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

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.