STING Activation Research Articles

Human papillomaviruses sensitize cells to DNA damage induced apoptosis by targeting the innate immune sensor cGAS.

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.

IFI16-dependent STING signaling is a crucial regulator of anti-HER2 immune response in HER2+ breast cancer

Relapse to anti-HER2 monoclonal antibody (mAb) therapies, such as trastuzumab in HER2+ breast cancer (BC), is associated with residual disease progression due to resistance to therapy. Here, we identify interferon-γ inducible protein 16 (IFI16)-dependent STING signaling as a significant determinant of trastuzumab responses in HER2+ BC. We show that down-regulation of immune-regulated genes (IRG) is specifically associated with poor survival of HER2+, but not other BC subtypes. Among IRG, IFI16 is identified as a direct target of EZH2, the underexpression of which leads to deficient STING activation and downstream CXCL10/11 expression in response to trastuzumab treatment. Dual inhibition of EZH2 and histone deacetylase (HDAC) significantly activates IFI16-dependent immune responses to trastuzumab. Notably, a combination of a novel histone methylation inhibitor with an HDAC inhibitor induces complete tumor eradication and long-term T cell memory in a HER2+ BC mouse model. Our findings demonstrate an epigenetic regulatory mechanism suppressing the expression of the IFI16-CXCL10/11 signaling pathway that provides a survival advantage to HER2+ BC to confer resistance to trastuzumab treatment.

Activation of STING Based on Its Structural Features.

The cGAS-cGAMP-STING pathway is an important innate immune signaling cascade responsible for the sensing of abnormal cytosolic double-stranded DNA (dsDNA), which is a hallmark of infection or cancers. Recently, tremendous progress has been made in the understanding of the STING activation mechanism from various aspects. In this review, the molecular mechanism of activation of STING protein based on its structural features is briefly discussed. The underlying molecular mechanism of STING activation will enable us to develop novel therapeutics to treat STING-associated diseases and understand how STING has evolved to eliminate infection and maintain immune homeostasis in innate immunity.

Taking the STING out of aging?

By preventing STING activation, the transcriptional coactivators YAP/TAZ suppress cellular senescence.

Abstract 6056: Activation of Sting in response to partial-tumor radiation exposure

Abstract Purpose: It has been shown that ionizing radiation can mediate antitumor immunity via the activation of the cytosolic DNA sensor cGAS/STING pathway. Therefore, the purpose of this study was to determine whether STING activation is involved in partial volume radiation therapy (RT). Materials/Methods: We investigated 67NR murine orthotopic breast tumors in Balb/c mice and LLC cells injected in the flank of C57Bl/6, cGAS, or STING KO mice. RT was delivered to 50% or 100% of the tumor volume using a 2X2 cm collimator on a microirradiator allowing precise irradiation. Tumors were collected at different time points post-RT and assessed for different measurements. Results: We previously showed that a single dose of radiation delivered to half of the tumor (50% RT) activated an anti-tumor immune response comparable to the response in a fully-irradiated tumor in the immunogenic 67NR murine breast carcinoma tumor model and in the less immunogenic and more radioresistant Lewis lung carcinoma (LLC) tumor model. We have also demonstrated that this immune response was due to the infiltration of CD8+ T cells along with an increased expression of ICAM adhesion molecules. Treatment with either anti-CD8 or anti-ICAM antibodies abrogated the hemi-RT response. Furthermore, a significant abscopal effect was observed after partial irradiation with a single dose of 10Gy in a bilateral 67NR tumors model. In this study, we tested whether the hemi-irradiation-mediated immune response involves the cGAS/STING canonical pathway, or a non-canonical activation of STING, in the 67NR or LLC tumor models. It has been reported that STING can be activated, independently of cGAS, via non-canonical activation of STING, involving ATM and TRAF6, among other factors. We found significant activation of the cGAS/STING pathway in the hemi-irradiated tumors as compared to control and to 100% exposed 67NR tumors. Interestingly, the increased expression of the cGAS/STING pathway was found in the hemi-irradiated tumor but, also in the non-irradiated part of this hemi-irradiated tumor. In the LLC model, a non-canonical activation of STING was involved. Using both cGAS and STING KO mice, we demonstrated that the partial exposure RT-mediated immune response is dependent on STING activation in the host while cGAS is dispensable. Conclusions: Upstream pathways responsible for STING activation are tumor type-specific. Identifying the upstream pathways responsible for STING activation in the partial RT-mediated immune response in different tumor types would improve this therapy and its potential combination with immune checkpoint blockade. Citation Format: Mickael Mathieu, Prerna R. Nepali, Sadna Budhu, Simon N. Powell, John Humm, Joseph Deasy, Adriana Haimovitz-Friedman. Activation of Sting in response to partial-tumor radiation exposure [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 6056.

