Regulation of the cGAS-STING Pathway: Non-Agonist Activation and Cellular Trafficking

The cGAS-STING pathway in the innate immune system plays an important role in infection and cancer. Exogenous or endogenous DNA and cyclic dinucleotides acutely activate cGAS-STING, leading to type I interferon (IFN) signaling. STING-mediated IFN signaling is dynamically regulated by STING trafficking from the ER to the Golgi then to the lysosome. Cofactors and cellular mechanisms that facilitate STING trafficking and signaling are incompletely understood. In the settings of cancer immunotherapy, STING agonists induce strong anti-tumor immunity in preclinical models and synergize with radiation, chemotherapy, and immune checkpoint inhibitor therapy. Evaluation of STING agonists in human clinical trials is ongoing, although early results did not yield satisfying prospects. Thus, novel approaches to activate STING signaling are needed. One non-agonist approach for activating the cGAS-STING pathway is to inhibit cytosolic DNase TREX1, which would lead to the build-up of self-DNA in the cytosol and activation of cGAS. TREX1 expression is upregulated during radiation therapy and TREX1 knockdown promotes anti-tumor immunity. I developed a cell-free DNase assay that quantitatively monitors the enzymatic activity of TREX1. As a proof of concept, inhibition of TREX1 by an existing small molecule inhibitor Compound I induced type I IFN response in many cell types in vitro. I performed an HTS screen of 300K small molecules with the TREX1 DNase assay and identified several candidate TREX1 inhibitors that exhibit over 10-fold higher potency compared to Compound I. Top candidates were well tolerated in a cell-based toxicity assay, making them promising small molecules to activate the cGAS-STING pathway as a non-agonist approach (Chapter 2). I also performed a proteomic screen to identify spatial-temporal regulators of STING during trafficking. Knockdown of several post-Golgi cofactors of STING activates tonic IFN signaling, suggesting that the cGAS-STING pathway is operational at homeostasis. This 'basal-flux' mode of STING activation requires functional cGAS and STING to maintain a basal immune state. Blocking of post-Golgi STING trafficking extends STING Golgi-dwell time and enhances STING signaling. I identified trans-Golgi coiled-coil protein GCC2 and Rab GTPase Rab14 as key regulators of STING Golgi-exit. Moreover, I found increased autoantibodies production in Gcc2-/- mice in a STING-dependent manner. As another non-agonist approach to activate the cGAS-STING pathway, I found that Gcc2KO and Rab14KO tumor cells elicited T cell- and type I IFN-dependent anti-tumor immunity in mice (Chapter 3). In summary, studies in this thesis describe detailed cellular mechanisms of STING trafficking as well as signaling regulation and novel non-agonist approaches to activate the cGAS-STING pathway for cancer immunotherapy.