Leveraging epigenetic vulnerabilities of the stem cell-related HOX -signature in glioblastoma

Background

Glioblastoma (GB) is the most aggressive primary brain tumor, characterized by therapy resistance, attributed to a multitude of epi-genetic changes resulting in phenotypic plasticity with altered cell states. To uncover druggable epigenetic vulnerabilities, we disturbed GB-derived spheres and observed coordinated repression of the aberrantly activated hemopoietic stem-like cell signature, dominated by HOXA genes. This signature has been associated with poor prognosis and resistance to therapy in GB. Here we investigate biological vulnerabilities associated with the deregulated epigenetic landscape in high- HOX GB.

Methods

GB-derived spheres (GS) were treated with an inhibitor of Bromodomain and extra-terminal motif proteins (BETi) (JQ1) or transduced with inducible constructs to genetically modulate HOXA10 expression (shRNA for knockdown, ectopic HOXA10 ). Functional effects were evaluated through proliferation, neurosphere formation, and senescence assays. Epigenomic profiling incorporated RNA-seq, ChIP-seq, ATAC-seq, promoter capture MicroC, and DNA methylation.

Results

BETi-mediated rapid, coordinated downregulation of the HOX- signature, suggested direct transcriptional regulation. Knockdown of HOXA10 alone yielded similar effects, decreasing expression of HOXA genes, reducing proliferation, self-renewal capacity, and triggering senescence. Conversely, ectopic HOXA10 expression was ineffective in reactivating the HOXA cluster, or reverse BETi-mediated biological effects. Integrative epigenomic analysis of high- HOX -GS revealed concerted activation of the HOXA region, with broad domains of H3K27ac/H3K4me3 associated with super-enhancer activity, open chromatin (ATAC) and focal DNA hypomethylation. Architectural changes included altered CTCF interactions and increased promoter-anchored looping.

Conclusion

These results position the HOX- signature as a potential therapeutic target and offer a mechanistic rationale for disrupting BET-dependent transcriptional regulation in high- HOX GB.

Key points

• Epigenetic activation of stem cell-related high- HOX signature in GB is associated with a super-enhancer encompassing the HOXA locus.

• Targeting this vulnerability by BETi or HOXA10 knockdown results in concerted repression and loss of stemness features.

Graphical Abstract

Created in BioRender. Chiesi, D. (2026) https://BioRender.com/eknk0ez

Importance of study

Glioblastoma (GB) are the most aggressive brain tumors in adults that are difficult to treat, due to their high plasticity resulting invariably to resistance to therapies. Here we report on the identification of epigenetic vulnerabilities that may be leveraged in combination therapies. Disturbing GB-derived stem-like cells with epigenetic drugs, we uncovered that a HOXA gene dominated hematopoietic stem cell-related signature, previously associated with aggressiveness and treatment resistance, can be repressed in a coordinated manner, resulting in loss of stem cell features. Analysis of the underlying epigenetic landscape revealed that the HOXA region was activated in high- HOX glioblastoma through the formation of a super-enhancer. This feature presents a particular vulnerability that may be leveraged by BETi as strategy of a combination therapy.