Abstract 3512: Immunotherapy by STING activation in a CRISPR induce mouse model of GBM decrease tumor progression by CD8 T-cells and altered vascularity

Abstract Intrinsic malignant brain tumors, such as glioblastoma (GBM) are frequently resistant to immunotherapy with few hyper-mutated glioblastomas showing response. Here we have applied a long-term treatment with STING agonist and PD-1 inhibitors alone and in combination towards eliciting enhanced host immune response to combat GBM progression. To address these questions we generated mouse models of GBM by intracranial implantation of a murine GBM stem cell line or orthotopic CRISPR induced mutation to astrocytes. Systemic administration of STING agonist crosses the blood-brain barrier and induces a type-1 IFN signature in the brain and this was further enhanced in the tumor milieu. Treatment outcomes were addressed by MRi and treated animals showed a significant reduction in tumor volume and hence improved survival in comparison to the animals in the control group. Histochemical evaluation of tumor samples from the animals that received treatment showed elevated necrosis and aberrant/leaky vasculature in comparison to the control tumor samples. The presence of cytotoxic T-cells were similar between the groups but activated PD-1 positive T-cells were increased in treated tumors. Ex-vivo analysis of isolated T-cells from tumor baring mice confirms cytotoxic T-cells targeting the GBM cells. In conclusion, application of STING agonist promotes alteration of vasculitis and priming of T-cells in GBM results in increased survival and decreased tumor progression. These results reveal that GBM can be targeted by immune therapy through systemic activation of STING/IFN pathway. Citation Format: Justin V. Joseph, Huiqiang Cai, Brian Hansen, Soren R. Paludan, Mikkel Vendelbo, Martin K. Thomsen. Immunotherapy by STING activation in a CRISPR induce mouse model of GBM decrease tumor progression by CD8 T-cells and altered vascularity [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 3512.

Abstract 382: MTL-STING restores endogenous STING expression for improving efficacy of cancer therapeutics

Abstract cGAS-cGAMP-STING is essential for sensing foreign DNA from pathogens or self-DNA from dying cancer cells. Activation of this pathway is critical for the innate immune response to cancer and is necessary for the full efficacy of various cancer treatments including checkpoint and PARP inhibitors, radiotherapy, and CAR T-cells. Intense efforts have focused on triggering this pathway with cGAMP analogs, which are small-molecule activators of STING. However, recent reports show that many cancer cells downregulate STING by promoter methylation. Furthermore, T-cells activated by cGAMP undergo apoptosis, which limits the efficacy of T-cells based tumor eradication. Indeed, emerging evidence suggests that the critical cells in which STING activation achieves maximal anti-cancer efficacy is myeloid cells in the tumor microenvironment (TME). saRNAs are small double-stranded complementary RNA oligos that increase target mRNA expression, they directly modulate traditional "undruggable" targets such as STING. Using single cell RNA sequencing data, we demonstrated that STING expression is decreased in myeloid cells within the TME of cancer patients. Therefore, our hypothesis was that saRNAs targeted against STING would restore its expression to levels that are necessary for pathway activation either alone or in combination with other drugs, including cGAMP analogs. Using our proprietary algorithm, we designed saRNA sequences that are complementary to positions +2000 to -2000 nucleotides around the transcriptional start site of STING. The lead sequence (named saSTING) was further optimized by our discovery pipeline. Upregulation of STING mRNA was detected up to 168 hours post-transfection of saSTING in multiple cell lines, with an increase in the range of 4 to 11 fold. The upregulation of STING expression was further validated at the protein level. Strand and sequence specificity of saSTING was demonstrated by mutational analysis. Concerning the mechanism of transcriptional activation via saSTING, we show that saSTING regulates STING expression by increasing both nascent pre-mRNA and mature mRNA. In essence, saSTING causes transcriptional activation across the whole STING locus, which is recapitulated in an upregulation of spliced and polyadenylated (mature) STING mRNA. Development candidate MTL-STING encapsulates saSTING in NOV340 liposomes for in vivo administration. NOV340 liposomes have previously been clinically validated to efficiently deliver saRNA to myeloid cells, which is the critical cellular subset to benefit from STING activation from an anti-tumor perspective. MTL-STING is pre-clinical development as a combination treatment administered intra-tumorally and is expected to enter Phase 1 in 2023. Citation Format: Choon Ping Tan, Konstantina Skourti Stathaki, Brid M Ryan, Valentí Gomez, Laura Sinigaglia, Siv Anita Hegre, Robert Habib, John J Rossi, Nagy A Habib. MTL-STING restores endogenous STING expression for improving efficacy of cancer therapeutics [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 382.

