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.