Activation Of Stimulator Of Interferon Genes Pathway Research Articles

Engineering mesoporous polydopamine-based potentiate STING pathway activation for advanced anti-biofilm therapy

The biofilm-induced “relatively immune-compromised zone” creates an immunosuppressive microenvironment that is a significant contributor to refractory infections in orthopedic endophytes. Consequently, the manipulation of immune cells to co-inhibit or co-activate signaling represents a crucial strategy for the management of biofilm. This study reports the incorporation of Mn2+ into mesoporous dopamine nanoparticles (Mnp) containing the stimulator of interferon genes (STING) pathway activator cGAMP (Mncp), and outer wrapping by M1-like macrophage cell membrane (m-Mncp). The cell membrane enhances the material's targeting ability for biofilm, allowing it to accumulate locally at the infectious focus. Furthermore, m-Mncp mechanically disrupts the biofilm through photothermal therapy and induces antigen exposure through photodynamic therapy-generated reactive oxygen species (ROS). Importantly, the modulation of immunosuppression and immune activation results in the augmentation of antigen-presenting cells (APCs) and the commencement of antigen presentation, thereby inducing biofilm-specific humoral immunity and memory responses. Additionally, this approach effectively suppresses the activation of myeloid-derived suppressor cells (MDSCs) while simultaneously boosting the activity of T cells. Our study showcases the efficacy of utilizing m-Mncp immunotherapy in conjunction with photothermal and photodynamic therapy to effectively mitigate residual and recurrent infections following the extraction of infected implants. As such, this research presents a viable alternative to traditional antibiotic treatments for biofilm that are challenging to manage.

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 associated with 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 the elevated levels of type I interferon and proinflammatory cytokines in primary macrophages derived from Trex1-/- mice and systemic inflammation in Trex1-/- mice. Overall, our work facilitates the development of specific inhibitors of STING as potential therapies in the treatment of cGAS-STING associated autoinflammatory diseases.

Amino Acid Metabolism-Regulated Nanomedicine for Enhanced Tumor Immunotherapy through Synergistic Regulation of Immune Microenvironment.

The reprogramming of tumor metabolism presents a substantial challenge for effective immunotherapy, playing a crucial role in developing an immunosuppressive microenvironment. In particular, the degradation of the amino acid L-tryptophan (Trp) to kynurenine (Kyn) by indoleamine-pyrrole 2,3-dioxygenase 1 (IDO1) is one of the most clinically validated pathways for immune suppression. Thus, regulating the Trp/Kyn metabolism by IDO1 inhibition represents a promising strategy for enhancing immunotherapy. Herein, metabolism-regulated nanoparticles are prepared through metal coordination-driven assembly of an IDO1 inhibitor (NLG919) and a stimulator of interferon genes (STING) agonist (MSA-2) for enhanced immunotherapy. After intravenous administration, the assembled nanoparticles could efficiently accumulate in tumors, enhancing the bioavailability of NLG919 and down-regulating the metabolism of Trp to Kyn to remodel the immunosuppressive tumor microenvironment. Meanwhile, the released MSA-2 evoked potent STING pathway activation in tumors, triggering an effective immune response. The antitumor immunity induced by nanoparticles significantly inhibited the development of primary and metastatic tumors, as well as B16 melanoma. Overall, this study provided a novel paradigm for enhancing tumor immunotherapy through synergistic amino acid metabolism and STING pathway activation.

Abstract 6758: Eradication of tumors and development of immunity from pHLIP-targeted intracellular delivery of STINGa

