Deubiquitylating Enzyme USP1 As Therapeutic Target in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3290-3290
Author(s):  
Deepika Sharma Das ◽  
Yan Song ◽  
Arghya Ray ◽  
Mehmet K. Samur ◽  
Nikhil C. Munshi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dna Repair ◽  
Cell Viability ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cysteine Proteases ◽  
Id Proteins ◽  
Healthy Donors ◽  
Equity Ownership

Abstract 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 anti-tumor therapies. Our prior studies have identified a role of deubiquitylating enzymes (DUBs) USP7, USP14, and UCHL5 in MM pathogenesis, and provided the rationale for targeting these DUBs in MM (Chauhan et al., Cancer Cell 2012, 11:345-358). Among DUBs, USP1 regulates DNA repair and the Fanconi anemia pathway by deubiquitylating two critical DNA repair proteins, FANCD2-Ub and PCNA-Ub. Additionally, USP1 stabilizes tumor-promoting inhibitor of DNA binding (ID) proteins. Here we examined the role of USP1 DUB in MM using both biochemical and RNA interference strategies. Methods We utilized MM cell lines, patient cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors. Cell viability was assessed using WST and CellTiter-Glo assays. MM.1S cells were transiently transfected with control short interfering RNA (siRNA) USP1 ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. A biochemical inhibitor of USP1 SJB3-019A (SJB) was purchased from Medchem Express, USA. In vitro DUB enzymatic activity was assessed using Ubiquitin-AMC and Ubiquitin-Rhodamine assay kits, as well as Ub-CHOP-reporter and K48-linked Ubiquitin tetramers. Competitive Ub-VS probe labeling was performed, as previously described (Chauhan et al., Cancer Cell 2012, 11:345-358). Signal transduction pathways were evaluated using immunoblotting. Statistical significance of data was determined using a Student's t test. Results Gene expression profiling (GEP) analysis of USP1 showed significantly higher USP1 levels in patient MM cells versus normal plasma cells (p < 0.05).We found a statistically significant inverse correlation between USP1 levels and overall patient survival (p =0.036). Immunoblot blot analysis show higher USP1 levels in MM cell lines and patient cells compared to normal cells.USP1 knock-down reduced MM cell viability (p < 0.05).To validate our siRNA data, we utilized a novel USP1 inhibitor SJB3-019A (SJB). Analysis using Ub-Rhodamine, Ub-AMC and Ub-EKL reporter assays showed that SJB is a potent, specific, and selective inhibitor of cellular USP1 DUB activity (EC50=1μM), and does not inhibit other DUBs (USP2/USP5/USP7/USP14) or other families of cysteine proteases (UCH37) (EC50 >10 μM). SJB blocks labeling of USP1 with HA-Ub-VS probe in a concentration-dependent manner, but did not alter labeling of other DUBs with HA-Ub-VS. SJB inhibits USP1-mediated cleavage of K48-linked Tetra-ubiquitin chains, but not that mediated by USP2 or USP7. Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, Dox-40, ARP1, KMS11, U266, ANBL6.WT, ANBL6.BR and LR5) and primary patient cells for 24h significantly decreases their viability (IC50 range 100nM to 500nM) (p< 0.05; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting selective anti-MM activity and a favorable therapeutic index for SJB. Tumor cells from 4 patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies were sensitive to SJB. Furthermore, SJB3-019A inhibits proliferation of MM cells, even in the presence of BM stromal cells and plasmacytoid dendritic cells. Mechanistic studies show that SJB3-019A- triggered apoptosis is associated with 1) induction of cell cycle arrest via p21 upregulation; 2) activation of caspase 3, caspase-8 and caspase-9; 3) decreased homologous recombination activity and increased levels of Ub-FANCD2, Ub-FANCI, and Ub-PCNA; 4) defective DNA repair, evident by reduced RAD51; 5) degradation of USP1 and ID proteins; and 6) downregulation of Notch-1, Notch-2, SOX-4, and SOX-2 proteins. Finally, combination of SJB with lenalidomide, pomalidomide, HDACi ACY-241, or bortezomib induces synergistic anti-MM activity and overcomes drug resistance. Conclusion Our preclinical studies provide the framework for clinical evaluation of USP1 inhibitors, alone or in combination, as a potential novel MM therapy. Disclosures Munshi: Celgene Corporation: Consultancy; Pfizer: Consultancy; Merck: Consultancy; Oncopep: Consultancy, Equity Ownership; Takeda: Consultancy. Chauhan:Stemline Therapeutics, Inc.: Consultancy; C4 Therapeutics: Equity Ownership; Oncopeptide AB: Consultancy; Epicent Rx: Consultancy. Anderson:Celgene Corporation: Consultancy; Millennium Pharmaceuticals: Consultancy; Novartis AG: Consultancy; Bristol-Myers Squibb:: Consultancy; Oncopep: Other: Scientific Founder; Acetylon: Other: Scientific Founder.

