Identification and validation of driver kinases from next-generation sequencing data

Abstract

It is well appreciated that activating mutations in kinase genes result in kinome reprogramming that leads to altered downstream signaling networks that drive tumor progression. Indeed small-molecule inhibition of activated kinases has heralded the wave of precision medicine in the past decade. The advent of next-generation sequencing has identified a plethora of potentially activating mutations and fusion genes in previously unreported kinase genes that can potentially be developed as targeted therapies. However, the bottleneck in the translation of these alterations into clinically useful therapies lies in their functional validation. Here we describe a set of in vitro functional assays we have optimized to assess whether mutations in kinases are activating. Through overexpression of wild-type and mutant kinase cDNA constructs, we described growth assays in two and three dimensions to ascribe functionality using breast cancer as a model system.