Tyrosine kinases targeted with small molecule inhibitors, Imatinib, Dasatinib, Nilotinib, Erlotinib and Gefitinib are prone to resistance due to mutation in the gatekeeper threonine residue. The first imatinib resistant mutation described in CML patients was an isoleucine substitution at the gatekeeper residue_T315. Mutation at the analogous position to T315 in other imatinib targets like c-KIT (T670) and PDGFRA (T674) have been linked to imatinib resistance in patients with GIST and HES, respectively. Interestingly, the T315I mutation has also been detected in imatinib-naïve CML patients. Similarly, the gatekeeper mutation T790M in EGFR causes resistance to Erlotinib and Gefitinib, and has been detected in patients prior to drug treatment and in the germ line of a family pedigree with several cases of lung cancer. Moreover, the sequence of v-SRC from several independent strains of avian Rous Sarcoma Virus (RSV) differ at up to ten amino acid positions from chicken c-SRC, but only one of these mutations is conserved in every v-SRC gene_a substitution of isoleucine for the gatekeeper threonine residue. Based on the strong correlation between substitution of the gatekeeper threonine and oncogenic activation of v-SRC, and on structural considerations gleaned from our previous studies with dual SRC-ABL kinase inhibitors, we reasoned that substitution of the gatekeeper threonine residue with bulkier residues would be a common mechanism of activation of tyrosine kinases. We evaluated the mutation of threonine in the native cellular forms of c-ABL, c-SRC, PDGFRA, PDGFRB, and EGFR, and found in all cases that substitution of a bulky hydrophobic residue activated kinase activity and promoted malignant transformation of the hematopoietic cell line, BaF3. Furthermore, mice injected with BAF3 cell expressing cABL-T315I, developed robust leukemia and died with in four weeks due to leukocytosis and splenomegaly. However, activated versions of cSRC were unable to induce leukemia in mice. These data demonstrate that mutation of the gatekeeper threonine is a common mechanism of activation for tyrosine kinases. Furthermore, molecular modeling studies of the gatekeeper mutants revealed that the isoleucine substitution widens the ATP-binding pocket and stabilize the DFG-motif in the active conformation. We speculate that this phenomenon may be a generic feature of kinase regulation. Therefore, in future drug design, structures that block the ability of the activation loop to adopt a “DFG-in” conformation should be favored, as they will represent an inhibitor class that is less susceptible to resistance and will retain activity against gatekeeper mutations.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA