Stimulator of interferon genes (STING) activation requires coat protein complex II (COPII)-mediated endoplasmic reticulum (ER) exit, but the mechanism remains elusive. Here, we identify EEΦxΦ (339EEVTV343 in human STING) as the ER-exit motif recognized by SEC24 homolog C (SEC24C). Using AlphaFold3, we present a predicted structure of SEC24C binding to a STING dimer, revealing the EEΦxΦ motif in a previously structurally unresolved region. Mutations in this motif or the SEC24C cargo-binding site disrupt STING trafficking and signaling. Our findings support a STING oligomerization and avidity threshold model that explains regulated ER exit. The EEΦxΦ motif is conserved in vertebrate STING homologs and is sufficient to mediate ER exit of unrelated proteins. Interestingly, the STING ER-exit motif is suboptimal compared with known SEC24C cargos, which is crucial for preventing immune overactivation. An engineered “super-ER-exit” STING is constitutively active and induces potent antitumor immunity. Tandem repeats of this motif competitively inhibit endogenous STING signaling. Collectively, this study elucidates the STING-ER-exit mechanism and presents strategies for modulating STING signaling.

Abstract Background The cGAS-STING pathway is a central sensor of cytosolic DNA that activates type I interferon (IFN-I) responses. In cancer, cGAS-STING signaling can promote antitumor immunity by enhancing antigen presentation and T cell priming. However, intrinsic STING activation in T cells also triggers T cell death within the tumor microenvironment in an IFN-independent manner, thereby limiting antitumor T cell immunity. The precise mechanisms underlying this dichotomous behavior remain unknown. Recent work has revealed that, beyond its canonical role in IFN induction, STING also acts as a proton channel that mediates IFN-independent processes such as autophagy, lysosome biogenesis, and cell death. Yet the physiological relevance of STING proton channel in vivo—particularly in shaping T cell fate in tumors—has remained unclear due to the lack of genetic models that selectively disrupt it. Methods We performed targeted mutagenesis of conserved residues in mouse and human STING to identify mutations that selectively abolish proton channel activity while preserving IFN-I signaling. The effects of the mutations on STING trafficking, signaling, autophagy induction, and antiviral responses were evaluated using biochemical assays, imaging, and flow cytometry. To define the physiological role of STING’s proton channel in vivo, we generated a knock-in mouse model carrying the channel mutation. T cell death and antitumor function were assessed using syngeneic tumor models. Results We identified a conserved STING mutation in mouse and human that eliminated proton channel activity and Golgi deacidification while preserving STING trafficking, TBK1-IRF3 activation, and IFN-I induction. Functional assays revealed that the channel mutation abolished STING-mediated autophagy, lysosome biogenesis, and impaired antiviral defense. Splenic T cells from the channel-deficient-STING knock-in mice lost channel-dependent activities and were resistant to STING-induced cell death in vitro. In vivo, STING channel deficiency protected CD8+ T cells from STING-driven cell death in an IFN-independent manner. We further showed that STING channel deficiency enhanced T cell persistence in the tumor microenvironment and significantly reduced tumor growth in the MC38 colon adenocarcinoma model, demonstrating a previously unrecognized role for the STING proton channel in modulating T cell survival and antitumor immunity. Conclusions We establish the first genetic model that selectively disrupts STING proton channel activity while preserving IFN-I signaling. This model uncovers a critical IFN-independent role for the STING proton channel in driving T cell death and promoting tumor immune evasion. Our findings demonstrate distinct, separable outputs of STING signaling and identify the proton channel activity of STING as a potential therapeutic target to improve T cell-mediated antitumor immunity. Citation Format: Cong Xing, Kun Song, Zhen Tang, Antonina Araszkiewicz, Nicole Dobbs, Wanwan Huai, Nan Yan. STING proton channel function controls T cell survival and tumor immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7401.

A signaling pathway that inhibits DNA virus replication is also protective against RNA viruses.

