Zhijian “James” Chen
UT Southwestern Medical Center
For the discovery of the cGAS enzyme that senses foreign and self DNA, solving the mystery of how DNA stimulates immune and inflammatory responses
The 2024 Albert Lasker Basic Medical Research Award honors a scientist for the discovery of the cGAS enzyme that senses foreign and self DNA. With original thinking and tour-de-force experimentation, Zhijian “James” Chen (UT Southwestern Medical Center) solved the mystery of how DNA stimulates immune and inflammatory responses. cGAS underpins a major mechanism by which mammals combat microbial invaders and it fosters antitumor immunity. In certain physiological settings, inappropriate cGAS activity contributes to autoimmune and inflammatory disorders. The enzyme provides a pharmaceutical target for diverse human maladies, and the signaling molecule that it generates holds promise for fighting infectious diseases and cancer.
A DNA-based alarm
DNA blazes in textbooks as the canonical blueprint for life, but it serves other functions too. Normally, DNA in animals is confined to nuclei or mitochondria. DNA outside of these compartments—in the cytoplasm—warns that a microbial intruder has gained access, that malignant cells are present, or that some other pathological process is unfolding.
In 1908, Ilya Mechnikov noted in his Nobel Prize lecture that nucleic acid, comprised primarily of DNA, recruits a “protective army of phagocytes” to ward off microbes, though no one knew how. This cellular army belongs to the innate immune system, which mobilizes quickly when it spots pathogens, fending them off in general ways. It also awakens the B cells and T cells of the adaptive immune system, which take longer to deploy but zero in on the specific interloper at hand and “remember” what they have seen.
In 2006, researchers showed that delivering double-stranded DNA (dsDNA) to the cytoplasm of mammalian cells causes quantities of powerful components of the innate immune system to spike. These signaling molecules include type I interferons such as interferon-β, so named because they interfere with viral infection.
The discovery launched a race to unearth a cytoplasmic dsDNA detector that prompts manufacture of type I interferons. Several candidates were announced, but under scrutiny, they all faltered.
In 2008, Glen N. Barber (University of Miami School of Medicine) and Hong-Bing Shu (Wuhan University) independently identified a linchpin in the interferon pathway. This protein, which Barber named stimulator of interferon genes (STING), spurs cells to crank up production of type I interferons, and the following year, he reported that DNA instigates the process. However, despite STING’s importance, it does not directly sense DNA.
Biochemical magnum opus
Through a series of imaginative, elegant, and incisive studies, Chen unmasked the elusive sensor. He tracked it down using an open-ended approach that made no assumptions about its identity or properties, demanding only that it perform the function that he sought: dsDNA-dependent activation of STING.
First, he wanted to trap elements of the pathway that operate before STING swings into motion. Toward that end, Chen, together with postdoctoral fellow Lijun Sun and graduate student Jiaxi Wu, obliterated STING in mouse cells so that he would collect only stimulators, not downstream elements. The team put dsDNA into these STING-deficient cells and extracted their innards, reasoning that they would contain the presumptive activator. The researchers then applied this material to a second set of cells and measured STING mobilization by tracking the status of a protein that it fires up: a key interferon-β regulator, IRF3.
The cellular contents successfully galvanized IRF3, so the investigators knew that the substance they sought was somewhere in the crude mixture. To tease it out, they employed chromatographic techniques to separate the components; then they checked which ones could goad STING. Through sequential purification steps, they homed in on the chemical of interest.
Subsequent analysis identified this compound as cyclic GMP-AMP (cGAMP), a type of molecule never before seen in mammalian cells, in which two nucleotides—in this case, GMP and AMP—join together in a circle through two phosphodiester bonds. Chen’s group reported this finding in 2012 and confirmed that cGAMP possesses definitive properties of a STING activator: A synthetic version of the molecule induces interferon-β production inside mammalian cells grown in culture dishes, and infection of mammalian cells with a DNA virus but not an RNA virus generates elevated quantities of cGAMP. Furthermore, the cyclic dinucleotide binds to STING and depends on STING to rev up IRF3. From these experiments, Chen concluded that cytoplasmic DNA kicks off a cascade of events: cGAMP appears and prods STING, which propels IRF3 to turn on type I interferon and related genes.
