Investigating epigenetic perturbations associated with DNMT3A and BCOR mutations in normal and malignant haematopoiesis

Abstract

Despite recent advances in cancer detection, molecular classification and treatment modalities, the 5-year survival rate of acute myeloid leukaemia (AML) has not changed in 30 years. The unique genetic context created by the mutational landscape of AML may dictate prognosis and treatment sensitivity, therefore a greater understanding of the molecular aetiologies underlying leukaemia initiation and maintenance will likely guide the development and use of more effective targeted therapeutics. Under this premise, the roles of two frequently mutated epigenetic regulators, BCOR and DNNMT3A, were investigated in the initiation of AML and maintenance of the disease. A combination of genetically engineered mouse models and next generation sequencing were used to interrogate the molecular and cellular consequences of each mutant protein in vivo. This analysis found that loss of Bcor function lead to a loss of H2AK119ub at the Hoxa7/9 locus and caused a concurrent transcriptional upregulation of important HSC-associated master transcription factors. A CRISPR/Cas9 screen confirmed that Hoxa9 upregulation was essential for the proliferative phenotype of Bcor mutant leukaemia. Studies investigating the frequently occurring DNMT3AR882H dominant negative oncoprotein revealed that sustained expression of the mutant was essential for maintaining the leukaemic transcriptional program in established leukaemias. Surprisingly, further analysis of the methylome of these tumours using reduced representation bisulfite sequencing led to the discovery that the DNA methylation patterns in established AML were not significantly dependent on DNMT3AR882H expression. This molecular analysis successfully identified key drivers of the transcriptional programs governing leukaemic cell identity in these distinct genetic contexts. The findings of this thesis emphasise that future research should focus on the development of therapeutic strategies specifically targeting these key drivers of leukaemic cell identity. Together this research provides valuable insight into the transcriptional and epigenetic mechanisms underlying leukaemogenesis, and proposes potential therapeutic targets for AML with BCOR or DNMT3AR882H mutations.