Poly-ADP-ribose polymerase (PARP) family proteins are involved in a wide range of cellular processes. Several PARPs are targeted by inhibitors as treatments for cancer based on their biochemical functions; however, the physiological functions of most PARPs and the potential adverse effects of PARP inhibition are unknown. Here, we show that PARP7 is important for lung physiology. Loss of PARP7 in mice increases susceptibility to chemically induced diffuse alveolar hemorrhaging (DAH) and pristane-induced lupus. Single-nucleus RNA-seq reveals that PARP7 is selectively expressed in alveolar type I cells and PARP7 loss increases immune cell infiltration within the lung, indicating a loss of epithelial barrier integrity. Further, PARP7 inhibition in human bronchial epithelial cells in air-liquid interface culture leads to increased barrier permeability after cigarette smoke challenge or bacterial infection. Mechanistically, we show that PARP7 target, aryl hydrocarbon receptor (AHR), mediates diverse cellular responses to cigarette smoke challenge, including loss of tight junction protein Occludin and increased expression of xenobiotic metabolizing genes and proinflammatory genes. Together, our study uncovers PARP7 as a key player in maintaining the epithelial barrier integrity within the lung, which may have important implications for pulmonary diseases and for guiding PARP7 inhibitor use in the clinic.

The 2',5'-oligoadenylate synthetase (OAS)-RNase L pathway is a classical antiviral innate immune pathway. Upon sensing dsRNA, OAS produces 2',5'-oligoadenylate (2-5A) as a second messenger to activate RNase L. Whether 2-5A can be transported to extend the reach of innate immune signaling has not been established. Here, we showed that 2-5A was transferred from cell to cell through connexin (CX43/CX45) gap junctions. 2-5A was also transferred through importers and exporters, allowing OAS to remotely activate RNase L and protect neighboring cells from viral infection. We identified ABCC10 as a 2-5A exporter. Loss of ABCC10 had no effect on 2-5A production but reduced 2-5A export and protection of neighboring cells. Furthermore, OAShi tumors such as MC38 naturally produced 2-5A in vivo, which was secreted via ABCC10 to activate host-not tumor-RNase L-mediated antitumor response. Therefore, 2-5A is an immunotransmitter that mediates short-range communication between cells in infection and cancer.

Three-prime repair exonuclease 1 (TREX1) is the major DNase in mammalian cells that degrades cytosolic DNA to prevent activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. Genotoxic stress, DNA damage, and radiotherapy induce TREX1 expression in cancer cells, allowing them to evade innate immune activation of type I IFN-mediated antitumor response. Therefore, targeting TREX1 could represent a potential approach to stimulate antitumor immunity. In this study, we conducted a high-throughput small-molecule inhibitor screen of TREX1 using a cell-free DNase assay. Compound 296 specifically inhibited TREX1 DNase activity at low micromolar concentrations, induced type I IFN signaling in cancer cells, and inhibited tumor growth in mice in an inteferon alpha/beta receptor (IFNAR)-dependent manner. Treatment with compound 296 also stimulated T-cell infiltration into tumors and synergized with immune checkpoint blockade. Trex1 knockout cancer cells elicited robust systemic antitumor immunity through tumor-intrinsic cGAS-STING activation and functioned as autologous cancer vaccines that protected against tumor challenge and metastasis. An inducible whole-body Trex1 knockout mouse model was established to simulate "on-demand" systemic TREX1 inactivation in adult mice. Sustained TREX1 loss suppressed a broad range of solid and metastatic tumors in adult mice without incurring severe immune toxicity, even when combined with immune checkpoint blockade, demonstrating the feasibility of an immune-safe therapeutic window. Together, these data demonstrate the antitumor efficacy and immune safety of multiple therapeutic modalities targeting TREX1, including targeting small-molecule inhibitors of TREX1 and employing TREX1 knockout tumor cells as an autologous cancer vaccine. These approaches should pave the way for developing TREX1-targeted cancer immunotherapies. SIGNIFICANCE:Therapeutic modalities targeting TREX1 can activate cGAS-STING signaling and can be incorporated into autologous cancer vaccine designs to improve cancer treatment, supporting the potential of inactivating TREX1 to harness innate immunity. See related commentary by Hanks, p. 2778.

