Stimulator Of Interferon Genes Protein Research Articles

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 T cells have received clinical approval for monotherapy and combination treatment use 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 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 or clinical stages.

The journey of STING: Guiding immune signaling through membrane trafficking

Stimulator of Interferon Genes (STING) serves as a pivotal mediator in the innate immune signaling pathway, transducing signals from various DNA receptors and playing a crucial role in natural immune processes. During cellular quiescence, STING protein resides in the endoplasmic reticulum (ER), and its activation typically occurs through the cGAS-STING signaling pathway. Upon activation, STING protein is transported to the Golgi apparatus, thereby initiating downstream signaling cascades. Vesicular transport serves as the primary mechanism for STING protein trafficking between the ER and Golgi apparatus, with COPII mediating anterograde transport from the ER to Golgi apparatus, while COPI is responsible for retrograde transport. Numerous factors influence these transport processes, thereby exerting either promoting or inhibitory effects on STING protein expression. Upon reaching the Golgi apparatus, to prevent over-activation, STING protein is transported to post-Golgi compartments for degradation. In addition to the conventional lysosomal degradation pathway, ESCRT has also been identified as one of the degradation pathways for STING protein. This review summarizes the recent findings on the membrane trafficking pathways of STING, highlighting their contributions to the regulation of cytokine production, the activation of immune cells, and the coordination of immune signaling pathways.

Stimulator of Interferon Genes Protein (STING) Expression in Cancer Cells: A Tissue Microarray Study Evaluating More than 18,000 Tumors from 139 Different Tumor Entities.

Stimulator of interferon genes protein (STING) activates the immune response in inflammatory cells. STING expression in cancer cells is less well characterized, but STING agonists are currently being evaluated as anticancer drugs. A tissue microarray containing 18,001 samples from 139 different tumor types was analyzed for STING by immunohistochemistry. STING-positive tumor cells were found in 130 (93.5%) of 139 tumor entities. The highest STING positivity rates occurred in squamous cell carcinomas (up to 96%); malignant mesothelioma (88.5%-95.7%); adenocarcinoma of the pancreas (94.9%), lung (90.3%), cervix (90.0%), colorectum (75.2%), and gallbladder (68.8%); and serous high-grade ovarian cancer (86.0%). High STING expression was linked to adverse phenotypes in breast cancer, clear cell renal cell carcinoma, colorectal adenocarcinoma, hepatocellular carcinoma, and papillary carcinoma of the thyroid (p < 0.05). In pTa urothelial carcinomas, STING expression was associated with low-grade carcinoma (p = 0.0002). Across all tumors, STING expression paralleled PD-L1 positivity of tumor and inflammatory cells (p < 0.0001 each) but was unrelated to the density of CD8+ lymphocytes. STING expression is variable across tumor types and may be related to aggressive tumor phenotype and PD-L1 positivity. The lack of relationship with tumor-infiltrating CD8+ lymphocytes argues against a significant IFN production by STING positive tumor cells.

BRD4 promotes LPS-induced endothelial cells senescence via activating and cooperating STING-IRF3 pathway

Endothelial cells (ECs) senescence is closely associated with the initiation and development of multiple age-related cardiovascular diseases. It is necessary to explore the underlying molecular mechanisms of ECs senescence, which is not only the basis to decipher cellular senescence, but also a novel therapeutic target for the endothelial senescence-related diseases. BRD4, a key epigenetic regulator, is universally related to gene expression regulation and has been reported to accelerate cell senescence. Besides, emerging evidence has suggested that the stimulator of interferon genes protein (STING) can regulate inflammatory and senescence-related diseases. However, whether STING pathway activation is regulated by BRD4 in the context of ECs senescence remains largely unclear. Here, we observed that elevated BRD4 and activated STING-IRF3 signaling pathway during ECs senescence and further confirmed that BRD4 could abolish STING activation. We demonstrated that BRD4 could inhibit E3 ubiquitin ligase HRD1-mediated ubiquitination degradation of STING via inhibiting HRD1 transcription. In addition to the direct regulatory effect of BRD4 on STING activation, we have confirmed that BRD4 cooperates with IRF3 and P65 to promote SASP gene expression, thereby accelerating ECs senescence. Here, we proposed a novel mechanism underlying BRD4’ key dual role in activating the STING pathway during ECs senescence.

