Abstract 3152: Acquired resistance to the third-generation EGFR inhibitor ASP8273 is associated with MET or NRAS gene amplifications in preclinical models
Authors: Ninomiya Kiichiro , Ohashi Kadoaki , Tomida Shuta , Kayatani Hiroe , Tamura Tomoki , Higo Hisao , Makimoto Go , Ninomiya Takashi , Kubo Toshio , Ichihara Eiki , Sato Akiko , Hotta Katsuyuki , Tabata Masahiro , Kiura Katsuyuki 
Published: July 2017

Abstract
Background. Third-generation EGFR tyrosine kinase inhibitors (TKIs) such as osimertinib and ASP8273 demonstrated biologic activity in patients with non-small cell lung cancer (NSCLC) harboring the EGFR T790M mutation; however, acquired resistance inevitably develops and the mechanisms of have not been fully elucidated. This study evaluated potential mechanisms of resistance to ASP8273.
Materials and Methods. ASP8273 was kindly provided by Astellas Pharma Inc. PC-9 cells harboring EGFR 19del and RPC-9 cells harboring EGFR 19del +T790M were used as cell models to explore alternative molecular mechanisms of acquired resistance to ASP8273. Resistant cell lines (PC-9ASR and RPC-9ASR) were established from each cell line after continuous exposure to ASP8273 for 6 months.
Results. Other EGFR-TKIs, including osimertinib, afatinib, or gefitinib, did not inhibit cell growth and acquired EGFR mutations were not detected in either of ASP8273 resistant cell lines. The PC-9-based model was first analyzed. The 50% inhibitory concentration (IC50) of ASP8273 was 45-fold higher in PC-9ASR cells (1,216 nmol/L) than in the parental PC-9 cells (27 nmol/L). A receptor tyrosine kinase array showed that MET phosphorylation was increased in PC-9ASR cells relative to PC-9 cells. Single-cell cloning revealed that PC-9ASRc2 cells harbored MET gene amplification, but other clones (c1, c3, c4, and c5) did not; these results suggest that heterogeneous cells comprised the resistant cell lines. Consistently, the combination of EGFR inhibitors (ASP8273 or gefitinib) with MET inhibitors (crizotinib or SGX-523) inhibited cell proliferation in vitro. In vivo, concomitant dosing of ASP8273 with crizotinib consistently inhibited this resistant tumor xenograft model. The RPC-9-based model was then assessed. The IC50 of ASP8273 was 40-fold greater in RPC-9ASR cells (1,393 nmol/L) than in the parental RPC-9 cells (32 nmol/L). Next-generation sequencing showed significant NRAS gene amplification and no NRAS mutations in RPC-9ASR cells. EGFR 19del +T790M was maintained in the resistant cells. Western blot analysis and an active RAS pull-down kit confirmed that NRAS protein was significantly overexpressed and that NRAS-GTPase activity was much higher in RPC-9ASR cells than in the RPC-9 parental cells. As expected, inhibition of RAS signaling using a MEK inhibitor (selumetinib or trametinib) in addition to ASP8273 overcame the resistance in vitro. Interestingly, a combination of MEK inhibitors with another third-generation EGFR-TKI, osimertinib, had no effect on the RPC-9 resistant cells.
Conclusions. These studies suggest that a bypass signaling pathway, such as MET gene amplification, or activation of the RAS signaling pathway plays a role in ASP8273 resistance in lung cancer cells harboring EGFR mutations. (This work was supported by KAKEN 16K19454 and 15H04830.)
Citation Format: Kiichiro Ninomiya, Kadoaki Ohashi, Shuta Tomida, Hiroe Kayatani, Tomoki Tamura, Hisao Higo, Go Makimoto, Takashi Ninomiya, Toshio Kubo, Eiki Ichihara, Akiko Sato, Katsuyuki Hotta, Masahiro Tabata, Katsuyuki Kiura. Acquired resistance to the third-generation EGFR inhibitor ASP8273 is associated with MET or NRAS gene amplifications in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3152. doi:10.1158/1538-7445.AM2017-3152