Human SLC46A2 is the dominant cGAMP importer in extracellular cGAMP-sensing macrophages and monocytes

Abstract

Administration of exogenous CDNs to activate the cGAMP-STING pathway is a promising therapeutic strategy to unleash the full potential of cancer immunotherapy. This strategy mirrors the role of endogenous extracellular cGAMP, which we recently described as an immunotransmitter exported by cancer cells and imported into local responder cells of unknown identities to promote anti-tumoral immunity, with irradiation enhancing this effect. Here, in low-dose irradiated murine tumors, we identified CD4 + T cells, M1 macrophages, and NKG2D Low NK cells as cGAMP responder cells that have decreased STING activation upon depletion of extracellular cGAMP. At higher doses of radiation, extracellular cGAMP promoted the death of T cells and macrophages. Furthermore, we identified the orphan protein SLC46A2 as the dominant importer of cGAMP and select bacterial CDNs in human macrophages and monocytes. Together, we provide the first cellular and molecular mechanisms of cGAMP as an immunotransmitter, paving the way for effective STING pathway therapeutics.

Identification of extracellular membrane protein ENPP3 as a major cGAMP hydrolase, cementing cGAMP’s role as an immunotransmitter

“…Other second messengers such as cAMP and cGMP are only reported to have intracellular functions and intracellular regulation. In contrast, cGAMP has only been found to have extracellular regulation with a large number of known cGAMP transporters 9,11,21,[39][40][41][42] and hydrolases that are all extracellular. It thus seems reasonable that the grand majority of cGAMP regulation would occur in the extracellular space.…”

“…It thus seems reasonable that the grand majority of cGAMP regulation would occur in the extracellular space. Indeed, we have shown that the presence of cytosolic DNA leads to the production of extracellular cGAMP to mediate immunity in a STING-dependent manner 8,11,13 . Together, we propose a model in which cGAMP’s main physiological function is that of a paracrine immunotransmitter 8,13,43,44 , and the differential expression of Enpp1 and Enpp3 regulate how much cGAMP cells emit and receive.…”

“…cGAMP potently activates its intracellular receptor, Stimulator of Interferon Genes (STING), leading to the production of type-I interferons and other interferon-induced genes to mount an innate immune response to the detected threat 2,6,7 . In addition to signaling within the producing cell, cGAMP is also known to be exported from some producer cells, such as cancer cells, and activate the STING pathway in other cell types within the nearby vicinity [8][9][10][11][12] . Activation of STING in responder cells, including fibroblasts, macrophages, and endothelial cells, elicit downstream anti-cancer 8,11,12 and anti-viral immune responses, but also can exacerbate systemic inflammation and autoimmunity 13 .…”

“…In addition to signaling within the producing cell, cGAMP is also known to be exported from some producer cells, such as cancer cells, and activate the STING pathway in other cell types within the nearby vicinity [8][9][10][11][12] . Activation of STING in responder cells, including fibroblasts, macrophages, and endothelial cells, elicit downstream anti-cancer 8,11,12 and anti-viral immune responses, but also can exacerbate systemic inflammation and autoimmunity 13 . Interestingly, while STING activation in cancer cells can promote metastasis 14,15 , STING activation in bystander cells of the tumor microenvironment delays primary tumor growth and metastasis and is responsible for the curative effect of radiation treatment 8,12,[16][17][18][19][20][21] .…”

Regulation and inhibition of the DNA sensor cGAS

“…The only mammalian protein that degrades cGAMP is ENPP1, a cell membrane‐bound esterase with extracellular activity (Li et al , 2014). Recent evidence suggests that cGAMP is transported across membranes by channel proteins and can thus be exported from and/or imported into cells (Luteijn et al , 2019; Ritchie et al , 2019; Carozza et al , 2020; preprint: Cordova et al , 2020; preprint: Lahey et al , 2020; Zhou et al , 2020a; Zhou et al , 2020b). Specifically, cGAMP exported by cancer cells may have important roles in anti‐tumour immunity (Marcus et al , 2018; Carozza et al , 2020).…”