Abstract 2483: Detection of STING-induced immune activation via [18F]FDG-PET imaging

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.

Abstract 2067: NL-201, a de novo engineered IL2/IL15 mimic, synergizes with radiation to generate potent antitumor immunity

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.

Abstract 6301: DNA methyltransferase inhibitors increase ERV reactivation and STING-dependent interferon/inflammasome signaling in TP53 mutant AML

Abstract DNA methyltransferase inhibitors (DNMTis), which transcriptionally activate hypermethylated genes in cancers and leukemias, also activate endogenous retroviruses (ERVs), leading to increased cytosolic double-stranded (ds) RNA and interferon (IFN) signaling, in a process termed viral mimicry. The tumor suppressor TP53 has been reported to cooperate with DNA methylation and IFN signaling to maintain transcriptional silencing of various repeat sequences, including ERVs. We now report that DNMTis, used to treat acute myeloid leukemia (AML) patients unfit for chemotherapy, robustly induce expression of ERVs and STING-dependent IFN/inflammasome signaling in AML with mutations in TP53(approximately 10%), a subset with poor prognosis. First, our studies of ERV transcripts (N=13) levels in TP53 mutant (KASUMI, KG1-1A, U937) and wild-type (WT; MOLM-14, OCI/AML2, and OCI/AML3) AML cell lines and primary cells (N=6 mutant, N=6 WT) show that, while TP53 mutant cells have low baseline ERV expression, DNMTi treatment significantly increases ERV expression, compared with WT TP53 cells (p<0.05). Moreover, inhibiting TP53 in WT TP53 AML cell lines with the specific TP53 inhibitor pifithrin significantly upregulates ERVs, validating the role of TP53 in suppressing ERVs in AML cells. Second, while TCGA data analysis showed that baseline STING transcript are low in TP53 mutant vs WT AML, phospho-STING (activated) is actually increased in TP53 mutant AML cells and DNMTi treatment further activates STING. Increased expression of downstream IFN/inflammasome genes in TP53 mutant AML treated with DNMTi, compared with results in WT cells, validates these results. Moreover, DNMTi combination with poly (ADP-ribose) polymerase inhibitors (PARPis), which we previously reported to activate STING in breast and ovarian cancer though increasing cytosolic dsDNA and dsRNA, further augments STING activation and IFN/inflammasome signaling (p<0.05). Third, the IFN/inflammasome signaling response to DNMTI/PARPI signaling is directly linked with decreased expression of DNA ds break repair (DSBR) genes and activity, leading to homologous recombination defects (HRD). Fourth, the mechanism for both IFN/inflammasome signaling and HRD induction is dependent on STING, since both CRISPR/CAS KO of STING or treatment with the STING inhibitor H-151 can rescue these effects in TP53 mutant and WT AML cells. Our findings increase understanding of the therapy potential for combining DNMTi+PARPi treatment in AML and suggest use of this paradigm to treat mutant TP53 AML. These results also suggest that activating STING may be a key strategy for increasing IFN/inflammasome signaling in treatment of TP53 mutant AML and that immune responses may be further augmented with immune therapy in AML and other cancers. Citation Format: Aksinija A. Kogan, Michael J. Topper, Lora Stojanovic, Lena J. McLaughlin, Tammy J. Kingsbury, Maria R. Baer, Michael Kessler, Stephen B. Baylin, Feyruz V. Rassool. DNA methyltransferase inhibitors increase ERV reactivation and STING-dependent interferon/inflammasome signaling in TP53 mutant AML [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 6301.

