Function Of STING Research Articles

STING induces HOIP-mediated synthesis of M1 ubiquitin chains to stimulate NFκB signaling.

STING activation by cyclic dinucleotides in mammals induces IRF3- and NFκB -mediated gene expression, and the lipidation of LC3B at Golgi-related membranes. While mechanisms of the IRF3 response are well understood, the mechanisms of NFκB activation mediated by STING remain unclear. We report that STING activation induces linear/M1-linked ubiquitin chain (M1-Ub) formation and recruitment of the LUBAC E3 ligase, HOIP, to LC3B-associated Golgi membranes where ubiquitin is also localized. Loss of HOIP prevents formation of M1-Ub ubiquitin chains and reduces STING-induced NFκB and IRF3-mediated signaling in human monocytic THP1 cells and mouse bone marrow derived macrophages, without affecting STING activation. STING-induced LC3B lipidation is not required for M1-Ub chain formation or the immune-related gene expression, however the recently reported function of STING to neutralize the pH of the Golgi may be involved. Thus, LUBAC synthesis of M1 ubiquitin chains mediates STING-induced innate immune signaling.

The Foxo1-YAP-Notch1 axis reprograms STING-mediated innate immunity in NASH progression

Innate immune activation is critical for initiating hepatic inflammation during nonalcoholic steatohepatitis (NASH) progression. However, the mechanisms by which immunoregulatory molecules recognize lipogenic, fibrotic, and inflammatory signals remain unclear. Here, we show that high-fat diet (HFD)-induced oxidative stress activates Foxo1, YAP, and Notch1 signaling in hepatic macrophages. Macrophage Foxo1 deficiency (Foxo1M-KO) ameliorated hepatic inflammation, steatosis, and fibrosis, with reduced STING, TBK1, and NF-κB activation in HFD-challenged livers. However, Foxo1 and YAP double knockout (Foxo1/YAPM-DKO) or Foxo1 and Notch1 double knockout (Foxo1/Notch1M-DKO) promoted STING function and exacerbated HFD-induced liver injury. Interestingly, Foxo1M-KO strongly reduced TGF-β1 release from palmitic acid (PA)- and oleic acid (OA)-stimulated Kupffer cells and decreased Col1α1, CCL2, and Timp1 expression but increased MMP1 expression in primary hepatic stellate cells (HSCs) after coculture with Kupffer cells. Notably, PA and OA challenge in Kupffer cells augmented LIMD1 and LATS1 colocalization and interaction, which induced YAP nuclear translocation. Foxo1M-KO activated PGC-1α and increased nuclear YAP activity, modulating mitochondrial biogenesis. Using chromatin immunoprecipitation (ChIP) coupled with massively parallel sequencing (ChIP-Seq) and in situ RNA hybridization, we found that NICD colocalizes with YAP and targets Mb21d1 (cGAS), while YAP functions as a novel coactivator of the NICD, which is crucial for reprogramming STING function in NASH progression. These findings highlight the importance of the macrophage Foxo1–YAP–Notch1 axis as a key molecular regulator that controls lipid metabolism, inflammation, and innate immunity in NASH.

Elucidating the function of STING in systemic lupus erythematosus through the STING Goldenticket mouse mutant

The complexity of systemic lupus erythematosus (SLE) arises from intricate genetic and environmental interactions, with STING playing a pivotal role. This study aims to comprehend the function of STING using the pristane-induced lupus (PIL) model in Sting missense mutant mice (Goldenticket or StingGt), which contrasts with previous research using Sting knockout mice. Investigating two-month-old StingGt mice over six months post-PIL induction, we observed a profound reduction in autoimmune markers, including antinuclear and anti-dsDNA antibodies, germinal center B cells, and plasma cells, compared to their wild-type counterparts. A pivotal finding was the marked decrease in IL-17-producing T cells. Notably, the severity of lupus nephritis and pulmonary hemorrhages was significantly diminished in StingGt mice. These findings demonstrate that different genetic approaches to interfere with STING signaling can lead to contrasting outcomes in SLE pathogenesis, which highlights the need for a nuanced understanding of the role of STING in drug development for SLE. In summary, the loss of Sting function in Goldenticket mutant mice rescued autoimmune phenotypes in PIL. This study reveals the critical nature of STING in SLE, suggesting that the method of STING modulation significantly influences disease phenotypes and should be a key consideration in developing targeted therapies.

