Synergism between 17-AAG and Imatinib in Imatinib-Resistant CML Cells: Inhibition of P-Glycoprotein by 17-AAG as a New Mechanism of Increasing Imatinib Activity.

Chronic myelogenous leukemia (CML) has gained outstanding importance for targeted cancer therapy. Inhibition of the BCR-ABL tyrosine kinase by imatinib (STI571, Glivec, Gleevec) leads to reduced proliferation of CML cells in vitro and sustained hematological and cytogenetic responses in vivo. However, resistance has been observed after variable periods of imatinib monotherapy especially in advanced stages of disease and overexpression of the BCR-ABL protein is one of the mechanisms of imatinib resistance. As combination therapy may allow to overcome drug resistance, we were interested in the effect of combination treatment with imatinib and 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of the heat shock protein 90 (Hsp90) chaperone complex. Furthermore, a new mechanism of action of the heat Hsp90 inhibitor 17-AAG is brought to light giving implications for an additional benefit of a combination treatment of imatinb-resistant chronic myelogenous leukemia. In imatinib-sensitive CML cell lines, combination index values (CI) obtained using the method of Chou and Talalay indicated additive (CI = 1) or slightly antagonistic (CI > 1) effects following simultaneous treatment with imatinib and 17-AAG. In contrast, the agents acted synergistically in imatinib-resistant BCR-ABL overexpressing LAMA84-R cells (CI = 0.6 at 75 % growth inhibition level). Growth inhibition of CFU-GM colonies of primary CML cells obtained from 3 patients is stronger after combination treatment than after monotherapy and annexin V / propidium iodide staining showed a strong increase of the apoptotic cell fraction in CML cells treated for 48 hrs by the combination treatment as compared to treatment with each drug alone. In imatinib-resistant cells BCR-ABL mRNA levels and protein expression were increased compared to the imatinib-sensitive parental cell line which is consistent to our cytogenetic and multicolor FISH analyses revealing multiple genomic BCR-ABL copies in imatinib-resistant cells. Furthermore, in imatinib-resistant cells P-glycoprotein activity was increased. Single treatment with 17-AAG lowered BCR-ABL and increased Hsp70 protein levels in both cell lines as expected whereas combination treatment was even more effective in this respect. Interestingly, single treatment with 17-AAG also decreased P-glycoprotein activity in a dose-dependent fashion as confirmed by a rhodamine-123 exclusion assay. The synergistic effect of both drugs in imatinib-resistant cells may thus be explained by increased intracellular levels of imatinib following 17-AAG treatment. The relevance of this additional mechanism warrants further exploration in clinical studies.