Xiaomin Niu¹, Fatao Liu², Yi Zhou², Daizhan Zhou², Ting Wang³, Ziming Li¹, Xiangyun Ye¹, Yongfeng Yu¹, Zhen Zhou¹, Meilin Liao¹, Yun Liu⁴, Zhiwei Chen¹, Shun Lu¹

 ¹Department of ShanghaiLung Cancer Center, Shanghai Chest Hospital, Shanghai

Jiao Tong University, 241 Huaihai West Road, Shanghai200030, PR China,

 ²Institute forNutritional Sciences, Shanghai Institutes for Biological Sciences,

Chinese Academy of Sciences, Shanghai, PR China, ³Bio-X Institutes, KeyLaboratory

for the Genetics of Developmental and NeuropsychiatricDisorders (Ministry of Education), Shanghai Jiao Tong University, Shanghai200030, PR China,

 ⁴Institutes of BiomedicalSciences, Fudan University,

Shanghai 200032, PR China

Objective:Lung cancer is the leading cause of cancerrelated death in the world. The last decade has witnessed the rapid developmentof epidermal growth factor receptor (EGFR) directed targeted therapies thathave significantly changed the treatment of non-small-cell lung cancer (NSCLC).The challenge of acquired resistance to EGFR tyrosine kinase inhibitors (TKIs)will be an issue with talking with activating EGFR mutations, which makes itcrucial to elucidate the mechanism of EGFR-TKI targeted drug resistance. Method:Methyl-sensitive cut counting sequencing (MSCC), one of the most commonlyused whole-genome DNA methylation sequencing technology, was applied toinvestigate the changes of paired tissue DNA methylation before and after TKI(erlotinib) treatment lasting two cycles with partial response (PR) for stageIIIa (N2) lung adenocarcinoma patients (screening subgroup, N=2) withactivating EGFR 19 deletion. Sequenom EpiTYPER assay method was furtheranalyzed to double confirm the changed methylated candidate genes in screeningsubgroup and another independent validation subgroup (N=10), through comparingthe methylated changes in paired tissues before and after TKI treatment.Western blotting, cell cycle and apoptosis analysis by the candidate gene(GABBR2) knockout and overexpression using siRNA technology were performed inthree lung adenocarcinoma cell lines, A549 (EGFR wild type), HCC4006 (EGFR 19deletion, DelL747-E749) and HCC827 (EGFR 19 deletion, DelE746-A750), toelucidate the mechanism of GABBR2 gene in the regulation of EGFR-TKI treatment.Result: Sixty aberrant methylated genes were first screened using MSCCmethod in the screening subgroup. Two methylated genes, CBFA2T3 and GABBR2,were clearly validated by the Sequenom EpiTYPER assay in the screening subgroupand another validation subgroup, subsequently. GABBR2 was significantly downregulated in HCC4006 and HCC827 cells but remained conservative in A549 cellsafter erlotinib treatment by Western blotting. The phenomenon was in line withthe obvious apoptosis of HCC4006 and HCC827 cell lines harboring EGFR 19mutations, but not in A549 cells with EGFR wild-type. GABBR2 was furtherinduced down regulation after erlotinib exposure through apoptosis methodsilenced by siRNA using RNAi technology. Meanwhile, GABBR2 gene wasdemonstrated up regulation rescued the apoptosis significantly, whenoverexpressing GABBR2 in HCC827 cell lines along with erlotinib treatment. Conclusion:Genome-wide screen of DNA methylation changes demonstrated that GABBR2 genemight be as a novel potential treatment target for stage IIIa (N2) EGFR 19deletion adenocarcinoma with erlotinib treatment. Our research provides a newtheoretical basis for the epigenetic study of EGFR mutated lung adenocarcinomatreatment.

Key Words: lung adenocarcinoma  epidermal growth factor receptor (EGFR)  tyrosine kinase inhibitor (TKI)  methyl-sensitive cut counting sequencing(MSCC)  methylation  GABB