Articles de revues : « STING Knockout »

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Kato, Yasuhiro, JeongHoon Park, Hyota Takamatsu, Hachirou Konaka, Wataru Aoki, Syunsuke Aburaya, Mitsuyoshi Ueda et al. « Apoptosis-derived membrane vesicles drive the cGAS–STING pathway and enhance type I IFN production in systemic lupus erythematosus ». Annals of the Rheumatic Diseases 77, n^o 10 (26 juin 2018) : 1507–15. http://dx.doi.org/10.1136/annrheumdis-2018-212988.

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ObjectiveDespite the importance of type I interferon (IFN-I) in systemic lupus erythematosus (SLE) pathogenesis, the mechanisms of IFN-I production have not been fully elucidated. Recognition of nucleic acids by DNA sensors induces IFN-I and interferon-stimulated genes (ISGs), but the involvement of cyclic guanosine monophosphate (GMP)–AMP synthase (cGAS) and stimulator of interferon genes (STING) in SLE remains unclear. We studied the role of the cGAS–STING pathway in the IFN-I-producing cascade driven by SLE serum.MethodsWe collected sera from patients with SLE (n=64), patients with other autoimmune diseases (n=31) and healthy controls (n=35), and assayed them using a cell-based reporter system that enables highly sensitive detection of IFN-I and ISG-inducing activity. We used Toll-like receptor-specific reporter cells and reporter cells harbouring knockouts of cGAS, STING and IFNAR2 to evaluate signalling pathway-dependent ISG induction.ResultsIFN-I bioactivity and ISG-inducing activities of serum were higher in patients with SLE than in patients with other autoimmune diseases or healthy controls. ISG-inducing activity of SLE sera was significantly reduced in STING-knockout reporter cells, and STING-dependent ISG-inducing activity correlated with disease activity. Double-stranded DNA levels were elevated in SLE. Apoptosis-derived membrane vesicles (AdMVs) from SLE sera had high ISG-inducing activity, which was diminished in cGAS-knockout or STING-knockout reporter cells.ConclusionsAdMVs in SLE serum induce IFN-I production through activation of the cGAS–STING pathway. Thus, blockade of the cGAS–STING axis represents a promising therapeutic target for SLE. Moreover, our cell-based reporter system may be useful for stratifying patients with SLE with high ISG-inducing activity.

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Sellaththurai, Sarithaa, Sumi Jung, Myoung-Jin Kim, Kishanthini Nadarajapillai, Subothini Ganeshalingam, Joon Bum Jeong et Jehee Lee. « CRISPR/Cas9-Induced Knockout of Sting Increases Susceptibility of Zebrafish to Bacterial Infection ». Biomolecules 13, n^o 2 (8 février 2023) : 324. http://dx.doi.org/10.3390/biom13020324.

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Stimulator of interferon genes (STING) is an adapter protein that is activated when cyclic dinucleotides (CDNs) are present. CDNs originate from the cytosolic DNA of both pathogens and hosts. STING activation promotes efficient immune responses against viral infections; however, its impact in bacterial infections is unclear. In this study, we investigated the role of Sting in bacterial infections by successfully creating a sting-deficient (sting(−/−) with a 4-bp deletion) knockout zebrafish model using CRISPR/Cas9. The transcriptional modulation of genes downstream of cGAS (cyclic GMP-AMP synthase)-Sting pathway-related genes was analyzed in seven-day-old wild-type (WT) and sting(−/−) embryos, as well as in four-day-old LPS-stimulated embryos. The expression of downstream genes was higher in sting(−/−) than in healthy WT fish. The late response was observed in sting(−/−) larvae following LPS treatment, demonstrating the importance of Sting-induced immunity during bacterial infection by activating the cGAS–STING pathway. Furthermore, adult sting(−/−) fish had a high mortality rate and significantly downregulated cGAS–STING pathway-related genes during Edwardsiella piscicida (E. piscicida) infection. In addition, we assessed NF-κB pathway genes following E. piscicida infection. Our results show fluctuating patterns of interleukin-6 (il6) and tumor necrosis factor-α (tnfα) expression, which is likely due to the influence of other NF-κB pathway-related immune genes. In summary, this study demonstrates the important role of Sting against bacterial infection.

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Froechlich, Guendalina, Carmen Caiazza, Chiara Gentile, Anna Morena D’Alise, Maria De Lucia, Francesca Langone, Guido Leoni et al. « Integrity of the Antiviral STING-mediated DNA Sensing in Tumor Cells Is Required to Sustain the Immunotherapeutic Efficacy of Herpes Simplex Oncolytic Virus ». Cancers 12, n^o 11 (17 novembre 2020) : 3407. http://dx.doi.org/10.3390/cancers12113407.

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The dichotomic contribution of cancer cell lysis and tumor immunogenicity is considered essential for effective oncovirotherapy, suggesting that the innate antiviral immune response is a hurdle for efficacy of oncolytic viruses. However, emerging evidence is resizing this view. By sensing cytosolic DNA, the cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) axis can both counteract viral spread and contribute to the elicitation of adaptive immunity via type I interferon responses. In this paper, we analyzed the tumor-resident function of Sting-mediated DNA sensing in a combined approach of oncovirotherapy and PD-1 immune checkpoint blockade, in an immunocompetent murine model. While supporting increased lytic potential by oncolytic HER2-retargeted HSV-1 in vitro and in vivo, Sting-knockout tumors showed molecular signatures of an immunosuppressive tumor microenvironment. These signatures were correspondingly associated with ineffectiveness of the combination therapy in a model of established tumors. Results suggest that the impairment in antiviral response of Sting-knockout tumors, while favoring viral replication, is not able to elicit an adequate immunotherapeutic effect, due to lack of immunogenic cell death and the inability of Sting-knockout cancer cells to promote anti-tumor adaptive immune responses. Accordingly, we propose that antiviral, tumor-resident Sting provides fundamental contributions to immunotherapeutic efficacy of oncolytic viruses.

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Sun, Shaohua, Yulin Xu, Ming Qiu, Sen Jiang, Qi Cao, Jia Luo, Tangjie Zhang et al. « Manganese Mediates Its Antiviral Functions in a cGAS-STING Pathway Independent Manner ». Viruses 15, n^o 3 (28 février 2023) : 646. http://dx.doi.org/10.3390/v15030646.

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The innate immune system is the first line of host defense sensing viral infection. Manganese (Mn) has recently been found to be involved in the activation of the innate immune DNA-sensing cGAS-STING pathway and subsequent anti-DNA virus function. However, it is still unclear whether Mn2+ mediates host defense against RNA viruses. In this study, we demonstrate that Mn2+ exhibited antiviral effects against various animal and human viruses, including RNA viruses such as PRRSVs and VSV, as well as DNA viruses such as HSV1, in a dose-dependent manner. Moreover, cGAS and STING were both investigated in the Mn2+ mediated antiviral roles using the knockout cells made by the CRISPR-Cas9 approach. Unexpectedly, the results revealed that neither cGAS knockout nor STING knockout had any effect on Mn2+-mediated antiviral functions. Nevertheless, we verified that Mn2+ promoted the activation of the cGAS-STING signaling pathway. These findings suggest that Mn2+ has broad-spectrum antiviral activities in a cGAS-STING pathway independent manner. This study also provides significant insights into redundant mechanisms participating in the Mn2+ antiviral functions, and also indicates a new target for Mn2+ antiviral therapeutics.

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Anghelina, Daniela, Eric Lam et Erik Falck-Pedersen. « Diminished Innate Antiviral Response to Adenovirus Vectors in cGAS/STING-Deficient Mice Minimally Impacts Adaptive Immunity ». Journal of Virology 90, n^o 13 (13 avril 2016) : 5915–27. http://dx.doi.org/10.1128/jvi.00500-16.

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ABSTRACTInfection by adenovirus, a nonenveloped DNA virus, induces antiviral innate and adaptive immune responses. Studies of transformed human and murine cell lines using short hairpin RNA (shRNA) knockdown strategies identified cyclic guanine adenine synthase (cGAS) as a pattern recognition receptor (PRR) that contributes to the antiadenovirus response. Here we demonstrate how the cGAS/STING cascade influences the antiviral innate and adaptive immune responses in a murine knockout model. Using knockout bone marrow-derived dendritic cells (BMDCs) and bone marrow-derived macrophages (BMMOs), we determined that cGAS and STING are essential to the induction of the antiadenovirus response in these antigen-presenting cells (APCs)in vitro. We next determined how the cGAS/STING cascade impacts the antiviral response following systemic administration of a recombinant adenovirus type 5 vector (rAd5V). Infection of cGAS−/−and STING−/−mice results in a compromised early antiviral innate response compared to that in wild-type (WT) controls: significantly lower levels of beta interferon (IFN-β) secretion, low levels of proinflammatory chemokine induction, and reduced levels of antiviral transcript induction in hepatic tissue. At 24 h postinfection, levels of viral DNA and reporter gene expression in the liver were similar in all strains. At 28 days postinfection, clearance of infected hepatocytes in cGAS or STING knockout mice was comparable to that in WT C57BL/6 mice. Levels of neutralizing anti-Ad5V antibody were modestly reduced in infected cGAS mice. These data support a dominant role for the cGAS/STING cascade in the early innate antiviral inflammatory response to adenovirus vectors. However, loss of the cGAS/STING pathway did not affect viral clearance, and cGAS deficiency had a modest influence on the magnitude of the antiviral humoral immune response to adenovirus infections.IMPORTANCEThe detection of viral infection by host sentinel immune cells contributes to the activation of a complex and varied antiviral innate and adaptive immune response, which limits virus replication, spread, and susceptibility to infection. In this study, we have characterized how the cGAS/STING DNA-sensing cascade contributes to early detection of adenovirus infections. cGAS influences APC activation and early innate antiviral inflammatory immune responses, but adaptive immune pathways associated with virus clearance and anti-Ad antibody production were minimally influenced by the loss of the cGAS PRR signaling cascade.

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Aoki, Masaya, Licun Wu, Junichi Murakami, Yidan Zhao, Hana Yun et Marc de Perrot. « IRF3 Knockout Results in Partial or Complete Rejection of Murine Mesothelioma ». Journal of Clinical Medicine 10, n^o 21 (7 novembre 2021) : 5196. http://dx.doi.org/10.3390/jcm10215196.

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Background: Malignant pleural mesothelioma (MESO) has a poor prognosis despite aggressive treatment with surgery, radiation and chemotherapy, and novel therapeutic approaches are needed. IRF3 is a downstream molecule of the cGAS/STING signaling pathway, but its roles have not been investigated in MESO. Methods: Various murine mesothelioma cell lines were inoculated into wild type (WT) and IRF3 knockout (IRF3KO) mice to compare tumor growth. AE17-bearing mice were treated with local radiotherapy (LRT) to evaluate the effect on tumor growth, and immune cell infiltration was analyzed by flow cytometry 20 days after tumor inoculation. TCGA data were used to examine the relationship between mRNA expression of IRF3 and genes of the cGAS/STING signaling cascade on prognosis in MESO. Correlations between gene expression of IRF3, cGAS/STING signaling pathway, and immune checkpoints were analyzed in TCGA MESO and our scRNA-Seq data from MESO patients. Results: In mouse mesothelioma models, AK7, RN5 and ZiP3 were completely rejected in IRF3KO mice 20 days after the tumor challenge. AE17tumor volume was slightly larger than WT mice around day 10 before shrinking and becoming significantly smaller than WT mice on day 20. LRT accelerated tumor shrinkage of AE17 tumors in IRF3KO mice. Compared with WT mice, the number of macrophages infiltrating the tumor of IRF3KO mice was significantly reduced, and CD4+ T cells and CD8+IFNγ+ T cells were significantly increased. TCGA data showed that IRF3 expression was an unfavorable prognostic factor in MESO patients. IRF3 expression, the cGAS/STING signaling pathway, and immune checkpoints were positively correlated. Conclusion: IRF3 could play a critical role in the tumor immune microenvironment of MESO.

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Xu, Yulin, Youwen Zhang, Shaohua Sun, Jia Luo, Sen Jiang, Jiajia Zhang, Xueliang Liu et al. « The Innate Immune DNA Sensing cGAS-STING Signaling Pathway Mediates Anti-PRRSV Function ». Viruses 13, n^o 9 (14 septembre 2021) : 1829. http://dx.doi.org/10.3390/v13091829.

