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

The Philadelphia (Ph) chromosome, a shortened version of chromosome 22, results from a reciprocal translocation between chromosomes 9q34 and 22q11 [1,2,3]. The Ph translocation positions the c-ABL gene of chromosome 9 downstream from the breakpoint cluster region (BCR) on chromosome 22; the resulting fusion gene produces a 190- or 210-kDa hybrid protein with constitutive kinase activity associated with chronic myelogenous leukemia (CML). The impressive clinical efficacy of imatinib mesylate, a selective and effective ABL kinase inhibitor, has revolutionized the treatment of CML. However, the development of resistance to imatinib, which occurs over months to years, constitutes a major drawback in the treatment of advanced CML [4, 5]. Mechanisms leading to drug resistance include amplification of the BCR-ABL gene, acquired additional genomic alterations, and most importantly, specific mutations within the ABL kinase domain that impede drug binding [2, 3,6,7,8]. The ATP-binding site is usually formed between the two lobes of the tyrosine kinase domain. Because the ATP-binding motif is highly conserved, most tyrosine kinase inhibitors generated have been ATP mimetics. Imatinib and other newer agents, such as nilotinib and dasatinib, bind to the ATP-binding cleft within the activation loop (A-loop) of the ABL kinase, establishing extensive contacts with residues lining the cleft and blocking access of ATP to the cleft. Thus, subsequent tyrosine phosphorylation of the substrate is inhibited [5,6,7,8,9]. These inhibitors differ from one another in their molecular structure, how they bind to the BCR-ABL protein, and what other tyrosine kinases they target. These differences lead to different patterns of activity and resistance, resulting in distinct profiles of resistance mutations that are likely to evolve within the kinase domain. Interrogation of the imatinib database indicates that 136 amino acid changes at 100 different ABL residues have been reported to date, and that the 16 most commonly mutated amino acids account for about 87% of all reported mutations; these include mutations at T315 (12.1%), E255 (10.7%), Y253 (9.3%), M351 (9.2%), and G250 (8.5%) [9]. Other types of mutations, such as deletions and insertions, have only recently been described. Reported in-frame deletions in exon 4 of the ABL kinase include Δ184–274 and Δ248–274, both of which display a phenotype of inactive kinase, lack of growth factor independence, and increased sensitivity to imatinib, nilotinib, and dasatinib [10,11,12]. An in-frame deletion skipping the first half of exon 8 has also been documented [13]. In addition, a 35-nt insertion derived from intron 8 was found positioned between the junction of ABL exons 8 and 9 in a patient with chronic CML resistant to imatinib [14]. The resulting truncated BCR-ABL protein lacks the C-terminal nuclear localization, DNA binding, and actin-binding domains. The key structures of the ABL kinase, part of the BCR-ABL leukemogenic molecule, consist of SH3, SH2 and kinase domains, proline-rich regions (P), as well as a nuclear localization signal (NLS), and DNA- and actin-binding (DB and AB) sites (fig. ​(fig.1a).1a). The core kinase domain is organized into an N-lobe, which carries the highly conserved nucleotide-phosphate-binding site for ATP (the P-loop), and a large carboxyl-terminal C-lobe containing the flexible activation loop (the A-loop), a regulatory subunit for kinase activity [13, 15, 16]. Frequency studies of BCR-ABL mutations detected in clinical CML samples revealed that mutations mainly cluster in four distinct regions of the kinase domain. Mutations in the P-loop (amino acids 244–255) are most common, followed by the T315I mutation, which causes global conformational changes. M351, the activation loop hinge, interacts with the SH2 domain and participates in autoregulation of kinase activity. The fourth cluster encompasses the A-loop from residues 381–402 [13]. Imatinib resistance is associated with at least 15 single amino acid mutations at 13 distinct positions within the ABL kinase domain; the most frequently involved positions are T315 and E255, known to be crucial for drug binding [2, 13, 17, 18]. P-loop as well as T315 mutations disrupt and shift the conformational equilibrium of the kinase to favor the active state and allosterically prevent imatinib and other kinase inhibitor binding. Accordingly, most of these point mutations confer resistance to imatinib, nilotinib, and dasatinib and are associated with a worse prognosis than are mutations elsewhere [16, 19]. Open in a separate window Open in a separate window Fig. 1. Schematic diagram and sequence alignment of wild-type and four mutant Bcr-Abl proteins resulting from premature termination mutations. a The organization of wild-type and four mutant protein domains is shown. b Amino acid sequences are aligned and labeled in black (wild type), red (exon 7 Del), pink (exon 7 2-nt Del), green (exon 6 997C→T) and blue (exon 6 4-nt Ins). Sequence differences caused by frameshift mutations are underlined. Premature stop codons are indicated by asterisks, and the position of T315 is marked in yellow.