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Title: Molecular analysis of leukaemia genomes during malignant transformation and leukaemia progression
Author: May, Philippa Charlotte
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
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This thesis details a tripartite approach to understanding genetic and epigenetic modifications in human leukaemia. First, an in vitro mouse model of precursor B-cell leukaemia was established by retroviral transduction of primary B-cell precursors with the oncogenes BCR-ABL1 or MYC. The model was used to study DNA damage incurred during the initial phase of oncogene activation i.e. oncogenic stress. Analysis of γH2AX (a mark of DNA damage) and the H3K27 acetylation histone modification (a mark of active enhancers and promoters) by chromatin immunoprecipitation sequencing (ChIP-seq) and mRNA expression (RNA-seq) were combined to establish that lineage specific transcription predisposes lineage specific genes to DNA damage. This was functionally validated with a lineage-switch model and technically validated using FISH. Second, the same cells were maintained during their recovery from the period of acute oncogenic stress until stably transformed. Re-analysis of the transformed populations using the same H3K27Ac ChIP-seq and RNA-seq techniques showed that the stable transformation of these cells is paralleled by activation of a subset of intergenic enhancers which induce an aberrant transcriptome enriched for cell activation and metabolic pathways. Finally, the techniques used in these in vitro experiments and the knowledge of oncogenic enhancers gained from them were then applied to the clinical setting. EVI1, a key haematopoietic transcription factor that regulates differentiation, is aberrantly expressed in a subset of myeloid leukaemia where it is universally associated with a poor prognosis. A patient sample biobank was established and suitable samples were filtered using a combination of RT-qPCR and FISH. Thus, H3K27AC ChIP-seq and newly optimised chromosome conformation capture sequencing (3C-seq or 4C) was used to understand the epigenetic interactions which result in aberrant expression. This has revealed several interactions between the EVI1 promoter with up- and down-stream intergenic regions which must be further investigated.
Supervisor: Feldhahn, Niklas ; Reid, Alistair ; Porter, Andy Sponsor: European Commission
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral