Cooperative DNA binding mediated by KicGAS/ORF52 oligomerization allows inhibition of DNA-induced phase separation and activation of cGAS.

Cyclic GMP-AMP synthase (cGAS) is a key DNA sensor that detects aberrant cytosolic DNA arising from pathogen invasions or genotoxic stresses. Upon binding to DNA, cGAS is activated and catalyzes the synthesis of cyclic GMP-AMP (cGAMP), which induces potent antimicrobial and antitumor responses. Kaposi sarcoma-associated herpesvirus (KSHV) is a human DNA tumor virus that causes Kaposi sarcoma and several other malignancies. We previously reported that KSHV inhibitor of cGAS (KicGAS) encoded by ORF52, inhibits cGAS enzymatic activity, but the underlying mechanisms remained unclear. To define the inhibitory mechanisms, here we performed in-depth biochemical and functional characterizations of KicGAS, and mapped its functional domains. We found KicGAS self-oligomerizes and binds to double stranded DNA cooperatively. This self-oligomerization is essential for its DNA binding and cGAS inhibition. Interestingly, KicGAS forms liquid droplets upon binding to DNA, which requires collective multivalent interactions with DNA mediated by both structured and disordered domains coordinated through the self-oligomerization of KicGAS. We also observed that KicGAS inhibits the DNA-induced phase separation and activation of cGAS. Our findings reveal a novel mechanism by which DNA viruses target the host protein phase separation for suppression of the host sensing of viral nucleic acids.

cGAS restricts colon cancer development by protecting intestinal barrier integrity.

The DNA-sensing enzyme cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) regulates inflammation and immune defense against pathogens and malignant cells. Although cGAS has been shown to exert antitumor effects in several mouse models harboring transplanted tumor cell lines, its role in tumors arising from endogenous tissues remains unknown. Here, we show that deletion of cGAS in mice exacerbated chemical-induced colitis and colitis-associated colon cancer (CAC). Interestingly, mice lacking cGAS were more susceptible to CAC than those lacking stimulator of interferon genes (STING) or type I interferon receptor under the same conditions. cGAS but not STING is highly expressed in intestinal stem cells. cGAS deficiency led to intestinal stem cell loss and compromised intestinal barrier integrity upon dextran sodium sulfate-induced acute injury. Loss of cGAS exacerbated inflammation, led to activation of STAT3, and accelerated proliferation of intestinal epithelial cells during CAC development. Mice lacking cGAS also accumulated myeloid-derived suppressive cells within the tumor, displayed enhanced Th17 differentiation, but reduced interleukin (IL)-10 production. These results indicate that cGAS plays an important role in controlling CAC development by defending the integrity of the intestinal mucosa.

Phosphorylation and chromatin tethering prevent cGAS activation during mitosis.

The cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) detects microbial and self-DNA in the cytosol to activate immune and inflammatory programs. cGAS also associates with chromatin, especially after nuclear envelope breakdown when cells enter mitosis. How cGAS is regulated during cell cycle transition is not clear. Here, we found direct biochemical evidence that cGAS activity was selectively suppressed during mitosis in human cell lines and uncovered two parallel mechanisms underlying this suppression. First, cGAS was hyperphosphorylated at the N terminus by mitotic kinases, including Aurora kinase B. The N terminus of cGAS was critical for sensing nuclear chromatin but not mitochondrial DNA. Chromatin sensing was blocked by hyperphosphorylation. Second, oligomerization of chromatin-bound cGAS, which is required for its activation, was prevented. Together, these mechanisms ensure that cGAS is inactive when associated with chromatin during mitosis, which may help to prevent autoimmune reaction.

DNA-induced liquid phase condensation of cGAS activates innate immune signaling.

The binding of DNA to cyclic GMP-AMP synthase (cGAS) leads to the production of the secondary messenger cyclic GMP-AMP (cGAMP), which activates innate immune responses. We have shown that DNA binding to cGAS robustly induced the formation of liquidlike droplets in which cGAS was activated. The disordered and positively charged cGAS N terminus enhanced cGAS-DNA phase separation by increasing the valencies of DNA binding. Long DNA was more efficient in promoting cGAS liquid phase separation and cGAS enzyme activity than short DNA. Moreover, free zinc ions enhanced cGAS enzyme activity both in vitro and in cells by promoting cGAS-DNA phase separation. These results demonstrated that the DNA-induced phase transition of cGAS promotes cGAMP production and innate immune signaling.

A STING-activating nanovaccine for cancer immunotherapy.

The generation of tumour-specific T cells is critically important for cancer immunotherapy. A major challenge in achieving a robust T-cell response is the spatiotemporal orchestration of antigen cross-presentation in antigen-presenting cells with innate stimulation. Here, we report a minimalist nanovaccine, comprising a simple physical mixture of an antigen and a synthetic polymeric nanoparticle, PC7A NP, which generates a strong cytotoxic T-cell response with low systemic cytokine expression. Mechanistically, the PC7A NP achieves efficient cytosolic delivery of tumour antigens to antigen-presenting cells in draining lymph nodes, leading to increased surface presentation while simultaneously activating type I interferon-stimulated genes. This effect is dependent on stimulator of interferon genes (STING), but not the Toll-like receptor or the mitochondrial antiviral-signalling protein (MAVS) pathway. The nanovaccine led to potent tumour growth inhibition in melanoma, colon cancer and human papilloma virus-E6/E7 tumour models. The combination of the PC7A nanovaccine and an anti-PD-1 antibody showed great synergy, with 100% survival over 60 days in a TC-1 tumour model. Rechallenging of these tumour-free animals with TC-1 cells led to complete inhibition of tumour growth, suggesting the generation of long-term antitumour memory. The STING-activating nanovaccine offers a simple, safe and robust strategy in boosting anti-tumour immunity for cancer immunotherapy.