USP14 modulates stem-like properties, tumorigenicity, and radiotherapy resistance in glioblastoma stem cells through stabilization of MST4-phosphorylated ALKBH5.

: Glioblastoma (GBM) is the most aggressive type of primary brain cancer and contains self-renewing GBM stem cells (GSCs) that contribute to tumor growth and therapeutic resistance. However, molecular determinants governing therapeutic resistance of GSCs are poorly understood. : We performed genome-wide analysis of deubiquitylating enzymes (DUBs) in patient-derived GSCs and used gene-specific shRNAs to identify an important DUB gene contributing to GSC survival and radioresistance. Subsequently, we employed mass spectrometry and immunoprecipitation to show the interaction between USP14 and ALKBH5, and identified the upstream kinase MST4, which is essential for the deubiquitylation and stabilization of ALKBH5. Additionally, we performed integrated transcriptome and mA-seq analyses to uncover the key downstream pathways of ALKBH5 that influence GSC radioresistance. : Our study demonstrates the essential role of the deubiquitinase USP14 in maintaining the stemness, tumorigenic potential, and radioresistance of GSCs. USP14 stabilizes the mA demethylase ALKBH5 by preventing its K48-linked ubiquitination and degradation through HECW2. The phosphorylation of ALKBH5 at serine 64 and 69 by MST4 increases its interaction with USP14, promoting ALKBH5 deubiquitylation. Furthermore, ALKBH5 directly interacts with the USP14 transcript in a manner dependent on YTHDF2, establishing a positive feedback loop that sustains the overexpression of both proteins in GSCs. The MST4-USP14-ALKBH5 signaling pathway is crucial for enhancing stem cell-like traits, facilitating homologous recombination repair of DNA double-strand breaks, and promoting radioresistance and tumorigenicity in GSCs. This signaling cascade is further stimulated in GSCs following exposure to ionizing radiation (IR). Inhibiting USP14 with the small molecule IU1 disrupts ALKBH5 deubiquitylation and increases the effectiveness of IR therapy on GSC-derived brain tumor xenografts. Our results identify the MST4-USP14-ALKBH5 signaling pathway as a promising therapeutic target for treating GBM.