VPS34-IN1 potentiates STING-dependent activation in human CAL-1 cells.

BackgroundWhile stimulator of interferon genes (STING) activation in innate immune cells of the tumor microenvironment can result in CD8 T cell-dependent antitumor immunity, whether STING signaling affects CD4 T-cell responses...

The induction of type I interferons through the transcription factor interferon regulatory factor 3 (IRF3) is considered a major outcome of stimulator of interferon genes (STING) activation that drives immune responses against DNA viruses and tumors. However, STING activation can also trigger other downstream pathways such as nuclear factor κB (NF-κB) signaling and autophagy, and the roles of interferon (IFN)-independent functions of STING in infectious diseases or cancer are not well understood. Here, we generated a STING mouse strain with a mutation (S365A) that disrupts IRF3 binding and therefore type I interferon induction but not NF-κB activation or autophagy induction. We also generated STING mice with mutations that disrupt the recruitment of TANK-binding kinase 1 (TBK1), which is important for both IRF3 and NF-κB activation but not autophagy induction (L373A or ∆CTT, which lacks the C-terminal tail). The STING-S365A mutant mice, but not L373A or ∆CTT mice, were still resistant to herpes simplex virus 1 (HSV-1) infections and mounted an antitumor response after cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) treatment despite the absence of STING-induced interferons. These results demonstrate that STING can function independently of type I interferons and autophagy, and that TBK1 recruitment to STING is essential for antiviral and antitumor immunity.

Removing the B (cell) STING to improve cancer immunotherapy.

Background: The stimulator of interferon genes (STING) plays a central role in innate immune response against infection and cancer. Naturally, the cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS)-STING pathway adopts polyvalent interactions to form higher-order assemblies to achieve a specific and rapid response while limiting unnecessary stimulation by host DNA. Several cyclic di-nucleotide (CDN) and non-CDN small molecule STING agonists have been studied in clinical trials showing limited therapeutic efficacy. ONM-501, a dual-activating STING agonist employs PC7A, a synthetic polymer that induces polyvalent STING condensation and prolongs innate immune activation. Mechanistically, PC7A binds to STING through a non-competitive surface site that is distinct from the binding pocket of CDN and non-CDN small molecule STING agonists. ONM-501 encapsulates the endogenous STING agonist cGAMP with the PC7A micelles offering dual ‘burst' and ‘sustained' STING activation. Effectiveness of using ONM-501 for immunotherapy against solid tumors has been demonstrated. Methods: Polyvalent interaction between PC7A and STING was studied and characterized by several different methods including ITC, a FRET assay between fluorophore labeled STING and PC7A and Nile-Red assay. The binding valency was investigated using a series of PC7A polymers with an increasing number of repeating units. The PC7A-STING binding site was elucidated using STING mutants produced by site-directed mutagenesis. STING activation was evaluated by measuring Ifnb1 and Cxcl10 expression using RT-qPCR. STING activation by PC7A and ONM-501 was also investigated using freshly resected human tissue. ONM-501 antitumor efficacy was evaluated in 6 different murine syngeneic tumor models. Results: PC7A activates STING through a non-canonical biomolecular condensation. It binds to a non-competitive surface site on the α5 helix of STING which is different from the CDN binding pocket. This binding was also retained with cGAMP-resistant STING variants (e.g., R232H). The formation of STING-PC7A condensate and the downstream activation were dependent on polymer repeating units. ONM-501 achieves a synergistic, rapid and sustained (6-24h) STING activation in vivo compared to cGAMP which peaked at 6h. Injection of ONM-501 in fresh human tissue resulted in >100-fold increase in cytokine expression while free CDN only showed marginal effect. Antitumor efficacy was demonstrated in MC38, CT26, B16F10, 4T1, A20 and TC-1 models after ONM-501 treatment. Complete response was observed when combined with anti-PD1 checkpoint blockade therapy. Conclusions: PC7A polymer achieves prolonged innate immune activation by polyvalent STING condensation through a distinct binding site. ONM-501 combines endogenous cGAMP with PC7A that potentially offers a synergistic strategy in spatiotemporal orchestration of immune environment for a highly effective immunotherapy against cancer. Citation Format: Suxin Li, Min Luo, Zhaohui Wang, Qiang Feng, Jonathan Wilhelm, Xu Wang, Wei Li, Jian Wang, Qingtai Su, Gaurav Bharadwaj, Jason Miller, Katy Torres, Stephen Gutowski, Agnieszka Cholka, Yang-xin Fu, Tian Zhao, Baran Sumer, Hongtao Yu, Jinming Gao. ONM-501 ― A synthetic polyvalent STING agonist for cancer immunotherapy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P049.

