Ubiquitin Receptor PSMD4/Rpn10 Is a Novel Therapeutic Target in Myeloma

Background and Rationale 19S proteasome-associated Ubiquitin Receptor (UbR) PSMD4/Rpn10 chaperones ubiquitinated proteins to 20S proteasome for their degradation. Our earlier study showed that blockade of UbR Rpn13 triggers MM cell death and overcomes proteasome-inhibitor (PI)-resistance (Yan et al., Leukemia 2016; Yan et al., BCJ 2021). In this study, we utilized biochemical and genetic strategies as well as animal models to show that targeting UbR PSMD4 triggers anti-MM activity, including against PI-resistant MM cells.

Materials and Methods Cell viability, drug sensitivity, and apoptosis assays were performed using WST/ CellTiter-Glo assay and Annexin V staining. MM cells were transiently transfected with control short interfering RNA (siRNA) PSMD4 ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. PSMD4 knockout HEK293 cell line was generated using CRISPR/Cas9. Doxcycycline (Dox)-inducible PSMD4-knockdown (KD) MM cell line was generated using short hairpin RNA (shRNA). Cell signaling and caspase activity were determined by western blotting. Compound X (Cpd X) was purchased from EMD Millipore, USA; and bortezomib was purchased from Selleck chemicals, USA. Alpha-assay kit was purchased from PerkinElmer, USA. In vivo studies were performed using a human MM xenograft model. Statistical significance was assessed with Student's t test.

Results 1) GEP database on MM patients 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) PSMD4-siRNA decreased viability of PI-resistant MM cells; 4) HEK293 cells carrying stable CRISPR/Cas9-PSMD4-knockout (KO), as well as MM cell lines (MM.1S, AMO-1) with Dox-inducible PSMD4-KD, showed increased polyubiquitinated proteins and reduced cell growth in both in vitro and animal models; 5)We designed an in vitro AlphaScreen® binding assay in HTS format to screen a library of 12K compounds, and identified novel PSMD4 prototype inhibitor Cpd X; 6) Binding and inhibition between compound Cpd X and PSMD4 was confirmed by pull-down, MS and FEB assays; 7) Treatment of MM and leukemic cell lines, as well as primary MM patient cells, with Cpd X significantly decreased their viability (IC₅₀range 1.6µM to 9µM; p < 0.001 for all cell lines; n=3) without markedly affecting the viability of normal PBMCs, suggesting a favorable therapeutic index; 8) Cpd X inhibits proliferation of MM cells even in the presence of BM stromal or plasmacytoid dendritic cells (pDCs); 9) Cpd X inhibits PSMD4 without blocking 20S proteasomal catalytic activities or 19S proteasome-associated deubiquitinating enzymatic activities; 10) PSMD4 blockade via either Cpd X or siRNA/shRNA triggered MM cell death associated with induction of caspase-dependent- and unfolded protein response-related apoptosis; and finally, 11) Animal model studies show that Cpd X is well-tolerated, inhibits tumor growth, and prolongs survival.

Conclusion Our preclinical data validates targeting UbR PSMD4/Rpn10 to enhance cytotoxicity and overcome PI resistance in MM. They provide the basis for further optimization studies of PSMD4 inhibitors for clinical application to improve MM patient outcome.