Experimental congenital diaphragmatic hernia features an alteration of DNA sensing targets cGAS and STING.

The pathogenesis of congenital diaphragmatic hernia (CDH) depends on multiple factors. Activation of the DNA-sensing cyclic-GMP-AMP-synthase (cGAS) and Stimulator-of-Interferon-Genes (STING) pathway by double-stranded DNA (dsDNA) links environmental stimuli and inflammation. We hypothesized that nitrofen exposure alters cGAS and STING in human bronchial epithelial cells and fetal rat lungs. We used the Quant-IT™-PicoGreen™ assay to assess dsDNA concentration in BEAS-2B cells after 24 h of nitrofen-exposure and performed immunofluorescence of cGAS/STING. We used nitrofen to induce CDH and harvested control and CDH lungs at embryonic day E15, E18 and E21 for cGAS/STING immunofluorescence, RT-qPCR and RNA-Scope™ in-situ-hybridization (E18, E21). We found a higher concentration of dsDNA following nitrofen treatment. Nitrofen-exposure to BEAS-2B cells increased cGAS and STING protein abundance. cGAS abundance was higher in nitrofen lungs at E15, E18 and E21. RNA-Scope in-situ-hybridization showed higher cGAS and STING expression in E18 and E21 lungs. RT-qPCR revealed higher mRNA expression levels of STING in E21 nitrofen-induced lungs. Our data suggest that nitrofen-exposure increases dsDNA content which leads to stimulation of the cGAS/STING pathway in human BEAS-2B cells and the nitrofen rat model of CDH. Consequently, DNA sensing and the cGAS-STING-pathway potentially contribute to abnormal lung development in CDH. We found an alteration of DNA sensing targets cGAS and STING in human BEAS-2B cells and experimental congenital diaphragmatic hernia with higher protein abundance and mRNA expression in cells and lung sections of nitrofen-treated rat pups. This is the first study to investigate DNA sensing, a potential link between environmental stimuli and inflammation, in experimental CDH. Our study extends the knowledge on the pathogenesis of experimental CDH.

Advances in the prerequisite and consequence of STING downstream signalosomes

Abstract The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is an evolving DNA-sensing mechanism involved in innate immunity and pathogen defense that has been optimized while remaining conserved. Aside from recognizing pathogens through conserved motifs, these receptors also detect aberrant or misplaced self-molecules as possible signs of perturbed homeostasis. Upon binding external or self-derived DNA, a mobile secondary messenger 2′3′-cyclic GMP-AMP (cGAMP) is produced by cGAS and in turn activates its adapter STING in the endoplasmic reticulum (ER). Resting-state or activated STING protein is finely restricted by multiple degradation machineries. The post-translational changes of the STING protein, along with the regulatory machinery of the secret routes, limit the onset, strength and sustention of STING signal. STING experiences a conformational shift and relocates with TBK1 from the ER to perinuclear vesicles containing transcription factors, provoking the transcription activity of IRF3/IFN-I and NF-κB pathways, as well as to initiate a number of cellular processes that have been shown to alter the immune landscape in cancer, such as autophagy, NLRP3 inflammasome, ER stress, and cell death. STING signal thus serves as a potent activator for immune mobilization yet also triggers immune-mediated pathology in tissues. Recent advances have established the vital role of STING in immune surveillance as well as tumorigenic process. This review provides an overview of the disparate outcomes of cancer attributed to the actions of pleiotropic and coordinated STING downstream signalosomes, along with the underlying mechanisms of STING function in pathologies, providing therapeutic implications for new approaches in hunt for the next generation of cancer immunotherapy base on STING.

