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

Abstract

Chronic myeloid leukemia is effectively treated with imatinib, but reactivation of BCR-ABL frequently occurs through acquisition of kinase domain mutations. The additional approved ABL tyrosine kinase inhibitors (TKIs) nilotinib and dasatinib, along with investigational TKIs such as ponatinib (AP24534) and DCC-2036, support the possibility that mutationmediated resistance in chronic myeloid leukemia can be fully controlled; however, the molecular events underlying resistance in patients lacking BCR-ABL point mutations are largely unknown. We previously reported on an insertion/ truncation mutant, BCR-ABL 35INS IntroductionImatinib is an inhibitor of BCR-ABL, the tyrosine kinase that causes chronic myeloid leukemia (CML). Most newly diagnosed patients achieve durable remissions on imatinib therapy, 1,2 but 10%-15% fail to respond or relapse. The leading cause of imatinib resistance is reactivation of BCR-ABL because of kinase domain point mutations. Most BCR-ABL mutants are susceptible to alternative ABL tyrosine kinase inhibitor (TKI) therapies. [3][4][5][6][7][8] Sequencing of the BCR-ABL kinase domain in patients exhibiting signs of TKI treatment failure has also revealed the presence of alternatively spliced variants, including BCR-ABL 35INS , in which retention of 35 intronic nucleotides at the exon 8/9 splice junction introduces a stop codon after 10 intron-encoded residues. 9-13 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. 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. Methods IL-3 withdrawalBa/F3 cells cultured in standard media (RPMI 1640 media, 10% FBS, L-glutamine, penicillin-streptomycin; Invitrogen) containing IL-3 from WEHI-conditioned media were infected with retrovirus expressing BCR-ABL, BCR-ABL 35INS , or BCR-ABL K271P/35INS (MSCV-IRES-GFP), and stable cell lines were sorted for GFP (FACSAria II; BD Biosciences). (E) Equimolar amounts...

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 itself was found to not contribute to TKI resistance because the BCR-ABL1 protein derived from the mRNA of BCR-ABL1 35INS lacked the kinase domain necessary for BCR-ABL1 kinase activity [32]. We detected BCR-ABL1 35INS in 34 out of 60 patients (56.7%) by direct sequencing.…”

“…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].…”

Compound mutations in BCR-ABL1 are not major drivers of primary or secondary resistance to ponatinib in CP-CML patients

“…Missense mutations outside the KD were excluded (N 5 55) (supplemental Table 1 available on the Blood Web site), as were all polymorphisms (N 5 3), deletions (N 5 5), frameshift (N 5 12), nonsense (N 5 18), and synonymous (N 5 60) mutations because these are not considered relevant for TKI resistance. [17][18][19] In samples in which multiple BCR-ABL1 missense mutations were detected, the phase (whether present on the same or different alleles) and frequency of all possible mutation combinations were calculated as described in the supplemental Methods.…”

Relapsed Philadelphia Chromosome-Positive Pre-B-ALL after CD19-Directed CAR-T Cell Therapy Successfully Treated with Combination of Blinatumomab and Ponatinib

“…Six months after CAR-T therapy, he presented with relapsed disease. TKI mutation analysis identified a BCR-ABL 35INS insertion mutation, which has not been associated with TKI resistance [17]. He received vincristine and prednisone as bridging salvage therapy to a second anti-CD19 CAR-T cell infusion.…”