Abstract 3822: Chromosomal instability shapes the tumor microenvironment through a chronic ER-stress response

Abstract Chromosomal instability (CIN) is a cancer hallmark associated with cancer metastasis and immune evasion. Yet, it is unclear how CIN modulates the tumor-microenvironment (TME). Here we show that CIN results in a protumor TME with enrichment of immune-suppressive macrophages, a granulocytic infiltrate, and exhausted T cells. Using ContactTracing, a newly developed computational tool to infer conditionally dependent cell-cell interactions from single cell RNA sequence data, we identify tumor ligands induced by the ER stress response in cancer cells as central mediators of immune suppression. Mechanistically, CIN-dependent chronic activation of the cytosolic DNA sensing cGAS-STING pathway promotes ER-stress-dependent transcription. Suppression of CIN or depletion of cancer cell STING reduces ER-stress and restores CIN-induced changes on the TME. Correspondingly, chronic STING activation in human breast cancer patients is associated with reduced tumor infiltrating lymphocytes and increased metastasis. Remarkably, pharmacologic inhibition of chronically active STING or depletion of downstream ER stress signaling suppresses metastasis in syngeneic models of melanoma, breast, and colorectal cancers, thereby demonstrating a viable therapeutic strategy for chromosomally unstable cancers. Citation Format: Jun Li, Melissa Hubisz, Ethan Earlie, Mercedes A. Duran, Emanuele Lettera, Su M. Phyu, Amit D. Amin, Matthew Deyell, Erina Kamiya, Karolina Budre, Julie-Ann Cavallo, Christopher Garris, Hannah Wen, Benjamin Izar, Eileen Parkes, Ashley Laughney, Samuel Bakhoum. Chromosomal instability shapes the tumor microenvironment through a chronic ER-stress response [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 3822.

Abstract 1877: Measurement of cytosolic DNA sensing cGAS-STING pathway functional activity using in vitro phenotypic assay models

Abstract Innate immunity plays a vital role in detecting cytoplasmic nucleic acids resulting from viral infection or the presence of tumor cells. Cytosolic DNA is sensed by the cyclic-GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, and in the case of tumors, activation of this pathway has potential to both positively and negatively modulate cancer development. Activation of the STING pathway is a key prerequisite for type I IFN production that is needed for either endogenous or treatment-induced cancer immune responses, and reduced cGAS-STING expression is associated with poorer survival of patients with lung adenocarcinoma, invasive breast ductal carcinoma, and gastric cancer. Intrinsic activation of the cGAS-STING pathway in the tumor microenvironment (TME) promotes cancer cell-intrinsic senescence and recruitment of immune cells, resulting in antitumor effects. For cancers that are difficult to treat and characterized by immune tolerance, combination therapies of STING agonists with immune checkpoint inhibitors represents a new paradigm in disease management. To date, only two early clinical trials of the STING agonist ADU-S100 in combination with checkpoint inhibitors are underway (NCT02675439 and NCT03172936). To progress clinical trials more rapidly and with better safety outcomes for patients with advanced treatment-refractory metastatic solid tumors or lymphomas, being able to reliably screen a variety of STING agonists in different types of in vitro cancer models is of paramount importance. We have previously reported a novel functional assay that is capable of demonstrating the potency level of different STING agonists, based on the IFNβ response in THP-1 monocytic leukemia cells. We further investigated the role of newly developed STING agonists and clinical compounds in phenotypic in vitro cancer cell and TME models to inform on oncotherapeutic development. STING-expressing cancer cell lines, chosen based on microarray mRNA expression, were treated with agonists and assayed for cGAS-STING activation status using a fully automated platform for high-content imaging. In vitro analysis demonstrated high phospho-STING activation at 4 hours. In the BioMAP® Oncology Colorectal TME model, STING activation increased IL-6 release and the effect on other primary immune and tissue remodeling biomarkers will be discussed. Preclinical studies indicate that STING agonists, used as adjuvants in combination with other agents or radiation therapy, suppress tumor progression, reduce cellular toxicity, and eliminate metastases in breast and pancreatic cancer models. Phenotypic assays that provide human, translational data early in discovery are a valuable tool to accelerate progress in this area. Citation Format: Satheesh K. Sainathan, Justin H. Lipner, Jennifer I. Drake, Brogan A. Epkins, Brianna M. Roux, Alastair J. King. Measurement of cytosolic DNA sensing cGAS-STING pathway functional activity using in vitro phenotypic assay models [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 1877.