The role of HPV11 E7 in modulating STING-dependent interferon β response in recurrent respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP) is a rare benign tumor caused mainly by the infection of the respiratory tract epithelial cells by the human papillomavirus (HPV) type 6/11. However, the specific mechanisms underlying the inhibition of the host's innate immune response by HPV remain unclear. For this purpose, we employed single-cell RNA sequencing to analyze the states of various immune cells in RRP samples post-HPV infection and utilized a cellular model of HPV infection to elucidate the mechanisms by which HPV evades the innate immune system in RRP. The results revealed distinct immune cell heterogeneity in RRP and demonstrated that HPV11 E7 can inhibit the phosphorylation of the stimulator of interferon genes protein, thereby circumventing the body's antiviral response. In vitro co-culture experiments demonstrated that stimulation of macrophages to produce interferon-beta induced the death of HPV-infected epithelial cells, also reducing HPV viral levels. In summary, our study preliminarily identifies the potential mechanisms by which HPV evades the host's antiviral immune response, as well as the latent antiviral functions exhibited by activated macrophages. This research serves as an initial exploration of antiviral immune evasion in RRP, laying a solid foundation for investigating immunotherapeutic approaches for the disease.IMPORTANCESurgical tumor reduction is the most common treatment for recurrent respiratory papillomatosis (RRP). One of the characteristics of RRP is its persistent recurrence, and multiple surgeries are usually required to control the symptoms. Recently, some adjuvant therapies have shown effectiveness, but none of them can completely clear human papillomavirus (HPV) infection, and thus, a localized antiviral immune response is significant for disease control; after all, HPV infection is limited to the epithelium. Inhibition of interferon-beta (IFN-β) secretion by HPV11 E7 viral proteins in epithelial cells by affecting stimulator of interferon genes phosphorylation may account for the persistence of low-risk HPV replication in the RRP. Moreover, suppression of the IFN-I pathway in RRP cell types might provide clues regarding the hyporeactive function of local immune cells. However, activation of macrophage groups to produce IFN-β can still destroy HPV-infected cells.

Abstract LB324: STING protein expression is increased in intermediate luminal cells in proliferative inflammatory atrophy of the prostate (PIA) and is absent in the majority of primary prostatic adenocarcinomas

Abstract Unresolved chronic inflammation has been postulated to drive prostate cancer development. We have proposed that proliferative inflammatory atrophy (PIA) can lead to the development of preneoplastic high grade prostatic intraepithelial neoplasia (PIN) lesions and/or directly to adenocarcinoma at times. Additionally, some PIA cells harbor molecular alterations seen in PIN and invasive carcinoma lesions, with a low frequency of PIA cells showing MYC over-expression, partial promoter hypermethylation and silencing of GSTP1, and formation of ETS gene fusions. PIA appears to result from inflammatory injury and regeneration in which proliferative and atrophic epithelial cells have an intermediate cell phenotype with both luminal and basal cell features, including basal cell markers, keratin 5 and BCL2, as well as varying levels of prostate luminal enriched genes such as androgen receptor, PSA and NKX3.1. Also, a number of gene products that are apparently stress induced, which are not expressed in normal prostate are induced in at least a subset of intermediate luminal cells in PIA, including GSTA1, PTGS2, LTF and PIGR (PMIDs:17384581, 37550827). Stimulator of interferon genes (STING), encoded by TMEM173, is a multipass transmembrane ER associated protein, and is a critical hub for innate responses directed against numerous bacterial, viral and parasitic pathogens, and is important for the induction and maintenance of adaptive immunity in a number of situations. Here, we examined STING expression by IHC, using a genetically validated assay, and show that the majority of atrophic prostatic luminal epithelial cells in all PIA lesions (N=20) consistently express high levels of STING, which is present in normal prostatic basal epithelial cells yet absent in normal-appearing prostatic luminal cells. In primary prostatic adenocarcinomas (&amp;gt; 260 tumors), STING protein was either completely negative , or extremely low in ~ 95% of cases. That STING in PIA is activate is supported by single cell RNA sequencing (scRNAseq) data from human prostatectomy tissues that show cells with the phenotype of intermediate luminal cells, indicative of PIA, express a number of interferon response genes that are not expressed in normal luminal cells (PMID 37905029). To our knowledge this is the first systematic report of STING protein staining in human prostate cancer and benign tissues. We postulate that upregulation of STING in human intermediate cells in PIA represents an inflammatory response that can drive interferon stimulated gene expression and inflammation in PIA. This innate immune response is absent, potentially by gene silencing, in prostatic adenocarcinoma, which may help neoplastic cells evade the immune response and could help contribute to the long standing finding that most prostate cancers are immunologically cold. Citation Format: Thomas M. Steele, Alan K. Meeker, Mindy K. Graham, Rulin Wang, Carolina Gomes-Alexandre, William G. Nelson, Fernanda Caramella Pereiria, Sushant Kachhap, Srinivasan Yegnasubramanian, Angelo Michael De Marzo. STING protein expression is increased in intermediate luminal cells in proliferative inflammatory atrophy of the prostate (PIA) and is absent in the majority of primary prostatic adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB324.

