Three-dimensional regulatory hubs support oncogenic programs in glioblastoma

Dysregulation of enhancer-promoter communication in the context of the three-dimensional (3D) nucleus is increasingly recognized as a potential driver of oncogenic programs. Here, we profiled the 3D enhancer-promoter networks of primary patient-derived glioblastoma stem cells (GSCs) in comparison with neuronal stem cells (NSCs) to identify potential central nodes and vulnerabilities in the regulatory logic of this devastating cancer. Specifically, we focused on hyperconnected 3D regulatory hubs and demonstrated that hub-interacting genes exhibit high and coordinated expression at the single-cell level and strong association with oncogenic programs that distinguish IDH-wt glioblastoma patients from low-grade glioma. Epigenetic silencing of a recurrent 3D enhancer hub--with an uncharacterized role in glioblastoma--was sufficient to cause concordant downregulation of multiple hub-connected genes along with significant shifts in transcriptional states and reduced clonogenicity. By integrating published datasets from other cancer types, we also identified both universal and cancer type-specific 3D regulatory hubs which enrich for varying oncogenic programs and nominate specific factors associated with worse outcomes. Genetic alterations, such as focal duplications, could explain only a small fraction of the detected hyperconnected hubs and their increased activity. Overall, our study provides computational and experimental support for the potential central role of 3D regulatory hubs in controlling oncogenic programs and properties. HIGHLIGHTS- 3D regulatory "hubs" in glioblastoma enrich for highly coregulated genes at a single-cell level and expand oncogenic regulatory networks. - Targeted perturbation of a highly recurrent 3D regulatory hub in GSCs results in altered transcriptional states and cellular properties. - 3D regulatory hubs across cancer types associate with tumor-specific and universal oncogenic programs and worse outcomes. - The majority of hyperconnected hubs do not overlap with structural variants, suggesting epigenetic mechanisms. eTOCHere we profile the 3D enhancer connectomes of primary patient-derived human glioblastoma stem cells (GSCs), identify hyperconnected 3D regulatory "hubs", and examine the impact of 3D hub perturbation on the transcriptional program and oncogenic properties.