Abstract 768: A kinome-wide siRNA screen identifies modifiers of sensitivity to the EGFR T790M-targeted tyrosine kinase inhibitor (TKI), AZD9291, in EGFR mutant lung adenocarcinoma
Authors: Ichihara Eiki , Bauer Joshua A. , Lu Pengcheng , Ye Fei , Cross Darren , Pao William , Lovly Christine M. 
Published: August 2015

New epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) that are targeted against EGFR T790M, such as AZD9291, are among the most promising agents for the treatment of EGFR mutant lung adenocarcinomas, as they can overcome a key mechanism of acquired resistance to first or second generation EGFR-TKIs. Emerging clinical data indicate that these drugs achieve partial responses in ∼60% of patients and demonstrate variable duration of benefit (Yang J et al. Ann Oncol 2014;25 (suppl 4): iv149). However, most responding patients are expected to develop eventually progressive disease. Mechanisms of resistance for this class of mutant specific EGFR TKIs are only beginning to be elucidated.

To identify modifiers of sensitivity to AZD9291 in EGFR mutant lung adenocarcinoma, we performed a kinome-wide short-interfering (si) RNA screen using clonal PC-9/BRc1 (EGFR exon19 deletion/T790M) cells and siRNA triplicates directed against 714 phylogenetically-related kinases (GE Dharmacon). The cells were derived from PC-9 cells chronically exposed to afatinib (Chmielecki J et al. Sci Transl Med. 2011;3:90ra59). The screen was performed in the absence and presence of AZD9291 (5 nM). Among the top 20 hits, siRNAs against the serine threonine kinases, MAPK1 (ERK2) and BRAF, sensitized cells to AZD9291. Both genes encode products involved in the RAS-MAP kinase signaling pathway. Confirming the significance of these hits, combinations of AZD9291 and the MEK inhibitor, selumetinib, were more effective at inhibiting cell growth than AZD9291 alone. Consistent with these findings, we found that ERK1/2 is readily dephosphorylated upon exposure to AZD929, however, ERK1/2 becomes re-phosphorylated after continuous exposure to AZD9291 for 96 hours. These data suggest that reactivation of ERK signaling may provide an early escape mechanism mediating drug resistance. ERK reactivation after AZD9291 treatment and stronger growth inhibition by AZD9291 + selumetinib combination therapy were also seen in multiple other EGFR mutant lines. The SRC kinase is known to activate RAS-MAP kinase pathway signaling, and pharmacologic or genetic ablation of SRC partially inhibited ERK reactivation, suggesting that SRC is involved in this escape mechanism.

In conclusion, through a kinome wide siRNA screen, we identified that gene products in the MAP kinase signaling pathway modify sensitivity to AZD9291. Such sensitivity may be associated with ERK re-phosphorylation within 96h of drug treatment. Collectively, these data suggest rational drug combinations that could be used to forestall resistance to AZD9291. Additional hits from the screen are currently under investigation.

This study is supported by AstraZeneca Oncology Innovative Medicines, National Institutes of Health (NIH) NCI grants R01-CA121210, P01-CA129243, U54-CA143798, and the Uehara Memorial Foundation.

Citation Format: Eiki Ichihara, Joshua A. Bauer, Pengcheng Lu, Fei Ye, Darren Cross, William Pao, Christine M. Lovly. A kinome-wide siRNA screen identifies modifiers of sensitivity to the EGFR T790M-targeted tyrosine kinase inhibitor (TKI), AZD9291, in EGFR mutant lung adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 768. doi:10.1158/1538-7445.AM2015-768