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

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

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

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

Combining VPS34 inhibitors with STING agonists enhances type I interferon signaling and anti-tumor efficacy.

An immunosuppressive tumor microenvironment promotes tumor growth and is one of the main factors limiting the response to cancer immunotherapy. We have previously reported that inhibition of vacuolar protein sorting 34 (VPS34), a crucial lipid kinase in the autophagy/endosomal trafficking pathway, decreases tumor growth in several cancer models, increases infiltration of immune cells and sensitizes tumors to anti-programmed cell death protein 1/programmed cell death 1 ligand 1 therapy by upregulation of C-C motif chemokine 5 (CCL5) and C-X-C motif chemokine 10 (CXCL10) chemokines. The purpose of this study was to investigate the signaling mechanism leading to the VPS34-dependent chemokine increase. NanoString gene expression analysis was applied to tumors from mice treated with the VPS34 inhibitor SB02024 to identify key pathways involved in the anti-tumor response. We showed that VPS34 inhibitors increased the secretion of T-cell-recruitment chemokines in a cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes protein (STING)-dependent manner in cancer cells. Both pharmacological and small interfering RNA (siRNA)-mediated VPS34 inhibition increased cGAS/STING-mediated expression and secretion of CCL5 and CXCL10. The combination of VPS34 inhibitor and STING agonist further induced cytokine release in both human and murine cancer cells as well as monocytic or dendritic innate immune cells. Finally, the VPS34 inhibitor SB02024 sensitized B16-F10 tumor-bearing mice to STING agonist treatment and significantly improved mice survival. These results show that VPS34 inhibition augments the cGAS/STING pathway, leading to greater tumor control through immune-mediated mechanisms. We propose that pharmacological VPS34 inhibition may synergize with emerging therapies targeting the cGAS/STING pathway.

High FLT3 expression increases immune-cell infiltration in the tumor microenvironment and correlates with prolonged disease-free survival in patients with non-small cell lung cancer.