Targeting Deubiquitylating Enzyme USP1 in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1813-1813
Author(s):  
Deepika Sharma Das ◽  
Yan Song ◽  
Arghya Ray ◽  
Paul Richardson ◽  
Dharminder Chauhan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dna Repair ◽  
Cell Lines ◽  
Cancer Cell ◽  
Therapeutic Index ◽  
Cysteine Proteases ◽  
Dependent Manner ◽  
Healthy Donors ◽  
Reporter Assays

Abstract Introduction Proteasome inhibitor Bortezomib is effective therapy of relapsed/refractory and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit its long-term utility. Importantly, the ability of bortezomib to overcome resistance to conventional therapies has validated therapeutically targeting the Ubiquitin Proteasome System (UPS), and suggested potential utility of inhibitors of other components of the UPS including deubiquitylating enzymes (DUBs). Therapeutic strategies directed against DUBs may allow for more specific targeting of the UPS, and therefore be less likely to have off-target activities with associated toxicities. Our prior studies have identified a role of USP7, USP14, and UCHL5 in MM pathogenesis, and provided the rationale for targeting these DUBs in MM (Chauhan et al., Cancer Cell 2012, 11:345-358; Tian et al., Blood 2014, 123:706-716). Among DUBs, USP1 regulates DNA repair and the Fanconi anemia pathway through its association with its WD40 binding partner UAF1, and through its deubiquitylation of two critical DNA repair proteins, FANCD2-Ub and PCNA-Ub. Here we examined the role of USP1 DUB in MM using both biochemical and RNA interference strategies. Methods We utilized MM cell lines, patient cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors. Cell viability was assessed using WST and CellTiter-Glo assays. MM.1S MM cells were transiently transfected with control short interfering RNA (siRNA), USP1 ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. A biochemical inhibitor of USP1 SJB3-019A (SJB) was purchased from Medchem Express, USA. In vitro DUB enzymatic activity was assessed using Ubiquitin-AMC and Ubiquitin-Rhodamine assay kits, as well as Ub-CHOP-reporter and K48-linked Ubiquitin tetramers. Competitive Ub-VS probe labeling was performed, as previously described (Chauhan et al., Cancer Cell 2012, 11:345-358). Signal transduction pathways were evaluated using immunoblotting. Statistical significance of data was determined using a Student's t test. Results Immunoblot analyses show higher USP1 levels in MM cell lines and patient cells than normal cells.USP1-siRNA inhibited MM cell proliferation, which was rescued by transfection of USP1 (WT). Using Ub-Rhodamine, Ub-AMC, and Ub-EKL reporter assays, we found higher USP1 deubiquitylating activity in patient MM cells versus normal cells, suggesting a favorable therapeutic index for targeting USP1. Importantly, siRNA-knockdown of USP1 both promoted degradation of tumorigenic ID1 protein, and inhibited proliferation of bortezomib-resistant (ANBL-6.BR) MM cells, suggesting that novel agents targeting USP1 may overcome bortezomib resistance. We next examined the effects of USP1 inhibitor SJB3 on MM cell growth and survival in our models of MM. Analysis using Ub-Rhodamine, Ub-AMC, and Ub-EKL reporter assays in a panel of MM cell lines showed that SJB is a potent, specific, and selective inhibitor of USP1 DUB activity (EC50 = 50 ± 5.7 nM), which does not inhibit other DUBs (USP2/USP5/USP7/USP14) or other families of cysteine proteases (EC50>100 μM). SJB blocks labeling of USP1 with HA-Ub-VS probe in a concentration-dependent manner, but did not alter labeling of other DUBs with HA-Ub-VS. SJB inhibits USP1-mediated cleavage of K48 linked polyubiquitin chains, but not that mediated by USP2 or USP7. Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, Dox-40, ARP1, KMS11, U266, ANBL6.WT, ANBL6.BR, and LR5) and primary patient cells for 24h significantly decreases their viability (IC50 range 100nM to 500nM) (p < 0.05; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for SJB. Tumor cells from 3 of 5 patients were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Mechanistic studies show that SJB-triggered apoptosis is associated with degradation of USP1 and Id1 protein. Finally, combination of SJB with lenalidomide, pomalidomide, HDACi ACY-1215, or bortezomib both induces synergistic anti-MM activity and overcomes drug resistance. Conclusion Our preclinical studies provide the framework for clinical evaluation of USP1 inhibitors, alone or in combination, as a potential MM therapy. Disclosures Chauhan: Stemline Therapeutics: Consultancy.

