EGFR with L858R and H870R double mutations showed reduced sensitivity to Gefitinib

Abstract

Tyrosine kinase inhibitor (TKI) treatment directed against epidermal growth factor receptor (EGFR) can lead to dramatic shrinkage of lung cancers that harbor common EGFR mutations such as L858R or exon 19 deletions. The T790M second mutation can confer TKI resistance but the effects of other types of double mutation, which can occur in up to 10% of lung cancers, are unclear. This study analyzed a double EGFR mutation (L858R+H870R) found in the leptomeningeal metastasis of a male Chinese patient whose primary lung cancer showed initial response to TKI. The functional role of the novel H870R mutation and its effect on L858R in terms of phosphorylation and TKI-induced apoptosis were examined in transfection studies. Vectors expressing full length EGFR cDNA with mutants L858R, H870R and L858R+H870R were constructed by site directed mutagenesis and transfected into COS7 cells. Vectors expressing wild type (WT) EGFR, delE746_A750 and the empty expression vector were used for comparison.

Cells in regular cultures were analyzed for survival by Hoechst staining. Compared to untreated cells, 512nM gefitinib increased apoptosis in L858R- (3.951 fold) and L858R+H870R- (2.334 fold) mutant cells, but not H870R- (1.154 fold) mutant cells, suggesting that H870R is not sensitive to gefitinib-induced apoptosis. Sensitivity to gefitinib is the highest in L858R but it is reduced in combined L858R+H870R mutation. >To further explore the correlation between phosphorylation profiles and gefitinib sensitivity, serum starved cells were treated with gefitinib in serial dilutions followed by EGF stimulation. The phosphorylation status of EGFR Y869 (Y845), Y1016 (Y992), Y1110 (Y1086) and Y1197 (Y1173) were investigated by western blot analysis. Differential phosphorylation profiles were observed. For pY1016, pY1110 and pY1197, L858R was the most sensitive to gefitinib inhibition. H870R and H870R+L858R showed lower sensitivity, requiring 16-32 folds higher concentration of gefitinib to effect similar degrees of inhibition at these sites. For pY869, a reversed pattern was observed with H870R and H870R+L858R showing similar sensitivity to gefitinib inhibition, while L858R showed relative resistance, requiring 16 fold higher concentration for the same effect. >In the EGFR TK domain, Y869 is important for keeping EGFR in an active conformation by van der Waals interaction with K860 and H-bond with E872. For H870R, the linear and basic side chain of arginine has replaced the ring structure of histidine, increasing the negative charges near Y869. The close proximity of H870R to Y869 may affect the H-bond between Y869 and E872, as well as phosphate-transfer along the signaling pathway and response to TKI. In summary, our results suggested that the presence of H870R could reduce the gefitinib sensitivity of L858R.