ATR inhibition potentiates ionizing radiation‐induced interferon response via cytosolic nucleic acid‐sensing pathways
2020; Springer Nature; Volume: 39; Issue: 14 Linguagem: Inglês
10.15252/embj.2019104036
ISSN1460-2075
AutoresXu Feng, Anthony Tubbs, Chunchao Zhang, Mengfan Tang, Sriram Sridharan, Chao Wang, Dadi Jiang, Dan Su, Huimin Zhang, Zhen Chen, Litong Nie, Yun Xiong, Min Huang, André Nussenzweig, Junjie Chen,
Tópico(s)NF-κB Signaling Pathways
ResumoArticle2 June 2020free access Source DataTransparent process ATR inhibition potentiates ionizing radiation-induced interferon response via cytosolic nucleic acid-sensing pathways Xu Feng orcid.org/0000-0003-1603-7070 Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Anthony Tubbs Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD, USA Search for more papers by this author Chunchao Zhang Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Mengfan Tang Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Sriram Sridharan Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD, USA Search for more papers by this author Chao Wang orcid.org/0000-0001-6191-5261 Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Dadi Jiang Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Dan Su orcid.org/0000-0003-1468-3538 Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Huimin Zhang orcid.org/0000-0001-8987-6014 Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Zhen Chen Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Litong Nie Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Yun Xiong Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Min Huang Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author André Nussenzweig orcid.org/0000-0003-0037-7898 Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD, USA Search for more papers by this author Junjie Chen Corresponding Author [email protected] orcid.org/0000-0002-1493-2189 Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Xu Feng orcid.org/0000-0003-1603-7070 Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Anthony Tubbs Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD, USA Search for more papers by this author Chunchao Zhang Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Mengfan Tang Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Sriram Sridharan Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD, USA Search for more papers by this author Chao Wang orcid.org/0000-0001-6191-5261 Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Dadi Jiang Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Dan Su orcid.org/0000-0003-1468-3538 Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Huimin Zhang orcid.org/0000-0001-8987-6014 Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Zhen Chen Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Litong Nie Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Yun Xiong Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Min Huang Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author André Nussenzweig orcid.org/0000-0003-0037-7898 Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD, USA Search for more papers by this author Junjie Chen Corresponding Author [email protected] orcid.org/0000-0002-1493-2189 Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Author Information Xu Feng1, Anthony Tubbs2, Chunchao Zhang1, Mengfan Tang1, Sriram Sridharan2, Chao Wang1, Dadi Jiang3, Dan Su1, Huimin Zhang1, Zhen Chen1, Litong Nie1, Yun Xiong1, Min Huang1, André Nussenzweig2 and Junjie Chen *,1 1Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 2Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD, USA 3Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA *Corresponding author. Tel: +1 713 792 4863; E-mail: [email protected] EMBO J (2020)39:e104036https://doi.org/10.15252/embj.2019104036 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mechanistic understanding of how ionizing radiation induces type I interferon signaling and how to amplify this signaling module should help to maximize the efficacy of radiotherapy. In the current study, we report that inhibitors of the DNA damage response kinase ATR can significantly potentiate ionizing radiation-induced innate immune responses. Using a series of mammalian knockout cell lines, we demonstrate that, surprisingly, both the cGAS/STING-dependent DNA-sensing pathway and the MAVS-dependent RNA-sensing pathway are responsible for type I interferon signaling induced by ionizing radiation in the presence or absence of ATR inhibitors. The relative