The Bcr-Abl mutations T315I and Y253H do not confer a growth advantage in the absence of imatinib

Abstract

Mutations in the Bcr-Abl kinase domain are a frequent cause of imatinib resistance in patients with advanced CML or Ph þ ALL. The impact of these mutations on the overall oncogenic potential of Bcr-Abl and on the clinical course of the disease in the absence of imatinib is presently unclear. In this study, we analyzed the effects of the Bcr-Abl P-loop mutation Y253H and the highly imatinib resistant T315I mutation on kinase activity in vitro and transforming efficiency of Bcr-Abl in vitro and in vivo. Immunoprecipitated Bcr-Abl Y253H and Bcr-Abl T315I proteins displayed similar kinase activities and substrate phosphorylation patterns as Bcr-Abl wildtype. We directly compared the proliferative capacity of mutant to wildtype Bcr-Abl in primary BM cells in vitro and in a murine transplantation model of CML by using a competitive repopulation assay. The results implicate that in the absence of imatinib, there is no growth advantage for cells carrying Bcr-Abl T315I or Bcr-Abl Y253H compared to Bcr-Abl wt , whereas imatinib treatment clearly selects for leukemic cells expressing mutant Bcr-Abl both in vitro and in vivo. Thus, the analysed Bcr-Abl mutants confer imatinib resistance, but do not induce a growth advantage in the absence of imatinib.

BCR-ABL mutants spread resistance to non-mutated cells through a paracrine mechanism

“…In some studies but not others, E255K and T315I increase BCR-ABL phosphorylation and target downstream proteins. [19][20][21] Increased or decreased clonogenicity, oncogenesis and growth in low serum conditions have also been reported. Our data indicate that in BCR-ABL cell lines the mutations principally confer resistance to a specific kinase inhibitor.…”

Novel pathway in Bcr-Abl signal transduction involves Akt-independent, PLC-γ1-driven activation of mTOR/p70S6-kinase pathway

“…The mice were followed up for disease development judged by symptoms such as abnormal gait and labored breathing as described earlier (Miething et al, 2006). Moribund animals were killed by cervical dislocation.…”

“…The sequences of oligonucleotides required for cloning of the PLC-g1 shRNAs into pLMP vector are available on request. Preparation of retroviruses and transduction of Ba/F3 and Ba/F3-p185 cells were performed as described earlier (Miething et al, 2006).…”

Targeting survival cascades induced by activation of Ras/Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways for effective leukemia therapy

“…Two recent frontiers in leukemia research are the emergence of imatinib-resistant CMLs [61][62][63][64][65][66][67][68][69][70][71][72][73][74][75][76][77] and the discovery of mutations at the Jak2 kinase in certain myeloproliferative diseases. These topic areas document the importance of genetic approaches in leukemia and abnormal hematopoiesis research as they were discovered upon identification of the genetic mutations responsible for the hematopoietic neoplasia.…”

“…117 The BCR-ABL T315I (threonine to isoleucine) mutation does not confer a growth advantage in the absence of imatinib but clearly does bestow imatinib, dasatinib and nilotinib resistance. 118 In one study, which examined the diversity of BCR-ABL KD mutations, 119 in persistent CML patients who received a second-generation BCR-ABL inhibitor after imatinib failure, 67 different KD mutations were observed before the start of therapy with the second BCR-ABL inhibitor, 15% of them had the T315I mutation. Upon treatment with dasatinib, nilotinib or a combination of dasatinib, nilotinib, and imatinib 26% of the patients developed additional BCR-ABL KD mutations.…”