant correlation between PAK4 expression and the inhibition concentration (IC50s) of PAMs in proliferation assays. Moreover, inhibition of PAK4 induced receptor and mitochondrial-mediated apoptotic pathways via Caspase-3, -8, and -9 activation. PAMs had no significant effect on normal PBMCs, suggesting a favorable therapeutic index in MM treatment. Finally, in two murine models of human myeloma, orally bioavailable KPT-8752 given daily was able to inhibit tumor growth in vivo and prolong overall survival. In summary, PAK4 plays an important cellular and molecular function in myeloma and its inhibition with a new class of PAK4 allosteric modulators provides a novel therapeutic approach for the treatment of MM. DisclosuresSenapedis:Karyopharm: Employment. Baloglu:Karyopharm: Employment. Anderson:Celgene: Consultancy; Sanofi-Aventis: Consultancy; Onyx: Consultancy; Acetylon: Scientific Founder, Scientific Founder Other; Oncoprep: Scientific Founder Other; Gilead Sciences: Consultancy.

Targeting Proteasome Ubiquitin Receptor RPN13/ADRM1 in Multiple Myeloma

Background and Rationale Deregulation of the ubiquitin-proteasome system (UPS) is linked to pathogenesis of various human diseases, including cancer. Targeting the proteasome is an effective therapy in multiple myeloma (MM) patients. Recent research efforts led to the discovery of newer agents that target enzymes modulating protein ubiquitin-conjugation/deconjugation rather than the proteasome itself, with the goal of generating more specific and less toxic antitumor therapies. Ubiquitylation is a dynamic reversible process coordinated by many enzymes: ubiquitin ligases attach ubiquitin to proteins allowing for their degradation, whereas deubiquitylating enzymes deconjugate ubiquitin from target proteins, thereby preventing their proteasome-mediated degradation. RPN13 is ubiquitin receptor within the 19S regulatory particle lid of the proteasome that recognizes ubiquitylated proteins marked for degradation by 20S core particle. Here we examined the role of RPN13 in MM using both biochemical and RNA interference strategies.Materials and Methods We utilizedMM cell lines, patient tumor cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors. Drug sensitivity/cell viability and apoptosis were assessed using XTT/MTT and Annexin V staining, respectively. MM.1S cells were transiently transfected with control short interfering RNA (siRNA), RPN13 siRNA ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. Synergistic/additive anti-MM activity was assessed by isobologram analysisusing “CalcuSyn” software program. Signal transduction pathways were evaluated using immunoblotting. Proteasome activity was measured as previously described (Chauhan et al., Cancer Cell 2005, 8:407-419). Statistical significance of data was determined using a Student’s t test. RA190 was purchased from Xcess Biosciences, USA; and bortezomib, lenalidomide, and pomalidomide were purchased from Selleck chemicals, USA.Results Analysis of RPN13/ADRM1 expression showed a significantly higher level in primary patient MM cells (n=73) versus normal plasma cells (n=15) (p < 0.004). Similarly, immunoblot analysis showed elevated RPN13 in MM cells versus normals. RPN13 siRNA knockdown significantly decreased MM cell viability (p < 0.001; n=3). To further validate our siRNA data, we utilized recently reported novel agent RA190 (bis-benzylidine piperidone) that targets RPN13. RA190 inhibits recognition of polyubiquitylated proteins and their deubiquitylation, which in turn prevents their degradation (Anchoori et al., Cancer Cell 2013, 24:791). Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, ARP-1, ANBL6.WT, and ANBL6.BR) and primary patient cells for 48h significantly decreased their viability (IC50 range 200nM to 600nM; p < 0.001 for all cell lines; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for RA190. Tumor cells were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Moreover, the cytotoxicity of RA190 was observed in MM cell lines sensitive and resistant to conventional (dex) and novel (bortezomib) therapies. Furthermore, RA190 inhibits proliferation of MM cells even in the presence of BM stromal cells. Mechanistic studies show that RA190-triggered MM cell death is associated with 1) accumulation of cells in early and late apoptotic phase; 2) increase in polyubiquinated proteins; and 3) activation of caspases mediating both intrinsic and extrinsic apoptotic pathways. Importantly, RA190-induced apoptosis in MM cells occurs in a p53-independent manner, since RA190 triggered significant apoptosis in both p53-null (ARP-1) and p53-mutant (RPMI-8226) MM cells (p < 0.004). Finally, combining RA190 with lenalidomide, pomalidomide, or bortezomib induces synergistic/additive anti-MM activity, and overcomes drug resistance.Conclusion Our preclinical data showing efficacy of RA190 in MM disease models validates targeting ubiquitin receptors upstream of the proteasome in the ubiquitin proteasomal cascade to overcome proteasome inhibitor resistance, and provides the framework for clinical evaluation of RPN13 inhibitors to improve patient outcome in MM. DisclosuresNo relevant conflicts of interest to declare.