Aspartate deficiency amplifies cGAS-STING signaling in antitumor immunity
Yuheng Liao, Hanze Wang, Hengxin Liu, Xi Chen, Renqiang Sun, Xie Li, Zhen Yang, Chenying Liu, Wei Wu, Ziqian He, Yuzheng Zhao, Ying Mao, Dan Ye, Hui Yang
Yuheng Liao, Hanze Wang, Hengxin Liu, Xi Chen, Renqiang Sun, Xie Li, Zhen Yang, Chenying Liu, Wei Wu, Ziqian He, Yuzheng Zhao, Ying Mao, Dan Ye, Hui Yang
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Research Article Metabolism Oncology

Aspartate deficiency amplifies cGAS-STING signaling in antitumor immunity

Abstract

Metabolic signals critically shape innate immune responses. Through pharmacological screening of metabolic pathways, we identified aspartate metabolism as a key regulator of cyclic GMP-AMP synthase (cGAS)–stimulator of interferon genes (STING) signaling. Genetically or aminooxyacetic acid–mediated (AOA-mediated) pharmacologically reducing aspartate levels markedly potentiated the cGAS-STING pathway, leading to stronger upregulation of type I interferons and interferon-stimulated genes. Mechanistically, disruption of de novo pyrimidine synthesis, a major downstream pathway of aspartate, induced mtDNA replication stress and increased mtDNA double-strand breaks, promoting mtDNA release into the cytosol. Cytosolic mtDNA synergized with cGAS-STING agonists to upregulate Z-DNA binding protein 1 (ZBP1), which recruits RIPK1/3 to sustain IRF3 phosphorylation, forming a positive feedback loop that amplifies innate immune signaling. In immunocompetent mouse models, AOA enhanced the antitumor efficacy of STING agonists, chemotherapy, or radiotherapy, whereas aspartate supplementation abrogated these effects. Consistently, aspartate levels negatively correlated with antitumor immunity in colorectal cancer patient samples. Together, our study identifies aspartate–pyrimidine metabolism as a critical metabolic checkpoint that licenses STING signaling by enabling mtDNA stress to cooperate with agonist stimulation, driving type I interferon–dependent ZBP1 induction and feed-forward amplification of STING signaling, thus offering a promising strategy to enhance antitumor immunity.

Authors

Yuheng Liao, Hanze Wang, Hengxin Liu, Xi Chen, Renqiang Sun, Xie Li, Zhen Yang, Chenying Liu, Wei Wu, Ziqian He, Yuzheng Zhao, Ying Mao, Dan Ye, Hui Yang

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Figure 3

AOA amplifies IFN response via disrupting aspartate-dependent pyrimidine synthesis.

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AOA amplifies IFN response via disrupting aspartate-dependent pyrimidine...
(A) Schematic illustration of cellular aspartate uptake and catabolism. (B) Schematic showing 13C4-aspartate flux in TCA cycle and asparagine synthesis. (C) L929 cells were treated with or without AOA in medium containing 20 mM 13C4-aspartate. The abundances of metabolites labeled with the stable isotope 13C in cells were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). The heatmap shows the percentage of isotope-labeled metabolite relative to the total content of each metabolite. (D) Mass isotopologue distribution (MID) of cellular metabolites in TCA and amino acids after AOA+13C4-aspartate treatment. (E) Relative abundance of N-carbamoyl-aspartate (n = 3 independent cultures). L929 cells were treated with AOA in the absence or presence of 20 mM aspartate followed by HT-DNA transfection and then collected for LC–MS/MS analysis. (F) Heatmap of log2 (fold-change) indicates nucleotide levels of L929 cells treated as E performed by LC–MS/MS. Heatmap of log2 (fold-change) in the indicated nucleotide levels in treatment groups compared with mock. Each square represents an individual replicate (n = 3 independent cultures). (G) Schematic diagram shows 13C4-aspartate as a tracer to pyrimidines. (H) Pyrimidines from 13C4-aspartate in L929 cells treated with or without AOA followed by HT-DNA transfection (n = 3 independent cultures). (I) Heatmap of log2 (fold-change) of the indicated nucleotide levels in AOA- and AOA+Asp–treated groups compared with the mock group after HT-DNA stimulation in Cad-knockdown L929 cells (n = 3 independent cultures). (J) ISGs expression in Scr and Cad-knockdown L929 cells treated as E. Data are represented as means ± SEM. Representative data are shown from 2 or 3 independent experiments. Statistical analysis was performed by unpaired t test (H) and 1-way ANOVA (E) or 2-way ANOVA followed by Tukey’s test (J). **P < 0.01; ***P < 0.001; ****P < 0.0001.