Next, Chen wanted to track down the enzyme that manufactures cGAMP. With Sun and Wu, he mashed up cells that make cGAMP when provoked by DNA and segregated their constituents based on size, charge, and other characteristics. The investigators then tested each sample for the enzymatic behavior that they sought: the ability to make cGAMP if DNA is present. By choosing the most active fractions and subjecting them to further separation steps, they gradually enriched for the desired protein, casting off others in the process.
Eventually, Chen zeroed in on three proteins whose quantities peaked in the same fractions that exhibited maximum enzymatic strength. The predicted amino acid sequence of one was especially exciting: It resembled 2´-5´-oligoadenylate synthase, which belongs to the same family as adenylate cyclase, the enzyme that makes the classic signaling entity cyclic AMP. Perhaps, Chen reasoned, this newly discovered protein would function analogously to adenylate cyclase; instead of marrying two atoms within ATP to form cyclic AMP, it would attach atoms within GTP and ATP to each other and create cGAMP.
Chen isolated the gene that encodes the protein, which he named cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS), and showed that cGAS overproduction induces interferon-β in cells that contain STING, but not in those that lack it. Furthermore, altering portions of cGAS that are predicted to be crucial for catalysis, based on analogy with its molecular relatives, obliterates the ability to foment IRF3 activation.
By several strategies, Chen demonstrated that cGAS is essential in human and mouse cells for making cGAMP and inducing interferon-β upon encountering DNA or a DNA virus. Furthermore, purified cGAS binds DNA and performs the expected reaction, fabricating cGAMP from ATP and GTP if DNA is present. Together, the results established that cGAS senses dsDNA in the cytoplasm and generates cGAMP in response; cGAMP, in turn, rouses STING, which triggers type I interferon production through IRF3 (see Figure).
Sounding a DNA siren
Microbial intruders or leakage from mitochondria or nuclei can introduce DNA into the cytoplasm. Once there, it binds to cGAS, which sparks production of the cyclic dinucleotide cGAMP from ATP and GTP. cGAMP prompts STING to migrate from the endoplasmic reticulum (ER) membrane to the Golgi, where it activates TANK-binding kinase 1 (TBK1) and IκB kinase (IKK). Subsequent events lead to activation of interferon regulatory factor 3 (IRF3) and NF-κB, respectively. Consequently, IRF3 and NF-κB enter the nucleus, where they turn on genes that encode proinflammatory proteins, including type I interferons, that incite the innate immune system. This pathway fends off dangerous microbes and cancer, but it also can instigate harmful autoimmune and inflammatory disorders. Illustration: Cassio Lynm / © Amino Creative
Blasting off with cGAS
This work broke open a new field and, in short order, several groups confirmed and extended Chen’s conclusions. Within months, scientists including Chen worked out the structure of cGAS, and they elucidated how DNA switches on cGAS and cGAMP switches on STING, necessary steps in harnessing the pathway for therapeutic use.
Chen expanded our understanding of cGAS’s reach when he determined that the cGAS-cGAMP-STING pathway detects not only DNA viruses, but also retroviruses such as HIV, whose genetic material is composed of RNA. Inside host cells, the viral RNA is converted to DNA. Retroviruses are notorious for evading the innate immune response, but certain manipulations—compromising the integrity of the viral capsid, for instance, which makes it leak—can render HIV able to stir type I interferons and related molecules. Chen exploited such tricks and showed that cGAMP and STING underlie the process. Thus, under some conditions, cGAS can sense HIV and other retroviruses. These observations raised the possibility that administration of cGAMP might help bypass HIV’s knack for sidestepping the immune system.
