Imatinib binding and cKIT inhibition is abrogated by the cKIT kinase domain I missense mutation Val654Ala.

Several activating mutations in the cKIT receptor tyrosine kinase are associated with the development and progression of gastrointestinal stromal tumors (GIST). Treatment of GIST with the tyrosine kinase inhibitor imatinib (Gleevec, STI571; Novartis, Basel, Switzerland) increases patient survival. However, many patients develop resistance to imatinib following initial responses. We sequenced cKIT exons from two patients with GIST after the development of imatinib resistance, revealing a point mutation in kinase domain I (exon 13), Val654Ala, which has been associated previously with relapse and resistance. Molecular modeling of cKIT-imatinib complexes shows that this residue is located in the drug-binding site and that the Val654Ala mutation disrupts drug binding by removing hydrophobic contacts with the central diaminophenyl ring of imatinib. Loss of these contacts results in a destabilizing effect on two key hydrogen bonds between imatinib and Asp310 and Thr670 of cKIT. Calculations based on published crystallography data show an estimated destabilization energy of 2.25 kcal/mol in the Val654Ala cKIT compared with wild type. When present on the same cKIT allele as an oncogenic mutation, the Val654Ala mutation abolishes imatinib-mediated inhibition of cKIT phosphoactivation in vitro. These results highlight some of the structural and functional consequences of the Val654Ala mutation in relapsing imatinib-resistant GIST and emphasize the importance of tumor genetics in drug development and patient-specific cancer treatment regimens.