Lingyin Li

All Publications

• A cGAMP-Containing Hydrogel for Prolonged SARS-CoV-2 Receptor-Binding Domain Subunit Vaccine Exposure Induces a Broad and Potent Humoral Response ADVANCED NANOBIOMED RESEARCH Bohnert, V., Gale, E. C., Lahey, L. J., Yan, J., Powell, A. E., Ou, B. S., Carozza, J. A., Li, L., Appel, E. A. 2024

  View details for DOI 10.1002/anbr.202400077

  View details for Web of Science ID 001364928000001

• PTER is a N-acetyltaurine hydrolase that regulates feeding and obesity. Nature Wei, W., Lyu, X., Markhard, A. L., Fu, S., Mardjuki, R. E., Cavanagh, P. E., Zeng, X., Rajniak, J., Lu, N., Xiao, S., Zhao, M., Moya-Garzon, M. D., Truong, S. D., Chou, J. C., Wat, L. W., Chidambaranathan-Reghupaty, S., Coassolo, L., Xu, D., Shen, F., Huang, W., Ramirez, C. B., Jang, C., Li, L., Svensson, K. J., Fischbach, M. A., Long, J. Z. 2024

  Abstract

  Taurine is a conditionally essential micronutrient and one of the most abundant amino acids in humans1-3. In endogenous taurine metabolism, dedicated enzymes are involved in the biosynthesis of taurine from cysteine and in the downstream metabolism of secondary taurine metabolites4,5. One taurine metabolite is N-acetyltaurine6. Levels of N-acetyltaurine are dynamically regulated by stimuli that alter taurine or acetate flux, including endurance exercise7, dietary taurine supplementation8 and alcohol consumption6,9. So far, the identities of the enzymes involved in N-acetyltaurine metabolism, and the potential functions of N-acetyltaurine itself, have remained unknown. Here we show that the body mass index associated orphan enzyme phosphotriesterase-related (PTER)10 is a physiological N-acetyltaurine hydrolase. In vitro, PTER catalyses the hydrolysis of N-acetyltaurine to taurine and acetate. In mice, PTER is expressed in the kidney, liver and brainstem. Genetic ablation of Pter in mice results in complete loss of tissue N-acetyltaurine hydrolysis activity and a systemic increase in N-acetyltaurine levels. After stimuli that increase taurine levels, Pter knockout mice exhibit reduced food intake, resistance to diet-induced obesity and improved glucose homeostasis. Administration of N-acetyltaurine to obese wild-type mice also reduces food intake and body weight in a GFRAL-dependent manner. These data place PTER into a central enzymatic node of secondary taurine metabolism and uncover a role for PTER and N-acetyltaurine in body weight control and energy balance.

  View details for DOI 10.1038/s41586-024-07801-6

  View details for PubMedID 39112712

  View details for PubMedCentralID 3501277

• PELI2 is a negative regulator of STING signaling that is dynamically repressed during viral infection. Molecular cell Ritchie, C., Li, L. 2024

  Abstract

  The innate immune cGAS-STING pathway is activated by cytosolic double-stranded DNA (dsDNA), a ubiquitous danger signal, to produce interferon, a potent anti-viral and anti-cancer cytokine. However, STING activation must be tightly controlled because aberrant interferon production leads to debilitating interferonopathies. Here, we discover PELI2 as a crucial negative regulator of STING. Mechanistically, PELI2 inhibits the transcription factor IRF3 by binding to phosphorylated Thr354 and Thr356 on the C-terminal tail of STING, leading to ubiquitination and inhibition of the kinase TBK1. PELI2 sets a threshold for STING activation that tolerates low levels of cytosolic dsDNA, such as that caused by silenced TREX1, RNASEH2B, BRCA1, or SETX. When this threshold is reached, such as during viral infection, STING-induced interferon production temporarily downregulates PELI2, creating a positive feedback loop allowing a robust immune response. Lupus patients have insufficient PELI2 levels and high basal interferon production, suggesting that PELI2 dysregulation may drive the onset of lupus and other interferonopathies.

