Altered Oncogenic Fitness of Imatinib- and Dasatinib-Resistant BCR-ABL Mutants Is Due to Differential Intrinsic Kinase Activity and Signaling Pathway Selection Defined by Phosphoproteome Profiling.

Point mutations in the kinase domain of BCR-ABL are the most common cause of resistance to imatinib therapy in CML. E255K and T315I are detectable before the initiation of imatinib therapy in a small population of CML patients, suggesting that certain imatinib-resistant BCR-ABL mutants display altered biological fitness relative to wild-type p210 BCR-ABL. To test this hypothesis, we performed primary murine bone marrow transformation assays using BCR-ABL-expressing retroviruses. In a survey of six clinically imatinib-resistant mutations (Y253F, Y253H, E255K, E255V, T315I, M351T) as well as five additional mutations that confer in vitro resistance to the dual SRC/ABL inhibitor dasatinib (L248R, V299L, T315A, F317L, F317V), we identified several that altered oncogenic fitness either positively or negatively relative to p210. To elucidate the molecular basis for altered transformation potential, we examined the kinase activity of p210 versus mutant full-length BCR-ABL. Enzyme kinetics studies with an optimized Abl peptide substrate as well as gel-based in vitro kinase assays illustrated that intrinsic kinase activity does explain some, but not all, of the mutant oncogenic potency phenotypes. The imatinib- and dasatinib-resistant mutant T315I, for instance, was a gain-of-fitness mutant in the bone marrow assay but exhibited a three-fold lower Vmax than p210. In contrast, T315A, a dasatinib-resistant mutation at the T315 gatekeeper residue, exhibits reduced transforming potential relative to wt p210 and is completely unable to phosphorylate Abl substrates in the kinase assays, although it does retain the ability to autophosphorylate. Subsequent kinase assays on immunoprecipitations from stably expressing IL-3-independent Ba/F3 cells illustrated that certain mutants bind and phosphorylate distinct substrates from p210. We further delineated signaling differences between p210 and the mutant panel by utilizing a mass spectrometry-based global phosphorylation profiling technique. Trypsinized proteins from stably expressing p210 and mutant Ba/F3 cells were immunoprecipitated using anti-phosphotyrosine antibodies, then identified and quantified by two dimensional mass spectrometry. Phosphorylation of specific substrates correlates with gain- or loss-of fitness observed in the bone marrow assay. We conclude that certain BCR-ABL kinase domain mutations, in addition to conferring drug resistance, also affect the oncogenic potency of BCR-ABL by altering intrinsic kinase activity as well as downstream targets. Elucidation of alternative signaling pathways activated by these mutants by the combination of biochemical and mass spectrometric methods may provide novel therapeutic targets for imatinib- and dasatinib-resistant CML.