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 5

AOA prolongs IRF3 phosphorylation via the ZBP1–RIPK1/3 axis.

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AOA prolongs IRF3 phosphorylation via the ZBP1–RIPK1/3 axis. (A) L929 ce...
(A) L929 cells were treated with 0.5 mM AOA for 1 hour followed by HT-DNA stimulation in the absence or presence of aspartate, and then cells were harvested for qPCR analysis of Zbp1 gene expression (top) or Western blot for detecting p-IRF3 levels. (B) qPCR analysis of Scr and Ripk1- or Ripk3-knockdown L929 cells with indicated treatment. (C) Western blot of Scr and Ripk1- or Ripk3-knockdown L929 cells with indicated treatment. (D) Endogenous co-IP in L929 cells with indicated treatment. (E) An in vitro kinase assay was performed by purified indicated proteins from L929 cells, revealing that RIPK1/3 directly phosphorylated S396 residues of IRF3. (F–H) Schematic representation of mouse IRF3 (F), mouse RIPK1 (G), mouse RIPK3 (H) full-length and truncations (top). Co-IP showing interactions between mouse IRF3 (F) and mouse RIPK1 (G) or mouse RIPK3 (H) full-length and truncations in L929 cells upon HT-DNA+AOA stimulation (bottom). (I) The proposed model: aspartate deficiency–induced mtDNA release initiates the ZBP1–RIPK1/RIPK3 module to prolong IRF3 phosphorylation at Ser396. Statistical analysis was performed by 1-way ANOVA (A) or 2-way ANOVA followed by Tukey’s test (B). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.