cGAS Activation Research Articles

Tandem‐Controlled Dynamic DNA Assembly Enables Temporally‐Selective Orthogonal Regulation of cGAS–STING Stimulation

Despite advances in the controlled reconfiguration of DNA structures for biological applications, the dearth of strategies that allow for orthogonal regulation of immune pathways remains a challenge. Here, we report for the first time an endogenous and exogenous tandem‐regulated DNA assembly strategy that enables orthogonally controlled stimulation of the cyclic GMP‐AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway. A DNA motif containing two palindromic sequences is engineered with an abasic site (AP)‐connected blocking sequence to inhibit its self‐assembly function, while apurinic/apyrimidinic endonuclease 1 (APE1)‐triggered enzymatic cleavage of the AP site enables the reconfiguration and self‐assembly of DNA motif into long double‐stranded structures, thus realizing allosteric activation of the catalytic activity of cGAS to produce 2’3’‐cyclic‐GMP‐AMP for STING stimulation. Importantly, we demonstrate that APE1‐regulated DNA assembly allows for cell‐selective activation of cGAS–STING signaling. Furthermore, by re‐engineering the DNA motif with a photocleavable group, enzyme‐triggered DNA assembly allows the cGAS–STING stimulation to operate (switched "ON''), whereas light‐mediated fragmentation of the double‐stranded DNA enables termination of such stimulation (switched "OFF"), thereby achieving orthogonal control over immune regulation. This work highlights an endogenous and exogenous tandem regulated strategy to modulate the cGAS–STING pathway in an orthogonally controlled manner.

Tandem-Controlled Dynamic DNA Assembly Enables Temporally-Selective Orthogonal Regulation of cGAS-STING Stimulation.

Despite advances in the controlled reconfiguration of DNA structures for biological applications, the dearth of strategies that allow for orthogonal regulation of immune pathways remains a challenge. Here, we report for the first time an endogenous and exogenous tandem-regulated DNA assembly strategy that enables orthogonally controlled stimulation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. A DNA motif containing two palindromic sequences is engineered with an abasic site (AP)-connected blocking sequence to inhibit its self-assembly function, while apurinic/apyrimidinic endonuclease 1 (APE1)-triggered enzymatic cleavage of the AP site enables the reconfiguration and self-assembly of DNA motif into long double-stranded structures, thus realizing allosteric activation of the catalytic activity of cGAS to produce 2'3'-cyclic-GMP-AMP for STING stimulation. Importantly, we demonstrate that APE1-regulated DNA assembly allows for cell-selective activation of cGAS-STING signaling. Furthermore, by re-engineering the DNA motif with a photocleavable group, enzyme-triggered DNA assembly allows the cGAS-STING stimulation to operate (switched "ON''), whereas light-mediated fragmentation of the double-stranded DNA enables termination of such stimulation (switched "OFF"), thereby achieving orthogonal control over immune regulation. This work highlights an endogenous and exogenous tandem regulated strategy to modulate the cGAS-STING pathway in an orthogonally controlled manner.

Age-Dependent Pathogenesis of Influenza A Virus H7N9 Mediated Through PB1-F2-Induced Mitochondrial DNA Release and Activation of cGAS-STING-NF-κB Signaling.

