H2AX phosphorylation driven by the SET-PP2A axis maintains glioma stem cell properties.

Glioblastoma harbors a subpopulation of glioma stem cells (GSCs) that drive tumor relapse and invasion. Although GSC properties are known to be regulated by key transcription factors and chromatin remodeling, the mechanisms governing stemness-associated gene expression and the role of histone proteins in this process remain poorly understood. Here, we revealed that GSCs possessed an increased level of histone variant H2AX and phosphorylated H2AX at Ser 139 (γH2AX). CRISPR-Cas9-mediated deletion of H2AX demonstrated its critical role in maintaining GSC self-renewal, tumorigenicity, and the expression of multiple stemness-associated genes. Reintroduction of wild-type H2AX, but not the phosphorylation-deficient S139A mutant, restored GSC properties in H2AX-knockout cells, underscoring the importance of H2AX phosphorylation. We further identified that the elevated γH2AX levels in GSCs result from suppressed PP2A phosphatase activity, driven by increased expression of the SET oncoprotein. Pharmacological reactivation of PP2A effectively reduced γH2AX levels in GSCs. Collectively, our findings uncover a novel mechanism for GSC maintenance and highlight the SET-PP2A axis as a key regulator of γH2AX levels, offering a potential therapeutic target to disrupt GSC-driven tumor growth.