Acute myeloid leukaemia (AML) is a heterogeneous clonal disorder that affects the development of haematopoietic cells and has an incidence of 2300 cases / year in the UK. Whilst treatment with conventional cytotoxic agents has significantly increased cure rates in AML, only 35-50% patients under the age of 60 years will be long term survivors. There is a need to develop novel agents targeting molecular lesions or dysregulated pathways to improve clinical responses. This study identified commonly dysregulated pathways and genes using Affymetrix gene expression profiling of AML patients. Analysis of gene expression data using MetacoreTM online gene ontology pathway analysis identified WNT proteins to be one of the most frequently dysregulated processes associated with AML. TCF7L2 was the most significantly dysregulated WNT transcription factor gene and was subsequently examined in greater detail. The expression of TCF7L2 was validated by RQPCR. These data significantly correlated with the normalised array expression data (R=0.748; P<0.01) which in turn correlated with overall protein expression determined and quantified by western blotting. TCF7L2 mRNA overexpression was found to be independently prognostic for reduced complete remission rate (P<0.05), OR=5.19 [95% C.I.=1.39 - 19.39]. The TCF7L2 gene undergoes exon splicing which yields multiple isoforms which have been reported to yield functionally distinct proteins. TCF7L2 mRNA isoform expression in AML patients was compared with that in normal human bone marrow and a human cord blood derived from CD34+ haematopoeitic progenitor cells. Extensive variability of TCF7L2 splicing at the 3’ end of the gene (exons 13-18) was identified. AML patients were heterogeneous in the isoform expression pattern, but aberrant exon composition (compared to normal cells) was not observed. At the protein level, expression of the 58 kDa isoform (exons 1-14 and 18) and 56 kDa isoform (exons 1-13 and 18) were detected. In both normal and AML cells, expression of the 56 kDa and 58 kDa isoforms were dominant. To determine the functional significance of TCF7L2 overexpression lentiviral shRNA vectors targeting TCF7L2 was transduced in leukaemic cell lines coexpressing a TCF-GFP reporter which enabled simultaneous analysis of the effect on TCFdependent transcription. Reporter activity was inhibited by shRNA vectors targeting TCF7L2, and the cells became non-responsive to WNT agonists (WNT3A and BIO) demonstrating that canonical WNT signalling is dependent on TCF7L2 expression in these cells. Phenotypically, shRNA expression was found to strongly inhibit proliferation and to promote apoptosis indicating that TCF7L2 expression is required for the proliferation and survival of myeloid leukaemia cells. Paradoxically, overexpression of individual TCF7L2 isoforms (72 kDa, 58 kDa and 56 kDa) suppressed WNT agonist responses in myeloid leukaemia cell lines but not in epithelial cells. Overexpression of TCF7L2 in normal CD34+ cells was found to promote monocytic differentiation. In summary, this study presents novel data identifying WNT signalling as the most commonly dysregulated process in AML. TCF7L2, a WNT transcription factor was found to be significantly overexpressed in AML and associated with poor clinical outcome. This protein was found to be required to maintain the proliferation and viability of myeloid leukaemia cells suggesting that targeting TCF7L2 maybe a valid approach in AML therapy.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:600599 |
| Date | January 2014 |
| Creators | Daud, Siti Sarah |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/57554/ |
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