Abstract Activation of the stimulator of interferon genes (STING) pathway causes the release of factors that trigger the immune response in the tumor microenvironment (TME). Transient strong activation leads to immune activation and tumor suppression, while prolonged weak activation leads to immuno-suppression promoting tumor development. Reprogramming of M2-type tumor-associated macrophages (M2-TAMs) toward an M1 phenotype, suppression of cancer-associated fibroblasts (CAFs) and myeloid-derived suppressor cells (mMDSCs), as well as activation of dendritic cells (DCs) to train T-cells are advantageous, while the activation of STING pathway in T-cells is leads to their apoptosis. Thus, targeted delivery of STINGa to specific cells with TME to induce strong transient activation may be a key to therapy. We have developed a delivery approach that uses one or two pH Low Insertion Peptides (pHLIPs) that collaborate in the targeted intracellular delivery of a STINGa. STINGa were conjugated with pHLIPs via S-S cleavable self-immolating linkers. Biophysical studies were performed to ensure proper interactions of pHLIP-STINGa agents with membrane lipid bilayers. pHLIPs extend the lifetime of STINGa in the blood and target them to acidic cancer and stromal cells (CAFs), as well as M2-TAMs, mMDSCs and DCs within TME. The targeting results in selective cytokine activation in tumors with no systemic activation, which triggers the eradication of small (100 mm3) and large (400-700 mm3) CT26 tumors in mice after one or two doses of pHLIP-STINGa. The tumor stroma was destroyed, intratumoral hemorrhage developed, and the level of acidity within the TME was reduced. No tumors developed in mice re-challenged by an additional injection of cancer cells in a 90-day experiment. Thus, targeted delivery of STINGa to cancer cells, tumor stroma and TAMs induces activation of signaling, potentially resulting in the recruitment and infiltration of T- and NK-cells, which gain access to the tumor core. The cytotoxic activity of T- and NK-cells is not impaired by the acidic environment and immune memory is developed. The pHLIP technology may allow transformation of immuno-activating agents into more potent therapeutics, since pHLIP can target and deliver these agents to cancer cells, tumor stroma and myeloid cells. Citation Format: Michael DuPont, Anna Moshnikova, Marissa Iraca, Craig Klumpp, Hannah Visca, Dana Allababidi, Phoebe Pelzer, Donald Engelman, Oleg Andreev, Yana Reshetnyak. Eradication of tumors and development of immunity from pHLIP-targeted intracellular delivery of STINGa [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 6758.

MnO2 nanozyme with lanthanide-based radiosensitization for advanced radiotherapy by tumor microenvironment triggering STING pathway activation

Radiotherapy, being a primary approach for cancer treatment, is plagued by systemic toxicity resulting from high-dose radiation, as well as radioresistance due to the unique characteristics of tumor microenvironment (TME), including hypoxia, high expression of reducing substances and immunosuppression. The collaboration between radiosensitization and manipulation of the TME has the potential to surmount the challenges associated with advanced radiotherapy. Here, we propose a radiosensitization approach that involves enhancing X-ray deposition through physical means, while simultaneously reducing radioresistance and activating the stimulator of interferon genes (STING) pathway using a nanozyme that mimics natural enzymatic activity. This is achieved by fabricating a lanthanide ion radiosensitizer-based multifunctional enzyme activity nanoplatform, MnO2@NaGdF4:5%Nd@NaYbF4:20%Nd(MN). In the TME, the NaGdF4:5%Nd@NaYbF4:20%Nd radiosensitizer sensitizes cancer cells to X-ray. The MnO2 exhibits catalase-like (CAT) activity, enabling it to catalyze the conversion of H2O2 into O2, thereby alleviating tumor hypoxia. Additionally, MnO2 reacts with overexpressed glutathione (GSH) by glutathione oxidase-like (GSHOx) activity to generate Mn2+ ions. This reaction optimizes the function of ·OH, which can also be produced by the released Mn2+ through a Fenton-like reaction. The DNA damage caused by ·OH can be detected by cyclic GMP-AMP synthase (cGAS) to activate STING pathway, which also can be activated by the released Mn2+ ion. Through a two-pronged strategy with radical oxygen species overproduction and STING pathway activation, the lanthanide doped radiosensitizer-based MN can be relied upon to deliver highly effective radiotherapy.

Metal coordination‐driven assembly of stimulator of interferon genes‐activating nanoparticles for tumor chemo‐immunotherapy

AbstractActivating the stimulator of interferon genes (STING) signaling pathway is critical for enhancing antitumor immunity and remodeling the immunosuppressive tumor microenvironment (TME). Herein, we report the preparation of STING‐activating nanoparticles via metal coordination‐driven assembly of a synthetic STING agonist (i.e., SR717) and a chemotherapeutic drug (i.e., curcumin). After intravenous administration, the assembled nanoparticles could efficiently accumulate in tumors to improve the bioavailability of SR717 and trigger potent STING pathway activation for effective immune responses. Meanwhile, the released curcumin evokes immunogenic cell death in tumors and regulates amino acid metabolism by inhibiting the activation of indoleamine 2,3‐dioxygenase 1, leading to the reversal of the immunosuppressive TME. The antitumor immunity induced by nanoparticles significantly inhibits the growth of primary, recurrent, and metastatic tumors. The assembled nanoparticles are promising for the co‐delivery of STING agonists and drugs in improved tumor chemo‐immunotherapy.

PPM1G regulates hepatic ischemia/reperfusion injury through STING-mediated inflammatory pathways in macrophages.

