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Abuhantash, Mays (2023) Targeting HOXA in engineered iPSCs and pre-clinical leukaemia models. PhD thesis, University of Nottingham.

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Abstract

Haematopoiesis is a highly regulated process governed by a complex network of transcription factors and signalling molecules. HOXA genes are master regulators of embryonic development and haematopoiesis. In normal haematopoiesis, expression of HOXA is high in haematopoietic stem and progenitor cells (HSPCs) and decreases upon differentiation and maturation. Aberrant expression of the HOXA cluster, particularly HOXA9, is associated with malignant haematopoiesis.

To enable routine and efficient production of iPSC-derived HSPCs, two haematopoietic differentiation systems mimicking the stages of haematopoietic development and specification in vivo were tested. The monolayer-based differentiation was improved using a single-cell derived approach. Embryoid body (EB) differentiation was found to be more efficient and reproducible. Cells were characterised at key stages of the process by flow cytometry, qRT-PCR and methylcellulose colony assays. To monitor HOXA9 expression, an endogenous HOXA9-mScarlet reporter (eA9m) iPSC line was generated using CRISPR/Cas9 system and validated by Sanger sequencing. The functionality of eA9m was assessed using EB differentiation and confocal microscopy. Fluctuations in HOXA9 expression were coincident with mScarlet-H expression, particularly at early stages of the EB differentiation. Proof-of-principle studies revealed that exposure of eA9m iPSCs or EBs to oncogenic MLL::AF9 results in deregulation of key haematopoietic genes, including HOXA genes.

In malignant haematopoiesis, MLL-rearranged leukaemias are a hallmark for aggressive paediatric blood cancer. MLL fusion proteins, particularly MLL::AF9, contribute to disease pathogenesis through upregulation of transcription factors, in particular HOXA9, which is associated with poor clinical outcome and a high incidence of relapse. A HOXAdel signature from a conditional transgenic mouse model was used to identify and validate potential anti-HOXA FDA-approved drugs. These drugs were found to possess variable anti-leukaemic properties in vitro, as shown by RNA sequencing analysis which should be further investigated. This highlights the importance of using advanced models for drug discovery. Thus, eA9m iPSCs can be used to generate isogenic models of diseases involving dysregulated expression of HOXA9 such as leukaemia.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Thompson, Alexander Seedhouse, Claire
Keywords:	Haematopoiesis; Transcription factors; Isogenic models of disease; Leukaemia cell lines; Anti-HOXA drugs; Haematopoietic stem and progenitor cells
Subjects:	W Medicine and related subjects (NLM Classification) > WH Hemic and lymphatic system
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID:	73906
Depositing User:	Abuhantash, Mays
Date Deposited:	31 Jul 2023 04:41
Last Modified:	31 Jul 2023 04:41
URI:	https://eprints.nottingham.ac.uk/id/eprint/73906

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