Cytosolic Cyclic Dinucleotides Research Articles

467 Stimulator of Interferon Genes Protein (STING) Agonist Incited Sustained Antitumor Immunity in Murine Models of Glioblastoma

INTRODUCTION: Introduction: Stimulator of interferon genes (STING) is an innate pattern recognition receptor natively localized to the endoplasmic reticulum that detects both exogenous and endogenous pathogenic or damage-associated cytosolic cyclic dinucleotides (CDNs), activating IRF3 (interferon regulatory factor 3), NF-κB (nuclear factor κB), and STAT6 (signal transducer and activator of transcription 6) signaling pathways to induce a potent type I interferon and proinflammatory cytokine responses. Activation of the STING pathway has been recognized as a critical component of the innate immune sensing of tumors that is required for optimal cytotoxic T-cell priming against tumor antigens. Recently a highly potent cyclic dinucleotide STING agonist, IACS-8803, showed robust activation of the STING pathway resulting in systemic anti-tumor response in the B16 murine model of melanoma. Additionally, the STING agonists have been shown to achieve robust tumor regression at both injected and distal lesions and are reported to facilitate generation of long-lived immunologic memory. METHODS: Methods: We established murine gliomas with stereotactic implantation of GL261, a murine glioma cell line. Mice were assessed with luciferase imaging to ensure baseline tumor size and then randomized into treatment arms (IgG, IACS-8803, CD47, PD-1 and combinations thereof). Treatment was initiated one week after the tumor implantation. RESULTS: Results: All mice that received IACS-8803 had no detectable luciferase activity. Tumor regression was additionally verified via MR Imaging. Utilization of IACS-8803 led to long-term tumor-free survival (p < 0.01). Re-challenge with GL261 tumors ensured long term memory was sustained with a single dose of agonist. CONCLUSIONS: Conclusion: Engaging the STING pathway may be an ideal targeting strategy for GBM. Ongoing studies are delineating the mechanisms of activating this pathway with specific attention being paid to reprogramming and education of innate immunity.

CGAS facilitates sensing of extracellular cyclic dinucleotides to activate innate immunity.

Cyclic dinucleotides (CDNs) are important second messenger molecules in prokaryotes and eukaryotes. Within host cells, cytosolic CDNs are detected by STING and alert the host by activating innate immunity characterized by type I interferon (IFN) responses. Extracellular bacteria and dying cells can release CDNs, but sensing of extracellular CDNs (eCDNs) by mammalian cells remains elusive. Here, we report that endocytosis facilitates internalization of eCDNs. The DNA sensor cGAS facilitates sensing of endocytosed CDNs, their perinuclear accumulation, and subsequent STING-dependent release of type I IFN Internalized CDNs bind cGAS directly, leading to its dimerization, and the formation of a cGAS/STING complex, which may activate downstream signaling. Thus, eCDNs comprise microbe- and danger-associated molecular patterns that contribute to host-microbe crosstalk during health and disease.

Detection of Cyclic Dinucleotides by STING.

STING (stimulator of interferon genes) is an essential signaling adaptor protein mediating cytosolic DNA-induced innate immunity for both microbial invasion and self-DNA leakage. STING is also a direct receptor for cytosolic cyclic dinucleotides (CDNs), including the microbial secondary messengers c-di-GMP (3',3'-cyclic di-GMP), 3',3'cGAMP (3',3'-cyclic GMP-AMP), and mammalian endogenous 2',3'cGAMP (2',3'-cyclic GMP-AMP) synthesized by cGAS (cyclic GMP-AMP synthase). Upon CDN binding, STING undergoes a conformational change to enable signal transduction by phosphorylation and finally to active IRF3 (Interferon regulatory factor 3) for type I interferon production. Here, we describe some experimental procedures such as Isothermal Titration Calorimetry and luciferase reporter assays to study the CDNs binding and activity by STING proteins.

Abstract 2495: STING agonists formulated into cancer vaccines (STINGVAX) can cure established tumor resistant to immune checkpoint blockade by activating NK cells

Abstract Stimulator of Interferon Genes (STING) is a cytosolic receptor that senses both exogenous and endogenous cytosolic cyclic dinucleotides, activating TBK-1/IRF-3, NF-κB, and STAT6 signaling pathways to induce robust type I interferons and proinflammatory cytokines. Cyclic dinucleotide ligands were formulated with GM-CSF-producing cellular cancer vaccines - termed STINGVAX - that demonstrated potent in vivo anti-tumor efficacy in multiple therapeutic models of established cancer. We found that rationally designed synthetic CDN derivative molecules, including one with an Rp,Rp dithio diastereomer and non-canonical c[A(2′,5′)pA(3′,5′)p] phosphate bridge structure conferred significantly enhanced therapeutic anti-tumor efficacy in multiple aggressive therapeutic models of established cancer in mice. Anti-tumor activity was STING-dependent and correlated with increased activation of dendritic cells and tumor antigen-specific CD8+ T cells. Tumors from STINGVAX treated mice demonstrated dramatic PD-L1 upregulation, which was associated with tumor infiltrating CD8+IFNγ+ T-cells. When combined with PD-1 blockade, STINGVAX induced regression of palpable, poorly immunogenic tumors that did not respond to PD-1 blockade alone. We found that the STINGVAX's anti-tumor efficacy is NK cell dependent, and demonstrated that CDN are potent activators of IFNg in NK cells. Citation Format: Young Kim, Drew Pardoll, Juan Fu, Tom Dubensky. STING agonists formulated into cancer vaccines (STINGVAX) can cure established tumor resistant to immune checkpoint blockade by activating NK cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2495. doi:10.1158/1538-7445.AM2015-2495