Ischemia/reperfusion injury (IRI) is generally unavoidable following liver transplantation. Here, we investigated the role of protein phosphatase, Mg2+ /Mn2+ dependent 1G (PPM1G) in hepatic IRI. Hepatic IRI was mimicked by employing a hypoxia/reperfusion (H/R) model in RAW 264.7 cells and a 70% warm ischemia model in C57BL/6 mice, respectively. In vitro, expression changes of tumor necrosis factor-α and interleukin were detected by quantitative real-time polymerase chain reaction (qRT-PCR), western blot analysis, and enzyme-linked immunosorbent assay. The protein expressions of PPM1G and the stimulator of interferon genes (STING) pathway components were analyzed by western blot. Interaction between PPM1G and STING was verified by coimmunoprecipitation (CO-IP). Immunofluorescence was applied for detection of p-IRF3. Flow cytometry, qRT-PCR and western blot were utilized to analyze markers of macrophage polarization. In vivo, histological analyses of mice liver were carried out by TUNEL and H&E staining. Changes in serum aminotransferases were also detected. Following H/R intervention, a steady decline in PPM1G along with an increase in inflammatory cytokines in vitro was observed. Addition of plasmid with PPM1G sequence limited the release of inflammatory cytokines and downregulated phosphorylation of STING. CO-IP validated the interaction between PPM1G and STING. Furthermore, inhibition of PPM1G with lentivirus enhanced phosphorylation of STING and its downstream components; meanwhile, p65, p38, and Jnk were also surged to phosphorylation. Expression of INOS and CD86 was surged, while CD206, Arg-1, and IL-10 were inhibited. In vivo, PPM1G inhibition further promoted liver damage, hepatocyte apoptosis, and transaminases release. Selective inhibition of STING with C-176 partially reversed the activation of STING pathway and inflammatory cytokines in vitro. M1 markers were also suppressed by C-176. In vivo, C-176 rescued liver damage and transaminase release caused by PPM1G inhibition. PPM1G suppresses hepatic IRI and macrophage M1 phenotype by repressing STING-mediated inflammatory pathways.

Hollow metal-organic framework-based, stimulator of interferon genes pathway-activating nanovaccines for tumor immunotherapy.

Nanovaccines have emerged as promising agents for cancer therapy because of their ability to induce specific immune responses without off-target effects. However, inadequate cytotoxic T lymphocyte response and low antigen/adjuvant encapsulation remain major obstacles to vaccinating against cancer. Herein, we designed a stimulator of interferon genes (STING) pathway-activating nanovaccine based on hollow metal-organic frameworks (MOFs) for tumor treatment. The nanovaccine (OVA@HZIF-Mn) was constructed by encapsulating a model antigen ovalbumin (OVA) into zeolitic imidazolate framework-8, followed by etching with tannic acid and functionalizing with manganese ions. Studies have shown that the nanovaccine can effectively enhance antigen uptake, STING pathway activation and dendritic cell maturation, triggering a robust immune response to inhibit tumor growth. In addition, no infection or pathological signs were observed in mice organs after multiple administrations. This study combines a simple assembly approach and superior therapeutic effect, providing a promising strategy for engineering effective nanovaccines.

Sting-Mediated Interferon Signaling Exerts Potent Antileukemic Effects in High-Risk Myeloid and Monocytic Malignancies

