IDENTIFICATION OF FACTORS INVOLVED IN DOUBLE STRANDED RNA-INDUCED CELL DEATH THROUGH GENOME-WIDE CRISPR SCREENS

Double-stranded RNAs (dsRNAs) are potent immunostimulatory nucleic acids of viral origin but also physiologically produced by mammalian cells. Increased levels of dsRNAs, due to viral infection or failure of endogenous repression, trigger an immune response that leads to cytokines production or cell death. Recent studies demonstrated that several cancer types already contain sufficient quantities of dsRNA to activate immune responses. Moreover, some clinical trials proved the efficacy of Polyinosinic-polycytidylic acid (pI:C), a synthetic analog of dsRNA, as adjuvant treatment in breast cancer patients, in terms of overall survival and longer relapse-free survival. The 2′,5′-oligoadenylate synthetase (OAS)-RNASEL system is widely thought to be the main responsible for dsRNA-induced cell death, through mechanisms not fully understood. To elucidate these mechanisms, we employed a candidate-based manner investigation and an unbiased approach. As death-inducing stimulus we used the combination of pI:C, and Interferon-α (IFN-α), which upregulates OAS enzymes thus maximizing the efficiency of dsRNA sensing in IFN-saturating condition. RNASEL-KO cells were, as expected, refractory to IFN-α+pI:C induced cell death; interestingly however, interfering with protein synthesis or RNA transcription with Cycloheximide (CHX) or Actinomycin D (ActD), caused RNASEL-KO cells to undergo apoptosis although CHX/ActD alone had no cytotoxic effect. This finding suggests the activation of RNAseL-independent systems different from those activated by canonical dsRNA receptors. In view of the partial knowledge in this field and the strong impact that clarification of dsRNA-dependent cell death mechanisms could have in development of new therapeutical strategies, we performed a Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening and integrative analysis to prioritize gene targets that favour/inhibit dsRNA-dependent cell death. The screen revealed that RNA surveillance, RNA Polymerase II Transcription Initiation, Mitochondrial translation and respiratory chain and DNA Repair genes have a protective role against dsRNA increase. Among multiple candidates with synergistic/inhibitory role with dsRNA, we validated the most translationally relevant BRCA1, commonly mutated in hereditary forms of breast, ovarian and several other cancers. Thus, our work shed light on potential new mechanisms involved in dsRNA dependent cell death, tracing a connection with DNA damage and BRCA1. We, therefore, will explore the interaction between dsRNA sensing and BRCA1 as a target for synthetic lethal combinations in BRCA1-deficient tumours.