Human DNA-PK activates a STING-independent DNA sensing pathway

Abstract

Detection of intracellular DNA by the cGAS-STING pathway activates a type I interferon-mediated innate immune response that protects from virus infection and can be harnessed to promote anti-tumor immunity. Whether there are additional DNA sensing pathways, and how such pathways might function, remains controversial. We show here that humans -but not mice -have a second, potent, STINGindependent DNA sensing pathway that is blocked by the E1A viral oncogene of human adenovirus 5.We identify human DNA-PK as the sensor of this pathway and demonstrate that DNA-PK kinase activity drives a robust and broad antiviral response. We discover that the heat shock protein HSPA8/HSC70 is a unique target of DNA-PK. Finally, we demonstrate that detection of foreign DNA and DNA damage trigger distinct modalities of DNA-PK activity. These findings reveal the existence, sensor, unique target, and viral antagonists of a STING-independent DNA sensing pathway (SIDSP) in human cells. IntroductionThe cGAS-STING DNA sensing pathway has emerged as a key component of the innate immune response that is important for antiviral immunity (1), contributes to specific autoimmune diseases (2), and mediates important aspects of antitumor immunity (3). cGAS binds to double-stranded DNA and catalyzes the formation of cyclic GMP-AMP (4, 5), a diffusible cyclic dinucleotide that activates the endoplasmic adaptor protein STING (6). Activated STING then serves as a platform for the inducible recruitment of the TBK1 kinase, which phosphorylates and activates the transcription factor IRF3, leading to the induction of the type I interferon mediated antiviral response (7).Nearly all studies on the cGAS-STING pathway involve the use of mice and mouse cells.Knockouts of cGAS (Mb21d1) and STING (Tmem173) have clearly demonstrated that both are essential for the transcriptional and cell biological responses to foreign intracellular DNA, and that they mediate the pathology of specific autoimmune diseases (2,8,9). However, numerous additional sensors of intracellular DNA have been proposed, all of which are thought to act upstream of STING (10). Whether STING-independent DNA sensing exists is currently unknown.Here, we report the unexpected finding that the E1A oncogene of human adenovirus 5 blocks two distinct DNA sensing pathways in human cells: the well-known cGAS-STING pathway (11), and a second, STING-independent DNA sensing pathway (SIDSP). We identify the DNA damage response protein DNA-PK as the sensor of the SIDSP, along with the heat shock protein HSPA8 as a unique SIDSP target. We show that the SIDSP is potently activated in human and primate cells, but it is weak or absent from mouse cells. Our findings demonstrate that human cells have a second DNA sensing pathway, with implications for host defense, autoimmunity, and anti-tumor immunity. Results Human adenovirus 5 E1A blocks two DNA sensing pathways in human cellsWe previously demonstrated that the viral oncogenes of the DNA tumor viruses are potent antagonists of the cGAS-STING DNA sensing pathway ...

Cell Cycle Checkpoints Cooperate to Suppress DNA- and RNA-Associated Molecular Pattern Recognition and Anti-Tumor Immune Responses

“…In contrast, a low dose of 2-Gy irradiation, which induced less DNA damage and restored micronuclei formation and inflammatory signaling in MCF10A XRCC4 KO cells (Figures S1C and S1D). This observation is consistent with a model in which c-NHEJ deficiency impairs cell cycle progression as a consequence of excessive DNA damage after 10 Gy, rather than through the putative role of c-NHEJ factors (i.e., DNA-PK) in pattern recognition (Burleigh et al, 2020;Ferguson et al, 2012;Zhang et al, 2011). We further analyzed inflammatory gene expression in genetically defined MCF10A cells.…”

“…Loss of c-NHEJ prevented anti-tumor immune re-sponses in syngeneic melanoma models treated with radiotherapy and immune checkpoint blockade. Although c-NHEJ components DNA-PKcs and KU have been reported as DNA sensors for innate immunity (Burleigh et al, 2020;Ferguson et al, 2012;Zhang et al, 2011), we observed similar reductions in cells that have lost end ligation factors XRCC4 and Lig4, (Graham et al, 2016). XRCC4 and LIG4 are not required for DSB end recognition or signaling, making them unlikely candidates for Article ll OPEN ACCESS pattern recognition.…”

Nucleic Acid Immunity and DNA Damage Response: New Friends and Old Foes

“…Interaction between DNA-PKcs and the progenome of the Human Immunodeficiency Virus (HIV) retrovirus has also been shown, although the link to inflammation is unexplored ( 65 ). Interestingly, some DNA viruses have evolved proteins that counteract DNA-PKcs-dependent detection ( 20 ) while others hijack NHEJ to the benefit of their replication ( 66 ), highlighting the tight interplay between viral life cycles and DDR ( 67 ). Yet, there is as of today, limited knowledge concerning the ways in which DNA-PK-dependent inflammatory responses, IFI16- and cGAS-dependent STING activation are orchestrated.…”

“…Indeed, whether DNA-PK requires STING for production of Interferons remains debated ( 18 , 20 , 68 ) ( Figure 1C ). It was reported that DNA-PKcs is recruited to dsDNA in the cytoplasm of both human and murine cells through KU80, triggering IRF3-dependent inflammatory responses ( 18 ).…”

Animal Models for the Study of Nucleic Acid Immunity: Novel Tools and New Perspectives

“…However, recent work has questioned this paradigm. Indeed, it has been reported that the DNA-PK DNA repair pathway can operate in the detection of cytosolic dsDNA in human cells, while such DNA-PKdependent IFN production is not witnessed in murine cells [45]. inflammasome inhibits the cGAS-STING pathway [47].…”