Proteomic Analysis Identifies Mechanism(s) of Overcoming Bortezomib-Resistance Via Targeting Ubiquitin Receptor Rpn13

Introduction Our studies have focused on delineating ubiquitin proteasome pathway signaling with a goal to identify and validate novel therapeutic targets to overcome proteasome-inhibitor-resistance in multiple myeloma (MM). Our siRNA screening studies showed ubiquitin Receptor (UbR) Rpn13 as a mediator of MM cell growth. Rpn13 is localized on the 19S proteasome, and chaperone ubiquitinated proteins for downstream proteasomal degradation. We showed that blockade of Rpn13 trigger cytotoxicity in MM cells and overcomes bortezomib-resistance (Song Yan et al., Leukemia 2016, 30(9):1877-1886). However, the mechanism(s) whereby Rpn13-inhibition overcomes bortezomib-resistance remains undefined. Here, MM cells were treated with the Rpn13 inhibitor RA190 to identify proteomic alterations and specific downstream substrate proteins/signaling mechanism involved in overcoming bortezomib-resistance. We identified and validated the Copper/Zinc-superoxide dismutase (SOD1), implicated in the regulation of mitochondrial unfolded protein response, as a novel therapeutic target in MM.

Materials and Methods MM.1S cells were treated with DMSO or RA190 (250 nM) for 24h and subjected to multiplexed proteomics analysis using tandem mass spectrometry. Data were analyzed using uniprot composite database and SEQUEST-based software platform. MM.1S cells were transiently transfected with scr-siRNA, SOD1-siRNA, control (pCDNA3.1)- or SOD1 (WT)-plasmid using the Nucleofector Kit V. Rpn13 knockout cells were generated using CRISPR/Cas9. Cell viability was assessed using WST-1, and Cell Titer-Glo assay. Alterations in ROS, SOD1, superoxide levels and Proteasome activity were measured as previously described (Chauhan et al., Cancer Cell 2005, 8:407-419; Chauhan et al., Cancer Cell 2012, 22(3):345-358). Rpn13 inhibitor RA190 and Cu-ZnSOD1 inhibitor LCS-1 were purchased from Calbiochem and Sigma-Aldrich, respectively. Statistical significance was determined using a Student's t test.

Results 1) Rpn13 inhibitor RA190 decreases viability in bortezomib-resistant MM cells. 2) Proteomic analysis showed a marked decrease in superoxide dismutase (SOD1) levels (69% ± 2.1%) in RA190- vs DMSO-treated MM cells. No significant decrease in SOD1 expression was noted in bortezomib-treated cells. 3) Transfection with SOD1-WT rescued cells from growth-inhibitory activity of RA190, suggesting that SOD1 mediates Rpn13-inhibition triggered cell death. 4) Both patient MM cells and MM cell lines exhibit higher SOD1 protein levels vs normal plasma cells/PBMCs; 5) SOD1 levels are higher in bortezomib-resistant vs -sensitive MM cells. 6) Gene expression (GEP) analysis showed inverse correlation between SOD1 and overall patient survival (n=175) (p = 0.00054). 7) Direct targeting of SOD1 using siRNA showed significant decrease in viability of MM cell lines, including bortezomib-resistant MM cells. 8) As for SOD1-siRNA, treatment of MM cell lines and primary patient cells with the SOD1 inhibitor LCS-1 decreased their viability (p < 0.05; n=3) without affecting normal PBMCs. 9) LCS-1 inhibits the proliferation of MM cells even in the presence of tumor-promoting BMSCs and plasmacytoid dendritic cells. 10) Mechanistic studies show that similar to RA190, LCS-1-induced MM cell death is associated with: (i) inhibition of SOD1 enzymatic activity and increase in superoxide and reactive oxygen species (ROS) levels; (ii) activation of apoptotic signaling via caspases, p53/p21 axis, and cyto-c release; (iii) decrease in MCL-1, BCL2, CDC2, CDC25C, and cyclin B1; (iv) induction of ER stress response; and (v) accumulation of ubiquitinated proteins and proteasome disassembly. 11) ROS inhibitor N-acetyl-L-cysteine blocked LCS-1-induced cell death and associated signaling. 12) LCS-1, but not RA190, induces cytotoxicity in CRISPR/Cas9-Rpn13-KO cells, confirming our proteomic data showing SOD1 as a downstream target of Rpn13.

Conclusion Our proteomic and functional validation studies show that targeting UbR Rpn13 with RA190 overcomes bortezomib-resistance via decreasing cellular SOD1 levels, which therefore represents a target for novel therapeutics to improve patient outcome.