Effect of intrinsic STING of CD8+T cells on self-metabolic reprogramming and anti-tumor effects.

e20062 Background: Many preclinical studies have demonstrated that STING agonists have effective anti-tumor effects, but their adverse effects prevent practical use. Previous research by our team has demonstrated that STING has a more significant anti-tumor role in host immune cells than tumor cells, and the level of STING mRNA is associated with the outcome of immunotherapy in non-small cell lung cancer. Although STING is necessary for CD8+T cells to exert immunological activity, its effect on CD8+T cells is debatable. Therefore, we will concentrate on the particular roles of STING in CD8+T cells. Methods: We used in vitro and in vivo models to confirm the influence of STING on CD8+T cell function. We examined the impact of STING on CD8+T cell metabolic function using RNA-seq, Seahorse, flow, electron microscopy, and other techniques. STING knockout mice were used to study the effect of competitive inhibition of glycolytic products during immunotherapy. In addition, we collected peripheral blood immune cells from 30 immunotherapy patients to examine the correlation between immune cell glycolysis levels and immunotherapy efficacy. Results: RNA-seq showed significant changes in signaling pathways such as glycolysis/glycogen synthesis, oxidative phosphorylation, tricarboxylic acid cycle, and one carbon unit in STING-/- CD8+T cells. In vitro experiments revealed that the inactivation of the STING gene reduced the number of mitochondria, altered the choice of metabolic pathways, and impaired the anti-tumor ability. Flow results showed that the mean fluorescence intensity of mitochondria in STING-/- CD8+T decreased. Seahorse experiment showed that STING-/- CD8+T glycolysis was enhanced, and oxidative phosphorylation was decreased. Meanwhile, the content of lactic acid in the culture supernatant of STING-/- CD8+ T increased dramatically, the high level of lactic acid further inhibited the proliferation of CD8+ T and the secretion of IFN-γ. Competitive inhibition of the glycolysis process or lactic acid formation can save the function of STING-/- CD8+ T cells and the secretion of IFN-γ, save the immunotherapy effect of STING-/- mice. This provides a new therapeutic idea for patients with low STING expression and poor immunotherapy response. In the peripheral blood immune cells of immunotherapy patients, we observed a negative correlation between the mRNA levels of glycolysis-associated molecules and STING. Additionally, we found a correlation between the levels of glycolysis-associated molecules in immune cells and immunotherapy efficacy outcomes. Conclusions: Our study is the first to explore the effect of STING on the metabolic process in CD8+ T cells. Intervening in the metabolic process of STING-deficient CD8+ T can save the efficacy of immunotherapy.

Abstract 6534: Overcoming B regulatory cell induced STING resistance by dual functional albumin nanomedicine to prolong survival of pancreatic cancer

