Clonal haematopoiesis of indeterminate potential (CHIP) is part of the normal ageing process that results from the acquisition of mutations in genes commonly associated with blood cancers. These mutations occur in haematopoietic stem cells (HSCs), leading to the formation of genetically distinct subpopulations of blood cells which have a competitive advantage over cells without the mutations.
The incidence of CHIP increases with age, occurring in approximately 10% of people over the age of 65. People with these mutations are 10-15 times more likely to develop myelodysplastic syndrome (MDS)/acute myeloid leukaemia (AML). Progression to AML only occurs in a very small proportion of people with CHIP. Therefore our projects' focus is around the biology of cells carrying the mutations, as well as clinical and biological risk factors that contribute to which patients with CHIP are at greatest risk of MDS and AML.
Our aim is to be able to identify the patients most at risk of developing cancer, so that they can be targeted for early follow-up and treatment, and develop therapeutics that will permit treatment of them early in the disease process.
Zebrafish as a model organism
In the Payne Lab, we use zebrafish to model the haematopoietic disorders we study. Zebrafish have a similar haematopoietic system to humans, are easy to genetically manipulate and produce transparent embryos, allowing us to easily study their development. A pair of adult zebrafish can produce hundreds of embryos, which is ideal for high-throughput in vivo drug screening.
Automated, high-throughput drug screening for novel therapeutics for MDS/AML
The aim of this project is to use high-throughput automated drug screening in live zebrafish to discover novel therapeutics for MDS and AML. A number of driver mutations for these diseases have been identified including Dnmt3a and Asxl1, and pharmacological therapies that are synthetic lethal in haematopoietic stem cells (HSCs) carrying these mutations is an attractive avenue for treatment. We use CRISPR to generate zebrafish carrying these mutations, which also have green fluorescent HSCs.
We have used the Wiscan Hermes High Content Imaging System to develop a fast, easy-to-use automated screening procedure suitable for high-throughput drug screens of live zebrafish. In collaboration with IDEA Bio-Medical, a Deep Learning-based Artificial Intelligence-driven application was developed and trained to automatically detect fish in brightfield images, identify anatomical structures, partition the animal into regions and exclusively select the desired side-oriented fish. This allows us to automatically count GFP+ HSCs in the tails of embryos, to look for compounds that lead to a reduction in these cell numbers in the mutant animals, but not in the wild-type.
We are currently undertaking a small-molecule screen using Dnmt3a mutants.
Automatic detection of anatomical structures in the zebrafish embryo
Modelling clonal heterogeneity and evolution of MDS in zebrafish
The aim of this project is to understand the clonal heterogeneity of MDS and the evolution from CHIP to MDS/AML. In order to do this, we have developed a transgenic zebrafish which allows the generation of mosaic tissue-specific mutations in HSPCs (haematopoietic stem and progenitor cells). Mutating the most commonly occurring MDS-associated genes using CRISPR and studying these zebrafish over time will allow us to better understand clonal evolution and to identify which clones are relevant for targeting with therapeutics.
Blood circulating in a transgenic zebrafish embryo at 24 hours post fertilisation (hpf)
ELSA: English Longitudinal Study of Ageing
The Payne Lab has collaborated with the English Longitudinal Study of Ageing (ELSA) which collects biological samples and information, including physical and mental health, wellbeing, finances and attitudes, from participants aged 50 and over in order to gain insight into ageing. We are planning to extract DNA from thousands of blood samples, some of which have been collected from the same participants over several years, and sequence them for a panel of CHIP genes. The results of this sequencing, coupled with the additional information collected by ELSA, will provide more information about how CHIP develops within an individual.
Cancer and ageing: studying leukaemia & blood stem cell exhaustion in short-lived African killifish
The overall aim is to establish the turquoise killifish (Nothobranchius furzeri) model for the study of leukaemia in an ageing context. This fish has recently emerged as a potent vertebrate model system for ageing and associated complex diseases, facilitated by its rapid ageing and short lifespan of about 4 months. This new potent model system allows for its experimental manipulation, will enable unique advances in our understanding of leukaemia and open up fresh opportunities for its prevention and treatment.
In three objectives we want to:
1) generate killifish models for myelodysplastic syndrome/acute myeloid leukaemia (MDS/AML);
2) analyse the age-related decline of haematopoiesis;
3) study the contribution of cell-autonomous and cell-extrinsic factors to the development of MDS/AML.
Defining the mechanism of L-leucine alleviation of the anaemia in Diamond-Blackfan Anaemia
Diamond-Blackfan anaemia (DBA) is a rare, inherited bone marrow failure syndrome usually presenting in childhood with pure red cell aplasia. In over 75% of patients, the underlying genetic cause is heterozygous mutation or loss of one of 20 ribosomal protein (RP) genes. Treatments such as corticosteroids, transfusions, or stem cell transplants may be used but these carry associated potential morbidities. We have shown in a zebrafish model of DBA that administration of the amino acid L-leucine can result in improvements in anaemia and growth. More recently, L-leucine is showing promising results in clinical trials. However, the mechanism by which L-Leucine exerts its effects has not been defined. By employing metabolomic, ribosomal footprinting, and molecular biological analyses, we aim to understand the mechanism of L-leucine in DBA zebrafish models.
Towards understanding the selective pressure of CEBPA alleles during developmental haematopoiesis
CCAAT/enhancer binding protein alpha (CEBPA) is a transcription factor mutated in 10-15% of acute myeloid leukaemia (AML). CEBPA mutations are also observed in rare cases of germline predisposition to AML, confirming their role as initiator mutations. CEBPA mutations show a distinct pattern of distribution; in-frame C-terminal mutations (C-term) in the basic leucine zipper region or frame-shift N-terminal mutations (N-term). CEBPA-mutated AML typically involves one of each of these mutations suggesting a selective pressure from each mutation to develop the other.
We have developed a zebrafish model for CEBPA-mutated AML, with mutant lines modelling both C-term and N-term mutations. These animals are leukaemia prone in the biallelic state succumbing to leukaemia within 60 days of life. During developmental hematopoiesis, all biallelic mutant combinations aberrant but distinct phenotypes in myeloid and HSPCs. To further understand this we undertook RNA-seq from each mutant genotype combination and identified four genes differentially expressed in opposing vectors in biallelic C-term compared with biallelic N-term mutant fish. In our ongoing work we hypothesise that these genes represent the drivers of the selection pressure towards the biallelic mutations most frequently observed in patients and are conduction an F0 knock-down screen of these genes.