Type I IFN (IFN-I) induce hundreds of antiviral genes as well as negative regulators that limit IFN-I signaling. Here, we investigate the family of 16 PARPs and find that 11 PARPs are ISGs, of which 8 PARPs inhibit IFN-I production. PARP7 is the most potent negative feedback regulator of IFN-I production. Using Parp7-/- and Parp7H532A/H532A mice, we show that PARP7 loss leads to systemic autoimmunity characterized by splenomegaly and increased autoantibodies and inflammatory cytokines. PARP7 loss also results in perivascular immune infiltration in the lung that forms tertiary lymphoid structures. Mechanistically, PARP7 inhibits multiple innate immune pathways in a cell-intrinsic and MARylation-dependent manner. PARP7 interacts with IRF3 through the catalytic domain and disrupts the IRF3:CBP/p300 transcriptional holocomplex required for IFN-I production. Irf3-/- or Irf3S1/S1 (transcription defective) or Sting-/- rescues Parp7H532A/H532A mouse autoimmunity and lung disease. Together, our study reveals physiological functions of PARP7 as a negative feedback regulator of IFN-I production that maintains immune homeostasis particularly in the lung.

PARP7, a mono-ADP-ribosyl (MAR) transferase, is a key suppressor of the type I interferon (IFN-I) IFNβ in various tumor cells and a validated drug target. This negative regulation is reversed by small-molecule inhibitors of PARP7 catalytic activity, resulting in increased IFN-β expression. Yet, the mechanism of action of PARP7 inhibitors remains unclear because the relevant substrates of PARP7-mediated MARylation are unknown. Using an optimized analog- sensitive chemical genetic (ASCG) approach, we identified the co-activators, p300 and CBP, as nuclear PARP7 substrates. We identified an α-helical domain in PARP7 essential for p300/CBP interaction, MARylation, and proteasome degradation. Disrupting PARP7-p300/CBP interaction prevents PARP7's suppression of IFNβ in colorectal cancer cells. p300/CBP reciprocally regulate PARP7's activity and nuclear localization. Intriguingly, treatment with PARP7 inhibitors increased IFNβ expression more than PARP7 knockout in a p300/CBP-dependent manner. Our findings suggest that in some contexts, IFNβ induction by PARP7 inhibitors occurs via two mechanisms: inhibiting MARylation of p300/CBP (loss-of-function) and stabilizing the PARP7- p300/CBP complex (gain-of-function). Teaser:Chemical genetics discovery of p300 and CBP as substrates of PARP7 that are essential for PARP7-mediated regulation of IFNβ via a dual mechanism.

Lysosomes are essential organelles for cellular homeostasis. Defective lysosomes are associated with diseases like lysosomal storage disorders (LSDs). How lysosomal defects are detected and lysosomal function restored remain incompletely understood. Here, we show that STING mediates a neuroinflammatory gene signature in three distinct LSD mouse models, Galctwi/twi, Ppt1-/-, and Cln7-/-. Transcriptomic analysis of Galctwi/twi mouse brain tissue revealed that STING also mediates the expression of lysosomal genes that are regulated by transcriptional factor EB (TFEB). Immunohistochemical and single-nucleus RNA-sequencing (snRNA-seq) analysis show that STING regulates lysosomal gene expression in microglia. Mechanistically, we show that STING activation leads to TFEB dephosphorylation, nuclear translocation, and expression of lysosomal genes. This process requires STING's proton channel function, the V-ATPase-ATG5-ATG8 cascade, and is independent of immune signaling. Furthermore, we show that the STING-TFEB axis facilitates lysosomal repair. Together, our data identify STING-TFEB as a lysosomal quality control mechanism that responds to lysosomal dysfunction.

