Background:

Cytosolic DNA is a major signal to trigger immune activation and anti-viral immunity. One pathway leading to immune activation downstream of cytosolic DNA sensing in mammals is the cGAS-STING pathway. cGAS binds to cytosolic double-stranded DNA and synthesizes the second messenger 2’3’-cyclic-GMP-AMP (cGAMP), which activates STING, eventually leading to type I interferon production, innate immune activation, and a variety of anti-viral effects. STING agonists, including synthetic cyclic-dinucleotides (CDNs) are additionally under clinical investigation as novel cancer immunotherapies, owing to their ability to stimulate favorable anti-tumor responses in mouse models. Recent data also indicate that there may be a role for extracellular cGAMP as a diffusible second messenger, particularly in cancer models – however, the mechanisms by which cGAMP crosses the plasma membrane to trigger immune activation, and its properties outside the cell, remain poorly characterized. Three new preprints show roles for extracellular cGAMP and identify SLC19A1 as a transporter mediating uptake of extracellular cGAMP and other CDNs, allowing for immune activation.

 

Key findings:

• Luteijn et al and Cordova and Ritchie et al used loss-of-function CRISPR screens to identify SLC19A1 as the dominant cGAMP importer. The two preprints use distinct cell lines and readouts of CDN uptake to identify the cGAMP importer, but converge on SLC19A1. SLC19A1 is known to import folate, which – along with other known antagonists of SLC19A1 – is able to block uptake of CDNs and subsequent cellular activation. Bypassing the requirement for SLC19A1 by electroporation of CDNs or intracellular activation of cGAS resulted in normal activation in SLC19A1-deficient cells, confirming the role of SLC19A1 specifically in uptake of CDNs.
• Carozza et al developed a sensitive mass spectrometry approach to detect extracellular cGAMP. As they had previously shown that the extracellular enzyme ENPP1 (present in serum) cleaved cGAMP, they examined export by ENPP1-deficient cell lines in a serum-free culture. Cancer cell lines in an ENPP-1 free system constitutively exported cGAMP in a freely soluble form, at high levels. Inhibition of extracellular ENPP-1 with a novel cell-impermeable ENPP1 inhibitor increased extracellular cGAMP concentrations, with no effect on intracellular cGAMP. Finally, cGAMP secretion by cancer cells in vivo drove CD11c+ dendritic cell accumulation in the tumor, and inhibition of cGAMP degradation with the ENPP-1 inhibitor increased anti-tumor immunity.

 

Importance:

This trio of preprints greatly solidify our understanding of cGAMP activity and resolve important questions in the field regarding the activity of extracellular and exogenous cGAMP. This is particularly important as synthetic CDNs, and other mechanisms of perturbing the cGAS-STING pathway, move forward in clinical trials. Together, these papers help build a model of cGAMP export, extracellular stability, and import (Figure 1), providing multiple new avenues of study of CDN-mediated immunity and therapeutic axes to target this key immune pathway.

 

Figure 1: A model for extracellular cGAMP activity

Sensing of cytosolic dsDNA by cGAS leads to cGAMP synthesis in cancer cells. cGAMP is exported by an unknown mechanism, leading to free soluble extracellular cGAMP. This may be degraded by ENPP1, but can also be imported into immune cells such as macrophages by the transporter SLC19A1. There, it binds to STING, leading to IRF3 activation and downstream effects including type I IFN secretion.

 

 

Moving forward:

• The mechanism by which cGAMP becomes extracellular still remains unclear. Carozza et al suggest that cGAMP is efficiently exported from living cancer cells, rather than being released following cell death, but that suggests the existence of an exporter. The identification of this export mechanism, leading to freely soluble cGAMP (rather than transfer through gap junctions or membrane fusions, as has been previously demonstrated) will be important to improve our understanding of this pathway.
• In a similar vein, under what circumstances might CDN import be favorable in a non-tumor setting? It is demonstrated that SLC19A1 may also import bacterial CDNs, and Luteijn et al argue that pathogen- or microbiota-derived CDNs may play an important role in inflammatory diseases. Is cGAMP export common in viral infections, and might this be a pathway to avoiding viral STING antagonists (only operating in infected cells), or might bacterial CDNs be the “dominant” ligand driving the evolution of this pathway?
• Both Luteijn et al and Cordova and Ritchie et al show that SLC19A1 is the dominant cGAMP importer, however their data suggest the existence of other importers/ Luteijn et al examine other folate transporters, which show some ability to import CDNs but are still insufficient to explain the residual transport activity (although, the triple KD/KO of all three tested SLC transporters is not shown – perhaps that would lead to complete loss of CDN import?). Hopefully, further studies will identify the multiple pathways leading to CDN uptake.

 

doi: https://doi.org/10.1242/prelights.8492

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