By: Kajsa Fritzell, MBW, Stockholm University

Title: The ADAR editing enzymes as therapeutic targets

Abstract
Editing by deamination of adenosine to inosine (A-to-I) in double-stranded RNA is a common event in the human transcriptome. Altered levels of the essential ADAR1 activity are associated with disease. Children with mutations in the ADAR1 gene suffer from fatal Aicardi-Goutières syndrome (AGS) characterized by aberrant interferon expression. In contrast, ADAR1 overexpression is associated with increased malignancy in several cancers including breast cancer, lung cancer and liver cancer. ADAR1 silencing in breast cancer cell lines leads to a significant increase in apoptosis, suggesting that ADAR1 acts as an anti-apoptotic factor and promotes cancer progression. Yet, suitable high-throughput assays to monitor editing activity in human cells have limited research progress. Here we describe the development of a bioluminescent reporter system that facilitates the rapid and accurate determination of endogenous editing activity. The system is based on the highly sensitive and quantitative Nanoluciferase that is conditionally expressed upon reporter transcript editing. Stably introduced into cancer cell lines, we show that the system can measure elevated endogenous ADAR1 editing activity induced by externally provided interferon as well as knockdown of ADAR1 and ADAR2. In an optimized single-well setup we used the reporter in a stable HeLa cell line to screen a small molecule library of 33 000 compounds for potential inhibitors. This yielded a primary hit rate of 0.9% at 70% inhibition. Thus, we have generated a key tool for high-throughput identification of modifiers of A-to-I editing activity in cancer cells.