PSMC2 Overexpression Promotes Synthetic Lethality Sensitizing Multiple Myeloma Cells to p97 Inhibition

Multiple myeloma (MM) is the second most common hematologic malignancy and intrinsic and acquired drug resistance of cancer cells to standard drugs remains a major obstacle that precludes successful survival of patients treated on contemporary clinical regimens. Proteasome inhibitors (PIs) have transformed care of MM patients leading to improved overall quality of life and tripled the overall survival (OS) of certain patient populations. However, drug resistance inevitably emerges through molecular mechanisms that remain elusive.

The 26S proteasome consists of two major subcomplexes: a 19S regulatory particle (RP) that is responsible for unfolding and translocating ubiquitinylated substrates into the inner chamber of a 20S core particle (CP) that hydrolyzes substrates. PSMC2 belongs to the 19S heterohexameric AAA-ATPase ring of the 19S RP. Gene expression profiles from bone marrow CD138⁺ cells of MM patients enrolled on the phase III APEX trial revealed that PSMC2 overexpression correlated with reduced response to bortezomib therapy as well as inferior progression-free and OS (Median OS for PSMC2 high vs. PSMC2 low 300 days vs. 625 days n=380, Kaplan-Meier (KM) curves significant by log-rank test with p=7x10^-6) Similarly, results from the MMRF CoMMpass study demonstrated that PSMC2 overexpression correlated with reduced OS (Median OS for PSMC2 high v low 1300 days vs. not reached, n=221 Kaplan-Meier (KM) curves significant by log-rank test with p=0.014). Strikingly, PSMC2 copy number was increased in tumor cells from nearly 40% of MM patients compared to normal controls.

Lentiviral-engineered overexpression of PSMC2 in MM cell lines increased proteasome activity and promoted bortezomib (Btz)-resistance (LD₅₀ of 10 nM vs. 6 nM in U266 overexpressed vs. vector control cells respectively, LD₅₀ of 9 nM vs. 6 nM in ARH77 overexpressed vs. vector controls respectively, PSMC2 effect significant with p<0.0001 by two-way ANOVA). Upon Btz challenge, PSMC2-overexpressing cells suppressed induction of the endoplasmic reticulum (ER) chaperone GRP78/BiP, reduced activation of the stress marker XBP-1 and blocked the unfolded protein response, relative to pLKO vector-transduced controls. We then performed a high-throughput pharmacologic screen to identify synthetic lethal interactions specific to PSMC2-overexpressing MM cell, identifying inhibitors that targeted the ER stress response, including inhibitors of HSP90, inositol-requiring enzyme 1a and p97/VCP. p97 is a key ATPase that interacts with ERAD transport channels to unfold, translocate and deliver ubiquitinylated substrates to ER-bound proteasomes. Subcellular fractionation and co-immunoprecipitation experiments indicated that PSMC2 physically interacts with ER-associated p97 to facilitate ERAD.

To target this interaction therapeutically, we cotreated PI-exposed MM patient cells with Btz and the ATP-competitive p97 inhibitor CB-5339 (CB), revealing synergistic effects (Btz LD₅₀ 2.5 nM vs. 7.5 nM, 2 nM vs 8 nM, 2 vs. 8 nM for patient samples treated with the first two patients treated with 0 or 100 nM CB and the third patient treated with 0 and 50 nM CB, p< 0.0001 for CB effect by two-way ANOVA testing). CB co-treatment with Btz reduced the growth of aggressive PSMC2 overexpressing myeloma xenografts in murine models and significantly extended OS (median OS 24 days CB/Btz vs 19-20 days for vehicle, Btz or CB treated, Kaplan-Meier curves with p=0.0014 by log-rank test). Importantly, CB exhibits a favorable side effect profile as monotherapy in early phase AML clinical trials. Taken together, we have identified a prognostic role for PSMC2 and revealed a novel mechanism of Btz tolerance that can be therapeutically exploited to overcome chemoresistance.