Activation Of Stimulator Of Interferon Genes Research Articles

Assembly of 2′,3′-Cyclic guanosine Monophosphate-Adenosine monophosphate and their spontaneous intracellular disassembly for enhanced antitumor immunity via natural STING pathway activation

The stimulator of interferon genes (STING) pathway plays an important role in remodeling the immunosuppressive tumor microenvironment (TME). Cyclic dinucleotides (CDNs), the natural ligands of STING, activate innate immunity to enhance tumor immunogenicity. However, the anionic properties of CDNs result in poor membrane permeability, while their rapid metabolism by enzymes hinders the efficient targeting and activation of STING in vitro and in vivo. To improve the bioavailability and antitumor immunity of CDNs, we designed a hydrogen-bonding-based supramolecular approach for 2′,3′-cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) (cGAMP). Leveraging the synthetic molecules cytosine-uracil-hexane (CUH), we formulated supramolecular micelles of cGAMP via specific hydrogen bonds in aqueous solutions. Micelles comprising the CUH-cGAMP complex enhanced the biological efficacy of cGAMP by increasing its stability, thereby prolonging the activation of STING-mediated innate and adaptive immunity. We demonstrated that CUH-cGAMP stands as a promising candidate for immunotherapy, exhibiting significant inhibition of tumor growth in a CT26 syngeneic mouse model.

Cyclopeptide Inhibitors Target the N-Terminal Tail of STING and Alleviate Autoinflammation.

Cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway is a crucial component of innate immunity that plays a vital role in protecting against pathogen infections and cellular stress. However, aberrant activation of cGAS-STING pathway is related to inflammatory and autoimmune diseases. Here, we developed cyclopeptide STING inhibitors by cyclizing the N-terminal tail (NTT) of STING. These cyclopeptides selectively inhibited the activation of STING pathway in human or murine cell lines. Mechanistically, the inhibitors directly bound to STING, and subsequently blocked the aggregation and activation of STING. In addition, the optimal inhibitor STi-2 significantly suppressed proinflammatory cytokine production and systemic inflammation in Trex1-/- mice. Overall, our work facilitates the development of specific inhibitors of STING as potential therapies for cGAS-STING associated autoinflammatory diseases.

STING Promotes the Progression of ADPKD by Regulating Mitochondrial Function, Inflammation, Fibrosis, and Apoptosis.

Autosomal dominant polycystic kidney disease (ADPKD) is a predominant genetic disease, which is caused by mutations in PKD genes and is associated with DNA damage in cystic cells. The intrinsic stimulator of interferon genes (STING) pathway is crucial for recognizing damaged DNA in the cytosol, triggering the expression of inflammatory cytokines to activate defense mechanisms. However, the precise roles and mechanisms of STING in ADPKD remain elusive. In this study, we show that Pkd1 mutant mouse kidneys show upregulation of STING, which is stimulated by the DNAs of nuclear and mitochondrial origin. The activation of STING promotes cyst growth through increasing (1) the activation of NF-κB in Pkd1 mutant cells and (2) the recruitment of macrophages in the interstitial and peri-cystic regions in Pkd1 mutant mouse kidneys via NF-κB mediating the upregulation of TNF-α and MCP-1. Targeting STING with its specific inhibitor C-176 delays cyst growth in an early-stage aggressive Pkd1 conditional knockout mouse model and a milder long-lasting Pkd1 mutant mouse model. Targeting STING normalizes mitochondrial structure and function, decreases the formation of micronuclei, induces Pkd1 mutant renal epithelial cell death via p53 signaling, and decreases renal fibrosis in Pkd1 mutant mouse kidneys. These results support that STING is a novel therapeutic target for ADPKD treatment.

STING-Activating Small Molecular Therapeutics for Cancer Immunotherapy.

