BCR-ABL Kinase Domain Mutations & SNPs in Imatinib Resistant or Intolerant Chronic Myeloid Leukaemia Patients.

Abstract

The Philadelphia chromosome, is formed as a result of a reciprocal translocation between chromosomes 9 and 22 and results in the formation of the hybrid oncoprotein BCR-ABL which is pathognomic of Chronic Myeloid Leukaemia (CML). Imatinib Mesylate (IM), a tyrosine kinase inhibitor that specifically binds BCR-ABL in its inactive conformation and functions as a competitive inhibitor of ATP binding, has revolutionized therapy for patients with CML. However, resistance develops in a significant proportion of cases and is predominantly mediated by single base-pair substitutions within the BCR-ABL kinase domain leading to changes in the amino acid composition and conformational changes in the kinase domain that inhibit IM binding whilst retaining BCR-ABL phosphorylation capacity. Second generation tyrosine kinase inhibitors such as Dasatinib and Nilotinib retain activity in IM-resistant patients due to less stringent binding requirements and represent viable alternatives for IM-resistant or intolerant CML patients. In this study, we undertook to examine the molecular mechanisms underlying IM resistance. A cohort of 33 patients with either primary or acquired resistance (n=31) to IM or intolerance to IM (n=2) was identified by persistently high or increasing levels of BCR-ABL transcripts determined by standardised real-time quantitative PCR. An initial allele-specific PCR screen was used to sensitively detect the clinically significant T315I mutation, which renders patients insensitive to currently available tyrosine kinase inhibitors: four (11.8%) IM resistant/intolerant patients were T315I positive. To further characterise the molecular mechanisms of mutation induced resistance, the BCR-ABL kinase domain was then screened for the presence of a mutation using a sensitive denaturing high performance liquid chromatography (dHPLC) approach. dHPLC can detect a single base pair substitution within the BCR-ABL kinase domain based on hybridization to a non-mutated wild type control. Mutated samples display reduced hybridization capacity to the dHPLC column and elute at an earlier time-point. Sensitivity of dHPLC (0.1–10%) is significantly greater than that of sequencing (15–25%). Following dHPLC analysis, samples showing evidence of mutation were examined by direct sequencing to identify the mutation(s) present. Kinase domain mutations have been identified in 18 of the 33 (55%) patients examined to date and these include p-loop mutations (M244V, G250E, Q252H), IM-binding domain mutations (T315I & F317L), catalytic domain mutations (M351T & E355G), and an activation-loop mutation (L387M). Three previously unreported mutations that may be associated with IM resistance (T267A, L273M, K291Q) were identified. The L273M positive patient also has an M244V mutation and has shown primary resistance to IM & is currently being treated with Nilotinib; the T267A patient has rising BCR-ABL transcripts, while the K291Q patient has had a 3 log reduction of BCR-ABL transcripts following IM dose escalation. In addition to the above mutations, 2 SNPs were identified at E275E and at L248L, which may not be clinically relevant. The identification of clinically significant mutations facilitates selection of alternative approaches to therapy such as IM dose escalation, second generation tyrosine kinase inhibitors or allogeneic stem cell transplantation, if eligible, allowing patient specific approaches to therapy.