The low success rate of cancer nanomedicines has raised debate on the role of the enhanced permeability and retention (EPR) effect on tumor deposition of nanotherapeutics. Here, we report a bifunctional nanoscale coordination polymer (NCP), oxaliplatin (OX)/2',3'-cyclic guanosine monophosphate-adenosine monophosphate (GA), to overcome the EPR limitation through stimulator of interferon genes (STING) activation and enhance chemotherapeutic and STING agonist delivery for tumor eradication. OX/GA encapsulates GA and OX in the NCP to protect GA from enzymatic degradation and improve GA and OX pharmacokinetics. STING activation by OX/GA disrupts tumor vasculatures and increases intratumoral deposition of OX by 4.9-fold over monotherapy OX-NCP. OX/GA demonstrates exceptional antitumor effects with >95% tumor growth inhibition and high cure rates in subcutaneous, orthotopic, spontaneous, and metastatic tumor models. OX/GA induces immunogenic cell death of tumor cells and STING activation of innate immune cells to enhance antigen presentation. NCPs provide an excellent nanoplatform to overcome the EPR limitation for effective cancer therapy.

STING is an intrinsic checkpoint inhibitor that restrains the TH17 cell pathogenic program.

We demonstrate that exposure to the AB5 subtilase cytotoxin (SubAB) induces the unfolded protein response (UPR) in human peripheral blood mononuclear cells, concomitant with a proinflammatory response across distinct cell subsets. Notably, SubAB selectively induces type-I interferon (IFN) expression in plasmacytoid dendritic cells, acting synergistically with Toll-like receptor 7 stimulation. The induction of type-I IFN in response to SubAB relies on stimulator of interferon genes (STING) activation, coupled with protein synthesis inhibition mediated by protein kinase R-like endoplasmic reticulum kinase (PERK) and phosphorylation of the eukaryotic translation initiation factor 2 subunit-alpha. By impeding mRNA translation through the integrated stress response, SubAB precipitates the downregulation of the negative innate signaling feedback regulator Tax1-binding protein 1. This downregulation is necessary to unleash TANK-binding kinase 1 signaling associated with STING activation. These findings shed light on how UPR-inducing conditions may regulate the immune system during infection or pathogenesis.

ISGylation by HERCs facilitates STING activation

Despite potent preclinical antitumor activity, activation of stimulator of interferon genes (STING) has shown modest therapeutic effects in clinical studies. Many STING agonists, including 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), show poor pharmacokinetic properties for sustaining STING activation in tumors and achieving optimal antitumor efficacy. Improved delivery of STING agonists and their effective combination with other treatments are needed to enhance their therapeutic effects. Herein, a 2D nanoplatform, cGAMP/MOL, is reported via conjugating cGAMP to a nanoscale metal-organic layer (MOL) for simultaneous STING activation and radiosensitization. The MOL not only exhibits strong radiosensitization effects for enhanced cancer killing and induction of immunogenic cell death, but also retains cGAMP in tumors for sustained STING activation. Compared to free cGAMP, cGAMP/MOL elicits stronger STING activation and regresses local tumors upon X-ray irradiation. Further combination with an immune checkpoint inhibitor bridges innate and adaptive immune systems by activating the tumor microenvironment to elicit systemic antitumor responses.

