Abstract

Introduction: Our recent genome-wide CRISPR knockout screen revealed that the Adenosine deaminase acting on RNA-1 (ADAR1) gene is essential for the survival of a majority of oral squamous cell carcinoma (OSCC) cell lines. Given that deleting ADAR1 caused severe lethality in OSCC, targeting ADAR1 could have therapeutic benefits. ADAR1 catalyzes Adenosine-to-Inosine RNA editing, and suppresses dsRNA sensing-triggered cell death. ADAR1 has two isoforms, the constitutively expressed P110 and the interferon-inducible P150. It is also an interferon-stimulated gene (ISG) that is highly expressed in cancers including OSCC. In this study, we aimed to determine the molecular mechanisms underlying ADAR1 dependency which may afford an opportunity to develop better treatment strategies for OSCC. Methods: ADAR1 dependency is validated by competitive co-culture assay, apoptosis and colony formation assay using single-guide RNA (sgRNA) knockout in OSCC cell lines (ORL-48, ORL-214, ORL-174, ORL-195, SCC9 and BICR10). Changes in protein expression upon gene(s) knockout are determined by western blotting. Results: We confirmed that ADAR1 depletion significantly increased apoptosis by flow cytometry and inhibited colony formation in selected cell lines. For cell lines that are ADAR1-less dependent (BICR10 and ORL-174), IFN-? treatment increased the expression of ADAR1 and other ISGs, sensitizing these cells to cell death. Notably, overexpression of ADAR1-P150 but not ADAR1-P110, significantly rescued cell lethality in ADAR1 depleted cells suggesting that the ADAR1-P150 is important in the survival of OSCC. We also showed that cell death is mediated by dsRNA sensors protein kinase R (PKR) and melanoma differentiation-associated protein 5 (MDA5) whereby knocking-out either one of these genes did not reverse cell lethality, but co-deleting both genes partially rescued cell death in ADAR1-dependent cell lines. Conclusion: The present findings revealed that the interferon-inducible P150 of ADAR1 is essential for OSCC survival. Activation of the dsRNA-sensing pathways such as PKR and MDA5 underlies this dependency. 

Keywords: ADAR1 dependency, dsRNA sensing, CRISPR knockout, Interferon-stimulated gene, OSCC