Abstract LB198: Cellular selectivity of STING stimulation determines priming of anti-tumor T cell responses

Abstract T cells that recognize tumor antigens are crucial for anti-tumor immune responses. Induction of anti-tumor T cells in immunogenic tumors depends on STING, the intracellular innate immune receptor for cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) and related cyclic dinucleotides (CDN). However, the optimal way to leverage STING activation in non-immunogenic tumors is still unclear. Here, we show that cGAMP delivery by intra-tumoral injection of virus-like particles (cGAMP-VLP) leads to differentiation of tumor-specific T cells, decrease in tumor regulatory T cells (Tregs) and anti-tumoral responses that synergize with PD1 blockade. By contrast, intra-tumoral injection of synthetic CDN leads to tumor necrosis and systemic T cell activation but no differentiation of tumor-specific T cells, and a demise of immune cells in injected tumors. Analyses of cytokine responses and genetic models revealed that cGAMP-VLP preferentially targets STING in dendritic cells at a 1000-fold less dose than synthetic CDN. Sub-cutaneous administration of cGAMP-VLP showed synergy when combined with a tumor Treg-depleting antibody to elicit systemic tumor-specific T cells, leading to complete and lasting tumor eradication. These findings show that cell targeting of STING stimulation shapes the anti-tumor T cell response and reveal a therapeutic strategy with T cell modulators, which may address the current limitations of STING-based approaches in patients. Citation Format: Bakhos Jneid, Aurore Bochnakian, Fabien Delisle, Emeline Djacoto, Jordan Denizeau, Christine Sedlik, Frédéric Fiore, Robert Kramer, Ian Walters, Sylvain Carlioz, Bernard Malissen, Eliane Piaggio, Nicolas Manel. Cellular selectivity of STING stimulation determines priming of anti-tumor T cell responses [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 LB198.

Anticancer effects of PARP1 hyperactivation by a decoy oligodeoxynucleotide in vitro and in vivo.

