Professor Case Western Reserve University Cleveland, Ohio, United States
Introduction: Multiple myeloma (MM) remains an incurable malignancy characterized by plasma cells that synthesize and secrete massive amounts of immunoglobulin (Ig, paraprotein). Hence, MM cells are exquisitely sensitive to agents that disrupt proteostasis. The proteasome is a highly sophisticated proteolytic complex designed to carry out selective hydrolysis of client proteins. Proteasome consist of two subcomplexes: a 20S catalytic core particle (CP) capped at one or both ends by 19S regulatory particle(s) (RP) that exhibit ATPase activity. Inhibition of the 20S proteasome catalytic subunit PSMB5 is the backbone of modern MM chemotherapy and has dramatically improved patient overall survival (OS). However, drug resistance inevitably emerges through molecular mechanisms that remain elusive. We postulated that the expression of clinically relevant proteasome subunits from MM cells correlated with reduced progression-free survival (PFS) and OS.
Methods: We correlated the expression of proteasome genes with clinical outcomes using the APEX trial dataset, which compared the effect of bortezomib vs. steroid, and the MMRF COMMPASS trial. Identified genes were then overexpressed or knocked out in MM cell lines. Biological and biochemical assays were then performed to determine the effect on proteasome structure and localization, drug sensitivity and ER stress.
Results: Results indicated that greater expression of the proteasome 19S RP ATPases PSMC2 and PSMC6 correlated with reduced OS, even outperforming the PI target PSMB5. MM cell lines that overexpressed PSMC2 and PSMC6 were then engineered. Non-denaturing gel electrophoresis demonstrated that PSMC2 is readily incorporated into 26S proteasomes. These cells exhibit higher levels of proteasome chymotrypsin-like activity activity (1.6-fold increase in ARH77 and 3-fold increase in U266 cells compared to controls; p< 0.01). PSMC2 overexpression induced resistant to PI challenge (bortezomib LD50 30 nM vs. 15nM in U266 cells and LD50 18 nM vs. 10 nM in ARH77 cells). PIs increase ER stress and PSMC2 overexpressing cells are better able to mitigate PI mediated ER stress with PSMC2 overexpressing cells exhibiting less induction of ER stress sensors phospho-IRE1, ATF4, and phospho-PERK compared to PSMC2 KD cells. Mechanistically, PSMC subunits may function in as a key player in the localization of proteasomes to the ER, recognition and degradation of misfolded ER proteins, and ER-associated protein degradation.
Conclusions: Taken together, our results demonstrate that PSMC2 and PSMC6 expression correlates with reduced OS in MM patients. Knockout of PSMC subunits decreased proteasome activity and increased dependence on autophagy, while PSMC upregulation increased proteasome activity and decreased PI sensitivity. Our work highlights the complex interplay of proteasome subunits in MM biology and suggests a prognostic and therapeutic role for 19S RP subunits in the anti-myeloma armamentarium.
