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

Abstract

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