Exactly why human infection of avian influenza A virus H7N9 causes more severe disease in the elderly remains elusive. In this study, we found that H7N9 PB1-F2 is a pathogenic factor in 15-18-month-old BALB/C mice (aged mice) but not in 6-8-week-old young adult mice (young mice). Recombinant influenza A virus with H7N9 PB1-F2-knockout was less pathogenic in aged mice as indicated with delayed weight loss. In contrast, survival of young mice infected with this virus was diminished. Furthermore, tissue damage, inflammation, proinflammatory cytokine and 2'3'-cGAMP production in the lung were less pronounced in infected aged mice despite no change in viral titer. cGAS is known to produce 2'3'-cGAMP to boost proinflammatory cytokine expression through STING-NF-κB signaling. We found that H7N9 PB1-F2 promoted interferon β (IFNβ) and chemokine gene expression in cultured cells through the mitochondrial DNA-cGAS-STING-NF-κB pathway. H7N9 PB1-F2 formed protein aggregate and caused mitochondrial cristae collapse, complex V-dependent electron transport dysfunction, reverse electron transfer-dependent oxidized mitochondrial DNA release to the cytoplasm and activation of cGAS-STING-NF-κB signaling. PB1-F2 N57 truncation, which is frequently observed in human circulating strains, mitigated H7N9 PB1-F2-mediated mitochondrial dysfunction and cGAS activation. In addition, we found that PB1-F2 of pathogenic avian influenza viruses triggered more robust cGAS activation than their human-adapted descendants. Our findings provide one explanation to age-dependent pathogenesis of H7N9 infection.

Cell-free assays reveal that the HIV-1 capsid protects reverse transcripts from cGAS.

Retroviruses can be detected by the innate immune sensor cyclic GMP-AMP synthase (cGAS), which recognizes reverse-transcribed DNA and activates an antiviral response. However, the extent to which HIV-1 shields its genome from cGAS recognition remains unclear. To study this process in mechanistic detail, we reconstituted reverse transcription, genome release, and innate immune sensing of HIV-1 in a cell-free system. We found that wild-type HIV-1 capsids protect viral genomes from cGAS even after completing reverse transcription. Viral DNA could be "deprotected" by thermal stress, capsid mutations, or reduced concentrations of inositol hexakisphosphate (IP6) that destabilize the capsid. Strikingly, the capsid inhibitor lenacapavir also disrupted viral cores and dramatically potentiated cGAS activity, both in vitro and in cellular infections. Our results provide biochemical evidence that the HIV-1 capsid lattice conceals the genome from cGAS and that chemical or physical disruption of the viral core can expose HIV-1 DNA and activate innate immune signaling.

CGAS activation in classical dendritic cells causes autoimmunity in TREX1-deficient mice

Detection of cytosolic DNA by the cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of interferon genes (STING) pathway provides immune defense against pathogens and cancer but can also cause autoimmunity when overactivated. The exonuclease three prime repair exonuclease 1 (TREX1) degrades DNA in the cytosol and prevents cGAS activation by self-DNA. Loss-of-function mutations of the TREX1 gene are linked to autoimmune diseases such as Aicardi-Goutières syndrome, and mice deficient in TREX1 develop lethal inflammation in a cGAS-dependent manner. In order to determine the type of cells in which cGAS activation drives autoinflammation, we generated conditional cGAS knockout mice on the Trex1-/- background. Here, we show that genetic ablation of the cGAS gene in classical dendritic cells (cDCs), but not in macrophages, was sufficient to rescue Trex1-/- mice from all observed disease phenotypes including lethality, T cell activation, tissue inflammation, and production of antinuclear antibodies and interferon-stimulated genes. These results show that cGAS activation in cDC causes autoinflammation in response to self-DNA accumulated in the absence of TREX1.

In Vitro Cleavage Assay to Characterize DENV NS2B3 Antagonism of cGAS.

cGAS is a key cytosolic dsDNA receptor that senses viral infection and elicits interferon production through the cGAS-cGAMP-STING axis. cGAS is activated by dsDNA from viral and bacterial origins as well as dsDNA leaked from damaged mitochondria and nucleus. Eventually, cGAS activation launches the cell into an antiviral state to restrict the replication of both DNA and RNA viruses. Throughout the long co-evolution, viruses devise many strategies to evade cGAS detection or suppress cGAS activation. We recently reported that the Dengue virus protease NS2B3 proteolytically cleaves human cGAS in its N-terminal region, effectively reducing cGAS binding to DNA and consequent production of the second messenger cGAMP. Several other RNA viruses likely adopt the cleavage strategy. Here, we describe a protocol for the purification of recombinant human cGAS and Dengue NS2B3 protease, as well as the in vitro cleavage assay.

