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.