cyclic-guanosine-monophosphate-adenosine-monophosphate and Neoplasms

2'3'-cGAMP is a key molecule in the cGAS-STING pathway. This cyclic dinucleotide is produced by the cytosolic DNA sensor cGAS in response to the presence of aberrant dsDNA in the cytoplasm which is associated with microbial invasion or cellular damage. 2'3'-cGAMP acts as a second messenger and activates STING, the central hub of DNA sensing, to induce type-I interferons and pro-inflammatory cytokines necessary for responses against infection, cancer or cellular stress. Classically, detection of pathogens or danger by pattern recognition receptors (PRR) was thought to signal and induce the production of interferon and pro-inflammatory cytokines in the cell where sensing occurred. These interferon and cytokines then signal in both an autocrine and paracrine manner to induce responses in neighboring cells. Deviating from this dogma, recent studies have identified multiple mechanisms by which 2'3'-cGAMP can travel to neighboring cells where it activates STING independent of DNA sensing by cGAS. This observation is of great importance, as the cGAS-STING pathway is involved in immune responses against microbial invaders and cancer while its dysregulation drives the pathology of a wide range of inflammatory diseases to which antagonists have been elusive. In this review, we describe the fast-paced discoveries of the mechanisms by which 2'3'-cGAMP can be transported. We further highlight the diseases where they are important and detail how this change in perspective can be applied to vaccine design, cancer immunotherapies and treatment of cGAS-STING associated disease.

Topics: DNA; Humans; Interferon Type I; Membrane Proteins; Neoplasms; Nucleotidyltransferases; Signal Transduction

The cGAS-STING pathway discovered ten years ago is an important component of the innate immune system. Activation of cGAS-STING triggers downstream signalling, such as TBK1-IRF3, NF-κB and autophagy, which in turn leads to antipathogen responses, durable antitumour immunity or autoimmune diseases. 2',3'-Cyclic GMP-AMP dinucleotides (2',3'-cGAMP), the key second messengers produced by cGAS, play a pivotal role in cGAS-STING signalling by binding and activating STING. Thus, 2',3'-cGAMP has immunotherapeutic potential, which in turn has stimulated research on the design and synthesis of 2',3'-cGAMP analogues for clinical applications over the past ten years. This review presents the discovery, metabolism, and function of 2',3'-cGAMP in the cGAS-STING innate immune signalling axis. The enzymatic and chemical syntheses of 2',3'-cGAMP analogues as STING-targeting therapeutics are also summarized.

Topics: Humans; Immunotherapy; Membrane Proteins; Models, Molecular; Molecular Conformation; Neoplasms; Nucleotides; Nucleotides, Cyclic; Nucleotidyltransferases; Signal Transduction

Cyclic GMP-AMP synthase (cGAS) senses foreign DNA to produce 2'3'-cyclic GMP-AMP (2'3'-cGAMP). 2'3'-cGAMP is a second messenger that binds and activates the adaptor protein STING, which triggers the innate immune response. As a STING agonist, the small molecule 2'3'-cGAMP plays pivotal roles in antiviral defense and has adjuvant applications, and anti-tumor effects. 2'3'-cGAMP and its analogs are thus putative targets for immunotherapy and are currently being testedin clinical trials to treat solid tumors. However, several barriers to further development have emerged from these studies, such as evidence of immune and inflammatory side-effects, poor pharmacokinetics, and undesirable biodistribution. Here, we review the status of 2'3'-cGAMP research and outline the role of 2'3'-cGAMP in immune signaling, adjuvant applications, and cancer immunotherapy, as well as various 2'3'-cGAMP detection methods.

Topics: Humans; Immunity, Innate; Neoplasms; Nucleotides, Cyclic; Second Messenger Systems; Tissue Distribution

Cyclic GMP-AMP synthase (cGAS), second messenger 2'3'-cyclic GMP-AMP (cGAMP) and stimulator of interferon genes (STING) are fundamental for sensing cytoplasmic double stranded DNA. Radiotherapy treatment induces large amounts of nuclear and mitochondrial DNA damage and results in the presence of DNA fragments in the cytoplasm, activating the cGAS/STING pathway. Triggering of the cGAS/STING pathway in the tumor microenvironment (TME) results in the production of type I interferons (IFNs). Type I IFNs are crucial for an effective antitumor defense, with myeloid cells as key players. Many questions remain on how these myeloid cells are activated and in which cells (tumor versus myeloid) in the TME the signaling pathway is initiated. The significance of cGAS/STING signaling in the onco-immunology field is being recognized, emphasized by the frequent occurrence of mutations in or silencing of genes in this pathway. We here review several mechanisms of cGAS/STING signal propagation in the TME, focusing on tumor cells and myeloid cells. Cell-cell contact-dependent interactions facilitate the transfer of tumor-derived DNA and cGAMP. Alternatively, transport routes via the extracellular space such as extracellular vesicles, and channel-mediated cGAMP transfer to and from the extracellular space contribute to propagation of cGAS/STING signal mediators DNA and cGAMP. Finally, we discuss regulation of extracellular cGAMP. Altogether, we provide a comprehensive overview of cGAS/cGAMP/STING signal propagation from tumor to myeloid cells in the TME, revealing novel targets for combinatorial treatment approaches with conventional anticancer therapies like radiotherapy.

