Glioblastoma (GBM) is an aggressive primary brain tumor characterized by therapeutic resistance and frequent recurrence, with glioma stem cells (GSCs), a distinct subpopulation of tumor-initiating cells, contributing substantially to tumor progression and recurrence. Although transcription factors and chromatin remodeling are known to regulate GSC stemness, the specific roles of histone variants in maintaining stemness remain poorly understood.

In this study published in Genes & Diseases, researchers from The Ohio State University and Columbus Academy investigated the role of the histone variant H2AX and its Ser139-phosphorylated form, γH2AX, in maintaining GSC properties.

Proteomic profiling of GSCs and differentiated glioma cells (DGCs), followed by validation across multiple GBM models, showed that both H2AX and γH2AX were elevated in GSCs. Higher levels were also detected in CD133+ GSC-enriched populations.

CRISPR-Cas9-mediated deletion of H2AX demonstrated that while cell proliferation was unaffected, H2AX was critical for maintaining GSC self-renewal, neurosphere formation capacity, and in vivo tumorigenicity. Furthermore, intracranial GBM387 xenograft models showed that H2AX knockout substantially prolonged the survival of tumor-bearing mice. RNA-sequencing and expression analyses revealed that H2AX depletion altered the transcriptional landscape, including the downregulation of stemness- and neurodevelopment-associated genes CXCL12, KLF4, JAK3, and SALL4, as well as suppression of PI3K–AKT signaling, a critical pathway for GSC maintenance.

To determine whether H2AX phosphorylation was required for these effects, the authors conducted rescue experiments using wild-type H2AX and a phosphorylation-deficient S139A mutant. They observed that reintroduction of wild-type H2AX into H2AX-knockout cells successfully restored self-renewal, neurosphere formation, and tumor-initiating capacity, whereas the S139A phosphorylation-deficient mutant failed to effectively rescue these phenotypes, underscoring the functional importance of γH2AX in GSC maintenance.

Mechanistically, elevated γH2AX in GSCs was not accompanied by increased DNA double-strand breaks or heightened DNA damage responses, but rather by suppressed protein phosphatase 2A (PP2A) activity, which dephosphorylates γH2AX. Proteomic and biochemical analyses identified increased expression of the SET oncoprotein—a potent endogenous inhibitor of PP2A—in GSCs. Knockdown of SET or pharmacological activation of PP2A using DT-061 effectively reduced γH2AX levels, supporting a SET–PP2A–γH2AX regulatory axis in which elevated SET suppresses PP2A activity and promotes γH2AX accumulation.

In conclusion, this study demonstrated that H2AX phosphorylation regulates GSC stemness and linked elevated γH2AX levels to SET-mediated PP2A inhibition; however, the precise role of the PP2A–γH2AX axis in maintaining GSC stemness warrants further investigation. Nevertheless, targeting SET–PP2A signaling to reduce γH2AX may represent a potential therapeutic strategy for disrupting the GSC compartment and limiting GBM recurrence.

Reference

Title of the original paper: H2AX phosphorylation driven by the SET-PP2A axis maintains glioma stem cell properties
Journal: Genes & Diseases
Genes & Diseases is a journal for molecular and translational medicine. The journal primarily focuses on publishing investigations on the molecular bases and experimental therapeutics of human diseases. Publication formats include full length research article, review article, short communication, correspondence, perspectives, commentary, views on news, and research watch.
DOI: https://doi.org/10.1016/j.gendis.2025.101961

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Funding Information:
National Cancer Institute (USA) (No. R01CA169368, R01CA188228, R01CA227874, UG1CA233331 and P30CA016058)

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Genes & Diseases publishes rigorously peer-reviewed and high quality original articles and authoritative reviews that focus on the molecular bases of human diseases. Emphasis is placed on hypothesis-driven, mechanistic studies relevant to pathogenesis and/or experimental therapeutics of human diseases. The journal has worldwide authorship, and a broad scope in basic and translational biomedical research of molecular biology, molecular genetics, and cell biology, including but not limited to cell proliferation and apoptosis, signal transduction, stem cell biology, developmental biology, gene regulation and epigenetics, cancer biology, immunity and infection, neuroscience, disease-specific animal models, gene and cell-based therapies, and regenerative medicine.

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