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Porcine reproductive and respiratory syndrome virus (PRRSV) modulates host innate immunity which plays a key role against PRRSV infection. As a RNA virus, PRRSV is mainly sensed by innate immune RNA receptors, whereas the role of innate immune DNA sensors in the PRRSV infection has not been elucidated. Here, we investigated the roles of DNA sensing cGAS-STING pathway in both PRRSV infected Marc-145 cells and porcine macrophages. The results show that in Marc-145 cells, the stable expression of STING with or without stimulations exhibited anti-PRRSV activity, and STING knockout heightened PRRSV infection. In CD163-3D4/21 porcine macrophages, either expression of STING or stimulation of cGAS-STING signaling obviously suppressed PRRSV infection, whereas in STING knockdown macrophages, the PRRSV infection was upregulated. Our results clearly demonstrate that the host cGAS-STING signal exerts an important antiviral role in PRRSV infection.

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Langereis, Martijn A., Huib H. Rabouw, Melle Holwerda, Linda J. Visser et Frank J. M. van Kuppeveld. « Knockout of cGAS and STING Rescues Virus Infection of Plasmid DNA-Transfected Cells ». Journal of Virology 89, n^o 21 (26 août 2015) : 11169–73. http://dx.doi.org/10.1128/jvi.01781-15.

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It is well known that plasmid DNA transfection, prior to virus infection, negatively affects infection efficiency. Here, we show that cytosolic plasmid DNA activates the cGAS/STING signaling pathway, which ultimately leads to the induction of an antiviral state of the cells. Using a transient one-plasmid clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, we generated cGAS/STING-knockout cells and show that these cells can be infected after plasmid DNA transfection as efficiently as nontransfected cells.

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Li, Suxin, Jian Wang, Jonathan Wilhelm, Qingtai Su, Gaurav Bharadwaj, Jason Miller, Wei Li et al. « Abstract 4234 : ONM-501 : A polyvalent STING agonist for oncology immunotherapy ». Cancer Research 82, n^o 12_Supplement (15 juin 2022) : 4234. http://dx.doi.org/10.1158/1538-7445.am2022-4234.

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

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Neupane, Chiranjivi, Ramesh Sharma, Hyun Jin Shin, Su Eun Park et Jin Bong Park. « Alteration of GABA transporter impairs cognitive behavior in Sting knockout mice ». IBRO Reports 6 (septembre 2019) : S434. http://dx.doi.org/10.1016/j.ibror.2019.07.1379.

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Li, Yang, Sharmy J. James, David H. Wyllie, Claire Wynne, Agnes Czibula, Ahmed Bukhari, Katherine Pye et al. « TMEM203 is a binding partner and regulator of STING-mediated inflammatory signaling in macrophages ». Proceedings of the National Academy of Sciences 116, n^o 33 (25 juillet 2019) : 16479–88. http://dx.doi.org/10.1073/pnas.1901090116.

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Regulation of IFN signaling is critical in host recognition and response to pathogens while its dysregulation underlies the pathogenesis of several chronic diseases. STimulator of IFN Genes (STING) has been identified as a critical mediator of IFN inducing innate immune pathways, but little is known about direct coregulators of this protein. We report here that TMEM203, a conserved putative transmembrane protein, is an intracellular regulator of STING-mediated signaling. We show that TMEM203 interacts, functionally cooperates, and comigrates with STING following cell stimulation, which in turn leads to the activation of the kinase TBK1, and the IRF3 transcription factor. This induces target genes in macrophages, including IFN-β. Using Tmem203 knockout bone marrow-derived macrophages and transient knockdown of TMEM203 in human monocyte-derived macrophages, we show that TMEM203 protein is required for cGAMP-induced STING activation. Unlike STING, TMEM203 mRNA levels are elevated in T cells from patients with systemic lupus erythematosus, a disease characterized by the overexpression of type I interferons. Moreover, TMEM203 mRNA levels are associated with disease activity, as assessed by serum levels of the complement protein C3. Identification of TMEM203 sheds light into the control of STING-mediated innate immune responses, providing a potential novel mechanism for therapeutic interventions in STING-associated inflammatory diseases.

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Papinska, Joanna, Harini Bagavant, Grzegorz B. Gmyrek et Umesh S. Deshmukh. « Pulmonary Involvement in a Mouse Model of Sjögren’s Syndrome Induced by STING Activation ». International Journal of Molecular Sciences 21, n^o 12 (25 juin 2020) : 4512. http://dx.doi.org/10.3390/ijms21124512.

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Sjögren’s Syndrome (SS), a chronic autoimmune disorder affecting multiple organ systems, is characterized by an elevated type I interferon (IFN) response. Activation of Stimulator of Interferon Genes (STING) protein induces type I IFN and in mice, several features of SS, including anti-nuclear antibodies, sialadenitis, and salivary gland dysfunction. Since lung involvement occurs in one-fifth of SS patients, we investigated whether systemic activation of STING also leads to lung inflammation. Lungs from female C57BL/6 mice injected with the STING agonist 5, 6-Dimethylxanthenone-4-acetic acid (DMXAA), were evaluated for acute and chronic inflammatory responses. Within 4h of DMXAA injection, the expression of Ifnb1, Il6, Tnf, Ifng, and Mx1 was significantly upregulated. At 1 and 2 months post-treatment, lungs showed lymphocytic infiltration in the peri-bronchial regions. The lungs from DMXAA treated mice showed an increased expression of multiple chemokines and an increase in lymphatic endothelial cells. Despite STING expression in bronchial epithelium and cells lining the alveolar wall, bone marrow chimeras between STING knockout and wild type mice showed that STING expression in hematopoietic cells was critical for lung inflammation. Our results suggest that activation of the STING pathway might be involved in SS patients with concomitant salivary gland and lung disease.

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Bai, Juli, Christopher Cervantes, Juan Liu, Sijia He, Haiyan Zhou, Bilin Zhang, Huan Cai et al. « DsbA-L prevents obesity-induced inflammation and insulin resistance by suppressing the mtDNA release-activated cGAS-cGAMP-STING pathway ». Proceedings of the National Academy of Sciences 114, n^o 46 (30 octobre 2017) : 12196–201. http://dx.doi.org/10.1073/pnas.1708744114.

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Chronic inflammation in adipose tissue plays a key role in obesity-induced insulin resistance. However, the mechanisms underlying obesity-induced inflammation remain elusive. Here we show that obesity promotes mtDNA release into the cytosol, where it triggers inflammatory responses by activating the DNA-sensing cGAS-cGAMP-STING pathway. Fat-specific knockout of disulfide-bond A oxidoreductase-like protein (DsbA-L), a chaperone-like protein originally identified in the mitochondrial matrix, impaired mitochondrial function and promoted mtDNA release, leading to activation of the cGAS-cGAMP-STING pathway and inflammatory responses. Conversely, fat-specific overexpression of DsbA-L protected mice against high-fat diet-induced activation of the cGAS-cGAMP-STING pathway and inflammation. Taken together, we identify DsbA-L as a key molecule that maintains mitochondrial integrity. DsbA-L deficiency promotes inflammation and insulin resistance by activating the cGAS-cGAMP-STING pathway. Our study also reveals that, in addition to its well-characterized roles in innate immune surveillance, the cGAS-cGAMP-STING pathway plays an important role in mediating obesity-induced metabolic dysfunction.

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Liu, Yonghong, Yuanyuan Liu, Jiaming Wu, Bernard Roizman et Grace Guoying Zhou. « Innate responses to gene knockouts impact overlapping gene networks and vary with respect to resistance to viral infection ». Proceedings of the National Academy of Sciences 115, n^o 14 (20 mars 2018) : E3230—E3237. http://dx.doi.org/10.1073/pnas.1720464115.

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Analyses of the levels of mRNAs encoding IFIT1, IFI16, RIG-1, MDA5, CXCL10, LGP2, PUM1, LSD1, STING, and IFNβ in cell lines from which the gene encoding LGP2, LSD1, PML, HDAC4, IFI16, PUM1, STING, MDA5, IRF3, or HDAC 1 had been knocked out, as well as the ability of these cell lines to support the replication of HSV-1, revealed the following: (i) Cell lines lacking the gene encoding LGP2, PML, or HDAC4 (cluster 1) exhibited increased levels of expression of partially overlapping gene networks. Concurrently, these cell lines produced from 5 fold to 12 fold lower yields of HSV-1 than the parental cells. (ii) Cell lines lacking the genes encoding STING, LSD1, MDA5, IRF3, or HDAC 1 (cluster 2) exhibited decreased levels of mRNAs of partially overlapping gene networks. Concurrently, these cell lines produced virus yields that did not differ from those produced by the parental cell line. The genes up-regulated in cell lines forming cluster 1, overlapped in part with genes down-regulated in cluster 2. The key conclusions are that gene knockouts and subsequent selection for growth causes changes in expression of multiple genes, and hence the phenotype of the cell lines cannot be ascribed to a single gene; the patterns of gene expression may be shared by multiple knockouts; and the enhanced immunity to viral replication by cluster 1 knockout cell lines but not by cluster 2 cell lines suggests that in parental cells, the expression of innate resistance to infection is specifically repressed.

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Zhao, Qinglan, Murli Manohar, Yi Wei, Stephen J. Pandol et Aida Habtezion. « STING signalling protects against chronic pancreatitis by modulating Th17 response ». Gut 68, n^o 10 (31 janvier 2019) : 1827–37. http://dx.doi.org/10.1136/gutjnl-2018-317098.

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ObjectiveChronic pancreatitis (CP) is an inflammatory disease with progressive fibrosis leading to exocrine and endocrine dysfunction. Currently, there are no approved effective therapies for CP. Stimulator of interferon genes (STING) signalling is a key innate immune sensor of DNA. In this study, we evaluated the role of STING signalling in CP.DesignWe used an experimental model of CP to test the effect of STING signalling in STING wild-type and knockout mice as well as bone marrow chimaeras (BMCs). STING was activated using a pharmacological agent. Since we found changes in Th17 cells, we used neutralising and control antibodies to determine the role of IL-17A. The effect of STING signalling was further explored in IL-17A generation and we examined the effect of IL-17A on pancreatic stellate cells (PSCs). Human pancreas from patients with CP and without CP were also stained for IL-17A.ResultsSTING activation decreased CP-associated pancreatic inflammation and fibrosis, whereas absence of STING led to worsening of the disease. BMCs showed that leucocytes play an important role in STING signalling–mediated amelioration of experimental CP. STING deletion was associated with increased Th17 cell infiltration in the pancreas, whereas STING agonist limited this Th17 response. Importantly, anti-IL-17A antibody treatment mitigated the severity of CP in the absence of STING signalling. STING deficiency promoted Th17 polarisation and PSCs express functional IL-17 receptor by upregulating fibrosis genes. Compared with tumour margins, pancreas from patients with CP had significant increase in IL-17A+ cells.ConclusionUnlike acute pancreatitis, STING activation is protective in CP. STING signalling is important in regulating adaptive immune responses by diminishing generation of IL-17A during CP and presents a novel therapeutic target for CP.

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Ding, Qiang, Jenna M. Gaska, Florian Douam, Lei Wei, David Kim, Metodi Balev, Brigitte Heller et Alexander Ploss. « Species-specific disruption of STING-dependent antiviral cellular defenses by the Zika virus NS2B3 protease ». Proceedings of the National Academy of Sciences 115, n^o 27 (18 juin 2018) : E6310—E6318. http://dx.doi.org/10.1073/pnas.1803406115.

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The limited host tropism of numerous viruses causing disease in humans remains incompletely understood. One example is Zika virus (ZIKV), an RNA virus that has reemerged in recent years. Here, we demonstrate that ZIKV efficiently infects fibroblasts from humans, great apes, New and Old World monkeys, but not rodents. ZIKV infection in human—but not murine—cells impairs responses to agonists of the cGMP-AMP synthase/stimulator of IFN genes (cGAS/STING) signaling pathway, suggesting that viral mechanisms to evade antiviral defenses are less effective in rodent cells. Indeed, human, but not mouse, STING is subject to cleavage by proteases encoded by ZIKV, dengue virus, West Nile virus, and Japanese encephalitis virus, but not that of yellow fever virus. The protease cleavage site, located between positions 78/79 of human STING, is only partially conserved in nonhuman primates and rodents, rendering these orthologs resistant to degradation. Genetic disruption of STING increases the susceptibility of mouse—but not human—cells to ZIKV. Accordingly, expression of only mouse, not human, STING in murine STING knockout cells rescues the ZIKV suppression phenotype. STING-deficient mice, however, did not exhibit increased susceptibility, suggesting that other redundant antiviral pathways control ZIKV infection in vivo. Collectively, our data demonstrate that numerous RNA viruses evade cGAS/STING-dependent signaling and affirm the importance of this pathway in shaping the host range of ZIKV. Furthermore, our results explain—at least in part—the decreased permissivity of rodent cells to ZIKV, which could aid in the development of mice model with inheritable susceptibility to ZIKV and other flaviviruses.

