Targeting m⁶A reader YTHDF1 augments antitumour immunity and boosts anti-PD-1 efficacy in colorectal cancer.

Abstract

The role of N⁶-methyladenosine (m⁶A) in tumour immune microenvironment (TIME) remains understudied. Here, we elucidate function and mechanism of YTH N⁶-methyladenosine RNA binding protein 1 (YTHDF1) in colorectal cancer (CRC) TIME. Clinical significance of YTHDF1 was assessed in tissue microarrays (N=408) and TCGA (N=526) cohorts. <i>YTHDF1</i> function was determined in syngeneic tumours, intestine-specific <i>Ythdf1</i> knockin mice, and humanised mice. Single-cell RNA-seq (scRNA-seq) was employed to profile TIME. Methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNA sequencing (RNA-seq) and ribosome sequencing (Ribo-seq) were used to identify YTHDF1 direct targets. Vesicle-like nanoparticles (VNPs)-encapsulated <i>YTHDF1</i>-siRNA was used for <i>YTHDF1</i> silencing in vivo. <i>YTHDF1</i> expression negatively correlated with interferon-γ gene signature in TCGA-CRC. Concordantly, YTHDF1 protein negatively correlated with CD8⁺ T-cell infiltration in independent tissue microarrays cohorts, implying its role in TIME. Genetic depletion of <i>Ythdf1</i> augmented antitumour immunity in CT26 (MSS-CRC) and MC38 (MSI-H-CRC) syngeneic tumours, while <i>Ythdf1</i> knockin promoted an immunosuppressive TIME facilitating CRC in azoxymethane-dextran sulphate-sodium or <i>Apc^Min/+</i> models. scRNA-seq identified reduction of myeloid-derived suppressor cells (MDSCs), concomitant with increased cytotoxic T cells in <i>Ythdf1</i> knockout tumours. Integrated MeRIP-seq, RNA-seq and Ribo-seq revealed p65/Rela as a YTHDF1 target. YTHDF1 promoted p65 translation to upregulate CXCL1, which increased MDSC migration via CXCL1-CXCR2 axis. Increased MSDCs in turn antagonised functional CD8⁺ T cells in TIME. Importantly, targeting YTHDF1 by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) or VNPs-si<i>YTHDF1</i> boosted anti-PD1 efficacy in MSI-H CRC, and overcame anti-PD1 resistance in MSS CRC. YTHDF1 impairs antitumour immunity via an m⁶A-p65-CXCL1/CXCR2 axis to promote CRC and serves as a therapeutic target in immune checkpoint blockade therapy.