e15060 Background: Poly (ADP-ribose) polymerase (PARP) inhibitors mediate significant anticancer effects against homologous recombination defective (HRD) tumors but have limited efficacy against homologous recombination proficient (HRP) neoplasms. Moreover, HRD tumors often become resistant to PARP inhibitors during treatment, constituting a major limitation to the clinical use of these agents. Here, we describe the metabolic, antineoplastic and immunomodulatory effects of a first-in-class oligodeoxynucleotide that operates as a PARP1 decoy (OX413), resulting in constitutive hyperactivation of PARP1 and consequent exhaustion of the DNA damage response. Methods: OX413-induced PARP trapping and hyperactivation, NAD+ consumption and cell cytotoxicity were monitored using (HRP and HRD) breast, ovarian or prostate cancer cells as well as normal fibroblasts and leukocytes. DNA repair abrogation was monitored by analyzing repair protein recruitment to damage sites. Accumulation of cytoplasmic DNA fragments was monitored after DNA staining and microscopy analysis. Effects on the innate immune response was assessed by following STING pathway activation and T-cell mediated anti-tumor cytotoxicity. In a mouse xenograft model of breast cancer, OX413-induced PARP activation and tumor infiltration by immune cells were analyzed ex-vivo by flow cytometry. Antitumor efficacy of OX413 alone or combined to a PD1 blocker was assessed in MPA/DMBA-driven HR+ HER2- mouse mammary carcinomas. Results: At odds with conventional PARP inhibitors, OX413 bound to and hyperactivated PARP1 with high affinity and in a dose-dependent manner, resulting in elevated cytotoxicity in multiple cancer (but not normal) cells irrespective of HR status. OX413 impaired DNA repair due to PARP trapping and rapid NAD+ consumption, leading to the accumulation of cytoplasmic chromatin fragments (CCFs). Consistent with this, OX413 triggered activation of the CGAS/STING pathway, CCL5 secretion and potentiation of tumor-targeting T cell responses. PARP1 engagement and STING activation were corroborated in vivo in EMT6 mouse mammary carcinomas, correlating with anticancer effects coupled to tumor infiltration by innate and adaptive immune cells. In MPA/DMBA-driven tumors, OX413 mediated considerable anticancer effects that could be exacerbated by PD1 inhibition. Conclusions: Our results provide preclinical rationale for using OX413 to trigger metabolic exhaustion in cancer cells and initiate inflammatory responses that can be actioned by immune checkpoint inhibitors in patients bearing HRD as well as HRP tumors.

Cancer immunotherapy based on image-guided STING activation by nucleotide nanocomplex-decorated ultrasound microbubbles.

The cytosolic innate immune sensor cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is crucial for priming adaptive antitumour immunity through antigen-presenting cells (APCs). Natural agonists, such as cyclic dinucleotides (CDNs), activate the cGAS-STING pathway, but their clinical translation is impeded by poor cytosolic entry and serum stability, low specificity and rapid tissue clearance. Here we developed an ultrasound (US)-guided cancer immunotherapy platform using nanocomplexes composed of 2'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) electrostatically bound to biocompatible branched cationic biopolymers that are conjugated onto APC-targeting microbubbles (MBs). The nanocomplex-conjugated MBs engaged with APCs and efficiently delivered cGAMP into the cytosol via sonoporation, resulting in activation of cGAS-STING and downstream proinflammatory pathways that efficiently prime antigen-specific T cells. This bridging of innate and adaptive immunity inhibited tumour growth in both localized and metastatic murine cancer models. Our findings demonstrate that targeted local activation of STING in APCs under spatiotemporal US stimulation results in systemic antitumour immunity and improves the therapeutic efficacy of checkpoint blockade, thus paving the way towards novel image-guided strategies for targeted immunotherapy of cancer.

Targeting MUC1-C Suppresses Chronic Activation of Cytosolic Nucleotide Receptors and STING in Triple-Negative Breast Cancer.

Simple SummaryTriple-negative breast cancers (TNBCs) are recalcitrant tumors with limited therapeutic options. Cytotoxic agents, including platinum-based drugs, are a standard of care for advanced TNBCs. Olaparib is also used for the treatment of germline BRCA mutant TNBC tumors in the adjuvant and recurrent disease settings. Notably, however, the effectiveness of these genotoxic agents is often limited by intrinsic and adaptive DNA damage resistance. We demonstrate in TNBC cells that the oncogenic MUC1-C protein chronically activates the type I interferon (IFN) pathway, drives the cGAS/STING axis and induces expression of the DNA damage resistance gene signature (IRDS). Targeting MUC1-C inhibits activation of this pathway in the response to carboplatin and olaparib and sensitizes TNBC cells to these agents. These findings indicate that MUC1-C is a target, which is druggable, for overcoming the obstacle of DNA damage resistance in the treatment of TNBCs.The MUC1-C apical transmembrane protein is activated in the acute response of epithelial cells to inflammation. However, chronic MUC1-C activation promotes cancer progression, emphasizing the importance of MUC1-C as a target for treatment. We report here that MUC1-C is necessary for intrinsic expression of the RIG-I, MDA5 and cGAS cytosolic nucleotide pattern recognition receptors (PRRs) and the cGAS-stimulator of IFN genes (STING) in triple-negative breast cancer (TNBC) cells. Consistent with inducing the PRR/STING axis, MUC1-C drives chronic IFN-β production and activation of the type I interferon (IFN) pathway. MUC1-C thereby induces the IFN-related DNA damage resistance gene signature (IRDS), which includes ISG15, in linking chronic inflammation with DNA damage resistance. Targeting MUC1-C in TNBC cells treated with carboplatin or the PARP inhibitor olaparib further demonstrated that MUC1-C is necessary for expression of PRRs, STING and ISG15 and for intrinsic DNA damage resistance. Of translational relevance, MUC1 significantly associates with upregulation of STING and ISG15 in TNBC tumors and is a target for treatment with CAR T cells, antibody–drug conjugates (ADCs) and direct inhibitors that are under preclinical and clinical development.