  View details for DOI 10.1016/j.molcel.2024.06.001

  View details for PubMedID 38917796

• Identification of the extracellular membrane protein ENPP3 as a major cGAMP hydrolase and innate immune checkpoint. Cell reports Mardjuki, R., Wang, S., Carozza, J., Zirak, B., Subramanyam, V., Abhiraman, G., Lyu, X., Goodarzi, H., Li, L. 2024: 114209

  Abstract

  2'3'-Cyclic guanosine monophosphate (GMP)-AMP (cGAMP) is a second messenger synthesized upon detection of cytosolic double-stranded DNA (dsDNA) and passed between cells to facilitate downstream immune signaling. Ectonucleotide pyrophosphatase phosphodiesterase I (ENPP1), an extracellular enzyme, was the only metazoan hydrolase known to regulate cGAMP levels to dampen anti-cancer immunity. Here, we uncover ENPP3 as the second and likely the only other metazoan cGAMP hydrolase under homeostatic conditions. ENPP3 has a tissue expression pattern distinct from ENPP1's and accounts for all cGAMP hydrolysis activity in ENPP1-deficient mice. Importantly, we also show that, as with ENPP1, selectively abolishing ENPP3's cGAMP hydrolysis activity results in diminished cancer growth and metastasis of certain tumor types in a stimulator of interferon genes (STING)-dependent manner. Both ENPP1 and ENPP3 are extracellular enzymes, suggesting the dominant role that extracellular cGAMP must play as a mediator of cell-cell innate immune communication. Our work demonstrates that ENPP1 and ENPP3 non-redundantly dampen extracellular cGAMP-STING signaling, pointing to ENPP3 as a target for cancer immunotherapy.

  View details for DOI 10.1016/j.celrep.2024.114209

  View details for PubMedID 38749434

• Stimulating STING for cancer therapy: Taking the extracellular route. Cell chemical biology Li, L. 2024

  Abstract

  Ten years ago, the second messenger cGAMP was discovered as the activator of the anti-cancer STING pathway. The characterization of cGAMP's paracrine action and dominant extracellular hydrolase ENPP1 cemented cGAMP as an intercellular immunotransmitter that coordinates the innate and adaptive immune systems to fight cancer. In this Perspective, I look back at a decade of discovery of extracellular cGAMP biology and drug development aiming to supply or preserve extracellular cGAMP for cancer treatment. Reviewing our understanding of the cell type-specific regulatory mechanisms of STING agonists, including their transporters and degradation enzymes, I explain on a molecular and cellular level the successes and challenges of direct STING agonists for cancer therapy. Based on what we know now, I propose new ways to stimulate the STING pathway in a manner that is not only cancer specific, but also cell type specific to fully harness the anti-cancer effect of cGAMP while avoiding collateral damage.

  View details for DOI 10.1016/j.chembiol.2024.04.004

  View details for PubMedID 38723635

• ENPP1 is an innate immune checkpoint of the anticancer cGAMP-STING pathway in breast cancer. Proceedings of the National Academy of Sciences of the United States of America Wang, S., Böhnert, V., Joseph, A. J., Sudaryo, V., Skariah, G., Swinderman, J. T., Yu, F. B., Subramanyam, V., Wolf, D. M., Lyu, X., Gilbert, L. A., Van't Veer, L. J., Goodarzi, H., Li, L. 2023; 120 (52): e2313693120

  Abstract

  Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) expression correlates with poor prognosis in many cancers, and we previously discovered that ENPP1 is the dominant hydrolase of extracellular cGAMP: a cancer-cell-produced immunotransmitter that activates the anticancer stimulator of interferon genes (STING) pathway. However, ENPP1 has other catalytic activities and the molecular and cellular mechanisms contributing to its tumorigenic effects remain unclear. Here, using single-cell RNA-seq, we show that ENPP1 in both cancer and normal tissues drives primary breast tumor growth and metastasis by dampening extracellular 2'3'-cyclic-GMP-AMP (cGAMP)-STING-mediated antitumoral immunity. ENPP1 loss-of-function in both cancer cells and normal tissues slowed primary tumor growth and abolished metastasis. Selectively abolishing the cGAMP hydrolysis activity of ENPP1 phenocopied ENPP1 knockout in a STING-dependent manner, demonstrating that restoration of paracrine cGAMP-STING signaling is the dominant anti-cancer mechanism of ENPP1 inhibition. Finally, ENPP1 expression in breast tumors deterministically predicated whether patients would remain free of distant metastasis after pembrolizumab (anti-PD-1) treatment followed by surgery. Altogether, ENPP1 blockade represents a strategy to exploit cancer-produced extracellular cGAMP for controlled local activation of STING and is therefore a promising therapeutic approach against breast cancer.

