Comparative analysis of two clinically active BCR-ABL kinase inhibitors reveals the role of conformation-specific binding in resistance

Abstract

Structural studies suggest that most point mutations in the BCR-ABL kinase domain cause resistance to the ABL kinase inhibitor imatinib by impairing the flexibility of the kinase domain, restricting its ability to adopt the inactive conformation required for optimal imatinib binding, rather than by directly interfering with drug contact residues. BMS-354825, currently in clinical development for imatinib-resistant chronic myelogenous leukemia, is a dual SRC/ABL kinase inhibitor that binds ABL in both the active and inactive conformation. To examine the potential role of conformational binding properties in drug resistance, we mapped the mutations in BCR-ABL capable of conferring resistance to BMS-354825. Through saturation mutagenesis, we identified 10 such BCR-ABL mutations, 8 of which occurred at drug contact residues. Some mutants were unique to BMS-354825, whereas others also conferred imatinib resistance. Remarkably, the identity of the amino acid substitution at either of two contact residues differentially affects sensitivity to imatinib or BMS-354825. The combination of imatinib plus BMS-354825 greatly reduced the recovery of drug-resistant clones. Our findings provide further rationale for considering kinase conformation in the design of kinase inhibitors against cancer targets.

Fig. 1.

BMS-354825 works in concert with imatinib to reduce the number of resistant clones. (A) Graphical representation of the frequency of drug-resistant Ba/F3 clones obtained from four independent screens. For each experiment, 75–90 × 10⁶ Ba/F3 cells infected with mutagenized p210 BCR-ABL were divided into five treatment groups (50 nM BMS-354825, 25 nM BMS-354825, 10,000 nM imatinib, and combinations of each) and plated at a density of 5 × 10⁵ cells per well (30–36 wells per treatment group). The number of clones obtained per well was averaged for each experimental condition, and an error bar represents the standard error between the four screens. (B) WT p210 Ba/F3 line grown in increasing concentrations of BMS-354825 in the presence of 0, 100, 200, 400, 800, and 1600 nM imatinib, as indicated, for 3 days. Normalized viable cell counts of the average of the triplicates are plotted with respect to the no-drug control. An error bar represents the standard deviation of the mean for each dose.

Fig. 2.

The identity of the mutation at Thr-315 and Phe-317 determines the sensitivity or resistance of BCR-ABL to each of the kinase inhibitors. (A) Western blot analysis of stable Ba/F3 clones expressing the indicated BCR-ABL p210 isoform. In each case, cells were normalized by cell count and lysed in equal volumes of lysis buffer after exposure to the indicated concentration of drug for 3–4 h. Western blots were analyzed with antibody to antiphosphotyrosine (anti-PY), ABL, and β-actin. Comparison of growth inhibition by BMS-354825 and imatinib on mutations at Thr-315 (B) and Phe-317 (C). WT p210 is plotted as a control.

Fig. 3.

Structural modeling of the T315A mutation provides rationale for continued inhibition of BCR-ABL kinase activity by imatinib but not BMS-354825. WT ABL complexed with imatinib (A) and BMS-354825 (B) illustrates key hydrogen-bonding interactions between each inhibitor and the ATP-binding pocket of ABL. In C, mutation to T315A allows a water molecule to bridge the hydrogen-bonding interactions of imatinib and ABL. (D) In the case of BMS-354825 and T315A, the drug displaces the water molecule, loses the critical hydrogen bonds, and prevents binding.