Background and Significance Therapeutic inhibition of the proteasomal catalytic activity of the 20S core particle (CP) by proteasome inhibitors (PIs) has been effective in treating multiple myeloma (MM); however, the clinical benefits are often limited by the off-target toxicities and the eventual development of PI resistance, resulting in disease relapse. Situated upstream of the 20S CP, the 19S regulatory particle (RP) of proteasome is essential for recruitment and unfolding of protein substrates prior to proteolysis. Since the ubiquitin receptors (UbRs) on 19S RP initiate degradation by recognizing ubiquitinated substrates, we hypothesized that inhibition of UbRs on 19S RP could provide a novel strategy to overcome PI resistance. Our previous studies have demonstrated that targeting the UbR PSMD4 and ADRM1 (Du et al., Blood 2023; Song et al., Leukemia 2016) can effectively overcome PI resistance and inhibit MM cell growth. PSMD2/Rpn1 constitutes one of the three UbRs on 19S RP and remains poorly understood in the context of MM.

Results In this study, we identified and validated PSMD2 as a novel therapeutic target in MM. To confirm the obligate role of PSMD2 in MM, we performed Total Proteome-wide analysis following CRISPR-Cas9-mediated inducible knockout of PSMD2, PSMD4, and ADRM1, with each UbR knockout individually. GSEA enrichment analysis showed that inhibition of PSMD2, but not the other two UbRs, significantly upregulates the interferon (IFN) alpha response and UV response pathway. Further mechanistic investigation revealed that PSMD2 depletion induces DNA damage, which subsequently activates the cGAS–STING–TBK1 axis, triggering type I IFN signaling and immunogenic cell death (ICD). Selective depletion of individual UbRs also differentially modulates the composition and function of proteasome subunits. Interestingly, PSMD4 expression decreased with PSMD2 knockdown whereas PSMD2 expression increased with knockdown of PSMD4, indicating that PSMD2 compensates for PSMD4, but not vice versa. We next compared the effects of UbRs inhibition on MM growth. Even partial loss of PSMD2 results in an earlier and more robust suppression of MM cell growth compared to complete loss of PSMD4.

To assess the clinical relevance of PSMD2 in MM, we analyzed the publicly available datasets and found that PSMD2 expression inversely correlates with clinical outcome. Further analysis of RNA expression levels and dependency scores revealed that PSMD2 is expressed at higher levels across all stages of patient bone marrow samples compared to normal plasma cells, and that PSMD2 is essential for the survival of various MM cell lines. PSMD2 protein levels were also increased in MM cell lines and MM patient CD138+ cells compared to normal plasma cells.

To determine whether the PSMD2 inhibition can overcome PI resistance, we established stable inducible knockdown (KD) cell lines using paired PI-sensitive and -resistant MM cell lines. In both cell lines, PSMD2 depletion reduced cell viability and impaired proteasome function, as evidenced by the accumulation of K48-linked ubiquitinated proteins. To further confirm that proteasome activity was impaired independently of the 20S CP, we performed a 20S activity assay, which showed that 20S function remained intact despite proteasome function inhibition. Consequently, endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) were activated, followed by G0–G1 cell cycle arrest and caspase-mediated apoptosis. Taking together, these findings suggest that targeting PSMD2 can overcome PI resistance. Finally utilizing a murine xenograft model, we demonstrated that inhibiting PSMD2 effectively inhibited MM cell growth and prolonged host survival.

Conclusions Overall, our data highlight the unique role of PSMD2 among UbRs in MM, and validate the therapeutic potential of targeting PSMD2 to overcome PI resistance, induce ICD, and improve patient outcome in MM.

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2025
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