Mechanism of the antimyeloma activity of PU-H71, a novel purine scaffold HSP90 inhibitor.

Abstract

8141 Background: PU-H71, a novel purine scaffold HSP90 inhibitor, has demonstrated anti-tumor activity in triple-negative breast cancer and Bcl6 dependent B-cell lymphoma preclinical models. Its mechanism of action is thought to be via inhibition of the cytosolic HSP90A and down regulation of its client proteins. HSP90B1 (a.k.a gp96, grp94), the endoplasmic reticulum paralogue of HSP90A, serves as a master chaperone for most integrins, which play a very important role in myeloma cell-bone marrow stromal cell interactions, cell survival and proliferation, and drug resistance. We evaluated the in vitro anti-myeloma activity of PU-H71 in human myeloma cell lines (HMCLs) and its effects on HSP90B1. Methods: Characterization of HSP90B1 and its clientele was performed in human myeloma cell lines (HMCLs) by flow cytometry and Western blotting. Stable HSP90B1 knockdown and control HMCLs were created using shRNA transduction. Drug treatments (dexamethasone, melphalan, doxorubicin, thalidomide, 17-AAG, 17DMAG and PU-H71) were performed for cell viability (trypan blue staining) and induction of apoptosis (Western blotting for anti-caspase 3). Western blotting was also performed for the unfolded protein response markers (IRE1a, XBP1, eIF2a) and the cell cycle markers (Cyclin D1,p27kip1). Results: PU-H71 has potent anti-myeloma activity with IC50 achieved in HMCLs with concentrations ≤ 100 nM at 24 hours. PU-H71 activates the unfolded protein response and increases apoptosis of HMCLs. HMCLs abundantly express HSP90B1 and show variable expression of client integrins. HSP90B1 knock-down HMCLs show less susceptibility to death when treated with PU-H71 at 24 and 48 hours. HSP90B1 knock-down HMCLs, when compared to control, demonstrate increased cell survival at lower concentrations of conventional drugs whereas more susceptibility with higher concentrations. Conclusions: The in vitro studies demonstrate that (1) PU-H71 is highly active against HMCLs; (2) PU-H71 acts by inhibiting both HSP90A and HSP90B1; (3) HSP90B1 knockdown HMCLs are more susceptible to cell death when treated with conventional myeloma drugs. Based on the results, HSP 90B1 may serve as a more specific anti-myeloma target. No significant financial relationships to disclose.