Clinical outcomes of patients (pts) with high-risk (HR) myeloid neoplasms remain dismal despite available therapies. Interferons (IFN) regulate hematopoietic stem-progenitor cell (HSPC) differentiation, proliferation and self-renewal capabilities and preferentially impair clonal HSPC fitness. To evaluate if myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) HSPCs activate IFN responses we performed RNA sequencing in bone marrow (BM) CD34 + cells obtained from pts with MDS (n=58), CMML (n=27) and age-matched healthy donors (HDs, n=17). This analysis revealed significant deregulation of IFN pathway genes in HSPCs from pts with CMML and to a lesser extent in those of pts with MDS. Activation of the stimulator of interferon genes (STING) can inhibit stem-cell function and induce myeloid and monocytic cell death via IRF3-dependent and independent mechanisms. Therefore, we hypothesized that STING pathway activation might have therapeutic potential in high risk (HR) myeloid and monocytic (MyMo) neoplasms. In vitro treatment of monocytic MOLM13 and THP1 cell lines with the novel synthetic STING agonist dazostingag resulted in induction of IRF3 phosphorylation, caspase 3 cleavage and apoptosis. Dazostingag was also able to induce enhanced cell death in multiple myeloid cell line cultures. Given the ability of dazostingag to induce apoptosis of monocytic cell lines, we sought to evaluate the activity of monocyte-directed STING activation using TAK-500, an IgG1 anti-CCR2 antibody linked to dazostingag as a STING agonist payload. To do so, we first analyzed the expression of the monocyte-specific cysteine-cysteine chemokine receptor 2 (CCR2) in primary pts samples and cell lines. Both CCR2 RNA and protein expression levels were higher in CMML compared to HDs or MDS. In accordance with this finding, monocytic cell lines (MOLM13, THP1) had significantly higher CCR2 expression compared to non-monocytic cell lines (SKM1, HL60, U937, KG1). Treatment of MOLM13 and THP1 cell lines with TAK-500 induced strong IRF3 phosphorylation, caspase 3 cleavage and apoptosis. In vitro treatment of BM mononuclear cells (MNCs) from pts with MDS (n=10), CMML (n=11) or AML (n=8) in co-culture with mesenchymal stromal cells with TAK-500 (0.5 mM) or dazostingag (1 mM) resulted in increased cell death of MNCs from pts with CMML and AML. TAK-500 (0.5 mM) significantly depleted CCR2 + BM MNCs from all disease groups but did not affect CCR2 + cells from HDs. Next, to evaluate whether STING activation impairs repopulation potential of neoplastic HSPCs we performed colony-formation studies in CD34 + BM cells from pts with CMML (n=8), MDS (n=3) and AML (n=4). Treatment with TAK-500 (0.5mM) and dazostingag (1mM) resulted in significantly impaired myeloid colony-formation in all disease groups without significantly impairing erythroid colony-formation in MDS or CMML samples. Taken together, these data suggests that STING activation might induce aberrant myelomonocytic cell death and deplete aberrant HSPCs in HR-MyMo neoplasms. To evaluate the in vivo efficacy of TAK-500 and dazostingag, we established xenograft models by inoculating NOG-IL15 transgenic mice with MOLM13 cells. Treatment with TAK-500 and dazostingag resulted in reduction of tumor burden evaluated by bioluminescence with no observed toxicities. Administration of weekly doses of TAK-500 induced enhanced and sustained tumor burden suppression. To determine the in vivo effect of STING activation in an immune competent model of CMML, we then evaluated the effects of TAK-500 and dazostingag in Vav-KDM6B/Tet2 f/f/Vav-Cre mice, a previously reported (Wei et al Leukemia 2022) animal model with CMML phenotype. Treatment with TAK-500 led to enhanced apoptosis of Ccr2 + BM cells in these mice. Moreover, the number of colonies formed by lineage negative/Sca1 positive/cKit positive BM progenitor (LSK) cells isolated from TAK-500 treated mice was significantly lower than in vehicle treated control mice, suggesting a negative regulation of repopulating capacity of Tet2 deficient/ KDM6B overexpressing BM HSPCs by TAK-500. Evaluation of the efficacy of TAK-500 and dazostingag in patient-derived xenograft models of primary CMML samples is planned. Taken together, our data suggests that CCR2-directed STING activation might have therapeutic potential in HR-MyMo neoplasms by depleting CCR2 + cells and impairing clonal HSPC repopulation potential.

Injectable Zwitterionic Physical Hydrogel with Enhanced Chemodynamic Therapy and Tumor Microenvironment Remodeling Properties for Synergistic Anticancer Therapy.

Surgical resection is the first-line therapy for breast cancer. However, residual tumor cells and the highly immunosuppressive tumor microenvironment (TME) continue to have a serious impact on tumor recurrence and metastasis postresection. Implantation of an in situ hydrogel system postresection has shown to be an effective treatment with great clinical potential. Herein, an injectable zwitterionic hydrogel system was developed for local drug delivery with enhanced immune activation and prevention of tumor recurrence. Driven by electrostatic interactions, poly(sulfobetaine methacrylate) (PSBMA) self-assembles into a hydrogel in saline, achieving low protein adsorption and tunable biodegradability. The chemotherapy drug doxorubicin (DOX) was loaded into copper peroxide nanoparticles (CuO2/DOX), which were coated with macrophage membranes to form tumor-targeting nanoparticles (M/CuO2/DOX). Next, M/CuO2/DOX and the stimulator of interferon genes (STING) agonist 2',3'-cGAMP were coloaded into PSBMA hydrogel (Gel@M/CuO2/DOX/STING). The hydrophilic STING agonist was first released by diffusion from hydrogel to activate the STING pathway and upregulate interferon (IFN) signaling related genes, remodeling the immunosuppressive TME. Then, M/CuO2/DOX targeted the residual tumor cells, combining with DOX-induced DNA damage, immunogenic tumor cell death, and copper death. Hence, this work combines chemodynamic therapy with STING pathway activation in TME, encouraging residual tumor cell death, promoting the maturation of dendritic cells, enhancing tumor-specific CD8+ T cell infiltration, and preventing postoperative recurrence and metastasis.