CGAS Expression is Enhanced in Systemic Sclerosis Associated Interstitial Lung Disease and Stimulates Inflammatory Myofibroblast Activation.

The lungs of patients with Systemic Sclerosis Associated Interstitial Lung Disease (SSc-ILD) contain inflammatory myofibroblasts arising in association with fibrotic stimuli and perturbed innate immunity. The innate immune DNA binding receptor Cyclic GMP-AMP synthase (cGAS) is implicated in inflammation and fibrosis, but its involvement in SSc-ILD remains unknown. We examined cGAS expression, activity, and therapeutic potential in SSc-ILD using cultured fibroblasts, precision cut lung slices (PCLS), and a well-accepted animal model. Expression and localization of cGAS, cytokines, and type 1 interferons were evaluated in SSc-ILD lung tissues, bronchoalveolar lavage (BAL), and isolated lung fibroblasts. CGAS activation was assessed in a publicly available SSc-ILD single cell RNA sequencing dataset. Production of cytokines, type 1 interferons, and αSMA elicited by TGFβ1 or local substrate stiffness were measured in normal human lung fibroblasts (NHLFs) via qRT-PCR, ELISA, and immunofluorescence. Small molecule cGAS inhibition was tested in cultured fibroblasts, human PCLS, and the bleomycin pulmonary fibrosis model. SSc-ILD lung tissue and BAL are enriched for cGAS, cytokines, and type 1 interferons. The cGAS pathway shows constitutive activation in SSc-ILD fibroblasts and is inducible in NHLFs by TGFβ1 or mechanical stimuli. In these settings, and in human PCLS, cGAS expression is paralleled by the production of cytokines, type 1 interferons, and αSMA that are mitigated by a small molecule cGAS inhibitor. These findings are recapitulated in the bleomycin mouse model. cGAS signaling contributes to pathogenic inflammatory myofibroblast phenotypes in SSc-ILD. Inhibiting cGAS or its downstream effectors represents a novel therapeutic approach.

The role of cGAS in epithelial dysregulation in inflammatory bowel disease and gastrointestinal malignancies.

The gastrointestinal tract is lined by an epithelial monolayer responsible for selective permeability and absorption, as well as protection against harmful luminal contents. Recognition of foreign or aberrant DNA within these epithelial cells is, in part, regulated by pattern recognition receptors such as cyclic GMP-AMP synthase (cGAS). cGAS binds double-stranded DNA from exogenous and endogenous sources, resulting in the activation of stimulator of interferon genes (STING) and a type 1 interferon response. cGAS is also implicated in non-canonical pathways involving the suppression of DNA repair and the upregulation of autophagy via interactions with PARP1 and Beclin-1, respectively. The importance of cGAS activation in the development and progression of inflammatory bowel disease and gastrointestinal cancers has been and continues to be explored. This review delves into the intricacies of the complex role of cGAS in intestinal epithelial inflammation and gastrointestinal malignancies, as well as recent therapeutic advances targeting cGAS pathways.

Agonists and Inhibitors of the cGAS-STING Pathway.

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is pivotal in immunotherapy. Several agonists and inhibitors of the cGAS-STING pathway have been developed and evaluated for the treatment of various diseases. The agonists aim to activate STING, with cyclic dinucleotides (CDNs) being the most common, while the inhibitors aim to block the enzymatic activity or DNA binding ability of cGAS. Meanwhile, non-CDN compounds and cGAS agonists are also gaining attention. The omnipresence of the cGAS-STING pathway in vivo indicates that its overactivation could lead to undesired inflammatory responses and autoimmune diseases, which underscores the necessity of developing both agonists and inhibitors of the cGAS-STING pathway. This review describes the molecular traits and roles of the cGAS-STING pathway and summarizes the development of cGAS-STING agonists and inhibitors. The information is supposed to be conducive to the design of novel drugs for targeting the cGAS-STING pathway.

