The Proteasome Inhibitor PR-171 Inhibits Cell Growth, Induces Apoptosis, and Overcomes De Novo and Acquired Drug Resistance in Human Multiple Myeloma Cells.

Abstract

Proteasome inhibitors are a new class of drugs that have shown to be cytotoxic and induce apoptosis in tumor cells, particularly, multiple myeloma (MM). This led to the clinical evaluation and subsequent FDA approval of the proteasome inhibitor, bortezomib, for the treatment of refractory and relapsed multiple myeloma. However, continued occurrence of refractoriness, development of resistance, and the toxicity of bortezomib in treated patients has led to the recent development of novel agents with increased binding efficiency and specificity to the proteasome. PR-171 is a member of the epoxomicin-class of compounds that act as potent and selective irreversible inhibitors of the chymotrypsin-like protease activity of the 20S proteasome resulting in accumulation of ubiquinated proteins and apoptosis of tumor cells (Ming et al., PNAS 96:10403). In this study, we evaluated the effects of PR-171 on human MM cells' survival, proteasome activity and drug resistance. PR-171 induced growth inhibition and apoptosis in the drug sensitive human MM cell lines 8226/S, MM1, H929, and U266, with IC50 values ranging from 9 to 21nM. PR-171 retains activity on cells resistant to melphalan, mitoxantrone, and zarnestra, but has reduced activity on cells resistant to doxorubicin. Pretreatment with verapamil restored sensitivity of the doxorubicin resistant cells (8226/Dox40) to PR-171 back to levels seen in the parental, drug sensitive cell line (8226/S). Proteasome activity was inhibited by PR-171 in the drug sensitive and drug resistant MM cell lines, although much less inhibition was observed in the doxorubicin resistant cells, consistent with the reduced effects of PR-171 on cell viability. In freshly isolated MM cells from patients, PR-171 at doses of 10–250nM induced apoptosis in 9/9 samples tested from both newly diagnosed and relapsed refractory patients. To test the effects of PR-171 on Cell Adhesion Mediated Drug Resistance (CAM-DR), MM cell lines were treated with drug after adhesion to fibronectin. PR-171 at 1–100nM overcomes CAM-DR in 8226/S and H929 cell lines, but not in MM1. The ability of PR-171 to overcome CAM-DR was also seen in primary MM cells from patients in 6 out of 9 samples tested. In summary, we have shown that PR-171 directly inhibits proteasome activity, proliferation, and induces apoptosis in human MM sensitive and resistant cell lines, induces apoptosis in isolated MM cells from newly diagnosed and relapsed refractory patients, and overcomes de novo drug resistance. These studies provide the foundation for clinical investigation of PR-171 to improve the outcome of patients with MM and other hematological malignancies.