  View details for DOI 10.1073/pnas.2313693120

  View details for PubMedID 38117852

• Phage anti-CBASS protein simultaneously sequesters cyclic trinucleotides and dinucleotides. Molecular cell Cao, X., Xiao, Y., Huiting, E., Cao, X., Li, D., Ren, J., Fedorova, I., Wang, H., Guan, L., Wang, Y., Li, L., Bondy-Denomy, J., Feng, Y. 2023

  Abstract

  Cyclic-oligonucleotide-based anti-phage signaling system (CBASS) is a common immune system that uses cyclic oligonucleotide signals to limit phage replication. In turn, phages encode anti-CBASS (Acb) proteins such as Acb2, which can sequester some cyclic dinucleotides (CDNs) and limit downstream effector activation. Here, we identified that Acb2 sequesters many CDNs produced by CBASS systems and inhibits stimulator of interferon genes (STING) activity in human cells. Surprisingly, the Acb2 hexamer also binds with high affinity to CBASS cyclic trinucleotides (CTNs) 3'3'3'-cyclic AMP-AMP-AMP and 3'3'3'-cAAG at a distinct site from CDNs. One Acb2 hexamer can simultaneously bind two CTNs and three CDNs. Phage-encoded Acb2 provides protection from type III-C CBASS that uses cA3 signaling molecules. Moreover, phylogenetic analysis of >2,000 Acb2 homologs encoded by diverse phages and prophages revealed that most are expected to bind both CTNs and CDNs. Altogether, Acb2 sequesters nearly all known CBASS signaling molecules through two distinct binding pockets and therefore serves as a broad-spectrum inhibitor of cGAS-based immunity.

  View details for DOI 10.1016/j.molcel.2023.11.026

  View details for PubMedID 38103556

• Galectin-1 mediates chronic STING activation in tumors to promote metastasis through MDSC recruitment. Cancer research Nambiar, D. K., Viswanathan, V., Cao, H., Zhang, W., Guan, L., Chamoli, M., Holmes, B., Kong, C., Hildebrand, R., Koong, A. J., von Eyben, R., Plevritis, S., Li, L., Giaccia, A., Engleman, E., Le, Q. T. 2023

  Abstract

  The immune system plays a crucial role in the regulation of metastasis. Tumor cells systemically change immune functions to facilitate metastatic progression. Through this study, we deciphered how tumoral Galectin-1 (Gal1) expression shapes the systemic immune environment to promote metastasis in head and neck cancer (HNC). In multiple preclinical models of HNC and lung cancer in immunogenic mice, Gal1 fostered the establishment of a pre-metastatic niche through polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), which altered the local microenvironment to support metastatic spread. RNA sequencing of MDSCs from pre-metastatic lungs in these models demonstrated the role of PMN-MDSCs in collagen and extracellular matrix remodeling in the pre-metastatic compartment. Gal1 promoted MDSC accumulation in the pre-metastatic niche through the NF-κB signaling axis, triggering enhanced CXCL2-mediated MDSC migration. Mechanistically, Gal1 sustained NF-κB activation in tumor cells by enhancing STING protein stability, leading to prolonged inflammation-driven MDSC expansion. These findings suggest an unexpected pro-tumoral role of STING activation in metastatic progression and establish Gal1 as an endogenous positive regulator of STING in advanced-stage cancers.

  View details for DOI 10.1158/0008-5472.CAN-23-0046

  View details for PubMedID 37409887

• Discovery of VH domains that allosterically inhibit ENPP1. Nature chemical biology Solomon, P. E., Bracken, C. J., Carozza, J. A., Wang, H., Young, E. P., Wellner, A., Liu, C. C., Sweet-Cordero, E. A., Li, L., Wells, J. A. 2023

  Abstract

  Ectodomain phosphatase/phosphodiesterase-1 (ENPP1) is overexpressed on cancer cells and functions as an innate immune checkpoint by hydrolyzing extracellular cyclic guanosine monophosphate adenosine monophosphate (cGAMP). Biologic inhibitors have not yet been reported and could have substantial therapeutic advantages over current small molecules because they can be recombinantly engineered into multifunctional formats and immunotherapies. Here we used phage and yeast display coupled with in cellulo evolution to generate variable heavy (VH) single-domain antibodies against ENPP1 and discovered a VH domain that allosterically inhibited the hydrolysis of cGAMP and adenosine triphosphate (ATP). We solved a 3.2 Å-resolution cryo-electron microscopy structure for the VH inhibitor complexed with ENPP1 that confirmed its new allosteric binding pose. Finally, we engineered the VH domain into multispecific formats and immunotherapies, including a bispecific fusion with an anti-PD-L1 checkpoint inhibitor that showed potent cellular activity.

