Background We have reported that the RNA N6-methyladenosine (m6A) reader YTHDF1 plays an important role in the tumourigenesis of intrahepatic cholangiocarcinoma (ICC). However, whether YTHDF1 influences the tumour immune microenvironment (TIME) of ICC remains unknown. Here, we investigated the function and mechanism by which YTHDF1 regulates the TIME of ICC.

Methods Different mouse models, including the ICC orthotopic mouse model, ICC hydrodynamic injection mouse model, and liver-specific Ythdf1-overexpressing mice, were applied. Tumour-infiltrating immune cells were profiled with flow cytometry and immunohistochemistry staining. The clinical significance of YTHDF1 in ICC TIME was evaluated with immunostaining with human ICC tissue microarrays. Chemokine array and ELISA assays were used to identify the cytokines and chemokines regulated by YTHDF1. The downstream molecular targets of YTHDF1 were identified by integrating RNA-sequencing, MeRIP-sequencing, RNA immunoprecipitation-sequencing, and ribosome-profiling.

Results Ythdf1-knockdown orthotopic ICC mouse model and Ythdf1-overexpressed ICC hydrodynamic injection mouse model elucidated that YTHDF1 recruited myeloid-derived suppressor cells (MDSCs) to tumours and inhibited the infiltration and cytotoxic function of CD8⁺ T cells, thus promoting tumour progression. Immunostaining of human ICC tissue microarray (n = 188) verified that higher expression of YTHDF1 was positively related to increased MDSCs accumulation (P < 0.001) and decreased CD8⁺ T cells infiltration (P < 0.001). Mechanistically, YTHDF1 upregulated CXCL6 expression in ICC, which recruited and activated MDSCs by binding to its receptor CXCR2, leading to the dysfunction of CD8⁺ T cells. By integrating multi-omics sequencing data, we pinpointed FOSL2, a transcription factor, as the key downstream target of YTHDF1. YTHDF1 bound to m⁶A-modified FOSL2 mRNA and promoted its translation. In turn, FOSL2 is directly bound to the CXCL6 promoter and facilitates CXCL6 transcription. The liver-specific Ythdf1-overexpressing mice model also confirmed that YTHDF1 upregulated the protein expression of FOSL2 and CXCL6. Furthermore, Ythdf1-knockdown synergized with anti-PD-L1 treatment significantly enhanced the therapeutic effects of immunotherapy in ICC.

Conclusions YTHDF1 facilitates ICC immune escape via FOSL2-CXCL6-CXCR2 signaling, which recruits and activates MDSCs and induces cytotoxic CD8⁺ T-cell dysfunction. Targeting YTHDF1 may be a promising strategy for improving immunotherapy efficacy in ICC.