Chen wanted to pin down the physiological relevance of cGAS, so he engineered mice that lack the gene to test whether the effects that he had unveiled in test tubes and cells extend to live animals. When infected with a DNA virus, the cGAS-deficient rodents displayed an unusually weak interferon response and more of them died than did their counterparts that carried normal amounts of cGAS. Additional experiments with injected cGAMP demonstrated that the signaling dinucleotide dramatically boosts antibody and T-cell reactions to foreign proteins.
These studies and others established that cGAS senses DNA viruses in live animals and sparks the innate immune system. We now know that a wide spectrum of microbial pathogens, including many intracellular bacteria, induce interferons through the cGAS-cGAMP-STING pathway. The ability of cGAMP to potently stimulate STING underscores the possibility of wielding the chemical to battle microbial scourges as well as cancer, as type I interferons have long been known to guard the body against malignancies.
Fueling inflammation
Although the innate immune system benefits people when it foils invaders, its dark side manifests when the body attacks itself. Scientists had linked defects in an enzyme called Trex1, which chews up DNA in the cytoplasm, to human autoimmune diseases including Aicardi-Goutieres Syndrome and systemic lupus erythematosus that are characterized by elevated activity of interferon-stimulated genes. These reports implied that the failure to destroy cytoplasmic DNA spurs the interferon pathway. Mice that lack Trex1 similarly excite interferon-induced genes and die a few months after birth due to severe inflammation.
In 2015, Chen showed that eliminating cGAS in mice that lack Trex1 rescues the lethal effects of the missing enzyme and abolishes the tissue and molecular pathology that normally kills the animals. Independently, Daniel Stetson (University of Washington School of Medicine) published similar findings. These results indicated that cGAS drives the detrimental effects caused by the absence of Trex1, and they suggested that thwarting cGAS might curb autoimmune diseases such as lupus, in which self DNA agitates the interferon pathway.
cGAS has been implicated not only in autoimmune conditions, but in numerous inflammatory illnesses, including age-related macular degeneration and neurological disorders such as Parkinson’s disease, Alzheimer disease, and amyotrophic lateral sclerosis. Calming the cGAS-cGAMP-STING pathway might therefore provide benefit across a broad span of ailments. These possibilities have ignited intense interest by pharmaceutical companies, which are attempting to develop agents that inhibit cGAS. On the flip side, because cGAS furnishes the first line of defense against dangerous human pathogens and contributes to antitumor immunity, enhancing the pathway in other physiological situations might bolster health.
Through creative and elegant investigations, Chen has delivered foundational insights that explain how our bodies stay safe in the sea of microbes around us and that illuminate mechanistic details of manifold illnesses. The impact of his work reverberates far beyond clinical biomedical science. cGAS-like enzymes guard thousands of bacterial species against the spread of their own viral-like menaces, bacteriophages. The system shines as an ancient evolutionary scheme for repelling would-be cellular hijackers that threaten to co-opt another’s resources to further their own domination.
by Evelyn Strauss
Selected Publications – Zhijian (James) Chen
Sun L, Wu J, Du F, Chen X, and Chen ZJ. (2013). Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science. 339, 786-791.
Wu J, Sun L, Chen X, Du F, Shi H, Chen C, and Chen ZJ. (2013). Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science. 339, 826-830.
Gao D, Wu J, Wu YT, Du F, Aroh C, Yan N, Sun L, and Chen ZJ. (2013). Cyclic GMP-AMP synthase is an innate immune sensor of HIV and other retroviruses. Science. 341, 903-906.
Li X, Wu J, Gao D, Wang H, Sun L, and Chen, ZJ. (2013). Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects. Science. 341, 1390-1394.
Gao D, Li T, Li X-D, Chen X, Li Q-Z, Wight-Carter M, and Chen ZJ. (2015). Activation of cyclic GMP-AMP synthase by self DNA causes autoimmune diseases. Proc. Natl. Acad. Sci. USA. 112, E5699-E5705.
Ablasser A, and Chen ZJ. (2019). cGAS in action: Expanding roles in immunity and inflammation. Science. 363, doi: 10.1126/science.aat8657.
Jenson JM, Li T, Du F, Ea CK, and Chen ZJ. (2023) Ubiquitin-like conjugation by bacterial cGAS enhances anti-phage defence. Nature. 616, 326-331.