Abstract Pancreatic cancer is the most lethal form of cancer with a 5-year survival at 11% and lacks treatment options. Immunotherapy, specifically anti-PD-1/PD-L1 antibody, proves ineffective for pancreatic cancer patients due to the immunosuppressive tumor microenvironment (TME). STING agonist has emerged as the most effective immune modulator to regulate immunosuppressive TME to enhance the efficacy of immunotherapy across many different cancer types. However, two challenges hinder the use of STING agonist in pancreatic cancer: (A) STING agonist strongly stimulates induction of B regulatory cells (Bregs) in pancreatic tumor and lymph node, leading to an intrinsic resistance to STING agonist and a severe compromised efficacy. (B) Most STING agonists are used by local intra-tumor injection in clinical trials, which only shrinks the local tumors without inhibiting distal tumors or metastasis, whereas it is also not feasible for intra-tumor injection for pancreatic cancer in clinical trials. In response, we developed a first-in-class dual functional drug (SH-273) to overcome STING resistance by eliminating Bregs in both tumors and lymph nodes for long-term efficacy in pancreatic cancer. SH-273 has dual function to stimulates STING function (EC50 100 nM) and inhibits PI3Kγ (IC50 7 nM) that eliminate Bregs to overcome STING resistance. Interestingly, SH-273 achieved dual function with an opposite mechanism in regulating IRF3 phosphorylation in myeloid cells (increase) vs. in Bregs (decrease). In addition, we developed an albumin nanoparticle of SH-273 (Nano-273) for systemic delivery to enhance drug targeting to tumor and lymph node to activate systemic anticancer immunity. 3D imaging results indicated that albumin nano formulation greatly enhanced drug lymphoid draining and pancreatic tumor penetrating. Nano-273, combined with anti-PD-1, achieved long-term median survival of 200 days in LSL-KrasG12D; LSL-Trp53R172H/+; Pdx1cre/+ (KPC) mice (a substantial 67% increase from 120 days without treatment). The 80-day survival extension is significant as KPC mice mimic human pancreatic cancers with Kras/P53 mutations that are unresponsive to other available therapies. Single cell RNA-sequencing and flow cytometry revealed that Nano-273, combined with anti-PD-1, reduces Bregs by 5 to 7-fold while increasing other B cell subtypes by 4-fold in both pancreatic tumors and lymph nodes, compared to control group or group treated with STING agonist alone. These findings suggest that Nano-273 overcomes STING resistance by eliminating Bregs and induced systemic immunity to achieve long-term anti-tumor efficacy in pancreatic cancer. Citation Format: Chengyi Li, Shuai Mao, Hongyi Zhao, Djibo Mahamadou, Zhongwei Liu, Hanning Wen, Miao He, Meilin Wang, Jinsong Tao, Bo Wen, Trang Hoang, Binyamin Jacobovitz, Fei Wen, Wei Gao, Duxin Sun. Overcoming B regulatory cell induced STING resistance by dual functional albumin nanomedicine to prolong survival of pancreatic cancer [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 6534.

A conserved ion channel function of STING mediates noncanonical autophagy and cell death

The cGAS/STING pathway triggers inflammation upon diverse cellular stresses such as infection, cellular damage, aging, and diseases. STING also triggers noncanonical autophagy, involving LC3 lipidation on STING vesicles through the V-ATPase-ATG16L1 axis, as well as induces cell death. Although the proton pump V-ATPase senses organelle deacidification in other contexts, it is unclear how STING activates V-ATPase for noncanonical autophagy. Here we report a conserved channel function of STING in proton efflux and vesicle deacidification. STING activation induces an electron-sparse pore in its transmembrane domain, which mediates proton flux in vitro and the deacidification of post-Golgi STING vesicles in cells. A chemical ligand of STING, C53, which binds to and blocks its channel, strongly inhibits STING-mediated proton flux in vitro. C53 fully blocks STING trafficking from the ER to the Golgi, but adding C53 after STING arrives at the Golgi allows for selective inhibition of STING-dependent vesicle deacidification, LC3 lipidation, and cell death, without affecting trafficking. The discovery of STING as a channel opens new opportunities for selective targeting of canonical and noncanonical STING functions.

STING promotes invasion and migration of uveal melanoma through p38‑MAPK signaling.