Stimulator of interferon genes (STING) is an ER-localized transmembrane protein and the receptor for 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), which is a second messenger produced by cGAMP synthase (cGAS), a cytosolic double-stranded DNA sensor. The cGAS-STING pathway plays a critical role in the innate immune response to infection of a variety of DNA pathogens through the induction of the type I interferons. Pharmacological activation of STING is a promising therapeutic strategy for cancer, thus the development of potent and selective STING agonists has been pursued. Here we report that mouse STING can be activated by phenylarsine oxide (PAO), a membrane permeable trivalent arsenic compound that preferentially reacts with thiol group of cysteine residue (Cys). The activation of STING with PAO does not require cGAS or cGAMP. Mass spectrometric analysis of the peptides generated by trypsin and chymotrypsin digestion of STING identifies several PAO adducts, suggesting that PAO covalently binds to STING. Screening of STING variants with single Cys to serine residues (Ser) reveals that Cys88 and Cys291 are critical to the response to PAO. STING activation with PAO, as with cGAMP, requires the ER-to-Golgi traffic and palmitoylation of STING. Our results identify a non-nucleotide STING agonist that does not target the cGAMP-binding pocket, and demonstrate that Cys of STING can be a novel target for the development of STING agonist.Key words: STING agonist, cysteine modification, innate immunity, phenylarsine oxide.

Background: The stimulator of interferon genes (STING) plays a central role in innate immune response against infection and cancer. Several cyclic di-nucleotide (CDN) and non-CDN small molecule STING agonists have demonstrated effectiveness against cancer in preclinical animal models, however their clinical trials showed limited therapeutic efficacy. ONM-501, a dual-activating STING agonist employs PC7A, a synthetic polymer that induces polyvalent STING condensation and prolongs innate immune activation has been recently developed. ONM-501 encapsulates the endogenous STING agonist cGAMP with the PC7A micelles offering dual ‘burst’ and ‘sustained’ STING activation. The mechanism and effectiveness of intratumorally delivered ONM-501 as an immunotherapy against solid tumors has been demonstrated in preclinical models. Methods: ONM-501 was recently evaluated for STING activation across different species: STING related IFNB1 and CXCL10 gene expression after ONM-501 treatment was measured by RT-qPCR in PBMCs from rat, cynomolgus monkey, and human. ONM-501 antitumor efficacy was evaluated in murine syngeneic tumor models. Abscopal effect was demonstrated by studying antitumor efficacy using both a primary/distal model and a lung metastatic model. STING knockout mice and STING knockout cancer cells were used to clarify the dependence of STING status to the ONM-501 antitumor immunity. Immune cell dependence was further elucidated by depletion of specific immune cell populations. Pilot safety studies including major organ function and systemic cytokine levels were performed in immunocompetent mice. Results: STING activation was observed across different species by measuring IFNB1 and CXCL10 mRNA in PBMCs from rat, cynomolgus monkey, and human after ONM-501 treatment. Antitumor efficacy was demonstrated both as a monotherapy and in combination with anti-PD1 in six different syngeneic tumor models. ONM-501 also induced an abscopal effect - tumor inhibition was observed in both primary and distal MC38 tumors in the same animal. Reduction of lung metastasis in an immune “cold” triple negative orthotopic breast cancer 4T1 model further confirmed the systemic antitumor immunity. Knocking out host STING (i.e. STING KO mice) rather than cancer cell STING KO resulted in abrogation of tumor inhibition suggesting that the host STING status is responsible for ONM-501-mediated anti-tumor immunity. Immune cell depletion studies further clarified that the antitumor immunity is dependent on CD8+ T cell and dendritic cells (DCs). No alteration of main organ function or systemic cytokine storm were observed in the pilot safety study. Conclusions: ONM-501 demonstrated marked anti-tumor efficacy in a panel of syngeneic tumor models. The anti-tumor effect was mediated by host STING and dependent on CD8+ T cells and DCs. These results support further evaluation of ONM-501 as a clinical candidate for the potential treatment of solid tumors. Citation Format: Suxin Li, Jian Wang, Jonathan Wilhelm, Qingtai Su, Gaurav Bharadwaj, Jason Miller, Wei Li, Katy Torres, Ruolan Han, Tian Zhao, Jinming Gao. ONM-501: A polyvalent STING agonist for oncology immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4234.