Hyperoside mitigates photoreceptor degeneration in part by targeting cGAS and suppressing DNA-induced microglial activation.

Activated microglia play an important role in driving photoreceptor degeneration-associated neuroinflammation in the retina. Controlling pro-inflammatory activation of microglia holds promise for mitigating the progression of photoreceptor degeneration. Our previous study has demonstrated that pre-light damage treatment of hyperoside, a naturally occurring flavonol glycoside with antioxidant and anti-inflammatory activities, prevents photooxidative stress-induced photoreceptor degeneration and neuroinflammatory responses in the retina. However, the direct impact of hyperoside on microglia-mediated neuroinflammation during photoreceptor degeneration remains unknown. Upon verifying the anti-inflammatory effects of hyperoside in LPS-stimulated BV-2 cells, our results here further demonstrated that post-light damage hyperoside treatment mitigated the loss of photoreceptors and attenuated the functional decline of the retina. Meanwhile, post-light damage hyperoside treatment lowered neuroinflammatory responses and dampened microglial activation in the illuminated retinas. With respect to microglial activation, hyperoside mitigated the pro-inflammatory responses in DNA-stimulated BV-2 cells and lowered DNA-stimulated production of 2'3'-cGAMP in BV-2 cells. Moreover, hyperoside was shown to directly interact with cGAS and suppress the enzymatic activity of cGAS in a cell-free system. In conclusion, the current study suggests for the first time that the DNA sensor cGAS is a direct target of hyperoside. Hyperoside is effective at mitigating DNA-stimulated cGAS-mediated pro-inflammatory activation of microglia, which likely contributes to the therapeutic effects of hyperoside at curtailing neuroinflammation and alleviating neuroinflammation-instigated photoreceptor degeneration.

Micronuclei induced by radiation, replication stress, or chromosome segregation errors do not activate cGAS-STING

The cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway plays a pivotal role in innate immune responses to viral infection and inhibition of autoimmunity. Recent studies have suggested that micronuclei formed by genotoxic stress can activate innate immune signaling via the cGAS-STING pathway. Here, we investigated cGAS localization, activation, and downstream signaling from micronuclei induced by ionizing radiation, replication stress, and chromosome segregation errors. Although cGAS localized to ruptured micronuclei via binding to self-DNA, we failed to observe cGAS activation; cGAMP production; downstream phosphorylation of STING, TBK1, or IRF3; nuclear accumulation of IRF3; or expression of interferon-stimulated genes. Failure to activate the cGAS-STING pathway was observed across primary and immortalized cell lines, which retained the ability to activate the cGAS-STING pathway in response to dsDNA or modified vaccinia virus infection. We provide evidence that micronuclei formed by genotoxic insults contain histone-bound self-DNA, which we show is inhibitory to cGAS activation in cells.

Ca2+-dependent contractile signaling induced by dsDNA-cGAS ligation mediates type 1 interferon generation and vascular injury

Acute Lung injury (ALI) is an uncontrolled systemic inflammatory response from bacterial or viral infection. The cyclic GMP-AMP synthase (cGAS) detects cytosolic double stranded DNA (dsDNA) release from bacteria, viruses, or apoptotic cells and induces innate immunity by generating the second messenger cGAMP and activation of stimulator of interferon genes (STING). However, the mechanisms regulating cGAS activity upon binding dsDNA remain elusive. Here, we show for the first in-depth analysis of mechanisms inducing cGAS activation in using lung endothelial cells (EC) and establish its translational relevance in causing ALI using a mouse model. We show that within 15 min, dsDNA ligation with cGAS mediates its interaction with a motor protein, myosin light chain 6 (MYL6), leading to phosphorylation of MLC and actin stress fiber formation. cGAS-MYL6-mediated MLC phosphorylation required calcium release and organization of an ER-Ca2+ sensor, Stromal Interaction Molecule 1 (STIM1) as puncta. Activated STIM1 also induced cGAS tyrosine phosphorylation at Y215/242 residues via cSrc. Interfering with MYL6 or cSrc expression impaired cGAS condensation and cGAMP generation. Liposome delivery of phosphodeficient cGAS (Y215/Y242àF215/F242) in EC of cGAS null mice blocked type1-IFN production during bacterial pneumonia. Our finding suggests that STIM1 is crucial in inducing cGAS activity and type1-IFN production. cGAS thus represents a novel target for inhibiting STING activation, thereby suppressing tissue injury and inflammation. NIH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Keeping cGAS in check: SPSB3 promotes nuclear cGAS degradation for maintaining immune homeostasis