Investigating the Role of MK2 in Head and Neck Squamous Cell Carcinoma Growth, Metastasis and STING Pathway Activation

Our prior work demonstrated that inhibition of MAPKAPK2 (MK2) can enhance radiation (RT)-mediated in vivo head and neck squamous cell carcinoma (HNSCC) tumor control and survival in preclinical immune incompetent models. The cytosolic DNA sensor cyclic GMP-AMP synthetase (cGAS) and its downstream adaptor protein, stimulator of interferon genes (STING), are conserved proteins within the innate immune signaling pathways and are important for mediating host defense against microbial infection and can play a role in anti-cancer immunity. We hypothesized that loss of MK2 enhances radiation-induced cGAS-STING pathway activation leading to improved tumor control and survival. MK2 shRNA knockdown human (Tu167, CAL27) and MK2 Cas9/CRISPR knockout (KO) syngeneic murine (Ly2, MLM3) HNSCC cell lines were treated with 10 Gy irradiation. Micronuclei were quantitated by DAPI-immunofluorescence (IF). Protein changes in cGAS-STING were evaluated by immunoblot. Inflammatory cytokine production including Type I IFNβ1 were evaluated by RT-qPCR. Ly2 and MLM3 cells were orthotopically or flank engrafted into immune competent mice (Balb/c, C57Bl/6, respectively) for animal tumor control-survival studies. Tumor immune cell infiltrate was examined using FACS and immunohistochemistry. Selected drug studies using the MK2 inhibitor, ATI-450, were performed with RT. Loss of MK2 in HNSCC (Tu167, CAL27, Ly2, MLM3) treated with RT led to a significant increase in micronuclei formation compared to control cells. MK2-enhanced micronuclei generation following RT could be inhibited with the actin filament polymerization inhibitor, cytochalasin B. RT treatment of MK2 shRNA cells led to increased cGAS and phospho-STING levels compared to either treatment alone. IFNβ1 levels were significantly higher in HNSCC cell lines treated with RT and with MK2 inhibited by an MK2 inhibitor (ATI-450) or genetic reduction compared to either treatment alone. In-vivo implantation of MLM3 cells into C57Bl/6 comparing control vs MK2 KO tumors treated with ±8 Gy RT demonstrated improved mouse survival favoring RT+MK2 KO over RT, MK2 KO or parental (63, 58, 58.5, 35 days, respectively). FACS analysis of MLM3 WT v KO tumors 3 days post RT showed an overall increase in the number of CD3/CD8 T-cells infiltrating into the tumor in all groups except for parental tumors. Further analysis demonstrated that loss of MK2 reversed CD8 T-cell exhaustion and when combined with radiotherapy led to increased CD8 T-cell activation. Furthermore, activated CD4 and CD8 T cells were reduced in WT+RT cells compared to WT tumors whereas no reduction was seen in the KO or KO+RT. HNSCC tumor MK2 inhibition enhances RT-mediated micronuclei formation and subsequent cGAS-STING-IFNβ1 levels. Loss of HNSCC MK2 leads to increased CD4-CD8 T-cell infiltration into the tumor and this effect is enhanced following RT. Targeting tumor MK2 may facilitate improved tumor control.

Innate immune pathway modulator screen identifies STING pathway activation as a strategy to inhibit multiple families of arbo and respiratory viruses

RNA viruses continue to remain a threat for potential pandemics due to their rapid evolution. Potentiating host antiviral pathways to prevent or limit viral infections is a promising strategy. Thus, by testing a library of innate immune agonists targeting pathogen recognition receptors, we observe that Toll-like receptor 3 (TLR3), stimulator of interferon genes (STING), TLR8, and Dectin-1 ligands inhibit arboviruses, Chikungunya virus (CHIKV), West Nile virus, and Zika virus to varying degrees. STING agonists (cAIMP, diABZI, and 2',3'-cGAMP) and Dectin-1 agonist scleroglucan demonstrate the most potent, broad-spectrum antiviral function. Furthermore, STING agonists inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and enterovirus-D68 (EV-D68) infection in cardiomyocytes. Transcriptome analysis reveals that cAIMP treatment rescue cells from CHIKV-induced dysregulation of cell repair, immune, and metabolic pathways. In addition, cAIMP provides protection against CHIKV in a chronic CHIKV-arthritis mouse model. Our study describes innate immune signaling circuits crucial for RNA virus replication and identifies broad-spectrum antivirals effective against multiple families of pandemic potential RNA viruses.

