The presence of double-stranded DNA (dsDNA) in the cytosol is a danger signal associated with microbial infections and tumor microenvironment. The cytosolic DNA surveillance pathway (CDSP) is critical for sensing such signal to elicit type I interferon response, which facilitates infection clearance and tumor rejection. Uncontrolled engagement of CDSP is also linked to autoimmune disease. The key components of CDSP are the DNA sensor—cyclic GMP-AMP synthase (cGAS), the second messenger—cyclic GMP-AMP (cGAMP), and the endoplasmic reticulum localized adaptor—stimulator of interferon inducible genes (STING). While numerous efforts, including ours, are focusing on developing STING agonists formulated cancer vaccine, an analytical method that is sensitive and reliable for cGAMP and other cyclic dinucleotides is also in high demand to enable quantitative investigations. In this work, we developed and optimized a rapid quantification workflow that integrates solid phase extraction, hydrophilic liquid chromatography and multiple reaction monitoring mass spectrometry. This workflow enables detection of cGAMP in cells at sub-femtomole levels and is amenable for scaling up for high throughput analysis. A biochemically synthesized nonradioactive isotope labeled cGAMP standard enables absolute quantification. As a proof of principle, this method is first applied to probe cGAMP in macrophages infected with Mycobacterium tuberculosis, and to help establish cGAS as the innate immune sensor for the pandemic pathogen. Second, constitutive levels of cGAMP in organs of autoimmune mouse models, of Trex1 or Dnase2 deficiencies, are quantified. The result indicates a direct engagement of the cGAS-cGAMP-STING pathway in the autoimmune pathology. Finally, we show that cGAMP is produced when the apoptosis program is initiated in conjunction with pro-apoptotic caspase inhibition. This shows that mitochondria have the capacity to simultaneously expose a cell-intrinsic inducer of the IFN response and to inactivate this response in a caspase-dependent manner. Altogether, these studies demonstrated the usefulness of this analytical method for quantifying cGAMP in a high dynamic range under different biological contexts. We are currently applying this method to measure the activation of the cGAS pathway in tumor immunity and immunotherapy.
Citation Format: Tuo Li, Zhijian J. Chen. Developing an analytical method for second messenger cyclic GMP-AMP. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B003.
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