In a recent publication in Nature, Xu etal.1 discovered a role of CRL5-SPSB3 ubiquitin ligase in promoting ubiquitination and degradation of nuclear cGAS, which prevents aberrant cGAS activation by genomic DNA and contributes to the maintenance of immune homeostasis.

Enzymatic activity of cGAS in the presence of three types of DNAs: limited cGAS stimulation by single-stranded HIV-1 SL2 DNA.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is activated by binding to DNA. Activated cGAS produces 2'3'-cGAMP, which subsequently binds to the adaptor protein STING (stimulator of interferon genes). This interaction triggers the cGAS/STING signaling pathway, leading to the production of type I interferons. Three types of DNA, namely double-stranded DNA longer than 40 base pairs, a 70-nucleotide single-stranded HIV-1 DNA known as SL2, and Y-form DNA with unpaired guanosine trimers (G3 Y-form DNA), induce interferon production by activating cGAS/STING signaling. However, the extent of cGAS activation by each specific DNA type remains unclear. The comparison of cGAS stimulation by various DNAs is crucial for understanding the mechanisms underlying cGAS-mediated type I interferon production in the innate immune response. Here, we revealed that cGAS produces 2'3'-cGAMP at a significantly lower rate in the presence of single-stranded SL2 DNA than in the presence of double-stranded DNA or G3 Y-form DNA. Furthermore, the guanine-to-cytosine mutations and the deletion of unpaired guanosine trimers significantly reduced the 2'3'-cGAMP production rate and the binding of cGAS to Y-form DNA. These studies will provide new insights into the cGAS-mediated DNA-sensing in immune response.

Metformin normalizes mitochondrial function to delay astrocyte senescence in a mouse model of Parkinson’s disease through Mfn2-cGAS signaling

BackgroundSenescent astrocytes play crucial roles in age-associated neurodegenerative diseases, including Parkinson’s disease (PD). Metformin, a drug widely used for treating diabetes, exerts longevity effects and neuroprotective activities. However, its effect on astrocyte senescence in PD remains to be defined.MethodsLong culture-induced replicative senescence model and 1-methyl-4-phenylpyridinium/α-synuclein aggregate-induced premature senescence model, and a mouse model of PD were used to investigate the effect of metformin on astrocyte senescence in vivo and in vitro. Immunofluorescence staining and flow cytometric analyses were performed to evaluate the mitochondrial function. We stereotactically injected AAV carrying GFAP-promoter-cGAS-shRNA to mouse substantia nigra pars compacta regions to specifically reduce astrocytic cGAS expression to clarify the potential molecular mechanism by which metformin inhibited the astrocyte senescence in PD.ResultsWe showed that metformin inhibited the astrocyte senescence in vitro and in PD mice. Mechanistically, metformin normalized mitochondrial function to reduce mitochondrial DNA release through mitofusin 2 (Mfn2), leading to inactivation of cGAS-STING, which delayed astrocyte senescence and prevented neurodegeneration. Mfn2 overexpression in astrocytes reversed the inhibitory role of metformin in cGAS-STING activation and astrocyte senescence. More importantly, metformin ameliorated dopamine neuron injury and behavioral deficits in mice by reducing the accumulation of senescent astrocytes via inhibition of astrocytic cGAS activation. Deletion of astrocytic cGAS abolished the suppressive effects of metformin on astrocyte senescence and neurodegeneration.ConclusionsThis work reveals that metformin delays astrocyte senescence via inhibiting astrocytic Mfn2-cGAS activation and suggest that metformin is a promising therapeutic agent for age-associated neurodegenerative diseases.

