Proteomic analysis of HSP90 inhibitor effects on human leukemia cells

Abstract

Hsp90 inhibitors have potential use as anti-cancer drugs. The mechanisms of action of the Hsp90 inhibitor, SNX-7081, have been investigated using comprehensive proteomic analysis techniques. DIGE (two-dimensional fluorescence difference gel electrophoresis), iTRAQ (isobaric tag for relative and absolute quantitation), LC-MS/MS, and antibody microarrays have been used to identify the protein changes induced by Hsp90 inhibitors. The results have extended the list of Hsp90-regulated proteins (clients) and suggest mechanisms of action such as down-regulation of Lyn kinase, inhibition of DNA repair, and arrest of the cell cycle, with increases in the surface antigens CD19, CD20 and CD262. We have shown that Hsp90 inhibitors can overcome imatinib resistance in human chronic myeloid leukemia (CML) MYLR cells, and fludarabine resistance in human chronic lymphocytic leukemia (CLL) MEC1 cells. We have demonstrated that SNX-7081 and fludarabine are synergistic against three p53-mutated cell lines (MEC1, MEC2 and U266). The results of cytotoxicity experiments with cultured primary CLL cells from 23 patients support this proposal. Label-free quantitative shotgun proteomic analysis of extracts from drug treated cells indicate that the mechanism of synergy is that fludarabine causes DNA damage, and SNX-7081 down-regulates DNA repair proteins, and cells accumulate DNA damage and undergo apoptosis. Combination of an Hsp90 inhibitor with fludarabine may lead to more effective chemotherapy regimens, and decrease the high toxicity encountered in clinical trials of Hsp90 inhibitors.