Uveal melanoma (UM) is the most common intraocular malignant tumor in adults, with a lack of effective treatment for metastasis and a poor prognosis. Stimulator of interferon genes (STING, also known as TMEM173) plays an important role in tumor development by regulating cell proliferation, metastasis and other cellular processes. However, the function of STING in UM remains unclear and requires further investigation. The present study analyzed the expression status of STING to elucidate the mechanisms underlying UM. The correlation between STING and the prognosis of UM was evaluated based on UM RNA‑seq data and clinical information extracted from The Cancer Genome Atlas database. Quantification of STING in UM cell lines and tissues was performed using the Wes Separation protein immunoassay. The effects of STING on the proliferation, migration and invasion of UM cells were investigated using Cell Counting Kit‑8, Transwell and wound healing experiments. Survival analysis demonstrated that high levels of STING in UM tissues indicated a poor prognosis. The expression of STING in UM tissues was higher than that in the choroid membranes. Furthermore, it was found that downregulation of STING expression in UM cells suppressed migration and invasion, whereas overexpression of STING significantly promoted migration and invasion. Notably, STING had no significant effect on UM cell proliferation. It was also identified that STING positively upregulated the phosphorylation of p38 mitogen‑activated protein kinase (p38‑MAPK) in UM cells, enhancing cell migration and invasion, which the p38‑MAPK inhibitor SB203580 reversed. Finally, the results of the present study demonstrated that high STING expression in UM indicates a poor prognosis. STING was revealed to promote the migration and invasion of UM cells through p38‑MAPK signaling.

At the Crossroads of the cGAS-cGAMP-STING Pathway and the DNA Damage Response: Implications for Cancer Progression and Treatment.

The evolutionary conserved DNA-sensing cGAS-STING innate immunity pathway represents one of the most important cytosolic DNA-sensing systems that is activated in response to viral invasion and/or damage to the integrity of the nuclear envelope. The key outcome of this pathway is the production of interferon, which subsequently stimulates the transcription of hundreds of genes. In oncology, the situation is complex because this pathway may serve either anti- or pro-oncogenic roles, depending on context. The prevailing understanding is that when the innate immune response is activated by sensing cytosolic DNA, such as DNA released from ruptured micronuclei, it results in the production of interferon, which attracts cytotoxic cells to destroy tumors. However, in tumor cells that have adjusted to significant chromosomal instability, particularly in relapsed, treatment-resistant cancers, the cGAS-STING pathway often supports cancer progression, fostering the epithelial-to-mesenchymal transition (EMT). Here, we review this intricate pathway in terms of its association with cancer progression, giving special attention to pancreatic ductal adenocarcinoma and gliomas. As the development of new cGAS-STING-modulating small molecules and immunotherapies such as oncolytic viruses involves serious challenges, we highlight several recent fundamental discoveries, such as the proton-channeling function of STING. These discoveries may serve as guiding lights for potential pharmacological advancements.

Abstract PR006: STING is a vulnerability in RCC independent of its innate immunity function

Abstract STING is an innate immune sensor for immune surveillance of viral/bacterial infection and maintenance of an immune-friendly microenvironment to prevent tumorigenesis. However, if and how STING exerts innate immunity-independent function remains elusive. Recently, we find STING expression is increased in renal cell carcinoma (RCC) patients and governs tumor growth through non-canonical innate immune signaling involving mitochondrial ROS maintenance and calcium homeostasis. We identify mitochondrial voltage dependent anion channel VDAC2 as a new STING binding partner. STING depletion potentiates VDAC2/GRP75-mediated MERC (mitochondria-ER contact) formation to increase mitochondrial ROS/calcium levels, impairs mitochondria function and suppresses mTORC1/S6K signaling leading to RCC growth retardation. STING interaction with VDAC2 occurs through STING-C88/C91 palmitoylation and inhibiting STING palmitoyl-transferases ZDHHCs by 2-BP significantly impedes RCC cell growth alone or in combination with sorafenib. Together, our studies reveal an innate immunity-independent function of STING in regulating mitochondrial function and growth in RCC, providing a rationale to target the STING/VDAC2 interaction in treating RCC. Citation Format: Pengda Liu, Zhichuan Zhu. STING is a vulnerability in RCC independent of its innate immunity function [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR006.