  View details for DOI 10.1038/s41589-023-01368-5

  View details for PubMedID 37400538

  View details for PubMedCentralID 7990037

• Phage anti-CBASS protein simultaneously sequesters cyclic trinucleotides and dinucleotides. bioRxiv : the preprint server for biology Cao, X., Xiao, Y., Huiting, E., Cao, X., Li, D., Ren, J., Guan, L., Wang, Y., Li, L., Bondy-Denomy, J., Feng, Y. 2023

  Abstract

  CBASS is a common anti-phage immune system that uses cyclic oligonucleotide signals to activate effectors and limit phage replication. In turn, phages encode anti-CBASS (Acb) proteins. We recently uncovered a widespread phage anti-CBASS protein Acb2 that acts as a "sponge" by forming a hexamer complex with three cGAMP molecules. Here, we identified that Acb2 binds and sequesters many CBASS and cGAS-produced cyclic dinucleotides in vitro and inhibits cGAMP-mediated STING activity in human cells. Surprisingly, Acb2 also binds CBASS cyclic trinucleotides 3'3'3'-cyclic AMP-AMP-AMP (cA3) and 3'3'3'-cAAG with high affinity. Structural characterization identified a distinct binding pocket within the Acb2 hexamer that binds two cyclic trinucleotide molecules and another binding pocket that binds to cyclic dinucleotides. Binding in one pocket does not allosterically alter the other, such that one Acb2 hexamer can simultaneously bind two cyclic trinucleotides and three cyclic dinucleotides. Phage-encoded Acb2 provides protection from Type III-C CBASS that uses cA3 signaling molecules in vivo and blocks cA3-mediated activation of the endonuclease effector in vitro. Altogether, Acb2 sequesters nearly all known CBASS signaling molecules through two distinct binding pockets and therefore serves as a broad-spectrum inhibitor of cGAS-based immunity.

  View details for DOI 10.1101/2023.06.01.543220

  View details for PubMedID 37398474

  View details for PubMedCentralID PMC10312549

• ENPP1's regulation of extracellular cGAMP is a ubiquitous mechanism of attenuating STING signaling. Proceedings of the National Academy of Sciences of the United States of America Carozza, J. A., Cordova, A. F., Brown, J. A., AlSaif, Y., Bohnert, V., Cao, X., Mardjuki, R. E., Skariah, G., Fernandez, D., Li, L. 2022; 119 (21): e2119189119

  Abstract

  SignificanceThe immune system strikes a careful balance between launching a robust response to threats and avoiding overactivation. The molecule cGAMP is an immunotransmitter that activates innate immunity and signals extracellularly, where it is subject to degradation by the enzyme ENPP1. Here, we engineer ENPP1 to lose activity toward cGAMP but not other substrates, thus creating a biochemically precise tool to understand how ENPP1 regulates extracellular cGAMP and thus innate immunity. We uncover that ENPP1's degradation of extracellular cGAMP has a long evolutionary history, and that this mechanism is critical for controlling diverse immune threats, including viral infection and inflammation.

  View details for DOI 10.1073/pnas.2119189119

  View details for PubMedID 35588451

• Biochemistry, Cell Biology, and Pathophysiology of the Innate Immune cGAS-cGAMP-STING Pathway. Annual review of biochemistry Ritchie, C., Carozza, J. A., Li, L. 2022

  Abstract

  In the decade since the discovery of the innate immune cyclic GMP-AMP synthase (cGAS)- 2'3'-cyclic GMP-AMP (cGAMP)- stimulator of interferon genes (STING) pathway, its proper activation and dysregulation have been rapidly implicated in many aspects of human disease. Understanding the biochemical, cellular, and regulatory mechanisms of this pathway is critical to developing therapeutic strategies that either harness it to boost defense or inhibit it to prevent unwanted inflammation. In this review, we first discuss how the second messenger cGAMP is synthesized by cGAS in response to double-stranded DNA and cGAMP's subsequent activation of cell-type-dependent STING signaling cascades with differential physiological consequences. We then review how cGAMP as an immunotransmitter mediates tightly controlled cell-cell communication by being exported from producing cells and imported into responding cells via cell-type-specific transporters. Finally, we review mechanisms by which the cGAS-cGAMP-STING pathway responds to different sources of mislocalized double-stranded DNA in pathogen defense, cancer, and autoimmune diseases. Expected final online publication date for the Annual Review of Biochemistry, Volume 91 is June 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  View details for DOI 10.1146/annurev-biochem-040320-101629