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

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3211-3211
Author(s):  
Yan Song ◽  
Arghya Ray ◽  
Dharminder Chauhan ◽  
Kenneth Anderson
Keyword(s):  
Multiple Myeloma ◽  
Cell Viability ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Statistical Significance ◽  
Functional Characterization ◽  
Prolonged Treatment ◽  
Equity Ownership

Abstract 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. Disclosures Anderson: 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.

Role Of Sirtuin-6 In Maintenance Of Genomic Instability In Multiple Myeloma Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 276-276
Author(s):  
Michele Cea ◽  
Antonia Cagnetta ◽  
Mariateresa Fulciniti ◽  
Yu-Tzu Tai ◽  
Chirag Acharya ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dna Damage ◽  
Dna Repair ◽  
Genomic Instability ◽  
Cell Lines ◽  
Dsb Repair ◽  
Dna Damaging Agents ◽  
Genotoxic Agents ◽  
Equity Ownership

Abstract Background Deregulation of the DNA damage response (DDR) signaling machinery underlies genomic instability, leading to cancer development and clonal evolution. Multiple Myeloma (MM) remains an incurable disease characterized by a highly unstable genome, with aneuploidy observed in nearly all patients. The mechanism causing this karyotypic instability is largely unknown, but recent observations have correlated these abnormalities with dysfunctional DDR machinery. Mammalian NAD+-dependent deacetylase sirtuin-6 (SIRT6) is emerging as new protein involved in multiple pathways, including maintenance of genome integrity. Methods A panel of 18 MM cell lines, both sensitive and resistant to conventional and novel anti-MM therapies, was used in this study. Blood and BM samples from healthy volunteers and MM patients were obtained after informed consent and mononuclear cells (MNCs) separated by Ficoll-Paque density sedimentation. Patient MM cells were isolated from BM MNCs by CD138-positive selection. Lentiviral delivery was used for expression and knock-down of SIRT6 in MM cell lines. The biologic impact of SIRT6 phenotype was evaluated using cell growth, viability and apoptosis assays. DNA Double-Strand Breaks (DSB) repair occurring via homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways was assessed using a transient direct repeat (DR)-GFP/I-SceI system. Results A comparative gene expression analysis of 414 newly-diagnosed uniformly-treated MM patients showed high levels of SIRT6 mRNA in MM patients versus MGUS or normal donors; moreover, in active MM elevated SIRT6 expression correlated with adverse clinical outcome. Due to its prognostic significance, we further evaluated its role in MM biology. We found higher SIRT6 nuclear expression in MM cell lines and primary cells compared to PBMCs from healthy donors. Targeting SIRT6 by specific shRNA increased MM cell survival by reducing DNA repair efficiency (HR and NHEJ). Whole genome profiling of three different SIRT6 knockout (Sirt6-/-) MM cell lines identified a restricted effect of SIRT6 silencing on transcription of DNA damage genes, which also represented the most down-regulated genes. Consistent with these data, GSEA algorithm revealed that gene set regulating DNA repair were prominently enriched in SIRT6 depleted cells (p<0.0001 and FDR=0.003), confirming the role of SIRT6 in this pathway. We next examined the therapeutic relevance of SIRT6 inhibition in MM by evaluating the effect of SIRT6 depletion on cytotoxicity induced by genotoxic agents. SIRT6 shRNA impaired DNA DSB repair pathways triggered by DNA damaging agents, thereby enhancing overall anti-MM activity of these agents. Finally, in concert with our in vitro data, studies using our human MM xenograft model confirmed that SIRT6 depletion enhanced anti-MM activity of DNA-damaging agents. Conclusion Collectively, our data provide basis for targeting SIRT6 as a novel therapeutic strategy in combination with genotoxic agents to enhance cytotoxicity and improve patient outcome in MM. Disclosures: Tai: Onyx: Consultancy. Hideshima:Acetylon Pharmaceuticals: Consultancy. Chauhan:Vivolux: Consultancy. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