Jenson J, and Chen ZJ. (2024) cGAS goes viral: A conserved immune defense system from bacteria to humans. Mol. Cell. 84, 120-130.
The boogie-woogie approach to creativity in art and science
The Dutch painter Piet Mondrian, famous for his geometric grid paintings, was an ardent jazz fan. In the late stage of his career, he became intrigued with boogie-woogie music, which inspired him to paint his masterpiece, Broadway Boogie Woogie.
Award Presentation: Michael Brown
Ordinary science advances slowly. We scientists move forward like a couple on a dance floor doing a slow box step. One step forward. One step to the side. One step back. Every so often a graceful couple captures the dance floor. Like Ginger Rogers and Fred Astaire, they sweep across the room. In a single step they twirl across a threshold, leading us ordinary scientists into a new ballroom where we can resume our slow box steps.
James Chen and his students are like Rogers and Astaire. In 2013 they published two elegant papers in a single issue of Science. The papers taught us how our bodies detect invasion by DNA viruses, and how this activates antiviral defenses. Biologists had crossed a threshold. Today we honor James Chen for his discovery and for the graceful elegance with which he made it.
Our bodies have two systems to defend against viruses. One is the immune system that generates antibodies. This system is powerful but slow. It takes time for antibodies to appear. So, the body needs a rapid defense to hold the line until the immune system can react. This rapid defense is called innate immunity. When a virus invades a cell, the cell secretes a protein called interferon. Like Paul Revere, interferon speeds through our bodies shouting, “the viruses are coming”. Other cells respond by mobilizing antiviral armies called cytokines. The cytokine army keeps the virus from advancing until George Washington can arrive with his antibody artillery.
A fundamental question is “how does a cell know it is infected, and how does this knowledge trigger interferon production?” Viruses come in two categories. Some use RNA as their hereditary material. Others use DNA. Our cells use different mechanisms to detect RNA and DNA viruses. In earlier work James Chen helped unravel the sensors for RNA viruses, but that is not the discovery that we honor today. Today, we honor Chen for showing us how our cells recognize DNA viruses—the viruses that cause smallpox, chicken pox, hepatitis B, cervical cancer, and many other diseases.
Most Lasker Awards recognize the thing that was discovered. In Chen’s case we recognize not only WHAT he discovered, but HOW he discovered it. His methods are original and breathtaking so I must describe them in a little detail.
In the first of their two Science papers, Chen and his students, Lijun Sun and Jiaxi Wu, invented a clever system to use biochemistry to discover the sensor for viral DNA. They used animal cells in tissue culture. To activate the sensing mechanism, they introduced foreign DNA into the cytoplasm of one set of cells. Then they made extracts from the DNA-treated cells and added the extracts to other cells that were permeabilized to allow the signaling molecule to enter. They found that the extracts from the DNA-treated cells produced a substance that activated the interferon gene in recipient cells that had not seen DNA. They assumed that the signaling substance was a protein, and their next task was to purify it. Soon they discovered that the signaling substance was not a giant protein but a tiny chemical—a combination of two molecules called AMP and GMP linked together in a circle to form Cyclic AMP-Cyclic GMP. They named it cGAMP. Molecules like cGAMP had never been seen before in animal cells.
The discovery of cGAMP alone would be worthy of a Lasker Award. But Chen and his students went much farther. Their first paper told us that cGAMP warns that viruses are invading. But how does viral DNA trigger cells to produce cGAMP? They solved this problem in their second paper, whose biochemical virtuosity is even more breathtaking than the first.