Stimulator of Interferon Genes (STING) is a cytosolic sensor of cyclic dinucleotides (CDNs). The activation of dendritic cells (DC) via the STING pathway, and their subsequent production of type I interferon (IFN) is considered central to eradicating tumours in mouse models. However, this contribution of STING in preclinical murine studies has not translated into positive outcomes of STING agonists in phase I & II clinical trials. We therefore questioned whether a difference in human DC responses could be critical to the lack of STING agonist efficacy in human settings. This study sought to directly compare mouse and human plasmacytoid DCs and conventional DC subset responses upon STING activation. We found all mouse and human DC subsets were potently activated by STING stimulation. As expected, Type I IFNs were produced by both mouse and human plasmacytoid DCs. However, mouse and human plasmacytoid and conventional DCs all produced type III IFNs (i.e., IFN-λs) in response to STING activation. Of particular interest, all human DCs produced large amounts of IFN-λ1, not expressed in the mouse genome. Furthermore, we also found differential cell death responses upon STING activation, observing rapid ablation of mouse, but not human, plasmacytoid DCs. STING-induced cell death in murine plasmacytoid DCs occurred in a cell-intrinsic manner and involved intrinsic apoptosis. These data highlight discordance between STING IFN and cell death responses in mouse and human DCs and caution against extrapolating STING-mediated events in mouse models to equivalent human outcomes.

Stimulator of interferon genes (STING) contributes to anti-tumor immunity by activating antigen-presenting cells and inducing mobilization of tumor-specific T cells. A role for tumor-migrating neutrophils in the anti-tumor effect of STING-activating therapy has not been defined. We used mouse tumor transplantation models for assessing neutrophil migration into the tumor triggered by intratumoral treatment with STING agonist, 2'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Intratumoral STING activation with cGAMP enhanced neutrophil migration into the tumor in an NF-κB/CXCL1/2-dependent manner. Blocking the neutrophil migration by anti-CXCR2 monoclonal antibody impaired T cell activation in tumor-draining lymph nodes (dLNs) and efficacy of intratumoral cGAMP treatment. Moreover, the intratumoral cGAMP treatment did not show any anti-tumor effect in type I interferon (IFN) signal-impaired mice in spite of enhanced neutrophil accumulation in the tumor. These results suggest that both neutrophil migration and type I interferon (IFN) induction by intratumoral cGAMP treatment were critical for T-cell activation of dLNs and the anti-tumor effect. In addition, we also performed in vitro analysis showing enhanced cytotoxicity of neutrophils by IFN-β1. Extrinsic STING activation triggers anti-tumor immune responses by recruiting and activating neutrophils in the tumor via two signaling pathways, CXCL1/2 and type I IFNs.

