Proteasome Regulatory Atpases PSMC2 and PSMC6 Modulate Proteosome Inhibitor Sensitivity and Correlate with Survival in Multiple Myeloma

Background: Multiple myeloma (MM) cells synthesize and secrete massive amounts of immunoglobulins and thus are exquisitely sensitive to therapeutics that disrupt proteostasis. The 26S proteasome complex is a 2.5-MDa holoenzyme comprised of >32 individual subunits responsible for the bulk of intracellular protein degradation. 26S proteasomes are comprised of barrel-shaped proteolytic core complex, the 20S core particle (CP), capped at either or both ends by 19S regulatory particles (RPs), which confer ATPase activity and specificity for ubiquitinated proteins. Six distinct ATPases (PSMC1-6) form a heterohexameric ring-like structure that rests atop the 20S CP's α-subunits and is responsible for unfolding and shuttling target proteins for degradation within the 20S CP. The proteasome inhibitor (PI) bortezomib (BTZ) is a potent, selective, and reversible drug that dramatically improved the prognosis of multiple myeloma (MM) patients and remains the backbone of induction therapy for transplant-eligible new diagnosis MM (NDMM), but response to therapy remains highly variable. Here, we predicted that gene expression analysis of publically available datasets could identify components within the ubiquitin-proteasome system (UPS) that areprognostic in patients treated with BTZ, suggesting potential new targets in MM.

Methods: We interrogated gene expression profiles (GEP) from the randomized, phase 3 Assessment of Proteasome Inhibition for Extending Remissions (APEX) study that compared BTZ with high-dose dexamethasone (DEX) in RRMM patients who had received 1-3 prior therapies. APEX datasets served as a test cohort to identify genes that influenced survival in patients treated with BTZ. Patients consented to genomic analyses of pretreatment tumor samples. CD138+ cells were isolated from BM aspirates and analyzed by RNA microarrays to generate GEP datasets. We correlated gene expression with response to BTZ or DEX and overall survival (OS). We then validated individual genes identified from the APEX analysis using the MM Research Foundation (MMRF) CoMMpass study, a longitudinal observational analysis of the clinical and molecular data for >1000 NDMM patients receiving various standard approved treatments. Finally, we used shRNA to knockdown PSMC2 in RPMI8226 and ARH77 cells and tested their sensitivity to BTZ and carfilzomib (CFZ).

Results: Analysis of the APEX dataset revealed that increased expression of genes PSMC2 and PSMC6, members of the heterohexameric ATPase ring of the 19S RP, correlated with poorer OS (PSMC2- Figure 1A, PSMC6- Median OS 320 versus 620 days for upper and lower tertile PSMC6, respectively). Using the CoMMpass database, increased PSMC2 expression also correlated with a worse OS (Figure 1B), while increased PSMC6 correlated with worse OS as well as progression free survival (PFS) (OS- HR= 2.29 p=0.0014, PFS- HR=1.63 p=0.0109). PSMC6 displayed greater expression in 50 patients above the median RNA expression (Fragments per Kb transcript per million mapped reads (FPKM)= 1 to 100) while PSMC2 expression was elevated in 400 patients above the median (FPKM= 25 to 100).

shRNA-mediated PSMC2 knockdown (KD) increased PI susceptibility in ARH77 and the RPMI8226 MM cells. In ARH77 cells, the BTZ LD50 decreased from 11 nM to 2.5 nM and the CFZ LD50 decreased from 11 to 9 nM in control and KD cells, respectively. In RPMI8226 cells, the BTZ LD50 decreased from 30 nM to 20 nM and the CFZ LD50 decreased from 28 to 17 nM in control and KD cells, respectively.

Conclusion: Taken together, our results indicate that increased PSMC2 and PSMC6 correlates with OS in MM in both patients treated with BTZ in the APEX and with a variety of standard therapies in the CoMMpass database. Analysis in this validation cohort extends the clinical significance of PSMC2/PSMC6 beyond the landmark APEX trial and suggests they may inform survival in patients treated with modern triplet anti-myeloma therapies. In addition to the prognostic importance across our test and validation datasets, point mutations in each ATPase subunit have been associated with PI resistance. Consistent with these findings, PSMC2 KD increased sensitivity to PI therapy. Controlled inhibition or degradation of proteasome 19S ATPase subunits using small molecules, PROTACs or RNA-based modalities now provides an opportunity to therapeutically target proteostasis in MM and other malignancies.

Ignatz-Hoover:Sanofi: Consultancy. Malek:AdaptiveBio: Consultancy, Honoraria; Takeda: Consultancy, Speakers Bureau; Sanofi: Consultancy; janssen: Consultancy; BMS: Speakers Bureau; Amgen: Speakers Bureau; GSK: Speakers Bureau; Karyopharm: Consultancy, Speakers Bureau.