M6A Demethylase ALKBH5 Regulates PD-L1 Expression and Tumor Immunoenvironment in Intrahepatic Cholangiocarcinoma.

N6-methyladenosine (m6A) has been reported as an important mechanism of posttranscriptional regulation. Programmed death-ligand 1 (PD-L1) is a primary immune inhibitory molecule expressed on tumor cells that promotes immune evasion. Here we report ALKBH5 as an important m6A demethylase that orchestrates PD-L1 expression in intrahepatic cholangiocarcinoma (ICC). Regulation of PD-L1 expression by ALKBH5 was confirmed in human ICC cell lines. Sequencing of the m6A methylome identified PD-L1 mRNA as a direct target of m6A modification whose levels were regulated by ALKBH5. Furthermore, ALKBH5 and PD-L1 mRNA were shown to interact. ALKBH5 deficiency enriched m6A modification in the 3'UTR region of PD-L1 mRNA, thereby promoting its degradation in a YTHDF2-dependent manner. In vitro and in vivo, tumor-intrinsic ALKBH5 inhibited the expansion and cytotoxicity of T cells by sustaining tumor cell PD-L1 expression. 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 the tumor immune microenvironment by promoting the expression of PD-L1 on monocytes/macrophages and decreasing the infiltration of myeloid-derived suppressor-like cells. Analysis of specimens from patients receiving anti-PD1 immunotherapy suggested that tumors with strong nuclear expression patterns of ALKBH5 are more sensitive to anti-PD1 immunotherapy. Collectively, these results describe a new regulatory mechanism of PD-L1 by mRNA epigenetic modification by ALKBH5 and the potential role of ALKBH5 in immunotherapy response, which might provide insights for cancer immunotherapies. SIGNIFICANCE: This study identifies PD-L1 mRNA as a target of ALKBH5 and reveals a role for ALKBH5 in regulating the tumor immune microenvironment and immunotherapy efficacy. ©2021 American Association for Cancer Research.