contributions of these two pathways in type I interferon signaling depend on cell type and/or genetic background. We propose that DNA damage-elicited double-strand DNA breaks releases DNA fragments, which may either activate the cGAS/STING-dependent pathway or—especially in the case of AT-rich DNA sequences—be transcribed and initiate MAVS-dependent RNA sensing and signaling. Together, our results suggest the involvement of two distinct pathways in type I interferon signaling upon DNA damage. Moreover, radiation plus ATR inhibition may be a promising new combination therapy against cancer. Synopsis Type I interferon signaling plays key roles in cancer radiotherapy with ionizing radiation (IR). Here, exploration of combinatory effects of IR and DNA damage kinase inhibitors reveals surprising involvement of distinct cytosolic nucleic acid-sensing pathways in interferon response induction. Inhibition of DNA damage response kinase ATR (ATRi) significantly potentiates IR-induced type I interferon response in multiple human and murine cancer cells. MAVS-dependent RNA sensing pathway is indispensable for interferon signaling induced by combined IR+ATRi in some human cells. Both cGAS/STING-dependent cytosolic DNA- and MAVS-dependent cytosolic RNA-sensing pathways contribute to IR+ATRi-induced interferon signaling to varying extent in different cell lines. DNA damage-elicited AT-rich DNA mediates type I interferon signaling in a subset of human cells. Introduction Radiotherapy is widely used for cancer treatment owing to its ability to damage DNA and lead to cell death and/or senescence (Jonathan et al, 1999; Sharma et al, 2016). ATR (ataxia-telangiectasia-mutated (ATM) and Rad3-related protein kinase) is a critical regulator of cellular DNA damage response (Marechal & Zou, 2013) and has become an attractive therapeutic target (Karnitz & Zou, 2015; Brown et al, 2017). AZD6738, a highly selective ATR inhibitor (ATRi) (Charrier et al, 2011), has been shown to enhance radiotherapy in a phase I clinical trial (Karnitz & Zou, 2015). However, besides the G2/M checkpoint bypass, other cellular processes influenced by ATRi, especially when combined with ionizing radiation (IR), remain poorly understood. It is well established that the cyclic GMP-AMP synthase (cGAS)/stimulator of IFN genes (STING) pathway is the major pathway responsible for sensing pathogenic double-strand DNA (dsDNA) and the melanoma differentiation-associated gene 5 (MDA5)/retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral-signaling protein (MAVS) pathway is responsible for sensing pathogenic RNA (Wu & Chen, 2014). Once activated by cytosolic DNA, cGAS will catalyze the production of cyclic GMP-AMP (cGAMP), which functions as a second messenger to activate downstream adaptor protein STING. Cytosolic RNA, once recognized by RIG-I/MDA5, will induce a structural rearrangement to liberate their CARD domains to form oligomers; this oligomerized CARD domains of RIG-I/MDA5 interact with and promote the polymerization of MAVS CARD, which turns on the downstream signals. These two pathways share the same downstream signaling cascade: Both MAVS and STING can recruit TANK binding kinase-1 (TBK1) and inhibitor of kappa B kinase-ɛ (IKKɛ) to phosphorylate the transcription factor IRF3 and promote IRF3 dimerization and accumulation in the nucleus, where it can function with IRF7 and NF-κB to initiate the transcription of type I interferons (IFNs) and proinflammatory cytokines (Wu & Chen, 2014). Type I IFNs can be secreted, which induces the expression of IFN-stimulated genes against pathogen infection via IFNAR/JAK/STAT signaling in an autocrine or paracrine manner (Ivashkiv & Donlin, 2014). Recent studies demonstrated that cytosolic nucleic acid-sensing pathways not only mediate protective immune defenses against pathogen infection but also potentiate efficient antitumor immune responses. Loss of antitumor T-cell responses and tumor rejection were observed in STING−/− or IRF3−/− mouse models burdened with transplanted tumors (Woo et al, 2014). Similarly, the antitumor effect of radiation was greatly attenuated in IFNAR−/− mice or in the absence of a host STING pathway (Deng et al, 2014). These results strongly suggest that the cytosolic DNA-sensing pathway in the host (mouse) plays a very important role in antitumor immunity. Meanwhile, several studies proposed that inhibiting chromatin regulators, such as DNA methyltransferase or lysine-specific histone demethylase 1 (LSD1), can trigger cytosolic sensing of double-strand