Most of the currently used cancer immunotherapies inhibit the programmed cell death protein 1 (PD1)-programmed cell death 1 ligand 1 (PDL1) axis of T-cells. However, dendritic cells (DCs) controlled by natural killer (NK) cells via the FMS-related tyrosine kinase 3 (FLT3) axis are necessary for activation of T-cells. The aim of the study was to evaluate FLT3 as a prognostic factor and determine its role in immune infiltration (with emphasis on NK cells and DCs). Using The Cancer Genome Atlas (TCGA) database, we performed bioinformatic analysis of the gene expression datasets of 501 lung squamous cell carcinoma (LUSC) and 515 lung adenocarcinoma (LUAD) patient who had corresponding clinical data [analysis was performed in R (version 4.2.0)]. Disease-free survival (DFS) differed between the FLT3-low and FLT3-high expression groups, respectively, in LUSC (61.0 vs 71.3 months P = 0.075) and LUAD (32.7 vs 47.5 months P = 0.045). A tumor microenvironment (TME) with high immune infiltration and rich in T-cell exhaustion markers was observed in the FLT3-high group. We showed overexpression of NK cell and DC gene signatures in the FLT3-high expression group as well as overexpression of key effector genes of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes protein (STING) pathway, which is crucial in response to radiotherapy. High expression of FLT3 in the TME was associated with immune cell infiltration (especially of NK cells and DCs), increased expression of T-cell exhaustion markers and expression of effector genes of the cGAS-STING pathway, which may consequently increase susceptibility to immunotherapy and radiotherapy. High FLT3 expression correlated with prolonged DFS in the LUSC and LUAD cohorts.

CGAS Mediates the Inflammatory Responses of Human Microglial Cells to Genotoxic DNA Damage.

Genomic instability is a key driving force for the development and progression of many age-related neurodegenerative diseases and central nervous system (CNS) cancers. Recently, the cytosolic DNA sensor, cyclic GMP-AMP synthase (cGAS), has been shown to detect and respond to self-DNA accumulation resulting from DNA damaging insults in peripheral cell types. cGAS has been shown to be important in the responses of microglia to DNA viruses and amyloid beta, and we have reported that it underlies the responses of human microglia to exogenous DNA. However, the role of this cytosolic sensor in the detection of self-DNA by glia is poorly understood and its ability to mediate the cellular responses of human microglia to genotoxic DNA damage has not been established. Here, we describe the ability of ionizing radiation and oxidative stress to elicit genomic DNA damage in human microglial cells and to stimulate the production of key inflammatory mediators by these cells in an NF-kB dependent manner. Importantly, we have utilized CRISPR/Cas9 and siRNA-mediated knockdown approaches and a pharmacological inhibitor of the cGAS adaptor protein stimulator of interferon genes (STING) to demonstrate that the cGAS-STING pathway plays a critical role in the generation of these microglial immune responses to such genotoxic insults. Together, these studies support the notion that cGAS mediates the detection of cytosolic self-DNA by microglia, providing a potential mechanism linking genomic instability to the development of CNS cancers and neurodegenerative disorders.

Sequential Delivery of Aptamer and STING Agonist Shuttled by Graphene Oxide

Stimulator of interferon genes (STING) is a prospective target spot of immunological therapy. The development of STING is still in the preliminary stage and full of challenges. such as lack of tumor specificity, and high-dose-induced T-cell apoptosis. Herein, the STING agonists were selectively delivered and released in the tumor through an aptamer (Apt) modified graphene oxide (GO). The STING agonist was released controllably from GO on account of the acidic tumor microenvironment, and then binds specifically to the STING protein, aiming to active STING pathway and stimulate immunosuppressive factors secretion via phosphorylating TBK1 and IFN IRF3, and secreta high-level IL-6. The resulting immunotherapy exhibited significant inhibition of colorectal tumors, and the producing stimulus was sufficient to activate plentiful enough T-cells with adaptability to inhibit the growth and metastasis of tumors. This work helps to promote sustaining study on the targeted delivery of STING agonists and enhanced activation of the STING pathway, and the translation of novel understanding to clinical applications will enhance the efficiency of immunotherapy for cancer with STING agonists.

