Targeting Proteasome- Associated Ubiquitin Receptor PSMD4/Rpn10 in Multiple Myeloma

The LeBow Institute for Myeloma Therapeutics and Jerome Lipper Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA Background and Rationale Normal cellular homeostasis is maintained by a balanced regulation of protein synthesis and degradation via the ubiquitin proteasome system (UPS). Deregulation of the UPS is linked to pathogenesis of various human diseases, including cancer. Targeting the 20S proteasome in the UPS via proteasome inhibitors bortezomib, carfilzomib, or ixazomib has proven effective therapy for relapsed, and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit their long-term utility. Therapeutic strategies directed against ubiquitin receptors upstream of the 20S proteasome may allow for more specific targeting of the UPS, with fewer off-target activities and toxicities. Ubiquitin-tagged proteins are recognized by 19S proteasome-associated ubiquitin receptors; the ubiquitin moiety is then removed from the substrate protein via deubiquitylating (DUB) enzyme enzymatic activity; target protein is then unfolded by the AAA-ATPases; followed by degradation of proteins through 20S proteolytic activities. Here we examined the role of ubiquitin receptor Rpn10/PSMD4 in MM using RNA interference strategies. Materials and Methods We utilized MM cell lines, patient tumor cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors. MM.1S cells were transiently transfected with control short interfering RNA (siRNA) or Rpn10-siRNA ON TARGET plus SMART pool using the cell line Nucleofector Kit V. Signal transduction pathways were evaluated using immunoblotting. Cell viability were assessed using WST-1 assay. Proteasome activity was measured as previously described (Chauhan et al., Cancer Cell 2005, 8:407-419). Statistical significance of data was determined using a Student9s t test. Results1) A statistically significant inverse correlation was noted between Rpn10 levels and overall patient survival (n=175) (p= 0.00064). 2) Analysis of PSMD4/Rpn10 gene expression showed a significantly higher level in primary patient MM cells versus normal plasma cells or PBMCs (p = 0.023 or p = 0.001, respectively). 3) Rpn10 protein is more highly expressed in MM cells than in normal cells. 4) Functional studies showed that blockade of Rpn10 using RNA interference (siRNA and shRNA) significantly decreased MM cell viability (p Conclusion Our preclinical data validates targeting 19S proteasome ubiquitin receptor Rpn10 upstream of proteasome in the ubiquitin proteasomal cascade, and provides the framework for development of proof-of-concept inhibitors targeting Rpn10 for future evaluation in clinical trials. Disclosures Chauhan: Triphase accelerator: Consultancy. Anderson: Bristol-Myers Squibb: Membership on an entity9s Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity9s Board of Directors or advisory committees; MedImmune: Membership on an entity9s Board of Directors or advisory committees; Gilead Sciences: Membership on an entity9s Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Oncopep: Other: scientific founder.