Topics: DNA, Mitochondrial; Humans; Interferon Type I; Membrane Proteins; Myeloid Cells; Neoplasms; Nucleotides, Cyclic; Nucleotidyltransferases; Tumor Microenvironment

Innate immunity is regulated by a broad set of evolutionary conserved receptors to finely probe the local environment and maintain host integrity. Besides pathogen recognition through conserved motifs, several of these receptors also sense aberrant or misplaced self-molecules as a sign of perturbed homeostasis. Among them, self-nucleic acid sensing by the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway alerts on the presence of both exogenous and endogenous DNA in the cytoplasm. We review recent literature demonstrating that self-nucleic acid detection through the STING pathway is central to numerous processes, from cell physiology to sterile injury, auto-immunity and cancer. We address the role of STING in autoimmune diseases linked to dysfunctional DNAse or related to mutations in DNA sensing pathways. We expose the role of the cGAS/STING pathway in inflammatory diseases, neurodegenerative conditions and cancer. Connections between STING in various cell processes including autophagy and cell death are developed. Finally, we review proposed mechanisms to explain the sources of cytoplasmic DNA.

Topics: Adenosine Triphosphate; Adult; Autoimmune Diseases; Autophagy; Cytokines; Cytoplasm; DNA; Guanosine Triphosphate; Humans; Immunity, Innate; Infant; Inflammation; Interferon Type I; Membrane Proteins; Mitochondria; Neoplasms; Neurodegenerative Diseases; NF-kappa B; Nucleotides, Cyclic; Nucleotidyltransferases; Signal Transduction

Stimulator of interferon genes (STING) is an adaptor protein that induces the secretion of type I interferons and proinflammatory cytokines and is triggered by cytosolic DNA of pathogen and host origins. Given that STING is a mediator in the immune system, pharmacological modulation of STING has shown viable therapeutic effects for pathogen infection, cancer, and inflammatory diseases. In the past decade, the substantial development in this field has encouraged the discovery of STING modulators. Here, we will summarize the current understanding of STING structure, survey the status quo of STING modulators, compare established bioassay methods, review the chemical structures and bioactivities of agonists and inhibitors, and propose suggestions and insights for the future exploitation of STING modulators.

Topics: Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Autoimmune Diseases; Binding Sites; Chemistry, Pharmaceutical; Drug Delivery Systems; Humans; Membrane Proteins; Neoplasms; Protein Structure, Secondary; Signal Transduction

Cytosolic DNA sensing, the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, is an important novel role in the immune system. Multiple STING agonists were developed for cancer therapy study with great results achieved in pre-clinical work. Recent progress in the mechanical understanding of STING pathway in IFN production and T cell priming, indicates its promising role for cancer immunotherapy. STING agonists co-administrated with other cancer immunotherapies, including cancer vaccines, immune checkpoint inhibitors such as anti-programmed death 1 and cytotoxic T lymphocyte-associated antigen 4 antibodies, and adoptive T cell transfer therapies, would hold a promise of treating medium and advanced cancers. Despite the applications of STING agonists in cancer immunotherapy, lots of obstacles remain for further study. In this review, we mainly examine the biological characters, current applications, challenges, and future directions of cGAS-STING in cancer immunotherapy.

Topics: Adenylyl Cyclases; Adjuvants, Immunologic; Animals; Cancer Vaccines; Clinical Trials as Topic; CTLA-4 Antigen; DNA; DNA, Neoplasm; Drug Screening Assays, Antitumor; Humans; Immune Checkpoint Inhibitors; Immunotherapy; Immunotherapy, Adoptive; Membrane Proteins; Mice; Neoplasm Proteins; Neoplasms; Nucleotides, Cyclic; Oncolytic Virotherapy; Protein Multimerization; Signal Transduction; Therapies, Investigational

Cancer immunotherapy modulates and leverages the host immune system to treat cancer. The past decade has witnessed historical advancement of cancer immunotherapy. A myriad of approaches have been explored to elicit or augment anticancer innate immunity and/or adaptive immunity. Recently, activation of stimulator of interferon (IFN) genes (STING), an intracellular receptor residing in the endoplasmic reticulum, has shown great potential to enhance antitumor immunity through the induction of a variety of pro-inflammatory cytokines and chemokines, including type I IFNs. A number of natural and synthetic STING agonists have been discovered or developed, and tested in preclinical models and in the clinic for the immunotherapy of diseases such as cancer and infectious diseases. Cyclic dinucleotides (CDNs), such as cyclic dimeric guanosine monophosphate (c-di-GMP), cyclic dimeric adenosine monophosphate (c-di-AMP), and cyclic GMP-AMP (cGAMP), are a class of STING agonists that can elicit immune responses. However, natural CDNs are hydrophilic small molecules with negative charges and are susceptible to enzymatic degradation, leading to low bioavailability in target tissues yet unwanted toxicities and narrow therapeutic windows. Drug delivery systems, coupled with nucleic acid chemistry, have been exploited to address these challenges. Here, we will discuss the underlying immunological mechanisms and approaches to STING activation, with a focus on the delivery of STING agonists, for cancer immunotherapy.