scholarly journals Identification of a First-in-Class SETD2 Inhibitor That Shows Potent and Selective Anti-Proliferative Activity in t(4;14) Multiple Myeloma: T(4;14) Multiple Myeloma Cells Are Dependent on Both H3K36 Di and Tri-Methylation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3207-3207 ◽  
Author(s):  
Michael J Thomenius ◽  
Jennifer Totman ◽  
Kat Cosmopoulos ◽  
Dorothy Brach ◽  
Lei Ci ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
High Risk ◽  
Cell Lines ◽  
Cancer Cell ◽  
Proliferative Activity ◽  
Therapeutic Potential ◽  
Response To Treatment ◽  
Employment Equity ◽  
Equity Ownership

Abstract t(4;14) chromosome translocations are found in 15% of newly diagnosed multiple myeloma (MM) cases and are associated with high risk. MM cells with t(4;14) over-express the histone methyltransferase (HMT), WHSC1/MMSET/NSD2, which leads to deregulation of gene expression due to increased di-methylation of Histone H3 at lysine 36 (H3K36me2). This activity has been shown to be essential for the survival of t(4;14) MM cells. In addition to WHSC1, another HMT, SETD2, has been shown to methylate H3K36. SETD2 is the only known enzyme capable of tri-methylation of H3K36 and has been reported to play a role in transcriptional elongation and alternative splicing. CRISPR pooled screening has shown that SETD2 activity is required for viability of a variety of cancer cell lines. This led Epizyme to develop small molecule inhibitors of SETD2 enzyme activity in order to understand the role of SETD2 in tumorigenesis. Through our drug discovery efforts, we identified EPZ-040414, a potent and selective inhibitor of SETD2 with low nM cell biochemical activity and broad selectivity against a panel of other HMTs. The proposed role of SETD2 in H3K36 methylation led us to test a panel of MM cells, including 6 t(4;14) cell lines with EPZ-040414. Inhibition of SETD2 resulted in reduced global tri-methylation of H3K36 in t(4;14) bearing MM cell lines. In contrast, there was no effect on global di-methyl H3K36 levels, indicating that WHSC1 activity is not affected by SETD2 inhibition. Moreover, 5/6 t(4;14) MM cell lines showed a cytotoxic response to treatment with EPZ-040414 with IC50s ranging between 60 and 200 nM, while all non-t(4;14) MM cell lines showed limited responses between 1 and 8 μM. Moreover, screening of a 280 cancer cell line panel with a SETD2 inhibitor showed minimal anti-proliferative activity in most cell lines tested. These findings show that t(4;14) MM cell lines require SETD2 activity for survival, suggesting that SETD2 inhibitors are strong candidates for the treatment of this high risk subgroup of MM. Efforts to further understand the interaction between SETD2 and WHSC1 in the molecular pathogenesis of t(4;14) myeloma will be presented. The current chemical series represented by EPZ-040414 is potent, selective, orally available, and currently under further evaluation for its therapeutic potential. Figure. Figure. Disclosures Thomenius: Epizyme Inc.: Employment, Equity Ownership. Totman:Epizyme Inc.: Employment, Equity Ownership. Cosmopoulos:Epizyme Inc.: Employment, Equity Ownership. Brach:Epizyme Inc.: Employment, Equity Ownership. Ci:Epizyme Inc.: Employment, Equity Ownership. Farrow:Epizyme Inc.: Employment, Equity Ownership. Smith:Epizyme Inc.: Employment, Equity Ownership. Chesworth:Epizyme Inc.: Employment, Equity Ownership. Duncan:Epizyme Inc.: Employment, Equity Ownership. Tang:Epizyme Inc.: Employment, Equity Ownership. Riera:Epizyme Inc.: Employment, Equity Ownership. Lampe:Epizyme Inc.: Employment, Equity Ownership.