Abstract 1263: Hypoxia induces down-regulation of stimulator of interferon genes (STING) that is synergized with oncometabolites

Abstract The cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway, a component of the innate immune system, functions to detect the presence of cytosolic DNA, and STING signaling is necessary for the anti-cancer immune response. However, STING expression is suppressed or lost in the majority of cancers, which correlates with poor survival in cancer patients. Hypoxia is a key feature of solid tumors that adversely affects prognosis by altering gene expression, creating an immunosuppressive tumor microenvironment, promoting genome instability, and hampering therapy responses. In this study, we found that that prolonged moderate hypoxia induces downregulation of STING expression in multiple cancer cell lines, causing decreased STING pathway activation after treatment with a STING agonist, or with DNA damaging chemotherapy. Mechanically, we found that hypoxia suppressed histone 3 lysine 4 (H3K4) methylation at the STING promoter based on chromatin immunoprecipitation (ChIP) assays in H1975 cells, a non-small cell lung cancer cell line, and knockdown of the H3K4 demethylases, KDM5A or KDM1A, prior to hypoxic exposure prevented hypoxia-induced down-regulation of STING expression. Moreover, decreased STING expression in hypoxic cells was reversed by treatment with the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine. Next, we found that prolonged hypoxia increased both D-2-hydroxyglutarate (D-2-HG) and L-2-HG levels in H1975 cells, and that 2-HG treatment, by itself, decreased STING expression under normoxic conditions in H1975 cells. These findings are consistent with analysis of TCGA data showing that STING expression at the mRNA level is reduced in malignancies with mutations in isocitrate dehydrogenase 1 (IDH1) which causes elevated D-2-HG. Furthermore, we found that hypoxia increased Branched Chain Amino Acid Transaminase 1 (BCAT1) expression, and overexpression of BCAT1 accelerated STING downregulation in hypoxic condition. Functionally, expression of interferon (IFN)-stimulated genes (ISGs), CCL5 and interferon-β, was low in hypoxic H1975 cells compared to normoxic cells after treatment with poly(dA:dT), a small molecule STING agonist, and doxorubicin. Immunogenic cell death (ICD) was also reduced in hypoxic H1975 cells. In summary, the results suggest that hypoxia suppresses STING expression through epigenetic regulation, leading to STING silencing, and that this process synergizes with oncometabolites. The decreased STING caused by hypoxia further causes reduction of innate immune responses in cancer cells. Our study provides new understanding of the underlying mechanisms for STING silencing, which will help not only to study cancer immunity but also to develop novel therapeutic strategies. Citation Format: Yuhong Lu, Annali Yurkevicz, Yanfeng Liu, Jonathan Dow, Peter M. Glazer. Hypoxia induces down-regulation of stimulator of interferon genes (STING) that is synergized with oncometabolites [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1263.

Cancer cell genotype associated tumor immune microenvironment exhibits differential response to therapeutic STING pathway activation in high-grade serous ovarian cancer

BackgroundHigh-grade serous ovarian carcinoma (HGSC) is the most lethal gynecologic malignancy characterized by resistance to chemotherapy and high rates of recurrence. HGSC tumors display a high prevalence of tumor suppressor...

Biomineralized MnO2 Nanoplatforms Mediated Delivery of Immune Checkpoint Inhibitors with STING Pathway Activation to Potentiate Cancer Radio-Immunotherapy.

Radiotherapy (RT), as one of the main methods in the clinical treatment of various malignant tumors, would induce systemic immunotherapeutic effects by triggering immunogenic cell death (ICD) of cancer cells. However, the antitumor immune responses produced by RT-induced ICD alone usually are not robust enough to eliminate distant tumors and thus ineffective against cancer metastases. Herein, a biomimetic mineralization method for facile synthesis of MnO2 nanoparticles with high anti-programmed death ligand 1 (αPDL1) encapsulation efficiency (αPDL1@MnO2) is proposed to reinforce RT-induced systemic antitumor immune responses. This therapeutic nanoplatforms-mediated RT can significantly improve the killing of tumor cells and effectively evoke ICD by overcoming hypoxia-induced radio-resistance and reprogramming the immunosuppressive tumor microenvironment (TME). Furthermore, the released Mn2+ ions from αPDL1@MnO2 under acidic tumor pH can activate the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway and facilitate the dendritic cells (DCs) maturation. Meanwhile, αPDL1 released from αPDL1@MnO2 nanoparticles would further promote the intratumoral infiltration of cytotoxic T lymphocytes (CTLs) and trigger systemic antitumor responses, resulting in a strong abscopal effect to effectively inhibit tumor metastases. Overall, the biomineralized MnO2-based nanoplatforms offer a simple strategy for TME modulation and immune activation, which are promising for enhanced RT immunotherapy.

Amplifying STING activation by bioinspired nanomedicine for targeted chemo- and immunotherapy of acute myeloid leukemia.