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Pawaria, Sudesh, et Ann Marshak-Rothstein. « Endosomal RNA-sensing toll-like receptor stimulated T cells play a unique role in autoimmune manifestations in murine model of DNA accrual. » Journal of Immunology 198, n^o 1_Supplement (1 mai 2017) : 75.9. http://dx.doi.org/10.4049/jimmunol.198.supp.75.9.

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Abstract DNaseII is a lysosomal DNase that plays a critical role in immune homeostasis. DNaseII deficiency in mice causes embryonic lethality, due to severe anemia associated with excessive production of type I interferon (IFN), and can be rescued by IFN receptor knockout (IFNaR). The DNaseII/IFNaR double knockout (DKO) mice develop a late-onset inflammatory arthritis, dependent on the cytosolic DNA sensor STING (Stimulator of Interferon Genes). They also develop early-onset clinical manifestations of systemic autoimmunity including disrupted hematopoiesis and B cell lymphopoiesis, splenomegaly (due to increased accumulation of Ter119+ cells), and the production of anti-nuclear antibodies (ANA), through a mechanism that is STING-independent, Unc93B1-dependent and likely to result from endosomal RNA-sensing TLRs. Bone marrow (BM) chimera studies have shown that the DNaseII deficiency is essential in both radioresistant host cells and hematopoietic donor cells for all disease manifestations. Development of arthritis requires the presence of STING in both host and donor. The TLR-dependent defects require TLR expression in only the hematopoietic compartment but still need DNaseII−/− recipients. Remarkably transplantation of DKO spleen fragments or injection of DKO spleen cells alone into DNaseII-sufficient Rag−/− mice leads to splenomegaly and disrupted hematopoiesis and lymphopoiesis. Moreover DKO T cells are sufficient to transfer these TLR-associated defects to the Rag−/− mice. As activated T cells are known to be radioresistant, T cells activated in a DKO environment may account for the radioresistant host component required to precipitate the TLR-dependent disease manifestations.

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Cetinbas, Naniye Malli, Travis Monnell, Winnie Lee, Kalli Catcott, Chen-Ni Chin, Pamela Shaw, Kelly Slocum et al. « 620 Tumor cell-intrinsic STING pathway is activated in the presence of cues from immune cells and contributes to the anti-tumor activity of tumor cell-targeted STING agonist antibody-drug conjugates ». Journal for ImmunoTherapy of Cancer 8, Suppl 3 (novembre 2020) : A656. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0620.

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BackgroundSTING pathway agonism has emerged as a potential therapeutic mechanism to stimulate an innate anti-tumor immune response. While in principle systemic administration of a STING agonist would have many therapeutic benefits, including the delivery of STING to all tumor lesions, such an approach may be limited by toxicity. Antibody-drug conjugates (ADCs) constitute a proven therapeutic modality that is ideally suited to allow systemic administration while stimulating the innate immunity in a targeted manner. We have previously demonstrated that targeted delivery of a STING agonist with an ADC induces robust anti-tumor immune responses.MethodsHerein we investigated the mechanism of action of tumor cell-targeted STING agonist ADCs. We evaluated STING pathway activation and anti-tumor activity elicited by ADCs harboring either wild type (wt) or mutant Fc deficient in Fcγ receptor (FcγR) binding in wt or STING knockout (ko) cancer cell mono-cultures, immune cell co-cultures, and in in vivo tumor models.ResultsConsistent with previous reports, the majority of cancer cell lines tested failed to induce STING pathway following STING agonist payload treatment in mono-cultures. In cancer cell:THP1 monocytic cell co-cultures, tumor-targeted STING agonist ADCs with wt Fc exhibited robust STING activation, whereas Fc-mutant ADCs or non-targeted control ADCs had minimal activity. Similar results were obtained when THP1 cells were treated in plates coated with target antigen without cancer cells, demonstrating STING activation in THP1 cells following FcγR-mediated uptake of antigen-bound ADCs. Tumor-targeted Fc-wt ADCs led to marked induction of STING pathway and cancer cell-killing in cancer cell:PBMC or primary monocyte co-cultures, and complete tumor regressions in in vivo tumors. Surprisingly, while at reduced levels relative to the Fc-wt ADCs, Fc-mutant ADCs exhibited significant activity in these in vitro and in vivo models, suggesting that tumor cell-intrinsic STING pathway may be activated in the presence of cues from immune cells. Consistently, STING agonist payload treatment in the presence of conditioned media from PBMC and primary monocyte but not from THP1 cultures, led to STING activation in cancer cell mono-cultures. Moreover, Fc-mutant ADCs had diminished activity in STING ko cancer cell:PBMC or primary monocyte co-cultures, demonstrating the contribution of tumor cell-intrinsic STING activation to the anti-tumor activity elicited by tumor cell-targeted STING agonist ADCs.ConclusionsIn conclusion, we demonstrated that tumor cell-targeted STING agonist ADCs induce robust anti-tumor activity through mechanisms involving both FcγR and tumor antigen-mediated ADC internalization and subsequent induction of STING pathway in immune cells and tumor cells.

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Lei, Zhao, Meihong Deng, Zhongjie Yi, Qian Sun, Richard A. Shapiro, Hongbo Xu, Tunliang Li et al. « cGAS-mediated autophagy protects the liver from ischemia-reperfusion injury independently of STING ». American Journal of Physiology-Gastrointestinal and Liver Physiology 314, n^o 6 (1 juin 2018) : G655—G667. http://dx.doi.org/10.1152/ajpgi.00326.2017.

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Liver ischemia-reperfusion (I/R) injury occurs through induction of oxidative stress and release of damage-associated molecular patterns (DAMPs), including cytosolic DNA released from dysfunctional mitochondria or from the nucleus. Cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) synthase (cGAS) is a cytosolic DNA sensor known to trigger stimulator of interferon genes (STING) and downstream type 1 interferon (IFN-I) pathways, which are pivotal innate immune system responses to pathogen. However, little is known about the role of cGAS/STING in liver I/R injury. We subjected C57BL/6 (WT), cGAS knockout (cGAS−/−), and STING-deficient (STINGgt/gt) mice to warm liver I/R injury and that found cGAS−/− mice had significantly increased liver injury compared with WT or STINGgt/gt mice, suggesting a protective effect of cGAS independent of STING. Liver I/R upregulated cGAS in vivo and also in vitro in hepatocytes subjected to anoxia/reoxygenation (A/R). We confirmed a previously published finding that hepatocytes do not express STING under normoxic conditions or after A/R. Hepatocytes and liver from cGAS−/− mice had increased cell death and reduced induction of autophagy under hypoxic conditions as well as increased apoptosis. Protection could be restored in cGAS−/− hepatocytes by overexpression of cGAS or by pretreatment of mice with autophagy inducer rapamycin. Our findings indicate a novel protective role for cGAS in the regulation of autophagy during liver I/R injury that occurs independently of STING. NEW & NOTEWORTHY Our studies are the first to document the important role of cGAS in the acute setting of sterile injury induced by I/R. Specifically, we provide evidence that cGAS protects liver from I/R injury in a STING-independent manner.

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Ni, Huanhe, Huanling Zhang, Lin Li, He Huang, Hui Guo, Lin Zhang, Chunwei Li et al. « T cell-intrinsic STING signaling promotes regulatory T cell induction and immunosuppression by upregulating FOXP3 transcription in cervical cancer ». Journal for ImmunoTherapy of Cancer 10, n^o 9 (septembre 2022) : e005151. http://dx.doi.org/10.1136/jitc-2022-005151.

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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 usually linked to more robust adaptive immune responses to tumors, however the intracellular function of STING in regulatory T cells is largely unknown. In the present study, we aimed to explore the contribution of intracellular STING activation to regulatory T cell induction (iTreg) in cervical cancer (CC) microenvironments.MethodsBlood samples and tumor specimens were obtained from patients with CC. The intratumoral STING, CCL22, CD8 and forkhead box P3 (FOXP3) expression levels were measured by immunohistochemistry. T cell-specific STING conditional knockout mice (CD4-Cre/STINGflox/flox, TKO) were generated, and syngeneic TC-1 tumor model were investigated. The differentiation and molecular regulatory pathway of human and murine iTreg under different treatments were investigated by ex vivo assays, immunoblotting and quantitative PCR. Tumor-associated exosomes (T-EXO) were isolated from CC cell lines and exosomal contents were identified by ELISA and Western blot analysis. The impact of T-EXO on T cell differentiation was tested in in vitro cell culture.ResultsIncreased STING, CCL22 level, FOXP3+ cells but decreased CD8+ cells in tumor tissues predicted poor survival. Tumor-bearing CD4-Cre-STINGflox/flox (TKO) mice displayed slower tumor growth tendencies as well as fewer FOXP3+ cells but higher CD8+ cell proportion in tumor tissues than wild-type (WT) mice. Activating of STING signaling cooperated with T cell receptor, interleukin-2 receptor and transforming growth factor-beta (TGF-β) signals to promote CD4+CD25highFOXP3+ iTreg differentiation from both human and murine CD4+-naïve T cells from WT and IFNAR−/− mice but not TKO or IRF3−/− mice in vitro. Ectopic STING, TBK1 or IRF3 expression promoted iTreg differentiation from human CD4+-naïve T cells. T cell-intrinsic STING activation induced FOXP3 transcription through TBK1-IRF3-mediated SMAD3 and STAT5 phosphorylation independent of interferon-β. In CC, tumor-derived exosomes activated STING signaling in tumor-infiltrated T cells by exosomal TGF-β, cyclic GMP-AMP synthase and 2’-3’-cGAMP, leading to iTreg expansion.ConclusionsThese findings highlight a novel mechanism for iTreg expansion mediated by tumor-derived exosome-activated T cell-intrinsic STING signal, and provide a rationale for developing immunotherapeutic strategies targeting STING signal in CC.

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Lee, Hailey R., Evan R. Abt, Khalid Rashid, Amanda L. Creech, Keke Liang, Liu Wei, Arthur Cho et al. « Abstract 2483 : Detection of STING-induced immune activation via [18F]FDG-PET imaging ». Cancer Research 82, n^o 12_Supplement (15 juin 2022) : 2483. http://dx.doi.org/10.1158/1538-7445.am2022-2483.

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Abstract Stimulator of interferon genes (STING) coordinates innate and adaptive immune responses by mediating type I interferon and NF-κB signaling and is therefore a promising cancer immunotherapy target. While first generation STING agonists have advanced to multiple clinical trials, their application is restricted to intratumoral administration and these agents have shown limited therapeutic efficacy. In contrast, recently developed second generation STING agonists allow for systemic administration and have been shown to exert highly potent anti-tumor effects in animal models of cancer. However, STING is expressed in a wide array of cell types and tissues, and the effects of systemic activation of this pattern recognition receptor are poorly understood. This highlights the need for the development of pharmacodynamic companion biomarkers that can be used to guide the clinical development of newly developed systemic STING agonists. Here, we show that systemic administration of the STING agonist diABZI triggers alterations in glucose metabolism in immune cells which can be detected non-invasively using [18F]Fluorodeoxyglucose (FDG) and positron emission tomography (PET). Accordingly, systemic STING activation significantly upregulated [18F]FDG uptake in secondary lymphoid organs, such as the spleen and lymph nodes, as indicated by PET imaging in vivo and confirmed by gamma counter measurements of isolated splenocytes ex vivo. Importantly, STING agonist-induced changes in [18F]FDG uptake in the spleen and lymph nodes were highly predictive of therapeutic responses to STING agonist mono- and combination therapies in animal models of pancreatic cancer. Using single-cell RNA sequencing we found that systemic STING activation triggers profound transcriptional alterations in T and B lymphocytes indicative of a metabolic shift towards enhanced glycolysis. We then explored whether metabolic alterations indicated by increased [18F]FDG uptake are associated with changes in the functional phenotypes of T and B cells. We found that systemic STING activation induced the upregulation of the early activation marker CD69 and immune checkpoint ligand PD-L1. Furthermore, CD69 and PD-L1 upregulation occurred in a STING agonist dose-dependent manner that correlated with [18F]FDG uptake as measured non-invasively by PET. In interferon receptor knockout mice, CD69 and PD-L1 expression was partially attenuated; however, [18F]FDG uptake was still induced following STING activation, suggesting that an interferon-independent mechanism mediates the observed glycolytic switch in lymphocytes. In summary, [18F]FDG-PET provides a novel non-invasive and widely applicable pharmacodynamic biomarker which has potential to guide and accelerate the clinical development of systemic STING agonist-based immunotherapies in immunologically cold malignancies. Citation Format: Hailey R. Lee, Evan R. Abt, Khalid Rashid, Amanda L. Creech, Keke Liang, Liu Wei, Arthur Cho, Willy Hugo, Timothy R. Donahue, Johannes Czernin, Caius G. Radu, Thuc M. Le. Detection of STING-induced immune activation via [18F]FDG-PET imaging [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 2483.