The common H232 STING allele shows impaired activities in DNA sensing, susceptibility to viral infection, and in monocyte cell function, while the HAQ variant possesses wild-type properties

Different innate immune pathways converge to Stimulator of interferon genes (STING) and trigger type I interferon responses after recognition of abnormal nucleic acids in the cells. This non-redundant function renders STING a major player in immunosurveillance, and an emerging target for cancer and infectious diseases therapeutics. Beyond somatic mutations that often occur in cancer, the human gene encoding STING protein, TMEM173 (STING1), holds great genetic heterogeneity; R232, HAQ (R71H-G230A-R293Q) and H232 are the most common alleles. Although some of these alleles are likely to be hypomorphic, their function is still debated, due to the available functional assessments, which have been performed in biased biological systems. Here, by using genetic background-matched models, we report on the functional evaluation of R232, HAQ and H232 variants on STING function, and on how these genotypes affect the susceptibility to clinically relevant viruses, thus supporting a potential contributing cause to differences in inter-individual responses to infections. Our findings also demonstrate a novel toll-like receptor-independent role of STING in modulating monocytic cell function and differentiation into macrophages. We further supported the interplay of STING1 variants and human biology by demonstrating how monocytes bearing the H232 allele were impaired in M1/M2 differentiation, interferon response and antigen presentation. Finally, we assessed the response to PD-1 inhibitor in a small cohort of melanoma patients stratified according to STING genotype. Given the contribution of the STING protein in sensing DNA viruses, bacterial pathogens and misplaced cancer DNA, these data may support the development of novel therapeutic options for infectious diseases and cancer.

Plasmacytoid Dendritic Cells, the Expression of the Stimulator of Interferon Genes Protein (STING) and a Possible Role of Th17 Immune Response in Cervical Lesions Mediated by Human Papillomavirus.

Human papillomavirus (HPV) is a virus with a DNA structure that specifically targets squamous epithelial cells. In individuals with a healthy immune system, HPV infection is typically resolved naturally, leading to spontaneous regression. However, when the viral genetic material integrates into the host DNA, it can disrupt the immune response and eventually give rise to neoplastic manifestations. Remarkably, HPV infection appears to activate a protein called Stimulator of Interferon genes (STING), which contributes to the infiltration of Treg Foxp3 + cells. This cellular response acts as a predisposing factor in patients with HPV, potentially exacerbating the progression of the infection. The STING is versatile in different circumstances and can play a role in the immune response as an anti-tumour therapeutic target in HPV-related carcinogenesis. The function of Th17 cells is ambiguous, depending on the microenvironment in the tumour. In this study, 46 biopsies of the uterine cervix of human immunodeficiency virus (HIV) positive patients were divided into three groups: I-cervicitis (10); II-low-grade intraepithelial neoplasia (26); III-moderate or severe intraepithelial neoplasia (10) and it was performed an immunohistochemical technique with the specific antibodies to HPV, CD123, STING and IL17. Immunostained cells were quantified and statistically analysed. Antigens of HPV were detected in the cervical intraepithelial neoplasia (CIN) groups and were absent in cervicitis group. The expression of CD123 was positive in 10.87% of the casuistic, with no statistical difference among groups. STING was present in the three groups. Group 1 presented an area fraction that varied from 3 to 20%, group 2 presented a variation of 3-23% and group 3 presented an area fraction between 4 and 12%. Cells expressing IL17 were present in three groups, more frequent in cervicitis. Considering that the casuistic is composed of women carrying HIV, this infectious agent could influence the numerical similarities of the cells studied among three groups, even in the absence of HPV.

Sting mutation attenuates acetaminophen-induced acute liver injury by limiting NLRP3 activation

Acetaminophen (N-acetyl-p-aminophenol; APAP), a widely used effective nonsteroidal anti-inflammatory drug, leads to acute liver injury at overdose worldwide. Evidence showed that the severity of liver injury associated with the subsequent involvement of inflammatory mediators and immune cells. The innate immune stimulator of interferon genes protein (STING) pathway was critical in modulating inflammation. Here, we show that STING was activated and inflammation was enhanced in the liver in APAP-overdosed C57BL/6J mice, and Sting mutation (Stinggt/gt) mice exhibited less liver damage. Multiplexing flow cytometry displayed that Sting mutation changed hepatic recruitment and replacement of macrophages/monocytes in APAP-overdosed mice, which was inclined to anti-inflammation. In addition, Sting mutation limited NLRP3 activation in the liver in APAP-overdosed mice, and inhibited the expression of inflammatory cytokines. Finally, MCC950, a potent and selective NLRP3 inhibitor, significantly ameliorated APAP-induced liver injury and inflammation. Besides, pretreatment of MCC950 in C57 mice resulted in changes of immune cells infiltration in the liver similar to Stinggt/gt mice. Our study revealed that STING played a crucial role in APAP-induced acute liver injury, possibly by maintaining liver immune cells homeostasis and inhibiting NLRP3 inflammasome activation, suggesting that inhibiting STING-NLRP3 pathway might be a potential therapeutic strategy for acute liver injury.