Immuno-oncology has become a revolutionary strategy for cancer treatment. Therapeutic interventions based on adaptive immunity through immune checkpoint therapy or chimeric antigen receptor (CAR) T cells have received clinical approval for monotherapy and combination treatment in various cancers. Although these treatments have achieved clinical successes, only a minority of cancer patients show a response, highlighting the urgent need to discover new therapeutic molecules that could be exploited to improve clinical outcomes and pave the way for the next generation of immunotherapy. Given the critical role of the innate immune system against infection and cancer, substantial efforts have been dedicated to developing novel anticancer therapeutics that target these pathways. Targeting the stimulator of interferon genes (STING) pathway is a powerful strategy to generate a durable antitumor response, and activation of the adaptor protein STING induces the initiation of transcriptional cascades, thereby producing type I interferons, pro-inflammatory cytokines and chemokines. Various STING agonists, including natural or synthetic cyclic dinucleotides (CDNs), have been developed as anticancer therapeutics. However, since most CDNs are confined to intratumoral administration, there has been a great interest in developing non-nucleotide agonists for systemic treatment. Here, we review the current development of STING-activating therapeutics in both preclinical and clinical stages.

LARP7 Contributes to Glucose-Induced Cardiac Dysfunction, Apoptosis and Fibrosis by Inhibiting the Degradation of STING.

Diabetic cardiomyopathy (DCM) is an important cause of heart failure in diabetic patients. The aim of this study was to investigate the pathogenesis of DCM and to identify potential therapeutic targets. A mouse model of type 1 DCM was constructed by continuous intraperitoneal injection of streptozotocin (STZ). Systolic and diastolic functions were measured by ultrasound. The expression of La-related protein 7 (LARP7), the stimulator of interferon genes (STING) pathway and light chain 3 (LC3) in myocardial tissue was detected by Western blot and immunofluorescence analyses. Neonatal mouse ventricular cardiomyocytes (NMVCMs) were isolated and cultured. An in vitro type 1 diabetes mellitus (T1DM) model was established by treatment with high glucose. Knockdown/overexpression of LARP7 and STING was achieved by adenovirus transduction, C-176 (a potent covalent inhibitor of STING), and plasmid transfection. The expression, activation, and localization of STING and LARP7 in cardiomyocytes was evaluated, as well as the interaction between the two. The effect of this interaction on the STING-dependent autophagy‒lysosomal pathway was also explored. In addition, the fibrosis and apoptosis of cardiomyocytes were evaluated. High glucose was found to increase the expression and activation of STING and LARP7 in mouse myocardial tissue. This was accompanied by myocardial fibrosis, impaired autophagy degradation function and impaired cardiac function. These findings were further confirmed by in vitro experiments. High glucose caused LARP7 to translocate from the nucleus to the cytoplasm, where it interacted with accumulated STING to inhibit its degradation. Inhibition of STING or LARP7 expression significantly improved myocardial injury induced by high glucose. Targeted inhibition of LARP7 or STING expression may be a potential therapeutic strategy for the treatment of DCM.

Activation of STING in pancreatic cancer-associated fibroblasts exerts an antitumor effect by enhancing tumor immunity

Pancreatic ductal adenocarcinoma (PDAC) has a high mortality rate; therefore, the development of effective treatments is a priority. The stimulator of interferon genes (STING) pathway enhances tumor immunity by inducing the production of type 1 interferon (IFN) and proinflammatory cytokines and chemokines and promoting the infiltration of cytotoxic T cells. To assess the function of STING on pancreatic tumorigenesis, Ptf1aER-Cre/+ LSL-KrasG12D/+ p53loxP/loxP mice (KPC mice) and Ptf1aER-Cre/+ LSL-KrasG12D/+ p53loxP/loxP/STING−/− mice (KPCS mice) were generated. However, STING deletion did not affect pancreatic tumorigenesis in mice. Because STING is expressed not only in immune cells but also in cancer-associated fibroblasts (CAFs), we evaluated the STING function in PDAC CAFs. A mouse STING agonist 5,6-Dimethyl-9-oxo-9H-xanthene-4-acetic acid (DMXAA) was administered to KPC mice and CAFs from KPC mice and the resulting immune response was evaluated. DMXAA activated STING in PDAC CAFs in KPC mice, promoting cytotoxic T cell infiltration by secreting proinflammatory cytokines and enhancing tumor immunity. We next generated STING-deficient PDAC cells and subcutaneous tumors in which STING was expressed only in CAFs by performing bone marrow transplantation and assessed the antitumor effect of STING-activated CAFs. The administration of DMXAA to subcutaneous tumors expressing STING only in CAFs sustained the antitumor effect of DMXAA. About half of human PDACs lacked STING expression in the cancer stroma, suggesting that STING activation in PDAC CAFs exerts an antitumor effect, and STING agonists can be more effective in tumors with high than in those with low STING expression in the stroma.