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Ghosh, Arundhati, Lulu Shao, Padmavati Sampath, Nidhi Patel, Jianzhong Zhu, Viet Hornung, Stephen Thorne et Saumendra N. Sarkar. « 2′-5′ Oligoadenylate like (OASL) inhibits interferon induction by inhibiting cGAS activity during DNA virus infection. » Journal of Immunology 198, n^o 1_Supplement (1 mai 2017) : 129.9. http://dx.doi.org/10.4049/jimmunol.198.supp.129.9.

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Abstract Oligoadenylate Synthetases-like (OASL) is a type I interferon (IFN)-stimulated gene (ISG). During RNA virus infection OASL promotes IFN induction by augmenting RIG-I activation and inhibits virus replication. Here we report a completely new role of OASL in the context of DNA virus infection. Unlike RNA viruses, human OASL or mouse Oasl2 knockout cells showed reduced replication when infected with DNA viruses such as Vaccinia (VV) and Herpes simplex virus (HSV). Consequently, Oasl2 knockout mice showed delayed and reduced VV spread and mortality as opposed to enhanced virus multiplication and spread with RNA virus such as Vesicular Stomatitis virus. Mechanistically, OASL negatively regulates DNA-sensor-mediated IFN induction in vitro and in vivo. Stimulation of both mouse and human OASL-deficient cells with intracellular DNA showed increased induction of IFN and ISG compared to the Wt cells. However, OASL-deficient cells did not exhibit any difference in IFN induction compared to Wt cells when stimulated with the STING ligand cGAMP, indicating the upstream effect of OASL on cGAS and not on STING stimulation. In vitro immunoprecipitation experiments showed OASL specifically interacted with cGAS, and this interaction is mediated by the OAS domain of OASL protein. Furthermore, this OASL-cGAS interaction led to the inhibition of cGAS enzyme activity and reduced cGAMP production. Together these results establish the unique ability of OASL to differentially modulate RNA and DNA virus replication and IFN induction.

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Young, Elizabeth "Betsy", Courtney R. Schott et E. Alejandro Sweet-Cordero. « Abstract A005 : STING activation overcomes immune escape in osteosarcoma metastasis ». Clinical Cancer Research 28, n^o 18_Supplement (15 septembre 2022) : A005. http://dx.doi.org/10.1158/1557-3265.sarcomas22-a005.

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Abstract Chromosomal instability (CIN) has been known to be a hallmark of cancer for decades, but how CIN influences the tumor microenvironment (TME) and defines the ability of tumors to metastasize is a fundamental question in cancer biology that remains unanswered. Osteosarcoma (OS) is a highly malignant bone tumor characterized by high CIN and an immunosuppressive TME and is thus an excellent model system to address the role of CIN in metastasis and TME programming. The cGAS-STING-ENPP1 pathway links CIN to immune evasion and metastasis, functioning to produce an immune response to infection by sensing cytoplasmic double-stranded DNA (dsDNA) and transducing this signal via the second messenger cGAMP, which activates interferon gene expression. CIN in cancer cells also results in cytosolic dsDNA which can also activate the STING pathway. However, cGAMP can also be hydrolyzed by ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which negatively regulates STING activation in the TME, enabling cancer cells to survive CIN and avoid immune surveillance. Our studies of this pathway in human-in-mouse metastasis models of OS indicate that either ENPP1 overexpression (OE) or STING knockout (KO) enhance metastasis, nominating the STING pathway as an important regulator metastasis in OS and suggesting that STING agonists or ENPP1 inhibitors may have therapeutic benefit. Indeed, OS patient-derived xenograft cell lines that are least aggressive in vivo show strong STING activation in response to transfection with dsDNA, while cell lines with a higher metastatic capacity do not, suggesting that metastatic capacity may be directly linked to STING pathway downregulation. Given that a fully competent murine immune system is crucial for evaluation of the STING pathway in mediating antitumor immunity, a major current focus of this work is development and use of syngeneic murine OS models for further in vivo dissection of the role of the STING-ENPP1 axis in OS. Future studies will also evaluate if ENPP1 inhibition constitutes a novel immune-targeted therapeutic that may overcome an immunosuppressive TME and activate the immune system in response to the cell-intrinsic consequences of CIN. Moreover, elucidating the immune cell types that drive STING-mediated anti-tumor immunity will inform new combinatorial approaches together with ENPP1 inhibition. Given the therapeutic relevance of TME reprogramming and the limited success that immunotherapy has garnered in many solid tumors, this line of investigation has broad applicability beyond OS and may enable rational design of novel combination immunotherapies to treat metastatic sarcomas and other CIN-high cancers. Citation Format: Elizabeth "Betsy" Young, Courtney R. Schott, E. Alejandro Sweet-Cordero. STING activation overcomes immune escape in osteosarcoma metastasis [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A005.

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Young, Elizabeth P., Courtney R. Schott, Amanda Koehne, Christine Johnson et E. Alejandro Sweet-Cordero. « Abstract A51 : STING activation overcomes immune escape in osteosarcoma metastasis ». Cancer Immunology Research 10, n^o 12_Supplement (1 décembre 2022) : A51. http://dx.doi.org/10.1158/2326-6074.tumimm22-a51.

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Abstract Chromosomal instability (CIN) has been known to be a hallmark of cancer for decades, but how CIN influences the tumor microenvironment (TME) and defines the ability of tumors to metastasize is a fundamental question in cancer biology that remains unanswered. Osteosarcoma (OS) is a highly malignant bone tumor characterized by high CIN and an immunosuppressive TME and is thus an excellent model system to address the role of CIN in metastasis and TME programming. The cGAS-STING-ENPP1 pathway links CIN to immune evasion and metastasis, functioning to produce an immune response upon sensing cytoplasmic double-stranded DNA (dsDNA) and transducing this signal via the second messenger cGAMP, which activates interferon gene expression. CIN in cancer cells also results in cytosolic dsDNA which can also activate the STING pathway. However, cGAMP can also be hydrolyzed by ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which negatively regulates STING activation in the TME, enabling cancer cells to survive CIN and avoid immune surveillance. Our studies of this pathway in human-in-mouse metastasis models of OS indicate that either ENPP1 overexpression (OE) or STING knockout (KO) enhance metastasis, nominating the STING pathway as an important regulator metastasis in OS and suggesting that STING agonists or ENPP1 inhibitors may have therapeutic benefit. Indeed, OS patient-derived xenograft cell lines that are least aggressive in vivo show strong STING activation in response to transfection with dsDNA, while cell lines with a higher metastatic capacity do not, suggesting that metastatic capacity may be directly linked to STING pathway downregulation. Given that a fully competent murine immune system is crucial for evaluation of the STING pathway in mediating antitumor immunity, a major current focus of this work is development and use of syngeneic murine OS models for further in vivo dissection of the role of the STING-ENPP1 axis in OS. Future studies will also evaluate if ENPP1 inhibition constitutes a novel immune-targeted therapeutic that may overcome an immunosuppressive TME and activate the immune system in response to the cell-intrinsic consequences of CIN. Moreover, elucidating the immune cell types that drive STING-mediated anti-tumor immunity will inform new combinatorial approaches together with ENPP1 inhibition. Given the therapeutic relevance of TME reprogramming and the limited success that immunotherapy has garnered in many solid tumors, this line of investigation has broad applicability beyond OS and may enable rational design of novel combination immunotherapies to treat metastatic sarcomas and other CIN-high cancers. Citation Format: Elizabeth "Betsy" P Young, Courtney R Schott, Amanda Koehne, Christine Johnson, E. Alejandro Sweet-Cordero. STING activation overcomes immune escape in osteosarcoma metastasis [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A51.

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Schwertner, Barbara, Georg Lindner, Camila Toledo Toledo Stauner, Elisa Klapproth, Clara Magnus, Anette Rohrhofer, Stefanie Gross et al. « Nectin-1 Expression Correlates with the Susceptibility of Malignant Melanoma to Oncolytic Herpes Simplex Virus In Vitro and In Vivo ». Cancers 13, n^o 12 (19 juin 2021) : 3058. http://dx.doi.org/10.3390/cancers13123058.

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Talimogene laherparepvec (T-VEC), an oncolytic herpes simplex virus, is approved for intralesional injection of unresectable stage IIIB/IVM1a melanoma. However, it is still unclear which parameter(s) predict treatment response or failure. Our study aimed at characterizing surface receptors Nectin-1 and the herpes virus entry mediator (HVEM) in addition to intracellular molecules cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) as potential bio-markers for oncolytic virus treatment. In 20 melanoma cell lines, oncolytic activity of T-VEC was correlated with the expression of Nectin-1 but not HVEM, as evaluated via flow cytometry and immunohistochemistry. Knockout using CRISPR/Cas9 technology confirmed the superior role of Nectin-1 over HVEM for entry and oncolytic activity of T-VEC. Neither cGAS nor STING as evaluated by Western Blot and immunohistochemistry correlated with T-VEC induced oncolysis. The role of these biomarkers was retrospectively analyzed for the response of 35 cutaneous melanoma metastases of 21 patients to intralesional T-VEC injection, with 21 (60.0%) of these lesions responding with complete (n = 16) or partial regression (n = 5). Nectin-1 expression in pretreatment biopsies significantly predicted treatment outcome, while the expression of HVEM, cGAS, and STING was not prognostic. Altogether, Nectin-1 served as biomarker for T-VEC-induced melanoma regression in vitro and in vivo.

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Dang, Sheng, Wanyang Li, Shubo Wen, Yang Song, Meirong Bai, Shuyan Li, Zeliang Chen et Jingbo Zhai. « Ag85a-S2 Activates cGAS-STING Signaling Pathway in Intestinal Mucosal Cells ». Vaccines 10, n^o 12 (16 décembre 2022) : 2170. http://dx.doi.org/10.3390/vaccines10122170.

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Brucellosis is a zoonotic disease caused by Gram-negative bacteria. Most of the brucellosis vaccines in the application are whole-bacteria vaccines. Live-attenuated vaccines are widely used for brucellosis prevention in sheep, goats, pigs, and cattle. Thus, there is also a need for an adjuvanted vaccine for human brucellosis, because the attenuated Brucella vaccines now utilized in animals cause human illness. Here, we developed a live-attenuated Brucella suis strain 2 vaccine (S2) adjuvanted with Ag85a (Ag85a-S2). We found that Ag85a-S2 activated cGAS-STING pathways both in intestinal mucosal cells in vivo and in the BMDM and U937 cell line in vitro. We demonstrated that the cGAS knockout significantly downregulated the abundance of interferon and other cytokines induced by Ag85a-S2. Moreover, Ag85a-S2 triggered a stronger cellular immune response compared to S2 alone. In sum, Ag85a-S2-mediated enhancement of immune responses was at least partially dependent on the cGAS-STING pathway. Our results provide a new candidate for preventing Brucella pathogens from livestock, which might reduce the dosage and potential toxicity compared to S2.

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Kelleher, Alan, Yin Wang, Luke Broses, Yu Leo Lei et Phillip Palmbos. « Abstract A27 : TRIM29-mediated immune evasion in bladder cancer invasive progression ». Cancer Immunology Research 10, n^o 12_Supplement (1 décembre 2022) : A27. http://dx.doi.org/10.1158/2326-6074.tumimm22-a27.