  View details for PubMedID 35287475

• Therapeutic Interventions Targeting Innate Immune Receptors: A Balancing Act CHEMICAL REVIEWS Cao, X., Cordova, A. F., Li, L. 2022; 122 (3): 3414-3458

  Abstract

  The innate immune system is an organism's first line of defense against an onslaught of internal and external threats. The downstream adaptive immune system has been a popular target for therapeutic intervention, while there is a relative paucity of therapeutics targeting the innate immune system. However, the innate immune system plays a critical role in many human diseases, such as microbial infection, cancer, and autoimmunity, highlighting the need for ongoing therapeutic research. In this review, we discuss the major innate immune pathways and detail the molecular strategies underpinning successful therapeutics targeting each pathway as well as previous and ongoing efforts. We will also discuss any recent discoveries that could inform the development of novel therapeutic strategies. As our understanding of the innate immune system continues to develop, we envision that therapies harnessing the power of the innate immune system will become the mainstay of treatment for a wide variety of human diseases.

  View details for DOI 10.1021/acs.chemrev.1c00716

  View details for Web of Science ID 000767145700008

  View details for PubMedID 34870969

• Human SLC46A2 Is the Dominant cGAMP Importer in Extracellular cGAMP-Sensing Macrophages and Monocytes. ACS central science Cordova, A. F., Ritchie, C., Bohnert, V., Li, L. 2021; 7 (6): 1073-1088

  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, 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.

  View details for DOI 10.1021/acscentsci.1c00440

  View details for PubMedID 34235268

• LRRC8A:C/E Heteromeric Channels Are Ubiquitous Transporters of cGAMP. Molecular cell Lahey, L. J., Mardjuki, R. E., Wen, X., Hess, G. T., Ritchie, C., Carozza, J. A., Bohnert, V., Maduke, M., Bassik, M. C., Li, L. 2020

  Abstract

  Extracellular 2'3'-cyclic-GMP-AMP (cGAMP) is an immunotransmitter exported by diseased cells and imported into host cells to activate the innate immune STING pathway. We previously identified SLC19A1 as a cGAMP importer, but its use across human cell lines is limited. Here, we identify LRRC8A heteromeric channels, better known as volume-regulated anion channels (VRAC), as widely expressed cGAMP transporters. LRRC8A forms complexes with LRRC8C and/or LRRC8E, depending on their expression levels, to transport cGAMP and other 2'3'-cyclic dinucleotides. In contrast, LRRC8D inhibits cGAMP transport. We demonstrate that cGAMP is effluxed or influxed via LRRC8 channels, as dictated by the cGAMP electrochemical gradient. Activation of LRRC8A channels, which can occur under diverse stresses, strongly potentiates cGAMP transport. We identify activator sphingosine 1-phosphate and inhibitor DCPIB as chemical tools to manipulate channel-mediated cGAMP transport. Finally, LRRC8A channels are key cGAMP transporters in resting primary human vasculature cells and universal human cGAMP transporters when activated.

  View details for DOI 10.1016/j.molcel.2020.10.021

  View details for PubMedID 33171122

• Diversity Is a Strength of Cancer Research in the US CANCER CELL Merad, M., Posey, A. D., Olivero, O., Singh, P. K., Mouneimne, G., Li, L., Wallace, L. M., Hayes, T. K. 2020; 38 (3): 297–300

  Abstract

  We stand against racism and discrimination in cancer research in the U.S. By sharing the stories of scientists from different ethnicities, identities, and national origins, we want to promote change through mentoring, active participation, and policy changes and to inspire the next generation of cancer researchers: we make better science together.

  View details for Web of Science ID 000581017400001

  View details for PubMedID 32931736

  View details for PubMedCentralID PMC7489360

• Structure-Aided Development of Small-Molecule Inhibitors of ENPP1, the Extracellular Phosphodiesterase of the Immunotransmitter cGAMP. Cell chemical biology Carozza, J. A., Brown, J. A., Bohnert, V., Fernandez, D., AlSaif, Y., Mardjuki, R. E., Smith, M., Li, L. 2020

  Abstract

  Cancer cells initiate an innate immune response by synthesizing and exporting the small-molecule immunotransmitter cGAMP, which activates the anti-cancer Stimulator of Interferon Genes (STING) pathway in the host. An extracellular enzyme, ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), hydrolyzes cGAMP and negatively regulates this anti-cancer immune response. Small-molecule ENPP1 inhibitors are much needed as tools to study the basic biology of extracellular cGAMP and as investigational cancer immunotherapy drugs. Here, we surveyed structure-activity relationships around a series of cell-impermeable and thus extracellular-targeting phosphonate inhibitors of ENPP1. In addition, we solved the crystal structure of an exemplary phosphonate inhibitor to elucidate the interactions that drive potency. This study yielded several best-in-class inhibitors with Ki< 2nM and excellent physicochemical and pharmacokinetic properties. Finally, we demonstrate that an ENPP1 inhibitor delays tumor growth in a breast cancer mouse model. Together, we have developed ENPP1 inhibitors that are excellent tool compounds and potential therapeutics.

