Inhibition of drug-resistant mutants of ABL, KIT, and EGF receptor kinases

Abstract

To realize the full potential of targeted protein kinase inhibitors for the treatment of cancer, it is important to address the emergence of drug resistance in treated patients. Mutant forms of BCR-ABL, KIT, and the EGF receptor (EGFR) have been found that confer resistance to the drugs imatinib, gefitinib, and erlotinib. The mutations weaken or prevent drug binding, and interestingly, one of the most common sites of mutation in all three kinases is a highly conserved "gatekeeper" threonine residue near the kinase active site. We have identified existing clinical compounds that bind and inhibit drug-resistant mutant variants of ABL, KIT, and EGFR. We found that the Aurora kinase inhibitor VX-680 and the p38 inhibitor BIRB-796 inhibit the imatinib- and BMS-354825-resistant ABL(T315I) kinase. The KIT/FLT3 inhibitor SU-11248 potently inhibits the imatinib-resistant KIT(V559D/T670I) kinase, consistent with the clinical efficacy of SU-11248 against imatinib-resistant gastrointestinal tumors, and the EGFR inhibitors EKB-569 and CI-1033, but not GW-572016 and ZD-6474, potently inhibit the gefitinib- and erlotinib-resistant EGFR(L858R/T790M) kinase. EKB-569 and CI-1033 are already in clinical trials, and our results suggest that they should be considered for testing in the treatment of gefitinib/erlotinib-resistant non-small cell lung cancer. The results highlight the strategy of screening existing clinical compounds against newly identified drug-resistant mutant variants to find compounds that may serve as starting points for the development of next-generation drugs, or that could be used directly to treat patients that have acquired resistance to first-generation targeted therapy.

Fig. 1.

BIRB-796 and VX-680 inhibit imatinib-resistant BCR-ABL(T315I). Ba/F3 cells expressing BCR-ABL(T315I) were treated for 2 hours with compound, whole-cell lysates prepared, and total protein analyzed by Western blot using anti-ABL and anti-phosphotyrosine antibodies as described (28). (A) Inhibition of BCR-ABL(T315I) by BIRB-796. (B) Inhibition of BCR-ABL(T315I) by VX-680.

Fig. 2.

SU-11248 inhibits imatinib-resistant KIT. (A) Binding affinities of known inhibitors of KIT with the activating V559D mutation compared to the imatinib-resistant V559D/T670I double mutant variant. Quantitative binding constants are shown in Table 1. (B) Cell-based assays confirm the results from in vitro binding experiments. KIT variants were expressed in HEK-293 cells and KIT autophosphorylation levels determined after treating for 2 h with each compound. Results for KIT(V559D) are shown in black, and for KIT(V559D/T670I) in gray. Binding and inhibition constants are plotted as -log(K_d) or -log(IC₅₀), such that higher bars indicate higher affinity binding or more potent inhibition. K_d and IC₅₀ values were measured at least twice and average values are plotted. Error bars represent the range of values obtained in independent replicate measurements.

Fig. 3.

EKB-569 and CI-1033 inhibit gefitinib-resistant EGFR. (A) Cellular proliferation assay. For each concentration point, viable cells were quantitated after 48 h of exposure to compound. (B) Autophosphorylation assay. EGFR autophosphorylation levels in H1975 cells were determined by ELISA after 2 h of exposure to compound followed by EGF stimulation. Results for both the proliferation assay and the autophosphorylation assay were normalized relative to vehicle control. IC₅₀ values were measured at least in duplicate, and average values are shown. The graphs show the average of replicate measurements for each concentration point. Error bars represent the range of values obtained.

Fig. 4.

Kinase interaction maps for BMS-354825 and VX-680. Each red circle indicates a kinase that binds the compound, and larger circles indicate higher affinity binding (see scale at right). Complete quantitative results are shown in Table 6. With sponsorship from Cell Signaling Technology and Sugen, the dendogram was originally presented as a poster in Science to accompany the first analysis of the human kinkome (52). [Figure adapted with permission from ref. (Copyright 2002, Cell Signaling Technology).]