Delineation of inhibitor-sensitive/resistant transforming mutations by mutagenesis of EGFR

Proc Amer Assoc Cancer Res, Volume 47, 2006 4028 (Introduction) Somatic mutations in the epidermal growth factor receptor gene, EGFR , are common in lung adenocarcinoma and are associated with clinical responsiveness to EGFR inhibitors such as gefitinib (Iressa®) and erlotinib (Tarceva®). A secondary substitution, T790M in EGFR, has recently been reported as responsible for acquired resistance to these inhibitors. To date, more than 50 different mutations in the kinase domain were documented. To determine the drug sensitivity of those mutations, we chose several mutations that cover more than 75% of the patient with mutations and examined their transforming activity and their drug sensitivity to EGFR inhibitors using the Ba/F3 cell system. Moreover, to explore further the possible range of inhibitor-resistant EGFR mutations, we have engineered mutant EGFR gene homologous to the most highly imatinib-resistant mutations found in the Abl1 tyrosine kinase with the G719S or L858R tumor-derived mutant backgrounds and examined their transforming activity and their drug sentitivities. Retroviruses expressing these mutants were tested for the ability to transform Ba/F3 cells to IL3-independent growth, at various concentrations of EGFR inhibitors. (Results) All lung cancer derived mutant EGFR gene could transform Ba/F3 cells into IL-3 independent on cell growth with the exception of L858R-R776C double-mutant EGFR. All but the insertions in exon 20 showed sensitivity to clinically approved EGFR inhibitors, both gefitinib and erlotinib, with IC50s ranging from 6.5 nM to 107 nM, while the insertions in exon 20 showed strong resistance to both gefitinib and erlotinib, with IC50s ranging from 3.28 to 4.37 μM. The introduction of T790M mutation to those sensitive mutations led to resistance with IC50s of greater than 10 μM regardless of the background mutation. Transformed mutant EGFR homologous to the resistance mutation in Abl tyrosine kinase with the L858R and G719S backgrounds, also showed resistance. Several EGFR mutants homologous to imatinib-resistant Abl, at L718, and M825, disrupted the transforming activity of the activated EGFR mutants. Surprisingly, the T790M single mutant EGFR, without known activating mutations, was also able to transform Ba/F3 cells. (Conclusion) We could successfully survey the transforming activity and drug sensitivity of mutant EGFR derived from lung cancer samples, and other aligned mutants from imatinib-resistance Abl mutations using Ba/F3 cells. This system might be useful as a tool for the screening of the newly developed EGFR inhibitors. The lack of transformation by EGFR mutants analogous to imatinib-resistant Abl mutants suggests that the mutation spectrum of inhibitor-resistant EGFR may be more limited than for Abl. Finally transforming ability of the T790M single mutant provides an explanation for its presence in pre-treatment lung adenocarcinomas.