ALKBH5 regulates PD-L1 expression and tumor immunoenvironment in intrahepatic cholangiocarcinoma

M6A has been reported as an important post-transcriptional regulation mechanism. Programmed death-ligand 1 (PD-L1) is a primary immune inhibitory molecule expressing on tumor cells to promote immune evasion. Here, we reported that ALKBH5 as an important m6A demethylase orchestrated PD-L1 expression in intrahepatic cholangiocarcinoma (ICC). Firstly, the regulation of PD-L1 expression by ALKBH5 was confirmed in human ICC cell lines. The sequence of m6A methylomes identified PD-L1 mRNA as a direct target of m6A modification and its level was regulated by ALKBH5. Further, the interaction between ALKBH5 and PD-L1 mRNA was verified. Mechanistic analysis suggested that ALKBH5 deficiency enriched m6A modification on the 3UTR region of PD-L1 mRNA, thereby promoting the degradation of PD-L1 mRNA in YTHDF2 dependent manner. Moreover, we verified that tumor-intrinsic ALKBH5 inhibited the expansion and cytotoxicity of T cells by sustaining tumor cell PD-L1 expression in vitro and in vivo. The ALKBH5-PD-L1 regulating axis was further confirmed in human ICC specimens. Single-cell mass cytometry analysis unveiled a complex role of ALKBH5 in tumor immune microenvironment by promoting the expression of PD-L1 on monocyte/macrophage cells and decreasing the infiltration of MDSC-like cells. At last, studies on specimens from patients receiving anti-PD1 immunotherapy suggested that tumors with ALKBH5 strong nuclear expression pattern are more sensitive to anti-PD1 immunotherapy. Our results mainly reported a new regulation mechanism of PD-L1 on mRNA epigenetic level by ALKBH5 and the potential role of ALKBH5 in anti-PD-L1 immunotherapy, which might provide potential insights for cancer immunotherapies.