Topics: Animals; Humans; Immunity, Innate; Immunotherapy; Membrane Proteins; Neoplasms; Nucleic Acids; Nucleotides, Cyclic

Detection of microbial DNA is an evolutionarily conserved mechanism that alerts the host immune system to mount a defense response to microbial infections. However, this detection mechanism also poses a challenge to the host as to how to distinguish foreign DNA from abundant self-DNA. Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) is a DNA sensor that triggers innate immune responses through production of the second messenger cyclic GMP-AMP (cGAMP), which binds and activates the adaptor protein STING. However, cGAS can be activated by double-stranded DNA irrespective of the sequence, including self-DNA. Although how cGAS is normally kept inactive in cells is still not well understood, recent research has provided strong evidence that genomic DNA damage leads to cGAS activation to stimulate inflammatory responses. This review summarizes recent findings on how genomic instability and DNA damage trigger cGAS activation and how cGAS serves as a link from DNA damage to inflammation, cellular senescence, and cancer.

Topics: Animals; Cellular Senescence; DNA Damage; Humans; Inflammation; Neoplasms; Nucleotides, Cyclic; Nucleotidyltransferases

Cyclic GMP-AMP (cGAMP) is a second messenger that activates the stimulator of interferon genes (STING) innate immune pathway to induce the expression of type I IFNs and other cytokines. Pharmacologic activation of STING is considered a potent therapeutic strategy in cancer. In this study, we used a cell-based phenotypic screen and identified podophyllotoxin (podofilox), a microtubule destabilizer, as a robust enhancer of the cGAMP-STING signaling pathway. We found that podofilox enhanced the cGAMP-mediated immune response by increasing STING-containing membrane puncta and the extent of STING oligomerization. Furthermore, podofilox changed the trafficking pattern of STING and delayed trafficking-mediated STING degradation. Importantly, the combination of cGAMP and podofilox had profound antitumor effects on mice by activating the immune response through host STING signaling. Together, these data provide insights into the regulation of cGAMP-STING pathway activation and demonstrate what we believe to be a novel approach for modulating this pathway and thereby promoting antitumor immunity.

Topics: Animals; Immunity, Innate; Membrane Proteins; Mice; Neoplasms; Podophyllotoxin; Signal Transduction

Interferon (IFN)-β induction via activation of the stimulator of interferon genes (STING) pathway has shown promising results in tumor models. STING is activated by cyclic dinucleotides such as cyclic GMP-AMP dinucleotides with phosphodiester linkages 2'-5' and 3'-5' (cGAMPs), that are produced by cyclic GMP-AMP synthetase (cGAS). However, delivery of STING pathway agonists to the tumor site is a challenge. Bacterial vaccine strains have the ability to specifically colonize hypoxic tumor tissues and could therefore be modified to overcome this challenge. Combining high STING-mediated IFN-β levels with the immunostimulatory properties of. We have engineered. Expression of cGAS in

Topics: Dendritic Cells; Ectopic Gene Expression; Humans; Interferon Type I; Macrophages; Neoplasms; Nucleotidyltransferases; Salmonella typhimurium; Tumor Microenvironment

Topics: 14-alpha Demethylase Inhibitors; Humans; Imidazoles; Immunity, Innate; Immunotherapy; Membrane Proteins; Neoplasms

Stimulator of interferon genes (STING) is an endoplasmic reticulum-membrane protein that plays important roles in cancer immunotherapy by activating innate immune responses. We designed and synthesized STING modulators and characterized compounds

Topics: Animals; Immunologic Factors; Immunotherapy; Interferons; Membrane Proteins; Mice; Neoplasms; Receptors, Interferon

Stereochemically and structurally complex cyclic dinucleotide-based stimulator of interferon genes (STING) agonists were designed and synthesized to access a previously unexplored chemical space. The assessment of biochemical affinity and cellular potency, along with computational, structural, and biophysical characterization, was applied to influence the design and optimization of novel STING agonists, resulting in the discovery of MK-1454 as a molecule with appropriate properties for clinical development. When administered intratumorally to immune-competent mice-bearing syngeneic tumors, MK-1454 exhibited robust tumor cytokine upregulation and effective antitumor activity. Tumor shrinkage in mouse models that are intrinsically resistant to single-agent therapy was further enhanced when treating the animals with MK-1454 in combination with a fully murinized antimouse PD-1 antibody, mDX400. These data support the development of STING agonists in combination with pembrolizumab (humanized anti-PD-1 antibody) for patients with tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 therapy.

Topics: Animals; Cytokines; Humans; Immunotherapy; Interferons; Membrane Proteins; Mice; Neoplasms

Recently, the emergence of immunotherapy has revolutionized traditional tumour treatment. However, effective treatments for patients exhibiting αPD-1 resistance are still lacking. In our study, a combination of cytosine-phosphate-guanine oligodeoxynucleotides (CpG-ODNs), anti-OX40 and cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) injection in situ systematically generated a robust antitumour immune response in TC1 and B16 cells, which are αPD-1-resistant malignancies. More precisely, this method activates both adaptive and innate immunity. Additionally, in situ vaccination with CpG/αOX40/cGAMP fully activates the production of cytokines. However, the combination of αPD-1 does not improve the efficacy of triple therapy, prompting further questions. Collectively, the combination of CpG/αOX40/cGAMP causes the regression of various αPD-1-resistant tumours through the full mobilization of innate and adaptive immunity. In addition, we explored the therapeutic effect of triple therapy on the αPD-1-sensitive cell line CT26. The results showed that triple therapy could significantly enhance the therapeutic effect of αPD-1, and some mice even achieved complete tumour regression after the combined application of αPD-1 and triple treatment.