scholarly journals Targeting Proteasome Ubiquitin Receptor RPN13/ADRM1 in Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 419-419
Author(s):  
Yan Song ◽  
Deepika Sharma Das ◽  
Arghya Ray ◽  
Durgadevi Ravillah ◽  
Nikhil C. Munshi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Viability ◽  
Tumor Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Statistical Significance ◽  
Healthy Donors ◽  
Rpmi 8226

Abstract 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. Disclosures No relevant conflicts of interest to declare.

scholarly journals Deubiquitylating Enzyme Rpn11/POH1/PSMD14 As Therapeutic Target in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4469-4469 ◽  
Author(s):  
Yan Song ◽  
Arghya Ray ◽  
Deepika Sharma Das ◽  
Mehmet K. Samur ◽  
Ruben D. Carrasco ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Viability ◽  
Board Of Directors ◽  
Cell Lines ◽  
20S Proteasome ◽  
Advisory Committees ◽  
Healthy Donors ◽  
Equity Ownership ◽  
Rpmi 8226

Abstract Introduction The ubiquitin proteasome pathway is a validated therapeutic target in multiple myeloma (MM), evidenced by the FDA approval of proteasome inhibitors bortezomib, carfilzomib, and ixazomib. However, these agents are associated with possible off-target toxicities and the eventual development of drug-resistance. Therapeutic strategies directed against deubiquitylating (DUB) enzymes upstream of the 20S proteasome may allow for more
specific targeting of the UPS, with fewer off-target activities
and toxicities. Rpn11 is a 19S-proteasome-associated DUB enzyme that facilitates protein degradation by the 20S proteasome core particle. Here we examined the role of Rpn11 in MM using both biochemical and RNA interference strategies. Materials and Methods Drug sensitivity, cell viability, and apoptosis assays were performed using WST, MTT, 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. In the xenograft mouse model, CB-17 SCID-mice were subcutaneously inoculated with MM.1S cells as previously described (Chauhan et al., Cancer Cell 2005, 8:407-419). Signal transduction pathways were evaluated using immunoblotting. Isobologram analysisand CalcuSyn software program were utilized to assesssynergistic/additive anti-MM activity. Statistical significance of observed differences were determined using a Student's t test. O-phenanthroline (OPA) was purchased from EMD Millipore, USA; and dex, lenalidomide, and pomalidomide were purchased from Selleck chemicals, USA. Results We found a statistically significant inverse correlation between Rpn11 levels and overall patient survival (p =0.022). Gene expression (GEP) analysisof Rpn11 showed a significantly higher level in patient MM cells versus normal plasma cells or PBMCs (p = 0.002 or p = 0.001 respectively). Immunohistochemical analysis of bone marrow biopsies from MM patients and normal healthy donors showed higher Rpn11 expression in MM cells than normal cells. Similarly, western blot analysis showed higher Rpn11 levels in MM cell lines and patient cells versus normal PBMCs.Rpn11 knockdown in MM cells significantly decreased cell viability (p < 0.001; n=3). To 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 patient MM cells with OPA significantly decreased cell viability (IC50 range 8µM to 60µM; p < 0.001 for all cell lines; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting selective anti-MM activity and a favorable therapeutic index for OPA. Importantly, the anti-MM activity of OPA was observed against tumor cells obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. In concert with these data, the cytotoxicity of OPA was observed in MM cell lines sensitive and resistant to conventional and novel therapies. Furthermore, OPA inhibits proliferation of MM cells even in the presence of BM stromal cells or plasmacytoid dendritic cells (pDCs). OPA inhibits Rpn11 DUB activity without blocking 20S proteasome activities. Mechanistic studies show that OPA-triggered MM cell apoptosis is associated with 1) activation of caspases; 2) accumulation of polyubiquitinated proteins; 3); induction of ER stress; and 4) induction of autophagy. OPA-induced apoptosis occurs in a p53-independent manner, since OPA triggered apoptosis in both p53-null (ARP-1) and p53-mutant (RPMI-8226) MM cells. OPA inhibits MM cell growth in vivo and prolongs survival in a MM xenograft mouse model. Finally, combining OPA with lenalidomide, pomalidomide, or dex induces synergistic/additive anti-MM activity, and overcomes drug resistance. Conclusion Our preclinical data showing efficacy of OPA in MM models both validates targeting 19S proteasome-associated DUB Rpn11, and provides the framework for clinical evaluation of Rpn11 inhibitors to overcome proteasome inhibitor resistance and improve patient outcome in MM. Disclosures Munshi: Celgene Corporation: Consultancy; Merck: Consultancy; Pfizer: Consultancy; Oncopep: Consultancy, Equity Ownership; Takeda: Consultancy. Chauhan:C4 Therapeutics: Equity Ownership; Epicent Rx: Consultancy; Oncopeptide AB: Consultancy; Stemline Therapeutics, Inc.: Consultancy. Anderson:Sonofi Aventis: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: Scientific Founder; Acetylon: Other: Scientific Founder; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Recurrent Non-Coding Mutations in BCL7A May Have Significant Functional Consequence in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 857-857
Author(s):  
Chandraditya Chakraborty ◽  
Eugenio Morelli ◽  
María Linares ◽  
Kenneth C. Anderson ◽  
Mehmet Kemal Samur ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Viability ◽  
Cell Lines ◽  
Chromatin Remodeling ◽  
Colony Formation ◽  
Ectopic Expression ◽  
Advisory Board ◽  
Mass Spectrometry Analysis ◽  
Rna Seq