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Abstract Background: Progression from noninvasive to muscle-invasive and then metastatic bladder cancer causes >17,000 deaths each year in the United States. Unfortunately, the molecular drivers of progression remain poorly understood and no strategies to prevent progression exist. We have identified an E3 ubiquitin ligase, TRIM29, which is highly expressed in 70% of muscle invasive bladder cancers (MIBC). Global overexpression of TRIM29 in a genetically engineered mouse model (GEMM) is sufficient to induce urothelial cancer initiation and invasive progression. TRIM29 has also been shown to bind and ubiquitinate STING (Stimulator of Interferon Genes), a regulator of anti-tumor cytokine production. However, the role of TRIM29-mediated suppression of STING and innate immune responses in invasive progression was not previously understood. Hypothesis: Given the importance of Trim29 in invasive progression demonstrated in our GEMM, we hypothesized TRIM29-mediated degradation of STING creates an immune-privileged niche required for muscle invasion. Methods: To determine the requirement for Trim29-mediated immune suppression and bladder cancer invasive progression, we generated a urothelial specific tamoxifen inducible Trim29 knockout (KO) GEMM. To induce bladder tumorigenesis, N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), a urothelial specific chemical carcinogen with similarities to tobacco, was delivered to mice via drinking water. Results: As expected, BBN promoted development of muscle-invasive urothelial tumors in 50% of mice after 24 weeks. Tamoxifen-induced Trim29 KO completely blocked progression to muscle-invasion, although these mice did demonstrate development of low grade, non-muscle invasive tumors. BBN upregulated Trim29 expression in the urothelium, but only modestly increased Sting and infiltrating macrophage and T cells. In contrast, Trim29 KO mice, which did not develop invasive tumors, harbored substantially elevated Sting levels in the urothelium and substantially increased macrophage and T cell infiltration in the bladder and tumor. In vitro, TRIM29 promoted STING ubiquitination and TRIM29 knockdown increased STING protein levels in bladder cancer cell lines. TRIM29 KO in these cells also resulted in increased expression of innate immune cytokines. Conclusion: These results suggest that TRIM29-mediated downregulation of STING in the bladder results in decreased immune surveillance in the tumor microenvironment, contributing to the development of an immune privileged niche required for MIBC progression. Citation Format: Alan Kelleher, Yin Wang, Luke Broses, Yu Leo Lei, Phillip Palmbos. TRIM29-mediated immune evasion in bladder cancer invasive progression [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A27.

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Bai, Lu, Jing Dai, Yuxuan Xia, Kaichuan He, Hongmei Xue, Qi Guo, Danyang Tian et al. « Hydrogen Sulfide Ameliorated High Choline-Induced Cardiac Dysfunction by Inhibiting cGAS-STING-NLRP3 Inflammasome Pathway ». Oxidative Medicine and Cellular Longevity 2022 (22 juillet 2022) : 1–12. http://dx.doi.org/10.1155/2022/1392896.

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Although it is an essential nutrient, high choline intake directly or indirectly via its metabolite is associated with increased risk of cardiovascular disease, the mechanism of which remains to be elucidated. The present study was performed to investigate whether hydrogen sulfide (H2S) was involved in high choline-induced cardiac dysfunction and explore the potential mechanisms. We found that ejection fraction (EF) and fractional shortening (FS), the indicators of cardiac function measured by echocardiography, were significantly decreased in mice fed a diet containing 1.3% choline for 4 months as compared to the control, while applying 3,3-dimethyl-1-butanol (DMB) to suppress trimethylamine N-oxide (TMAO, a metabolite of choline) generation ameliorated the cardiac function. Subsequently, we found that feeding choline or TMAO significantly increased the protein levels of cyclic GMP-AMP (cGAMP) synthase (cGAS), stimulator of interferon genes (STING), NOD-like receptor protein 3 (NLRP3), caspase-1, and interleukin-1β (IL-1β) as compared to the control, which indicated the activation of cGAS-STING-NLRP3 inflammasome axis. Moreover, the protein expression of cystathionine γ-lyase (CSE), the main enzyme for H2S production in the cardiovascular system, was significantly increased after dietary supplementation with choline, but the plasma H2S levels were significantly decreased. To observe the effect of endogenous H2S, CSE knockout (KO) mice were used, and we found that the EF, FS, and plasma H2S levels in WT mice were significantly decreased after dietary supplementation with choline, while there was no difference between CSE KO + control and CSE KO + choline group. To observe the effect of exogenous H2S, mice were intraperitoneally injected with sodium hydrosulfide (NaHS, a H2S donor) for 4 months, and we found that NaHS improved the cardiac function and reduced the protein levels of cGAS, STING, NLRP3, caspase-1, and IL-1β in mice receiving dietary choline. In conclusion, our studies revealed that high choline diet decreased plasma H2S levels and induced cardiac dysfunction via cGAS-STING-NLRP3 inflammasome axis while H2S treatment could restore the cardiac function by inhibiting cGAS-STING-NLRP3 inflammasome axis.

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Liang, Yaoji, Suqin Wu et Jiahuai Han. « RIP3 is involved in host defense against HSV-1 infection via enhancing STING mediated antiviral signaling (P1405) ». Journal of Immunology 190, n^o 1_Supplement (1 mai 2013) : 57.14. http://dx.doi.org/10.4049/jimmunol.190.supp.57.14.

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Abstract Herpes simplex virus type 1 (HSV-1), a double stranded DNA virus, is a widespread virus that infects approximately 80% of adults worldwide. HSV-1 infection is also the major cause of fatal childhood HSV-1 encephalitis (HSE). The mechanisms of how host cells defend against HSV-1 are still not fully known. Here we report that receptor interacting protein 3 (RIP3) enhances type I interferon production in response to HSV-1 infection. In our study of the functions of RIP3, we have found that the HSV-1 replication in RIP3 knockout L929 was unrestrained, and that HSV-1 induced TBK1 and IRF3 activations were impaired in RIP3 deficient cells. Further study has shown that over-expression of wild type RIP3, but not RHIM-mutant RIP3, enhances type I interferon production, especially when co-expressed with STING, a central signaling molecule in the innate immune response to DNA virus infection. Upon treating the cell with HSV-1, TBK1 was recruited to RIP3. In addition, RIP3 can promote the interaction between STING and TBK1 in a RHIM dependent manner. We propose that upon HSV-1 stimulation, RIP3 promotes the recruitment of TBK1 to STING, which leads to TBK1-mediated IRF3 activation and subsequent type I IFN secretion. Our study revealed that, in addition to its function in necrosis, RIP3 plays a role in type I interferon production. These results may also provide a new perspective for the exploration of childhood HSE pathogenesis.

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Hasan, Maroof, Vijay K. Gonugunta, Nicole Dobbs, Aktar Ali, Guillermo Palchik, Maria A. Calvaruso, Ralph J. DeBerardinis et Nan Yan. « Chronic innate immune activation of TBK1 suppresses mTORC1 activity and dysregulates cellular metabolism ». Proceedings of the National Academy of Sciences 114, n^o 4 (9 janvier 2017) : 746–51. http://dx.doi.org/10.1073/pnas.1611113114.

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Three-prime repair exonuclease 1 knockout (Trex1−/−) mice suffer from systemic inflammation caused largely by chronic activation of the cyclic GMP-AMP synthase–stimulator of interferon genes–TANK-binding kinase–interferon regulatory factor 3 (cGAS–STING–TBK1–IRF3) signaling pathway. We showed previously that Trex1-deficient cells have reduced mammalian target of rapamycin complex 1 (mTORC1) activity, although the underlying mechanism is unclear. Here, we performed detailed metabolic analysis in Trex1−/− mice and cells that revealed both cellular and systemic metabolic defects, including reduced mitochondrial respiration and increased glycolysis, energy expenditure, and fat metabolism. We also genetically separated the inflammatory and metabolic phenotypes by showing that Sting deficiency rescued both inflammatory and metabolic phenotypes, whereas Irf3 deficiency only rescued inflammation on the Trex1−/− background, and many metabolic defects persist in Trex1−/−Irf3−/− cells and mice. We also showed that Leptin deficiency (ob/ob) increased lipogenesis and prolonged survival of Trex1−/− mice without dampening inflammation. Mechanistically, we identified TBK1 as a key regulator of mTORC1 activity in Trex1−/− cells. Together, our data demonstrate that chronic innate immune activation of TBK1 suppresses mTORC1 activity, leading to dysregulated cellular metabolism.

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Avery, Joseph, Chi Ma, FuKun Hoffmann et Peter Hoffmann. « Selenoprotein I is important for phosphatidylethanolamine synthesis and autophagy during T cell activation ». Journal of Immunology 204, n^o 1_Supplement (1 mai 2020) : 150.8. http://dx.doi.org/10.4049/jimmunol.204.supp.150.8.

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Abstract Selenoprotein I (SELENOI) is a type I CDP-alcohol phosphatidyltransferase that synthesizes phosphatidylethanolamine (PE) in the final reaction of the Kennedy pathway. We have found that SELENOI deletion in the whole animal is embryonic lethal, which emphasizes the key role PE synthesis plays in cellular division and development. One important role for PE in cells is the role it plays as a ligand for the conversion of LC3I to the lipidated LC3II molecule, which in turn allows the progression of early autophagosomes to proceed to later stage autophagosomes. Using a T cell-specific knockout (KO) model of SELENOI, we investigated SELENOI loss-of-function studies in these immune cells. In particular, SELENOI KO T cells were stimulated with STING-dependent and STING-independent agonists to test the importance of this selenoenzyme in autophagy and downstream signaling events that depend on efficient autophagy. Our results found that PE levels were lower in TCR-activated T cells lacking SELENOI compared to wild-type (WT) controls. LC3I to LC3II conversion was impaired in SELENOI KO T compared to controls, and STING-dependent autophagosome formation was particularly decreased in the absence of SELENOI. Using bone marrow-derived macrophages (BMDMs) from a doxycycline-inducible transgenic mouse that expresses a short hairpin RNA (shRNA) that lowers SELENOI <50%, we found impaired LC3I to LC3II conversion as well as reduced autophagosome formation but not to the extent of that found with the KO T cell model. Our data suggest that SELENOI is an important enzyme for proper lipidation of LC3-I and autophagosome formation within immune cells.

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Gao, Daxing, Jiaxi Wu, You-Tong Wu, Fenghe Du, Chukwuemika Aroh, Nan Yan, Lijun Sun et Zhijian J. Chen. « Cyclic GMP-AMP Synthase Is an Innate Immune Sensor of HIV and Other Retroviruses ». Science 341, n^o 6148 (8 août 2013) : 903–6. http://dx.doi.org/10.1126/science.1240933.

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Retroviruses, including HIV, can activate innate immune responses, but the host sensors for retroviruses are largely unknown. Here we show that HIV infection activates cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) synthase (cGAS) to produce cGAMP, which binds to and activates the adaptor protein STING to induce type I interferons and other cytokines. Inhibitors of HIV reverse transcriptase, but not integrase, abrogated interferon-β induction by the virus, suggesting that the reverse-transcribed HIV DNA triggers the innate immune response. Knockout or knockdown of cGAS in mouse or human cell lines blocked cytokine induction by HIV, murine leukemia virus, and simian immunodeficiency virus. These results indicate that cGAS is an innate immune sensor of HIV and other retroviruses.

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Gusho, Elona, et Laimonis A. Laimins. « Human papillomaviruses sensitize cells to DNA damage induced apoptosis by targeting the innate immune sensor cGAS ». PLOS Pathogens 18, n^o 7 (25 juillet 2022) : e1010725. http://dx.doi.org/10.1371/journal.ppat.1010725.

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The cyclic GMP-AMP synthase (cGAS) is a critical regulator of the innate immune response acting as a sensor of double-strand DNAs from pathogens or damaged host DNA. Upon activation, cGAS signals through the STING/TBK1/IRF3 pathway to induce interferon expression. Double stranded DNA viruses target the cGAS pathway to facilitate infection. In HPV positive cells that stably maintain viral episomes, the levels of cGAS were found to be significantly increased over those seen in normal human keratinocytes. Furthermore the downstream effectors of the cGAS pathway, STING and IRF3, were fully active in response to signaling from the secondary messenger cGAMP or poly (dA:dT). In HPV positive cells cGAS was detected in both cytoplasmic puncta as well as in DNA damage induced micronuclei. E6 was responsible for increased levels of cGAS that was dependent on inhibition of p53. CRISPR-Cas9 mediated knockout of cGAS prevented activation of STING and IRF3 but had a minimal effect on viral replication. A primary function of cGAS in HPV positive cells was in response to treatment with etoposide or cisplatin which lead to increased levels of H2AX phosphorylation and activation of caspase 3/7 cleavage while having only a minimal effect on activation of homologous recombination repair factors ATM, ATR or CHK2. In HPV positive cells cGAS was found to regulate the levels of the phosphorylated non-homologous end-joining kinase, DNA-PK, which may contribute to H2AX phosphorylation along with other factors. Importantly cGAS was also responsible for increased levels of DNA breaks along with enhanced apoptosis in HPV positive cells but not in HFKs. This study identifies an important and novel role for cGAS in mediating the response of HPV positive cells to chemotherapeutic drugs.

