Selection of randomly mutated EGFR-libraries for erlotinib-resistance. Erlotinib-resistance selection

In this study a novel high-throughput drug resistance screen was developed, which was termed PhosphoFlowSeq. Briefly, the coding regions of human EGFR were randomly mutated by error prone PCR, followed by ligation of the resulting EGFR library into an appropriate plasmid. The EGFR-plasmid library was subsequently transfected into HEK293T cells, followed by incubation of the EGFR-expressing HEK293T cells with the EGFR-directed tyrosine kinase inhibitor (TKI) erlotinib. Next, cells showing EGFR phosphorylation despite the presence of erlotinib were selected by flow cytometry, followed by plasmid isolation and PCR amplification of the enriched EGFR genes, thereby closing the selection cycle. Finally, the enriched libraries, as well as EGFR-wt and the non-selected starting libraries as controls, were analyzed by deep sequencing in order to find mutations that were enriched during the erlotinib-resistance selection. We demonstrated that PhosphoFlowSeq enables robust and reproducible enrichment of the drug resistance mutation T790M (in four independent experiments, termed experiment I, II, III and IV), which has also been shown to be the primary resistance mechanism upon erlotinib treatment in the clinics. As a further control, the libraries were also screened for loss-of-function despite the presence of the EGFR-ligand EGF (this control was included in experiments II and IV only).