Transcriptional regulation and DNA damage response of extrachromosomal DNA in human glioblastoma stem cells
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Date
16/08/2023Item status
Restricted AccessEmbargo end date
16/08/2024Author
Purshouse, Karin Rita
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Abstract
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.