PSMD3 orchestrates metabolic reprogramming and immunogenic cell death in multiple myeloma

Abstract 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 responsible for recognizing and delivering ubiquitinated proteins to the 20S CP. We identified PSMD3/Rpn3, a scaffold subunit of the 19S RP essential for proteasome assembly, as a potential target in MM. Using preclinical in vitro and in vivo models, we demonstrated that targeting PSMD3 induces MM cell apoptosis and overcomes PI resistance. Mechanically, targeting PSMD3 disrupts protein homeostasis, impairs metabolic adaptation, and relieves immune suppression in MM. Results Bioinformatic analyses revealed that PSMD3 is highly expressed in MM patient samples and correlates with poor overall survival. IHC and immunoblotting confirmed elevated PSMD3 levels in MM patient plasma cells compared to healthy controls. siRNA-mediated PSMD3 knockdown significantly reduced the viability of multiple MM cell lines, including those resistant to bortezomib (ANBL6-BR), carfilzomib (AMO1-CFZR), and pomalidomide (H929-PomR), suggesting its role in overcoming drug resistance. Inducible PSMD3 knockout (PSMD3-iKO) in AMO1 and KMS11 cells suppressed proliferation, while re-expression of wild-type PSMD3 restored growth, confirming target specificity. PSMD3 silencing induced apoptosis and cell cycle arrest, validated by flow cytometry and immunoblotting. Given PSMD3's role in proteasome assembly, we evaluated proteasome function and found marked accumulation of K48-linked polyubiquitinated proteins and activation of ER stress pathways in PSMD3-depleted MM cells. A degron-linked reporter assay further confirmed impaired proteasome degradation. In-gel proteasome assays demonstrated that PSMD3 knockout disrupted 26S proteasome assembly without affecting 20S core activity, indicating a deficiency in 19S regulatory particle incorporation. Finally, in a xenograft mouse model, PSMD3 depletion significantly suppressed tumor growth and prolonged survival, validating its therapeutic relevance in vivo. To elucidate the mechanism underlying PSMD3 depletion–induced cell death, we performed quantitative proteomic analyses. Pathway enrichment analysis revealed significant alterations in mitochondrial-related processes including glycolysis and oxidative phosphorylation, indicating that PSMD3 knockdown disrupts mitochondrial homeostasis. Functional metabolic profiling using the Seahorse XF Analyzer demonstrated that PSMD3 silencing led to profound mitochondrial dysfunction, evidenced by elevated mitochondrial superoxide levels and a marked reduction in maximal respiratory capacity, spare respiratory capacity, and ATP-linked respiration. This mitochondrial impairment resulted in the release of mitochondrial double-stranded DNA (mtDNA) into the cytosol, which in turn activated the cGAS-STING pathway. Activation of this innate immune sensor triggered a robust type I interferon response, as evidenced by upregulation of interferon-stimulated genes (ISGs). Moreover, PSMD3 depletion induced hallmark features of immunogenic cell death (ICD), including surface exposure of calreticulin and release of high mobility group box 1 (HMGB1), both of which enhance the immunogenicity of MM cells and promote anti-tumor immune recognition. Conclusion Our in vitro and in vivo data highlight the therapeutic potential of targeting PSMD3, a central regulator of protein homeostasis which links mitochondrial metabolism to immune surveillance in multiple myeloma. Targeting PSMD3 not only abrogates protein degradation but also disrupts energy metabolism and induces immunogenic cell death via cGAS-STING activation. These findings provide a compelling rationale for developing PSMD3-targeted strategies to overcome drug resistance and promote immune-mediated clearance of MM cells.