Three Novel Alternative Splicing Mutations in BCR-ABL1 detected in CML Patients with Resistance to Kinase Inhibitors.

Abstract 1107

Poster Board I-129

The BCR-ABL1 tyrosine kinase is the target for kinase inhibitors in patients with chronic myeloid leukemia (CML). Multiple types of mutations in the BCR-ABL1 kinase domain have been reported. We previously reported a common alternatively spliced BCR-ABL mRNA with a 35-nucleotide insertion (35INS) between ABL1 kinase domain exons 8 and 9 that is expressed at various levels in CML patients with resistance to kinase inhibitors. Here we report 3 novel alternative splicing mutants expressed as the dominant transcripts in patient with CML and resistance to kinase inhibitors. We initially screened for ABL1 kinase domain mutations in approximately 200 patients with resistance to more than one of the three kinase inhibitors (imatinib, nilotinib, or dasatinib). Screening was conducted by first amplifying the fusion BCR-ABL1 transcript, to ensure that normal ABL1 was not amplified, then sequencing the ABL1 kinase domain coding region. We discovered 3 not previously described splice mutants, present in one patient each. All 3 showed >90% mutant transcript. The first resulted from the insertion of 79 nucleotides into the ABL1 exon 8-9 junction. The inserted sequence in fact contained a sequence from regions of intron 8, located 120 bp apart: the 35-nucleotide sequence previously described, and an additional 44-nucleotide segment downstream from 35INS. The combined 79-nucleotide insertion splice mutant showed the same protein change as 35INS (p C475YfsX11). The second splice mutation comprised an 84-nucleotide sequence from intron 7 inserted into the ABL1 exon7-8 junction, also causing a frameshift and protein truncation (p A424EfsX18). The third splice derived from a 231–nucleotide sequence from intron 4 retained in the ABL1 exon 4-5 junction. This insertion added 40 intron-encoded amino acids after K274, leading to a frameshift and early termination of BCR-ABL1 in the p-loop (p E275LfsX41). Exposure to kinase inhibitors may lead to the expression of alternatively spliced mRNA that is significantly elongated but causes a truncated fusion BCR-ABL1 that confers resistance to kinase inhibitors. These findings, when combined with the data on 35INS, support the concept that loss of the C-terminus of BCR-ABL1 is associated with significant resistance to kinase inhibitors; this mechanism appears to be a major source of resistance to kinase inhibitors.