ADAR1 masks the cancer immunotherapeutic promise of ZBP1-driven necroptosis

A disappointingly small proportion (10-30%) of patients with cancer show lasting responses to immune checkpoint blockade (ICB)-based monotherapies. The RNA-editing enzyme ADAR1 is an emerging determinant of resistance to ICB therapy, and prevents ICB responsiveness by repressing immunogenic double-stranded (ds)RNAs, such as those arising from the dysregulated expression of endogenous retroelements (EREs). These dsRNAs trigger an interferon (IFN)-dependent antitumor response by activating the A-form dsRNA (A-RNA) sensing proteins MDA-5, PKR, and OAS1. Here, we show that ADAR1 also prevents accrual of endogenous Z-form dsRNA elements (Z-RNAs) which were enriched in the 3’UTRs of IFN-stimulated mRNAs. Depleting ADAR1 resulted in Z-RNA accumulation and activation of the Z-RNA sensor ZBP1, culminating in RIPK3-mediated necroptosis. As no clinically viable ADAR1 inhibitors currently exist, we searched for a compound that can override the requirement for ADAR1 inhibition and directly activate ZBP1. We identified a small molecule, the curaxin CBL0137, which potently activates ZBP1 by triggering Z-DNA formation in cells. CBL0137 induced ZBP1-dependent necroptosis in cancer-associated fibroblasts and strongly reversed ICB unresponsiveness in mouse models of melanoma. Collectively, these results demonstrate that ADAR1 represses endogenous Z-RNAs and identifies ZBP1-mediated necroptosis as a new determinant of tumor immunogenicity masked by ADAR1. Therapeutic activation of ZBP1-induced necroptosis provides a readily-translatable avenue for rekindling immune responsiveness of ICB-resistant human cancers. Strand-specific total RNA-seq of IFNb-treated mouse embryonic fibroblasts (Zbp1-/-, Adar1-/- MEFs) at three time points: 0h(untreated), 24h, 48h. Each experiment includes four biological replicas.