The cGAS–STING signalling axis, comprising the synthase for the second messenger cyclic GMP–AMP (cGAS) and the cyclic GMP–AMP receptor stimulator of interferon genes (STING), detects pathogenic DNA to trigger an innate immune reaction involving a strong type I interferon response against microbial infections. Notably however, besides sensing microbial DNA, the DNA sensor cGAS can also be activated by endogenous DNA, including extranuclear chromatin resulting from genotoxic stress and DNA released from mitochondria, placing cGAS–STING as an important axis in autoimmunity, sterile inflammatory responses and cellular senescence. Initial models assumed that co-localization of cGAS and DNA in the cytosol defines the specificity of the pathway for non-self, but recent work revealed that cGAS is also present in the nucleus and at the plasma membrane, and such subcellular compartmentalization was linked to signalling specificity of cGAS. Further confounding the simple view of cGAS–STING signalling as a response mechanism to infectious agents, both cGAS and STING were shown to have additional functions, independent of interferon response. These involve non-catalytic roles of cGAS in regulating DNA repair and signalling via STING to NF-κB and MAPK as well as STING-mediated induction of autophagy and lysosome-dependent cell death. We have also learnt that cGAS dimers can multimerize and undergo liquid–liquid phase separation to form biomolecular condensates that could importantly regulate cGAS activation. Here, we review the molecular mechanisms and cellular functions underlying cGAS–STING activation and signalling, particularly highlighting the newly emerging diversity of this signalling pathway and discussing how the specificity towards normal, damage-induced and infection-associated DNA could be achieved. The cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway senses DNA in the cytoplasm, whether of pathogenic or endogenous (chromatin or mitochondrial) origin, and triggers the interferon response. The mechanisms of DNA recognition and cGAS–STING activation and signalling are now coming into focus, providing insights into the cellular functions of this pathway, including interferon-independent roles.

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Molecular mechanisms and cellular functions of cGAS–STING signalling

Currently, pyroptosis has received more and more attention because of its association with innate immunity and disease. The research scope of pyroptosis has expanded with the discovery of the gasdermin family. A great deal of evidence shows that pyroptosis can affect the development of tumors. The relationship between pyroptosis and tumors is diverse in different tissues and genetic backgrounds. In this review, we provide basic knowledge of pyroptosis, explain the relationship between pyroptosis and tumors, and focus on the significance of pyroptosis in tumor treatment. In addition, we further summarize the possibility of pyroptosis as a potential tumor treatment strategy and describe the side effects of radiotherapy and chemotherapy caused by pyroptosis. In brief, pyroptosis is a double-edged sword for tumors. The rational use of this dual effect will help us further explore the formation and development of tumors, and provide ideas for patients to develop new drugs based on pyroptosis.

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Pyroptosis: mechanisms and diseases.

Chemotherapy, radiation therapy, as well as targeted anticancer agents can induce clinically relevant tumor-targeting immune responses, which critically rely on the antigenicity of malignant cells and their capacity to generate adjuvant signals. In particular, immunogenic cell death (ICD) is accompanied by the exposure and release of numerous damage-associated molecular patterns (DAMPs), which altogether confer a robust adjuvanticity to dying cancer cells, as they favor the recruitment and activation of antigen-presenting cells. ICD-associated DAMPs include surface-exposed calreticulin (CALR) as well as secreted ATP, annexin A1 (ANXA1), type I interferon, and high-mobility group box 1 (HMGB1). Additional hallmarks of ICD encompass the phosphorylation of eukaryotic translation initiation factor 2 subunit-α (EIF2S1, better known as eIF2α), the activation of autophagy, and a global arrest in transcription and translation. Here, we outline methodological approaches for measuring ICD markers in vitro and ex vivo for the discovery of next-generation antineoplastic agents, the development of personalized anticancer regimens, and the identification of optimal therapeutic combinations for the clinical management of cancer.

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Detection of immunogenic cell death and its relevance for cancer therapy

The development of immune checkpoint inhibitors (ICIs) is revolutionizing the way we think about cancer treatment. Even so, for most types of cancer, only a minority of patients currently benefit from ICI therapies. Intrinsic and acquired resistance to ICIs has focused research towards new combination therapy approaches that seek to increase response rates, the depth of remission and the durability of benefit. In this Review, we describe how radiotherapy, through its immunomodulating effects, represents a promising combination partner with ICIs. We describe how recent research on DNA damage response (DDR) inhibitors in combination with radiotherapy may be used to augment this approach. Radiotherapy can kill cancer cells while simultaneously triggering the release of pro-inflammatory mediators and increasing tumour-infiltrating immune cells - phenomena often described colloquially as turning immunologically 'cold' tumours 'hot'. Here, we focus on new developments illustrating the key role of tumour cell-autonomous signalling after radiotherapy. Radiotherapy-induced tumour cell micronuclei activate cytosolic nucleic acid sensor pathways, such as cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING), and propagation of the resulting inflammatory signals remodels the immune contexture of the tumour microenvironment. In parallel, radiation can impact immunosurveillance by modulating neoantigen expression. Finally, we highlight how tumour cell-autonomous mechanisms might be exploited by combining DDR inhibitors, ICIs and radiotherapy.

