Abstract

Immunotherapies targeting adaptive immune response have demonstrated unprecedented clinical efficacy in cancer treatment, especially with immune checkpoint inhibitors blocking CTLA4 or PD1. Now new drugs, which act on the innate immune system to boost the effects of the adaptive system, are being developed. In this area, the cGAS–STING pathway has been proposed to enhance anti tumor immunogenicity. We know that Tumor-derived DNA activates the cyclic GMP-AMP synthase (cGAS). This leads to the production of 2’3’ cGAMP, a cyclic dinucleotide (CDN), which then binds to STING proteins. Phosphorylated STING next interacts with TBK1, leading to the recruitment and activation of the IRF3 dimer. Nuclear translocation of IRF3 leads to the transcription of genes encoding IFN-a/b. In addition, the STING pathway controls NF-kB dependent inflammatory cytokine expression. As a negative feedback loop, the DNA-stimulated cGAS-STING-TBK1 pathway also triggers STING protein degradation through a p62 SQSTM1 associated autophagy process.

Here we provide a detailed pharmacological evaluation of the well-known STING binders 2’3’cGAMP, c-diGMP, and c-diAMP, using the new HTRF human STING biochemical assay. We also demonstrate that the new HTRF phospho and total STING assays, along with the phospho-TBK1 and the phospho-IRF3 assays, enable an in-depth understanding of STING signaling. Finally, we show a dose-dependent IFNb secretion upon cGAMP-induced STING activation using the HTRF IFNb assay.