The Allelic Context of the C 797 S Mutation Acquired upon Treatment with Third-Generation EGFR Inhibitors Impacts Sensitivity toSubsequent Treatment Strategies

Purpose: A secondary EGFR mutation, T790M, is the most common resistance mechanism in EGFR-mutant adenocarcinomas that have progressed on erlotinib. Third-generation EGFR inhibitors capable of inhibiting mutant EGFR with T790M produce responses innearly two thirds of patients.However, acquired resistance mechanisms in patients treated with these drugs are yet to be described. Experimental Design: To study acquired resistance to thirdgeneration EGFR inhibitors, T790M-positive cells derived from an erlotinib-resistant cancer were made resistant to a thirdgeneration TKI and then characterized using cell and molecular analyses. Results: Cells resistant to a third-generation TKI acquired an additional EGFRmutation, C797S, which prevented suppression of EGFR. Our results demonstrate that the allelic context in which C797S was acquired may predict responsiveness to alternative treatments. If the C797S and T790M mutations are in trans, cells will be resistant to third-generation EGFR TKIs, but will be sensitive to a combination of firstand third-generation TKIs. If the mutations are in cis, no EGFR TKIs alone or in combination can suppress activity. If C797S develops in cells wild-type for T790 (when third-generation TKIs are administered in the first-line setting), the cells are resistant to third-generation TKIs, but retain sensitivity to first-generation TKIs. Conclusions: Mutation of C797S in EGFR is a novel mechanism of acquired resistance to third-generation TKIs. The context in which the C797S develops with respect to the other EGFR alleles affects the efficacy of subsequent treatments. Clin Cancer Res; 21(17); 3924–33. 2015 AACR. See related commentary by Ayeni et al., p. 3818 Introduction The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib, and afatinib are effective treatments for EGFR-mutant non–small cell lung cancers (NSCLCs; refs. 1–5). Although most patients with EGFR-mutant NSCLC respond to these therapies, the responses are not permanent, and patients typically develop resistance after an average of 1 year on treatment (6). There are several mechanisms of acquired resistance to erlotinib, including the development of a "gatekeeper" point mutation, T790M, which prevents the TKI from effectively inhibiting EGFR (7, 8), reactivation of downstream signaling pathways via bypass tracks (9–14), and phenotypic/histological changes such as epithelial-to-mesenchymal transition (EMT) or small cell lung cancer (SCLC) transformation (12, 14, 15). T790M is the most common resistance mechanism in these cancers and is observed in over 50% of resistant biopsies (12, 14). Second-generation EGFR inhibitors, including afatinib (BIBW2992) and dacomitinib (PF00299804), are irreversible EGFR inhibitors that bind to Cys797 and have been shown in preclinical experiments to effectively inhibit EGFRwith activating mutations (exon 19 deletion or L858R) as well as those with the T790M resistance mutation (16, 17). However, their activity in patients with erlotinib-resistant cancers harboring T790M has been minimal (18, 19). The discordance between laboratory and clinical results is likely due to a poor therapeutic window. These drugs are equally potent against wild-type EGFR and EGFR T790M, and thus the toxicity resulting from inhibiting wild-type EGFR (rash anddiarrhea) precludes the use of doses thatwould be needed to effectively suppress T790M (20). More recently, thirdgeneration EGFR TKIs, including WZ4002, CO-1686, AZD-9291, and EGF816, have been developed to target mutant EGFR harboring T790M (21–24). This class of inhibitor also binds covalently to Cys797, and largely spares WT EGFR, thereby decreasing toxicity andpermitting the use of doses that fully suppress T790M. This large therapeutic window likely underlies the greater than 50% response rates observed in clinical trials with CO-1686 and AZD-9291 in erlotinib-resistant, T790M-positive NSCLCs (25, 26). Based on these promising results, both drugs have received FDA "breakthrough therapy designation," and this class of inhibitors is on the verge of becoming widely implemented for treatment of this patient population. Previous studies have generated acquired resistance to third-generation inhibitors in well-studied cell lines, and have identified mechanisms that have also been observed in cancers with acquired resistance to Massachusetts General Hospital Cancer Center, Massachusetts General Hospital, Boston, Massachusetts. Department of Medicine, Harvard Medical School, Boston, Massachusetts. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Jeffrey A. Engelman, 149 13th Street 7th Floor, Charlestown, MA 02129. Phone: 617-724-7298; Fax: 617-724-9648; E-mail: jengelman@mgh.harvard. edu doi: 10.1158/1078-0432.CCR-15-056