Dasatinib Resistance in CML Patients. Identification of Novel BCR-ABL Kinase Domain Mutation.

Dasatinib is a dual specific SRC-ABL kinase inhibitor which has been increasingly used in treatment of CML and Ph + ALL patients. Although it has excellent activity, a proportion of patients, particularly those with advanced disease had developed evidence of dasatinib resistance. In order to assess the reasons for resistance we have analyzed BCR-ABL kinase domain utilizing single step PCR reaction followed by amplification of ABL kinase domain with subsequent subcloning (≥10 clones) from 23 CML patients who demonstrated clinical evidence of dasatinib failure. We have found mutations in the BCR-ABL domain in 19 out of 23 patients. 14 were mapped to ATP “binding pocket”, of those, seven had pan-resistant T315I and five had F317L with one F317I mutation. In addition, three of 19 patients had V299L mutation. All of these mutations were previously reported in association with dasatinib resistance. We also observed that one of 8 patients in chronic phase and three of 15 patients in advanced phase had two different mutations in separate clones_§. Interestingly, four of 19 patients were found to have novel V304D mutation, which is located outside of dasatinib binding site in β 4–5 loop structure and may not be directly involved in dasatinib resistance. It appears that BCR-ABL mutations in advanced phase CML are more common, but occur at lower frequency in dasatinib resistant patients in chronic phase, 93% vs. 62%. In conclusion, our clinical sample suggests that selection for T315I and F317L mutations occurs frequently in dasatinib resistant patients which was predicted from preclinical study. Both mutations were proposed to directly and indirectly affect dasatinib binding. Two additional mutations have been identified with incidence that exceeds random occurrence. The significance of these mutations (V299L, D304D) will be further studied.