YES1 amplification as a mechanism of acquired resistance (AR) to EGFR tyrosine kinase inhibitors (TKIs) identified by a transposon mutagenesis screen and clinical genomic testing.

Abstract

9043 Background: Overcoming AR to EGFR TKIs remains challenging, and in many cases the mechanisms are still unclear. To identify novel mechanisms of resistance to EGFR TKIs, we performed a forward genetic screen using transposon mutagenesis in EGFR-mutant lung adenocarcinoma cells. Methods: EGFR TKI-sensitive PC9 cells were co-transfected with plasmids encoding a mutagenic piggyBactransposon and hyperactive piggyBac transposase. Transposon-tagged, afatinib-resistant clones were generated by sequential selection of transfected cells with puromycin and 1µM afatinib. Transposon insertion sites were mapped using a modified TraDIS-type method and next-generation sequencing (NGS). Selected clones were characterized using Western blots, receptor tyrosine kinase (RTK) arrays, and viability assays following treatment with TKIs or siRNA-mediated gene knockdowns. We reviewed MSK-IMPACT™ NGS data on 100 patient tumors with EGFR TKI AR. Available tumor samples were analyzed by fluorescence in situ hybridization (FISH). Results: In 187/188 afatinib-resistant clones, transposon insertion sites consistent predominantly with gene upregulation were found in MET, the Src family kinase (SFK) member YES1, or both. Clones with activating YES1 insertions exhibited resistance to all three generations of EGFR TKIs; high levels of expression of tyrosine-phosphorylated YES1; sensitivity to the SFK TKI dasatinib and to siRNA-mediated knockdown of YES1; and tyrosine phosphorylation of YAP1 and ERBB3. A query of the MSK-IMPACT™ data on EGFR TKI AR patients revealed amplification of YES1 and no alteration of MET, ERBB2 or BRAF in 3/54 T790M-negative (95% CI 1 to 16%) and 1/46 (95% CI 1 to 12%) T790M-positive cases. Amplification of YES1was confirmed by FISH in 2/2 cases, and was absent in matched pre-TKI samples in 2/2 cases. Conclusions: YES1 amplification is found in 4% of patients with acquired resistance to EGFR TKIs and is potentially targetable by Src family kinase inhibitors. Forward genetic screens using transposon mutagenesis and routine clinical NGS of patient samples can identify novel mechanisms of resistance to targeted therapies.