Multiple myeloma (MM) is a complex hematological malignancy characterized by gene pathway deregulations. Initial sequencing approaches have failed to identify any single frequent (&gt;25%) mutation in the coding genome. We, therefore performed a deep (average coverage &gt; 80X) whole genome sequencing (WGS) on 260 MM samples (208 newly diagnosed and 52 first relapse after uniform treatment) to comprehensively identify recurrent somatic alterations in non-coding regions. We have identified the most frequently involved genes affected by perturbation in neighboring non-coding region and integrate their expression using our matching deep RNA-seq data from the same patients. One of the most prominent examples is mutations in the 5' untranslated region and intron 1 of the BCL7A gene in 76% of myeloma patients. Integration of WGS with RNA-seq data confirmed significant downregulation of its expression (p values &lt; 1e-5) in the MM cells as compared to normal plasma cells (PC). This led us to investigate the consequences of BCL-7A loss in MM. To evaluate the role of BCL7A in MM, using gain of- (GOF) and loss-of-function (LOF) approaches, we have utilized a large panel of MM cell lines with differential expression of BCL7A at the RNA and protein levels. Ectopic expression of BCL7A in a panel of 3 MM cell lines with low basal levels of BCL7a significantly reduced cell viability and colony formation over time. Inhibition of cell viability was associated with induction of apoptotic cell death in the BCL7A overexpressing cells compared to control cells. LOF studies in 3 MM cell lines with relatively higher expression of BCL7a using 3 BCL7A-specific shRNA constructs showed a more proliferative phenotype, with increased growth and viability and enhanced colony formation. The effects of BCL7A loss in MM cells were further confirmed using CRISPR-Cas9 system. BCL7a-KO cells had higher proliferative rate compared to WT cells and add back of lentiviral BCL7a plasmid reversed this effect. BCL7A is part of the SWI/SNF chromatin remodeling complex. Mutations in the genes encoding m-SWI/SNF subunits are found in more than 20% of human cancers, with subunit- and complex-specific functions. We confirmed that when expressed, BCL7A interacts with BCL11A into the SWI/SNF complex in MM cells. Comparative, mass spectrometry analysis in fact revealed SMARCC2 (BAF170), an integral subunit of SWI/SNF complex, to bind with BCL7A-BCL11A complex. However, BCL7A loss causes decreased SMARCC2 incorporation into SWI/SNF, thus suggesting that presence of BCL7A is crucial in the formation of SWI/SNF complex in MM cells and might play an important role in chromatin remodeling. Interestingly, oncogenes DEK (DNA binding oncogene) and TPD52 (tumor protein D52) involved in cancer cell proliferation and chromatin remodeling formed complex with BCL11A in BCL7A KO MM cells. Additionally, several anti-apoptotic proteins such as ANXA-1 and BCL2 are in complex with BCL11A when BCL7A is lost, suggesting the formation of an anti-apoptotic complex with consequences on MM cell survival. Currently ongoing studies are investigating the molecular mechanism of non-coding mutations impacting BCL7A expression and pathways affected by its downregulation with impact on MM cell growth and survival. In conclusion, we report biological consequences of a frequent (&gt;75% patients) non-coding mutation in MM with cellular and molecular effects of BCL7A loss in which implicates a functional role of the m-SWI/SNF complex in driving a MM cell proliferative phenotype. Disclosures Anderson: Gilead Sciences: Other: Advisory Board; Janssen: Other: Advisory Board; Sanofi-Aventis: Other: Advisory Board; C4 Therapeutics: Other: Scientific founder ; OncoPep: Other: Scientific founder . Munshi:Abbvie: Consultancy; Abbvie: Consultancy; Amgen: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Adaptive: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Oncopep: Consultancy; Celgene: Consultancy.

