Role of the cGAS-STING pathway in systemic and organ-specific diseases

Abstract

Cells are equipped with numerous sensors that recognize nucleic acids, which probably evolved for defence against viruses. Once triggered, these sensors stimulate the production of type I interferons and other cytokines that activate immune cells and promote an antiviral state. The evolutionary conserved enzyme cyclic GMP-AMP synthase (cGAS) is one of the most recently identified DNA sensors. Upon ligand engagement, cGAS dimerizes and synthesizes the dinucleotide second messenger 2',3'-cyclic GMP-AMP (cGAMP), which binds to the endoplasmic reticulum protein stimulator of interferon genes (STING) with high affinity, thereby unleashing an inflammatory response. cGAS-binding DNA is not restricted by sequence and must only be >45 nucleotides in length; therefore, cGAS can also be stimulated by self genomic or mitochondrial DNA. This broad specificity probably explains why the cGAS-STING pathway has been implicated in a number of autoinflammatory, autoimmune and neurodegenerative diseases; this pathway might also be activated during acute and chronic kidney injury. Therapeutic manipulation of the cGAS-STING pathway, using synthetic cyclic dinucleotides or inhibitors of cGAMP metabolism, promises to enhance immune responses in cancer or viral infections. By contrast, inhibitors of cGAS or STING might be useful in diseases in which this pro-inflammatory pathway is chronically activated.

Fig. 1. Involvement of cGAS and/or STING in disease.

The cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway has been implicated in systemic disease processes, including autoimmunity, autoinflammation, ageing and cancer, as well as organ-specific diseases that target the eyes, brain, lungs, heart, kidneys, skin and intestines. cGAS–STING is also involved in the response to many viral infections, which can be systemic or local. AKI, acute kidney injury; COPA, coatomer protein subunit-α; IBD, inflammatory bowel disease; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; SAVI, STING-associated vasculopathy with onset in infancy.

Fig. 2. Signalling pathways downstream of cGAS and of endosomal TLRs.

Binding to double-stranded DNA induces dimerization of cyclic GMP–AMP synthase (cGAS), which, in turn, leads to the synthesis of the second messenger 2′,3′-cyclic GMP–AMP (cGAMP). cGAMP binds with high affinity to stimulator of interferon genes (STING) leading to STING oligomerization and exit from the endoplasmic reticulum (ER); coatomer protein (COP) complexes facilitate the transport of STING. In the ER–Golgi intermediate compartment (ERGIC), STING recruits serine/threonine-protein kinase TBK1. The transcription factor interferon regulatory factor 3 (IRF3), which is a key TBK1 substrate, enters the nucleus following phosphorylation and promotes the transcription of IFNβ. Oligomerization of STING also enables activation of the inhibitor of nuclear factor-κB (NF-κB) kinase (IKK) complex, which promotes activation of the NF-κB transcription factor. NF-κB translocates to the nucleus where it promotes transcription of inflammatory cytokines tumour necrosis factor (TNF), IL-6 and IL-1β. By contrast, endosomal Toll-like receptors (TLRs) use the MyD88 or TRIF to generate platforms for downstream signal transduction. The DNA sensor TLR9 and the single-stranded RNA sensors, TLRs 7 and 8, activate IRF5 and IRF7 via Myd88 and IL-1 receptor-associated kinase 4 (IRAK4), which results in the synthesis of IFNα. The double-stranded RNA sensor TLR3, engages the TRIF platform, which leads to the activation of TBK1 and IKK, leading to IFNβ transcription. In non-classical pathway activation (dashed arrows), activated STING molecules on the ERGIC can bind to microtubule-associated protein 1 light chain 3 (MAP1LC3; also known as LC3) on the phagophore, which leads to STING degradation through autophagy. Moreover, STING activation in the ER might lead to PKR-like ER kinase (PERK) activation and subsequent cell senescence or fibrosis.

Fig. 3. Mechanisms of activation of the cGAS–STING pathway.

In the cytoplasm, cyclic GMP–AMP synthase (cGAS) can be activated by microbial or host DNA. The best-studied sources of cGAS-activating nucleic acids are DNA viruses and host mitochondria. However, several other DNA sources and cGAS-activating stimuli, such as endogenous retroelement DNA, micronuclei generated in senescent cells or owing to genomic instability, and the formation of chromatin bridges, have been reported^,,,. Microbe-induced cell fusion also enables the release of chromatin into the cytosol and RNA viruses can promote cGAS activation through ribosomal collision^,. Finally, certain DNA-binding proteins such as polyglutamine-binding protein 1 (PQBP1) might co-activate cGAS in combination with the HIV capsid protein or Tau.

Fig. 4. Transport and metabolism of cGAMP.

Following activation, cyclic GMP–AMP synthase (cGAS) produces 2′,3′-cyclic GMP–AMP (cGAMP), which binds to stimulator of interferon genes (STING) within cells. The fate of intracellular cGAMP depends on its rate of synthesis, the cell type in which it is produced and the biological context. Excess cGAMP is transported out of cells and is hydrolysed by membrane-bound or soluble ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) to yield AMP and GMP in the extracellular space^,. When production and export of cGAMP exceeds the catabolic capacity of ENPP1, cGAMP is imported into adjacent or distal cells through specific transporters, some of which can mediate bidirectional transport (LRRC8). The best-studied transporter in vascular endothelial cells is the volume-regulated anion (VRAC) channel LRRC8A:C heteromer, whereas the solute carrier SLC19A1 is functional in human cell lines and peripheral blood mononuclear cells, and SLC46A2 is active in CD14⁺ monocytes and pro-inflammatory macrophages^,–. In tumours, cGAMP can be transported between adjoining cells through gap junctions (for example, connexins 43 and 45)^,,, which can be blocked by the compounds shown. Several chemical compounds can block ENPP1 and cGAMP import as indicated. Sphingosine 1-phosphate can activate LRRC8 to potentiate cGAMP transport.

Fig. 5. Targeting the cGAS–STING pathway.

The cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway can be inhibited at many stages. Reverse transcriptase inhibitors (RTI) block the synthesis of cGAS-activating complementary DNA from endogenous retroelements. Aminoquinoline-type antimalarial drugs (AMDs) block DNA binding to cGAS, whereas aspirin acetylates the cGAS residues Lys414, Lys384 and Lys394, which leads to its inactivation. Active site inhibitors block the synthesis of the second messenger 2′,3′-cyclic GMP–AMP (cGAMP). For STING inhibition, both naturally occurring nitro-fatty acids (NO2-FAs) and the nitrofuran derivative, H-151, block palmitoylation of crucial cysteine residues on STING, preventing oligomerization of the protein. A number of different TBK1 inhibitors, mostly belonging to the aminopyrimidine class, such as compound II, have been generated and used to treat autoimmune disorders in mice. In contrast to attenuation of the cGAS–STING pathway, STING can be activated by synthetic cyclic dinucleotides (CDNs), which might be useful in enhancing antitumour immunity.