Epigenetic state determines the in vivo efficacy of STING agonist therapy

While STING-activating agents have shown limited efficacy in early-phase clinical trials, multiple lines of evidence suggest the importance of tumor cell-intrinsic STING function in mediating antitumor immune responses. Although STING signaling is impaired in human melanoma, its restoration through epigenetic reprogramming can augment its antigenicity and T cell recognition. In this study, we show that reversal of methylation silencing of STING in murine melanoma cell lines using a clinically available DNA methylation inhibitor can improve agonist-induced STING activation and type-I IFN induction, which, in tumor-bearing mice, can induce tumor regression through a CD8+ T cell-dependent immune response. These findings not only provide mechanistic insight into how STING signaling dysfunction in tumor cells can contribute to impaired responses to STING agonist therapy, but also suggest that pharmacological restoration of STING signaling through epigenetic reprogramming might improve the therapeutic efficacy of STING agonists.

STING Suppresses Mitochondrial VDAC2 to Govern RCC Growth Independent of Innate Immunity.

STING is an innate immune sensor for immune surveillance of viral/bacterial infection and maintenance of an immune-friendly microenvironment to prevent tumorigenesis. However, if and how STING exerts innate immunity-independent function remains elusive. Here, the authorsreport that STING expression is increased in renal cell carcinoma (RCC) patients and governs tumor growth through non-canonical innate immune signaling involving mitochondrial ROS maintenance and calcium homeostasis. Mitochondrial voltage-dependent anion channel VDAC2 is identified as a new STING binding partner. STING depletion potentiates VDAC2/GRP75-mediated MERC (mitochondria-ER contact) formation to increase mitochondrial ROS/calcium levels, impairs mitochondria function, and suppresses mTORC1/S6K signaling leading to RCC growth retardation. STING interaction with VDAC2 occurs through STING-C88/C91 palmitoylation and inhibiting STING palmitoyl-transferases ZDHHCs by 2-BP significantly impedes RCC cell growth alone or in combination with sorafenib. Together, these studies reveal an innate immunity-independent function of STING in regulating mitochondrial function and growth in RCC, providing a rationale to target the STING/VDAC2 interaction in treating RCC.

T cell-intrinsic STING signaling promotes regulatory T cell induction and immunosuppression by upregulating FOXP3 transcription in cervical cancer

BackgroundStimulator of interferon genes (STING) is an innate immune sensor of cytoplasmic double-stranded DNA originating from microorganisms and host cells. The activation of cytosolic DNA-STING pathway in tumor microenvironments is...

Abstract 1506: APOBEC3A a causative agent in cancer-related chromosomal instability and STING driven metastasis

Abstract Chromosomal instability (CIN), a hallmark of cancer, has been associated with a highly metastatic phenotype in numerous cancers. Mechanisms by which these genomic events occur during the lifespan of a neoplasm have yet to be fully delineated. In our study we identified a novel function for APOBEC3A (A3A), a primate-specific cytidine deaminase upregulated in multiple cancers including Pancreatic ductal adenocarcinoma (PDAC), in the initiation of CIN.We demonstrate that A3A is not routinely detected in non-diseased pancreata; however, expression was detected in the epithelial and stromal compartments from patients with chronic pancreatitis and in precursor lesions of PDAC and is further significantly increased in invasive neoplasia. Strikingly, we found that A3A expression but not A3A-induced mutagenesis, was associated with a significantly reduced overall survival in PDAC. By employing a variety of genetic tools, we identified a novel function for A3A in initiating CIN, underscoring its potential role in driving the observed early metastatic propensity in PDAC. Using a series of in vivo and in vitro models, we demonstrate that A3A-induced CIN leads to aggressive cancer, featuring enhanced, STING-dependent, distant organ seeding and metastatic growth. As a consequence of aberrant A3A function and CIN-mediated upregulation of STING, NfkB and Stat3 pathways were activated. More importantly, all these effects were independent of the deaminase domain, underscoring a novel role of A3A beyond its established canonical function. Remarkably, in a novel autochthonous murine model of PDA expressing human A3A, we identified numerous copy number changes, homologous to those altered in A3A overexpressing patient tumors, including deletions in DNA repair pathway genes. While we showed that A3A-expressing PDAC cells do not fully phenocopy a BRCA mutation-like phenotype, A3A-expressing PDAC cells are exceptionally sensitive to PARP inhibition and DNA cross linking agents, recapitulating a state of “chemical BRCAness”. The combined effect of deletions in these genes has yet to be ascertained, and we hypothesize that these losses further add to ongoing DNA damage overall in PDAC. In summary, our study shows, that A3A drives metastasis and specific deletions through a deaminase-independent initiation of CIN, with potential implications for targeted treatment strategies in pancreatic cancer. Citation Format: Sonja Maria Woermann, Amy Zhang, Fredrik I. Thege, Chris Gates, Reuben S. Harris, Susan R. Ross, Faiyaz Notta, Anirban Maitra, Andrew D. Rhim. APOBEC3A a causative agent in cancer-related chromosomal instability and STING driven metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1506.

