Bromodomain and Extra-terminal Tail (BET) proteins are epigenetic transcription regulators and potential therapeutic targets in glioblastoma (GB), the most aggressive primary malignant brain tumor in adults. A differential gene expression analysis of GB-derived sphere (GS) lines demonstrated that the BET inhibitor JQ1 significantly downregulates cancer-relevant gene signatures, including our previously defined HOX signature. HOX genes are crucial for limb segmentation during embryonic development and are associated with treatment resistance in a subset of GB patients characterized by a high-HOX signature. The HOXA gene cluster, located on chromosome 7 (Chr7), is a CpG-rich region and the expression of the HOXA-dominated signature correlates with Chr7 gain and widespread DNA hypermethylation. Previous findings indicated a significant negative correlation between DNA hypermethylation at the alternative HOXA10 promoter and HOX gene expression. This study aims to investigate the oncogenic role of HOXA10 and the epigenetic mechanisms underlying HOXA cluster deregulation in high-HOX GB.

To explore these mechanisms, we used various assays: (1) comparative methylation analysis at the HOXA locus in a high-HOX GS line vs original tumor, along with RNA- and ChIP-seq analysis after JQ1 treatment; (2) BETi treatment and modulation of HOXA10/HOXA5 expression using inducible shRNA/ectopic expression systems to examine HOXA gene regulation; (3) functional assays post-HOXA10 modulation to assess cancer-relevant biological impact.

We confirmed that BETi concertedly decreases HOXA gene expression in high-HOX GS lines. Remarkably, knockdown of HOXA10 in high-HOX GS lines is associated with a concerted downregulation of other HOXA genes; however, ectopic HOXA10 expression suggests that HOXA10 proteins may not be the sole factor contributing to HOX signature expression. Specificity tests using shHOXA5 models affirmed the unique role of HOXA10, as HOXA5 knockdown did not alter the expression of other HOXA genes. Functionally, HOXA10 knockdown in high-HOX GS lines results in potently decreased cell proliferation and stemness, accompanied by an emergence of senescence-like traits.

Our results set HOXA10 as a key player of the stem cell-related HOX signature in high-HOX GB, likely enabling tumor progression by its association with pro-proliferative and stem-like features. BETi-mediated downregulation of HOX signature genes presents a promising therapeutic avenue for targeting high-HOX GB. Ongoing research focuses on differential gene expression, aiming to identify HOXA10-regulated genes and reveal its role in BETi-mediated effects. Besides, we are also assessing the potential changes in 3D chromatin architecture and epigenetic landscape around the HOXA cluster that may be involved HOX signature expression.