Xiao, Zhijie

Abstract Lung cancer has the highest mortality across all cancers in the world. Epidermal growth factor receptor (EGFR) is commonly mutated in lung adenocarcinomas from Asian non-smoking females. These tumors usually respond to EGFR tyrosine kinase inhibitors (EGFR-TKI), but the outcomes vary in different patients. Though EGFR T790M, amplification of MET and other mechanisms have been shown to confer EGFR-TKI resistance, the molecular mechanisms in around 30% of patients are still unknown. To uncover novel candidate pathways of EGFR-TKI resistance, we analyzed the whole exome mutation profiles of pre-treatment, EGFR T790M-free lung adenocarcinomas with typical activating mutations (L858R, exon 19 deletions). Amongst 19 non-responding (NR) tumors without significant shrinkage after gefitinib or erlotinib treatment, 67 cancer-related genes showed coding region non-synonymous single nucleotide variations (SNV) or insertion-deletions (indels) but they were not detected in 22 responding tumors. Gene set enrichment analysis (GSEA) showed 14 of the 67 genes were involved in the EGFR and WNT signaling network (false discovery rate q=1.14 e-8 for EGFR and q=4.3 e-9 for WNT), including well-known TKI resistance-related genes PIK3CA, PTEN and NF1. Notably, ARID1A, which encodes a subunit of the chromatin remodeling complex, showed nonsense mutations in 2 NR but not responding tumors, which were validated by Sanger sequencing. More than 40% of ARID1A mutations in lung adenocarcinomas recorded in COSMIC were nonsense mutations or frameshift indels. Loss of function mutations of ARID1A have also been reported in various cancers, together suggesting ARID1A might be a tumor suppressor but its involvement in EGFR mutant lung cancers is unknown. To investigate the potential role of ARID1A, its expression was first studied using Kaplan-Meier plotter. Low expression of ARID1A is associated with significantly better overall survival than high expression level in lung adenocarcinoma patients (p=8.5 e-7). To test whether loss of ARID1A could cause TKI resistance, ARID1A was knocked down by shRNA in HCC827 lung cancer cell line harboring EGFR exon19 deletion. MTT cell viability assay using gefitinib showed increase of IC50. Western blot assay further showed ARID1A knockdown increased phosphorylation of AKT at Ser473. Together, the findings suggest loss of ARID1A could lead to EGFR bypass and promote activation of the downstream PI3K/AKT pathway, leading to EGFR TKI resistance. Overall, our findings suggest EGFR-TKI resistance mutations could be detected before TKI drug selection. Resistant gene mutations present in pre-treatment tumors could help to predict the treatment response. More specifically, our study identified a promising gene list involved in EGFR-TKI resistance, and suggested ARID1A loss could be a novel EGFR-TKI resistance mechanism. Citation Format: Xuyuan Gao, Hang Xu, James CM Ho, Oscar SH Chan, Feng Xu, Junwen Wang, Victor HF Lee, Vicky PC Tin, Zhijie Xiao, Siqi Wang, Judy WP Yam, Maria P Wong. EGFR-mutant lung adenocarcinomas mutation profiles reveal ARID1A might be a novel tyrosine kinase resistance pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-196.

Abstract Lung cancer is the most lethal malignancy around the world. In Hong Kong, chronic tobacco use is prevalent in male but <20% of female patients smoke. Epidermal growth factor receptor (EGFR) mutations affect 30% and 67% of male and female lung adenocarcinomas, respectively. Treatment responses to tyrosine kinase inhibitors (TKI) are not uniform and all patients eventually develop drug resistance. Mutation screening of immediate pre-treatment tumors is the most ideal for assessing drug targets but in practice, excision specimens of primary or metastatic sites are more efficient in terms of tumor quantity, timeliness and patient acceptance. However, information on the applicability of this approach and correlation with the clinical outcome is limited. For this purpose, we compared the whole exome mutation profiles of 39 excised EGFR mutant cancers treated with first line TKI with the clinical response type, including 15 stage I/II and 24 stage III/IV tumors. They included 16 non-responders (NR) defined clinically as those with stable or progressive disease, and 23 responders (R) defined by partial or complete tumor shrinkage. Considering only the non-synonymous SNV and INDEL mutations of coding regions of known actionable targets and genes listed in both the CGC and COSMIC cancer databases, the tumors harbored 54 mutated genes including 26 recurrently mutated and 28 genes involving only 1 tumor. Excluding EGFR, TP53 was the most commonly mutated gene occurring in 25/39 (64.1%) cases. Most known resistant genes involved in the EGFR and bypass signaling network showed mutations only in the NR group including recurrent mutations of PTEN (3/16), PIK3CA (2/16) and NF1 (2/16), and single case mutations of AKT1, ALK, RAF1 and KDR. Two EGFR network candidates showed mutations in both the NR and R groups, including HGF (2/16 NR, 1/23 R) and ROS1 (1 case in either group). Four post-treatment (post-TKI) tumors of acquired resistance were also analyzed, all of which showed EGFR T790M while no pre-treatment NR or R tumor harbored this mutation. Notably, mutations in the β-catenin pathway were prominent in the NR and post-TKI tumors, including APC (2/16 NR), CTNNB1 (1/16 NR, 1/4 post-TKI) and c-MYC (1/16 NR, 1/4 post-TKI) while they were not detected in the R tumors. Also, nonsense mutations of ARID1A was observed in 2/16 NR but none of the R tumors. In summary, our findings revealed candidate TKI resistant mutations involving the EGFR and bypass signaling networks in pre-treatment excision specimens particularly in non-responding patients. The EGFR T790M was not detectable in pre-treatment samples but was prevalent in post-TKI treated cancers. While this study is limited by its small cohort size, the findings indicate deep sequencing analysis of pre-treatment excision specimens of EGFR-mutant lung adenocarcinomas is warranted for detection of resistant mutations and predicting treatment response. Note: This abstract was not presented at the meeting. Citation Format: Xu-yuan Gao, Hang Xu, James CM Ho, Oscar SH Chan, Feng Xu, Junwen Wang, Victor HF Lee, Vicky PC Tin, Zhijie Xiao, Siqi Wang, Judy WP Yam, Maria P. Wong. Whole-exome sequencing reveals tyrosine kinase-resistant mutations in pretreatment EGFR-mutant lung adenocarcinomas [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 LB-274. doi:10.1158/1538-7445.AM2017-LB-274

Tumor-initiating cells (TIC) are dynamic cancer cell subsets that display enhanced tumor functions and resilience to treatment but the mechanism of TIC induction or maintenance in lung cancer is not fully understood. In this study, we show the calcium pathway transcription factor NFATc2 is a novel regulator of lung TIC phenotypes, including tumorspheres, cell motility, tumorigenesis, as well as in vitro and in vivo responses to chemotherapy and targeted therapy. In human lung cancers, high NFATc2 expression predicted poor tumor differentiation, adverse recurrence-free and cancer-specific overall survivals. Mechanistic investigations identified NFATc2 response elements in the 3’ enhancer region of SOX2, and NFATc2/SOX2 coupling upregulates ALDH1A1 by binding to its 5’ enhancer. Through this axis, oxidative stress induced by cancer drug treatment is attenuated, leading to increased resistance in a mutation-independent manner. Targeting this axis provides a novel approach for the long-term treatment of lung cancer through TIC elimination.