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Bayer, Abraham L., Jodie Pietruska, Jaymes Farrell, Siobhan McRee, Pilar Alcaide et Philip W. Hinds. « AKT1 Is Required for a Complete Palbociclib-Induced Senescence Phenotype in BRAF-V600E-Driven Human Melanoma ». Cancers 14, n^o 3 (23 janvier 2022) : 572. http://dx.doi.org/10.3390/cancers14030572.

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Cellular senescence is a carefully regulated process of proliferative arrest accompanied by functional and morphologic changes. Senescence allows damaged cells to avoid neoplastic proliferation; however, the induction of the senescence-associated secretory phenotype (SASP) can promote tumor growth. The complexity of senescence may limit the efficacy of anti-neoplastic agents, such as CDK4/6 inhibitors (Cdk4/6i), that induce a senescence-like state in tumor cells. The AKT kinase family, which contains three isoforms that play both unique and redundant roles in cancer progression, is commonly hyperactive in many cancers including melanoma and has been implicated in the regulation of senescence. To interrogate the role of AKT isoforms in Cdk4/6i-induced cellular senescence, we generated isoform-specific AKT knockout human melanoma cell lines. We found that the CDK4/6i Palbociclib induced a form of senescence in these cells that was dependent on AKT1. We then evaluated the activity of the cGAS-STING pathway, recently implicated in cellular senescence, finding that cGAS-STING function was dependent on AKT1, and pharmacologic inhibition of cGAS had little effect on senescence. However, we found SASP factors to require NF-κB function, in part dependent on a stimulatory phosphorylation of IKKα by AKT1. In summary, we provide the first evidence of a novel, isoform-specific role for AKT1 in therapy-induced senescence in human melanoma cells acting through NF-κB but independent of cGAS.

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Ghosh, Arundhati, Padmavathi Sampath, Jianzhong Zhu, Veit Hornung, Stephen Thorne et Saumendra N. Sarkar. « Modulation of cellular immune response by 2′-5′ Oligoadenylate Synthetase - like (OASL) proteins during DNA virus infection ». Journal of Immunology 196, n^o 1_Supplement (1 mai 2016) : 61.6. http://dx.doi.org/10.4049/jimmunol.196.supp.61.6.

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Abstract Oligoadenylate Synthetase-like (OASL) is a type I interferon (IFN) stimulated gene, strongly induced following virus infection, but lacks enzymatic activity to synthesize 2′–5′ oligoadenylates. We show that human OASL, and its functional ortholog mouse Oasl2 are critical modulators of cellular antiviral signaling. During RNA virus infection, OASL binds the RNA-sensor, RIG-I, and enhances RIG-I signaling by mimicking one of the RIG-I-ligands, K63-linked polyubiquitin [p(Ub)], thus exerting an antiviral activity. In contrast, by binding the DNA-sensor, cyclic GMP-AMP synthase (cGAS), OASL negatively regulates IFN induction and restricts inflammation in the context of DNA virus infections, some of which can establish persistent infection. Human OASL or mouse Oasl2 knockout cells showed enhanced IFN induction when stimulated either with intracellular DNA or DNA viruses such as Vaccinia and Herpes simplex virus. Consequently, Oasl2 knockout mice showed delayed and reduced Vaccinia virus spread and mortality. Additionally, OASL or Oasl2 affected IFN induction when stimulated with intracellular DNA, but not with cGAMP, indicating the upstream effect of OASL is on cGAS stimulation and not on STING stimulation. Mechanistically, OASL specifically interacted with cGAS, and inhibited its activity. Together these results establish the unique ability of OASL to differentially modulate RNA and DNA virus replication and IFN induction.

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Gong, Yan, Guangwei Li, Jun Tao, Ne N. Wu, Machender R. Kandadi, Yaguang Bi, Shuyi Wang, Zhaohui Pei et Jun Ren. « Double knockout of Akt2 and AMPK accentuates high fat diet-induced cardiac anomalies through a cGAS-STING-mediated mechanism ». Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1866, n^o 10 (octobre 2020) : 165855. http://dx.doi.org/10.1016/j.bbadis.2020.165855.

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Jiang, Xiaoyi, Jian Wang, Xichen Zheng, Zhida Liu, Xinyu Zhang, Yuwei Li, Jonathan Wilhelm et al. « Intratumoral administration of STING-activating nanovaccine enhances T cell immunotherapy ». Journal for ImmunoTherapy of Cancer 10, n^o 5 (mai 2022) : e003960. http://dx.doi.org/10.1136/jitc-2021-003960.

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BackgroundCancer vaccines are able to achieve tumor-specific immune editing in early-phase clinical trials. However, the infiltration of cytotoxic T cells into immune-deserted tumors is still a major limiting factor. An optimized vaccine approach to induce antigen-specific T cells that can perform robust tumor infiltration is important to accelerate their clinical translation. We previously developed a STING-activating PC7A nanovaccine that produces a strong anti-tumor T cell response on subcutaneous injection. This study systematically investigated the impact of administration methods on the performance of nanovaccines.MethodsTumor growth inhibition by intratumoral delivery and subcutaneous delivery of nanovaccine was investigated in TC-1 human papillomavirus-induced cancer model and B16-OVA melanoma model. Nanovaccine distribution in vivo was detected by clinical camera imaging, systemic T cell activation and tumor infiltration were tested by in vivo cytotoxicity killing assay and flow cytometry. For mechanism analysis, T cell recruitment was investigated by in vivo migration blocking assay, multiplex chemokine array, flow cytometry, RT-qPCR, chemotaxis assay and gene knockout mice.ResultsNanovaccine administration was found to alter T cell production and infiltration in tumors. Intratumoral delivery of nanovaccines displayed superior antitumor effects in multiple tumor models compared with subcutaneous delivery. Mechanistic investigation revealed that intratumoral administration of the nanovaccine significantly increased the infiltration of antigen-specific T cells in TC-1 tumors, despite the lower systemic levels of T cells compared with subcutaneous injection. The inhibition of tumor growth by nanovaccines is primarily dependent on CD8+ cytotoxic T cells. Nanovaccine accumulation in tumors upregulates CXCL9 expression in myeloid cells in a STING dependent manner, leading to increased recruitment of IFNγ-expressing CD8+ T cells from the periphery, and IFNγ reciprocally stimulates CXCL9 expression in myeloid cells, resulting in positive feedback between myeloid-CXCL9 and T cell-IFNγ to promote T cell recruitment. However, the STING agonist alone could not sustain this effect in the presence of a systemic deficiency in antigen-specific T cells.ConclusionsOur results demonstrate that intratumoral administration of PC7A nanovaccine achieved stronger antitumor immunity and efficacy over subcutaneous injection. These data suggest intratumoral administration should be included in the therapeutic design in the clinical use of nanovaccine.

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Wang, Dang, Ruixue Wang et Kui Li. « Impaired Antiviral Responses to Extracellular Double-Stranded RNA and Cytosolic DNA, but Not to Interferon-α Stimulation, in TRIM56-Deficient Cells ». Viruses 14, n^o 1 (5 janvier 2022) : 89. http://dx.doi.org/10.3390/v14010089.

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The physiologic function of tripartite motif protein 56 (TRIM56), a ubiquitously expressed E3 ligase classified within the large TRIM protein family, remains elusive. Gene knockdown studies have suggested TRIM56 as a positive regulator of the type I interferon (IFN-I) antiviral response elicited via the Toll-like receptor 3 (TLR3) and cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) pathways, which detect and respond to danger signals—extracellular double-stranded (ds) RNA and cytosolic dsDNA, respectively. However, to what extent these pathways depend on TRIM56 in human cells is unclear. In addition, it is debatable whether TRIM56 plays a part in controlling the expression of IFN-stimulated genes (ISGs) resulting from IFN-I based antiviral treatment. In this study, we created HeLa-derived TRIM56 null cell lines by gene editing and used these cell models to comprehensively examine the impact of endogenous TRIM56 on innate antiviral responses. Our results showed that TRIM56 knockout severely undermined the upregulation of ISGs by extracellular dsRNA and that loss of TRIM56 weakened the response to cytosolic dsDNA. ISG induction and ISGylation following IFN-α stimulation, however, were not compromised by TRIM56 deletion. Using a vesicular stomatitis virus-based antiviral bioactivity assay, we demonstrated that IFN-α could efficiently establish an antiviral state in TRIM56 null cells, providing direct evidence that TRIM56 is not required for the general antiviral action of IFN-I. Altogether, these data ascertain the contributions of TRIM56 to TLR3- and cGAS–STING-dependent antiviral pathways in HeLa cells and add to our understanding of the roles this protein plays in innate immunity.

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Mathsyaraja, Haritha, Benjamin Wolf, Chari Cortez, Adam Kashishian, Alison Karst, Madelyn Cueva, Min Wang, Kathleen S. Keegan et Jennifer Cain. « Abstract 2079 : Tumor cell intrinsic inactivation of TREX1 increases type I IFN signaling and immune cell recruitment ». Cancer Research 82, n^o 12_Supplement (15 juin 2022) : 2079. http://dx.doi.org/10.1158/1538-7445.am2022-2079.

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Abstract Tumor cells employ mechanisms to evade anti-tumor immune responses that can include the downregulation or silencing of cytosolic nucleic acid sensing pathway components to dampen Type I IFN signaling. It has been documented that genomic instability and mitotic stress in malignant cells can lead to aberrant cytosolic DNA accumulation and activation of the cGAS-STING pathway. A key negative regulator of cytosolic DNA is the DNA exonuclease TREX1. TREX1 assists in removing DNA from the cytosol, in turn reducing cGAS-STING mediated type I IFN induction. Not surprisingly, TREX1 is overexpressed in several tumor types when compared to normal tissue. The functional importance of TREX1 in cytoplasmic nucleic acid surveillance is evidenced by inactivating TREX1 mutations having an association with Type I inteferonopathies such as Aicardi-Goutières syndrome and SLE. Specifically, most of these mutations impact the enzymatic function of TREX1, underscoring the importance of its exonuclease function. To directly assess TREX1 function in cancer, we inactivated TREX1 in a panel of tumor cell lines. Loss of TREX1 resulted in reduced proliferation and increased expression of the Type I IFN-stimulated gene CXCL10. This finding was validated with CRISPR knockout of TREX1, which also led to the activation of IRF3 and a Type I IFN associated gene signature. To characterize the role of TREX1 function in curbing anti-tumor immunity in vivo, we inactivated Trex1 in the murine syngeneic B16F10 melanoma model. TREX1 knockout together with PD1 blockade resulted in slower tumor growth when compared to PD1 blockade alone. This was accompanied by an increase in CD8+ T and NK cell infiltration in tumors. In addition, gene expression profiling of whole tumors revealed that TREX1 loss resulted in increased type I IFN signaling within the tumor microenvironment. In summary, our data strongly suggest a critical role for TREX1 in suppressing anti-tumor immunity and indicate it would be an attractive target for therapeutic intervention. Citation Format: Haritha Mathsyaraja, Benjamin Wolf, Chari Cortez, Adam Kashishian, Alison Karst, Madelyn Cueva, Min Wang, Kathleen S. Keegan, Jennifer Cain. Tumor cell intrinsic inactivation of TREX1 increases type I IFN signaling and immune cell recruitment [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 2079.

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Liu, Shuaitong, Gregory Mazo, Tuo Zhang, Ning Yang, Yi Wang, Adrian Tan, Jiahu Wang et al. « 696 Single-cell RNA-seq reveals the critical roles of the STING- and MDA5-mediated cytosolic nucleic acid-sensing pathways as well as IFNAR/STAT2 signaling in recombinant MVA-induced antitumor immunity ». Journal for ImmunoTherapy of Cancer 9, Suppl 2 (novembre 2021) : A724. http://dx.doi.org/10.1136/jitc-2021-sitc2021.696.

