Investigation of the role and mechanism of beta-catenin activation in acute myeloid leukaemia.

Abstract

Aberrant activation of β-catenin is a common event in Acute Myeloid Leukaemia (AML), and accumulating evidence indicates this pathway plays a critical role in the establishment and maintenance of myeloid neoplasms. In AML, increased β-catenin signalling has been associated with activating mutations in the FLT3 receptor, and the oncogenic AML1-ETO and PML-RARα translocation products. In the absence of these lesions, however, it remains unclear which mechanisms may activate β-catenin in AML more broadly. Here we have explored a potential role for the multipotent haematopoietic cytokine, interleukin-3 (IL-3) in the regulation of β-catenin signalling in myeloid and leukaemic cells. We show that IL-3 can induce the dose dependent stabilisation of β-catenin in a myeloid model of Hox oncogenesis, and that β-catenin is required for IL-3 driven colony formation and growth. Enforced expression of β-catenin in this system allows cell survival at sub-optimal concentrations of IL-3 which may contribute to leukaemic transformation by providing a survival advantage to blast cells in the haematopoietic niche. We also demonstrate that IL-3 can promote β-catenin activation in the IL-3 dependent human erythroleukaemia cell line, TF-1.8, and in primary AML cells. Furthermore, Affymetrix gene expression analysis of bone marrow cells from four AML patients treated ± IL-3 revealed a strong correlation between the IL-3 induced signature and Wnt/β-catenin gene networks. Interestingly, the IL-3 receptor alpha subunit (IL-3Rα) has been previously shown to be overexpressed in AML leukaemic stem cells and progenitors compared to normal counterparts, and elevated levels of IL-3Rα are associated with poor prognosis and overall survival. Consistent with the regulation of β-catenin by IL-3, we show that a neutralising monoclonal antibody (7G3) which targets IL-3Rα, inhibits IL-3 mediated activation of β-catenin in TF-1.8 and primary AML cells. Modified versions of 7G3 are currently undergoing clinical trials for patients with AML, and our data indicates that this therapy may be more effective for patients with elevated levels of oncogenic β-catenin. As previous studies have demonstrated that cytokines can induce the inhibitory phosphorylation of GSK3β via activation of the PI3K/AKT pathway, we have also made use of pharmacological PI3K and AKT small molecule inhibitors to determine the importance of this axis in the IL-3 mediated regulation of β-catenin. On the whole, the work in this thesis reveals a novel mechanism which may contribute to β-catenin activation in AML, and provides further insight into the amplitude of IL-3 signalling in normal and malignant haematopoiesis.