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

SUMMARYExternal and intrinsic factors regulate the transcriptional profile of T helper 17 (TH17) cells, thereby affecting their pathogenic potential and revealing their context-dependent plasticity. The stimulator of interferon genes (STING), a component of the intracellular DNA-sensing pathway, triggers immune responses but remains largely unexplored in T cells. Here, we describe an intrinsic role of STING in limiting the TH17 cell pathogenic program. We demonstrate that non-pathogenic TH17 cells express higher levels of STING than those activated under pathogenic conditions. Activation of STING induces interleukin-10 (IL-10) production in TH17 cells, decreasing IL-17A and IL-23R expression in a type I interferon (IFN)-independent manner. Mechanistically, STING-induced IL-10 production partially requires aryl hydrocarbon receptor (AhR) signaling, while the decrease of IL-17A expression occurs due to a reduction of Rorγt transcriptional activity. Our findings reveal a regulatory function of STING in the TH17 cell activation program, proposing it as a valuable target to limit TH17-cell-mediated inflammation.

Abstract 2038: A non-canonical, cell-autonomous STING function protects breast cancer cells from intrinsic and genotoxic-induced DNA instability

Abstract Several studies have recently indicated the activation of the immune system against tumor cells as well as the targeting of cancer cell DNA damage repair mechanisms as effective strategies to target tumor growth. STING is a well-known DNA sensor of innate immunity mostly characterized as a transmembrane protein of various cytoplasmic organelles that senses cytosolic DNA as a danger signal and triggers inflammatory responses. A current cancer immunotherapy strategy relies on the use of STING agonists to boost the patient's immune system through a cytokine-mediated recruitment of immune cells that infiltrate and kill tumor cells. However, the role of the STING pathway in cancer is far to be fully understood as there is otherwise accumulating evidence that activation of the cGAS-STING pathway can have a deleterious outcome. We recently showed that genotoxic treatment of breast cancer PDXs and cell lines triggered the STING pathway. Genetic inhibition of this pathway in MCF7 cells increased genotoxic treatment efficacy by promoting cell death and delaying cell colony regrowth, indicating that STING pathway intrinsically promotes cell resistance to treatment. In this study, we show that STING silencing decreased cell viability in a panel of classical or PDX-derived breast cancer cell lines irrespective of their ER status and of the genotoxic treatment received. Cell fractionation indicates that part of the STING pool intrinsically resides in the nucleus of various malignant and non-malignant cells. Fluorescence and electron microscopy show that STING partly resides at the inner membrane of the nucleus, and mass-spectrometry analysis revealed that STING interacts with core proteins of the non-homologous end joining (NHEJ) DNA damage repair (DDR) complex. STING promotes NEHJ-related protein assembly with chromatin, and its silencing decreases DDR and cell viability, while STING overexpression protects cancer cells from genotoxic treatment. STING involvement in DDR is independent of the classical STING-TBK1-IFN inflammatory response, thus identifying a new functional pathway for STING. STING nuclear localization was confirmed in a panel of breast cancer patient-derived xenografts and in surgical samples from breast cancer patients that received neoadjuvant chemotherapy. Evaluation of the impact of STING expression on patient outcome via the Kaplan Meier plotter show that overall STING expression level is positively correlated with favorable outcome in breast cancer patients, however high STING expression in breast and ovarian cancer patients treated with adjuvant chemotherapy is associated with poor prognosis. These findings place STING at the crossroad of DDR and immune surveillance, two major pathways for tumorigenesis and tumor survival. Citation Format: Laura Cheradame, Ida Chiara Guerrera, Julie Gaston, Alain Schmitt, Vincent Jung, Marion Pouillard, Nina Radosevic-Robin, Mauro Modesti, Jean-Gabriel Judde, Vincent Goffin, Stefano Cairo. A non-canonical, cell-autonomous STING function protects breast cancer cells from intrinsic and genotoxic-induced DNA instability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2038.

