2’3’-cGAMP is an immunotransmitter produced by cancer cells and regulated by ENPP1

Carozza, Jacqueline A.; Boehnert, V.; Shaw, Kelsey E.; Nguyen, Khanh Cong; Skariah, Gemini; Brown, Jenifer A.; Rafat, Marjan; Eyben, Rie von; Graves, Edward E.; Glenn, Jeffrey S.; Smith, Mark A.; Li, Lingyin

doi:10.1101/539312

Cited by 19 publications

(36 citation statements)

References 59 publications

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“…2C). The ENPP1 inhibitor, STF-1084, (Carozza et al, 2019) led to a significantly greater induction in individual ISG expression as well as the cumulative IFN scores, compared to both the vehicle-treated skin exposed to UVB as well as to the unexposed skin treated with the ENPP1 inhibitor ( Fig. 2D-E).…”

Section: Cgas Is Essential For the Early Cutaneous Ifn-i Response Aftmentioning

confidence: 89%

“…Since export of the cGAS product, cGAMP, plays a role in the induction of IFN-I following radiation therapy (Ablasser et al, 2013;Bakhoum et al, 2018;Chen et al, 2016), we asked whether release of cGAMP following UVB radiation injury impacts IFN-I stimulation in the skin. To address this question, we inhibited the cell surface membrane enzyme ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), which hydrolyzes cGAMP (Carozza et al, 2019;Li et al, 2014), shortly before skin exposure to UVB (Fig. 2C).…”

Section: Cgas Is Essential For the Early Cutaneous Ifn-i Response Aftmentioning

confidence: 99%

“…50µl 100 µM STF1084 (two injections per mouse) (generously provided by Yinling Li at Stanford University), as used by Carozza et al for intra-tumoral injections (Carozza et al, 2019). After 30 minutes, mice were irradiated with a single dose of UVB light as described above.…”

Section: In Vivo Treatment With Hydroxycloroquine (Hcq) and Enpp1 Inhmentioning

confidence: 99%

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The early local and systemic Type I interferon responses to ultraviolet B light exposure are cGAS dependent

Skopelja-Gardner

Tai

et al. 2019

Preprint

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AbstractMost systemic lupus erythematosus (SLE) patients are photosensitive and ultraviolet B light (UVB) exposure worsens cutaneous disease and precipitates systemic flares of disease. The pathogenic link between skin disease and systemic exacerbations in SLE remains elusive. In an acute model of UVB-triggered inflammation, we observed that a single UV exposure triggered a striking IFN-I signature not only in the skin, but also in the blood and kidneys. The early IFN-I signature was significantly higher in female compared to male mice. The early IFN-I response in the skin was almost entirely, and in the blood partly, dependent on the presence of cGAS, as was skin inflammatory cell infiltration. Inhibition of cGAMP hydrolysis augmented the UVB-triggered IFN-I response. UVB skin exposure leads to cGAS-activation and both local and systemic IFN-I signature and could contribute to acute flares of disease in susceptible subjects such as patients with SLE.

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Section: Cgas Is Essential For the Early Cutaneous Ifn-i Response Aftmentioning

confidence: 89%

Section: Cgas Is Essential For the Early Cutaneous Ifn-i Response Aftmentioning

confidence: 99%

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The early local and systemic Type I interferon responses to ultraviolet B light exposure are cGAS dependent

Skopelja-Gardner

Tai

et al. 2019

Preprint

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“…In this way, cGAMP can signal viral infections as well as cancerous cells in proliferation. Although the charged nature of cGAMP is not conducive to passive diffusion across cell membranes, gap junctions permit intercellular transfer (Wu & Chen, 2014;Ablasser et al, 2013) and transporters pump cGAMP both into and out of the cell (Carozza et al, 2020;Ritchie et al, 2019;Zhou et al, 2020). cGAMP binds and activates the endoplasmic reticulum surface receptor stimulator of interferon genes (STING) to activate the production of type 1 interferons.…”

Section: Introductionmentioning

confidence: 99%

Crystal structures of human ENPP1 in apo and bound forms

Dennis

Newman

Dolezal

et al. 2020

Acta Crystallogr D Struct Biol

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Cancer is one of the leading causes of mortality in humans, and recent work has focused on the area of immuno-oncology, in which the immune system is used to specifically target cancerous cells. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is an emerging therapeutic target in human cancers owing to its role in degrading cyclic GMP-AMP (cGAMP), an agonist of the stimulator of interferon genes (STING). The available structures of ENPP1 are of the mouse enzyme, and no structures are available with anything other than native nucleotides. Here, the first X-ray crystal structures of the human ENPP1 enzyme in an apo form, with bound nucleotides and with two known inhibitors are presented. The availability of these structures and a robust crystallization system will allow the development of structure-based drug-design campaigns against this attractive cancer therapeutic target.

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“…SLC19A1. Similarly, STF-1084, another ENPP1 inhibitor we developed 5 , only weakly inhibited [ 3 H] MTX uptake through SLC19A1 (Fig. 1c).…”

mentioning

confidence: 82%

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

Ritchie

Cordova

2019

Preprint

Self Cite

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We previously reported that SLC19A1 is an importer of the immunotransmitter 2'3'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 cells 2 . 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 [ 32 P] cGAMP uptake assay. Using this assay, they showed that inhibition of SLC19A1 abolishes [ 32 P] 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 [ 32 P] 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. ResultscGAMP is not stable in serum-containing tissue culture media (hereafter referred to simply as media) due to the presence of its hydrolase ENPP1 in serum 3 , and it is degraded into AMP and Pi, both of which are reported substrates of SLC19A1 4 . Our analysis using thin layer chromatography (TLC) confirmed that in media a small percentage of [ 32 P] cGAMP is degraded into [ 32 P] AMP and [ 32 P] Pi while STF-1623, an ENPP1 inhibitor we developed 5 , efficiently blocks degradation ( Fig. 1a). Therefore, an uptake assay without ENPP1 inhibitors could be measuring uptake of cGAMP degradation products. This may explain why Luteijn et al. were able to measure [ 32 P] uptake for up to five hours without reaching equilibrium 2 , despite reports that uptake of other SLC19A1 substrates reaches equilibrium within an hour 4,6 . To investigate further, we found that preincubation of [ 32 P] cGAMP in media before performing an uptake assay increased [ 32 P] uptake, with a linear correlation between the preincubation time and the amount of [ 32 P] uptake. Furthermore, adding STF-1623 to the preincubation abolished the increase in [ 32 P] uptake, indicating that the increased [ 32 P] uptake is due to degradation of [ 32 P] cGAMP ( Fig. 1b). Importantly, STF-1623 did not inhibit uptake of [ 3 H] methotrexate (MTX), a model substrate of SLC19A1, indicating that STF-1623 does not inhibit cGAMP uptake through [ 3...

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2’3’-cGAMP is an immunotransmitter produced by cancer cells and regulated by ENPP1

Cited by 19 publications

References 59 publications

The early local and systemic Type I interferon responses to ultraviolet B light exposure are cGAS dependent

The early local and systemic Type I interferon responses to ultraviolet B light exposure are cGAS dependent

Crystal structures of human ENPP1 in apo and bound forms

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

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