Defining heterogeneity in core regulatory circuitry reveals HOXB3 condensation as a potential target in glioblastoma.

Glioblastoma (GBM) exhibits marked heterogeneity, yet therapeutic strategies effectively targeting this variability remain inadequately developed. Here we employed single-cell CUT&Tag analysis to investigate H3K27ac modifications, uncovering pronounced heterogeneity within the core regulatory circuitry (CRC) of GBM. Notably, we observed heterogeneous condensation states of CRC factors, particularly HOXB3, which are shaped by its intrinsically disordered regions and interactions with RUNX1, driving the phenotypic manifestations. Leveraging these findings, we synthesized the peptide P621-R9, which effectively disrupted HOXB3 condensation, altered chromatin structure and reduced transcription at super-enhancer-associated oncogenic sites in GBM cells exhibiting HOXB3 condensation. Treatment with P621-R9 selectively diminished tumourigenic potential in GBM patient-derived xenograft models characterized by HOXB3 condensates, but showed no efficacy in the models lacking these condensates. These results highlight the critical role of CRC condensation in GBM heterogeneity and suggest that peptide-based targeting of distinct GBM subpopulations could represent an avenue for therapeutic exploration.