CGAS–STING Cytosolic DNA Sensing Pathway Is Suppressed by JAK2-STAT3 in Tumor Cells

Deficiencies in DNA repair and DNA degrading nucleases can lead to accumulation of cytosolic DNA and the activation of STING pathways in tumor cells. The DNA sensors that recognize cytosolic DNA in tumor cells are not known. Here we show that double-stranded DNA and RNA:DNA hybrids bind cGAS and activate the cGAS-STING pathway in TRAMP-C2 prostate and other cancer cells. STING function in TRAMP-C2 cells was critical for rejection and immune cell infiltration of TRAMP-C2 tumors. In contrast, STING agonists failed to active STING and induce type I interferon (IFN) expression in many tested tumor cells. Inhibition of JAK2 and STAT3 restored type I IFN expression in these tumor cells. Intratumoral injections of IL-6, a known activator of JAK2 and STAT3, significantly reduced the anti-tumor effects of cGAMP. In summary, we show that IL-6 suppresses recognition of cytosolic DNA by the cGAS-STING pathway in tumor cells, which contributes to anti-tumor responses. Funding Statement: This work was supported by the NRF grant HUJ-CREATE-Cellular and Molecular Mechanisms of Inflammation and a ministry of education grant. Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: Mice were housed according to the IACUC guidelines of the National University of Singapore (R14-0204).

Targeting the STING pathway in tumor-associated macrophages regulates innate immune sensing of gastric cancer cells.

Rationale:STING is a critical player in the innate and adaptive immune system, sensing cytosolic DNA to activate the expression of interferon genes and regulate T lymphocytes, which drives immunogenic responses to cancer cells. Tumor-associated macrophages (TAMs), abundantly present in the tumor microenvironment, play a key role in cancer development. Gastric cancer is one of the most common and leading causes in cancer-related death worldwide. However, studies on the function and regulation of STING in TAMs and their roles in gastric cancer progression are still limited.Methods: We analyzed STING and CD68 expression of 200 pairs of gastric cancer and adjacent normal tissues by immunohistochemistry to identify the prognostic values of STING, as well as the correlations between STING and CD68 in gastric cancer. The characteristics of STING-altered macrophages, as well as their effects on cancer cell apoptosis and T cell differentiation were examined by flow cytometry. Cytokines secreted by STING-altered macrophages were identified by the Human Inflammation Array3 kit. Concentrations of soluble IL24 and IFN-β were measured by ELISA. In vivo models, including spontaneous gastric cancer in p53+/- mice and cell line-based xenografts, were established, and clinical benefits of STING-altered macrophages were examined.Results: Our study identifies STING as a prognostic factor for gastric cancer, and for the first time demonstrated that knocking-down STING and STING activation by 2'3'-c-GAMP both promote TAMs polarizing into pro-inflammatory subtype and induce apoptosis of gastric cancer cells, mechanistically through IL6R-JAK-IL24 pathway.Conclusions: This study evaluated effects of targeting STING in TAMs in anti-gastric-cancer therapies. Moreover, we unveil a novel function of STING to activate the IL6R-JAK-IL24 pathway in macrophages.