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BackgroundOncolytic viruses are promising therapeutic agents for advanced cancers because of their ability to induce antitumor innate and adaptive immunity. Modified vaccinia virus Ankara (MVA) is an attenuated, replication-deficient poxvirus safe for human use, making it a favorable platform for cancer immunotherapy. Previously we discovered the E5R gene in MVA encodes an inhibitor of the cGAS/STING-mediated cytosolic DNA-sensing pathway. The engineered MVA deleting the E5R gene and expressing a dendritic cell (DC) growth factor Flt3L together with a T cell co-stimulator OX40L generates strong antitumor immunity when delivered intratumorally. We also demonstrated that intratumoral (IT) injection of 1st generation rMVA promotes CD8+ activation in a cGAS/STING- and STAT1/STAT2-dependent manner and depletes the OX40hi regulatory T cells (Tregs) via OX40L/OX40 interaction and IFNAR signaling. The E3L gene from MVA encodes an inhibitor of the cytosolic dsRNA-sensing pathways. The purpose of this study is to investigate whether rMVAΔE3L, with the deletion of E3L from rMVA (MVAΔE5R-hFlt3L-mOX40L), further improves its antitumor effects. We also focused on utilizing single-cell RNA-seq (scRNA-seq) to investigate the alteration of the immunosuppressive tumor microenvironment by IT delivery of rMVAΔE3L.MethodsWe engineered rMVAΔE3L by deleting the E3L gene from rMVA. We investigated the innate immune responses of bone marrow-derived DCs (BMDCs) and tumor cell lines to rMVAΔE3L and rMVA. We also compared the antitumor efficacy of the two viruses in murine B16-F10 melanoma and MC38 colon cancer models. In addition, we performed scRNA-seq of sorted tumor-infiltrating CD45+ immune cells from tumors harvested two days after IT injection with rMVAΔE3L or PBS in wild-type, MDA5/STING-, STAT2-, or IFNAR1-deficient mice.ResultsCompared with rMVA, rMVAΔE3L more potently activates both the cGAS/STING- and MDA5/MAVS-mediated nucleic acid-sensing pathways in DCs and tumor cell lines. IT rMVAΔE3L generates stronger antitumor effects than rMVA. Our results showed that after rMVAΔE3L treatment, there was an influx of inflammatory monocytes into the tumors in WT mice, absent in STAT2 or IFNAR knockout mice. Evaluation of viral transcriptomes at a single-cell level revealed that macrophages and monocytes were more susceptible to rMVAΔE3L than other cell types. The transcriptomic changes on DC subsets are largely dependent on IFNAR/STAT2 signaling. By contrast, MDA5/STING-mediated pathway determines the transcriptome profile of inflammatory monocytes in response to rMVAΔE3L.ConclusionsTaken together, our results demonstrate that scRNA-seq is a powerful approach to interrogate the host immune responses to immunogenic viruses, which would guide future designs of more effective viral-based cancer immunotherapy.AcknowledgementsThis work was supported that the Society of Memorial Sloan Kettering (MSK) research grant (L.D.), MSK Technology Development Fund (L.D.), Parker Institute for Cancer Immunotherapy Career Development Award (L.D.), sponsored research award from IMVAQ Therapeutics. This work was supported in part by the Swim across America (J.D.W., T.M.), Ludwig Institute for Cancer Research (J.D.W., T.M.), National Cancer Institute grants R01 CA56821 (J.D.W). This research was also funded in part through the NIH/NCI Cancer Center Support Grant P30 CA008748.

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Temple, Rachel Mary, Eyal Amiel et Joshua J. Obar. « When signals get crossed : the consequences of dual allergen- and influenza-mediated signaling in mast cells ». Journal of Immunology 196, n^o 1_Supplement (1 mai 2016) : 123.4. http://dx.doi.org/10.4049/jimmunol.196.supp.123.4.

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Abstract Despite the many advances in our understanding of viral infections, influenza A virus (IAV) continues to impose a significant heath burden and presents a serious threat for a worldwide pandemic. Severe IAV infection is characterized by a cytokine storm. Recent evidence indicates mast cell activation is a key initiator of this cytokine storm during severe IAV infection. During the 2009 H1N1 pandemic, asthma was the most common co-morbidity among hospitalized individuals suggesting a potential interplay between pre-existing mast cell disease and IAV infection. Experimental data examining the cross-talk that can occur during dual FcɛRI and IAV stimulation of mast cells is lacking. Here we show that signaling through FcɛRI concurrent with IAV infection (A/WSN/33) augments the mast cell response resulting in a highly synergist enhancement of cytokine synthesis and release by these cells in vitro. Surprisingly, while IAV induced cytokine production by A/WSN/33 is dependent on the MAVS pathway, using bone marrow cultured mast cells from MAVS and STING knockout mice, we determined this synergistic effect can occur independent of MAVS signaling and the STING adaptor. Furthermore, the synergy can be observed as early as 1 hour PI, prior to significant viral replication. These data suggest an early signaling event is responsible for the synergy. Our data supports a role for the lipid mediator sphingosine-1-phosphate, which is known to be important during allergen stimulation, and as our data demonstrates, is essential for proper IAV activation of mast cells. Taken together, our data suggest that IAV may incite asthmatic exacerbation through enhanced cytokine production from mast cells receiving two distinct stimuli simultaneously.

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Nemazee, David, Linghang Peng, Tanya Blane, Deli Huang et Amanda Gavin. « Cleavage of DNA and RNA by PLD3 and PLD4 limits autoinflammatory triggering by multiple sensors, including endolysosomal TLRs and a STING dependent sensing pathway. » Journal of Immunology 208, n^o 1_Supplement (1 mai 2022) : 158.05. http://dx.doi.org/10.4049/jimmunol.208.supp.158.05.

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Abstract We have studied the biochemistry and genetics of Phospholipase D3 (PLD3) and PLD4 because polymorphisms of these proteins have been associated with several important inflammatory and autoimmune diseases, including rheumatoid arthritis, systemic sclerosis, systemic lupus erythematosus and Alzheimer’s disease. We found that both PLD3 and PLD4 are 5’ exonucleases able to degrade both ssDNA and ssRNA. Deficiency in PLD3, PLD4 or both results in a variety of inflammatory defects, with the most serious being lethal hemophagocytic lymphohistiocytosis (HLH) in double knockout mice. Pld3−/−Pld4−/− mice represent a unique spontaneous model of HLH apparently independent of microbes, whereas Pld4−/− mice develop a range of phenotypes depending upon genetic background ranging from macrophage activation syndrome to lupus like disease. Evidence will be presented showing that the diseases in these mice result from autorecognition of nucleic acids and can be reversed by mutation of nucleic acid sensing pathways. PLD3 and PLD4 tend to limit TLR9 recognition but have a more complex effect on recognition by TLR7 and TLR8. Unc93b13d/3dPld3−/−Pld4−/− mice spontaneously signal through STING suggesting that PLD3 and PLD4 also may regulate cytoplasmic nucleic acid sensing. Supported by NIH grants R01 AI142945 and R56AG070775

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Kazmierski, Julia, Carina Elsner, Katinka Döhner, Shuting Xu, Aurélie Ducroux, Fabian Pott, Jenny Jansen et al. « A Baseline Cellular Antiviral State Is Maintained by cGAS and Its Most Frequent Naturally Occurring Variant rs610913 ». Journal of Immunology 209, n^o 3 (1 août 2022) : 535–47. http://dx.doi.org/10.4049/jimmunol.2100685.

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Abstract Upon recognition of aberrantly located DNA, the innate immune sensor cyclic GMP-AMP synthase (cGAS) activates stimulator of IFN genes (STING)/IFN regulatory factor (IRF)3–driven antiviral responses. In this study, we characterized the ability of a specific variant of the human cGAS-encoding gene MB21D1, rs610913, to alter cGAS-mediated DNA sensing and viral infection. rs610913 is a frequent G>T polymorphism resulting in a P261H exchange in the cGAS protein. Data from the International Collaboration for the Genomics of HIV suggested that rs610913 nominally associates with HIV-1 acquisition in vivo. Molecular modeling of cGAS(P261H) hinted toward the possibility for an additional binding site for a potential cellular cofactor in cGAS dimers. However, cGAS(wild-type [WT]) or cGAS(P261H)-reconstituted THP-1 cGAS knockout cells shared steady-state expression of IFN-stimulated genes, as opposed to cells expressing the enzymatically inactive cGAS(G212A/S213A). Accordingly, cGAS(WT) and cGAS(P261H) cells were less susceptible to lentiviral transduction and infection with HIV-1, HSV-1, and Chikungunya virus as compared with cGAS knockout or cGAS(G212A/S213A) cells. Upon DNA challenge, innate immune activation appeared to be mildly reduced upon expression of cGAS(P261H) compared with cGAS(WT). Finally, DNA challenge of PBMCs from donors homozygously expressing rs610913 provoked a trend toward a slightly reduced type I IFN response as compared with PBMCs from GG donors. Taken together, the steady-state activity of cGAS maintains a baseline antiviral state rendering cells more refractory to IFN-stimulated gene–sensitive viral infections. rs610913 failed to grossly differ phenotypically from the WT gene, suggesting that cGAS(P261H) and WT cGAS share a similar ability to sense viral infections in vivo.

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Ghonim, Mohamed A., Salome V. Ibba, Abdelmetalab F. Tarhuni, Youssef Errami, Hanh H. Luu, Matthew J. Dean, Ali H. El-Bahrawy et al. « Targeting PARP-1 with metronomic therapy modulates MDSC suppressive function and enhances anti-PD-1 immunotherapy in colon cancer ». Journal for ImmunoTherapy of Cancer 9, n^o 1 (janvier 2021) : e001643. http://dx.doi.org/10.1136/jitc-2020-001643.

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BackgroundPoly(ADP-ribose) polymerase (PARP) inhibitors (eg, olaparib) are effective against BRCA-mutated cancers at/near maximum tolerated doses by trapping PARP-1 on damaged chromatin, benefitting only small patient proportions. The benefits of targeting non-DNA repair aspects of PARP with metronomic doses remain unexplored.MethodsColon epithelial cells or mouse or human bone marrow (BM)-derived-myeloid-derived suppressor cells (MDSCs) were stimulated to assess the effect of partial PARP-1 inhibition on inflammatory gene expression or immune suppression. Mice treated with azoxymethane/four dextran-sulfate-sodium cycles or APCMin/+ mice bred into PARP-1+/− or treated with olaparib were used to examine the role of PARP-1 in colitis-induced or spontaneous colon cancer, respectively. Syngeneic MC-38 cell-based (microsatellite instability, MSIhigh) or CT-26 cell-based (microsatellite stable, MSS) tumor models were used to assess the effects of PARP inhibition on host responses and synergy with anti-Programmed cell Death protein (PD)-1 immunotherapy.ResultsPartial PARP-1 inhibition, via gene heterozygosity or a moderate dose of olaparib, protected against colitis-mediated/APCMin-mediated intestinal tumorigenesis and APCMin-associated cachexia, while extensive inhibition, via gene knockout or a high dose of olaparib, was ineffective or aggravating. A sub-IC50-olaparib dose or PARP-1 heterozygosity was sufficient to block tumorigenesis in a syngeneic colon cancer model by modulating the suppressive function, but not intratumoral migration or differentiation, of MDSCs, with concomitant increases in intratumoral T cell function and cytotoxicity, as assessed by granzyme-B/interferon-γ levels. Adoptive transfer of WT-BM-MDSCs abolished the protective effects of PARP-1 heterozygosity. The mechanism of MDSC modulation involved a reduction in arginase-1/inducible nitric oxide synthase/cyclo-oxygenase-2, but independent of PARP-1 trapping on chromatin. Although a high-concentration olaparib or the high-trapping PARP inhibitor, talazoparib, activated stimulator of interferon gene (STING) in BRCA-proficient cells and induced DNA damage, sub-IC50 concentrations of either drug failed to induce activation of the dsDNA break sensor. STING expression appeared dispensable for MDSC suppressive function and was not strictly required for olaparib-mediated effects. Ironically, STING activation blocked human and mouse MDSC function with no additive effects with olaparib. A metronomic dose of olaparib was highly synergistic with anti-PD-1-based immunotherapy, leading to eradication of MSIhigh or reduction of MSS tumors in mice.ConclusionsThese results support a paradigm-shifting concept that expands the utility of PARP inhibitor and encourage testing metronomic dosing of PARP inhibitor to enhance the efficacy of checkpoint inhibitor-based immunotherapies in cancer.

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Dean, Matthew J., Liqin Zheng, Phaethon Philbrook, Augusto C. Ochoa et A. Hamid Boulares. « Abstract 2081 : Poly ADP-ribose polymerase inhibition enhances T cell cytotoxicity and anti-tumor function by altering NAD+ levels ». Cancer Research 82, n^o 12_Supplement (15 juin 2022) : 2081. http://dx.doi.org/10.1158/1538-7445.am2022-2081.