RING finger protein 13 protects against nonalcoholic steatohepatitis by targeting STING-relayed signaling pathways

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disorder worldwide. Recent studies show that innate immunity-related signaling pathways fuel NAFLD progression. This study aims to identify potent regulators of innate immunity during NAFLD progression. To this end, a phenotype-based high-content screening is performed, and RING finger protein 13 (RNF13) is identified as an effective inhibitor of lipid accumulation in vitro. In vivo gain- and loss-of-function assays are conducted to investigate the role of RNF13 in NAFLD. Transcriptome sequencing and immunoprecipitation-mass spectrometry are performed to explore the underlying mechanisms. We reveal that RNF13 protein is upregulated in the liver of individuals with NASH. Rnf13 knockout in hepatocytes exacerbate insulin resistance, steatosis, inflammation, cell injury and fibrosis in the liver of diet-induced mice, which can be alleviated by Rnf13 overexpression. Mechanically, RNF13 facilitates the proteasomal degradation of stimulator of interferon genes protein (STING) in a ubiquitination-dependent way. This study provides a promising innate immunity-related target for NAFLD treatment.

Loops Mediate Agonist-Induced Activation of the Stimulator of Interferon Genes Protein.

The stimulator of interferon genes (STING) is an important therapeutic target for cancer diseases. The activated STING recruits downstream tank-binding kinase 1 (TBK1) to trigger several important immune responses. However, the molecular mechanism of how agonist molecules mediate the STING-TBK1 interactions remains elusive. Here, we performed molecular dynamics simulations to capture the conformational changes of STING and TBK1 upon agonist binding. Our simulations revealed that multiple helices (α5-α7) and especially three loops (loop 6, loop 8, and C-terminal tail) of STING participated in the allosteric mediation of the STING-TBK1 interactions. Consistent results were also observed in the simulations of the constitutive activating mutant of STING (R284S). We further identified α5 as a key region in this agonist-induced activation mechanism of STING. Free-energy perturbation calculations of multiple STING agonists demonstrated that an alkynyl group targeting α5 is a determinant for agonist activities. These results not only offer deeper insights into the agonist-induced allosteric mediation of STING-TKB1 interactions but also provide a guidance for future drug development of this important therapeutic target.

Discovery of novel amidobenzimidazole derivatives as orally available small molecule modulators of stimulator of interferon genes for cancer immunotherapy

Stimulator of interferon genes (STING) agonists show promise as immunomodulatory agents for cancer therapy. In this study, we report the discovery of a novel orally available STING agonist, SAP-04, that exhibits potent immunomodulatory effects for cancer therapy. By optimizing the amidobenzimidazole core with various pyridine-based heterocyclic substituents, we identified a monomeric variant that displayed more efficient STING agonistic activity than the corresponding dimer. SAP-04 efficiently induced cytokine secretion related to innate immunity by directly binding of the compound to the STING protein, followed by sequential signal transduction for the STING signaling pathway and type I interferon (IFN) responses. Further pharmacological validation in vitro and in vivo demonstrated the potential utility of SAP-04 as an immunomodulatory agent for cancer therapy in vivo. The in vivo anticancer effect was observed in a 4T1 breast tumor syngeneic mouse model through oral administration of the compound. Our findings suggest a possible strategy for developing synthetically accessible monomeric variants as orally available STING agonists.