Stimulator of interferon genes (STING) is an essential adaptor protein of the innate DNA-sensing signaling pathway, which recognizes genomic DNA from invading pathogens to establish antiviral responses in host cells. STING activity is tightly regulated by several posttranslational modifications, including phosphorylation. However, specifically how the phosphorylation status of STING is modulated by kinases and phosphatases remains to be fully elucidated. In this study, we identified protein phosphatase 6 catalytic subunit (PPP6C) as a binding partner of Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 48 (ORF48), which is a negative regulator of the cyclic GMP-AMP synthase (cGAS)-STING pathway. PPP6C depletion enhances double-stranded DNA (dsDNA)-induced and 5'ppp double-stranded RNA (dsRNA)-induced but not poly(I:C)-induced innate immune responses. PPP6C negatively regulates dsDNA-induced IRF3 activation but not NF-κB activation. Deficiency of PPP6C greatly inhibits the replication of herpes simplex virus 1 (HSV-1) and vesicular stomatitis virus (VSV) as well as the reactivation of KSHV, due to increased type I interferon production. We further demonstrated that PPP6C interacts with STING and that loss of PPP6C enhances STING phosphorylation. These data demonstrate the important role of PPP6C in regulating STING phosphorylation and activation, which provides an additional mechanism by which the host responds to viral infection.IMPORTANCE Cytosolic DNA, which usually comes from invading microbes, is a dangerous signal to the host. The cGAS-STING pathway is the major player that detects cytosolic DNA and then evokes the innate immune response. As an adaptor protein, STING plays a central role in controlling activation of the cGAS-STING pathway. Although transient activation of STING is essential to trigger the host defense during pathogen invasion, chronic STING activation has been shown to be associated with several autoinflammatory diseases. Here, we report that PPP6C negatively regulates the cGAS-STING pathway by removing STING phosphorylation, which is required for its activation. Dephosphorylation of STING by PPP6C helps prevent the sustained production of STING-dependent cytokines, which would otherwise lead to severe autoimmune disorders. This work provides additional mechanisms on the regulation of STING activity and might facilitate the development of novel therapeutics designed to prevent a variety of autoinflammatory disorders.

The immune system’s ability to respond to foreign DNA is a highly conserved mechanism. One crucial part of this mechanism is Stimulator of Interferon Genes (STING), a central innate immune protein located in the endoplasmic reticulum. It is a pattern recognition receptor that is activated in response to intracellular infection and, ultimately, mediates type 1 interferon (IFN) production. Encoded by the gene TMEM173, STING is expressed in T‐cells, monocytes, natural killer cells, and dermal fibroblasts.. It is composed of four transmembrane helices and a cytoplasmic ligand binding and signaling domain, which form a domain‐swapped dimer assembly. The STING pathway is initiated when double‐stranded DNA (dsDNA) is sensed, causing cyclic‐GMP‐AMP‐synthase (cGAS) to cyclize GTP and ATP and produce 2′3′‐cyclic‐GMP‐AMP (cGAMP), the main ligand that activates STING. Upon STING activation, the ligand‐binding domain closes, inducing a 180 degree rotation of the ligand‐binding domain relative to the transmembrane domain. The C‐terminal tail of STING has been found to autoinhibit the protein by blocking the polymerization interface of inactive STING, preventing auto activation. The conformational change made in response to cGAMP releases the CTT, exposing the polymer interface and allowing disulfide link formation. Alternatively, STING can be activated by bacterial cyclic‐di‐GMP in a cooperative manner, with cyclic‐di‐GMP serving as a partial antagonist of the cGAMP signaling pathway. Structural and biochemical studies have provided elegant insight into the mechanism of differential of STING by cGAMP and cDi‐GMP. STING activates tank binding kinase 1 (TBK1) which phosphorylates transcription factor IRF‐3 and causes it to translocate to the nucleus. Gain‐of‐function mutations of TMEM173 causing elevated transcription of IFNs have been proven to cause autoimmune syndromes such as systemic lupus erythematosus and, most recently, three mutations of TMEM173 have been found to directly cause STING‐associated vasculopathy with onset in infants (SAVI). New research proposes a therapeutic strategy that uses molecules that induces a different STING conformation than cGAMP‐bound STING as a STING inhibitor. This method would prevent constitutive STING activation in autoimmune syndromes while being less immunosuppressive than previously proposed therapies involving STING inhibition. The Walton High School MSOE Center for Biomolecular Modeling SMART Team has designed a 3‐D model of STING using a recently published cryo‐electron microscopy structure that reveals the transmembrane domain structure to further investigate and examine the structure‐function relationship of STING.Support or Funding InformationMSOE Center for Biomolecular Modeling