Lingyin Li

All Publications

• 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

• 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

• 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

• 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