Defective mitophagy in aged macrophages promotes mitochondrial DNA cytosolic leakage to activate STING signaling during liver sterile inflammation.

Macrophage‐stimulator of interferon genes (STING) signaling mediated sterile inflammation has been implicated in various age‐related diseases. However, whether and how macrophage mitochondrial DNA (mtDNA) regulates STING signaling in aged macrophages remains largely unknown. We found that hypoxia‐reoxygenation (HR) induced STING activation in macrophages by triggering the release of macrophage mtDNA into the cytosol. Aging promoted the cytosolic leakage of macrophage mtDNA and enhanced STING activation, which was abrogated upon mtDNA depletion or cyclic GMP‐AMP Synthase (cGAS) inhibition. Aged macrophages exhibited increased mitochondrial injury with impaired mitophagy. Mechanistically, a decline in the PTEN‐induced kinase 1 (PINK1)/Parkin‐mediated polyubiquitination of mitochondria was observed in aged macrophages. Pink1 overexpression reversed the inhibition of mitochondrial ubiquitination but failed to promote mitolysosome formation in the aged macrophages. Meanwhile, aging impaired lysosomal biogenesis and function in macrophages by modulating the mTOR/transcription factor EB (TFEB) signaling pathway, which could be reversed by Torin‐1 treatment. Consequently, Pink1 overexpression in combination with Torin‐1 treatment restored mitophagic flux and inhibited mtDNA/cGAS/STING activation in aged macrophages. Moreover, besides HR‐induced metabolic stress, other types of oxidative and hepatotoxic stresses inhibited mitophagy and promoted the cytosolic release of mtDNA to activate STING signaling in aged macrophages. STING deficiency protected aged mice against diverse types of sterile inflammatory liver injuries. Our findings suggest that aging impairs mitophagic flux to facilitate the leakage of macrophage mtDNA into the cytosol and promotes STING activation, and thereby provides a novel potential therapeutic target for sterile inflammatory liver injury in aged patients.

The Chlamydia trachomatis Inclusion Membrane Protein CTL0390 Mediates Host Cell Exit via Lysis through STING Activation.

The obligate intracellular bacterium Chlamydia trachomatis is the causative agent of the most frequently reported bacterial sexually transmitted disease. Upon internalization into host cells, C. trachomatis remains within a membrane-bound compartment known as an inclusion, where it undergoes its developmental cycle. After completion of this cycle, bacteria exit the host cell. One mechanism of exit is lysis, whereby the inclusion and host cell rupture to release bacteria; however, the mechanism of lysis is not well characterized. A subset of C. trachomatis effectors, known as inclusion membrane proteins (Inc), are embedded within the inclusion membrane to facilitate host cell manipulation. The functions of many Inc proteins are unknown. We sought to characterize the Inc protein CTL0390. We determined that CTL0390 is expressed throughout the developmental cycle and that its C-terminal tail is exposed to the cytosol. To investigate the function of CTL0390, we generated a ctl0390 mutant complemented with ctl0390 on a plasmid. Loss of CTL0390 did not affect infectious progeny production but resulted in a reduction in lysis. Overexpression of CTL0390 induced premature lysis and host nuclear condensation, the latter of which could be reduced upon inhibition of the cGAS-STING DNA sensing pathway. Infection with the clt0390 mutant led to reduced Golgi translocation of STING, and chemical and genetic approaches to inactivate STING revealed that STING plays a role in lysis in a CTL0390-dependent manner. Together, these results reveal a role for CTL0390 in bacterial exit via lysis at late stages of the Chlamydia developmental cycle and through STING activation.

