Abstract A114: In vitro acquired resistance to the mutant selective EGFR inhibitor CO-1686 is associated with epithelial-mesenchymal transition (EMT).

Abstract Non-small cell lung cancer (NSCLC) patients with activating epidermal growth factor receptor (EGFR) mutations initially respond well to first generation reversible EGFR tyrosine kinase inhibitors. However, clinical efficacy is limited by acquired resistance driven by the primary drug-resistance T790M mutation in EGFR. CO-1686 is a novel, irreversible and orally delivered kinase inhibitor that specifically targets the most common primary and acquired mutant forms of EGFR while exhibiting minimal activity towards the wild-type (WT) receptor, and has shown evidence of clinical efficacy in on-going phase I/II clinical trials in NSCLC. To assess the mechanisms of acquired resistance to CO-1686, we continuously exposed NCI-H1975 (L858R/T790M), HCC827 (del19) and PC-9 (del19) NSCLC cell lines to several months of increasing doses of CO-1686 until resistance (IC50 >100x over parental) developed. Drug resistance in the CO-1686 resistant (COR) clones extended to additional EGFR TKIs including erlotinib and afatinib. To determine if CO-1686 resistance in the COR cell clones was dependent on EGFR signaling, we examined the functional effects of EGFR siRNA knockdown in NCI-H1975 parental cells and CO-1686 resistant clones. Compared to the parental NCI-H1975 cell line, the resistant clones demonstrated a reduced dependence on EGFR expression for viability. Analysis of genes differentially expressed in the COR clones compared to the parental cell line demonstrated a significant enrichment of genes associated with EMT. Further genetic mutation or copy number alteration in the EGFR gene were not observed in COR clones. Consistent with a mesenchymal cell signature in the COR clones, vimentin expression was up-regulated and E-cadherin down-regulated in the CO-1686 resistant clones at both the protein and RNA level. qRT-PCR analysis of additional markers further supported EMT including the up-regulation of AXL, ZEB1, CDH5, FN1 and the down-regulation of the epithelial markers MIR200B, CLDN4, EPCAM and CLDN7. Higher basal levels of pAKT were also observed in the COR cell clones as compared to the parental NCI-H1975 cell line. Although not effective when used as a single agent, the allosteric AKT inhibitor MK-2206 restored partial drug sensitivity to one of the COR clones when used together in an equimolar fashion with CO-1686. Taken together, these preclinical data suggest that CO-1686 resistance, unlike first and second generation EGFR TKIs, is not mediated by further mutation of EGFR. Resistance to CO-1686 is associated with EMT and increased levels of pAKT and pAXL, suggesting potential actionable targeted combination therapies to be explored in the clinic. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A114. Citation Format: Henry Haringsma, Annette O. Walter, Robert Tjin Tham Sjin, Andrew Allen, Thomas C. Harding, Andrew D. Simmons. In vitro acquired resistance to the mutant selective EGFR inhibitor CO-1686 is associated with epithelial-mesenchymal transition (EMT). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A114.