The Mechanisms of Cytokine Dependent IFN-Production and Grown Inhibition in ADAR1 Loss

Abstract

A previously published, pooled in vivo loss-of-function CRISPR genetic screen in the B16 mouse melanoma model revealed Adenosine deaminase acting on RNA (ADAR1) as a potential immunotherapy target1. Our lab subsequently showed that the loss of ADAR1 in transplanted B16 tumor cells led to a strong immune-mediated, anti-tumor effect and an inflamed tumor microenvironment with increased CD8+ T-cell infiltration. This antitumor effect depended on two Interferon(IFN)-primed in vitro phenotypes of ADAR1-deficient B16 cells: MDA5-dependent secretion of IFN-β and PKR-dependent growth arrest. This work focuses on elucidating the mechanisms of both in vitro phenotypes. Given ADAR1’s role in editing endogenous dsRNA to prevent sensing by cellular RNA sensors, we used Cross-linking Immunoprecipitation followed by high throughput sequencing (CLIP-Seq) to identify which endogenous ligands were sensed by MDA5 in the absence of ADAR1 to contribute to the IFN production phenotype. We found that the MDA5-bound peaks from ADAR1-null samples were enriched for Short interspersed nuclear elements (SINEs), which are known ADAR1 substrates, compared to peaks from wild-type controls. We also interrogated the PKR-dependent growth arrest phenotype to determine whether the translational shutdown that is frequently associated with PKR was also accompanied by cell death in this tumor system. We found that ADAR1-deficient cells were dying by apoptosis at significantly higher levels when stimulated with Type 1 or Type 2 IFN. Taken together, these results suggest endogenous dsRNA that is enriched for SINEs, when unedited in ADAR1 null tumors, can get sensed by MDA5, subsequently leading to a positive feedback loop of IFN production, PKR hyperactivation and tumor-cell intrinsic death.