Topics: Animals; Humans; Immunity, Innate; Immunotherapy; Mice; Neoplasms; Nucleotides, Cyclic

Immunotherapy is an effective strategy to control and eliminate primary and metastatic tumor by restarting and restoring the specific anti-tumor immune response. However, tumor immunotherapy often showed limited efficacy due to the poor T cell responses in vivo and the tumor suppressive microenvironments. Herein, we constructed polyethyleimine modified gold nanorods (GNRs-PEI) by conjugating PEI to GNRs via SAu bonds. GNRs-PEI/cGAMP nanoparticles were formed via electrostatic interaction and then loaded by macrophages. The GNRs-PEI/cGAMP-laden macrophages (GPc-RAWs) were intravenously injected into the tumor bearing mice and the in situ tumor vaccines were obtained after NIR irradiation. Besides, anti-PD-L1 antibody, an immune checkpoint inhibitor, was introduced to reverse immunosuppressive microenvironment and assisted to achieve the synergistic anti-tumor immunotherapy. GNRs-PEI/cGAMP-laden macrophages with NIR irradiation could effectively inhibit the primary tumors, while little effect for the contralateral tumors. When combined with anti-PD-L1 antibody, the combined strategy not only inhibited the growth of primary tumor, but also significantly delayed the proliferation of the contralateral tumors. More importantly, this strategy reversed immunosuppressive microenvironment without obvious side effects. Therefore, this study provided a great immunotherapy platform for the efficient treatment of primary and metastatic tumors.

Topics: Animals; Cancer Vaccines; Immune Checkpoint Inhibitors; Immunotherapy; Macrophages; Mice; Neoplasms; Nucleotides, Cyclic; Tumor Microenvironment

With the advent of bioinformatic tools in efficiently predicting neo-antigens, peptide vaccines have gained tremendous attention in cancer immunotherapy. However, the delivery of peptide vaccines remains a major challenge, primarily due to ineffective transport to lymph nodes and low immunogenicity. Here, a strategy for peptide vaccine delivery is reported by first fusing the peptide to the cytosolic domain of the stimulator of interferon genes protein (STINGΔTM), then complexing the peptide-STINGΔTM protein with STING agonist 2'3' cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). The process results in the formation of self-assembled cGAMP-peptide-STINGΔTM tetramers, which enables efficient lymphatic trafficking of the peptide. Moreover, the cGAMP-STINGΔTM complex acts not only as a protein carrier for the peptide, but also as a potent adjuvant capable of triggering STING signaling independent of endogenous STING protein-an especially important attribute considering that certain cancer cells epigenetically silence their endogenous STING expression. With model antigen SIINFEKL, it is demonstrated that the platform elicits effective STING signaling in vitro, draining lymph node targeting in vivo, effective T cell priming in vivo as well as antitumoral immune response in a mouse colon carcinoma model, providing a versatile solution to the challenges faced in peptide vaccine delivery.

Topics: Animals; Cancer Vaccines; Membrane Proteins; Mice; Neoplasms; Nucleotides, Cyclic; Peptides; Vaccines, Subunit

An immunosuppressive tumour microenvironment is a major obstacle in the control of pancreatic and other solid cancers

Topics: Animals; B-Lymphocytes, Regulatory; Humans; Immunity, Innate; Immunotherapy; Interferon Regulatory Factor-3; Interferon Type I; Interleukins; Killer Cells, Natural; Membrane Proteins; Mice; Neoplasms; Nucleotides, Cyclic; Tumor Microenvironment

Stimulator of Interferon Genes (STING) plays an important role in innate immunity by inducing type I interferon production upon infection with intracellular pathogens. STING activation can promote increased T-cell activation and inflammation in the tumor microenvironment, resulting in antitumor immunity. Natural and synthetic cyclic dinucleotides (CDNs) are known to activate STING, and several synthetic CDN molecules are being investigated in the clinic using an intratumoral administration route. Here, we describe the identification of STING agonist

Topics: Administration, Intravenous; Animals; Binding Sites; Cell Line, Tumor; Half-Life; Humans; Immunotherapy; Membrane Proteins; Mice; Molecular Docking Simulation; Neoplasms; Nucleotides, Cyclic; Phosphates; Pyrimidines; Rats; Structure-Activity Relationship; Transplantation, Heterologous

Cytosolic DNA is characteristic of chromosomally unstable metastatic cancer cells, resulting in constitutive activation of the cGAS-STING innate immune pathway. How tumors co-opt inflammatory signaling while evading immune surveillance remains unknown. Here, we show that the ectonucleotidase ENPP1 promotes metastasis by selectively degrading extracellular cGAMP, an immune-stimulatory metabolite whose breakdown products include the immune suppressor adenosine. ENPP1 loss suppresses metastasis, restores tumor immune infiltration, and potentiates response to immune checkpoint blockade in a manner dependent on tumor cGAS and host STING. Conversely, overexpression of wild-type ENPP1, but not an enzymatically weakened mutant, promotes migration and metastasis, in part through the generation of extracellular adenosine, and renders otherwise sensitive tumors completely resistant to immunotherapy. In human cancers, ENPP1 expression correlates with reduced immune cell infiltration, increased metastasis, and resistance to anti-PD-1/PD-L1 treatment. Thus, cGAMP hydrolysis by ENPP1 enables chromosomally unstable tumors to transmute cGAS activation into an immune-suppressive pathway. SIGNIFICANCE: Chromosomal instability promotes metastasis by generating chronic tumor inflammation. ENPP1 facilitates metastasis and enables tumor cells to tolerate inflammation by hydrolyzing the immunotransmitter cGAMP, preventing its transfer from cancer cells to immune cells.

