The Impact of Radiation-Induced DNA Damage on cGAS-STING-Mediated Immune Responses to Cancer

Abstract

Radiotherapy is a major modality used to combat a wide range of cancers. Classical radiobiology principles categorize ionizing radiation (IR) as a direct cytocidal therapeutic agent against cancer; however, there is an emerging appreciation for additional antitumor immune responses generated by this modality. A more nuanced understanding of the immunological pathways induced by radiation could inform optimal therapeutic combinations to harness radiation-induced antitumor immunity and improve treatment outcomes of cancers refractory to current radiotherapy regimens. Here, we summarize how radiation-induced DNA damage leads to the activation of a cytosolic DNA sensing pathway mediated by cyclic GMP-AMP (cGAMP) synthase (cGAS) and stimulator of interferon genes (STING). The activation of cGAS-STING initiates innate immune signaling that facilitates adaptive immune responses to destroy cancer. In this way, cGAS-STING signaling bridges the DNA damaging capacity of IR with the activation of CD8+ cytotoxic T cell-mediated destruction of cancer-highlighting a molecular pathway radiotherapy can exploit to induce antitumor immune responses. In the context of radiotherapy, we further report on factors that enhance or inhibit cGAS-STING signaling, deleterious effects associated with cGAS-STING activation, and promising therapeutic candidates being investigated in combination with IR to bolster immune activation through engaging STING-signaling. A clearer understanding of how IR activates cGAS-STING signaling will inform immune-based treatment strategies to maximize the antitumor efficacy of radiotherapy, improving therapeutic outcomes.

Keywords: DNA damage; antitumor immunity; cGAS–STING signaling; cancer; nucleic acid sensing; radiation; type I interferon.

Figure 1

Radiation-induced DNA damage activates cGAS–STING signaling, type I interferon production, and immune activation within the tumor microenvironment. Radiation-induced DNA damage leads to accumulation of cytosolic dsDNA within irradiated tumor cells. Micronuclei, CCFs, and mtDNA have been reported as sources of cytosolic dsDNA. cGAS detects cytosolic dsDNA and produces cGAMP, a secondary messenger molecule formed by phosphodiester linkages between ATP and GTP molecules. cGAMP binding of STING induces conformational changes that enable the recruitment of TBK1, which then phosphorylates STING. Phosphorylated STING recruits IRF3, which is next phosphorylated by TBK1. Phosphorylated IRF3 dimerizes and translocates to the nucleus where it functions as a transcription factor for the expression of type I IFNs. Following irradiation of tumor cells, DCs acquire tumor-derived dsDNA within the cytosol via internalizing tumor-derived exosomes or engulfment of irradiated tumor cells. Furthermore, cGAMP produced within irradiated tumor cells is transferred to the cytosol of DCs via gap junctions. Tumor-derived dsDNA or cGAMP activates cGAS–STING signaling and subsequent production type I IFNs within DCs as described above. DCs produce greater amounts type I IFNs compared to other cellular compartments in the TME. Type I IFNs are secreted and act on IFNAR receptors initiating a variety of responses including induction of IFN-stimulated genes, DC activation and maturation, as well as CD8+ T cell activation. Activated DCs cross-present TA on MHC I molecules which is recognized by tumor-reactive CD8+ T cells via their TCR. Activated CD8+ cytotoxic T cells recognize TA–MHC I complexes presented on the surface of target tumor cells and induce apoptosis through the release of perforin and granzyme B, or death receptor signaling involving FasL/FasR. Treatment with IR leads to the accumulation of TA-specific CD8+ T cells, specialized cross-presenting DCs, and type I IFNs within the TME—all of which are paramount for optimal antitumor immune responses induced by radiotherapy. Abbreviations: cytoplasmic chromatin fragments (CCFs); dendritic cells (DCs); double-stranded DNA (dsDNA); interferon (IFN); IFN-α/β-receptor (IFNAR); ionizing radiation (IR); mitochondrial DNA (mtDNA); T cell receptor (TCR); tumor-antigen (TA); tumor microenvironment (TME).

Figure 2

Radiation-induced MAVS signaling leads to the production of type I interferons in cancer cells. Following IR treatments, cytosolic RNA species have been reported to activate MAVS signaling and subsequent production of type I IFNs. MDA5 and RIG-I are cytosolic RNA sensors upstream of the adaptor protein, MAVS. Upon detection of cytosolic RNA, MDA5 and/or RIG-I initiate MAVS-mediated signaling leading to expression of type I IFNs. Specifically, the presence of sncRNAs or ERV-derived dsRNA in the cytoplasm are detected by RIG-I or MDA5, respectively, and initiate MAVS signaling post-IR treatments. Furthermore, AT-rich dsDNA released following irradiation may be transcribed by RNA polymerase III to produce cytosolic dsRNA which activates RIG-I–MAVS signaling. In this way, MAVS signaling plays a role in the production of type I IFNs following radiotherapy, although the precise contribution to overall antitumor immune responses remains to be determined. Abbreviations: double-stranded DNA (dsDNA); double-stranded RNA (dsRNA); endogenous retrovirus (ERV); interferon (IFN); ionizing radiation (IR); small non-coding RNAs (sncRNAs).

Figure 3

Pathways antagonizing radiation-induced cGAS-STING-type I interferon signaling. Several pathways hinder radiation-induced production of type I IFNs via cGAS–STING signaling and downstream antitumor immune responses. Trex1, a DNA exonuclease, degrades cytoplasmic DNA reducing the amount of ligand available for activation of cGAS–STING signaling in irradiated tumor cells. Induction of Trex1 in tumor cells following IR is also associated with reducing dsDNA cargo within exosomes, reducing the amount of substrate available for DC-mediated production of type I IFNs. Caspase 3 and caspase 9 are associated with reducing the production of type I IFNs of irradiated cancer cells and blunting the efficacy of IR in preclinical models. Caspase 3 is suggested to reduce production of type I IFN by facilitating cellular breakdown of irradiated cells. Caspase 9 is implicated in suppression of radiation-induced cGAS-STING signaling, however the exact mechanisms at play are not yet elucidated. Lastly, the efficacy of IR is reduced by non-canonical NF-κB signaling (involving the p52/RelB NF-κB complex) within DCs. Non-canonical NF-κB signaling inhibits RelA binding to the Ifnb promoter in DCs to regulate expression of type I IFN. Abbreviations: dendritic cells (DCs); double-stranded DNA (dsDNA); interferon (IFN); IFN-α/β-receptor (IFNAR); ionizing radiation (IR).