RNA (dsRNA) and induce type I IFN response in cancer cells. This in turn stimulates antitumor T-cell immunity and restrains tumor growth, especially when combined with immune checkpoint blockade (Chiappinelli et al, 2015; Roulois et al, 2015; Sheng et al, 2018). These data indicate that the intrinsic RNA-sensing pathway in tumor cells is also crucial for antitumor immunity in some circumstances. Type I IFNs induced by IR are well documented, and type I IFN-dependent innate and adaptive immunity is essential for the efficacy of radiotherapy (Burnette et al, 2011). However, it remains unclear how radiation induces cell-intrinsic type I IFN signaling and which cytosolic nucleic acid-sensing pathways are involved in this process. In the current study, we sought to address this gap in knowledge by examining the pathways involved in IR-induced type I IFN signaling to determine how best to maximize the efficacy of radiotherapy. Results ATR inhibition significantly potentiates IR-induced inflammatory signals To achieve a comprehensive understanding of cellular responses following ATRi therapy in combination with radiotherapy, we performed total RNA sequencing (RNA-seq) using MCF10A cells treated with DMSO, IR, the ATRi AZD6738, or AZD6738 + IR. Hierarchical clustering analysis of mRNA data separated them into three clusters based on 3,463 differentially expressed genes (Fig 1A). Compared with DMSO, treatment with AZD6738 alone did not lead to any significant change in mRNA expression (Fig 1A). However, cluster 1, comprising the commonly upregulated genes in the IR and AZD6738 + IR groups, was mainly enriched in pathways including antimicrobial peptides, metal sequestration by antimicrobial proteins, and the innate immune system (Fig 1A and B). Cluster 3, comprising the commonly downregulated genes shared by the IR and AZD6738 + IR groups, was enriched in pathways involved in the cell cycle and M phase (Fig 1A and B). These data are consistent with the well-known function of ATR in DNA damage-induced G2/M checkpoint control. However, cluster 2, comprising the genes slightly upregulated in the IR group and possibly further amplified in the AZD6738 + IR group, was mainly enriched in the IFN and cytokine signaling pathways (Fig 1A and B), suggesting that ATR inhibition greatly enhances IFN response when combined with IR. Figure 1. ATR inhibition significantly potentiates ionizing radiation (IR)-induced inflammatory signals Hierarchical clustering analysis of RNA sequencing data from cells treated with DMSO, 20 Gy IR, 250 nM AZD6738, or AZD6738 + IR (n = 2 biological replicates). Top enriched pathway analysis of differentially expressed genes in each cluster as shown in A. Heat map of 33 representative inflammatory genes regulated by AZD6738 + IR. MCF10A WT cells were pretreated with DMSO or AZD6738 (250 nM) for 2 h, and then irradiated with 20 Gy, cells were harvested at indicated time points, and immunoblotting was performed with indicated antibodies. h, hours; pSTAT1, pSTAT1(Y701); pIRF3, pIRF3(S396); pTBK1, pTBK1(S172). MCF10A WT cells were pretreated with DMSO or AZD6738 (250 nM) for 2 h, and then irradiated with indicated dose. 72 h later, cells were harvested for immunoblotting with indicated antibodies. Same condition as E except the irradiation dose with 20 Gy, mRNA levels of inflammatory genes as indicated were assessed by real-time quantitative PCR, presented as mean ± standard error of the mean of three biological replicates. Download figure Download PowerPoint We next examined the effect of AZD6738 on IR-induced inflammatory signaling. As shown in Fig 1C, treatment with AZD6738 significantly enhanced the expression of all 33 classic inflammatory genes. Using STAT1 phosphorylation at Y701 (pSTAT1) as a surrogate marker for type I IFN signaling (Harding et al, 2017), we confirmed that AZD6738 + IR induced STAT1 activation in a time- and IR dose-dependent manner (Fig 1D and E). STAT1 activation following IR alone or AZD6738 + IR normally happened after 2 days and when the IR dose was higher than 2 Gy. This activation was not only shown by pSTAT1 level but also shown by STAT1 protein level (Fig 1D and E), which is consistent with a previous study showing that prolonged IFN signal stimulation could greatly increase the protein expression of STAT1 in certain cell types (Cheon et al, 2013). The addition of type I IFN-neutralizing antibody remarkably diminished pSTAT1 signals induced by AZD6738 + IR (Appendix Fig S1A), suggesting that pSTAT1 is a reliable readout for