The orphan nuclear receptor EAR-2 (NR2F6) is a gene that I previously found to be expressed at a higher level in clonogenic leukemia single cells than in leukemia cells that can not divide. For this thesis I undertook to perform the first investigations of the roles EAR-2 may play in normal haematopoiesis and in the pathogenesis of acute myelogenous leukaemia. Here, I show that EAR-2 is overexpressed in the bone marrow of patients with MDS, AML and CMML compared to healthy controls and that EAR-2 is a gatekeeper to hematopoietic differentiation. Over-expression of EAR-2 prevents the differentiation of cell lines, while knock down induces their spontaneous differentiation. In vitro, primary bone marrow cells that over-express EAR-2 do not differentiate into granulocytes in suspension culture, but have greatly extended replating capacity in colony assays. In vivo, overexpression of EAR-2 in a chimeric mouse model leads to a condition that resembles myelodysplastic syndrome characterised by hypercellular bone marrow, an increase in blasts, abnormal localization of immature progenitors, morphological dysplasia of the erythroid lineage and a competitive advantage over wild-type cells, that eventually leads to AML in a subset of the mice. Furthermore, animals that are transplanted with grafts of sorted bone marrow develop a rapidly fatal leukemia that is characterized by pancytopenia, enlargement of the spleen, infiltration of blasts into the spleen, liver and peripheral blood. Interestingly, development of leukemia is preceded by expansion of the stem cell compartment. Overexpression of EAR-2 increases the maintenance of KSL primitive bone marrow cells in ex vivo suspension culture, while knockdown of EAR-2 induces rapid differentiation of KSL cells into granulocytes. These data establish that EAR-2 is a novel oncogene that regulates hematopoietic cell differentiation. Furthermore, I show that EAR-2 is also a novel negative regulator of T-cell lymphopoiesis, and demonstrate that down-regulation of EAR-2 is important for the survival, proliferation and differentiation of T-cell progenitors. Overall, this work establishes that expression of EAR-2 is an important determinant of cell fate decisions in the hematopoietic system.

The orphan nuclear receptor EAR-2 (NR2F6) is a gene that I previously found to be expressed at a higher level in clonogenic leukemia single cells than in leukemia cells that can not divide. For this thesis I undertook to perform the first investigations of the roles EAR-2 may play in normal haematopoiesis and in the pathogenesis of acute myelogenous leukaemia. Here, I show that EAR-2 is overexpressed in the bone marrow of patients with MDS, AML and CMML compared to healthy controls and that EAR-2 is a gatekeeper to hematopoietic differentiation. Over-expression of EAR-2 prevents the differentiation of cell lines, while knock down induces their spontaneous differentiation. In vitro, primary bone marrow cells that over-express EAR-2 do not differentiate into granulocytes in suspension culture, but have greatly extended replating capacity in colony assays. In vivo, overexpression of EAR-2 in a chimeric mouse model leads to a condition that resembles myelodysplastic syndrome characterised by hypercellular bone marrow, an increase in blasts, abnormal localization of immature progenitors, morphological dysplasia of the erythroid lineage and a competitive advantage over wild-type cells, that eventually leads to AML in a subset of the mice. Furthermore, animals that are transplanted with grafts of sorted bone marrow develop a rapidly fatal leukemia that is characterized by pancytopenia, enlargement of the spleen, infiltration of blasts into the spleen, liver and peripheral blood. Interestingly, development of leukemia is preceded by expansion of the stem cell compartment. Overexpression of EAR-2 increases the maintenance of KSL primitive bone marrow cells in ex vivo suspension culture, while knockdown of EAR-2 induces rapid differentiation of KSL cells into granulocytes. These data establish that EAR-2 is a novel oncogene that regulates hematopoietic cell differentiation. Furthermore, I show that EAR-2 is also a novel negative regulator of T-cell lymphopoiesis, and demonstrate that down-regulation of EAR-2 is important for the survival, proliferation and differentiation of T-cell progenitors. Overall, this work establishes that expression of EAR-2 is an important determinant of cell fate decisions in the hematopoietic system.