Investigation of co-operating factors in the development of MLL::AF4 infant acute lymphoblastic leukaemia

Abstract

The MLL::AF4 fusion gene is prevalent in infant acute lymphoblastic leukaemia (iALL) and associated with early onset and absence of additional mutations. The fusion gene has been detected in new-born blood samples, suggesting a foetal progenitor cell origin. This PhD project aimed to study the interaction between epigenetic changes and the MLL::AF4 fusion gene in iALL. It involved mutating PHF3 and CHD4, inhibiting HDAC1 and HDAC2, identifying CHD4 binding sites, and tracing the fusion gene's origins in iALL samples. The project developed two gene-editing approaches to manipulate NuRD complex members PHF3 and CHD4 in MLL::AF4 ALL cells. Neither approach was able to successfully introduce precise base substitutions. Augmentation of the CRISPR system with HDAC inhibition resulted in alterations in CD19 and CD33 expression. However, limited single-cell cloning efficiency hindered the further examination of modified cells. Inhibition of HDAC1 and HDAC2, core members of the NuRD complex, resulted in significant H3K9 acetylation in MLL-rearranged and MLL germline ALL cell lines. However it was not associated with an alteration in their lineage-specific immunophenotype, indicating no impact on lineage development. The project investigated CHD4 binding on dysregulated genes linked to MLL::AF4 lineage switched relapse cases (ALL to AML), revealing its presence at genes involved in haematopoietic lineage development. It is important to clarify that the results obtained are of a preliminary nature and require further validation. Finally, this project optimised flow cytometric sorting and single-cell culture of HSPCs to investigate the cellular and phylogenetic origins of the MLL::AF4 fusion by whole genome sequencing. Although none of the initial 38 colonies tested positive, the focus now revolves around bulk sequencing of B blasts, offering insights into their characteristics and position within the derived phylogenetic tree. This methodology has gone on to be used to investigate additional cases and many more colonies.