Background: The Bcr-Abl tyrosine kinase inhibitor imatinib is frontline therapy for chronic myeloid leukemia (CML). Resistance to imatinib is a significant problem, especially for CML patients treated in advanced phases of the disease. The most prevalent cause of imatinib resistance is restoration of Bcr-Abl kinase activity via kinase domain mutations that decrease imatinib sensitivity (IC50 WT Bcr-Abl: 300 nM; Bcr-Abl mutants: ∼1 to >10 μM). While dose escalation may recapture response in the case of weakly resistant mutants such as M351T (IC50: 930 nM), it is not effective for highly imatinib-resistant mutants. The need for a more potent Bcr-Abl inhibitor that retains target specificity and favorable pharmacokinetic properties led to synthesis of AMN107, an aminopyrimidine related to imatinib. To assess the potential of AMN107 for treating kinase domain imatinib-resistant CML, we evaluated AMN107 in cellular and biochemical assays against a panel of sixteen kinase domain mutants that comprise >90% of clinically observed mutations. Methods: Cell proliferation assays, Bcr-Abl tyrosine phosphorylation immunoblot analyses, and apoptosis assays were performed comparing AMN107 and imatinib. Cells studied were: parental Ba/F3 cells, Ba/F3 cells expressing WT Bcr-Abl or one of the following Bcr-Abl mutants: M244V, G250E, Q252H, Y253F, Y253H, E255K, E255V, F3llL, T315I, F317L, M351T, F359V, V379I, L387M, H396P, or H396R. In addition, the sensitivity of mutants was tested in autophosphorylation and peptide substrate kinase assays using purified WT and mutant Abl kinase domains. Results: AMN107 inhibited growth of cells expressing WT Bcr-Abl with ∼23-fold higher potency than imatinib (IC50: 13 nm vs 300 nM). Sensitivity of Bcr-Abl mutants to AMN107 segregated into four categories: high (IC50 ≤ 70 nM; IC90 ≤ 165 nM): [10/16: M244V, G250E, Q252H, F3llL, F317L, M351T, V379I, L387M, H396P, H396R], medium (IC50 ≤ 200 nM; IC90 ≤ 485 nM): [3/16: Y253F, E255K, F359V], low (IC50 ≤ 450 nM; IC90 ≤ 2 μM): [2/16: Y253H, E255V], and insensitive (IC50 >2 μM): [T315I]. Analogous results were obtained for cellular immunoblot and apoptosis assays. Autophosphorylation and peptide substrate kinase assays using purified WT and mutant Abl kinase domains confirmed cellular assay results and established Bcr-Abl as a direct AMN107 target. Conclusions: AMN107 is a potent inhibitor of WT Bcr-Abl and most imatinib-resistant Bcr-Abl mutants and is currently in phase I clinical trials. If AMN107 trough levels are similar to the levels achieved by imatinib (1.5 μM), WT Bcr-Abl and 13/16 mutants would be predicted to be sensitive to AMN107, while three mutants require significantly higher AMN107 levels or an alternate inhibitor. These data indicate that AMN107 is a highly active Bcr-Abl inhibitor that may have clinical utility in patients with imatinib-refractory CML.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]