STING activation under pre-existing inflammatory conditions causes severe skin disease

Abstract The activation of the stimulator of interferon genes 1 (Sting1) pathway causes rapid induction of type I IFNs. Recently, different STING agonists are being proposed as therapeutics for controlling viral infections like those caused by the SARS-CoV-2. Considering that excessive STING activation leads to autoimmunity, this study was undertaken to investigate the effects of STING activation under pre-existing systemic inflammatory conditions induced by a viral infection. Female BALB/c mice were treated (Tx) with TLR3 agonist poly(IC), which was followed 24h later by subcutaneous injection of STING agonist diamidobenzimidazole (diAbZi). Only diAbZi or vehicle Tx mice were used as controls. Mice Tx with poly(IC) and diAbZi showed the highest systemic levels of type I IFNs, IL-6, and TNFα. Within 10 days, the salivary glands showed increased ILC1, monocytes, and CD8+ T cells. However, glandular function was not affected. Five days after Tx, except for the vehicle controls, all mice showed differing extents of redness, hair loss, and ulceration at the injection site. By day 10, diABZi alone Tx mice showed either normal skin or a small scar formation. In contrast, mice Tx with poly IC and diABZi, showed extensive hair loss, erythema, and ulceration. In addition, hematoxylin and eosin-stained skin sections showed extensive neutrophilic infiltration at the site of ulceration, epidermal thickening, and patchy lymphocytic infiltrate that extended into the subdermal layers. Our data demonstrate that STING activation under systemic inflammatory conditions, such as a viral infection, can lead to severe skin disease. Therefore, caution needs to be exercised in employing STING agonists as therapeutics for treating SARS-CoV-2.

Radioresistant cells in STING gain-of-function mice initiate lymphocyte dependent lung inflammation and IFNγ dependent mortality

Abstract Pediatric patients with gain-of-function (GOF) mutations in the cytosolic dsDNA sensing adaptor STING develop an autoinflammatory syndrome known as STING Associated Vasculopathy with onset in Infancy (SAVI). SAVI patients show elevated interferon signature genes and suffer from interstitial lung disease (ILD) with lymphocyte predominate bronchus associated lymphoid tissue (BALT). We genetically engineered SAVI mice (STING V154M or VM) that recapitulates human disease, including lymphocyte rich ILD, and found by genetic ablation that lymphocytes are required for lung pathology. However, while ablation of either T or B lymphocytes prolongs survival, lung immune aggregates persist, indicating either can independently be recruited as BALT. Using radiation chimeras, we found that hematopoietic stem cell (HSC) derived STING GOF was neither sufficient nor required for VM lung disease, suggesting a role for lung radioresistant parenchymal and stromal cell STING GOF in SAVI. We identified lung endothelia as putative radioresistant cells which express STING and confirmed expression by IF microscopy and FACS. FACS analysis also revealed SAVI lung endothelia upregulate MHCII, but only when sufficient for IFNgR. VM T cells produce IFNg, and IFNgR deficiency was sufficient to prevent endothelial MHCII upregulation and attenuate mortality. However, HSC STING GOF was not required for endothelial MHCII upregulation, suggesting stromal intrinsic STING GOF initiates this outcome. These findings suggest that activation of STING in stromal and parenchymal cells play a key role the initiation of lung inflammation in patients with connective tissue disease ILD. Supported by: T32 GM107000 (MSTP); T32 AI132152 (KMG); F30 HL154674 (KMG); AI128358 (AMR/KAF); Lupus Research Alliance