type I IFN signaling. Please note that STAT1 protein level positively correlates with pSTAT1 level, especially under prolonged treatment conditions, which is the consequence of a positive feedback loop resulting in STAT1 upregulation. In addition, VE-822, another highly specific ATRi, also significantly enhanced IR-induced STAT1 activation (Appendix Fig S1B). Moreover, inhibition of CHK1, the direct downstream target of ATR, via its specific inhibitors LY2603618 or AZD7762, also potentiated IR-induced STAT1 activation (Appendix Fig S1C and D). Real-time PCR results showed that the mRNA levels of multiple inflammatory genes, such as IFNB1, ISG54, CCL5, and IL6, were robustly induced by AZD6738 + IR compared with IR alone (Fig 1F), which is consistent with the RNA-seq data (Fig 1C). Moreover, this synergistic effect was observed not only in MCF10A cells but also in another human mammary epithelial cell line (HMLE), breast cancer cell line T-47D, and colon cancer cell line HCT 116 (Appendix Fig S1E–H). Taken together, these data indicate that inhibition of the ATR-CHK1 axis robustly synergizes IR-induced type I IFN signaling. G2/M DNA damage checkpoint abrogation is required for IFN signaling induced by ATRi + IR Cell cycle progression through mitosis has been proposed as essential step in IR-induced type I IFN signaling (Harding et al, 2017). To examine the role of cell cycle progression in type I IFN signaling induced by AZD6738 + IR, we treated cells with the CDK1 inhibitor RO-3306 or the CDK4/6 inhibitor PD-0332991, which can induce cell cycle arrest in the late G2 phase or G1 phase, respectively (Appendix Fig S2). Both inhibitors significantly impaired STAT1 activation induced by AZD6738 + IR (Fig 2A and B), indicating that active cell cycle progression is required for this process. Figure 2. G2/M DNA damage checkpoint abrogation is required for interferon signaling induced by ATR inhibition + ionizing radiation (IR) MCF10A WT cells were pretreated with AZD6738 (250 nM) and RO-3306 (9 μM) for 2 h, and then irradiated with 20 Gy; 3 days later, cells were harvested for immunoblotting with indicated antibodies. Same condition as described in A, except that RO-3306 is replaced by PD-0332991 (1 μM). MCF10A WT cells were pretreated with DMSO or AZD6738 (250 nM) for 2 h, and then irradiated with 20 Gy; 24 h later, cell cycle distribution was measured by flow cytometry. EdU (10 μM) was added into medium 2 h before harvesting for flow cytometry. MCF10A WT cells were pretreated with DMSO or AZD6738 (250 nM) for 2 h, and then irradiated with 20 Gy, and cells were harvested at indicated time points for immunoblotting. MCF10A control cells and CDC25C-WT/constitutively active mutant CDC25C-S216A overexpressed cells were pretreated with DMSO or AZD6738 (250 nM) for 2 h, and then irradiated with 20 Gy; 24 h later, cells were harvested for immunoblotting with indicated antibodies. Same condition as indicated in E, except that cells were harvested 72 h later. Download figure Download PowerPoint The ATR/CHK1/CDC25C-dependent G2/M DNA damage checkpoint is known to often become activated after DNA damage to block the cell cycle at the G2 phase, allowing cells to repair damaged DNA (Liu et al, 2000). Abrogation of G2/M DNA damage checkpoint by AZD6738 allowed the cells to bypass IR-induced G2 arrest, pushing more cells into mitosis and the following G1 phase, as shown by FACS and immunoblotting probed with antibodies of phos-H3 (S10) and phos-CDC2 (Y15) (Fig 2C and D), which are mitosis markers. Consistently, overexpression of wild-type (WT) CDC25C or constitutively active mutant CDC25C-S216A (Peng et al, 1997) maintained CDK1 activity even after exposure to IR (Fig 2E). As a result, overexpression of these proteins also significantly enhanced IR-induced STAT1 activation, to a level similar to that of treatment with AZD6738 (Fig 2F). These data indicate that abrogation of the ATR/CHK1/CDC25C-dependent G2/M DNA damage checkpoint is required for type I IFN signaling induced by ATRi + IR. The cGAS/STING-dependent cytosolic dsDNA-sensing pathway is dispensable for type I IFN signaling induced by ATRi + IR in MCF10A cells Previous studies showed that micronuclei formation and micronuclei disruption are well correlated with DNA damage-induced STAT1 activation (Harding et al, 2017; Mackenzie et al, 2017). Those studies proposed that the dsDNA sensor cGAS could enter micronuclei after the rupture of micronuclei envelopes, sense dsDNA, and then initiate inflammatory signaling through the production