The Wnt/β-catenin signalling pathway is involved in regulating cellular transcription of numerous target genes in chronic myeloid leukaemia (CML). A previous report using granulocyte-macrophage progenitors (GMPs) showed that elevated levels of unphosphorylated (active) β-catenin, alongside mis-splicing of glycogen synthase kinase 3β (GSK3β), correlated with disease progression. It was of interest to determine if using the more readily available peripheral blood mononuclear cells (MNCs) also showed any correlation with patient outcome when β-catenin and GSK3β were measured in this source material. Further investigation in these cells addressed GSK3β activity regulation, possible regulation of c-Myc degradation by GSK3β, and investigation of Wnt transcription factors in CML. Results indicated that levels of β-catenin (and its phosphorylated variants) could not be used to distinguish patient treatment outcomes using CML MNCs. However the levels of BCR-ABL1 induced β-catenin-Tyr654 phosphorylation showed a significant decrease in patients in blast crisis compared to chronic phase (P=0.012), as did the levels of GSK3β – which demonstrated significantly decreased activity in blast crisis patients via a significant increase in Ser9 phosphorylation and a significant decrease in Tyr216 phosphorylation in blast crisis compared to chronic phase (P=0.026 and <0.001 respectively). This decrease in GSK3β activity was not reflected by changes in the levels of β-catenin and, as such, this led to the question of whether alternative mechanisms were influencing regulation of GSK3β activity other than Wnt/β-catenin signalling. Results showed that protein phosphatase 2A (PP2A) was regulating the inhibition of GSK3β in CML. This is a dual mechanism with GSK3β also affecting PP2A inhibition. GSK3β is known to have numerous substrates, which include c-Myc. Due to the role of c-Myc as an oncogene in CML and the role of GSK3β in c-Myc degradation, it was of interest to determine if the changes in GSK3β activity were reflected in the degradation of c-Myc. Results showed that neither GSK3β nor PP2A (also involved in the mechanism) were the rate limiting components of c-Myc degradation. Analysis of the Wnt signalling pathway revealed up regulation of WNT1, WNT8A, WNT9A and FZD7 mRNAs in chronic phase MNCs (P=0.011, 0.045, 0.037 and 0.037 respectively) - which was inconsistent with β-catenin findings. These unexpected results can be explained by internal regulation of the pathway by the negative regulator NKD1, which is also significantly higher in samples from chronic phase patients (P=0.013). Finally, investigation into Wnt transcription factors showed that factors TCF1, TCF4 and LEF1 mRNA levels were significantly higher in blast crisis samples compared to chronic phase (P=0.001, 0.016, and 0.003 respectively). This suggests a possible mechanism for Wnt-independent activation of these transcription factors in the blast crisis phase of the disease. In conclusion, it was determined that β-catenin levels cannot be used to either; distinguish between patient response cohorts, or be a factor of disease progression in CML MNCs. The activity of GSK3β is significantly down-regulated in blast crisis. PP2A is involved in this regulation. However when investigating c-Myc degradation, neither GSK3β nor PP2A act as the regulating factors in CML. There may be an internal regulation of the pathway by which NKD1 prevents an upregulation of pathway activity via WNT1, WNT8A, WNT9A and FZD7 reaching β-catenin (CTNNB1). Finally, the transcription factors mRNA expression is significantly upregulated in blast crisis, but they could be acting independently of the Wnt pathway.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:664367 |
| Date | January 2014 |
| Creators | Fowler, Rachael |
| Publisher | University of Liverpool |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://livrepository.liverpool.ac.uk/2007740/ |
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