TBHQ mitigates fatty liver ischemia–reperfusion injury by activating Nrf2 to attenuate hepatocyte mitochondrial damage and macrophage STING activation

BackgroundLiver ischemia–reperfusion (IR) injury is an inevitable pathophysiological process in various liver surgeries. Previous studies have found that IR injury is exacerbated in fatty liver due to significant hepatocellular damage and macrophage inflammatory activation, though the underlying mechanisms are not fully understood. In this study, we aim to explore the role and mechanism of Nrf2 (Nuclear factor erythroid 2-related factor 2) signaling in regulating hepatocellular damage and macrophage immune response in fatty liver IR injury. MethodsThe study used high-fat diet-induced fatty liver mice to establish an IR model, alongside an in vitro co-culture system of primary hepatocytes and macrophages. This approach was used to examine mitochondrial dysfunction, oxidative stress, mitochondrial DNA (mtDNA) release, and activation of macrophage STING (Stimulator of interferon genes) signaling. We also conducted recovery verification using H-151 (a STING inhibitor) and tBHQ (an Nrf2 activator). ResultsCompared to the control group, mice on a high-fat diet demonstrated more severe liver IR injury, as evidenced by increased histological damage, elevated liver enzyme levels, and heightened inflammatory markers. The HFD group showed significant oxidative stress and mitochondrial dysfunction and damage post-IR, as indicated by elevated levels of ROS and lipid peroxidation markers, and decreased antioxidant enzyme activity. Elevated mtDNA release from hepatocytes post-IR activated macrophage STING signaling, worsening inflammation and liver damage. However, STING signaling inhibition with H-151 in vivo or employing STING knockout macrophages significantly reduced these injuries. In-depth mechanism studies have found that the transfer of Nrf2 protein into the nucleus of liver cells after IR in fatty liver is reduced. Pre-treatment with tBHQ ameliorated liver oxidative stress, mitochondrial damage and suppressed the macrophage STING signaling activation. ConclusionsOur study reveals a novel mechanism where the interaction between hepatocellular damage and macrophage inflammation intensifies liver IR injury in fatty liver. Enhancing Nrf2 activation to protect mitochondrial from oxidative stress damage and inhibiting macrophage STING signaling activation emerge as promising strategies for clinical intervention in fatty liver IR injury.

STING orchestrates the neuronal inflammatory stress response in multiple sclerosis

Inflammation-induced neurodegeneration is a defining feature of multiple sclerosis (MS), yet the underlying mechanisms remain unclear. By dissecting the neuronal inflammatory stress response, we discovered that neurons in MS and its mouse model induce the stimulator of interferon genes (STING). However, activation of neuronal STING requires its detachment from the stromal interaction molecule 1 (STIM1), a process triggered by glutamate excitotoxicity. This detachment initiates non-canonical STING signaling, which leads to autophagic degradation of glutathione peroxidase 4 (GPX4), essential for neuronal redox homeostasis and thereby inducing ferroptosis. Both genetic and pharmacological interventions that target STING in neurons protect against inflammation-induced neurodegeneration. Our findings position STING as a central regulator of the detrimental neuronal inflammatory stress response, integrating inflammation with glutamate signaling to cause neuronal cell death, and present it as a tractable target for treating neurodegeneration in MS.

Spatiotemporal release of non-nucleotide STING agonist and AKT inhibitor from implantable 3D-printed scaffold for amplified cancer immunotherapy

Immunotherapy through the activation of the stimulator of interferon genes (STING) signaling pathway is increasingly recognized for its robust anti-tumor efficacy. However, the effectiveness of STING activation is often compromised by inadequate anti-tumor immunity and a scarcity of primed immune cells in the tumor microenvironment. Herein, we design and fabricate a co-axial 3D-printed scaffold integrating a non-nucleotide STING agonist, SR-717, and an AKT inhibitor, MK-2206, in its respective shell and core layers, to synergistically enhance STING activation, thereby suppressing tumor recurrence and growth. SR-717 initiates the STING activation to enhance the phosphorylation of the factors along the STING pathway, while MK-2206 concurrently inhibits the AKT phosphorylation to facilitate the TBK1 phosphorylation of the STING pathway. The sequential and sustained release of SR-717 and MK-2206 from the scaffold results in a synergistic STING activation, demonstrating substantial anti-tumor efficacy across multiple tumor models. Furthermore, the scaffold promotes the recruitment and enrichment of activated dendritic cells and M1 macrophages, subsequently stimulating anti-tumor T cell activity, thereby amplifying the immunotherapeutic effect. This precise and synergistic activation of STING by the scaffold offers promising potential in tumor immunotherapy.