Inflammatory microenvironment remodelling by tumour cells after radiotherapy.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a novel coronavirus that has caused a worldwide pandemic of the human respiratory illness COVID-19, resulting in a severe threat to public health and safety. Analysis of the genetic tree suggests that SARS-CoV-2 belongs to the same Betacoronavirus group as severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). Although the route for viral transmission remains a mystery, SARS-CoV-2 may have originated in an animal reservoir, likely that of bat. The clinical features of COVID-19, such as fever, cough, shortness of breath, and fatigue, are similar to those of many acute respiratory infections. There is currently no specific treatment for COVID-19, but antiviral therapy combined with supportive care is the main strategy. Here, we summarize recent progress in understanding the epidemiological, virological, and clinical characteristics of COVID-19 and discuss potential targets with existing drugs for the treatment of this emerging zoonotic disease.

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The hallmarks of COVID-19 disease.

The detection of pathogens through nucleic acid sensors is a defining principle of innate immunity. RNA-sensing and DNA-sensing receptors sample subcellular compartments for foreign nucleic acids and, upon recognition, trigger immune signalling pathways for host defence. Over the past decade, our understanding of how the recognition of nucleic acids is coupled to immune gene expression has advanced considerably, particularly for the DNA-sensing receptor cyclic GMP-AMP synthase (cGAS) and its downstream signalling effector stimulator of interferon genes (STING), as well as the molecular components and regulation of this pathway. Moreover, the ability of self-DNA to engage cGAS has emerged as an important mechanism fuelling the development of inflammation and implicating the cGAS-STING pathway in human inflammatory diseases and cancer. This detailed mechanistic and biological understanding is paving the way for the development and clinical application of pharmacological agonists and antagonists in the treatment of chronic inflammation and cancer.

DNA sensing by the cGAS–STING pathway in health and disease

Activated macrophages undergo a metabolic switch to aerobic glycolysis, accumulating Krebs' cycle intermediates that alter transcription of immune response genes. We extended these observations by defining fumarate as an inhibitor of pyroptotic cell death. We found that dimethyl fumarate (DMF) delivered to cells or endogenous fumarate reacts with gasdermin D (GSDMD) at critical cysteine residues to form S-(2-succinyl)-cysteine. GSDMD succination prevents its interaction with caspases, limiting its processing, oligomerization, and capacity to induce cell death. In mice, the administration of DMF protects against lipopolysaccharide shock and alleviates familial Mediterranean fever and experimental autoimmune encephalitis by targeting GSDMD. Collectively, these findings identify GSDMD as a target of fumarate and reveal a mechanism of action for fumarate-based therapeutics that include DMF, for the treatment of multiple sclerosis.

https://science.sciencemag.org/content/sci/early/2020/08/19/science.abb9818.full.pdf

Succination inactivates gasdermin D and blocks pyroptosis

The cGAS-STING pathway is the major cytosolic DNA sensing pathway to induce downstream innate immune responses against viral infection as well as tumorigenesis. Recently it has been demonstrated that cGAS-STING pathway takes central stage in the immune sensing of tumor. STING activation induces the production of type I interferons (IFNα and β) and pro-inflammatory cytokines that collectively stimulate tumor antigen cross presentation to CD8 T cells and primes the adaptive immune response against tumor. In order to evaluate the potential of STING agonism to activate the anti-tumor immune response, we developed GSK532, a novel cyclic dinucleotide (CDN). Unlike cGAMP, the endogenous CDN ligand for STING, GSK532 potently activates STING both in vitro and in vivo. In vitro functional assays demonstrated the ability of GSK532 to induce cytokine responses in a panel of human PBMC samples with various STING haplotypes. Furthermore, GSK532 was shown to activate STING orthologs from preclinical species including cyno, minipig, dog, rat and mice. GSK532 demonstrated high stability in human whole blood with minimal degradation observed over 52 hrs. When dosed intratumorally, GSK532 induced a strong anti-tumor effect in the CT26 murine syngeneic model in both the injected (primary) and uninjected (distal) tumors. The cured mice were resistant to rechallenge with same tumor cell line indicating the involvement of an adaptive immune response. Further evidence of the development of anti-tumor adaptive immune response comes from the observation that the efficacy of GSK532 was significantly attenuated when CD8+ T cells were depleted in the murine model. Consistent with the anti-tumor efficacy, pharmacokinetic analysis demonstrated high exposure and long retention of GSK532 in both injected and distal tumors and pharmacodynamic studies showed induction of IFNβ and several inflammatory cytokines (TNFα, IL-6, etc) in both tumors. Currently, GSK532 is being progressed towards clinical investigation. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. Citation Format: Jingsong Yang, Michael Adam, Jim Clemens, Katrina Creech, Jess Schneck, Kishore Pasikanti, Jean-Luc Tran, Devika Joglekar, Chris Hopson, Scott Pesiridis, Joshi Ramanjulu, Yiqian Lian, Jerry L. Adams, James Smothers, Axel Hoos. Preclinical characterization of GSK532, a novel STING agonist with potent anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5554.

Scott Pesiridis

Papers

DNA sensing by the cGAS–STING pathway in health and disease

Succination inactivates gasdermin D and blocks pyroptosis

Abstract 5554: Preclinical characterization of GSK532, a novel STING agonist with potent anti-tumor activity