DNA damage repair kinase DNA-PK and cGAS synergize to induce cancer-related inflammation in glioblastoma.

Cytosolic DNA promotes inflammatory responses upon detection by the cyclic GMP-AMP (cGAMP) synthase (cGAS). It has been suggested that cGAS downregulation is an immune escape strategy harnessed by tumor cells. Here, we used glioblastoma cells that show undetectable cGAS levels to address if alternative DNA detection pathways can promote pro-inflammatory signaling. We show that the DNA-PK DNA repair complex (i) drives cGAS-independent IRF3-mediated type I Interferon responses and (ii) that its catalytic activity is required for cGAS-dependent cGAMP production and optimal downstream signaling. We further show that the cooperation between DNA-PK and cGAS favors the expression of chemokines that promote macrophage recruitment in the tumor microenvironment in a glioblastoma model, a process that impairs early tumorigenesis but correlates with poor outcome in glioblastoma patients. Thus, our study supports that cGAS-dependent signaling is acquired during tumorigenesis and that cGAS and DNA-PK activities should be analyzed concertedly to predict the impact of strategies aiming to boost tumor immunogenicity.

Harnessing the cooperation between DNA-PK and cGAS in cancer therapies: The cooperation between DNA-PK and cGAS shapes tumour immunogenicity.

The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is central for the initiation of anti-tumoural immune responses. Enormous effort has been made to optimise the design and administration of STING agonists to stimulate tumour immunogenicity. However, in certain contexts the cGAS-STING axis fuels tumourigenesis. Here, we review recent findings on the regulation of cGAS expression and activity. We particularly focus our attention on the DNA-dependent protein kinase (DNA-PK) complex, that recently emerged as an activator of inflammatory responses in tumour cells. We propose that stratification analyses on cGAS and DNA-PK expression/activation status should be carried out to predict treatment efficacy. We herein also provide insights into non-canonical functions borne by cGAS and cGAMP, highlighting how they may influence tumourigenesis. All these parameters should be taken into consideration concertedly to choose strategies aiming to effectively boost tumour immunogenicity.

The unexpected role of the STING protein in lipid metabolism.

Detection of cytosolic pathological nucleic acids is a key step for the initiation of innate immune responses. In the past decade, the stimulator of interferon genes (STING) adaptor protein has emerged as a central platform enabling the activation of inflammatory responses in the presence of cytosolic DNAs. This has prompted a plethora of approaches aiming at modulating STING activation in order to boost or inhibit inflammatory responses. However, recent work has revealed that STING is also a direct regulator of metabolic homeostasis. In particular, STING regulates lipid metabolism directly, a function that is conserved throughout evolution. This indicates that STING targeting strategies must take into consideration potential metabolic side effects that may alter disease course, but also suggests that targeting STING may open the route to novel treatments for metabolic disorders. Here we discuss recent work describing the metabolic function of STING and the implications of these findings.

La détection des acides nucléiques pathologiques cytosoliques est une étape clé pour le déclenchement des réponses immunitaires innées. Au cours de la dernière décennie, la protéine adaptatrice STING (stimulator of interferon genes) est apparue comme une plateforme centrale permettant l'activation des réponses inflammatoires en présence d'ADN cytosolique. Cela a donné lieu à une multitude d'approches visant à moduler l'activation de STING afin de stimuler ou d'inhiber les réponses inflammatoires. Cependant, des travaux récents ont révélé que STING est également un régulateur direct de l'homéostasie métabolique. En particulier, STING régule directement le métabolisme des lipides, une fonction qui est conservée au cours de l'évolution. Cela indique que les stratégies de ciblage de STING doivent prendre en compte les effets secondaires métaboliques potentiels qui peuvent modifier l'évolution de la maladie, mais suggère également la possibilité que le ciblage de STING puisse ouvrir la voie à de nouvelles façons de traiter les pathologies présentant une composante métabolique. Nous discutons ici les travaux récents décrivant la fonction métabolique de STING et les implications de ces résultats.

Multiplexed targeted analysis of polyunsaturated fatty acids and oxylipins using liquid chromatography-tandem mass spectrometry.

Polyunsaturated fatty acids (PUFAs) and their oxidized products (oxylipins) are important mediators in intra- and extra-cellular signaling. We describe here the simultaneous quantification of 163 PUFAs and oxylipins using liquid chromatography-mass spectrometry (LC-MS). The protocol details steps for PUFA purification from various biological materials, the conditions for LC-MS analysis, as well as quantitative approaches for data evaluation. We provide an example of PUFA quantification in animal tissue along with the bioinformatic protocol, enabling efficient inter-sample comparison and statistical analysis. For complete details on the use and execution of this protocol, please refer to Vila et al.,1 Costanza et al.,2 Blomme et al.,3 and Blomme et al.4.

Dietary restriction induces a sexually dimorphic type I interferon response in mice with gene-environment interactions.

Intermittent fasting (IF) is an established intervention to treat the growing obesity epidemic. However, the interaction between dietary interventions and sex remains a significant knowledge gap. In this study, we use unbiased proteome analysis to identify diet-sex interactions. We report sexual dimorphism in response to intermittent fasting within lipid and cholesterol metabolism and, unexpectedly, in type I interferon signaling, which was strongly induced in females. We verify that secretion of type I interferon is required for the IF response in females. Gonadectomy differentially alters the every-other-day fasting (EODF) response and demonstrates that sex hormone signaling can either suppress or enhance the interferon response to IF. IF fails to potentiate a stronger innate immune response when IF-treated animals were challenged with a viral mimetic. Lastly, the IF response changes with genotype and environment. These data reveal an interesting interaction between diet, sex, and the innate immune system.

