P-093: Ubiquitin receptor PSMD4/Rpn10 as therapeutic target in Multiple Myeloma

Abstract

<h3>Background</h3> Our prior studies showed that targeting Ubiquitin Receptor (UbR) Rpn13 upstream of the 20S proteasome signaling cascade overcomes proteasome-inhibitor (PI)-resistance (Yan et al. Leukemia 2016; Yan et al, 2021 BCJ–please complete reference details). Besides Rpn13, 19S-associated UbR PSMD4/Rpn10 also plays a key role in chaperoning ubiquitinated substrate proteins for downstream 20S proteasomal degradation. In the present study, we show that inhibition of PSMD4 triggers potent anti-MM activity using both in in vitro and in vivo models of MM, including against PI-resistant MM cells. <h3>Methods</h3> Cell viability, and apoptosis assays were performed using WST/ CellTiter-Glo assay, and Annexin V staining, respectively. PSMD4 knockout 293 cell line was generated using CRISPR/Cas9. Doxcycycline (Dox)-inducible PSMD4-knockdown (KD) MM cell line was generated using short hairpin RNA (shRNA). A xenograft human MM model was used to characterize the role of PSMD4 on tumor progression. Statistical significance was assessed with Student's t test. <h3>Results</h3> 1) MM patient gene expression profiling database showed that PSMD4 expression inversely correlates with overall survival (n=175; p=0.00064). 2) RT-PCR, immunoblotting, and immunohistochemistry of MM patient BM showed higher PSMD4 levels in patient MM cells and MM cell lines versus normal cells. 3) Transient transfection of MM cells including, bortezomib-resistant MM cells with PSMD4-siRNA decreased their viability; conversely, transfection with PSMD4-WT rescued cells from growth-inhibitory activity of PSMD4-siRNA. Western blotting confirmed knockdown of PSMD4 by PSMD4-siRNA versus scrambled (scr)-siRNA, and restoration of PSMD4 levels in cells transfected with PSMD4-WT versus PSMD4-siRNA. 4) CRISPR/Cas9-PSMD4-KO showed reduced cell growth. 5) Both PSMD4-KO and PSMD4-KD cells showed elevated levels of polyubiquitylated proteins, indicating blockade of proteasome-mediated protein degradation; 6) PSMD4 blockade triggered apoptosis, cell-cycle arrest, activation of caspases, and endoplasmic reticulum stress response signaling in MM cells; and finally, 7) Transplantation of Dox-inducible PSMD4-KD MM.1S cells in mice correlated with significantly reduced tumor growth. <h3>Conclusion</h3> Our preclinical data validates targeting UbR PSMD4/Rpn10 to enhance cytotoxicity and overcome PI resistance in MM, as well as provides the basis for the development and clinical evaluation of PSMD4 inhibitors to improve patient outcome.