Abstract LB-398: Identification of novel mechanisms of resistance to EGFR tyrosine kinase inhibitors in non-small cell lung cancer (2011)

Abstract Most lung adenocarcinoma patients with activating mutations in the Epidermal Growth Factor Receptor (EGFR) gene initially respond to EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib and gefitinib. However, most patients eventually acquire resistance to this class of drugs. In approximately half of all resistant tumors this is caused by a secondary EGFR mutation (T790M) and/or by amplification of the MET tyrosine kinase receptor, yet the cause of resistance in the remainder of cases is largely unknown. In this study, we aim to identify novel determinants of resistance to EGFR TKIs in order to find new drug targets that may increase the effectiveness of EGFR targeted therapy. We performed a genome-wide siRNA screen examining cell viability in the presence and absence of erlotinib. We used a lung adenocarcinoma cell line (PC9) with activated EGFR that is highly sensitive to erlotinib. We selected hits as genes that upon knockdown showed increased cell viability in drug treated versus untreated conditions. Within the list of candidate genes is the tumor suppressor Neurofibromin (NF1). Using multiple shRNA constructs, we confirmed the enhanced survival in the presence of erlotinib upon NF1 knockdown in a number of EGFR mutant lung adenocarcinoma cell lines using different cell viability and cell competition assays. NF1 is a known negative regulator of RAS activity. We observed increased RAS activity and MAPK signaling in cells where NF1 was knocked down using RNA interference and found that this was resistant to inhibition by erlotinib. Moreover, treatment with low doses of MEK inhibitors resensitized the NF1 knockdown cells to erlotinib. To assess the relevance of this resistance mechanism in vivo, we made use of a tetracycline-inducible mouse model of EGFR-dependent lung cancer. Prolonged erlotinib treatment of lung tumor-bearing mice leads to the outgrowth of resistant tumors. Evaluation of the expression levels of Nf1 in erlotinib-resistant tumors in this preclinical model showed low Nf1 mRNA levels in about half of all erlotinib resistant tumors when compared to the levels in normal adjacent lung. Interestingly, tumors bearing the EGFR-T790M mutation do not show decreased Nf1 expression. These observations suggest that decreased NF1 might be a novel resistance mechanism in EGFR-driven lung tumors. Together, our results show that reduced NF1 expression leads to resistance to erlotinib in EGFR mutant lung adenocarcinomas via enhanced RAS and MAPK pathway activity. Since NF1 is known to be mutated in a subset of lung adenocarcinomas, it is possible that NF1 mutations or loss of expression in EGFR mutant lung tumors could result in de novo or acquired resistance to EGFR inhibitors and that these patients may benefit from treatment with a MEK inhibitor in combination with continued EGFR tyrosine kinase inhibitor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-398. doi:10.1158/1538-7445.AM2011-LB-398