Chen and his students set out to purify the enzyme that detects DNA and makes cGAMP. Their only source was tissue culture cells. Instead of adding DNA to cells, this time they first made extracts of the cells and then added DNA to directly activate the enzyme that produces cGAMP. To detect cGAMP they added the activated solution to permeabilized cells that had not seen DNA, and again they measured interferon activation. To isolate the enzyme from the DNA-treated cell extracts, they separated the proteins into fractions and incubated each fraction with DNA to see if that fraction contained the enzyme. Here they faced a problem. Tissue culture cells contain tiny amounts of protein. To identify the enzyme, they had to separate it from the many thousands of proteins in this tiny amount of starting material. They used standard methods of protein purification, but their most purified solution still contained too many proteins. They couldn’t tell which protein was the enzyme. They tried two other methods to purify the enzyme and again each purified solution contained too many proteins. Then they hit upon their brilliant idea. Each of their three purified solutions contained a different mixture of proteins, but they all produced cGAMP so they all must contain the cGAMP enzyme. The cGAMP enzyme might be the only protein that was present in all three purified preparations. So, they used mass spectrometry to identify the proteins. Only three proteins were present in all three solutions. When they analyzed the sequence of these three proteins, the correct one was obvious. One protein resembled known enzymes that work on nucleotides. This must be the culprit. They named their enzyme cGAS which stands for cGAMP synthetase.
Using recombinant DNA techniques, they produced pure cGAS. They showed that DNA binds to cGAS and activates it to produce cGAMP. But there was a problem. cGAS is activated by animal DNA as well as viral DNA. How does it distinguish the two? The answer lies in real estate. Location. Location. Location. cGAS is in the cytoplasm where viral DNA is located. Animal cell DNA is restricted to nuclei or mitochondria. cGAS is activated only by cytoplasmic viral DNA.
Once Chen had crossed the DNA sensing threshold it was time for the box steppers to step in. Chen and the box steppers showed that cGAMP binds to a protein called STING that transmits the signal to activate the interferon gene. The implications for medicine are profound. To increase the immune killing of cancer cells, pharmaceutical companies are testing drugs that activate cGAS. On the other hand, cGAS is sometimes activated abnormally by cellular DNA that leaks out of the nucleus. Activated cGAS produces cGAMP which triggers interferon production and causes inflammation and tissue damage. The result is autoimmune diseases like lupus and rheumatoid arthritis. Drugs that inhibit cGAS are effective in animal models of autoimmune diseases. Soon trials in humans will begin.
In summary, James Chen is the Fred Astaire of viral immunity. He is a worthy recipient of the Lasker Basic Science Award.
Acceptance remarks: James Chen
As a kid growing up in a remote mountain in southern China, I often looked up and gazed the stars. But never had I ever imagined that I would be gazing at my stars so closely, now at the Lasker Luncheon. I want to thank the Lasker Jury and the foundation for selecting me to receive this tremendous honor.
When I was young, I learned that the wind is created by mixing hot air and cold air. Based on this, I drew a wind machine, attempting to blow away the mountains surrounding me, because I thought the mountains kept us poor and isolated. After living in Dallas for nearly three decades, now I miss those mountains. Obviously, my wind machine never worked, but my parents found a better way, which was to send me to school. Now I am living proof that education is the greatest equalizer. And my message to the young people is this: if I can get so close to the stars, you can too.
My love for science, and for biochemistry in particular, was aroused by the late Cecile Pickart, my PhD advisor who taught me the art of protein purification. My interest in applying biochemical approach to solving biological problems was encouraged by Tom Maniatis, who taught me how to choose important problems to work on.
In 1997, I started my lab at UTSW, right next to the lab of Eric Olson, who recruited me to join the new department of Molecular Biology. This was the best move in my life, because UTSW is a scientific oasis built and cultivated by my scientific heroes and leaders who are here today. In Dallas, I was fortunate to recruit talented students and postdocs who believe in the power of biochemistry in making discoveries even in this post-genomic era. This effort culminated in the discovery of cGAS and cGAMP by a dream team led by Josh Sun and Jiaxi Wu. It has been gratifying to see the rapid progress of understanding the cGAS pathway in the past decade, thanks to spectacular efforts by many scientists around the globe.
Today I feel honored to receive the Lasker award for basic research together with the clinical awardees who discovered GLP1 and developed the medicine that has benefited millions of people. It is my dream that one day our discovery of cGAS will have a similar clinical impact.