of second messenger cGAMP to activate STING and downstream events (Harding et al, 2017; Mackenzie et al, 2017). We therefore generated cGAS and STING knockout (KO) clones in MCF10A cells. As anticipated, cGAMP stimulation could induce STING and IRF3 phosphorylation and STAT1 activation in WT or cGAS KO cells, but not in STING KO cells (Fig 3A). Furthermore, cGAS or STING KO could block herring testis DNA (HT-DNA)-induced IRF3 phosphorylation and STAT1 activation (Fig 3B). These data demonstrated that the cGAS/STING pathway is functional in MCF10A cells and is abolished in our KO cells. Figure 3. The cGAS/STING-dependent cytosolic dsDNA-sensing pathway is dispensable for interferon (IFN) signaling induced by ATR inhibition + ionizing radiation (IR) A. MCF10A wild-type (WT), cGAS knockout (KO), and STING KO cells were treated with cGAMP at indicated concentrations for 4 h, and cells were harvested for immunoblotting. B. MCF10A WT, cGAS KO, and STING KO cells were transfected with herring testis DNA (HT-DNA, 2 μg/ml) for 6 h with Lipofectamine 3000. Immunoblotting was performed with indicated antibodies. C–F. (C, D, and F) MCF10A WT or two independent clones for cGAS, STING, and IRF3 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 h, and then irradiated with 20 Gy; 3 days later, cells were harvested for immunoblotting with indicated antibodies. cGAS, STING, and IRF3 KO were verified by immunoblotting and Sanger DNA sequencing (Appendix Fig S7). (E) Same condition as described in C, except that cells were harvested for real-time quantitative PCR. IFNB1 mRNA level was measured and statistical analyzed by two-way ANOVA (*** means P < 0.001). Data are presented as mean ± standard error of the mean of three biological replicates. Download figure Download PowerPoint However, cGAS or STING KO showed only modest or undetectable effects on STAT1 activation induced by IR alone or ATRi + IR (Fig 3C and D, Appendix Fig S3A). Consistently, IFN-β induction was statistically significantly, but very slightly decreased in cGAS or STING KO cells upon treatment with ATRi + IR (Fig 3E). In addition, knockdown of STING did not interfere with IR-induced STAT1 activation in HCT 116 cells (Appendix Fig S3B), which are already defective in cGAS expression and IFN stimulatory DNA (ISD) sensing (Appendix Fig S3C and D). As mentioned above, ATRi significantly enhanced IR-induced STAT1 activation in T-47D cells, which are reported to be defective in both cGAS expression and STING expression (Zierhut et al, 2019). Furthermore, overexpression of the DNA exonuclease TREX1, which degrades cytosolic dsDNA (Grieves et al, 2015; Vanpouille-Box et al, 2017), could not abolish STAT1 activation induced by ATRi + IR (Appendix Fig S3E). Please note that one needs to be careful when interpreting results from these overexpression experiments, since it is possible that overexpressed TREX1 may not have access and/or ability to degrade all types of DNA fragments. Taken together, these data strongly suggest that the cGAS/STING-dependent cytosolic dsDNA-sensing pathway plays a very minor role in type I IFN signaling induced by IR alone or ATRi + IR in some human cells, e.g., MCF10A, HCT 116, and T-47D cells. However, IRF3 KO blocked IFN-β expression and STAT1 activation induced by ATRi + IR (Fig 3E and F), indicating that other pathways, and not the cGAS/STING pathway, are mainly responsible for type I IFN signaling induced by ATRi + IR, which is mediated by IRF3 activation. MAVS-dependent cytosolic RNA-sensing pathway is indispensable for IFN signaling induced by ATRi + IR in MCF10A cells As mentioned above, IRF3 activation is crucial for ATRi plus IR-induced type I IFN signaling. Apart from the cGAS/STING-dependent cytosolic dsDNA-sensing pathway, the RIG-I/MDA5/MAVS-dependent cytosolic RNA-sensing pathway is also important for IRF3 activation, upon sensing of cytosolic immunogenic RNAs by RIG-I or MDA5 (Wu & Chen, 2014). To test whether the cytosolic RNA-sensing pathway is involved in STAT1 activation induced by ATRi + IR, we generated KO cells for MAVS and its upstream RNA sensors RIG-I and MDA5 and verified the KO using RNA Sendai virus (SeV) infection. Consistent with previous studies (Yoneyama et al, 2004; Desai et al, 2005; Kawai et al, 2005; Meylan et al, 2005; Seth et al, 2005; Xu et al, 2005), SeV-induced IRF3 phosphorylation and STAT1 activation were significantly abrogated in RIG-I KO, MAVS KO, and IRF3 KO cells, but not in cGAS KO, STING KO, or MDA5 KO cells (Appendix Fig S4A). MAVS KO nearly