Dissecting the genomic and epigenomic alterations underlying glioblastoma

Glioblastoma (GBM) is the most common intrinsic primary brain tumour, characterised by structural complexity, tumoural heterogeneity and poor prognosis. GBM tumours reactivate neurodevelopmental programmes and they are driven by GBM stem-like cells (GSCs), displaying phenotypic similarities to neural stem cells (NSCs). Although much is known about the recurrent coding alterations and the role of GSCs in GBM, we still have a poor understanding of the contribution of non-coding alterations and complex structural variants (SVs) to GBM pathogenesis, as well as the role of key NSC transcription factors such as SOX2 and SOX9, in regulating self-renewal activity in GSCs and gliomagenesis. To study non-coding alterations and complex SVs, we compiled a large (n = 230) cohort of GBM tumours with whole genome sequencing data. We uncovered a lack of recurrent non-coding somatic small mutations (SSMs) apart from TERT promoter mutations. We identified loci under putative selection for SV recurrence, including those harbouring focal amplications and deletions, associated with complex SVs such as extrachromosomal DNA (ecDNA) and chromothripsis. We discovered underappreciated features of ecDNAs in GBM, including a high prevalence of ecDNAs generated via episomal exclusion, and uncovered the extent by which ecDNAs shape tumoural heterogeneity via seismic amplification and putative chromosomal re-integration. Notably, despite the relatively high SSM- and SV-derived neoantigen burdens, ecDNA+ samples exhibited higher transcriptional immune suppression compared to ecDNA- samples, an effect not seen in other complex SV types, suggesting that ecDNAs may modulate the tumour-immune microenvironment to evade immune clearance. Last, contrary to our expectations, ecDNA and chromothripsis were not associated with poor overall survival; rather, patients whose GBMs sustained high levels of overall complex SV burden were associated with significantly shorter overall survival, independent of other available prognostic factors. To complement these genomic analyses, we also explored transcriptional regulatory programs in GBM, particularly the role of SOX2 and SOX9 in regulating GSC self-renewal. We used chromatin immunoprecipitation followed by sequencing (ChIP-seq) data across 7 patient-derived GSCs. We found that SOX9 binds exclusively and at close proximity to SOX2, and co-bound SOX2/SOX9 sites are enriched with GSC-specific super-enhancers (SEs). We identified a dual-mode of SOX2/SOX9 co-binding that was regulatory element-dependent – as a monomer at promoters and a dimer at enhancers, reminiscent of SOX9 activity in cartilage development. Co-bound SOX2/SOX9 SEs target genes regulating NSC identity, including QKI, PTPRZ1 and CDK6. Intriguingly, SOX2/SOX9 co-binding also target their own and each other’s regulatory regions, suggesting the existence of an auto- and cross-regulatory feed-forward axis. Importantly, we observed no increased in mutational burden across SOX2/SOX9 co-binding sites, arguing against mutation-driven reactivation processes at these neurodevelopmental enhancers. These findings altogether suggest that SOX2 and SOX9 cooperate at shared enhancers and target genes, and their increased co-activity may contribute to the core programs of self-renewal displayed by GSCs. In summary, we present a thorough computational genomic analysis of the non-coding alterations and structural complexity shaping the GBM genome, revealing a complex interplay between ecDNA, immune evasion pathways and complex SV burden during gliomagenesis. Furthermore, we identify putative downstream targets of SOX2 and SOX9 that may underlie self-renewal activity in GSCs. It is hoped that these findings may lead to an increased understanding of GBM pathogenesis and ultimately result in new therapeutic avenues for GBM patients.

Grantor dc:publisher

The University of Edinburgh

Year dc:date.issued

2025

Author dc:creator

• Hamdan, Alhafidz

Advisors dc:contributor.advisor

• Pollard, Steven
• Semple, Colin

• glioblastoma
• structural variant
• ecDNA
• chromothripsis
• genomic instability
• prognosis
• GBM
• amplification
• sox2
• sox9
• axis
• GSC
• stem cell
• feed-forward
• whole genome sequencing

Licence dc:rights.uri

• https://creativecommons.org/licenses/by/4.0/

Language dc:language.iso

en

Identifier URI

http://dx.doi.org/10.7488/era/6513

OAI identifier oai:identifier

oai:era.ed.ac.uk:1842/43983