The LRRC8A:C Heteromeric Channel Is a cGAMP Transporter and the Dominant cGAMP Importer in Human Vasculature Cells

Abstract

Extracellular 2'3'-cyclic-GMP-AMP (cGAMP) is an immunotransmitter secreted by cancer cells and taken up by host cells to activate the anti-cancer STING pathway. No cGAMP exporter has been identified, and SLC19A1, a recently identified cGAMP importer, does not account for the import activity in most cell types. Here, we identify the LRRC8A:C heteromeric channel, a volume-regulated anion channel (VRAC), as a cGAMP transporter. This channel mediates cGAMP import or export depending on the cGAMP chemical gradient, and channel activation or inhibition modulates cGAMP transport. Other 2'3'-cyclic dinucleotides are also transported by LRRC8A:C channels, including the investigational cancer therapeutic ADU-S100. Furthermore, we demonstrate that the LRRC8A-containing channel is the dominant cGAMP importer in primary human vasculature cells. Given tumor vasculature's regulation of immune infiltration and its disruption in response to STING agonists, we have uncovered a leading molecular mechanism for extracellular cGAMP signaling in this important anticancer target.

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

“…SLC46A2 is the third human cGAMP transporter we identified after SLC19A1, a minor importer in CD14 + monocytes, and the LRRC8 channels, which are used by primary endothelial cells (Lahey et al, 2020) (Figure 7E). We hypothesize that different cell types in the tumor microenvironment express different levels of these and other cGAMP transporters.…”

“…The bacterial CDNs 3'3'-cGAMP and 3'3'-CDA are associated with pathogenic bacteria (Corrigan et al, 2011;Davies et al, 2012;Woodward et al, 2010), while 3'3'-CDG is produced by a wide variety of bacteria, including commensals (Ryan et al, 2006). The ability of SLC46A2 and other CDN transporters (Lahey et al, 2020;Ritchie et al, 2019) to selectively import certain CDNs (such as cGAMP and 3'3'-CDA) but not others (3'3'-CDG) suggests that CDN transporters could regulate how the immune system differentially responds to pathogenic and commensal bacteria. Once the tumors reached 100 mm 3 , the tumors were irradiated with 0, 12, or 20 Gy.…”

“…Custom plasmids (pTwist-CMV) containing the CDSs of mouse Slc19a1 and Slc46a2 were purchased from Twist Bioscience. To generate doxycycline inducible lentiviral plasmids, the transporter CDS was amplified from the appropriate plasmid using the primers listed in Supplemental Table 1 and cloned into EcoRI/BamHI linearized pLVX-TetOne-FLAG-Hydro plasmid (Lahey et al, 2020) by isothermal Gibson assembly (Gibson et al, 2009). Mouse Models 7-9 week-old female BALB/c mice (Jackson Laboratories) were inoculated with 5 x 10 4 4T1luciferase cells suspended in 50 μL of PBS.…”

“…Furthermore, the cGAMP import mechanism of these responder cells is also unknown. It is clear that import mechanisms differ by cell type and context, and although multiple cGAMP transporters have been identified (Lahey et al, 2020;Luteijn et al, 2019;Ritchie et al, 2019;Zhou et al, 2020a), it is unknown which ones are used by cGAMP responder cells. Identifying the responder cells to endogenous extracellular cGAMP and characterizing their cGAMP transport mechanisms will be crucial to understanding the role of the STING pathway in the antitumoral immune response and to rationally design more effective CDN-based therapeutics.…”

The clinical and molecular significance associated with STING signaling in estrogen receptor-positive early breast cancer

“…While we observed STING staining in the stroma of tumors, we were unable to delineate expression in fibroblasts v. immune cells, for example. Recently, specific efflux and/or influx channels for 2'3'cGAMP have been identified for monocytes, macrophages, fibroblasts and endothelial cells [53][54][55][56][57] . As STING activation within host immune cells is key for anti-cancer responses, expression of these channels within cancers may drastically alter the nature of STING responses in response to either endogenous or exogeneous STING activation.…”