Acquired MET Exon 14 Alteration Drives Secondary Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor in EGFR-Mutated Lung Cancer.

Abstract

Novel resistance mechanisms to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant lung cancer continue to be defined.1 With the recent approval of the third-generation EGFR TKI osimertinib as first-line treatment, and expanded use of large-panel next-generation sequencing (NGS)–based testing, many novel resistance mechanisms are emerging. Resistance to osimertinib can result from on-target mechanisms, such as the acquisition of second-site EGFR mutations, or from the activation of off-target mechanisms or bypass pathways, including acquired oncogenic fusions of RET, ALK, BRAF, and FGFR1,2 and amplification or mutation of HER2, BRAF, MEK, KRAS, PIK3CA, and MET.3 MET amplification is reported in 5% to 22% of EGFR TKI resistance.1,4 Preclinical studies have shown that acquired resistance to EGFR TKIs resulting from MET amplification can be reversed by combined therapy with EGFR and MET inhibitors.4 MET exon 14 skipping alteration (METex14) is present in 3% to 4% of lung adenocarcinomas,5 and the MET receptor lacking exon 14 shows decreased protein turnover because of loss of the ubiquitination site encoded by exon 14, resulting in aberrant MET activation and oncogenesis.6 In preclinical and clinical studies, responses to MET inhibitors, such as crizotinib, have been reported in patients with lung cancer with METex14 as a primary driver.6-11 However, it has not been previously implicated in acquired resistance to EGFR-TKIs. In this study, we used targeted NGS with Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT),12 immunohistochemistry, cell-free DNA testing, and fluorescence in situ hybridization to evaluate acquired resistance mediated by METex14. Furthermore, we used in vitro functional studies to establish METex14 as a novel mechanism of acquired resistance to EGFR TKIs. We used MSK-IMPACT, a large-panel NGS assay, to detect mutations, copy-number alterations, and select gene fusions involving up to 468 cancer-associated genes.12 METex14 was introduced into PC9 and H1975 cells as follows. Briefly, full-length METex14 was polymerase chain reaction amplified and subcloned into pLenti-CMV-blast lentiviral vector (plasmid 17451; Addgene, Cambridge, MA). The lentiviral plasmids were cotransfected with packaging plasmids into HEK 293 T cells using FuGENE HD (Promega, Madison, WI), and lentiviruses were generated. Cells were infected with lentivirus-expressing METex14 cDNA, followed by selection with blasticidin (20 µg/mL) for 8 days. The Data Supplement provides more detailed methods.