Topics: Animals; Humans; Hydrolysis; Immunotherapy; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasms; Nucleotides, Cyclic; Tumor Escape

The stimulator of interferon genes (STING) is an endoplasmic reticulum transmembrane protein that is a target of therapeutics for infectious diseases and cancer. However, early-phase clinical trials of small-molecule STING agonists have shown limited antitumour efficacy and dose-limiting toxicity. Here, we show that a polyvalent STING agonist-a pH-sensitive polymer bearing a seven-membered ring with a tertiary amine (PC7A)-activates innate-immunity pathways through the polymer-induced formation of STING-PC7A condensates. In contrast to the natural STING ligand 2',3'-cyclic-GMP-AMP (cGAMP), PC7A stimulates the prolonged production of pro-inflammatory cytokines by binding to a non-competitive STING surface site that is distinct from the cGAMP binding pocket. PC7A induces antitumour responses that are dependent on STING expression and CD8

Topics: Animals; CD8-Positive T-Lymphocytes; Female; HEK293 Cells; HeLa Cells; Humans; Immunity, Innate; Lymph Nodes; Membrane Proteins; Mice; Neoplasms; Nucleotides, Cyclic; Polymers; THP-1 Cells

Stimulator of interferon genes (STING) promotes anti-tumour immunity by linking innate and adaptive immunity, but it remains unclear how intratumoural treatment with STING agonists yields anti-tumour effects. Here we demonstrate that intratumoural injection of the STING agonist cGAMP induces strong, rapid, and selective apoptosis of tumour endothelial cells (ECs) in implanted LLC tumour, melanoma and breast tumour, but not in spontaneous breast cancer and melanoma. In both implanted and spontaneous tumours, cGAMP greatly increases TNFα from tumour-associated myeloid cells. However, compared to spontaneous tumour ECs, implanted tumour ECs are more vulnerable to TNFα-TNFR1 signalling-mediated apoptosis, which promotes effective anti-tumour activity. The spontaneous tumour's refractoriness to cGAMP is abolished by co-treatment with AKT 1/2 inhibitor (AKTi). Combined treatment with cGAMP and AKTi induces extensive tumour EC apoptosis, leading to extensive tumour apoptosis and marked growth suppression of the spontaneous tumour. These findings propose an advanced avenue for treating primary tumours that are refractory to single STING agonist therapy.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Disease Models, Animal; Drug Resistance, Neoplasm; Female; Gene Knockdown Techniques; Human Umbilical Vein Endothelial Cells; Humans; Immunity, Innate; Injections, Intralesional; Male; Membrane Proteins; Mice; Mice, Transgenic; Neoplasms; Nucleotides, Cyclic; Phosphorylation; Proto-Oncogene Proteins c-akt; Receptors, Tumor Necrosis Factor, Type I; Signal Transduction; Tumor Microenvironment; Tumor Necrosis Factor-alpha

Intracellular DNA triggers interferon release during the innate immune response. Cyclic GMP-AMP synthase (cGAS) senses intracellular double-stranded DNA not only in response to viral infection but also under autoimmune conditions. Measuring the levels of cyclic GMP-AMP (cGAMP) as a second messenger of cGAS activation is important to elucidate the physiological and pathological roles of cGAS. Therefore, we generated monoclonal antibodies against cGAMP using hybridoma technology to test antibody specificity and establish methods to detect intracellular cGAMP. The resulting cGAMP-specific antibody enabled the development of a time-resolved fluorescence energy transfer assay with a quantifiable range of 0.1 nM to 100 nM cGAMP. Using this assay, we detected cellular and tissue cGAMP. We confirmed that the cGAMP antibody successfully targeted intracellular cGAMP through immunocytochemical analyses. These results demonstrated that the cGAMP antibody is a powerful tool that allows determining cGAS involvement in autoimmunity and disease pathology at the cell and tissue levels.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Autoimmune Diseases of the Nervous System; Autoimmunity; Biomarkers; Caco-2 Cells; Disease Models, Animal; Enzyme Activation; Exodeoxyribonucleases; Fluorescence Resonance Energy Transfer; HEK293 Cells; High-Throughput Screening Assays; HL-60 Cells; Humans; Immunohistochemistry; Mice, Inbred C57BL; Mice, Knockout; Neoplasms; Nervous System Malformations; Nucleotides, Cyclic; Nucleotidyltransferases; Phosphoproteins; Predictive Value of Tests; Reproducibility of Results