  View details for DOI 10.1016/j.chembiol.2020.07.007

  View details for PubMedID 32726585

• cGAMP as an Adjuvant in Antiviral Vaccines and Cancer Immunotherapy BIOCHEMISTRY Ritchie, C., Li, L. 2020; 59 (18): 1713–15

  View details for DOI 10.1021/acs.biochem.0c00226

  View details for Web of Science ID 000535232800002

  View details for PubMedID 32301606

• Structural Insights into STING Signaling. Trends in cell biology Ergun, S. L., Li, L. 2020; 30 (5): 399–407

  Abstract

  Since its discovery 12 years ago, the stimulator of interferon genes (STING) pathway has attracted the intense focus of top cell biologists, biochemists, and structural biologists, due to its unique activation mechanisms and broad implications in cancer, aging, and autoimmunity. The STING pathway is an essential innate immune signaling cascade responsible for the sensing of aberrant cytosolic double-stranded DNA (dsDNA), which is a hallmark of cancer and viral infection. Erroneous STING activation can exacerbate many autoimmune and inflammatory syndromes. Therefore, it is remarkable how rapidly, effectively, and specifically the STING pathway responds to a myriad of threats while generally maintaining immune homeostasis. Here we review high-impact structural work that collectively paints a picture of STING signaling with atomic resolution. The elegant molecular mechanisms not only give clues to how STING has evolved to distinguish between self and foreign, but they also enable development of novel therapeutics to treat STING-related diseases.

  View details for DOI 10.1016/j.tcb.2020.01.010

  View details for PubMedID 32302551

• Extracellular cGAMP is a cancer cell-produced immunotransmitter involved in radiation-induced anti-cancer immunity. Nature cancer Carozza, J. A., Böhnert, V., Nguyen, K. C., Skariah, G., Shaw, K. E., Brown, J. A., Rafat, M., von Eyben, R., Graves, E. E., Glenn, J. S., Smith, M., Li, L. 2020; 1 (2): 184-196

  Abstract

  2'3'-cyclic GMP-AMP (cGAMP) is an intracellular second messenger that is synthesized in response to cytosolic double-stranded DNA and activates the innate immune STING pathway. Our previous discovery of its extracellular hydrolase ENPP1 hinted at the existence of extracellular cGAMP. Here, we detected that cGAMP is continuously exported but then efficiently cleared by ENPP1, explaining why it has previously escaped detection. By developing potent, specific, and cell impermeable ENPP1 inhibitors, we found that cancer cells continuously export cGAMP in culture at steady state and at higher levels when treated with ionizing radiation (IR). In mouse tumors, depletion of extracellular cGAMP decreased tumor-associated immune cell infiltration and abolished the curative effect of IR. Boosting extracellular cGAMP with ENPP1 inhibitors synergized with IR to delay tumor growth. In conclusion, extracellular cGAMP is an anti-cancer immunotransmitter that could be harnessed to treat cancers with low immunogenicity.

  View details for DOI 10.1038/s43018-020-0028-4

  View details for PubMedID 33768207

  View details for PubMedCentralID PMC7990037

• Extracellular cGAMP is a cancer-cell-produced immunotransmitter involved in radiation-induced anticancer immunity NATURE CANCER Carozza, J. A., Bohnert, V., Nguyen, K. C., Skariah, G., Shaw, K. E., Brown, J. A., Rafat, M., von Eyben, R., Graves, E. E., Glenn, J. S., Smith, M., Li, L. 2020; 1 (2): 184-+

  View details for DOI 10.1038/s43018-020-0028-4

  View details for Web of Science ID 000608021200012

• Development of cGAMP-Luc, a sensitive and precise coupled enzyme assay to measure cGAMP in complex biological samples. The Journal of biological chemistry Mardjuki, R. E., Carozza, J. A., Li, L. n. 2020