Abstract 2457: Brain derived endothelial cells as a mediator of cGAS-STING driven anti-tumor efficacy in glioblastoma

Abstract The median survival time for newly diagnosed patients with glioblastoma (GBM) is 15 months, highlighting the need for improved therapies. Current treatment for newly diagnosed and recurrent GBM consists of tumor resection and chemoradiation therapy. The immunosuppressive tumor microenvironment (TME) is a central obstacle in the treatment of GBM. Lack of NK cell and CD8+ T cell infiltration prevents efficacy with immunotherapies like immune checkpoint blockade (ICB).One approach our lab is pursuing to enhance tumor infiltrating lymphocytes is through harnessing the anti-viral cGAS-STING pathway. Upon cGAS activation by cytosolic double stranded DNA, the TBK1 kinase is phosphorylated, initiating a subsequent type I interferon (IFN) response and the recruitment of immune cells. This pathway is commonly altered in many cancer types to evade immune activation. Our lab has demonstrated survival benefits with STING agonist treatment in immunocompetent GBM mouse models. We have also shown strong STING localization in the vasculature, implicating endothelial cells as a potential treatment target for anti-tumor efficacy. Here we investigated the potential role of tumor vasculature in mediating the effects of STING agonists in GBM therapy.We have shown robust cGAS-STING activation in brain endothelial cell lines through enhanced secretion of CXCL10, IFN-β, and RANTES upon STING agonist treatment in vitro. Western blotting also shows pathway activation in response to treatment through increased phospho-TBK1 levels. Activation was further potentiated by manganese.To investigate the effects of STING activation in brain endothelial cells in detail, we performed RNA sequencing. This showed 262 differentially expressed genes in response to pharmacologic cGAS-STING activation, with 183 of these being upregulated. The top gene ontology terms enriched include type I interferon signaling pathway, defense response to virus, and antigen processing and presentation of exogenous peptide antigen via MHC class I, TAP-independent. In addition, gene expression of tight junction protein Claudin 5 was also enhanced. We have also shown increased resistance in response to activation, through transendothelial electrical resistance (TEER) assays. Taken together, these results suggest that cGAS-STING activation in endothelial cells leads to tight junction remodeling in addition to immunomodulatory changes, implicating roles for endothelial cells in barrier function and immune cell interactions respectively. Current research in our lab focuses on elucidating the mechanisms behind these changes. Our goal is to identify beneficial endothelial cell changes which promote anti-tumor immunity in order to inform future treatment strategies. We are currently performing studies aimed at demonstrating that cGAS-STING activation in endothelial cells is critical for the efficacy of STING agonist treatment. Citation Format: Andrea Schmidt, Sandra Remson, Philippa Vaughn-Beaucaire, Jorge L. Jimenez-Macias, Praveen Srinivasan, Maximillian Pinho-Schwermann, Sean Lawler. Brain derived endothelial cells as a mediator of cGAS-STING driven anti-tumor efficacy in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2457.

Abstract 2057: Monitoring cGAMP Biochemically and in cell using homogenous HTS formatted bioluminescent assay