Clearance of apoptotic cells by macrophages prevents excessive inflammation and supports immune tolerance. Here, we examined the effect of blocking apoptotic cell clearance on anti-tumor immune response. We generated an antibody that selectively inhibited efferocytosis by phagocytic receptor MerTK. Blockade of MerTK resulted in accumulation of apoptotic cells within tumors and triggered a type I interferon response. Treatment of tumor-bearing mice with anti-MerTK antibody stimulated T cell activation and synergized with anti-PD-1 or anti-PD-L1 therapy. The anti-tumor effect induced by anti-MerTK treatment was lost in Sting

Topics: Adenosine Triphosphate; Animals; Apoptosis; B7-H1 Antigen; c-Mer Tyrosine Kinase; Cells, Cultured; Female; Immunity, Innate; Immunotherapy; Interferon Type I; Macrophages; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Neoplasms; Nucleotides, Cyclic; Nucleotidyltransferases; Phagocytosis; Programmed Cell Death 1 Receptor; Receptors, Purinergic P2X7; Signal Transduction; Xenograft Model Antitumor Assays

2',5'/3',5'-cGMP-AMP (cGAMP) is a second messenger produced in response to cytosolic dsDNA that activates the stimulator of interferon genes (STING) pathway. We recently discovered that cGAMP is exported by cancer cells and that this extracellular signal is an immunotransmitter key to tumor detection and elimination by the innate immune system. The enhancement of extracellular cGAMP levels therefore holds great promise for managing cancer. However, there is still much more to understand about the basic biology of cGAMP before its full therapeutic potential can be realized. To answer these questions, we must be able to detect and quantitate cGAMP with an assay that is high-throughput, sensitive, and precise. Existing assays fall short of these needs. Here, we describe the development of cGAMP-Luc, a coupled enzyme assay that relies on the degradation of cGAMP to AMP by ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) and an optimized assay for the detection of AMP by luciferase. We also developed STING-CAP, a STING-mediated method to concentrate and purify cGAMP from any type of biological sample. We conclude that cGAMP-Luc is an economical high-throughput assay that matches the accuracy of and surpasses the detection limit of MS, the current gold standard of cGAMP quantitation. We propose that cGAMP-Luc is a powerful tool that may enable discoveries that advance insights into extracellular cGAMP levels in healthy and diseased tissues, such as cancer.

Topics: Enzyme Assays; HEK293 Cells; HeLa Cells; Humans; Luciferases; Membrane Proteins; Neoplasms; Nucleotides, Cyclic; Phosphoric Diester Hydrolases; Pyrophosphatases; Signal Transduction

Topics: Antiviral Agents; Biomimetics; Humans; Immunotherapy; Influenza, Human; Nanoparticles; Neoplasms; Nucleotides, Cyclic; Pulmonary Surfactants; Vaccines

Activation of innate immune signaling in the tumor microenvironment is central to a successful anti-tumor immune response, and it is in large part mediated by cytosolic double-stranded DNA sensing. Here, Carozza et al. (2020b) report potent and selective inhibitors of ENPP1, a negative regulator of innate immune signaling, which are shown to potentiate anti-tumor immune responses.

Topics: Humans; Immunity, Innate; Neoplasms; Nucleotides, Cyclic; Phosphoric Diester Hydrolases; Signal Transduction; Tumor Microenvironment

2'3'-cyclic GMP-AMP (cGAMP) is an intracellular second messenger that is synthesized in response to cytosolic double-stranded DNA and activates the innate immune STING pathway. Our previous discovery of its extracellular hydrolase ENPP1 hinted at the existence of extracellular cGAMP. Here, we detected that cGAMP is continuously exported but then efficiently cleared by ENPP1, explaining why it has previously escaped detection. By developing potent, specific, and cell impermeable ENPP1 inhibitors, we found that cancer cells continuously export cGAMP in culture at steady state and at higher levels when treated with ionizing radiation (IR). In mouse tumors, depletion of extracellular cGAMP decreased tumor-associated immune cell infiltration and abolished the curative effect of IR. Boosting extracellular cGAMP with ENPP1 inhibitors synergized with IR to delay tumor growth. In conclusion, extracellular cGAMP is an anti-cancer immunotransmitter that could be harnessed to treat cancers with low immunogenicity.

Topics: Animals; Mice; Neoplasms; Nucleotides, Cyclic; Second Messenger Systems

Sensing of cytoplasmic DNA by cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) results in production of the dinucleotide cGAMP and consecutive activation of stimulator of interferon genes (STING) followed by production of type I interferon (IFN). Although cancer cells contain supra-normal concentrations of cytoplasmic DNA, they rarely produce type I IFN spontaneously. This suggests that defects in the DNA-sensing pathway may serve as an immune escape mechanism. We find that cancer cells produce cGAMP that is transferred via gap junctions to tumor-associated dendritic cells (DCs) and macrophages, which respond by producing type I IFN in situ. Cancer-cell-intrinsic expression of cGAS, but not STING, promotes infiltration by effector CD8

Topics: Animals; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Dendritic Cells; Disease Progression; DNA Damage; Humans; Immunotherapy; Interferon Type I; Membrane Proteins; Mice, Inbred C57BL; Microsatellite Repeats; Neoplasms; Nucleotides, Cyclic; Nucleotidyltransferases

