Figure 4.

The H3255DR cells contain an amplification in EGFR T790M. A. The H3255GR (L858R/T790M) cells were made resistant to PF00299804 by growing them in increasing concentrations of PF00299804. Parental and resistant H3255DR cells were treated with PF00299804 at the indicated concentrations, and viable cells were measured after 72 hours of treatment. B. The H3255DR cells have higher baseline EGFR phosphorylation than the H3255GR cells. C. Sequence tracing of EGFR exon 20 from H3255DR cells demonstrates that the mutant allele (T) is the predominant allele (wild type ACG (T); mutant ATG (M)). D. The H3255DR cells are EGFR dependent for growth. Control (NT; non-targeting), EGFR or ERBB3-specific shRNAs were introduced into H3255, H3255GR and H3255DR cells and cell viability was measured using an MTS assay 6 days later. Viability of cells is shown relative to cells expressing the control shRNA. Error bars indicate standard deviation.