26S Proteasome Non-Atpase Subunit 1, PSMD1/Rpn2, Is a Potential Therapeutic Target in Multiple Myeloma

Background and Rationale Multiple myeloma (MM) is a cancer of the plasma cells that involves the production of a large amount of immunoglobulins, making MM cells sensitive to disturbances in their proteolytic pathways. Yet, they often develop resistance to conventional proteasome inhibitors (PIs). The 26S proteosome is made of the 20S core particle (CP), which degrades proteins and is targeted by PIs, and the 19S regulatory particle (RP), which recognizes ubiquitinated proteins and feeds them into the 20S CP. Here, we focused on identifying and validating novel therapeutic targets within the 19S RP to overcome the PIs resistance in MM. PSMD1/Rpn2, the non-ATPase regulatory subunit 1 is a key structural component of 19S RP. In the current study, we utilized our preclinical in vitro and in vivo models to show that targeting Rpn2 triggers anti-MM activity and overcomes PI-resistance.

Results The largest non-ATPase subunit, Rpn2 which is a scaffolding protein and is responsible for the docking of other proteasome subunits within 19S RP. Our immunoblot analysis showed higher Rpn2 expression in MM versus normal peripheral blood mononuclear cells. Immunohistochemistry studies showed significantly higher Rpn2 expression in BM biopsies from MM patients than from healthy individuals. Knocking down Rpn2 using transiently transfected siRNA decreased the viability of various MM cell lines, as assessed by WST-1 assays, including cell lines that are PI-resistant (ANBL6.BR) or carry p53 alterations (JJN3, ARP1) (p< 0.01). Both stable inducible Rpn2-KD and inducible Rpn2-KO AMO1 cells had significantly reduced cell growth. Importantly, transfection with Rpn2-WT rescued cells from the growth-inhibitory activity of Rpn2 siRNA. Rpn2 blockade increased the level of k48-ubiquitinated proteins, reflecting inhibition of proteasome-mediated protein degradation, and caused cell death through cell cycle arrest, apoptosis associated with the activation of caspases, and endoplasmic reticulum stress response signaling. Finally, using Dox-inducible Rpn2-KD AMO1 MM cells in a xenograft mouse model, we found that Rpn2 depletion reduced tumor growth and prolonged mouse survival (p < 0.005).

Conclusion Our preclinical data demonstrate the therapeutic potential of targeting Rpn2 and provide the preclinical basis for developing Rpn2 inhibitors to overcome PI resistance in MM.

Munshi:Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Takeda Oncology: Consultancy; Bristol-Myers Squibb: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Adaptive Biotechnology: Consultancy; GSK: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Karyopharm: Consultancy; Pfizer: Consultancy; Abbvie: Consultancy; Legend: Consultancy. Chauhan:Stemline Therapeutics: Consultancy; Oncopeptides: Consultancy; C4 Therapeutics: Current equity holder in publicly-traded company. Anderson:Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees; Dynamic Cell Therapy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; NextRNA: Other: Scientific founder ; Raqia: Other: Scientific founder ; C4 Therapeutics: Other: Scientific founder ; OncoPep: Other: Scientific founder ; Starton: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Precision Biosciences: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Window: Membership on an entity's Board of Directors or advisory committees.