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Agonists of mouse STING (TMEM173) shrink and even cure solid tumors by activating innate immunity; human STING (hSTING) agonists are needed to test this therapeutic hypothesis in humans. The endogenous STING agonist is 2'3'-cGAMP, a second messenger that signals the presence of cytosolic double-stranded DNA. We report activity-guided partial purification and identification of ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP1) to be the dominant 2'3'-cGAMP hydrolyzing activity in cultured cells. The hydrolysis activity of ENPP1 was confirmed using recombinant protein and was depleted in tissue extracts and plasma from Enpp1(-/-) mice. We synthesized a hydrolysis-resistant bisphosphothioate analog of 2'3'-cGAMP (2'3'-cG(s)A(s)MP) that has similar affinity for hSTING in vitro and is ten times more potent at inducing IFN-β secretion from human THP1 monocytes. Studies in mouse Enpp1(-/-) lung fibroblasts indicate that resistance to hydrolysis contributes substantially to its higher potency. 2'3'-cG(s)A(s)MP is therefore improved over natural 2'3'-cGAMP as a model agonist and has potential as a vaccine adjuvant and cancer therapeutic.

Mounting evidence suggests that the tumor microenvironment is profoundly immunosuppressive. Thus, mitigating tumor immunosuppression is crucial for inducing sustained antitumor immunity. Whereas previous studies involved intratumoral injection, we report here an inhalable nanoparticle-immunotherapy system targeting pulmonary antigen presenting cells (APCs) to enhance anticancer immunity against lung metastases. Inhalation of phosphatidylserine coated liposome loaded with STING agonist cyclic guanosine monophosphate–adenosine monophosphate (NP-cGAMP) in mouse models of lung metastases enables rapid distribution of NP-cGAMP to both lungs and subsequent uptake by APCs without causing immunopathology. NP-cGAMP designed for enhanced cytosolic release of cGAMP stimulates STING signaling and type I interferons production in APCs, resulting in the pro-inflammatory tumor microenvironment in multifocal lung metastases. Furthermore, fractionated radiation delivered to one tumor-bearing lung synergizes with inhaled NP-cGAMP, eliciting systemic anticancer immunity, controlling metastases in both lungs, and conferring long-term survival in mice with lung metastases and with repeated tumor challenge. Successful anticancer immunotherapy should induce robust systemic immunity against metastases. Here, the authors engineer an inhalable nano-STING agonist, which synergizes with fractionated radiation to control lung metastases and confers long-term systemic antitumor immunity in mice.

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.

Stimulator of interferon genes (STING) is an intracellular sensor of cyclic di-nucleotides involved in the innate immune response against pathogen- or self-derived DNA. STING trafficking is tightly linked to its function, and its dysregulation can lead to disease. Here, we systematically characterize genes regulating STING trafficking and examine their impact on STING-mediated responses. Using proximity-ligation proteomics and genetic screens, we demonstrate that an endosomal sorting complex required for transport (ESCRT) complex containing HGS, VPS37A and UBAP1 promotes STING degradation, thereby terminating STING-mediated signaling. Mechanistically, STING oligomerization increases its ubiquitination by UBE2N, forming a platform for ESCRT recruitment at the endosome that terminates STING signaling via sorting in the lysosome. Finally, we show that expression of a UBAP1 mutant identified in patients with hereditary spastic paraplegia and associated with disrupted ESCRT function, increases steady-state STING-dependent type I IFN responses in healthy primary monocyte-derived dendritic cells and fibroblasts. Based on these findings, we propose that STING is subject to a tonic degradative flux and that the ESCRT complex acts as a homeostatic regulator of STING signaling. STING is an intracellular sensor of pathogen- or host-derived DNA. In this study, the authors identify an ESCRT complex that regulates STING degradation, thus acting as a homeostatic regulator of STING signalling and type-I interferon responses.