The Novel Human Double Minute (HDM)-2 Inhibitor DS-5272 Is Active Both Alone, and In Combination With Other Novel Agents, Against Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1927-1927
Author(s):  
Dongmin Gu ◽  
Hua Wang ◽  
Zhiqiang Wang ◽  
Richard E. Davis ◽  
Shengli Cai ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Viability ◽  
Cell Lines ◽  
Annexin V ◽  
Myeloma Cell ◽  
Novel Agents ◽  
Type I ◽  
Employment Equity ◽  
Equity Ownership ◽  
Combination Regimens

Abstract Background The ubiquitin-proteasome pathway is now a validated target for myeloma therapy given the regulatory approvals of proteasome inhibitors such as bortezomib and carfilzomib. Another logical set of targets are the E3 ligases, whose role is to facilitate the protein poly-ubiquitination typically needed for recognition by the constitutive or immunoproteasome prior to proteolysis. One of the more attractive such targets is HDM-2, the E3 ligase best known for its role in turnover of the p53 tumor suppressor, in part because p53 deletion or mutation is less common in myeloma than in solid tumors, and because it has been possible to develop agents targeting the HDM-2 p53 binding pocket. Methods Activity of the specific HDM-2 inhibitor DS-5272 (Daiichi-Sankyo) was evaluated using a panel of p53 wild-type (wt) and mutant (mut) myeloma cell lines and also against primary patient samples. Studies were performed with DS-5272 both alone, and also in combination regimens with novel agents. Tetrazolium dye-based assays were employed to determine cell viability, Annexin V staining was used to examine apoptosis, and quantitative PCR and Western blotting were used to study selected transcripts and gene products, respectively. This study was supported in part by the M. D. Anderson Cancer Center SPORE in Multiple Myeloma. Results DS-5272 induced potent cytotoxicity in wt p53 MM1.S, H929, and MOLP-8 myeloma cell lines, with a median inhibitory concentration (IC50) in the single micromolar range, which was reproduced in studies of primary CD138+ plasma cells but not CD138- cells from myeloma patients. This cytotoxicity was both time- and concentration-dependent, and DS-5272 was more potent than the prototypical HDM-2 inhibitors, Nutlin-3a and MI-219. A dependence of DS-5272 activity on wt p53 was demonstrated by the much lower IC50 in the wt p53 than mut p53 cell lines, and the finding that suppression of p53 with a shRNA, as well as mutation of p53 with a sequence-specific zinc finger nuclease, significantly increased the IC50. Notably, DS-5272 remained active in the presence of conditioned media from stromal cells, or key myeloma cytokines such as IL-6. The reduced viability after exposure to DS-5272 was due at least in part to activation of type I cell death, as determined by increased staining for Annexin V, activation of caspases 9 and 3, and cleavage of PARP. Other downstream effects included induction of transcription of p21, Bax, HDM-2, NOXA, and PUMA. These proteins, as well as p27 and p53 were induced, while the abundance of Survivin, CHK1, Aurora A and B, PLK1, and KIF11 was reduced. Consistent with these latter effects, DS-5272 induced some accumulation of cells at the G2/M phase, and the appearance of cells with a disordered spindle. Suppression of KIF11 (kinesin spindle protein (KSP; Eg5)), which is responsible for centromere separation and bipolar spindle formation, seemed to occur through the binding of p21 to the cell cycle genes homology region (CHR) within the KIF11 promoter. Finally, combinations of DS-5272 with the specific KIF11 inhibitor Ispinesib produced enhanced G2/M arrest, as well as a synergistic reduction in cell viability with increased levels of apoptosis. Conclusion DS-5272 is a potent and novel agent with activity against multiple myeloma, and is active both alone and in rationally designed combination regimens with other drugs. These findings provide a rationale for the clinical translation of HDM-2 inhibitors as monotherapy, and possibly with other agents such as the KSP inhibitor ARRY-520, which is also active against myeloma, for patients with wt p53 relapsed and/or refractory myeloma. Disclosures: Cai: Daiichi-Sankyo Pharma Development: Employment, Equity Ownership. Seki:Daiichi Sankyo Co., Ltd.: Employment, Equity Ownership. Tse:Daiichi-Sankyo Pharma Development: Employment, Equity Ownership.

