Protocol to induce and assess cGAS-STING pathway activation in vitro

Chamma, Hanane; Guha, Soumyabrata; Laguette, Nadine; Vila, Isabelle K.

doi:10.1016/j.xpro.2022.101384

Cited by 8 publications

(5 citation statements)

References 14 publications

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“…To this end, we treated THP-1 and ARPE-19 cells with increasing concentrations of extracellular 2’-3’cGAMP and monitored the impact on cell viability and found exogenous 2’-3’cGAMP was well-tolerated in each cell type at the assessed concentrations and duration (Fig 1A). To determine the impact of adding 2’-3’cGAMP to the media, cells were treated with the cyclic dinucleotide for 24 hrs and quantified IFNB RNA levels by RT-qPCR as a surrogate readout for the activation of the STING pathway (27). In both THP-1 and ARPE-19 cells, there was a dose-dependent increase of IFNB RNA expression in response to treatment (Fig 1B), indicating the cells responded to exogenous 2’-3’cGAMP.…”

Section: Resultsmentioning

confidence: 99%

Herpes Simplex Virus-1 targets the 2’-3’cGAMP importer SLC19A1 as an antiviral countermeasure

Szemere,

Murphy

2024

Preprint

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To establish a successful infection, herpes simplex virus-1 (HSV-1), a virus with high seropositivity in the human population, must undermine host innate and intrinsic immune defense mechanisms, including the stimulator of interferon genes (STING) pathway. Recently it was discovered that not only de novo produced intracellular 2’-3’cGAMP, but also extracellular 2’-3’cGAMP activates the STING pathway by being transported across the cell membrane via the folate transporter, SLC19A1, the first identified extracellular antiporter of this signaling molecule. We hypothesized that the import of exogenous 2’-3’cGAMP functions to establish an antiviral state like that seen with the paracrine antiviral activities of interferon. Further, to establish a successful infection, HSV-1 must undermine this induction of the STING pathway by inhibiting the biological functions of SLC19A1. Herein, we report that treatment of the monocytic cell line, THP-1 cells, epithelial cells (ARPE-19) and SH-SY5Y neuronal cell line with exogenous 2’-3’cGAMP induces interferon production and establishes an antiviral state. Using either pharmaceutical inhibition or genetic knockout of SLC19A1 blocks the 2’-3’cGAMP-induced antiviral state. Additionally, HSV-1 infection results in the reduction of SLC19A1 transcription, translation, and importantly, the rapid removal of SLC19A1 from the cell surface of infected cells. Our data indicate SLC19A1 functions as a newly identified antiviral mediator for extracellular 2’-3’cGAMP which is undermined by HSV-1. This work presents novel and important findings about how HSV-1 manipulates the host’s immune environment for viral replication and discovers details about an antiviral mechanism which information could aid in the development of better antiviral drugs in the future.

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Section: Resultsmentioning

confidence: 99%

Herpes Simplex Virus-1 targets the 2’-3’cGAMP importer SLC19A1 as an antiviral countermeasure

Szemere,

Murphy

2024

Preprint

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“…CLCYT236-2) or dsDNA with kinase inhibitors as listed in Dataset EV1 . Double-stranded DNA was prepared as previously described (Chamma et al, 2022 ) using the following 45 bp ssDNAs:…”

Section: Methodsmentioning

confidence: 99%

An integrative epigenome-based strategy for unbiased functional profiling of clinical kinase inhibitors

Gualdrini,

Rizzieri,

Polletti

et al. 2024

Mol Syst Biol

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More than 500 kinases are implicated in the control of most cellular process in mammals, and deregulation of their activity is linked to cancer and inflammatory disorders. 80 clinical kinase inhibitors (CKIs) have been approved for clinical use and hundreds are in various stages of development. However, CKIs inhibit other kinases in addition to the intended target(s), causing both enhanced clinical effects and undesired side effects that are only partially predictable based on in vitro selectivity profiling. Here, we report an integrative approach grounded on the use of chromatin modifications as unbiased, information-rich readouts of the functional effects of CKIs on macrophage activation. This approach exceeded the performance of transcriptome-based approaches and allowed us to identify similarities and differences among CKIs with identical intended targets, to recognize novel CKI specificities and to pinpoint CKIs that may be repurposed to control inflammation, thus supporting the utility of this strategy to improve selection and use of CKIs in clinical settings.

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“…In contrast, there might be a depletion of total STING levels. Additionally, among the IFN-responsive genes, a robust upregulation of IFNβ, OAS1, C-X-C motif chemokine ligand 10 (CXCL10), and interferon-stimulated gene 15 (ISG15) can be detected [80]. Transgene expression, following transfection with naked DNA, may be augmented by inhibition of cGAS-STING [81].…”

Section: Irf3 Nf-κb and Downstream Signalingmentioning

confidence: 99%

At the Crossroads of the cGAS-cGAMP-STING Pathway and the DNA Damage Response: Implications for Cancer Progression and Treatment

Korneenko,

Pestov,

Nevzorov

et al. 2023

Pharmaceuticals

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The evolutionary conserved DNA-sensing cGAS-STING innate immunity pathway represents one of the most important cytosolic DNA-sensing systems that is activated in response to viral invasion and/or damage to the integrity of the nuclear envelope. The key outcome of this pathway is the production of interferon, which subsequently stimulates the transcription of hundreds of genes. In oncology, the situation is complex because this pathway may serve either anti- or pro-oncogenic roles, depending on context. The prevailing understanding is that when the innate immune response is activated by sensing cytosolic DNA, such as DNA released from ruptured micronuclei, it results in the production of interferon, which attracts cytotoxic cells to destroy tumors. However, in tumor cells that have adjusted to significant chromosomal instability, particularly in relapsed, treatment-resistant cancers, the cGAS–STING pathway often supports cancer progression, fostering the epithelial-to-mesenchymal transition (EMT). Here, we review this intricate pathway in terms of its association with cancer progression, giving special attention to pancreatic ductal adenocarcinoma and gliomas. As the development of new cGAS–STING-modulating small molecules and immunotherapies such as oncolytic viruses involves serious challenges, we highlight several recent fundamental discoveries, such as the proton-channeling function of STING. These discoveries may serve as guiding lights for potential pharmacological advancements.

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Protocol to induce and assess cGAS-STING pathway activation in vitro

Cited by 8 publications

References 14 publications

Herpes Simplex Virus-1 targets the 2’-3’cGAMP importer SLC19A1 as an antiviral countermeasure

Herpes Simplex Virus-1 targets the 2’-3’cGAMP importer SLC19A1 as an antiviral countermeasure

An integrative epigenome-based strategy for unbiased functional profiling of clinical kinase inhibitors

At the Crossroads of the cGAS-cGAMP-STING Pathway and the DNA Damage Response: Implications for Cancer Progression and Treatment

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