An unbiased in vitro screen for activating epidermal growth factor receptor mutations.

摘要: Cancer tissues harbor thousands of mutations, and a given oncogene may be mutated at hundreds sites, yet only few these mutations have been functionally tested. Here, we describe an unbiased platform for the functional characterization variants single receptor tyrosine kinase (RTK) gene in assay. Our vitro screen activating (iSCREAM) enabled rapid analysis conferring gain-of-function RTK activity promoting clonal growth. The screening strategy included somatic model cancer evolution utilized library 7,216 randomly epidermal growth factor (EGFR) single-nucleotide that were tested murine lymphoid Ba/F3 cells. These cells depend on exogenous interleukin-3 (IL-3) growth, but this dependence can compensated by ectopic EGFR overexpression, enabling selection mutants. Analysis enriched mutants revealed A702V, novel variant structurally stabilized dimer interface conferred sensitivity to inhibition afatinib. As proof concept our approach, recapitulated clinical observations identified L858R as major variant. Altogether, iSCREAM robust enrichment 21 from total mutations. findings indicate power identification are under pressure heterogeneity evolution.