Abstract 2562: Functional characterization of Polθ in esophageal squamous cell carcinoma

Abstract

Abstract Introduction: Wild-type (WT) Polθ, encoded by POLQ gene, is a specialized DNA polymerase which helps to protect cells against genomic instability by allowing DNA double-strand break (DSB) repairs through the alternative end-joining (AltEJ) pathway. In our previous targeted gene sequencing studies, we identified a high frequency of POLQ germline variants associated with esophageal squamous cell carcinoma (ESCC) in an endemic high-risk region of China. Here in this study, we aim to determine the functional impact of POLQ in ESCC, examine the synthetic lethality relationship of POLQ-mediated AltEJ DNA repair pathway and the canonical homologous recombination (HR) pathway, and eventually explore its potential roles as a biomarker or therapeutic target in ESCC. Methods: To determine the functional roles of Polθ, POLQ knock out (KO), FANCD2 KO and POLQ/FANCD2 double KO ESCC cell lines were generated using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique. MTT assay was used to assess cell viabilities with or without additional treatments that induced DNA damage or replication stress. DSB repair efficiency was examined by the H2AX foci recovery assay. The single cell gel electrophoresis/Comet assay was used to evaluate the genome stability. The expression level of POLQ was compared between ESCC patients’ samples and paired non-tumoral tissues using Q-PCR. Results: Among all ESCC cell lines we tested, 8 out of 13 (61.5%) exhibited higher levels of POLQ mRNA/protein than the normal immortalized esophageal epithelial cell line NE1. A 2-fold or more elevation of POLQ was found in 17 out of 28 Hong Kong ESCC patients’ tumor samples when compared with their paired non-tumoral tissues. Depletion of POLQ in high-POLQ expressing ESCC cell lines (KYSE180TS and SLMTorT3) only slightly diminished the cell viability. Double KO POLQ and FANCD2, one of the HR genes, significantly sabotaged cell proliferation, as compared with single KO POLQ or FANCD2, especially after treated with Cisplatin or Hydroxyurea. The percentage of H2AX foci positive cells increased considerably upon POLQ KO at 8 hours post 4Gy ionizing radiation, hinting the compromised DSB repair efficiency in POLQ depleted cells. The POLQ-deficient cells also showed a higher degree of genomic instability as suggested by the Comet assay. Conclusions: The results suggested that Polθ is overexpressed in ESCC tumors and cell lines. The Polθ mediated DSB repair pathway may work as a backup plan of HR to prevent the genomic instability in ESCC. Targeting Polθ might be a potential therapeutic approach for the better management of HR-deficient ESCC. Acknowledgments: Research Grants Council Collaborative Research Fund grant number 106150246 and Asian Cancer Research Fund to MLL. We thank Prof. Jean-Sébastien Hoffmann for providing the Polθ antibody. Citation Format: Jian Li, Josephine Ko, Wei Dai, Lvwen Ning, Hoi Yan Ng, Valen Zhuoyou Yu, Maria Lung. Functional characterization of Polθ in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2562.