Figure 6.

Selection for EGFR T790M expressing clones following exposure to PF00299804. A. HCC827 cells were transfected with either GFP or EGFR delE746_A750/T790M and the resulting cells were either untreated or exposed to 100 nM PF00299804. The cells were stained with crystal violet after 15 days of drug exposure. B. Quantification of colonies from A. Error bars indicate standard deviation. C. Sequence tracing of EGFR exon20 from untreated (control) or PF00299804 treated HCC827delE746_A750/T790M cells. The PF00299804 treated cells have a substantial increase in the mutant (T) allele. The delE746_A750/T790M transgene contains the G>A EGFR SNP (arrow) in cis and is concurrently amplified. D. The PF00299804 resistant HCC827delE746_A750/T790M cells express greater amount of EGFR T790M protein. The delE746_A750/T790M expression construct contains FLAG at the C-terminus. Cell extracts were immunoblotted to detect the indicated proteins. E. Sequence tracing of EGFR exon 20 from untreated (control) or PF00299804 treated xenografts. The PF00299804 treated tumors have an increase in the mutant (T) allele compared to the untreated tumors. F. Quantification of colonies using cells from A following continuous low dose (200 nM) or pulse (7hr) treatment with either 1 μM or 3 μM PF00299804. Error bars indicate standard deviation.