  Abstract

  2'5'/3'5' cyclic GMP-AMP (cGAMP) is a second messenger produced in response to cytosolic dsDNA that activates the stimulator of interferon genes (STING) pathway. We recently discovered that cGAMP is exported by cancer cells and that this extracellular signal is an immunotransmitter key to tumor detection and elimination by the innate immune system. The enhancement of extracellular cGAMP levels therefore holds great promise for managing cancer. However, there is still much more to understand about the basic biology of cGAMP before its full therapeutic potential can be realized. To answer these questions, we must be able to detect and quantitate cGAMP with an assay that is high throughput, sensitive, and precise. Existing assays fall short of these needs. Here, we describe the development of cGAMP-Luc, a coupled enzyme assay that relies on the degradation of cGAMP to AMP by ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) and an optimized assay for the detection of AMP by luciferase. We also developed STING-CAP, a STING-mediated method to concentrate and purify cGAMP from any type of biological sample. We conclude that cGAMP-Luc is an economical high throughput assay that matches the accuracy of and surpasses the detection limit of mass spectrometry, the current gold standard of cGAMP quantitation. We propose that cGAMP-Luc is a powerful tool that may enable discoveries that advance insights into extracellular cGAMP levels in healthy and diseased tissues, such as cancer.

  View details for DOI 10.1074/jbc.RA119.012170

  View details for PubMedID 32127400

• IFN-Independent STING Signaling: Friend or Foe? Immunity Böhnert, V. n., Ritchie, C. n., Li, L. n. 2020; 53 (1): 8–10

  Abstract

  In this issue, Wu et al. demonstrate the importance of the neglected interferon (IFN)-independent STING signaling axis in mice. They find that although this axis is important for antiviral HSV-1 resistance, it has a pro-cancer role by promoting T cell death.

  View details for DOI 10.1016/j.immuni.2020.06.021

  View details for PubMedID 32668231

• ENPP1 antagonists in combination with radiation or checkpoint inhibitors demonstrate antitumor activity in syngeneic mice models of pancreatic adenocarcinoma, neuroblastoma, TNBC, and colon cancer Li, L., Smith, M., Chang, B. BMC. 2019

  View details for Web of Science ID 000496473200157

• 2 ' 3 '-cGAMP is an immunotransmitter produced by cancer cells and regulated by ENPP1 Carozza, J., Bohnert, V., Shaw, K., Khanh Nyugen, Skariah, G., Brown, J., Rafat, M., von Eyben, R., Graves, E., Glenn, J., Smith, M., Li, L. AMER CHEMICAL SOC. 2019

  View details for Web of Science ID 000525061505479

• STING Polymer Structure Reveals Mechanisms for Activation, Hyperactivation, and Inhibition. Cell Ergun, S. L., Fernandez, D., Weiss, T. M., Li, L. 2019

  Abstract

  How the central innate immune protein, STING, is activated by its ligands remains unknown. Here, using structural biology and biochemistry, we report that the metazoan second messenger 2'3'-cGAMP induces closing of the human STING homodimer and release of the STING C-terminal tail, which exposes a polymerization interface on the STING dimer and leads to the formation of disulfide-linked polymers via cysteine residue 148. Disease-causing hyperactive STING mutations either flank C148 and depend on disulfide formation or reside in the C-terminal tail binding site and cause constitutive C-terminal tail release and polymerization. Finally, bacterial cyclic-di-GMP induces an alternative active STING conformation, activates STING in a cooperative manner, and acts as a partial antagonist of 2'3'-cGAMP signaling. Our insights explain the tight control of STING signaling given varying background activation signals and provide a therapeutic hypothesis for autoimmune syndrome treatment.

  View details for DOI 10.1016/j.cell.2019.05.036

  View details for PubMedID 31230712

• SLC19A1 Is an Importer of the Immunotransmitter cGAMP. Molecular cell Ritchie, C., Cordova, A. F., Hess, G. T., Bassik, M. C., Li, L. 2019

  Abstract

  2'3'-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 activateintracellular STING. Using a genome-wide CRISPRscreen, we identified SLC19A1 as the first knownimporter of cGAMP and other CDNs, including theinvestigational new drug 2'3'-bisphosphosphothioate-cyclic-di-AMP (2'3'-CDAS). These discoveries will provide insight into cGAMP's role as an immunotransmitter and aid in the development of more targeted CDN-based cancer therapeutics.