Chemotherapy resistance and the tumor immune microenvironment are dual reasons for the poor therapeutic efficacy of treating acute myeloid leukemia (AML), causing suboptimal clinical outcomes and high relapse rates. Activation of the stimulator of interferon genes (STING) pathway based on innate immunity can effectively improve antitumor immunity. However, traditional STING agonists are limited due to their easy degradation and difficult membrane transport. Here, a bioinspired nanomedicine synergizing chemo- and immunotherapy was developed by activating the STING pathway for targeted and systemic AML cell damage. We show that a leukemia cell membrane (LCM)-camouflaged hollow MnO2 nanocarrier (HM) with encapsulated doxorubicin (DOX) (denoted LHMD) could bind specifically to AML cells with a homologous targeting effect. Then, MnO2 was decomposed into Mn2+ in response to endosomal acid and glutathione (GSH), which improved the magnetic resonance imaging (MRI) signal for AML detection and activated the STING pathway. In mouse models, LHMD was confirmed to eradicate established AML and prevent the engraftment of AML cells. The percentages of T-helper 1 (Th1) and T-helper 17 (Th17) cells and the concentrations of type I interferon (IFN-Ⅰ) and proinflammatory cytokines increased, while the percentage of T-helper 2 (Th2) cells decreased, reflecting the anti-AML immune response induced by Mn2+ after treatment with LHMD. This nanotechnology-based therapeutic regimen may represent a generalizable strategy for generating an anti-leukemia immune response. STATEMENT OF SIGNIFICANCE: Relapse and chemotherapy refractoriness are main causes for the dismal prognosis of AML, making it urgent to develop more effective anti-AML therapies. This study proposes an innovative strategy to combat this issue by designing a biomimetic BM-targeted nanomedicine based on a MnO2 nano-carrier to rationally deliver chemotherapeutic agents and to trigger Mn2+ mediated STING pathway activation for potent immune- and chemotherapy against AML cells. Hence, the nanomedicine design addresses the challenges associated with AML therapy and proposes a promising strategy to improve the therapeutic efficacy against AML.

Inhibition of Stimulator of Interferon Genes Protects Against Myocardial Ischemia-Reperfusion Injury in Diabetic Mice

Background: Although the past decade has witnessed substantial scientific progress with the advent of cardioprotective pharmacological agents, most have failed to protect against myocardial ischemia/reperfusion (I/R) injury in diabetic hearts. This study was aimed at investigating the role of stimulator of interferon genes (STING) in I/R injury in diabetic mice and further exploring the underlying mechanisms. Methods: Type 2 diabetic mice were subjected to I/R or sham operation to investigate the role of STING. STING knockout mice were subjected to 30 minutes of ischemia followed by reperfusion for 24 hours. Finally, myocardial injury, cardiac function, and inflammation levels were assessed. Results: STING pathway activation was observed in diabetic I/R hearts, as evidenced by increased p-TBK and p-IRF3 expression. STING knockout significantly decreased the ischemic area and improved cardiac function after I/R in diabetic mice. STING knockout also elicited cardio-protective effects by decreasing serum cardiac troponin T and lactate dehydrogenase levels, thus diminishing the inflammatory response in the heart after I/R in diabetic mice. In vitro, STING inhibition decreased the expression of hypoxia-re-oxygenation-induced inflammatory cytokines. Conclusions: Targeting STING inhibits inflammation and prevents I/R injury in diabetic mice. Thus, STING may be a potential novel therapeutic target against myocardial I/R injury in diabetes.

SAMHD1 silencing cooperates with radiotherapy to enhance anti-tumor immunity through IFI16-STING pathway in lung adenocarcinoma

BackgroundSterile alpha motif domain and histidine-aspartate domain-containing protein 1 (SAMHD1) is a DNA end resection factor, which is involved in DNA damage repair and innate immunity. However, the role of SAMHD1 in anti-tumor immunity is still unknown. This study investigated the effects of SAMHD1 on stimulator of interferon genes (STING)-type I interferon (IFN) pathway and radiation-induced immune responses.MethodsThe roles of SAMHD1 in the activation of cytosolic DNA sensing STING pathway in lung adenocarcinoma (LUAD) cells were investigated with flow cytometry, immunofluorescence, immunoblotting and qPCR. The combined effects of SAMHD1 silencing and radiation on tumor cell growth and STING pathway activation were also evaluated with colony formation and CCK8 assay. The Lewis lung cancer mouse model was used to evaluate the combined efficiency of SAMHD1 silencing and radiotherapy in vivo. Macrophage M1 polarization and cytotoxic T cell infiltration were evaluated with flow cytometry.ResultsThe single-stranded DNA (ssDNA) accumulated in the cytosol of SAMHD1-deficient lung adenocarcinoma (LUAD) cells, accompanied by upregulated DNA sensor IFN-γ-inducible protein 16 (IFI16) and activated STING pathway. The translocation of IFI16 from nucleus to cytosol was detected in SAMHD1-deficient cells. IFI16 and STING were acquired in the activation of STING-IFN-I pathway in SAMHD1-deficient cells. SAMHD1 silencing in LUAD cells promoted macrophage M1 polarization in vitro. SAMHD1 silencing synergized with radiation to activate ssDNA-STING-IFN-I pathway, inhibit proliferation, promote apoptosis and regulate cell cycle. SAMHD1 silencing cooperated with radiotherapy to inhibit tumor growth and increase CD86+MHC-IIhigh M1 proportion and CD8+ T cell infiltration in vivo.ConclusionsSAMHD1 deficiency induced IFN-I production through cytosolic IFI16-STING pathway in LUAD cells. Moreover, SAMHD1 downregulation and radiation cooperated to inhibit tumor growth and enhance anti-tumor immune responses through macrophage M1 polarization and CD8+ T cell infiltration. Combination of SAMHD1 inhibition and radiotherapy may be a potentially therapeutic strategy for LUAD patients.

