Activity of ABT-737, an Inhibitor of BCL-2 Family Proteins, in Imatinib- or Dasatinib-Resistant CML Cells: Involvement of BCL-2 Family Proteins for Drug Resistance.

Abstract

Imatinib and dasatinib are two clinically active ABL kinase inhibitors that serve as paradigm for the study of emergence of resistance in targeted cancer therapy. We have developed the imatinib- or dasatinib-resistant cell lines. K562 cells and TF1 BCR-ABL cells were cultured with 0.1 μM of imatinib or 0.5 nM of dasatinib for 2 weeks, and then the concentration of imatinib or dasatinib was gradually increased. Finally imatinib-resistant K562-IMR cells and TF1 BCR-ABL-IMR cells can grow in the presence of 10 μM of imatinib. Also dasatinib- resistant K562-BMSR cells and TF1 BCR-ABL-BMSR cells can grow in the presence of 10 nM of dasatinib. These resistant cell lines do not have the point mutations of BCR-ABL kinase domain determined by PCR analysis. We used DNA microarrays to identify genes whose transcription was altered in imatinib- or dasatinib-resistant K562 cells. The gene expression data was obtained from three independent microarray hybridizations. We used 1.5-fold cut off which is a reliable limit for the identification of differentially expressed genes. Imatinib- or dasatinib-resistant K562 cells up-regulate the expression of several MAP kinase genes (MAP2K7, MAP3K1, and MAP3K7), apoptosis-regulated genes (BCL2L1, BCL6, and MCL1). Immunoblotting confirmed that the imatinib-resistant cell lines and dasatinib-resistant cell lines expressed the increased amount of BCR-ABL proteins compared to parental cell lines. p42/p44 MAP kinase is more activated in both inatinib-resistant cell lines and dasatinib-resistant cell lines. Up-regulation of Lyn kinase is observed in dasatinib-resistant cell lines but not in imatinib-resistant cell lines. Up-regulation of Bcl-XL and Mcl-1 is also observed in dasatinib-resistant cell lines. We determined the sensitivity of imatinib and dasatinib in these drug resistant cell lines. Cells were treated with 10 μM of imatinib or 5 nM of dasatinib for 72 hrs. The induction of apoptosis was analyzed by APO2.7. In TF-1 BCR-ABL cells, the treatment with 10 μM of imatinib or 5 nM of dasatinib effectively induced apoptosis. In imatinib-resistant TF1 BCR-ABL cells, treatment with 5 nM of dasatinib significantly increased the APO2.7-positive cells compared with imatinib-treatment. Also treatment with 10 μM of imatinib increased the apoptotic cells in BMS-resistant TF1 BCR-ABL cells. These results suggest that relatively high dose of imatinib or dasatinib still has biological activity in each compound resistant cells. By using these drug resistant cell lines, we determined the activity of ABT-737, an inhibitor of Bcl-2 family proteins. AST-737 is a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-XL, and Bcl-w with high affinity (Nature 435: 677, 2005). ABT-737 did not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with imatinib or dasatinib in these resistant cell lines. Treatment of 1μM of ABT-737 exhibited more sensitive in dasatinib-resistant K562 cells as single agent mechanism-based killing compared to parental K562 cells or imatinib-resistant K562 cells. Taken together, these finding indicate that co-treatment with ABT-737 enhances cytotoxic effects of imatinib or dasatinib and may have activity against imatinib- or dasatinib-resistant BCR-ABL-positive leukemia.