Abstract 766: Suppression of gefitinib-induced EMT in EGFR mutant NSCLC preferentially selects for acquired T790M

Abstract

Abstract Activating EGFR mutations in non-small lung cancer (NSCLC) confer sensitivity to reversible EGFR tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib. Despite promising initial response acquired resistance develops mediated by the emergence of the secondary T790M mutation or by focal amplification of MET. An epithelial-to-mesenchymal transition (EMT) is clinically linked to NSCLCs with acquired EGFR TKI resistance. The exact mechanisms of EGFR TKI resistance with EMT phenotype remain elusive; therefore, we attempted to develop a strategy to prevent the emergence of EGFR TKI resistance with EMT phenotype. In order to mimic the development of acquired EGFR TKI resistance in NSCLC patients, TKI-sensitive HCC4006 cells harboring mutated-EGFR were exposed to increasing concentrations of gefitinib to generate resistant cells with mesenchymal phenotype. After 6 months, the cells became resistant to gefitinib (HCC4006GeR) with no MET copy number increase and with no apparent gain of secondary T790M mutation. Subsequent genomics and proteomics analyses of HCC4006GeR confirmed enrichment of genes distinctive to mesenchymal cells. Multiplex Luminex growth factor assays identified increased secretion of TGFβ1 from HCC4006Ge-R cells. We discovered that the depletion of EGFR by shRNA or inactivation of mutated EGFR activity by EGFR TKI promoted TGFβ1 secretion and subsequent induction of EMT, which modulate signaling and apoptosis pathways contributing to the development of the resistance. Consequently, we hypothesized that concurrent inhibition of EGFR and TGFβ receptor in HCC4006 cells should prevent the emergence of gefitinib resistance with mesenchymal phenotype. After culturing HCC4006 cells in EGFR (gefitinib) and TGFβ receptor (SB431542) inhibitors, we successfully prevented EMT, although those cells were still resistant to gefitinib (HCC4006GeSB-R). Interestingly, the cells remain sensitive to irreversible EGFR TKI AZD9291 and CO-1686, suggesting the presence of T790M mutation. DNA sequencing of EGFR expressed in HCC4006GeSB-R detected secondary T790M mutation. Droplet digital PCR analysis detected that the frequencies of EGFR allele coding for T790M in HCC4006Ge-R and HCC4006GeSB-R cells are 1.2% and 18.3%, respectively, which were increased from 0.015% in HCC4006 cells harboring heterozygous Del L747-E749+A750P/T790M mutation. The frequency of T790M allele in HCC4006 was higher than the frequency in PC9 cells (0.001%) which reproducibly develop EGFR T790M as a mechanism of EGFR TKI resistance. Taken together, we discovered that suppression of gefitinib-induced EMT in EGFR mutant HCC4006 NSCLC cells preferentially selects for the previously unreported and rare subpopulation of HCC4006 harboring T790M. These results also underscore heterogeneity within HCC4006 cells that give rise to the divergent resistance mechanisms according to treatment. Citation Format: Margaret Soucheray, Marzia Capelletti, Ines Pulido, Yunan Kuang, Cloud P. Paweletz, Jeffrey H. Becker, Eiki Kikuchi, Chunxiao Xu, Tarun B. Patel, Fatima Al-shahrour, Julian Carretero, Kwok-Kin Wong, Pasi A. Janne, Geoffrey I. Shapiro, Takeshi Shimamura. Suppression of gefitinib-induced EMT in EGFR mutant NSCLC preferentially selects for acquired T790M. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 766. doi:10.1158/1538-7445.AM2015-766