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Mechanism of Drug Resistance to Dasatinib (BMS-354825) and Imatinib in Chronic Myelogenous Leukemia Cells.

Seiichi Okabe, Tetsuzo Tauchi, Hal E. Broxmeyer and Kazuma Ohyashiki

Abstract

The selective tyrosine kinase inhibitor, imatinib has shown remarkable clinical activity in patients with chronic myelogenous leukemia (CML), however, imatinib does not completely eradicate BCR/ABL-expressing cells, and cells resistant to imatinib develop. It has been reported that mutations in the kinase domain (KD) of BCR/ABL impairs binding of imatinib and causes resistance to it. So, additional therapy is necessary to overcome imatinib resistance in patients with an acute form of CML. The novel BCR/ABL inhibitor, dasatinib (BMS-354825), which A dual inhibitor of both Src and Abl kinases, suppresses the activity of these kinases at subnanomolar concentrations in imatinib resistance cells. Dasatinib is presently being evaluated in a clinical trial in CML patients with imatinib resistance, but drug resistance to dasatinib and imatinib are not fully evaluated. In this study, we used TF-1 BCR/ABL cell lines, which were transfected with the BCR/ABL gene, as well as parental TF-1 cells and K562 cell lines and established dasatinib resistant TF-1 BCR-ABL BMS-R and K562 BMS-R cells and imatinib resistant TF-1 BCR-ABL IM-R and K562 IM-R cells. We show here that dasatinib potently induced apoptosis of TF-1 BCR-ABL and K562 cells in 72 hours treatment. IC50 of dasatinib was 0.75nM (TF-1 BCR/ABL) and 1nM (K562) and IC50 of imatinib was 500nM (TF-1 BCR/ABL) and 750nM (K562). In the resistant cell lines, IC50 of dasatinib was 15μM (TF-1 BCR/ABL BMS-R) and 25μM (K562 BMS-R). TF-1 BCR/ABL BMS-R and K562 BMS-R cells were also resistant to imatinib (IC50: more than 10μM). TF-1 BCR-ABL IM-R and K562 IM-R cells were also resistant to dasatinib (IC50: 7.5 nM (TF-1 BCR/ABL IM-R) and the value was more than 10nM for K562 IM-R). There was no mutation in Abl kinase in these resistant cell lines suggesting that BCR/ABL mutation-independent mechanism is involved in resistance to imatinib and dasatinib. Because the correlation of parameters with defined protein expression patterns and protein signatures would predict disease progression and drug resistant, we investigated the protein expression pattern by using antibody microarray system (Antibody Microarray: Lab Vision Corporation). We found that dasatinib resistant cells had reduced protein levels of BCR/ABL. In TF-1 BCR/ABL BMS-R cells, Zap-70 protein was increased and activated. In contrast, the src kinase, lck, is increased and activated in K562 BMS-R cells. Moreover, mitogen-activated protein kinase (MAPK) and Akt were also activated in K562 BMS-R cells. We also found that src kinases, especially Lyn, as well as MAPK and Akt were activated in K562 IM-R cells. The phosphatase SHP-2 was decreased in dasatinib resistant cell lines. Signal-transducing activators of transcription 5 (STAT5) phosphorylation was reduced in K562 BMS-R cells. 24 hour removal of dasatinib from culture medium of K562 BMS-R led to apoptosis of the cells and activated caspase 3 and poly (ADP-ribose) polymerase (PARP). We also found that the phosphatase SHP-1 was increased after removal of dasatinib. The expression and protein activation signatures identified in this study provide insight into the mechanism of resistance to dasatinib and imatinib. Our study demonstrates development of dasatinib resistance and suggests that this information may be of therapeutic relevance.