51P - Enhancing the anti-breast tumour activity of STING through a novel sting transcriptional regulator

BackgroundSTING (STimulator of INterferon Genes) is a pattern recognition receptor detecting cytoplasmic nucleic acids and transmits signals that activate host innate immune responses. It has been found to be involved in anti‐microbial immunity, autoimmune disorder and tumorigenesis. Thus, the acknowledgement of the cellular regulation of STING is important. However, transcriptional regulation of STING and its role in tumor development has not been reported. MethodsSTNF expressing vector was constructed and used to identify the STNF regulatory function of STING. Small interfering RNA was used to silence STNF expression. For the first time, full length of STING promoter was constructed with luciferase reporter which enabled quick and semi-quantitative of STING promoter activity. The bc-GenExMiner v4.2 database was used to evaluate the expression and prognostic merit of STNF and STING in breast cancer (BC). Western blot, RT-qPCR and CCK-8 assay were used to detect STING expression and BC cell growth. ResultsWe have identified a novel negative regulator of STING (STNF). The effect of STNF appeared to be at the transcriptional level of STING since STNF could suppress the promoter activity of STING. STNF was up-regulated in breast cancer (BC) and associated with poor prognosis of BC patients. Silencing STNF or pharmacologically inhibiting STNF promoted STING expression and suppressed BC growth. STING down-regulation was observed in different types of BC and restoration of STING expression resulted in broad BC inhibition. ConclusionsOur study demonstrated an unprecedented strategy used by breast tumor to escape from STING mediated tumor suppression. The identification of a novel STING regulator will provide insights for novel anti-tumor strategy against BC. Legal entity responsible for the studyXiao-Fang Yu. FundingNational Youth Science Fund Project: 81701988(C0025181). DisclosureAll authors have declared no conflicts of interest.

Abstract 2661: Targeting endoplasmic reticulum-resident proteins for the treatment of B cell cancer

Abstract IRE-1 splices XBP-1 mRNAs, leading to activation of the functional XBP-1 transcription factor. We showed that genetic deletion of XBP-1 in chronic lymphocytic leukemia (CLL) cells decelerated malignant progression of CLL in mice. We synthesized and characterized a specific inhibitor, B-I09, which could block the RNase activity of IRE-1 with high potency and efficacy. B-I09 clearly suppressed activation of the IRE-1/XBP-1 pathway, as evidenced by the decreased mRNA and protein levels of XBP-1 in intact cells. B-I09 specifically targeted mouse CLL cells in vivo by inducing apoptosis. Because XBP-1 deficiency could compromise the BCR signaling, a crucial survival signal for CLL, we tested whether pharmacological inhibition of XBP-1 could enhance the effect of inhibitors of the BCR signaling by combining B-I09 with ibrutinib (a Bruton’s tyrosine kinase inhibitor) to treat human CLL cells. A strong pharmacological synergism was determined using the Chou-Talalay combination index method, suggesting that B-I09 could help ibrutinib to achieve higher cytotoxicity at a lower dose, addressing ibrutinib’s toxicity issue. Our studies also led us to discover that IRE-1 interacted with STING, an ER-resident protein critical for cytoplasmic DNA sensing and interferon production. We showed that the IRE-1/XBP-1 pathway was required for the interferon-producing function of STING, and that agonists of STING selectively triggered mitochondria-mediated apoptosis in malignant B cells. Upon stimulation, STING was degraded inefficiently in malignant B cells, implying that prolonged activation of STING could lead to apoptosis. In CLL-bearing mice, injection of the STING agonist, 3'3'-cGAMP, induced apoptosis and regression of leukemia. Similarly efficacious effects were elicited by 3'3'-cGAMP injection in syngeneic or immunodeficient NSG mice grafted with malignant B cells. These data suggested that STING agonists could directly eradicate CLL and other B cell malignancies in vivo. IRE-1 and STING are thus useful ER-resident protein targets for the treatment of B cell cancer. Citation Format: Chih-Hang Anthony Tang, Juan R. Del Valle, Chih-Chi Andrew Hu. Targeting endoplasmic reticulum-resident proteins for the treatment of B cell cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2661.

Autophagy induced by STING, an unnoticed and primordial function of cGAS.

Autophagy induced by STING, an unnoticed and primordial function of cGAS.