completely blocked STAT1 activation induced by IR alone or ATRi + IR (Fig 4A), as well as IFN-β expression (Fig 4B). In addition, IRF3 activation, IFN-β induction, and STAT1 activation were also dramatically suppressed in RIG-I or MDA5 KO cells, but not in unrelated CDK5 KO cells (Fig 4B–E, Appendix Fig S4B and C). Meanwhile, TBK1 activation or RIG-I upregulation induced by ATRi + IR was also abrogated in MDA5 KO cells (Fig 4D). However, MDA5 is generally believed to be activated by long dsRNA, whereas RIG-I is believed to be activated by short dsRNA and 5′-ppp single-strand RNA (Wu & Chen, 2014). Thus, it is hard to imagine such robust change in single MDA5 or RIG-I KO cells. It is possible but unlikely that one type of specific RNA needs both MDA5 and RIG-I at the same time to initiate type I IFN signaling. Given that MDA5 and RIG-I are well known IFN-stimulated genes (ISGs), they are significantly induced after ATRi plus IR treatment (Fig 4C and D). We speculate that MDA5/RIG-I upregulation could further amplify type I IFN signaling elicited by ATRi + IR, which may be the reason that even single KO of MDA5 or RIG-I could show robust change in type I IFN signaling. To further explore this positive feedback regulation on type I IFN signaling elicited by ATRi + IR, we then knocked out another transcription factor IRF7 (a classical ISG), which function with IRF3 to modulate type I IFN expression. As anticipated, STAT1 activation was significantly diminished in IRF7 KO cells (Fig 4F). However, attenuated STAT1 activation and MDA5 induction were not as evident in TBK1 KO cells as in MAVS KO cells (Appendix Fig S4D), potentially owing to redundant functions between TBK1 and IKKɛ (Wu & Chen, 2014). In addition, overexpression of dsRNA-specific endoribonuclease RNase III derived from E. coli drastically impaired STAT1 activation induced by ATRi + IR (Appendix Fig S4E). Consistently, knockdown of MAVS or RIG-I also impaired IR-induced STAT1 activation in HCT 116 cells (Appendix Fig S3B). Figure 4. The MAVS-dependent cytosolic RNA-sensing pathway is indispensable for interferon (IFN) signaling induced by ATR inhibition + ionizing radiation (IR) A–F. (A, C, D, and F) MCF10A WT or two independent clones for MAVS, RIG-I, MDA5, and IRF7 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 h, and then irradiated with 20 Gy; 3 days later, cells were harvested for immunoblotting with indicated antibodies. MAVS, RIG-I, MDA5, and IRF7 KO were verified by immunoblotting and Sanger DNA sequencing (Appendix Fig S7). (B and E) Same condition as described in A, except that cells were harvested for real-time quantitative PCR instead of immunoblotting. IFNB1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean ± standard error of the mean of three biological replicates. Source data are available online for this figure. Source Data for Figure 4 [embj2019104036-sup-0002-SDataFig4.pdf] Download figure Download PowerPoint Taken together, these data indicate that the cytosolic RNA-sensing pathway and positive feedback loop are required for optimized type I IFN signaling induced by ATRi + IR at least in MCF10A and HCT 116 cells. Cytosolic nucleic acid-sensing pathways involved in IR-induced type I IFN signaling differ between cell lines We sought to study the efficacy of ATRi + IR in tumor control in a syngeneic mouse tumor model. To this end, we chose a murine mammary carcinoma cell line, 4T1, and checked the synergistic effect of ATRi on IR-induced IFN signaling in these cells. As shown in Fig 5A, ATRi significantly enhanced IR-induced IFN-β expression in a dose-dependent manner. Figure 5. Cytosolic nucleic acid-sensing pathways involved in ionizing radiation (IR)-induced type I interferon (IFN) signaling differ in murine and human cells 4T1 cells were pretreated with AZD6738 at indicated concentrations for 2 h, and then irradiated with 10 Gy. 4 days later, Ifnb1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean ± standard error of the mean of three biological replicates. Same condition as described in A. Ifnb1 mRNA levels were measured by real-time quantitative PCR in WT, Sting KO, Mavs KO, and Irf3 KO 4T1 cells. Data are presented as mean ± standard error of the mean of three biological replicates. All KO cells were verified by immunoblotting and Sanger DNA sequencing (Appendix Fig S7). MC38 cells were pretreated with AZD6738 at indicated concentrations for 2 h, and then irradiated with 20 Gy. 3 days later, Ifnb1 mRNA level wa
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