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 + T-cell activity, and the combination of PC7A and cGAMP led to synergistic therapeutic outcomes (including the activation of cGAMP-resistant STING variants) in mice bearing subcutaneous tumours and in resected human tumours and lymph nodes. The activation of the STING pathway through polymer-induced STING condensation may offer new therapeutic opportunities. A polyvalent STING agonist prolongs the activation of innate-immunity pathways through the formation of STING condensates, and leads to synergistic therapeutic outcomes in vivo when combined with the STING ligand cGAMP.

Insect odorant receptors In many organisms, from worms to humans, olfactory cues are detected by large families of seven transmembrane-spanning receptors, which have until now been classified as G protein-coupled receptors. Insects, however, have evolved a surprisingly simple and efficient sense of smell in which the odorant receptors require a second component — the ion-channel-forming chaperone protein Or83b — for correct function. In the first of two related papers, Sato et al . show that these heteromeric receptors form ligand-gated cation channels that are not dependent on G protein-coupled second messengers, and speculate that other seven transmembrane-spanning proteins may show similar ion channel activity. Wicher et al . show that, in addition to direct channel activation, ligand binding to odorant receptors causes G protein-coupled channel activation. This work has implications for the search for insect odorant receptor inhibitors for possible use in controlling host seeking behaviour of disease carrying insects such as the mosquito. Olfactory cues are detected by large families of seven transmembrane-spanning receptors, which have until now been classified as G-protein-coupled receptors. In insects, these odorant receptors require a second protein (Or83b) for correct function. The second of two related papers shows that, in addition to direct channel activation, ligand binding to odorant receptors causes G-protein-coupled channel activation. From worm to man, many odorant signals are perceived by the binding of volatile ligands to odorant receptors 1 that belong to the G-protein-coupled receptor (GPCR) family 2 . They couple to heterotrimeric G-proteins, most of which induce cAMP production 3 . This second messenger then activates cyclic-nucleotide-gated ion channels to depolarize the olfactory receptor neuron, thus providing a signal for further neuronal processing. Recent findings, however, have challenged this concept of odorant signal transduction in insects, because their odorant receptors, which lack any sequence similarity to other GPCRs 4 , are composed of conventional odorant receptors (for example, Or22a), dimerized with a ubiquitously expressed chaperone protein 5 , such as Or83b in Drosophila 6 . Or83b has a structure akin to GPCRs, but has an inverted orientation in the plasma membrane 4 , 7 . However, G proteins are expressed in insect olfactory receptor neurons 8 , and olfactory perception is modified by mutations affecting the cAMP transduction pathway 9 . Here we show that application of odorants to mammalian cells co-expressing Or22a and Or83b results in non-selective cation currents activated by means of an ionotropic and a metabotropic pathway, and a subsequent increase in the intracellular Ca 2+ concentration. Expression of Or83b alone leads to functional ion channels not directly responding to odorants, but being directly activated by intracellular cAMP or cGMP. Insect odorant receptors thus form ligand-gated channels as well as complexes of odorant-sensing units and cyclic-nucleotide-activated non-selective cation channels. Thereby, they provide rapid and transient as well as sensitive and prolonged odorant signalling.

