Characterisation of genetic and epigenetic aberrations in paediatric high grade glioma

A thesis submitted in fulfilment of the requirements of the University of Wolverhampton for the degree of Doctor of Philosophy.Paediatric high grade glioma (HGG), including diffuse intrinsic pontine glioma (DIPG) are highly aggressive tumours with no effective cures. Lack of understanding of the molecular biology of these tumours, in part due to lack of well-characterised pre-clinical models, is a great challenge in the development of novel therapies. Analysis of paired cell culture/biopsy samples in this study revealed that paediatric HGG short-term cell cultures retain many of the tumour characteristics in vivo. Using a genome-wide approach, copy number, gene and miRNA expression, and methylation changes were characterised in 17 paediatric HGG-derived short-term cell cultures including 3 from DIPG. The majority of the genomic changes were unique from those arising in adult HGG. Approximately 65% (11/17) of paediatric HGG short-term cell cultures had balanced genetic profiles resembling normal karyotypes. The most frequent copy number gain and loss were detected at 14q11.2 (94%) and 8p11.23-p11.22 (59%), respectively. H3F3A (K27M) mutation was present in 2/17 (12%) cases and concurrent loss of CDKN2A and BRAFV600E in 1/17 (6%) case. Genes involved in reelin/PI3K signaling (DAB1), RTK signaling (PTPRE), and arginine biosynthesis (ASS1 and ASL) were frequently deregulated by methylation in these tumours. The anti-growth and anti-migratory properties of DAB1 and PTPRE were demonstrated in vitro. Preliminary investigations validated the therapeutic potential of ADI-PEG20 (arginine depletion), and PI-103 (PI3K/mTOR inhibition) in a subset of paediatric HGG short-term cell cultures. This study has identified novel genetic and epigenetic changes in paediatric HGG that may, following further validation, be translated into potential biomarkers and/or therapeutic targets

Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry.

A simple method for extraction of high quality RNA from cells that have been fixed, stained and sorted by flow cytometry would allow routine transcriptome analysis of highly purified cell populations and single cells. However, formaldehyde fixation impairs RNA extraction and inhibits RNA amplification. Here we show that good quality RNA can be readily extracted from stained and sorted mammalian cells if formaldehyde is replaced by glyoxal-a well-characterised fixative that is widely compatible with immunofluorescent staining methods. Although both formaldehyde and glyoxal efficiently form protein-protein crosslinks, glyoxal does not crosslink RNA to proteins nor form stable RNA adducts, ensuring that RNA remains accessible and amenable to enzymatic manipulation after glyoxal fixation. We find that RNA integrity is maintained through glyoxal fixation, permeabilisation with methanol or saponin, indirect immunofluorescent staining and flow sorting. RNA can then be extracted by standard methods and processed into RNA-seq libraries using commercial kits; mRNA abundances measured by poly(A)+ RNA-seq correlate well between freshly harvested cells and fixed, stained and sorted cells. We validate the applicability of this approach to flow cytometry by staining MCF-7 cells for the intracellular G2/M-specific antigen cyclin B1 (CCNB1), and show strong enrichment for G2/M-phase cells based on transcriptomic data. Switching to glyoxal fixation with RNA-compatible staining methods requires only minor adjustments of most existing staining and sorting protocols, and should facilitate routine transcriptomic analysis of sorted cells

The GALNT9, BNC1 and CCDC8 genes are frequently epigenetically dysregulated in breast tumours that metastasise to the brain.

Tumour metastasis to the brain is a common and deadly development in certain cancers; 18-30 % of breast tumours metastasise to the brain. The contribution that gene silencing through epigenetic mechanisms plays in these metastatic tumours is not well understood