Stimulator of Interferon Genes (STING) is a critical component of the innate immune system. Mechanisms of STING-mediated type I interferon (IFN-I) signaling during infection are well studied in myeloid cells. However, homeostatic STING expression patterns and their regulation, particularly in lymphoid cells, are unknown. We established a Sting1IRES-EGFP reporter mouse to systematically characterize STING expression spatially in tissues and temporally along development of immune cells. Using this reporter and conditional Sting1 transgenic mouse models, we show that STING expression is repressed in neutrophils and forced STING signaling and expression drives systemic inflammatory disease due to secretion of cytokines and chemokines by neutrophils. Additionally, we show that STING expression is temporally restricted during T lymphocyte development at the double positive stage. Forced STING expression and signaling severely impairs T lymphocyte development and reduces thymopoiesis independent of the type I IFN receptor (IFNAR1). Mechanistically, STING expression in the thymus is controlled via epigenetic silencing by DNA methyltransferase 1 (DNMT1). Forced STING signaling in the thymus favors lineage commitment to innate-like γδ T cells rather than adaptive αβ T cells, revealing a previously unanticipated role of STING in T lymphocyte fate choice. Using two syngeneic tumor models and a cohort of human colorectal cancer patients, we found that tumor-infiltrating CD8+ T lymphocytes gradually repress STING expression as a tumor grows and loss of STING expression strongly correlates with CD8+ T cell exhaustion. Together, our data demonstrates the physiological importance of controlled, rather than ubiquitous STING expression and uncovers STING expression dynamics as an important new dimension of STING pathobiology. Kennady Knox, Devon Jeltema, Nicole Dobbs, Kun Yang, Cong Xing, Kun Song, Zhen Tang, Gustavo Torres-Ramirez, Jiefu Wang, Shan Gao, Tuoqi Wu, Chen Yao, Jian Wang, Nan Yan. Dynamic STING repression orchestrates immune cell functionality across development and maturation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB209.

Flavivirus replication in mammalian cells requires host oligosaccharyltransferase (OST) complex, which is classically known to catalyze protein N-glycosylation. However, enzymatic activity of OST is not required for flavivirus infection, leaving the underlying mechanism puzzling. We show the STT3A sub-complex of OST, including STT3A and DC2, to be critically required for dengue virus (DENV) and Zika virus (ZIKV) infection. We find that STT3A nucleates a mega protein complex assembly during DENV infection as a scaffold through its interaction with other OST subunits, translocon proteins, and viral nonstructural proteins. The integrity of this mega protein complex is important for supporting flavivirus infection. We also identified a small-molecule compound NSC-323241 that disrupts STT3A-mediated mega protein complex assembly and potently blocks DENV and ZIKV infection. Together, our study reveals a scaffolding function of STT3A in flavivirus infection through comprehensive molecular dissection of the multi-subunit OST complex and associated host and viral proteins.

Three-prime repair exonuclease 1 (TREX1) is a cytosolic DNase that suppresses cGAS–STING–mediated type I interferon (IFN-I) responses by degrading self-DNA. While this function is essential for immune homeostasis—highlighted by the fact that TREX1 loss causes autoimmune disorders such as Aicardi-Goutières syndrome—many cancers exploit TREX1 upregulation to evade immune surveillance, particularly following genotoxic therapies. Therapeutically inhibiting TREX1 offers an opportunity to re-engage IFN-I–driven antitumor immunity, but it also raises concerns about triggering immune-related adverse events. Here, we aimed to define the therapeutic benefit and immune safety of targeting TREX1 in vivo. We employed a multimodal strategy to assess the therapeutic potential and safety of targeting TREX1. First, we conducted a high-throughput screen to identify small-molecule inhibitors of TREX1 and evaluated lead compound #296 in syngeneic mouse tumor models. Second, we tested TREX1-deficient tumor cells as autologous cancer vaccines. Finally, we generated inducible whole-body Trex1 knockout mice to model systemic TREX1 inhibition and assess its long-term immune safety. Lead compound #296 selectively inhibited TREX1 enzymatic activity, triggered IFN-I signaling in tumor cells, and significantly suppressed tumor growth in multiple syngeneic mouse models. Treatment with #296 enhanced CD8+ T cell infiltration and reversed resistance to anti–PD-1 therapy in poorly immunogenic B16-F10 melanoma, without inducing systemic inflammation or elevated CRP. TREX1-deficient tumor cells activated tumor-intrinsic cGAS–STING signaling and functioned as effective autologous cancer vaccines, eliciting durable systemic antitumor immunity that protects against both tumor rechallenge and metastasis. Importantly, systemic TREX1 deletion in adult mice using an inducible knockout model led to sustained tumor suppression across diverse tumor models, while maintaining immune safety over 6 months with minimal immune toxicity. Our study reveals TREX1 as a druggable target to stimulate IFN-I–driven antitumor immunity. Across three distinct therapeutic approaches—small-molecule inhibitor, cell-based vaccination, and systemic gene knockout—TREX1 targeting elicited durable tumor control with minimal autoimmune toxicity, supporting its translational potential as a safe and effective cancer immunotherapy. Cong Xing, Nan Yan. Therapeutic inhibition of TREX1 elicits type I interferon–mediated antitumor immunity with minimal autoimmune toxicity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Mechanisms of Cancer Immunity and Cancer-related Autoimmunity; 2025 Sep 24-27; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(9 Suppl):Abstract nr PR-16.