  View details for DOI 10.1016/j.molcel.2019.05.006

  View details for PubMedID 31126740

• STING Signaling Promotes Inflammation in Experimental AcutePancreatitis. Gastroenterology Zhao, Q., Wei, Y., Pandol, S. J., Li, L., Habtezion, A. 2018; 154 (6): 1822

  Abstract

  BACKGROUND & AIMS: Acute pancreatitis (AP) is characterized by severe inflammation and acinar cell death. Transmembrane protein 173 (TMEM173 or STING) is a DNA sensor adaptor protein on immune cells that recognizes cytosolic nucleic acids and transmits signals that activate production of interferons and the innate immune response. We investigated whether leukocyte STING signaling mediates inflammation in mice with AP.METHODS: We induced AP in C57BL/6J mice (control) and C57BL/6J-Tmem173gt/J mice (STING-knockout mice) by injection of cerulein or placement on choline-deficient DL-ethionine supplemented diet. In some mice, STING signaling was induced by administration of a pharmacologic agonist. AP was also induced in C57BL/6J mice with bone marrow transplants from control or STING-knockout mice and in mice with disruption of the cyclic GMP-AMP synthase (Cgas) gene. Pancreata were collected, analyzed by histology, and acini were isolated and analyzed by flow cytometry, quantitative polymerase chain reaction, immunoblots, and enzyme-linked immunosorbent assay. Bone-marrow-derived macrophages were collected from mice and tested for their ability to detect DNA from dying acinar cells in the presence and absence of deoxyribonuclease (DNaseI).RESULTS: STING signaling was activated in pancreata from micewith AP but not mice without AP. STING-knockout mice developed less severe AP (less edema, inflammation, and markers of pancreatic injury) than control mice, whereas mice given a STING agonist developed more severe AP than controls. In immune cells collected from pancreata, STING was expressed predominantly in macrophages. Levels of cGAS were increased in mice with vs without AP, and cGAS-knockout mice had decreased edema, inflammation, and other markers of pancreatic injury upon induction of AP than control mice. Wild-type mice given bone marrow transplants from STING-knockout mice had less pancreatic injury and lower serum levels of lipase and pancreatic trypsin activity following induction of AP than mice given wild-type bone marrow. DNA from dying acinar cells activated STING signaling in macrophages, which was inhibited by addition of DNaseI.CONCLUSIONS: In mice with AP, STING senses acinar cell death (by detecting DNA from dying acinar cells) and activates a signaling pathway that promotes inflammation. Macrophages express STING and activate pancreatic inflammation in AP.

  View details for PubMedID 29425920

• STING Signaling Promotes Inflammation in Experimental Acute Pancreatitis GASTROENTEROLOGY Zhao, Q., Wei, Y., Pandol, S. J., Li, L., Habtezion, A. 2018; 154 (6): 1822-+

  View details for DOI 10.1053/j.gastro.2018.01.065

  View details for Web of Science ID 000432464700046

• Activation of the STING-Dependent Type I Interferon Response Reduces Microglial Reactivity and Neuroinflammation NEURON Mathur, V., Burai, R., Vest, R. T., Bonanno, L. N., Lehallier, B., Zardeneta, M. E., Mistry, K. N., Do, D., Marsh, S. E., Abud, E. M., Blurton-Jones, M., Li, L., Lashuel, H. A., Wyss-Coray, T. 2017; 96 (6): 1290-+

  Abstract

  Brain aging and neurodegeneration are associated with prominent microglial reactivity and activation of innate immune response pathways, commonly referred to as neuroinflammation. One such pathway, the type I interferon response, recognizes viral or mitochondrial DNA in the cytoplasm via activation of the recently discovered cyclic dinucleotide synthetase cGAS and the cyclic dinucleotide receptor STING. Here we show that the FDA-approved antiviral drug ganciclovir (GCV) induces a type I interferon response independent of its canonical thymidine kinase target. Inhibition of components of the STING pathway, including STING, IRF3, Tbk1, extracellular IFNβ, and the Jak-Stat pathway resulted in reduced activity of GCV and its derivatives. Importantly, functional STING was necessary for GCV to inhibit inflammation in cultured myeloid cells and in a mouse model of multiple sclerosis. Collectively, our findings uncover an unexpected new activity of GCV and identify the STING pathway as a regulator of microglial reactivity and neuroinflammation.

  View details for PubMedID 29268096

  View details for PubMedCentralID PMC5806703

• Host-Pathogen interactions: Nucleotide circles of life and death. Nature chemical biology Li, L. 2017; 13 (2): 130-131

  View details for DOI 10.1038/nchembio.2289

  View details for PubMedID 28103224