Biology subjects

Cordova, A. F.

Publications and source records attributed to Cordova, A. F..

5 recordsLinked to original sources

bioRxiv2024

Toxicity of extracellular cGAMP and its analogs to T cells is due to SLC7A1-mediated import

STING agonists are promising innate immune therapies and can synergize with adaptive immune checkpoint blockade therapies for cancer treatment, but their effectiveness is limited by the toxicity to activated T cells. An important class of STING agonists are analogs of the endogenous STING agonist, cGAMP, and while transporters for these small molecules are known in some cell types, how they enter and kill T cells remains unknown. Here, we identify the cationic amino acid transporter SLC7A1 as the dominant transporter of cGAMP and its analogs in activated primary mouse and human T cells. T cells upregulate this transporter upon activation and rapid proliferation to meet their high metabolic demand, but this comes at the cost of enabling increased transport and toxicity of cGAMP. To circumvent the essentiality of SLC7A1 to proliferating T cells, we found that the residues responsible for cGAMP transport are separate from the arginine binding pocket allowing us to perturb cGAMP transport and STING-activation mediated killing without impacting arginine transport. These results suggest that SLC7A1 is a potential target for alleviating T cell toxicity associated with cGAMP and its analogs.

immunology↗

bioRxiv2021

Probing pathophysiology of extracellular cGAMP with substrate-selective ENPP1

The biology of the immune second messenger cGAMP depends on its cellular localization. cGAMP, which is synthesized in response to cytosolic double-stranded DNA, also exists in the extracellular space as a paracrine immunotransmitter that enhances the anticancer immune response. However, the role of extracellular cGAMP is unexplored outside of cancer due to a lack of tools to systemically manipulate it. The extracellular enzyme ENPP1, the only known hydrolase of cGAMP, is a promising target. However, because ENPP1 also degrades extracellular ATP, using genetic knockouts of ENPP1 to study extracellular cGAMP leads to confounding effects. Here we report the H362A point mutation in ENPP1, the dominant cGAMP hydrolase, which selectively abolishes ENPP1s ability to degrade cGAMP, while retaining activity toward other substrates. H362 is not necessary for binding cGAMP or the catalytically-essential zinc atoms but instead supports the in-line reaction geometry. H362 is evolutionarily conserved down to bacteria, suggesting an ancient origin for extracellular cGAMP biology. Enpp1H362A mice do not display the systemic calcification seen in Enpp1-/- mice, highlighting the substrate-specific phenotype of ENPP1. Remarkably, Enpp1H362A mice were resistant to HSV-1 infection, demonstrating the antiviral role of endogenous extracellular cGAMP. The ENPP1H362A mutation is the first genetic tool to enable exploration of extracellular cGAMP biology in a wide range of tissues and diseases.

immunology↗

bioRxiv2020

Murine M1 macrophages are among the direct responders to tumor-derived extracellular cGAMP and their human counterparts use SLC46A2 to import cGAMP

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, an immunotransmitter that is transferred from cancer cells to cGAMP-sensing cells in the host, promoting immunity. However, the CDN import mechanisms used by host cells within tumors remain unknown. Here we identified the protein SLC46A2 as the dominant cGAMP importer in primary human monocytes. Furthermore, we discovered that monocytes and M1-polarized macrophages directly sense tumor-derived extracellular cGAMP in murine tumors. Finally, we demonstrated that SLC46A2 is the dominant cGAMP importer in monocyte-derived macrophages. Together, we provide the first cellular and molecular mechanisms of cGAMP as an immunotransmitter, paving the way for effective STING pathway therapeutics.

cancer biology↗

bioRxiv2019

In response to Luteijn et al.: Concerns regarding cGAMP uptake assay and evidence that SLC19A1 is not the major cGAMP importer in human PBMCs

We previously reported that SLC19A1 is an importer of the immunotransmitter 23-cyclic-GMP-AMP (cGAMP)1 by performing a genome wide screen in U937 cells. Soon after, Lutejin et al. reported similar findings by conducting a screen in THP-1 cells2. While the conclusions of these two studies largely overlap, we arrived at significantly different conclusions regarding how broadly SLC19A1 is used by different cell types. Our study suggests that in addition to SLC19A1, many cultured and primary cell types use alternative, unidentified transporters to import cGAMP and other cyclic dinucleotides (CDNs). This conclusion was based on our findings that inhibition of SLC19A1 did not significantly reduce extracellular cGAMP signaling in multiple cell types, including primary CD14+peripheral blood mononuclear cells (PBMCs) from most donors. In contrast, Luteijn et al. concluded that SLC19A1 is the major CDN importer in humans, largely based on their use of a radiolabeled [32P] cGAMP uptake assay. Using this assay, they showed that inhibition of SLC19A1 abolishes [32P] uptake in total PBMCs. However, they did not test whether inhibition of SLC19A1 affects extracellular cGAMP signaling in these cells. Here, we highlight an important issue with the [32P] cGAMP uptake assay used by Luteijn et al. and demonstrate that measuring extracellular cGAMP signaling through the STING pathway is currently the best method for evaluating cGAMP import. We also show that inhibition of SLC19A1 has no effect on extracellular cGAMP signaling in total PBMCs, confirming that this cell type relies on other transport mechanisms for cGAMP import.

biochemistry↗

bioRxiv2019

SLC19A1 is an importer of the immunotransmitter cGAMP

23-cyclic-GMP-AMP (cGAMP) is a second messenger that activates the antiviral Stimulator of Interferon Genes (STING) pathway. We recently identified a novel role for cGAMP as a soluble, extracellular immunotransmitter that is produced and secreted by cancer cells. Secreted cGAMP is then sensed by host cells, eliciting an antitumoral immune response. Due to the antitumoral effects of cGAMP, other CDN-based STING agonists are currently under investigation in clinical trials for metastatic solid tumors. However, it is unknown how cGAMP and other CDNs cross the cell membrane to activate intracellular STING. Using a genome-wide CRISPR screen we identified SLC19A1 as the first known importer of cGAMP and other CDNs, including the investigational new drug 2'3'-bisphosphosphothioate-cyclic-di-AMP (2'3'-CDAS). These discoveries will provide insight into cGAMPs role as an immunotransmitter and aid in the development of more targeted CDN-based cancer therapeutics.

biochemistry↗