Structure-activity relationship study of amidobenzimidazole derivatives as stimulator of interferon genes (STING) agonists

Stimulator of interferon genes (STING) is a crucial adaptor protein that can regulate the innate immune response by inducing the secretion of type Ι interferons and other cytokines after recognizing endogenous or exogenous DNA. Due to the key role of STING in the innate immune system, the activation of STING pathway is expected to be an efficacious immunotherapeutic tactic to treat cancer. In this study, we performed a structure-activity relationship study of amidobenzimidazole monomer, led to a series of ABZI STING agonist derivatives with potent STING-activating effects. Among them, compound 72, as a representative compound, markedly activated the STING-TBK1-IRF3 signaling pathway and significantly increased the mRNA and protein levels of IFN-β, CXCL10 and IL-6 in both WT THP-1 cells and human peripheral blood mononuclear cells (hPBMCs). In addition, it was confirmed that compound 72 was highly selective for human STING, specifically targeting human STING signaling and showing no activation of m-STING.

Sting Agonists Activate Bone Marrow Macrophages and Synergise with Anti-CD47 Blockade Improving Survival in Animal Models of Acute Myeloid Leukemia

The bone marrow (BM) microenvironment regulates acute myeloid leukemia (AML) initiation, proliferation, and chemotherapy resistance. Previous research by our group has identified activation of the stimulator of interferon genes (STING) pathway within BM macrophages supresses leukemic growth. This contrasts with solid tumours where infiltrating macrophages release immunosuppressive signals, allowing cancer progression. AML blasts also regulate the action of macrophages by activating the 'don't eat me’ signal via interactions between CD47 and SIRPα on macrophages. Recent studies have led to clinical trials investigating the potential of blocking CD47 in patients with AML. Here we hypothesise and investigate whether activation of the STING pathway in macrophages of the AML tumor microenvironment will enhance the anti-leukemic effects of anti-CD47 antibody in disease models of AML. Initially we found that activation of STING pathway, using DMXAA or cGAMP, in bone marrow derived macrophages (BMDM) induced clearance (via phagocytosis) of MN1 and HOXA9/Meis1 cells (two murine AML models) from the BM of animals with AML. To determine if the STING agonist activated the STING pathway we performed in vitro and in vivo experiments. In vitro BMDM treated with DMXAA or cGAMP induced STING gene expression signature (upregulation of GBP, IFIT3 and IRF3). For in vivo assessment of STING activation in macrophages, C57Bl/6 animals were treated with DMXAA for 24h, followed by isolation by FACS of BM macrophages (GR1-, F4/80+/ CD115int). RNA extracted and qPCR showed upregulation of STING gene expression signature. Next using two established AML in vivo models (HOXA9/Meis1 and MN1) we investigated the effect of STING agonist on AML progression. Results show that treatment with STING agonist significantly improved survival of mice with AML. Moreover, the numbers and activity of cytotoxic T cells was not changed between treatment groups. Next, we explored the role of anti-CD47 blockade in combination with STING agonist. In vitro CD47 blockade synergised with DMXAA to significantly increase phagocytosis of AML by macrophages. In vivo we found that mice with AML receiving the combined treatments (DMXAA and anti-CD47) demonstrated significantly prolonged survival, over animals given either treatment alone. In summary, we report that BM macrophages activated via the STING pathway supresses AML expansion through phagocytosis and not T cell mediated cytotoxicity. A process which is enhanced by anti-CD47 blocked on AML blasts. The results reported here provide a molecular mechanistic rationale for the clinical evaluation of targeting STING and CD47 in patients with AML.