Abstract The STimulator of INterferon Genes (STING) is a transmembrane protein located at the endoplasmic reticulum, which serves as a molecular hub in response to cytoplasmic cyclic dinucleotide (CDN) second messengers such as cyclic GMP-AMP (cGAMP). Upon its generation via cyclic GMP-AMP synthase (cGAS) by a wide array of pathogens that provide extracellular microbial DNA and by intracellular (nuclear and mitochondrial) DNA, it binds directly to the STING protein resulting in the activation of the STING pathway. This activation by cytosolic DNA initiates a cascade of events, cGAS-STING-TBK1-IRF3 signaling, that induces type 1 IFN response, and eventually the signaling is terminated by degradation of pathway components and clearance of stimulatory DNA. Given its significant role as an adaptor for DNA sensors to pathogens and its involvement in immune sensing, tumor growth control, and control of autoinflammatory and autoimmune disorders, the STING pathway has become a very important drug target. Credible evidence suggests that the cGAS–cGAMP–STING pathway makes fundamental contributions to at least three major cancer therapies: radiation therapy, chemotherapy, and immunotherapy, and it thus represents a promising drug target. Therefore, there is a need to identify lead compounds that effectively modulate human STING for further drug development. Towards this goal, we have developed a bioluminescent assay to monitor the concentration of cGAMP in intracellular as well as extracellular compartments. The assay relies on the new concept of Protein Complementation Assay (PCA) using nanoluciferase (nanoluc) complementation technology where a small peptide (SmBiT) derived from nanoluc linked to cGAMP through a linker (cGAMP tracer) can be recognized by an anti cGAMP antibody that is linked to a nanoluc complementary fragment (LgBiT) resulting in bioluminescence upon the addition of nanoluc substrate. The presence of free cGAMP that is generated in the reaction competes with cGAMP tracer and decreases bioluminescence. We have optimized the assay for higher sensitivity down to less than 10 nM cGAMP and less than one ng of cGAS and thus, and it is suitable for testing compounds that modulate cGAS activity. We also improved cell lysis condition to increase recovery of cGAMP that is generated intracellularly using several cGAS activators and correlating its concentration with cytokine release. We envision using this assay to monitor cGAMP presence in sera and spinal fluids of patients who have ALS and Alzheimer diseases. The assay is homogenous, with minimal or no false hits using LOPAC screening and HTS formatted and can be completed in less than one hour. Thus, this assay provides valuable contribution for investigating modulators of the STING pathway and facilitates the search for novel therapeutics. Citation Format: Said A. Goueli, kevin Hsiao, Nate Murray, dareen Mikheil, Matt Larsen, Hui Wang. Monitoring cGAMP Biochemically and in cell using homogenous HTS formatted bioluminescent assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2057.

Abstract 6598: Inhibition of cGAS as a strategy to restore anti-tumor immunity response to STING agonist in chromosomally unstable tumors

Abstract The activation of STING (stimulator of interferon genes) has been widely shown to induce signaling pathways, triggering type I interferon and ultimately generating anti-tumor immunity. Whereas initial preclinical studies on STING agonists exhibited boost in anti-tumor response, clinical trials combining STING agonist and immune checkpoint blockade agent PD-1 reported that only small subset of patients benefited from the treatment. This study highlights a potential key mechanism underlying the lack of benefit from STING agonist observed in most clinical trial patients. We reveal a phenomenon known as chromosomal instability (CIN), characterized by persistent chromosome segregation errors over multiple rounds of mitosis, drives chronic activation of cGAS (cyclic GMP‒AMP synthase) through continuously supplying its substrate in the form of micronuclei. Chronic activation of cGAS stimulates degradation of its downstream target STING in a feedback-loop manner. This consequently attenuates type I interferon signaling upon STING activation, blunting its anti-tumor response. Remarkably, we show suppression of CIN, achieved through introduction of mitotic centromere-associated kinesin (MCAK), or inhibition of cGAS activity, either by pharmacological agent or total genetic ablation of cGAS using CRISPR-Cas9, restores type I interferon production and anti-tumor immunity upon the administration of STING agonist, MSA-2 in four of the five advanced tumor models. Furthermore, we show this restoration of type I interferon signaling and anti-tumor immunity is cancer cell STING-dependent as genetic deletion of STING in the tumor cells nullifies this effect. In summary, we provide a mechanistic insight underlying the limited anti-tumor response observed in STING agonist trials and propose potential strategies, through finetuning of CIN level or inhibition of cGAS activity, to illicit stronger anti-tumor immunity in combination with STING agonist treatment. Citation Format: Christy Hong, Lindsay Caprio, Mercedes Duran, Meri Rogava, Hsiang-His Ling, Sreeharsha Gurrapu, Leon Ebel, Melody Di Bona, Benjamin Izar, Samuel F. Bakhoum. Inhibition of cGAS as a strategy to restore anti-tumor immunity response to STING agonist in chromosomally unstable tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6598.

