Determining the Effect and Mechanism of EpoR Mutations on Erythroid Cell Differentiation and the Role of FOXO3 on Erythroid Differentiation

Student thesis: Doctoral ThesisDoctor of Philosophy (PhD)

Abstract

Erythropoietin receptor (EpoR) signalling is essential for the proliferation and survival of erythroid progenitors, with a recent report implicating the EpoR in cell cycle regulation in mice 1, but its other functions throughout erythropoiesis, especially human erythropoiesis, remain poorly defined. Studying the role of the EpoR in erythropoiesis is severely hindered by the death of EpoR null erythroid cells due to the critical anti-apoptotic role of EpoR signalling. Hence, a human cellular model is required. In this thesis, BEL-A cells were genome edited to truncate the EpoR, resulting in EpoR membrane retention and consequently hyperactivation, as found in patients with primary familial and congenital polycythaemia (PFCP). This enabled the role of EpoR signalling in erythroid cell cycle and differentiation in humans to be dissected.

BEL-A cells with a truncated EpoR (E6) had increased membrane abundance of the receptor which was thus hyperactivated, reflecting the phenotype of PFCP patients. E6 cells had both a delayed and defective differentiation and their morphology was abnormal, with an increased incidence of bi/multinuclear cells and an increase in cell size beyond the physiological norm, as well as abnormal super-sized cells. Comparative proteomic data revealed the cell cycle was an overall dysregulated picture. Levels of GATA1 and its downstream targets KLF1 and cyclin A2 were decreased in E6 cells, as well as kinesins, all of which contributing to the defective differentiation of E6 cells.

In considering how reported EpoR mutations cause hyperactivation of the EpoR, a recent study has questioned this being solely due to the truncation itself, leading to the loss of residues critical for EpoR internalisation 2. Instead, they report a specific new tail sequence following frameshift mutations to be causative. In accordance, this thesis also demonstrates the reason for EpoR activation was not due to the truncation of the EpoR itself, but due to the presence of a novel C-terminal MDTVP amino acid tail.

A standalone, additional project focused on the role of FOXO3 in β-thalassemia using a human model system. Though a couple of studies implicate FOXO3 in β-thalassemia 3,4, this is the first study to knockdown FOXO3 in β-thalassemia cells. FOXO3 knockdown exacerbated the ineffective erythropoiesis phenotype of β-thalassemia, indicating it plays a critical role in supporting erythroid maturation and survival, especially in β-thalassemia.

Overall, the work described in this thesis reveal a novel role for EpoR signalling in human erythroid cell differentiation and indicates the quaternary structure of the EpoR plays an important role in erythropoiesis. As well, that FOXO3 supports the survival of and maturation of β-thalassemia cells.
Date of Award7 May 2024
Original languageEnglish
Awarding Institution
  • University of Bristol
SupervisorJan Frayne (Supervisor)

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