Non-canonical STING activation by calcium influx paired to endoplasmic reticulum stress.

STING plays a pivotal role in initiating antiviral and proinflammatory responses in vertebrates. cGAS detection of cytosolic dsDNA, leads to the synthesis of 2'3'-cGAMP that subsequently binds to STING, mediating its canonical activation. Activation of STING triggers a signaling cascade that mediates the expression of antiviral and proinflammatory genes. Due to the known effects of STING activation, this protein has been related with several situations as antitumor immunity and managing cellular stress responses. While dsDNA is the best-known mediator of STING responses, recent studies have proposed alternative non-canonical activations of this protein. In this study, we explore the non-canonical activation of STING by thapsigargin, a known SERCA inhibitor commonly utilized to provoke ER stress and initiate the unfolded protein response (UPR). Our research reveals that thapsigargin can elicit functional antiviral and pro-inflammatory responses independently of other agonists. Remarkably, thapsigargin can induce Type I IFN and proinflammatory responses as early as 30 min post-treatment. This activation is STING-dependent but occurs independently of its well-known inducers, cGAS and IFI16. This activation also depends on SERCA inhibition and Ca2+ influx, and is inseparable from ER stress induction, which excludes an off-target interaction of this compound. However, it does not require ATF6, IRE1α, and PERK pathways of UPR signaling. Intriguingly, thapsigargin can activate STING even when canonical loss-of-function mutations are present, such as Goldenticket (I200N), 2'3'-cGAMP binding site (R238A/Y240A), or S366 phosphorylation, however TBK1-STING interaction is required for Type I Interferon induction. The observed activation was not exclusive of thapsigargin treatment, being able to be reproduced with a combination of Ca2+ influx and ER stress inducers. Investigating the mechanisms of non-canonical STING activation is crucial for advancing our understanding of innate immunity and developing novel therapeutic approaches.