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Abstract Poly ADP-ribose polymerase (PARP) is an NAD-dependent DNA repair enzyme. Inhibition of PARP in BRCA-mutant cancers has been a clinically approved therapy for several years. While the PARP inhibitor, olaparib, has been approved for the treatment of BRCA-mutant breast and ovarian cancers, it has not been used in other cancers because its mechanism of action relies on synthetic lethality. We have found that olaparib may also have use in other cancers due to its ability to modulate the immune system directly. Specifically, we have found that olaparib is able to increase the proliferation, activation, and cytotoxicity of T cells in vitro and reduce tumor growth in vivo at low doses. Recent papers have proposed that olaparib is able to enhance T cell function through the stimulator of interferon genes (STING) pathway by enhancing antigen presenting cell activity and resulting in more granzyme B expression and enhanced tumor killing. Using purified T cells from both wild type and STING knockout mice, we have shown that olaparib can also cause an increase in granzyme B and perforin expression, an increase in T cell proliferation, and enhanced tumor cell killing in a STING-independent manner. Because PARP is an NAD-consuming enzyme, we examined the role of NAD+ in the activation T cells and the production of granzyme B and perforin. By using an inhibitor of NAD+ recycling, FK866, we show that T cells have reduced NAD+ levels, reduced granzyme B and perforin expression, and even fail to activate in the presence of CD3 and CD28 when NAD+ is limited; however, adding olaparib to these cells rescues the effects of FK866 and allows proliferation and granzyme B production. In vivo we can see that even at olaparib doses that are significantly below those which induce synthetic lethality of tumor cells, olaparib is able to inhibit tumor growth in models of both colorectal and breast cancer. This suggests that olaparib can modulate the activity of T cells directly and has implications for enhancing the anti-tumor activity of these cells both alone and in combination with other forms of immunotherapy. Citation Format: Matthew J. Dean, Liqin Zheng, Phaethon Philbrook, Augusto C. Ochoa, A. Hamid Boulares. Poly ADP-ribose polymerase inhibition enhances T cell cytotoxicity and anti-tumor function by altering NAD+ levels [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 2081.

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Li, Jian, Josephine Mun-Yee Ko, Wei Dai, Valen Zhuoyou Yu, Hoi Yan Ng, Jean-Sébastien Hoffmann et Maria Li Lung. « Depletion of DNA Polymerase Theta Inhibits Tumor Growth and Promotes Genome Instability through the cGAS-STING-ISG Pathway in Esophageal Squamous Cell Carcinoma ». Cancers 13, n^o 13 (26 juin 2021) : 3204. http://dx.doi.org/10.3390/cancers13133204.

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Overexpression of the specialized DNA polymerase theta (POLQ) is frequent in breast, colon and lung cancers and has been correlated with unfavorable clinical outcomes. Here, we aimed to determine the importance and functional role of POLQ in esophageal squamous cell carcinoma (ESCC). Integrated analysis of four RNA-seq datasets showed POLQ was predominantly upregulated in ESCC tumors. High expression of POLQ was also observed in a cohort of 25 Hong Kong ESCC patients and negatively correlated with ESCC patient survival. POLQ knockout (KO) ESCC cells were sensitized to multiple genotoxic agents. Both rH2AX foci staining and the comet assay indicated a higher level of genomic instability in POLQ-depleted cells. Double KO of POLQ and FANCD2, known to promote POLQ recruitment at sites of damage, significantly impaired cell proliferation both in vitro and in vivo, as compared to either single POLQ or FANCD2 KOs. A significantly increased number of micronuclei was observed in POLQ and/or FANCD2 KO ESCC cells. Loss of POLQ and/or FANCD2 also resulted in the activation of cGAS and upregulation of interferon-stimulated genes (ISGs). Our results suggest that high abundance of POLQ in ESCC contributes to the malignant phenotype through genome instability and activation of the cGAS pathway.

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Haarer, Elena L., Corey J. Theodore, Shirley Guo, Ryan B. Frier et Kenneth G. Campellone. « Genomic instability caused by Arp2/3 complex inactivation results in micronucleus biogenesis and cellular senescence ». PLOS Genetics 19, n^o 1 (27 janvier 2023) : e1010045. http://dx.doi.org/10.1371/journal.pgen.1010045.

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The Arp2/3 complex is an actin nucleator with well-characterized activities in cell morphogenesis and movement, but its roles in nuclear processes are relatively understudied. We investigated how the Arp2/3 complex affects genomic integrity and cell cycle progression using mouse fibroblasts containing an inducible knockout (iKO) of the ArpC2 subunit. We show that permanent Arp2/3 complex ablation results in DNA damage, the formation of cytosolic micronuclei, and cellular senescence. Micronuclei arise in ArpC2 iKO cells due to chromatin segregation defects during mitosis and premature mitotic exits. Such phenotypes are explained by the presence of damaged DNA fragments that fail to attach to the mitotic spindle, abnormalities in actin assembly during metaphase, and asymmetric microtubule architecture during anaphase. In the nuclei of Arp2/3-depleted cells, the tumor suppressor p53 is activated and the cell cycle inhibitor Cdkn1a/p21 mediates a G1 arrest. In the cytosol, micronuclei are recognized by the DNA sensor cGAS, which is important for stimulating a STING- and IRF3-associated interferon response. These studies establish functional requirements for the mammalian Arp2/3 complex in mitotic spindle organization and genome stability. They also expand our understanding of the mechanisms leading to senescence and suggest that cytoskeletal dysfunction is an underlying factor in biological aging.

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Li, Xuefeng, Kristin Huntoon, Yifan Wang, Carl Walkey, Betty Y. Kim et Wen Jiang. « Abstract 2067 : NL-201, a de novo engineered IL2/IL15 mimic, synergizes with radiation to generate potent antitumor immunity ». Cancer Research 82, n^o 12_Supplement (15 juin 2022) : 2067. http://dx.doi.org/10.1158/1538-7445.am2022-2067.

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Abstract There is a growing interest in combining radiotherapy (RT), a standard of care treatment for many cancer types, with immunotherapeutic agents to improve efficacy. One such approach is RT in combination with high-dose recombinant interleukin 2 (IL-2), which elicits anti-tumor immune responses by stimulating T-cell and NK cell populations. Although effective for some patients, the overall clinical benefit of high-dose recombinant IL-2 is limited by toxicity and the expansion of regulatory T cells (Tregs) through signaling events mediated by IL2RΑ (CD25). In this study, we examined RT in combination with NL-201, a highly potent and stable CD25-independent IL-2 and IL-15 receptor agonist with enhanced affinity for IL2RΒ and IL2RΓ. Single-agent NL-201 was well-tolerated in mice, yielded tumoricidal activity, expanded peripheral T cells, and enhanced the infiltration of effector T cells and dendritic cells (DCs) into murine breast cancers. In combination with RT, NL-201 enhanced activation of the cytosolic DNA sensor cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, resulting in increased type I interferon (IFN) production in DCs and, consequently, greater tumor infiltration by T cells and more efficient priming of antigen-specific T cells by professional antigen-presenting cells (APCs). The immune stimulatory mechanisms triggered by the NL-201 and RT combination resulted in superior tumor growth inhibition and survival benefit in both localized and metastatic murine breast cancers, including those spread to the central nervous system. Genetic knockout of STING in host cells abrogated the activity of the NL-201 and RT combination, thus confirming the crucial role of innate immune sensors in the combination treatment’s antitumor responses. Additionally, when combined with PD-1 antibodies, NL-201 significantly improved growth inhibition of metastatic breast cancers that were previously resistant to checkpoint blockade. We demonstrated that the CD25-independent IL-2 and IL-15 receptor agonist, NL-201, in combination with RT is well-tolerated and elicits robust anti-tumor activity through both innate and adaptive responses, including in checkpoint resistant tumors and brain metastases. Furthermore, we identify a unique mechanism in which NL-201 synergizes with RT, likely via the direct activation of STING in DCs. Taken together, the results provided herein support further preclinical and clinical investigation of this novel combination regimen in both localized and metastatic settings. Citation Format: Xuefeng Li, Kristin Huntoon, Yifan Wang, Carl Walkey, Betty Y. Kim, Wen Jiang. NL-201, a de novo engineered IL2/IL15 mimic, synergizes with radiation to generate potent antitumor immunity [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 2067.

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Lejeune, Pascale, Véronique Cruciani, Axel Berg-Larsen, Andreas Schlicker, Anne Mobergslien, Lisa Bartnitzky, Sandra Berndt et al. « Immunostimulatory effects of targeted thorium-227 conjugates as single agent and in combination with anti-PD-L1 therapy ». Journal for ImmunoTherapy of Cancer 9, n^o 10 (octobre 2021) : e002387. http://dx.doi.org/10.1136/jitc-2021-002387.

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BackgroundTargeted thorium-227 conjugates (TTCs) are an emerging class of targeted alpha therapies (TATs). Their unique mode of action (MoA) is the induction of difficult-to-repair clustered DNA double-strand breaks. However, thus far, their effects on the immune system are largely unknown. Here, we investigated the immunostimulatory effects of the mesothelin-targeted thorium-227 conjugate (MSLN-TTC)in vitroandin vivoin monotherapy and in combination with an inhibitor of the immune checkpoint programmed death receptor ligand 1 (PD-L1) in immunocompetent mice.MethodsThe murine cell line MC38 was transfected with the human gene encoding for MSLN (hMSLN) to enable binding of the non-cross-reactive MSLN-TTC. The immunostimulatory effects of MSLN-TTC were studiedin vitroon human cancer cell lines and MC38-hMSLN cells. The efficacy and MoA of MSLN-TTC were studiedin vivoas monotherapy or in combination with anti-PD-L1 in MC38-hMSLN tumor-bearing immunocompetent C57BL/6 mice. Experiments were supported by RNA sequencing, flow cytometry, immunohistochemistry, mesoscale, and TaqMan PCR analyses to study the underlying immunostimulatory effects.In vivodepletion of CD8+ T cells and studies with Rag2/Il2Rg double knockout C57BL/6 mice were conducted to investigate the importance of immune cells to the efficacy of MSLN-TTC.ResultsMSLN-TTC treatment induced upregulation of DNA sensing pathway transcripts (IL-6,CCL20,CXCL10, and stimulator of interferon genes (STING)-related genes)in vitroas determined by RNASeq analysis. The results, including phospho-STING activation, were confirmed on the protein level. Danger-associated molecular pattern molecules were upregulated in parallel, leading to dendritic cell (DC) activationin vitro. MSLN-TTC showed strong antitumor activity (T:C 0.38, p<0.05) as a single agent in human MSLN-expressing MC38 tumor-bearing immunocompetent mice. Combining MSLN-TTC with anti-PD-L1 further enhanced the efficacy (T:C 0.08, p<0.001) as evidenced by the increased number of tumor-free surviving animals. MSLN-TTC monotherapy caused migration of CD103+ cDC1 DCs and infiltration of CD8+ T cells into tumors, which was enhanced on combination with anti-PD-L1. Intriguingly, CD8+ T-cell depletion decreased antitumor efficacy.ConclusionsThesein vitroandin vivodata on MSLN-TTC demonstrate that the MoA of TTCs involves activation of the immune system. The findings are of relevance for other targeted radiotherapies and may guide clinical combination strategies.

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Loew, Lisa, Niluka Goonawardane, Jeremy Ratcliff, Dung Nguyen et Peter Simmonds. « Use of a small DNA virus model to investigate mechanisms of CpG dinucleotide-induced attenuation of virus replication ». Journal of General Virology 101, n^o 11 (1 novembre 2020) : 1202–18. http://dx.doi.org/10.1099/jgv.0.001477.

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Suppression of the CpG dinucleotide is widespread in RNA viruses infecting vertebrates and plants, and in the genomes of retroviruses and small mammalian DNA viruses. The functional basis for CpG suppression in the latter was investigated through the construction of mutants of the parvovirus, minute virus of mice (MVM) with increased CpG or TpA dinucleotides in the VP gene. CpG-high mutants displayed extraordinary attenuation in A9 cells compared to wild-type MVM (>six logs), while TpA elevation showed no replication effect. Attenuation was independent of Toll-like receptor 9 and STING-mediated DNA recognition pathways and unrelated to effects on translation efficiency. While translation from codon-optimized VP RNA was enhanced in a cell-free assay, MVM containing this sequence was highly attenuated. Further mutational analysis indicated that this arose through its increased numbers of CpG dinucleotides (7→70) and separately from its increased G+C content (42.3→57.4 %), which independently attenuated replication. CpG-high viruses showed impaired NS mRNA expression by qPCR and reduced NS and particularly VP protein expression detected by immunofluorescence and replication in A549 cells, effects reversed in zinc antiviral protein (ZAP) knockout cells, even though nuclear relocalization of VP remained defective. The demonstrated functional basis for CpG suppression in MVM and potentially other small DNA viruses and the observed intolerance of CpGs in coding sequences, even after codon optimization, has implications for the use of small DNA virus vectors in gene therapy and immunization.

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