The STING pathway is increasingly recognized as a key regulator of neuroinflammation in neurodegenerative disease, but its role in noninflammatory conditions remains unclear. We generated a postnatal inducible whole-body Ngly1 knockout mouse (iNgly1-/-) to model NGLY1 deficiency, an early-onset neurodegenerative disorder. iNgly1-/- mice exhibit progressive motor deficits, Purkinje cell loss, and shortened lifespan without evidence of gliosis or immune activation. Cell type-specific deletion of Ngly1 in Purkinje cells or microglia failed to induce disease, suggesting multiple cell-intrinsic and cell-extrinsic signals are required. Genetic ablation of Sting1 in iNgly1-/- mice rescues Purkinje cell loss, improves motor function, and extends lifespan. Single-nucleus RNA sequencing reveals proteostasis disruption in Purkinje cells, altered cerebellar granule cell subpopulations, and STING-dependent suppression of cholesterol biosynthesis in glia. Pharmacological inhibition of STING with an orally bioactive antagonist, VS-X4, significantly mitigates neuropathology and motor disease. These findings identify STING as a key mediator of neuropathology in NGLY1 deficiency and implicate a role of STING in noninflammatory neurological disease.

The DNA exonuclease TREX1 acts as an innate immune checkpoint by degrading cytosolic DNA and suppressing cGAS–STING–IFN-I signaling. While cancers exploit TREX1 to evade innate immune sensing, its therapeutic potential has not been fully explored. We investigated multiple modalities for therapeutically targeting TREX1 and demonstrated their efficacy and immunologic safety. A high-throughput screen of 295,000 compounds using a cell-free DNase assay identified small-molecule inhibitors of TREX1. Lead compound #296 was evaluated for IFN-I induction, tumor growth inhibition, and synergy with immune checkpoint blockade (ICB). In parallel, TREX1-knockout tumor cells were tested as autologous cancer vaccines. An inducible whole-body Trex1 knockout mouse model was developed to mimic systemic TREX1 inhibition and assess therapeutic efficacy and immune safety. Compound #296 selectively inhibited TREX1 and robustly activated IFN-I signaling in cancer cells, resulting in CD8+ T cell infiltration and tumor regression. It synergized with anti–PD-1 therapy in resistant B16-F10 melanoma models without causing systemic inflammation or elevated CRP. TREX1-deficient tumor cells activated tumor-intrinsic cGAS–STING signaling and functioned as effective autologous cancer vaccines, eliciting durable systemic antitumor immunity. Systemic TREX1 deletion in adult mice using an inducible knockout model significantly inhibited growth of MC38, E0771, LLC, and B16-F10 tumors, and slowed tumor metastasis, while maintaining immune safety over 6 months with minimal immune-related toxicity. Our study reveals TREX1 as a druggable innate immune checkpoint and presents three distinct and complementary therapeutic strategies: small-molecule inhibitors, tumor-cell-based vaccines, and systemic inactivation. Each approach elicited potent antitumor responses with favorable immune safety, establishing TREX1 as a promising target to reprogram the tumor-immune microenvironment and overcome resistance in immunologically cold tumors. Cong Xing, Nan Yan. The innate immune checkpoint TREX1 is a safe, effective, and druggable target for cancer immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B025.

The stimulator of interferon gene (STING) is an important innate immune mediator of the cytoplasmic DNA sensing pathway. As a mediator known for its role in the immune response to infections, STING is also surprisingly at the center of a variety of non-infectious human diseases, including cancer, autoimmune diseases and neurodegenerative diseases. Recent studies have shown that STING has many signaling activities, including type I interferon (IFN-I) and other IFN-independent activities, many of which are poorly understood. STING also has the unique property of being continuous transported from the ER to the Golgi then to the lysosome. Mutations of STING or trafficking cofactors are associated with human diseases affecting multiple immune and non-immune organs. Here, we review recent advances in STING trafficking and signaling mechanisms based in part on studies of STING-associated monogenic inborn error diseases.