African swine fever virus MGF505-6R attenuates type I interferon production by targeting STING for degradation.

African swine fever (ASF) is an acute hemorrhagic and devastating infectious disease affecting domestic pigs and wild boars. It is caused by the African swine fever virus (ASFV), which is characterized by genetic diversity and sophisticated immune evasion strategies. To facilitate infection, ASFV encodes multiple proteins to antagonize host innate immune responses, thereby contributing to viral virulence and pathogenicity. The molecular mechanisms employed by ASFV-encoded proteins to modulate host antiviral responses have not been comprehensively elucidated. In this study, it was observed that the ASFV MGF505-6R protein, a member of the multigene family 505 (MGF505), effectively suppressed the activation of the interferon-beta (IFN-β) promoter, leading to reduced mRNA levels of antiviral genes. Additional evidence has revealed that MGF505-6R antagonizes the cGAS-STING signaling pathway by interacting with the stimulator of interferon genes (STING) for degradation in the autophagy-lysosomal pathway. The domain mapping revealed that the N-terminal region (1-260aa) of MGF505-6R is the primary domain responsible for interacting with STING, while the CTT domain of STING is crucial for its interaction with MGF505-6R. Furthermore, MGF505-6R also inhibits the activation of STING by reducing the K63-linked polyubiquitination of STING, leading to the disruption of STING oligomerization and TANK binding kinase 1 (TBK1) recruitment, thereby impairing the phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3). Collectively, our study elucidates a novel strategy developed by ASFV MGF505-6R to counteract host innate immune responses. This discovery may offer valuable insights for further exploration of ASFV immune evasion mechanisms and antiviral strategies.

RING finger protein 122-like (RNF122L) negatively regulates antiviral immune response by targeting STING in common carp (Cyprinus carpio L.)

Stimulator of interferon genes (STING), as an imperative adaptor protein in innate immune, responds to nucleic acid from invading pathogens to build antiviral responses in host cells. Aberrant activation of STING may trigger tissue damage and autoimmune diseases. Given the decisive role in initiating innate immune response, the activity of STING is intricately governed by several posttranslational modifications, including phosphorylation and ubiquitination. Here, we cloned and characterized a novel RNF122 homolog from common carp (named CcRNF122L). Expression analysis disclosed that the expression of CcRNF122L is up-regulated under spring viremia of carp virus (SVCV) stimulation in vivo and in vitro. Overexpression of CcRNF122L hampers SVCV- or poly(I:C)-mediated the expression of IFN-1 and ISGs in a dose-dependent way. Mechanistically, CcRNF122L interacts with STING and promotes the polyubiquitylation of STING. This polyubiquitylation event inhibits the aggregation of STING and the subsequent recruitment of TBK1 and IRF3 to the signaling complex. Additionally, the deletion of the TM domain abolishes the negative regulatory function of CcRNF122L. Collectively, our discoveries unveil a mechanism that governs the STING function and the precise adjustment of the innate immune response in teleost.

MDIVI-1 ALLEVIATES SEPSIS-INDUCED LIVER INJURY BY INHIBITING STING SIGNALING ACTIVATION.