Stimulator of IFN genes (STING) spontaneously contributes to anti-tumor immunity by inducing type I interferons (IFNs) following sensing of tumor-derived genomic DNAs in the tumor-bearing host. Although direct injection of STING ligands such as cyclic diguanylate monophosphate (c-di-GMP) and cyclic [G(2′,5′)pA(3′,5′)p] (cGAMP) into the tumor microenvironment exerts anti-tumor effects through strong induction of type I IFNs and activation of innate and adaptive immunity, the precise events caused by STING in the tumor microenvironment remain to be elucidated. We describe here our finding that a CD45 + CD11b mid Ly6C + cell subset transiently accumulated in mouse tumor microenvironment of 4T1 breast cancer, squamous cell carcinomas, CT26 colon cancer, or B16F10 melanoma tissue after intratumoral injection of cGAMP. The accumulated cells displayed a macrophage (M ) phenotype since the cells were positive for F4/80 and MHC class II and negative for Ly6G. Intratumoral cGAMP treatment did not induce Mφ accumulation in STING-deficient mice. Depletion of CD8 + T cell using anti-CD8 mAb impaired the anti-tumor effects of cGAMP treatment. Depletion of the Mφ using clodronate liposomes impaired the anti-tumor effects of cGAMP treatment. Functional analysis indicated that the STING-triggered tumor-migrating Mφ exhibited phagocytic activity, production of tumor necrosis factor alpha TNFα), and high expression levels of T cell-recruiting chemokines, Cxcl10 and Cxcl11 , IFN-induced molecules, MX dynamin-like GTPase 1 ( Mx1 ) and 2′-5′ oligoadenylate synthetase-like 1 ( Oasl1 ), nitric oxide synthase 2 ( Nos2 ), and interferon beta 1 ( Ifnb1 ). These results indicate that the STING-triggered tumor-migrating Mφ participate in the anti-tumor effects of STING-activating compounds.

Enterotoxigenic Escherichia coli (ETEC) in humans and animals colonizes the intestine and thereafter secrets heat-stable enterotoxin (ST) with or without heat-labile enterotoxin (LT), which triggers massive fluid and electrolyte secretion into the gut lumen. The crosstalk between the cyclic nucleotide-dependent protein kinase/cystic fibrosis transmembrane conductance regulator (cAMP or cGMP/CFTR) pathway involved in ETEC-induced diarrhea channels, and the canonical Wnt/β-catenin signaling pathway leads to changes in intestinal stem cell (ISC) fates, which are strongly associated with developmental disorders caused by diarrhea. We review how alterations in enterotoxin-activated ion channel pathways and the canonical Wnt/β-catenin signaling pathway can explain inhibited intestinal epithelial activity, characterize alterations in the crosstalk of cyclic nucleotides, and predict harmful effects on ISCs in targeted therapy. Besides, we discuss current deficits in the understanding of enterotoxin-intestinal epithelial cell activity relationships that should be considered when interpreting sequelae of diarrhea.

Immunotherapy is one of the key strategies for cancer treatment. The cGAS-cGAMP-STING-IRF3 pathway of cytosolic DNA sensing plays a pivotal role in antiviral defense. We report that the STING activator cGAMP possesses significant antitumor activity in mice by triggering the STING-dependent pathway directly. cGAMP enhances innate immune responses by inducing production of cytokines such as interferon-β, interferon-γ and stimulating dendritic cells activation, which induces the cross-priming of CD8 + T cells. The antitumor mechanism of cGAMP was verified by STING and IRF3, which were up-regulated upon cGAMP treatment. STING-deficiency dramatically reduced the antitumor effect of cGAMP. Furthermore, cGAMP improved the antitumor activity of 5-FU and clearly reduced the toxicity of 5-FU. These results demonstrated that cGAMP is a novel antitumor agent and has potential applications in cancer immunotherapy.

The microenvironment tends to be immunosuppressive during tumor growth and proliferation. Immunotherapy has attracted much attention because of its ability to activate tumor-specific immune responses for tumor killing. The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is an innate immune pathway that activates antitumor immunity by producing type I interferons. Cyclic dinucleotides (CDNs), produced by cGAS sensing cytoplasmic abnormal DNA, are major intermediate activating molecules in the STING pathway. Nowadays, CDNs and their derivatives have widely worked as powerful STING agonists in tumor immunotherapy. However, their clinical translation is hindered by the negative electrical properties, sensitivity to hydrolytic enzymes, and systemic toxicity. Recently, various CDN delivery systems have made significant progress in addressing these issues, either through monotherapy or in combination with other treatment modalities. This review details recent advances in CDNs-based pharmaceutical development or delivery strategies for enriching CDNs at tumor sites and activating the STING pathway.