As a first-year graduate student from the Chemistry Department, I found myself far from my comfort zone in the Biochemistry Department talking to my now-advisor, Dr. Lingyin Li, about potential projects. I imagine that she told me about the importance of the innate immune STING pathway in the anti-cancer response. She probably also described how cells respond to DNA in the cytosol, a danger signal marking damaged or infected cells, by making a cyclic dinucleotide second messenger called cGAMP, which eventually (via a signaling cascade) triggers the production of immune-activating interferons. However, I don’t remember much of that; it likely went above my head. But then Lingyin pitched me a project using mass spectrometry to look for cGAMP outside the cell and something clicked into place – cGAMP was an irresistible molecule to measure.
Why were we looking for extracellular cGAMP, when its role as an intracellular messenger was well established? In 2014, Lingyin discovered the enzyme that degrades cGAMP – ENPP1 (Li et al., Nat. Chem. Biol. 2014). The catch? ENPP1 was extracellular, motivating the search for extracellular cGAMP. Even if someone had looked for extracellular cGAMP, they were unlikely to see it because ENPP1 is abundant in the serum, including the serum used for routine cell culture experiments. To set up the experiment, we removed all sources of ENPP1 (using ENPP1-/- cells and serum-free cell medium). Then we addressed the other challenge - finding cGAMP among the trillions of other small molecules and proteins in the medium and cell lysate. We teamed up with Khanh Nguyen in Jeffrey Glenn’s lab at Stanford, who developed a robust and sensitive mass spectrometry method that enabled all of the cGAMP detection experiments in this paper.
With detection method in hand and ENPP1 removed, we were ready to look for extracellular cGAMP. Concerned we wouldn’t see anything, I did massive concentration and purification steps on the cell medium. In fact, there was a LOT of extracellular cGAMP - after 24 hours, there were as many molecules of cGAMP outside of the cell as inside. That is strong export.
We were eager to look for extracellular cGAMP produced by cancer cells. Work over the past century has shown that cancer cells have chromosomal instability and high cytosolic DNA, the stimulus for the STING pathway. The downstream effects of the STING pathway recruit tumor-killing immune cells. Could cancer cells export cGAMP as a signal to stimulate immune STING? First, we needed a way to inhibit ENPP1 quickly and easily in many cell settings – we collaborated with Mark Smith at the Stanford ChEM-H Medicinal Chemistry Knowledge Center to design a potent, extracellular ENPP1 inhibitor. With this chemical tool, we found extracellular cGAMP from cancer cells, again in large quantities. Not only that, but extracellular cGAMP also increased with exposure to ionizing radiation, a common cancer treatment that elicits DNA damage.
Here’s where we connected the dots: we knew cancer cells could export cGAMP. We also knew immune cells import cGAMP, from other work in Lingyin’s lab (Ritchie and Cordova et al., Mol. Cell. 2019; Lahey et al., bioRxiv 2020). I teamed up with postdoc Dr. Volker Böhnert, my coauthor, and depleted extracellular cGAMP from mice to see what would happen. We actually needed to produce our own cGAMP-neutralizing protein – in total, we purified over 1 gram of protein, which corresponds to 50 liters of protein-expressing bacteria!
When we depleted extracellular cGAMP in a triple-negative breast cancer mouse model after treating the radiation, the radiation-induced survival effect was abolished. Looking back to my first year as a chemistry graduate student, I never would have dreamed that I could contribute to a cancer biology experiment. But now I can appreciate this discovery as a breakthrough in our understanding of how extracellular cGAMP can mediate the effect of radiation and alert immune cells to the presence of cancerous cells.
This is a story of bringing together chemistry and biology to find new cancer-immune signaling that we are proud to tell in Nature Cancer. What is difficult for a biologist to do may be trivial for a chemist, and what is unfathomable to a chemist may be routine for a biologist. It’s a story of persistence in validating a scientific model that no one believed in but is now being incorporated into academic reviews and pharmaceutical company pipelines. And it’s a story that is ongoing as we continue to map the molecular details of extracellular cGAMP signaling and therapeutically harness this anti-cancer immunotransmitter.
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