Protocol to induce and assess cGAS-STING pathway activation in vitro.

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway plays a pivotal role in several cellular processes including pathogen recognition and inflammatory responses. We describe a protocol to activate the cGAS-STING pathway in murine cells using nucleic acids transfection. We describe how to prepare the nucleic acid probes and validate activation of the pathway by western blot and gene expression analysis. The protocol can be applied to investigate cGAS-STING signaling in both murine and human cell lines. For complete details on the use and execution of this protocol, please refer to Vila et al. (2022).

Activation of STING in the pancreatic tumor microenvironment: A novel therapeutic opportunity.

Pancreatic ductal adenocarcinoma (PDAC) is a cancer of poor prognosis that presents with a dense desmoplastic stroma that contributes to therapeutic failure. PDAC patients are mostly unresponsive to immunotherapy. However, hopes to elicit response to immunotherapy have emerged with novel strategies targeting the Stimulator of Interferon Genes (STING) protein, which is a major regulator of tumor-associated inflammation. Combination of STING agonists with conventional immunotherapy approaches has proven to potentiate therapeutic benefits in several cancers. However, recent data underscore that the output of STING activation varies depending on the cellular and tissue context. This suggests that tumor heterogeneity, and in particular the heterogeneity of the tumor microenvironment (TME), is a key factor determining whether STING activation would bear benefits for patients. In this review, we discuss the potential benefits of STING activation in PDAC. To this aim, we describe the major components of the PDAC TME, and the expected consequences of STING activation.

Alternative pathways driven by STING: From innate immunity to lipid metabolism.

The Stimulator of Interferon Genes (STING) is a major adaptor protein that is central to the initiation of type I interferon responses and proinflammatory signalling. STING-dependent signalling is triggered by the presence of cytosolic nucleic acids that are generated following pathogen infection or cellular stress. Beyond this central role in controlling immune responses through the production of cytokines and chemokines, recent reports have uncovered inflammation-independent STING functions. Amongst these, a rapidly growing body of evidence demonstrates a key role of STING in controlling metabolic pathways at several levels. Since immunity and metabolic homeostasis are tightly interconnected, these findings deepen our understanding of the involvement of STING in human pathologies. Here, we discuss these findings and reflect on their impact on our current understanding of how nucleic acid immunity controls homeostasis and promotes pathological outcomes.

STING orchestrates the crosstalk between polyunsaturated fatty acid metabolism and inflammatory responses.

Concerted alteration of immune and metabolic homeostasis underlies several inflammation-related pathologies, ranging from metabolic syndrome to infectious diseases. Here, we explored the coordination of nucleic acid-dependent inflammatory responses and metabolic homeostasis. We reveal that the STING (stimulator of interferon genes) protein regulates metabolic homeostasis through inhibition of the fatty acid desaturase 2 (FADS2) rate-limiting enzyme in polyunsaturated fatty acid (PUFA) desaturation. STING ablation and agonist-mediated degradation increased FADS2-associated desaturase activity and led to accumulation of PUFA derivatives that drive thermogenesis. STING agonists directly activated FADS2-dependent desaturation, promoting metabolic alterations. PUFAs in turn inhibited STING, thereby regulating antiviral responses and contributing to resolving STING-associated inflammation. Thus, we have unveiled a negative regulatory feedback loop between STING and FADS2 that fine-tunes inflammatory responses. Our results highlight the role of metabolic alterations in human pathologies associated with aberrant STING activation and STING-targeting therapies.

Nucleic Acid Immunity and DNA Damage Response: New Friends and Old Foes.

The maintenance of genomic stability in multicellular organisms relies on the DNA damage response (DDR). The DDR encompasses several interconnected pathways that cooperate to ensure the repair of genomic lesions. Besides their repair functions, several DDR proteins have emerged as involved in the onset of inflammatory responses. In particular, several actors of the DDR have been reported to elicit innate immune activation upon detection of cytosolic pathological nucleic acids. Conversely, pattern recognition receptors (PRRs), initially described as dedicated to the detection of cytosolic immune-stimulatory nucleic acids, have been found to regulate DDR. Thus, although initially described as operating in specific subcellular localizations, actors of the DDR and nucleic acid immune sensors may be involved in interconnected pathways, likely influencing the efficiency of one another. Within this mini review, we discuss evidences for the crosstalk between PRRs and actors of the DDR. For this purpose, we mainly focus on cyclic GMP-AMP (cGAMP) synthetase (cGAS) and Interferon Gamma Inducible Protein 16 (IFI16), as major PRRs involved in the detection of aberrant nucleic acid species, and components of the DNA-dependent protein kinase (DNA-PK) complex, involved in the repair of double strand breaks that were recently described to qualify as potential PRRs. Finally, we discuss how the crosstalk between DDR and nucleic acid-associated Interferon responses cooperate for the fine-tuning of innate immune activation, and therefore dictate pathological outcomes. Understanding the molecular determinants of such cooperation will be paramount to the design of future therapeutic approaches.