Cyclic dinucleotide (CDN) agonists of stimulator of interferon genes (STING) are a promising class of immunotherapeutics that activate innate immunity to increase tumour immunogenicity. However, the efficacy of CDNs is limited by drug delivery barriers, including poor cellular targeting, rapid clearance and inefficient transport to the cytosol where STING is localized. Here, we describe STING-activating nanoparticles (STING-NPs)-rationally designed polymersomes for enhanced cytosolic delivery of the endogenous CDN ligand for STING, 2'3' cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). STING-NPs increase the biological potency of cGAMP, enhance STING signalling in the tumour microenvironment and sentinel lymph node, and convert immunosuppressive tumours to immunogenic, tumoricidal microenvironments. This leads to enhanced therapeutic efficacy of cGAMP, inhibition of tumour growth, increased rates of long-term survival, improved response to immune checkpoint blockade and induction of immunological memory that protects against tumour rechallenge. We validate STING-NPs in freshly isolated human melanoma tissue, highlighting their potential to improve clinical outcomes of immunotherapy.

Topics: Animals; Cytosol; Endosomes; Female; Humans; Immunotherapy; Inflammation; Membrane Proteins; Mice; Mice, Inbred C57BL; Nanoparticles; Neoplasm Metastasis; Neoplasms; Nucleotides, Cyclic; Polymers; T-Lymphocytes; Tumor Microenvironment

Immunotherapies have significantly improved cancer patient survival, but response rates are still limited. Thus, novel formulations are needed to expand the breadth of immunotherapies. Pathogen associated molecular patterns (PAMPs) can be used to stimulate an immune response, but several pathogen recognition receptors are located within the cell, making delivery challenging. We have employed the biodegradable polymer acetalated dextran (Ace-DEX) to formulate PAMP microparticles (MPs) in order to enhance intracellular delivery. While treatment with four different PAMP MPs resulted in tumor growth inhibition, cyclic GMP-AMP (cGAMP) MPs were most effective. cGAMP MPs showed anti-tumor efficacy at doses 100-1000 fold lower than published doses of soluble cGAMP in two murine tumor models. Treatment with cGAMP MPs resulted in increased natural killer cell numbers in the tumor environment. Immune cell depletion studies confirmed that NK cells were responsible for the anti-tumor efficacy in an aggressive mouse melanoma model. NK cells and CD8

Topics: Acetylation; Animals; CD8-Positive T-Lymphocytes; Dextrans; Disease Models, Animal; Hydrodynamics; Immunity; Immunotherapy; Killer Cells, Natural; Melanoma; Membrane Proteins; Mice, Inbred C57BL; Microspheres; Neoplasms; Nucleotides, Cyclic; Pathogen-Associated Molecular Pattern Molecules; Triple Negative Breast Neoplasms; Tumor Burden

2'3'-cyclic-GMP-AMP (cGAMP) is a second messenger that activates the antiviral stimulator of interferon genes (STING) pathway. We recently identified a novel role for cGAMP as a soluble, extracellular immunotransmitter that is produced and secreted by cancer cells. Secreted cGAMP is then sensed by host cells, eliciting an antitumoral immune response. Due to the antitumoral effects of cGAMP, other CDN-based STING agonists are currently under investigation in clinical trials for metastatic solid tumors. However, it is unknown how cGAMP and other CDNs cross the cell membrane to activate intracellular STING. Using a genome-wide CRISPR screen, we identified SLC19A1 as the first known importer of cGAMP and other CDNs, including the investigational new drug 2'3'-bisphosphosphothioate-cyclic-di-AMP (2'3'-CDA

Topics: Cell Line, Tumor; Cell Membrane; CRISPR-Cas Systems; Genome, Human; Humans; Immunity, Innate; Membrane Proteins; Neoplasms; Nucleotides, Cyclic; Reduced Folate Carrier Protein; Signal Transduction

Type I interferon (IFN) production within the tumor microenvironment is important in shaping the immune response to the tumor. In this issue of Immunity, Marcus et al. (2018) reveal that tumor cells produce 2'3'-cGAMP, which activates the STING pathway in non-tumor cells and leads to type I IFN production and the priming of natural killer cells for tumor rejection.

Topics: Humans; Killer Cells, Natural; Membrane Proteins; Neoplasms; Nucleotides, Cyclic

Detection of cytosolic DNA by the enzyme cGAS triggers the production of cGAMP, a second messenger that binds and activates the adaptor protein STING, which leads to interferon (IFN) production. Here, we found that in vivo natural killer (NK) cell killing of tumor cells, but not of normal cells, depends on STING expression in non-tumor cells. Experiments using transplantable tumor models in STING- and cGAS-deficient mice revealed that cGAS expression by tumor cells was critical for tumor rejection by NK cells. In contrast, cGAS expression by host cells was dispensable, suggesting that tumor-derived cGAMP is transferred to non-tumor cells, where it activates STING. cGAMP administration triggered STING activation and IFN-β production in myeloid cells and B cells but not NK cells. Our results reveal that the anti-tumor response of NK cells critically depends on the cytosolic DNA sensing pathway, similar to its role in defense against pathogens, and identify tumor-derived cGAMP as a major determinant of tumor immunogenicity with implications for cancer immunotherapy.