The Biologic Relevance of PIM Kinases in the Context of Multiple Myeloma and Their Potential As Therapeutic Targets in Combination Drug Therapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2992-2992
Author(s):  
Janani Ramachandran ◽  
Loredana Santo ◽  
Homare Eda ◽  
Dharminder Chauhan ◽  
Ka Tat Siu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Dna Damage ◽  
Drug Therapy ◽  
Cell Viability ◽  
Cell Lines ◽  
Research Funding ◽  
Doxorubicin Treatment ◽  
Pim Kinases

Abstract The proto-oncogene proviral integration sites for moloney murine leukemia virus (PIM) are serine/threonine kinases currently under investigation as therapeutic targets in hematologic cancers, where they have important functional roles as mediators of apoptosis, cell migration and homing. Thus far in Multiple Myeloma (MM), the PIM Kinases have been shown to mediate cap-dependent and cap-independent translation through the PI3K/AKT pathway and the activation of mTOR signaling (Lu J et al. 2013). In the context of the bone marrow microenvironment, PIM kinase expression in MM cells is known to be elevated in the presence of bone marrow stromal cells, and is mediated by the IL-6/STAT3 pathway, as well as the TNFα/NFΚB pathway (activated by Osteoclast secretion) (Hiasa M et al. 2014). Here, we have further studied the role of PIM kinases in MM. We observed elevated expression of PIM 1, 2 and 3 in patient derived myeloma cells (CD138+) as opposed to the stromal compartment (CD138-), confirming that PIM expression is predominantly hematologic lineage-specific. Inhibition of all three kinases by a pan PIM inhibitor results in reduced cell viability in tested human-derived MM cell lines, reinforcing the importance of the kinases as targets in drug therapy, as shown previously. Because the expression of Pim2 is consistently higher in primary tumor cells as well as in human MM lines, compared to Pim1 and Pim3, we sought to identify their distinct biologic significance. Single knock down (KD) of each kinase resulted in varying effects on cell viability, suggesting, together with the PIM expression profile, that the three kinases play different roles in the biology of MM. As the Pim2 KD resulted in the most profound decrease in cell viability, we focused our efforts to dissect the mechanistic importance of Pim2. Since Pim1 and Pim2 regulate the DNA damage response (DDR) via checkpoint kinase 1 (Chk1) in other hematologic malignancies, we investigated the effect of Pim2 KD on major signaling factors involved in the DDR. Transient KD resulted in phosphorylation of DDR pathway markers including ATR, CHK1/2, P21 and H2AX, and mimicked the effects of Doxorubicin treatment (a known DNA Damage causing agent). Furthermore, Doxorubicin treatment downregulated Pim2 expression, suggesting that Pim2 functions as an upstream regulator of the DDR pathway in MM. Pim2 appears to be the most relevant target in MM; however, because of a lack of a specific PIM2 inhibitor we used the pan-PIM inhibitor as a tool compound. Although the pan PIM kinase inhibitor showed single agent activity, combination approaches were more efficacious. Combining the pan-PIM inhibitor with bortezomib shows a significant synergistic effect on cell viability in multiple MM cell lines (MM1S, U266, KMS-12BM). Based on our KD experiments Pim2 predominantly mediates cell viability, suggesting that specifically targeting Pim2 in combination with Bortezomib will have a more direct effect on MM cell survival. To confirm the role of Pim2, ongoing experiments aim to focus on the effects of ectopically expressing Pim2 in the context of myeloma. We propose that Pim2 overexpression will not only serve anti-apoptotic purposes, but will further protect against DNA Damage in human MM cell lines. Disclosures Chauhan: Stemline Therapeutics: Consultancy. Huszar:Astra Zeneca: Employment. Raje:AstraZeneca: Research Funding; Onyx: Consultancy; BMS: Consultancy; Acetylon: Research Funding; Millenium: Consultancy; Amgen: Consultancy; Novartis: Consultancy; Takeda: Consultancy; Eli Lilly: Research Funding; Celgene Corporation: Consultancy.

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