26S Proteasome Non-Atpase Subunit 3 (PSMD3/Rpn3) Is a Potential Therapeutic Target in Multiple Myeloma

Background and Rationale: Multiple myeloma (MM) is a cancer of the plasma cells characterized by excessive production of immunoglobulins and dependence on the protein degradation system, which makes proteasome inhibitors (PIs) an important treatment for MM patients. However, PI resistance remains an unsolved problem in MM. PIs directly target the 20S core particle (CP) of the proteasome, which is responsible for proteolysis. The 19S regulatory particle (RP) is another component of the 26S proteasome that identifies ubiquitinated proteins and directs them to the 20S CP for degradation. We hypothesized that targeting components of the 19S RP would shut down proteasome activity. We focused on PSMD3/Rpn3, a critical scaffold subunit of the 19S RP responsible for proteasome assembly. Using preclinical in vitro and in vivo models, we demonstrated that targeting PSMD3 induces anti-MM effects and overcomes PI resistance. Results: Bioinformatic analysis indicated that PSMD3 is highly expressed in MM patients, and its high expression correlated to poor MM patient survival. Our immunoblot analysis showed significantly higher PSMD3 expression in MM patients' plasma cells versus healthy donor plasma cells. Knocking down PSMD3 using siRNA decreased the viability of various MM cell lines, including cell lines that are PI-resistant (ANBL6.BR) or carry p53 mutation/deletion (ANBL6-WT, JJN3, ARP1, KMS11). Inducible PSMD3-knockout (PSMD3-iKO) significantly reduced the cell growth and led to cell death of AMO1 and KMS11 cells. Importantly, adding PSMD3-WT back to PSMD3 iKO cells restored cell growth, indicating that the repression was not due to off-target effects. We found that PSMD3 depletion led to cell apoptosis and cell cycle arrest, validated by both flow cytometry and immunoblot. Similar results were found in Inducible PSMD3-knockdown (PSMD3-iKD) JJN3 cells and H929 cells. Intriguingly, depleting PSMD3 in ANBL6-BR (PI resistant) and ANBL-WT (PI sensitive) cell lines led to cell death at a similar level, demonstrating that targeting PSMD3 could overcome PI resistance in MM. As PSMD3 plays a key role in proteasome assembly, we assessed proteasome function after PSMD3 depletion. First, K48 polyubiquitinated proteins accumulated and downstream endoplasmic reticulum stress response signaling was significantly activated in AMO1 and KMS11 PSMD3-iKO cell lines, as well as in ANBL6-BR and ANBL6-WT PSMD3 iKD cell lines. A similar accumulation is reflected by an increased GFP level in a PSMD3-siRNA-transfected reporter cell line expressing Ub-tagged GFP, which is constitutively targeted for proteasome degradation. Using an in-gel proteasome assay, we found that PSMD3 knockout triggered a robust decrease in 26S proteasome assembly without affecting 20S proteasome activity, which indicated that fewer 19S RP were assembled onto 20S CPs when PSMD3 was absent. These data validate PSMD3 as a scaffold component in the proteasome assembly process and that PSMD3 blockade, like PIs, inhibits proteasome-mediated protein degradation. Finally, using Dox-inducible JJN3 PSMD3-iKD MM cells in a xenograft murine model, we found that PSMD3 depletion significantly reduced tumor growth and prolonged mouse survival. Conclusion: Our in vitro and in vivo data highlight the therapeutic potential of targeting PSMD3 and provide a strong preclinical foundation for developing PSMD3 inhibitors as a strategy to overcome proteasome inhibitor resistance in MM.