Topics: Animals; Cell Line; Cell Line, Tumor; Gene Expression Regulation; Humans; Interferons; Killer Cells, Natural; Membrane Proteins; Mice, Inbred C57BL; Mice, Knockout; Neoplasms; Nucleotides, Cyclic; Nucleotidyltransferases; Signal Transduction

Agonists for TLR9 and Stimulator of IFN Gene (STING) act as vaccine adjuvants that induce type-1 immune responses. However, currently available CpG oligodeoxynucleotide (ODN) (K-type) induces IFNs only weakly and STING ligands rather induce type-2 immune responses, limiting their potential therapeutic applications. Here, we show a potent synergism between TLR9 and STING agonists. Together, they make an effective type-1 adjuvant and an anticancer agent. The synergistic effect between CpG ODN (K3) and STING-ligand cyclic GMP-AMP (cGAMP), culminating in NK cell IFN-γ (type-II IFN) production, is due to the concurrent effects of IL-12 and type-I IFNs, which are differentially regulated by IRF3/7, STING, and MyD88. The combination of CpG ODN with cGAMP is a potent type-1 adjuvant, capable of inducing strong Th 1-type responses, as demonstrated by enhanced antigen-specific IgG2c and IFN-γ production, as well as cytotoxic CD8(+) T-cell responses. In our murine tumor models, intratumoral injection of CpG ODN and cGAMP together reduced tumor size significantly compared with the singular treatments, acting as an antigen-free anticancer agent. Thus, the combination of CpG ODN and a STING ligand may offer therapeutic application as a potent type-II IFN inducer.

Topics: Adjuvants, Immunologic; Animals; Cell Line, Tumor; Drug Synergism; Female; Immunoglobulin G; Interferon Regulatory Factor-3; Interferon Regulatory Factor-7; Interferon Type I; Interferon-gamma; Interleukin-12; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Myeloid Differentiation Factor 88; Neoplasms; Nucleotides, Cyclic; Oligodeoxyribonucleotides; T-Lymphocytes, Cytotoxic; Toll-Like Receptor 9

Spontaneous CD8 T-cell responses occur in growing tumors but are usually poorly effective. Understanding the molecular and cellular mechanisms that drive these responses is of major interest as they could be exploited to generate a more efficacious antitumor immunity. As such, stimulator of IFN genes (STING), an adaptor molecule involved in cytosolic DNA sensing, is required for the induction of antitumor CD8 T responses in mouse models of cancer. Here, we find that enforced activation of STING by intratumoral injection of cyclic dinucleotide GMP-AMP (cGAMP), potently enhanced antitumor CD8 T responses leading to growth control of injected and contralateral tumors in mouse models of melanoma and colon cancer. The ability of cGAMP to trigger antitumor immunity was further enhanced by the blockade of both PD1 and CTLA4. The STING-dependent antitumor immunity, either induced spontaneously in growing tumors or induced by intratumoral cGAMP injection was dependent on type I IFNs produced in the tumor microenvironment. In response to cGAMP injection, both in the mouse melanoma model and an ex vivo model of cultured human melanoma explants, the principal source of type I IFN was not dendritic cells, but instead endothelial cells. Similarly, endothelial cells but not dendritic cells were found to be the principal source of spontaneously induced type I IFNs in growing tumors. These data identify an unexpected role of the tumor vasculature in the initiation of CD8 T-cell antitumor immunity and demonstrate that tumor endothelial cells can be targeted for immunotherapy of melanoma.

Topics: Animals; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cell Proliferation; CTLA-4 Antigen; Dendritic Cells; Disease Models, Animal; Dose-Response Relationship, Immunologic; Endothelial Cells; Immunity; Injections, Intralesional; Interferon Type I; Lymphocytes, Tumor-Infiltrating; Melanoma; Melanoma, Experimental; Membrane Proteins; Mice, Inbred C57BL; Neoplasms; Nucleotides, Cyclic; Receptor, Interferon alpha-beta; Signal Transduction

Ionizing radiation-mediated tumor regression depends on type I interferon (IFN) and the adaptive immune response, but several pathways control I IFN induction. Here, we demonstrate that adaptor protein STING, but not MyD88, is required for type I IFN-dependent antitumor effects of radiation. In dendritic cells (DCs), STING was required for IFN-? induction in response to irradiated-tumor cells. The cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthase (cGAS) mediated sensing of irradiated-tumor cells in DCs. Moreover, STING was essential for radiation-induced adaptive immune responses, which relied on type I IFN signaling on DCs. Exogenous IFN-? treatment rescued the cross-priming by cGAS or STING-deficient DCs. Accordingly, activation of STING by a second messenger cGAMP administration enhanced antitumor immunity induced by radiation. Thus radiation-mediated antitumor immunity in immunogenic tumors requires a functional cytosolic DNA-sensing pathway and suggests that cGAMP treatment might provide a new strategy to improve radiotherapy.

Topics: Adaptive Immunity; Adaptor Proteins, Vesicular Transport; Animals; Antineoplastic Agents; Cells, Cultured; Cross-Priming; Dendritic Cells; DNA; Immunity, Innate; Interferon-beta; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Myeloid Differentiation Factor 88; Neoplasms; Nucleotides, Cyclic; Nucleotidyltransferases; Radiation, Ionizing; Receptor, Interferon alpha-beta; RNA Interference; RNA, Small Interfering; Signal Transduction; Xanthones