Transcriptional regulation and DNA damage response of extrachromosomal DNA in human glioblastoma stem cells
Creators: name: Purshouse, Karin Rita
Publisher: name: The University of Edinburgh
Year: 2023
Subjects: Transcriptional regulation
DNA damage response
extrachromosomal DNA
human glioblastoma stem cells
Glioblastoma
Extrachromosomal DNA (ecDNA)
ecDNA oncogene transcription
DNA damage sites
random and targeted DNA damage

description: Glioblastoma is a cancer characterised by limited treatment options and poor prognosis. Glioblastoma is driven by neural stem cell-like cells and is characterised by intratumoral heterogeneity. Extrachromosomal DNA (ecDNA) are circular regions of DNA that are seen in many cancers and are particularly frequent in glioblastoma. They are an important means of oncogene amplification, and correlate with treatment resistance and poor prognosis. Due to their frequency and association with accessible chromatin, it has been proposed that ecDNA oncogene transcription is amplified by their clustering with each other and key components of the transcriptional machinery. Such a mechanism could lead to greater levels of oncogene transcription than expected from copy number amplification alone. A major mechanism of ecDNA generation is massive DNA damage (chromothripsis), but the impact of DNA damage on existing ecDNA is yet to be fully characterised. This thesis characterises ecDNA in five glioblastoma cell lines derived from patients using WGS and DNA FISH analysis. Super-resolution imaging and quantitative image analysis are used to evaluate the spatial organisation of ecDNA-resident oncogenes in glioblastoma cell lines. EcDNA are widely distributed throughout the nucleus, but a novel cluster analysis method demonstrates that ecDNA do not cluster closely with each other, nor do they closely engage with large transcriptional hubs. Focusing on the EGFR oncogene, transcriptional output is increased in cells harbouring ecEGFR. A combination of RNA:DNA FISH and RNAseq:WGS analysis is used to demonstrate that transcription per gene copy number is similar between chromosomal and ecDNA EGFR loci. This suggests increased ecDNA-resident oncogene transcription is primarily driven by copy number amplification rather than synergistic ecDNA regulatory processes and interactions. Glioblastoma cells have many active DNA damage sites not closely related to ecDNA. To explore the impact of random and targeted DNA damage on ecDNA characteristics and dynamics, ionising radiation (IR) and CRISPR/Cas9 are utilised respectively. Treatment of ecEGFR-harbouring cells with IR results in a reduction in EGFR foci copy number and EGFR expression. Preliminary experiments suggest EGFR expression reduces further with co-treatment with the PARP inhibitor olaparib, although ecEGFR copy number appears protected. In a glioblastoma cell line with ecEGFR, CRISPR/Cas9 targeting EGFR generates cells with reduced EGFR expression. Cells can be FACS-sorted into EGFR High, Low and Null pools, in which Low and Null pools lose ecEGFR by DNA FISH and WGS analysis. Repeating this in a glioblastoma cell line with EGFR as a chromosomal amplification and MYC ecDNA demonstrates minimal effect on EGFR, but marked rearrangement of ecMYC loci. Overall, these findings suggest that ecDNA are an important mechanism of oncogene amplification, the transcriptional effect of which is primarily driven by copy number. Understanding the impact of DNA damage on ecDNA and cell selection is important for development of new therapeutic strategies that successfully target genomic vulnerabilities.