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 4

Aspartate deficiency induces mtDNA DSBs, leading to its release into the cytoplasm.

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Aspartate deficiency induces mtDNA DSBs, leading to its release into the...
(A) Schematic showing that aspartate-mediated pyrimidine nucleotide synthesis is essential for intracellular DNA homeostasis. (B) Heatmap from RNA-seq revealing the expression of DNA repair enzymes in L929 cells with indicated treatment. The fold-change of expression was normalized to the mock group (n = 3 independent cultures). (C) Genome browser screenshots of END-seq on mitochondria from L929 cells with indicated treatment. The expanded view of the indicated region shows more detailed genomic features. (D) The proportion of END-seq reads in mitochondrial and nuclear chromosomal regions. (E) Representative immunofluorescence images of mitochondrial (TOM20, red) or dsDNA (green) in BJ-5ta cells treated with AOA in the absence or presence of 20 mM aspartate for 6 hours. Scale bars, 10 μm. Lower panel: quantification of cytosolic dsDNA in BJ-5ta cells with indicated treatment (n = 39–51 fields per group from 3 biological replicates). (F) Representative immunofluorescence images of BJ-5ta cells treated with ddC (100 μM) for 72 hours, followed by 6 hours of AOA treatment in the absence or presence of 20 mM aspartate. (G) 2D–structured illumination microscopy imaging of mtDNA externalization under the indicated conditions. (H) Western blot detected α-tubulin (cytosol), TOM20 (OMM), PDH (Matrix), COX IV (IMM), and histone H3 (nuclei) to validate protocol from Supplemental Figure 7A in L929 cells treated as indicated. wcl, whole cell lysate; pel, pellet; cyt, cytosolic fraction. (I) L929 cells were treated with AOA in the absence or presence of 20 mM aspartate followed by HT-DNA transfection, and then cells were harvested for qPCR analysis of mtDNA or nDNA levels in cytosolic fractions. Data are represented as means ± SEM. Representative data are shown from 2 or 3 independent experiments. Statistical analysis was performed by 1-way ANOVA followed by Tukey’s test (E and I). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. DSBs, double-strand breaks.