BCR-ABL truncation due to premature translation termination as a mechanism of resistance to kinase inhibitors

Abstract

7028 Background: The major mechanism underlying imatinib resistance in patients with chronic myeloid leukemia (CML) is clonal expansion of leukemic cells with point mutations in the BCR-ABL tyrosine kinase. We describe three novel ABL premature termination mutations leading to BCR-ABL truncation in leukemia patients with multidrug (imatinib/nilotinib/dasatinib) resistance. Methods: Peripheral blood or bone marrow samples from drug-resistant CML patients were collected. Total nucleic acids were purified and subjected to two rounds of PCR analysis, with the first PCR designed to eliminate amplification of the wild-type, non-translocated ABL gene. Bi-directional sequencing was then performed. HL60 cells (a Ph-negative myeloid leukemia cell line) and peripheral blood of healthy subjects were used as negative controls; a human CML cell line (K562) was used as a positive control. Results: We identified an exon 7 deletion in three CML patients, a 4-nt insertion (908insCAGG) near the exon 5/6 junction in one CML case, and an exon 6 point mutation (997C>T) in one patient with acute lymphoblastic leukemia (ALL). These mutations all create premature stop codons and cause termination at residues 381, 315, and 333, respectively, leading to truncated proteins with only the first quarter of the kinase domain (P-loop) or lacking the C-terminus of ABL including the A-loop. Conclusions: These novel mutations, and the previously documented 35-nt insertion in exon 8, may constitute a new class of mutations that 1) cause truncation of the BCR-ABL kinase; (2) abolish the regulatory element in the ABL kinase domain and the downstream C-terminal region; and (3) confer significant drug resistance. [Table: see text]