BET inhibition induces coordinated repression of the stem cell-related HOX signature in GB uncovering an epigenetic vulnerability

Abstract 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 with an inhibitor of Bromodomain and extra-terminal motif proteins (BETi) 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 the deregulated epigenetic landscape and potential vulnerabilities in high-HOX GB. Methods GB-derived spheres (GS) were treated with the 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 failed to reactivate 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.