Mutation Mediated Resistance to the Tyrosine Kinase Inhibitors Imatinib, Dasatinib & Nilotinib in Philadelphia Positive Leukaemia

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. It is observed in over 95% of Chronic Myeloid Leukaemia (CML) and approximately 30% of adult Acute Lymphoblastic Leukaemia (ALL) cases. Imatinib Mesylate (IM), a tyrosine kinase inhibitor that specifically binds BCR-ABL in its inactive conformation has revolutionized therapy for CML and Ph+ ALL. However, resistance develops in a significant proportion of patients and is predominantly mediated by single base-pair substitutions within the BCR-ABL kinase domain leading to changes in the amino acid composition 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 patients with a suitable molecular profile. In this study, we undertook to examine the molecular mechanisms underlying IM resistance. A cohort of 40 patients with either primary or acquired resistance or intolerance to IM was identified by persistent high or increasing levels of BCR-ABL transcripts determined by real-time quantitative PCR. An allele-specific PCR screen was used to sensitively detect the clinically significant T315I mutation, which renders patients insensitive to currently available tyrosine kinase inhibitors: five (12.5%) IM resistant/intolerant patients were T315I positive. To further elucidate the molecular mechanisms of mutation induced resistance, the BCR-ABL kinase domain was screened for the presence of a mutation using a sensitive denaturing high performance liquid chromatography (dHPLC) approach. Samples showing evidence of mutation were examined by direct sequencing to identify the mutation(s) present. Kinase domain mutations have been identified in 20 of the 40 (50%) patients examined to date and these include p-loop mutations (M244V, G250E, Q252H), IM-binding domain mutations (T315I), catalytic domain mutations (M351T), an activation-loop mutation (L387M). Three previously unreported mutations were identified in patients with indications of IM resistance (T267A, E275Q) and Nilotinib resistance (L273M). The L273 residue lies adjacent to a region of the BCR-ABL kinase domain bound by Nilotinib. Three patients were found to harbour mutations at two distinct kinase domain residues while one patient harboured mutations at three distinct residues, supporting the theory that patients who develop mutation-mediated resistance to one kinase inhibitor may become resistant to subsequent inhibitors by a similar mechanism. The identification of clinically significant mutations facilitates selection of alternative approaches to therapy such as dose escalation of IM, second generation tyrosine kinase inhibitors or allogeneic stem cell transplant, if eligible, at an early stage in a patient’s disease, tailoring patient specific approaches to therapy.