Abstract

In advanced-phase CML, resistance to imatinib mesylate is frequently associated with point mutations in the Bcr-Abl kinase domain. New, highly potent Abl kinase inhibitors such as AMN107 and BMS-354824, have recently entered clinical trials. Data from analyses of resistant patients will be available not before a large number of resistant patients will have been treated within clinical trials. Therefore, it will be important to generate specific resistance profiles for each compound prior to its therapeutic application. Using a cell-based screening method for resistance of Bcr-Abl positive leukemia to Abl kinase inhibitors, we generated a resistance profile for AMN107 and compared it to the resistance profile of imatinib mesylate. In contrast to imatinib, resistance to AMN107 was associated with a very limited spectrum of Bcr-Abl kinase mutations. While 26 exchanges at 21 positions occured with imatinib, the AMN107 screen revealed eight different exchanges at seven amino acid positions, with four exchanges affecting the P-loop. Novel mutations which have never been observed with imatinib, either in vitro or in resistant patients, emerged in the presence of AMN107 including an F359 exchange to isoleucine and a Q252H/S349L double mutant. In contrast to imatinib, the frequency of resistant colonies dramatically decreased with increasing AMN107 concentrations. Rarely emerging resistant colonies at a concentration of 400 nM AMN107 exclusively contained T315I. With the exception of T315I, all mutations that were identified were effectively suppressed when AMN107 was increased to 2000 nM, a concentration which is achieved in plasma in treated patients. Thus, in this system, increasing the AMN107 concentration to 400 nM prevented the emergence of resistant colonies, with the exception of T315I. Our findings suggest that AMN107 might be superior to imatinib in terms of the development of resistance. Also, AMN107 at clinically relevant concentrations may overcome imatinib resistant disease, including cases with expression of P-loop mutations. However, our study indicates that clinical resistance to AMN107 may be associated with the predominant emergence of T315I. Using this or similar approaches, it will be possible to provide information that translates into combinatorial and sequential treatment strategies and to determine critical plasma concentrations for mutations that might occur during treatment.

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