Proinflammatory hyperactivation of Kupffer cells (KCs) is foremost involved in the pathogenesis of sepsis-induced liver injury. Our previous study found that stimulator of interferon genes (STING) signaling was activated in KCs in response of lipopolysaccharide (LPS) and knocking down dynamin-related protein 1 (DRP1) in KCs effectively inhibited the activation of STING signaling and the subsequent production of proinflammatory cytokines. In this study, we demonstrated that in vivo treatment with mitochondrial division inhibitor 1 (Mdivi-1), a selective inhibitor of DRP1, alleviated cecal ligation and puncture (CLP)-induced liver injury with the improvement of liver pathology and function. Moreover, we found that STING in liver was mainly concentrated in KCs and STING signaling was significantly activated in KCs after CLP. The STING deficiency effectively ameliorated liver injury and decreased the mortality of septic mice, which were reversely worsened by the enhanced activation of STING with DMXAA. The further study showed that Mdivi-1 markedly attenuated STING signaling activation in KCs and inhibited systemic inflammatory response. Importantly, DMXAA application in CLP mice blunted Mdivi-1's liver protection effect. Taken together, our study confirmed Mdivi-1 effectively alleviated CLP-induced liver injury partially through inhibiting STING signaling activation in KCs, which provides new insights and a novel potential pharmacological therapeutic target for treating septic liver injury.

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.

Synthesis of Original Cyclic Dinucleotide Analogues Using the Sulfo-click Reaction.

The stimulator of interferon genes (STING) protein plays a crucial role in the activation of the innate immune response. Activation of STING is initiated by cyclic dinucleotides (CDNs) which prompted the community to synthesize structural analogues to enhance their biological properties. We present here the synthesis and biological evaluation of four novel CDN analogues composed of an N-acylsulfonamide linkage. These CDNs were obtained in high overall yields via the sulfo-click reaction as a key step.

STING activation in cardiomyocytes drives hypertrophy-associated heart failure via NF-κB-mediated inflammatory response

Accumulating evidence highlights the key importance of innate immunity in heart hypertrophy and failure. Though stimulator of interferon genes (STING) is an integral innate immunity regulator, whether cardiomyocyte-derived STING driving cardiac hypertrophy and failure has rarely been explored, nor has its underlying mechanism been clarified. Herein, we addressed these two questions through several mouse experiments. Our results revealed that cardiac tissues from patients exhibiting cardiac hypertrophy markedly increased STING expression. Myocardial tissues of mice challenged with angiotensin II (Ang II) or transverse aortic constriction (TAC) also showed that STING was consistently upregulated and activated. Activation of STING by cGAMP or DMXAA resulted in cardiomyocyte hypertrophy in vitro, which was abolished by STING knockout. Furthermore, deleting or pharmacologically inhibiting STING attenuated cardiac hypertrophy and dysfunction in TAC or Ang II-treated mice. In contrast, mice with cardiomyocyte-specific STING activation developed cardiac hypertrophy and failure. Mechanistically, NF-κB signaling but not TBK1 or autophagy formation was implicated in STING -induced cardiac hypertrophy and failure. Collectively, we identified that STING-NF-κB axis mediated inflammatory response to drive cardiac hypertrophy-associated heart failure, highlighting its promise as a potential therapeutic target in clinical practice.

ENPP1 is an innate immune checkpoint of the anticancer cGAMP-STING pathway in breast cancer.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) expression correlates with poor prognosis in many cancers, and we previously discovered that ENPP1 is the dominant hydrolase of extracellular cGAMP: a cancer-cell-produced immunotransmitter that activates the anticancer stimulator of interferon genes (STING) pathway. However, ENPP1 has other catalytic activities and the molecular and cellular mechanisms contributing to its tumorigenic effects remain unclear. Here, using single-cell RNA-seq, we show that ENPP1 in both cancer and normal tissues drives primary breast tumor growth and metastasis by dampening extracellular 2'3'-cyclic-GMP-AMP (cGAMP)-STING-mediated antitumoral immunity. ENPP1 loss-of-function in both cancer cells and normal tissues slowed primary tumor growth and abolished metastasis. Selectively abolishing the cGAMP hydrolysis activity of ENPP1 phenocopied ENPP1 knockout in a STING-dependent manner, demonstrating that restoration of paracrine cGAMP-STING signaling is the dominant anti-cancer mechanism of ENPP1 inhibition. Finally, ENPP1 expression in breast tumors deterministically predicated whether patients would remain free of distant metastasis after pembrolizumab (anti-PD-1) treatment followed by surgery. Altogether, ENPP1 blockade represents a strategy to exploit cancer-produced extracellular cGAMP for controlled local activation of STING and is therefore a promising therapeutic approach against breast cancer.

