MET-EGFR dimerization in lung adenocarcinoma is dependent on EGFR mtations and altered by MET kinase inhibition.

Elena Ortiz-Zapater,Gilbert Fruhwirth,Michael J Neat,Gregory Weitsman,Tony Ng,Frank Mccaughan,Richard W Lee,Peter Parker,William Owen,George Santis,Robert G Dunn

doi:10.1371/journal.pone.0170798

Abstract

Advanced lung cancer has poor survival with few therapies. EGFR tyrosine kinase inhibitors (TKIs) have high response rates in patients with activating EGFR mutations, but acquired resistance is inevitable. Acquisition of the EGFR T790M mutation causes over 50% of resistance; MET amplification is also common. Preclinical data suggest synergy between MET and EGFR inhibitors. We hypothesized that EGFR-MET dimerization determines response to MET inhibition, depending on EGFR mutation status, independently of MET copy number. We tested this hypothesis by generating isogenic cell lines from NCI-H1975 cells, which co-express L858R and T790M EGFR mutations, namely H1975L858R/T790M (EGFR TKI resistant); H1975L858R (sensitized) and H1975WT (wild-type). We assessed cell proliferation in vitro and tumor growth/stroma formation in derived xenograft models in response to a MET TKI (SGX523) and correlated with EGFR-MET dimerization assessed by Förster Resonance Energy Transfer (FRET). SGX523 significantly reduced H1975L858R/T790M cell proliferation, xenograft tumor growth and decreased ERK phosphorylation. The same was not seen in H1975L858R or H1975WT cells. SGX523 only reduced stroma formation in H1975L858R. SGX523 reduced EGFR-MET dimerization in H1975L858R/T790M but induced dimer formation in H1975L858R with no effect in H1975WT. Our data suggests that MET inhibition by SGX523 and EGFR-MET heterodimerisation are determined by EGFR genotype. As tumor behaviour is modulated by this interaction, this could determine treatment efficacy.

Highlights

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) have revolutionised treatment of non-small cell lung cancer (NSCLC) in patients with EGFR mutations
To assess if EGFR-MET interaction is modified by EGFR mutations, we first generated two novel cell lines by modification of the NCI-H1975 lung adenocarcinoma cell line that harbours L858R and T790M (L858R/T790M) mutant EGFR
Using Western blot (WB), we showed the total levels of EGFR in the generated cell lines (Fig 1B) and that the H1975L858R and H1975WT cells became sensitive to the EGFR TKI Erlotinib upon removal of the T790M EGFR sequence even at a low concentration of Erlotinib (Fig 1C)

Summary

Introduction

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) have revolutionised treatment of non-small cell lung cancer (NSCLC) in patients with EGFR mutations. These mutations cause constitutive kinase activity and are oncogenic drivers in 10–20% of Caucasian patients and up to 50% of eastern Asians.[1] Such mutations induce conformational changes in the receptor that alter the dimerization interface, destabilize the inactive state and increase kinase activity to 50 times that of the wild type (WT) EGFR.[2] The EGFR exon 21 L858R and in-frame exon 19 deletions account for 85% of such mutations.[3] Whilst responses are often impressive, resistance is inevitable. The commonest mechanism for resistance is acquisition or clonal expansion of the EGFR exon 20 T790M mutation

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Conclusion