We managed to study this issue in SKMG3 cells harboring the EGFR A289D mutant, because we had previously demonstrated that this mutant, unlike EGFRvIII, doesn’t abrogate the ability of EGFR to respond to EGF. Erlotinib, on the other hand, was stronger than lapatinib at curbing EGFR in lung cancer cell lines with the EGFR kinase domain mutants EGFR746 750 and EGFR L858R, consistent with previous studies. GW9508 clinical trial Akt and Erk, two well documented effector kinases of the examined EGFR kinase domain mutants, were also more potently inhibited by erlotinib in comparison to lapatinib in these lines. Curiously, inhibition of EGFR in SKMG3 GBM cells did not result in Akt or Erk inhibition, suggesting the A289D mutant uses other downstream effector pathways. We also examined the effects of lapatinib and erlotinib on cell death. Lapatinib, but not erlotinib, induced cell death in every examined GBM cell lines with EGFR ectodomain mutants. In EGFR mutant lung cancer cell lines, erlotinib induced cell death at lower levels than lapatinib. 3. Type-ii EGFR inhibitors efficiently displace ATP from EGFR EC mutants Our results with four different EGFR kinase inhibitors proposed that the catalytic domain of Lymph node EGFR ectodomain mutants may like a lazy like conformation that is more available to lapatinib or HKI 272 than to erlotinib or CI 1033. To help check this model, we developed an analysis that measures the potential of EGFR kinase inhibitors to compete entirely cell lysates with ATP for binding to the ATP cleft of the EGFR kinase domain. Coincubation of total cell lysates from A289D EGFR mutant SKMG3 cells with biotinylated ATP and erlotinib demonstrated reduced ATP presenting with increasing erlotinib concentrations. Coincubation of the sample of the same total cell lysate with increasing concentrations of lapatinib blocked ATP binding at lower concentrations Bortezomib MG-341 of lapatinib than erlotinib. As a specificity control, we observed no displacement of ATP binding by either lapatinib or erlotinib and decided ATP binding to the kinase domain of SRC. We also repeated these experiments with total cell lysates from H3255 lung cancer cells, and found that erlotinib blocked ATP binding to the EGFR kinase domain more effectively than lapatinib. Since differences in rates involving the reversible EGFR kinase inhibitors erlotinib and lapatinib might affect outcomes of the ATP competition analysis, we performed additional experiments with the irreversible EGFR kinase inhibitors CI 1033 and HKI 272. In whole mobile lysates from A289D EGFR SKMG3 cells, HKI 272 better blocked ATP binding to the EGFR kinase domain than CI 1033, in line with our model. Lastly, we explored whether a forced change in receptor conformation, induced by ligand binding, might alter the capability of EGFR inhibitors to get entry to the kinase domain and block EGFR phosphorylation.