Sting Activation in Bone Marrow Macrophages Targets AML Blasts for Phagocytosis in an ICAM-1 Dependent Mechanism

The initiation and proliferation of acute myeloid leukaemia (AML) is in part regulated by the bone marrow microenvironment. Previous work by our group has shown that the stimulator of interferon genes (STING) pathway is activated in macrophages in the AML bone marrow microenvironment (Moore et al., 2022) . We found that AML-derived mitochondrial damage-associated molecular patterns were processed by bone marrow macrophages (BMMs) via LC3-associated phagocytosis (LAP). Furthermore, activation of STING resulted in a suppression of AML growth through an LAP dependent mechanism. Here we have investigated how the activation of STING in bone marrow macrophages increases macrophage phagocytic activity and in turn supresses the growth of AML. To understand the role of STING activation in bone marrow derived macrophages (BMDMs) in AML we used various STING agonists including DMXAA, 2'3'-cGAMP, and CpG oligonucleotides. We showed STING activation was induced through these agonists by measuring the upregulation of STING related genes ( Gbp2, Irf7 and Ifit3). We demonstrated that STING activation caused increased phagocytic clearance of live MN1 cells (mouse AML cell line) in an in vitro phagocytosis assay. In order to investigate how STING activation increases the phagocytic clearance of AML cells by BMDMs, we used three complementary methods. First, we performed a receptor tyrosine kinase array to measure the phosphorylation of mouse kinases following STING activation. Second, we investigated the transcriptomic changes resulting from STING activation with mRNA-sequencing. Third, after combining these results we identified an upregulation of key phagocytic pathways that specifically led us to focus on the adhesion molecules, in particular ICAM-1. Further investigation using an anti-ICAM-1 blocking antibody showed a reduction in the phagocytic potential of the BMDMs demonstrating the role of ICAM-1 in the phagocytic clearance of AML. Next, we activated human macrophages (derived from peripheral blood monocytes collected from healthy donors with approval of the East of England - Cambridgeshire and Hertfordshire Research Ethics Committee and the UK Health Research Authority, IRAS Project ID 33753) through STING which depleted AML in vitro confirming the results obtained using the mouse system. To investigate the in vivo significance of these findings, we treated C57Bl/6 animals with DMXAA and observed a significantly decreased tumour burden and increased survival following engraftment with MN1 cells. Cytotoxic T cell activity and number in the bone marrow was unchanged by STING activation in this model. In summary, we have shown that the activation of STING in bone marrow macrophages directly supresses the growth and proliferation of AML by stimulating the phagocytic activity of the macrophages, and the clearance of the leukaemic cells.

Direct, Potent, and TP53-Independent Activity of Sting Agonists Against Acute Myeloid Leukaemia Enhances Venetoclax Efficacy In Vivo