Abstract 6630: Non-cell-autonomous cancer progression from chromosomal instability

Abstract Chromosomal instability (CIN) is a driver of cancer metastasis, yet the extent to which this effect depends on the immune system remains unknown. Using ContactTracing—a newly developed, validated and benchmarked tool to infer the nature and conditional dependence of cell-cell interactions from single-cell transcriptomic data—we show that CIN-induced chronic activation of the cGAS-STING pathway promotes downstream signal re-wiring in cancer cells, leading to a pro-metastatic tumor microenvironment. This re-wiring is manifested by type I interferon tachyphylaxis selectively downstream of STING and a corresponding increase in cancer cell-derived endoplasmic reticulum (ER) stress response. Reversal of CIN, depletion of cancer cell STING or inhibition of ER stress response signaling abrogates CIN-dependent effects on the tumor microenvironment and suppresses metastasis in immune competent, but not severely immune compromised, settings. Treatment with STING inhibitors reduces CIN-driven metastasis in melanoma, breast and colorectal cancers in a manner dependent on tumor cell-intrinsic STING. Finally, we show that CIN and pervasive cGAS activation in micronuclei are associated with ER stress signaling, immune suppression and metastasis in human triple-negative breast cancer, highlighting a viable strategy to identify and therapeutically intervene in tumors spurred by CIN-induced inflammation. Citation Format: Jun Li, Melissa Hubisz, Ethan Earlie, Mercedes Duran, Christy Hong, Austin Varela, Emanuele Lettera, Matthew Deyell, Bernardo Tavora, Jonathan Havel, Phyu Su, Amit Dipak Amin, Karolina Budre, Erina Kamiya, Julie-Ann Cavallo, Christopher Garris, Simon Powell, Jorge Reis-Filho, Hannah Wen, Sarah Bettigole, Atif Khan, Benjamin Izar, Eileen Parkes, Ashley Laughney, Samuel Bakhoum. Non-cell-autonomous cancer progression from chromosomal instability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6630.

Lamin B1 curtails early human papillomavirus infection by safeguarding nuclear compartmentalization and autophagic capacity

Human papillomavirus (HPV) infection is a primary cause of cervical and head-and-neck cancers. The HPV genome enters the nucleus during mitosis when the nuclear envelope disassembles. Given that lamins maintain nuclear integrity during interphase, we asked to what extent their loss would affect early HPV infection. To address this question, we infected human cervical cancer cells and keratinocytes lacking the major lamins with a HPV16 pseudovirus (HP-PsV) encoding an EGFP reporter. We found that a sustained reduction or complete loss of lamin B1 significantly increased HP-PsV infection rate. A corresponding greater nuclear HP-PsV load in LMNB1 knockout cells was directly related to their prolonged mitotic window and extensive nuclear rupture propensity. Despite the increased HP-PsV presence, EGFP transcript levels remained virtually unchanged, indicating an additional defect in protein turnover. Further investigation revealed that LMNB1 knockout led to a substantial decrease in autophagic capacity, possibly linked to the persistent activation of cGAS by cytoplasmic chromatin exposure. Thus, the attrition of lamin B1 increases nuclear perviousness and attenuates autophagic capacity, creating an environment conducive to unrestrained accumulation of HPV capsids. Our identification of lower lamin B1 levels and nuclear BAF foci in the basal epithelial layer of several human cervix samples suggests that this pathway may contribute to an increased individual susceptibility to HPV infection.Graphical abstract