The interferon inducible protein 16 (IFI16) is a prominent sensor of nuclear pathogenic DNA, initiating innate immune signaling and suppressing viral transcription. However, little is known about mechanisms that initiate IFI16 antiviral functions or its regulation within the host DNA-filled nucleus. Here, we provide in vitro and in vivo evidence to establish that IFI16 undergoes liquid-liquid phase separation (LLPS) nucleated by DNA. IFI16 binding to viral DNA initiates LLPS and induction of cytokines during herpes simplex virus type 1 (HSV-1) infection. Multiple phosphorylation sites within an intrinsically disordered region (IDR) function combinatorially to activate IFI16 LLPS, facilitating filamentation. Regulated by CDK2 and GSK3β, IDR phosphorylation provides a toggle between active and inactive IFI16 and the decoupling of IFI16-mediated cytokine expression from repression of viral transcription. These findings show how IFI16 switch-like phase transitions are achieved with temporal resolution for immune signaling and, more broadly, the multi-layered regulation of nuclear DNA sensors.
© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cytoplasmic DNA triggers a cGAS-mediated signaling cascade that promotes an innate immune response and is potentially actionable in cancer immunotherapy. Here we show that a cytoplasmic-localizing lupus anti-DNA autoantibody activates cGAS and facilitates an immune-mediated prolongation of survival in orthotopic models of glioblastoma (GBM). Mechanistically, cellular penetration and blood-brain barrier crossing by the anti-DNA autoantibody is linked to nucleoside transport. Pulldown, knockdown, signaling, and cytotoxicity assays demonstrate autoantibody association with and activation of cGAS. In orthotopic GBM models, the autoantibody localizes to brain tumor, increases tumor CD8+ T cell content, and prolongs survival in immunocompetent but not immunodeficient mice. This work introduces the new concept of a cGAS-activating anti-DNA autoantibody, which impacts theories on mechanisms of autoimmunity and has translational applications in cancer immunotherapy.

Long interspersed nuclear element-1 (L1)–mediated reverse transcription (RT) of Alu RNA into cytoplasmic Alu complementary DNA (cDNA) has been implicated in retinal pigmented epithelium (RPE) degeneration. The mechanism of Alu cDNA–induced cytotoxicity and its relevance to human disease are unknown. Here we report that Alu cDNA is highly enriched in the RPE of human eyes with geographic atrophy, an untreatable form of age-related macular degeneration. We demonstrate that the DNA sensor cGAS engages Alu cDNA to induce cytosolic mitochondrial DNA escape, which amplifies cGAS activation, triggering RPE degeneration via the inflammasome. The L1-extinct rice rat was resistant to Alu RNA–induced Alu cDNA synthesis and RPE degeneration, which were enabled upon L1-RT overexpression. Nucleoside RT inhibitors (NRTIs), which inhibit both L1-RT and inflammasome activity, and NRTI derivatives (Kamuvudines) that inhibit inflammasome, but not RT, both block Alu cDNA toxicity, identifying inflammasome activation as the terminal effector of RPE degeneration.

Oligonucleotide-based therapeutics have the capacity to engage with nucleic acid immune sensors to activate or block their response, but a detailed understanding of these immunomodulatory effects is currently lacking. We recently showed that 2'-O-methyl (2'OMe) gapmer antisense oligonucleotides (ASOs) exhibited sequence-dependent inhibition of sensing by the RNA sensor Toll-Like Receptor (TLR) 7. Here we discovered that 2'OMe ASOs can also display sequence-dependent inhibitory effects on two major sensors of DNA, namely cyclic GMP-AMP synthase (cGAS) and TLR9. Through a screen of 80 2'OMe ASOs and sequence mutants, we characterized key features within the 20-mer ASOs regulating cGAS and TLR9 inhibition, and identified a highly potent cGAS inhibitor. Importantly, we show that the features of ASOs inhibiting TLR9 differ from those inhibiting cGAS, with only a few sequences inhibiting both pathways. Together with our previous studies, our work reveals a complex pattern of immunomodulation where 95% of the ASOs tested inhibited at least one of TLR7, TLR9 or cGAS by ≥30%, which may confound interpretation of their in vivo functions. Our studies constitute the broadest analysis of the immunomodulatory effect of 2'OMe ASOs on nucleic acid sensing to date and will support refinement of their therapeutic development.
© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cyclic cGMP-AMP synthase (cGAS) is a pattern recognition cytosolic DNA sensor that is essential for cellular senescence. cGAS promotes inflammatory senescence-associated secretory phenotype (SASP) through recognizing cytoplasmic chromatin during senescence. cGAS-mediated inflammation is essential for the antitumor effects of immune checkpoint blockade. However, the mechanism by which cGAS recognizes cytoplasmic chromatin is unknown. Here we show that topoisomerase 1-DNA covalent cleavage complex (TOP1cc) is both necessary and sufficient for cGAS-mediated cytoplasmic chromatin recognition and SASP during senescence. TOP1cc localizes to cytoplasmic chromatin and TOP1 interacts with cGAS to enhance the binding of cGAS to DNA. Retention of TOP1cc to cytoplasmic chromatin depends on its stabilization by the chromatin architecture protein HMGB2. Functionally, the HMGB2-TOP1cc-cGAS axis determines the response of orthotopically transplanted ex vivo therapy-induced senescent cells to immune checkpoint blockade in vivo. Together, these findings establish a HMGB2-TOP1cc-cGAS axis that enables cytoplasmic chromatin recognition and response to immune checkpoint blockade.