Introduction TP53 mutation occurs in approximately 20% of patients with newly diagnosed acute myeloid leukaemia (AML). Treatment outcomes are universally poor and discovery of new therapies with TP53 independent activity represents an area of very high unmet need. Cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) sense cytoplasmic double-stranded DNA to elicit type I interferon production. Direct pharmacologic activation of STING has been investigated in clinical trials with the goal of enhancing immunogenicity. During apoptosis, activated caspases have been shown to suppress activation of the cGAS/STING pathway to render cell death immunologically silent. Whilst the canonical role of STING is stimulation of an inflammatory response, we now show that direct activation of STING by potent and clinically relevant agonists can induce apoptosis directly in cancerous cells by leveraging the high levels of expressed STING in a variety of blood cancers. Importantly, apoptosis induced by STING agonists may be enhanced through combination with diverse BH3-mimetic drugs in a TP53 independent manner (Diepstraten et al, submitted). In this abstract, we highlight the activity of STING agonists in AML and its potential for clinical testing, especially among patients with TP53 mutated disease. Methods We tested the clinically relevant small molecule STING agonist diABZI (purchased from SYNthesis Med Chem), which is currently in early phase clinical trials (NCT03843359, NCT05424380). diABZI was tested alone or in combination with the BCL-2 specific BH3 mimetic drug venetoclax (purchased from Chemgood). Drug sensitivity assays were performed using human AML derived cell lines and primary bone marrow or peripheral blood mononuclear cells from patients with AML or myelodysplastic syndrome with excess blasts 2 (MDS-EB2). Cells were cultured for 48 hours, and cell death assessed by PI or DAPI staining. In vivo experiments were conducted by transplanting NOD- scidIL2Rgamma null (NSG) mice with MOLM-13 TP53 wildtype (WT) or TP53 knockout(KO) cells. Mice then received either no treatment (control), diABZI alone (1.5 mg/kg intravenously twice a week), venetoclax alone (50 mg/kg orally Monday-Friday), or combination diABZI and venetoclax for two weeks ( n=5 per treatment arm). Survival analysis was performed using the log-rank (Mantel-Cox) test with Bonferroni correction. Results Single agent diABZI was active in 5/6 human AML cell lines tested, including cell lines with TP53 mutation. Isogenic MOLM-13 TP53 KO cells demonstrated the same sensitivity as the TP53 WT parental cells. BAX/BAK KO and STING KO cell lines were resistant to diABZI, confirming that cell death occurred by apoptosis being dependent on BAX/BAK as well as STING. Combination of diABZI and venetoclax resulted in additive killing in MOLM-13 and OCI-AML3 cells (Bliss scores of 7.62 and 8.06, respectively). Ex vivo testing was performed on 18 different primary specimens (16 AML and 2 MDS-EB2; 12/18 cases had high risk genetic or clinical features such TP53 mutation, complex karyotype, secondary disease, or relapse after prior therapy) (Figure 1). Strikingly, diABZI was active as a single agent in 16/18 cases, with marked efficacy in 11 cases (IC 50 <100 nM). The addition of venetoclax enhanced killing of AML cells, especially in cases resistant to STING agonist alone. In vivo, diABZI monotherapy and combination therapy with venetoclax was well tolerated without significant weight loss or hematologic toxicity. Treatment with diABZI prolonged survival in the highly aggressive MOLM-13 xenograft model (Figure 2). In both the TP53 WT and TP53 KO context, single agent diABZI was superior to single agent venetoclax ( p=0.012 and p=0.011, respectively), but survival was most prolonged in mice receiving combination therapy ( p=0.013and p=0.012, respectively). Conclusion Activation of STING can exert direct anti-tumour effects in blood cancers via induction of apoptosis. Human AML cells are highly sensitive to the pro-apoptotic effects of STING agonists. The combination of venetoclax and STING agonist shows marked efficacy in ex vivo and in vivo studies, including in models of TP53 defective AML. Combining STING agonists with venetoclax represents a highly promising and novel therapeutic approach for AML and should be considered for accelerated early phase clinical trials.

Activation of human STING by a molecular glue-like compound

Stimulator of interferon genes (STING) is a dimeric transmembrane adapter protein that plays a key role in the human innate immune response to infection and has been therapeutically exploited for its antitumor activity. The activation of STING requires its high-order oligomerization, which could be induced by binding of the endogenous ligand, cGAMP, to the cytosolic ligand-binding domain. Here we report the discovery through functional screens of a class of compounds, named NVS-STGs, that activate human STING. Our cryo-EM structures show that NVS-STG2 induces the high-order oligomerization of human STING by binding to a pocket between the transmembrane domains of the neighboring STING dimers, effectively acting as a molecular glue. Our functional assays showed that NVS-STG2 could elicit potent STING-mediated immune responses in cells and antitumor activities in animal models.

STING activation promotes autologous type I interferon-dependent development of type 1 regulatory T cells during malaria.

The development of highly effective malaria vaccines and improvement of drug-treatment protocols to boost antiparasitic immunity are critical for malaria elimination. However, the rapid establishment of parasite-specific immune regulatory networks following exposure to malaria parasites hampers these efforts. Here, we identified stimulator of interferon genes (STING) as a critical mediator of type I interferon production by CD4+ T cells during blood-stage Plasmodium falciparum infection. The activation of STING in CD4+ T cells by cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) stimulated IFNB gene transcription, which promoted development of IL-10- and IFN-γ-coproducing CD4+ T (type I regulatory [Tr1]) cells. The critical role for type I IFN signaling for Tr1 cell development was confirmed in vivo using a preclinical malaria model. CD4+ T cell sensitivity to STING phosphorylation was increased in healthy volunteers following P. falciparum infection, particularly in Tr1 cells. These findings identified STING expressed by CD4+ T cells as an important mediator of type I IFN production and Tr1 cell development and activation during malaria.