ABSTRACT
Mutations in the Bcr-Abl kinase domain are a frequent cause of imatinib resistance in patients with advanced CML or Ph+ 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-AblY253H and Bcr-AblT315I 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-AblT315I or Bcr-AblY253H compared to Bcr-Ablwt, 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.
Subject(s)
Antineoplastic Agents/pharmacology , Fusion Proteins, bcr-abl/genetics , Piperazines/pharmacology , Protein-Tyrosine Kinases/genetics , Pyrimidines/pharmacology , Amino Acid Substitution , Animals , Benzamides , Bone Marrow Cells/pathology , Bone Marrow Cells/virology , Cell Proliferation/drug effects , Cell Transformation, Viral , Disease Models, Animal , Enzyme Activation/drug effects , Fusion Proteins, bcr-abl/drug effects , Gene Transfer Techniques , Imatinib Mesylate , In Vitro Techniques , Male , Mice , Mice, Inbred BALB C , Mutation , NIH 3T3 Cells , Neoplasm Transplantation , Phosphorylation , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , Retroviridae/metabolism , Tumor Cells, CulturedABSTRACT
The Bcr-Abl fusion protein arising through the t(9;22)(q34;q11) reciprocal translocation is the causative agent in chronic myeloid leukemia and a subset of acute lymphocytic leukemia. Imatinib mesylate is a specific inhibitor of the Bcr-Abl kinase and has shown promising results in clinical studies. The structural relation between the Bcr-Abl oncogene and the tyrosine kinase inhibitor imatinib has recently been elucidated by an elegant crystal structure analysis, emphasizing the importance of dephosphorylated tyrosine 393 (Tyr393) in Bcr-Abl for access of the inhibitor to the kinase domain. By mutating this tyrosine to phenylalanine and thereby mimicking a constitutively dephosphorylated state, we now show that Ba/F3 cells transformed by this mutant demonstrate an increased sensitivity towards imatinib in vivo. This effect is not due to an impaired kinase activity of Bcr-Abl Y393F, since a synthetic substrate is phosphorylated with similar kinetics. Treatment of Ba/F3 cells transfected with Bcr-Abl wild type with a phosphatase inhibitor diminished the effect of imatinib, but did not influence the growth of Ba/F3 cells transfected with Bcr-AblY393F. The results support the findings of the crystal structure and indicate that Tyr393 indeed plays a significant role for the sensitivity of Bcr-Abl towards imatinib in vivo. These data implicate the regulation of Tyr393 phosphorylation as a potential mechanism of imatinib resistance.
