Abstract B002: A CRISPR/Cas9 screening strategy for identifying modifiers of the viral mimicry response

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Abstract Viral mimicry is the induction of a cellular antiviral response triggered by endogenous nucleic acids rather than exogenous viral infection. Such nucleic acids are sensed by cytosolic pattern recognition receptors and interpreted by the cell as an infection, leading to activation of an antiviral signaling cascade that results in interferon (IFN) signaling and subsequent upregulation of interferon-stimulated genes (ISGs). While downstream effects of ISGs are diverse and vary from direct induction of apoptosis to adaptive immune system engagement, the end result of viral mimicry is the culling of the ‘infected’ cancer cell. The viral mimicry response was first described as a mechanism of the FDA-approved DNA demethylating agents azacytidine (AZA) and decitabine (DAC). By removing transcriptionally repressive DNA methylation, these compounds allow normally-silenced repetitive elements of the genome to be expressed as double-stranded RNA (dsRNA) species that trigger viral mimicry. Since then, a body of literature has emerged implicating viral mimicry as a common mechanism underlying many different therapies and drug targets, including histone modifiers (e.g., LSD1, EZH2) as well as non-epigenetic targets such as cell cycle regulators (e.g., CDK4/6). The diversity of viral mimicry-inducing targets highlights the need for a systematic and discovery-based method to identify novel drug targets. Here, we accomplish this through the development of a viral mimicry cell reporter system composed of a green fluorescent protein (GFP) reporter coupled to the interferon-stimulated response element (ISRE) for CRISPR/Cas9-based pooled library screening. Using ISRE stimulation and subsequent GFP expression as a readout for viral mimicry activation, flow cytometry-based methods can be used to quantify the population of cells undergoing viral mimicry. Exogenous treatments of type I IFN or the synthetic dsRNA analogue polyinosinic-polycytidylic acid in the absence or presence of the JAK/STAT inhibitor ruxolitinib were used in conjunction with flow cytometry to validate the robustness and specificity of the reporter. To date, two preliminary screens in colorectal and lung cancer cell lines (LIM1215 and A549, respectively) have been performed using this system with a pooled single guide RNA library targeting epigenetic modifiers. These proof-of-principle experiments revealed two candidate hits – the histone methyltransferase SETDB1 and the lysine demethylase KDM5C. Follow-up validation experiments confirm GFP expression and viral mimicry activation in cells with single-gene knockouts for SETDB1 and KDM5C and demonstrate enhanced activation in combination with decitabine, as well as provide rationale for further study of these hits as therapeutic targets. Our work thus far demonstrates the feasibility of our approach and indicates the utility of our system as a novel tool for studying viral mimicry biology. Citation Format: Raymond Chen, Ilias Ettayebi, Daniel D. De Carvalho. A CRISPR/Cas9 screening strategy for identifying modifiers of the viral mimicry response. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B002.