Molecular characterisation of ASXL1-mutant clonal haematopoiesis

Thesis

Adult stem cells accumulate somatic mutations throughout life as a result of failure to repair DNA damage. If a somatic mutation confers a fitness advantage upon the stem cell, the resulting clone can expand to be over-represented in that tissue. In the haematopoietic system, this process is known as clonal haematopoiesis (CH). Whilst CH is a common condition amongst older healthy individuals, it has been associated with a greater risk of developing leukaemia. One of the genes that is most frequently found mutated in CH and myeloid malignancies is ASXL1- a regulator of histone modification distribution. Mutations in ASXL1 are associated with poor prognosis of blood cancers, and are likely to be acquired years before the onset of disease. Thus far, the mechanisms by which ASXL1 confers a clonal expansion upon haematopoietic stem and progenitor cells are poorly understood. Identification of these mechanisms could reveal novel therapeutic strategies for blood cancer.

In this study, the cellular and molecular foundations of ASXL1-mutant clonal haematopoiesis in humans are characterised. By applying TARGET-seq+ to 4 ASXL1- mutant CH samples and 5 age-matched healthy control samples, this study reveals that the ASXL1-mutant clone follows broadly similar differentiation trajectories to healthy cells. However, the ASXL1-mutant clone is greatly expanded within the HSC compartment. Additionally, mutant cells show an inefficiency of terminal maturation of the erythroid and lymphoid lineages. Compared to wildtype HSCs within the CH bone marrow, mutant HSCs exhibit a transcriptional shift towards a less-dormant, differentiation-primed state, and an upregulation of polycomb target genes. Interestingly, unlike recent reports linking inflammation to clonal expansion in CH, no significant difference in transcriptional response to inflammation was seen between ASXL1-mutant and wildtype HSCs. These results suggest a distinct mechanism of clonal expansion for ASXL1-mutant cells.

Here, I also present proof-of-concept results for a novel multiomic protocol- GCUT- capable of simultaneously profiling epigenomic component distribution and genotype of single cells. Through a cell mixing experiment, cell lines were clustered on the basis of histone mark distribution, and identified by high-fidelity genotyping of single nucleotide variants. Future use of this protocol on cells from human ASXL1-mutant CH samples may identify the direct genomic targets of mutant ASXL1, and reveal novel insights into mechanisms of clonal expansion.

Authors: Groom, AJ

• DOI: 10.5287/ora-bmw0vdbkj
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English