Abstract 2109: Clonal heterogeneity of EGFR TKI-resistant NSCLC revealed through single-cell dilutional cloning and high-throughput functional screens
Authors: Lau Dawn Pingxi , Rikka Shivaji , Leong Hui Sun , Tan Gek San , Christodoulou Eleni G , Krisna Sai Sakktee , Toh Shen Yon , Kwang Xue Lin , Chong Fui Teen , Liew Audrey Ann , Lim Tony Kiat Hon , Ramanuj DasGupta , Iyer N Gopalakrishna , Tan Daniel Shao-Weng 
Published: July 2016

Abstract
While EGFR tyrosine kinase inhibitors (TKIs) can elicit significant tumor shrinkage in the clinic, drug resistance inevitably ensues, likely due to a persistent subpopulation. T790M mutation is a common resistance mechanism found in 60% of tumors progressing on TKI. The aim of this study is to uncover novel mechanisms of gefitnib-resistant (GR) NSCLC by selecting TKI naïve PC9 single cell clones (SCC) differing in stemness and vulnerabilities to gefitinib (G), and inducing resistance through sublethal drug exposures. A high throughput drug screen was performed in 4 SCC to discover alternate therapies to target T790M negative GR clones.
231 SCC of PC9 were isolated and confirmed to be T790M negative (0.3% sensitivity) using Agena SABER assay. All clones were characterized by their vulnerability to G (viable after 48 hour exposure to 0.1uM) and ALDH1 activity using the ALDEFLUOR assay and flow cytometry. Four “diverse” clones (stratified according to ALDH1 and sensitivity to TKI) were selected to generate GR cell lines. Agglomerative hierarchical clustering of differentially expressed genes revealed that the clone exhibiting extreme low survival and ALDH1 signature clusters significantly apart from the rest. Subsequent iterations of this experiment (in total 5 per clone) revealed unique (and mostly stochastic) patterns of acquiring T790M across all clones.
GR clones were then subjected to mutational analysis using the Ion AmpliSeq™ Colon and Lung Cancer Panel v1, and showed that 11/20 (55%) PC9 SCC GR lines have acquired the T790M mutation. T790M positive (AF range: 2.4 - 41.0%) GR lines were sensitive to Afatinib and WZ4002, a 2nd and 3rd generation EGFR TKI, with EGFR1086 phosphorylation being completely absent in all non-responding lines. T790M negative GR lines further acquired NRAS Q61K, G12D mutation and KRAS Q22K mutation. A high throughput drug screen with kinase inhibitor library from Selleck Chemicals (n = 273 kinases, n = 349 anticancer library) confirmed the selectivity of MEK inhibitors, as well as identified a subset of T790M-ve lines that are sensitive to Aurora kinase inhibitors.
Our study highlights the potential for pEGFR to complement T790M status as a selection biomarker, particularly where low tumor cell concentration may yield false negative results. Taken together, we have uncovered potential therapeutically tractable subsets of T790M negative EGFR TKI resistant cell lines. pEGFR may identify a subset of T790M negative tumors that remain driven by and may be an alternate selection biomarker to 2nd/3rd gen TKI. Combinational approaches with MEK and aurora kinase inhibitors could be further explored in T790M negative/ pEGFR negative tumors.
Citation Format: Dawn Pingxi Lau, Shivaji Rikka, Hui Sun Leong, Gek San Tan, Eleni G Christodoulou, Sai Sakktee Krisna, Shen Yon Toh, Xue Lin Kwang, Fui Teen Chong, Audrey Ann Liew, Tony Kiat Hon Lim, DasGupta Ramanuj, N Gopalakrishna Iyer, Daniel Shao-Weng Tan. Clonal heterogeneity of EGFR TKI-resistant NSCLC revealed through single-cell dilutional cloning and high-throughput functional screens. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2109.