A Gene Polymorphism within the Kinase Domain of BCR-ABL and Its Effects on Sensitivity to Tyrosine Kinase Inhibitors.

Background: Point mutations that impair drug binding are the most important mechanism of acquired resistance to imatinib. Mutations within the ATP binding loop (P-loop) of BCR-ABL are associated with a poor prognosis in patients on imatinib. We have identified a single nucleotide polymorphism at position 247 (numbering according to ABL-B) that leads to the replacement of lysine by arginine. In CML patients, either the lysine or arginine allele of amino acid 247 becomes part of the BCR-ABL fusion gene. Due to its close proximity to the P-loop, we decided to investigate the allele frequency of K247R and whether the presence of the arginine allele affected sensitivity of Bcr-Abl to Abl kinase inhibitors.

Patients and Methods: We investigated the frequency of the arginine allele of K247R in 157 patients with CML and 213 healthy blood donors by conventional sequencing, restriction enzyme digestion and single strand conformational polymorphism analysis. We used Abl autophosphorylation and substrate phosphorylation assays, immunoblotting and cellular proliferation assays to examine the influence of K247R upon imatinib and dasatinib sensitivity.

Results: We found that frequency of the arginine allele of K247R was 1.6% in patients with CML and 0.2% in the controls (P = 0.11). In one patient analysis of CD3+ and CD33+ obtained at the time of complete cytogenetic response revealed the presence of K247R in both cell compartments, consistent with a polymorphism. Three out of 5 patients with the arginine allele of K247R expressed in BCR-ABL failed to achieve a major cytogenetic response to imatinib, suggesting reduced drug sensitivity. However, in vitro assays showed no alteration in sensitivity to imatinib or dasatinib compared to “wild type”.

Conclusions: K247R is a rare polymorphism within the kinase domain of Abl. There is no evidence that K247R is more frequent in CML patients, and the presence of K247R does not affect drug sensitivity. It is important that clinicians are aware that K247R does not reflect a kinase domain mutation, and that its presence does not signal a need to change therapeutic strategy unless there are other signs of inadequate response to drug therapy.