A chemical genetics approach to identify therapeutic vulnerabilities in Gefitinib resistant, EGFR T790M

Abstract

Background: The first generation EGFR inhibitor, Gefitinib, has been approved for the treatment of non-small cell lung carcinoma (NSCLC) with activating EGFR mutations. However resistance to Gefitinib quickly develops and is mainly driven by the EGFR T790M mutation in the kinase domain. On the other hand, while EGFR was found to be overexpressed in the majority of HNSCC, clinical trials on the use of Gefitinib for treatment of HNSCC has been disappointing thus far. This is mainly due to the lack of activating mutations in the EGFR kinase domain of HNSCC patients, as well as treatment resistance. In this study, we sought to identify alternative therapeutic vulnerabilities in Gefitinib resistant HNSCC that are negative for EGFR T790M mutation. Methods: We developed a pair of isogenic Gefitinib sensitive (TM17S) and resistant (TM17R) cell lines through stepwise chronic treatment of the TM17S cell line with Gefitinib. The TM17S and TM17R cell lines were characterized for their mutational profile using POLARIS Xplora cancer panel and through gene expression studies. High-throughput chemical screen with two annotated compound libraries was carried out to identify alternative therapeutic vulnerabilities in the TM17R cell line. Results: While the TM17R cell line was derived to be resistant to Gefitinib, a first generation EGFR tyrosine kinase inhibitor (TKI), TM17R cells were found to be resistant to all 3 generations of EGFR TKIs. Comparison of the mutational profiles of TM17S and TM17R cells did not reveal obvious mutational drivers of resistance. Strikingly the EGFR T790M mutation was not detected in both TM17S and TM17R cells. We also observed a down regulation of EGFR gene and protein expression. All these could account for the resistance of TM17R cells to all 3 generations of EGFR TKIs, and suggests an alternative mechanism of resistance. Results from the high-throughput chemical screen revealed an alternative therapeutic vulnerability of TM17R cells to Aurora kinase inhibitors. This therapeutic vulnerability was also observed in 2 other Gefitinib sensitive and resistant pairs of HNSCC cell lines. Conclusions: In EGFR T790M negative cohorts, a possible mechanism of resistance to EGFR TKIs would involve the downregulation of EGFR. While this could lead to resistance to all 3 generations of EGFR TKIs, resistant cells developed sensitivity to Aurora kinase inhibitors instead. Comparison of the gene expression profiles of these cell lines with similar therapeutic vulnerabilities could potentially identify biomarkers of Gefitinib resistance in HNSCC. Legal entity responsible for the study: NA Funding: The Agency for Science, Technology and Research (A*STAR) of Singapore Disclosure: All authors have declared no conflicts of interest.