BCR-ABLalternative splicing as a common mechanism for imatinib resistance: evidence from molecular dynamics simulations

Abstract

Rare cases of chronic myelogenous leukemia (CML) express high levels of alternatively spliced BCR-ABL mRNA with a 35-bp insertion (35INS) between ABL kinase domain exons 8 and 9. This insertion results in a frameshift leading to the addition of 10 residues and truncation of 653 residues due to early termination. Sensitive PCR-based testing showed that 32 of 52 (62%) imatinib-resistant CML patients in chronic phase and 8 of 38 (21%) in accelerated or blast crisis expressed varying levels of the alternatively spliced BCR-ABL mRNA. A three-dimensional structural model of the 35INS ABL kinase domain complexed with imatinib was built using homology modeling, followed by molecular dynamics simulations. Simulation results showed that the new residues cause a significant global conformational change, altering imatinib binding in a way similar to that of the T315I mutation and, therefore, providing resistance to imatinib that depends on the level of expression. [Mol Cancer Ther 2008;7(12):3834 -41]

The BCR-ABL35INS insertion/truncation mutant is kinase-inactive and does not contribute to tyrosine kinase inhibitor resistance in chronic myeloid leukemia

“…10,12 Notably, the reported frequency of detection of the BCR-ABL 35INS mutant in cases of imatinib resistance (including instances in which a point mutation is concurrently detected in the BCR-ABL kinase domain) as detected by direct sequencing is ϳ1%-2%, 10,14 although more sensitive quantitative assays have reported detection of very low levels of the mutant transcript at a considerably increased prevalence. 14 Although BCR-ABL truncated immediately after the ABL kinase domain is fully transforming in a murine model of CML, 15 we predicted BCR-ABL 35INS would lack kinase activity, because the mutation eliminates the last 2 helices of the ABL kinase domain and disrupts a complex set of interactions among noncontiguous residues. 10 By contrast, recent reports have suggested that BCR-ABL 35INS confers TKI resistance in CML 9,12,14,16 and have proposed a BCR-ABL 35INS tailored clinical trial, 16 but they have not addressed the mechanism for this or assessed BCR-ABL 35INS catalytic activity.…”

“…14 Although BCR-ABL truncated immediately after the ABL kinase domain is fully transforming in a murine model of CML, 15 we predicted BCR-ABL 35INS would lack kinase activity, because the mutation eliminates the last 2 helices of the ABL kinase domain and disrupts a complex set of interactions among noncontiguous residues. 10 By contrast, recent reports have suggested that BCR-ABL 35INS confers TKI resistance in CML 9,12,14,16 and have proposed a BCR-ABL 35INS tailored clinical trial, 16 but they have not addressed the mechanism for this or assessed BCR-ABL 35INS catalytic activity. We provide cell-based and biochemical studies of BCR-ABL 35INS and a retrospective analysis of its detection in the context of treatment and response in CML patients.…”

“…[9][10][11][12][13] The result is loss of the last 653 residues of BCR-ABL, including 22 native kinase domain residues. 10,12 Notably, the reported frequency of detection of the BCR-ABL 35INS mutant in cases of imatinib resistance (including instances in which a point mutation is concurrently detected in the BCR-ABL kinase domain) as detected by direct sequencing is ϳ1%-2%, 10,14 although more sensitive quantitative assays have reported detection of very low levels of the mutant transcript at a considerably increased prevalence. 14 Although BCR-ABL truncated immediately after the ABL kinase domain is fully transforming in a murine model of CML, 15 we predicted BCR-ABL 35INS would lack kinase activity, because the mutation eliminates the last 2 helices of the ABL kinase domain and disrupts a complex set of interactions among noncontiguous residues.…”

Chronic myeloid leukemia: mechanisms of blastic transformation

“…Notably, alternative splicing was also observed for BCR-ABL1. Aberrant BCR-ABL1 mRNA splicing results in the generation of transcripts harboring a 35-kb insertion between ABL1 domain exons 8 and 9, resulting in a frameshift with a truncation that, like IK6 expression, is associated with imatinib resistance (53,54).…”

Molecular analysis of the BCR-ABL1 kinase domain in chronic-phase chronic myelogenous leukemia treated with tyrosine kinase inhibitors in practice: Study by the Nagasaki CML Study Group

“…BCR-ABL1 35INS, a retention of 35 intronic nucleotides at the splice junction of exon 8/9, which results in a stop codon after 10 intron-encoded residues, was not included in the BCR-ABL1 KD mutations because BCR-ABL1 35INS was considered to be an alternative spliced variant, and not a point mutation [29][30][31][32].…”