The m6A Demethylase ALKBH5 Maintains Tumorigenicity of Glioblastoma Stem-Like Cells by Sustaining FOXM1 Expression and Cell Proliferation Programing

The dynamic and reversible N6-methyladenosine (m6A) RNA modification installed and erased by N6-methyltransferases and demethylases regulates gene expression and cell fate. Here, we show that the m6A demethylase ALKBH5 is highly expressed in glioblastoma stem-like cells (GSCs). Silencing ALKBH5 suppresses the proliferation of patient-derived GSCs in vitro and in vivo. Integrated transcriptome and m6A-seq analyses revealed altered expression of select ALKBH5 target genes, including FOXM1, a critical transcription factor for the ALKBH5-dependent cell cycle gene expression. ALKBH5 binds to and demethylates FOXM1 nascent transcripts, leading to enhanced FOXM1 expression. Further, a long noncoding RNA antisense to the FOXM1 (FOXM1-AS) interacts with ALKBH5 and FOXM1 nascent transcripts and promotes their interaction. Depleting ALKBH5 and FOXM1-AS disrupted GSC tumorigenesis through the FOXM1 axis. Our work uncovers a novel function for ALKBH5 in maintaining GSC tumorigenicity and provides insight into critical roles of the m6A RNA methylation in human brain tumor. Overall design: m6A-seq and anti-m6A RIP-seq of mRNA from control and shALKBH5 cells

动态且可逆的N6-甲基腺苷（N6-methyladenosine，m6A）RNA修饰由N6-甲基转移酶与去甲基化酶催化完成安装与移除过程，可调控基因表达与细胞命运。本研究证实，m6A去甲基化酶ALKBH5在胶质母细胞瘤干细胞样细胞（glioblastoma stem-like cells，GSCs）中呈高表达状态。敲低ALKBH5可在体外及体内抑制患者来源的GSCs增殖。整合转录组与m6A测序（m6A-seq）分析结果显示，部分ALKBH5靶基因的表达水平发生改变，其中包括FOXM1——一种调控ALKBH5依赖性细胞周期基因表达的关键转录因子。ALKBH5可结合并去甲基化FOXM1的新生转录本，从而提升FOXM1的表达水平。进一步研究发现，靶向FOXM1的长链非编码RNA反义转录本（FOXM1-AS）可与ALKBH5及FOXM1新生转录本发生相互作用，并促进二者的结合。同时敲低ALKBH5与FOXM1-AS可通过FOXM1通路破坏GSCs的致瘤能力。本研究揭示了ALKBH5在维持GSCs致瘤特性中的全新功能，并为阐明m6A RNA甲基化在人类脑肿瘤中的关键作用提供了新的见解。实验设计：对对照组与shALKBH5转染细胞的mRNA进行m6A测序（m6A-seq）与抗m6A RNA免疫沉淀测序（anti-m6A RIP-seq）。