Targeting the N-acetyltransferase 10/DKK2 axis enhances CD8+ T cell antitumor activity in colorectal cancer models
Mengmeng Li, Xiaoya Zhao, Jun Wu, Shimeng Zhou, Yao Fu, Chen Chen, Zhuang Ma, Jiawen Xu, Yun Qian, Zhangding Wang, Bo Wang, Qiang Wang, Qingqing Ding, Changyu Chen, Honggang Wang, Xiaozhong Yang, Weijie Dai, Wenjie Zhang, Shouyu Wang
Mengmeng Li, Xiaoya Zhao, Jun Wu, Shimeng Zhou, Yao Fu, Chen Chen, Zhuang Ma, Jiawen Xu, Yun Qian, Zhangding Wang, Bo Wang, Qiang Wang, Qingqing Ding, Changyu Chen, Honggang Wang, Xiaozhong Yang, Weijie Dai, Wenjie Zhang, Shouyu Wang
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Research Article Gastroenterology Immunology Oncology

Targeting the N-acetyltransferase 10/DKK2 axis enhances CD8+ T cell antitumor activity in colorectal cancer models

Abstract

Despite overexpression of N-acetyltransferase 10 (NAT10) in colorectal cancer (CRC), its immunomodulatory role in the tumor microenvironment remains elusive. Here, we reveal that NAT10 promotes immune evasion through N4-acetylcytosine–dependent (ac4C-dependent) mRNA stabilization. Using syngeneic mouse models (MC38/CT-26), intestinal epithelial-cell specific Nat10 conditional KO (Nat10cKO) mice, patient-derived organoids, and clinical specimens, we show that Nat10 ablation enhanced CD8+ T cell–mediated antitumor immunity. Single-cell RNA-seq revealed increased cytotoxic CD8+ T cell infiltration in Nat10cKO tumors, which was corroborated by the inverse correlation of tumoral NAT10 expression and CD8+ T cell number in clinical specimens. Multi-omics integration analysis identified DKK2 as the predominant NAT10-regulated transcript. NAT10 stabilized DKK2 mRNA via ac4C modification, leading to high expression of the DKK2 protein. Secreted DKK2 engaged LRP6 receptors to activate AKT-mTOR signaling, inducing cholesterol accumulation in CD8+ T cells and impairing their cytotoxicity. Pharmacological NAT10 inhibition (Remodelin treatment) or DKK2 neutralization restored CD8+ T cell function and synergized with anti–PD-1 therapy. Our findings establish the NAT10/DKK2/LRP6/AKT-mTOR/cholesterol axis as a critical regulator of CD8+ T cell dysfunction in CRC, positioning NAT10/DKK2 as a potential target to enhance immunotherapy efficacy.

Authors

Mengmeng Li, Xiaoya Zhao, Jun Wu, Shimeng Zhou, Yao Fu, Chen Chen, Zhuang Ma, Jiawen Xu, Yun Qian, Zhangding Wang, Bo Wang, Qiang Wang, Qingqing Ding, Changyu Chen, Honggang Wang, Xiaozhong Yang, Weijie Dai, Wenjie Zhang, Shouyu Wang

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Figure 4

NAT10 directly targets DKK2 mRNA for ac4C modification to stabilize its expression.

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NAT10 directly targets DKK2 mRNA for ac4C modification to stabilize its ...
(A) Consensus motif analysis of ac4C-modified transcripts by HOMER in MC38 WT/Nat10-KO cells. (B) Metagene plot showing the distribution of ac4C-containing peaks across mRNAs. (C) Gene ontology (GO) analysis highlighting pathways significantly enriched in ac4C-modified genes. (D) Venn diagram of candidate genes with altered ac4C peaks (acRIP-seq) and transcript levels (mRNA-seq) upon Nat10 KO. Dkk2 exhibited the most pronounced reduction in ac4C modification. (E) Integrative Genomics Viewer (IGV) tracks of ac4C peak on Dkk2 mRNA based on acRIP-seq data in both MC38 WT/Nat10-KO cells. (F) IGV diagrams displaying read distributions and chemical ac4C sequencing–derived ac4C altered abundance across the 3′-UTR of DKK2 mRNA. (G and H) acRIP-qPCR quantification of Dkk2 mRNA using anti-ac4C in WT/Nat10-KO cells. (I) Schematic of the dual-luciferase reporter containing the WT and mutant (MUT) Dkk2 ac4C motif. (J) 293T cells were cotransfected with Myc-Nat10 plasmid and Dkk2 WT or MUT reporters for 48 hours. Luciferase activity in each group was detected. (K and L) Luciferase activity of WT/MUT reporters in Nat10-KO versus control cells. (M) qRT–PCR analysis of Dkk2 mRNA levels in Nat10-KO/Nat10-overexpressing (OE) CRC cells. (N) qRT–PCR analysis of Dkk2 mRNA levels in MC38 Nat10-KO/OE cells treated with actinomycin D (2.5 μg/mL) at the indicated time points. The data are presented as the mean ± SD of 3 independent experiments (G, H, and J–N). Statistical analysis was performed by 2-way ANOVA (G, H, J, and N), 1-way ANOVA (M, left), and 2-tailed Student’s t test (K, L, and M, right). ns, P ≥ 0.05. P < 0.05 was considered to indicate statistical significance.