FUNCTIONAL PROTEOMIC ANALYSIS OF LEUKAEMOGENIC PROTEIN TYROSINE KINASE TARGETS

UoM administered thesis: Unknown

  • Authors:
  • Norhaida Che Azmi

Abstract

Myeloproliferative neoplasms (MPNs) are clonal proliferative disorders associated with JAK2 mutation (e.g JAK2 K539L, JAK2 V617F), MPL mutation (e.g MPL W515L) or product from reciprocal chromosomal translocations in many cases (e.g BCR/ABL). The mutated thrombopoietin receptor MPL W515L found in thrombocytosis and myelofibrosis is constitutively activated leading to a downstream signal transduction cascade activation including the JAK-STAT signalling pathway. MPL W515L induced JAK2 mutation is associated with polycythaemia vera. Using quantitative proteomics I have investigated the effects of the MPL W515L oncogene on the proteome. This was performed to delineate specific features of MPL W515L action with a view to identifying new therapeutic targets for MPN patients. Within the proteins identified as being differentially expressed as a consequence of MPL W515L expression I observed an enrichment of proteins involved in motility. This was associated with a MPL W515L induced increase in chemokinesis. Further investigation into this altered chemokinesis elucidated a pathway from CXCL12/CXCR4/CD45 mediated Src activation through to THOC5 Y225 phosphorylation that had been compromised by MPL W515L. The MPL W515L induced THOC5 phosphorylation was linked to elevated MYC expression. Either chemical inhibition of MYC or gene silencing reduced both the level of THOC5 Y225 phosphorylation and also the increased chemokinesis. Of interest, because of its reported role in both myelofibrosis and motility, the MPL W515L expressing cells were found to demonstrate increased release of transforming growth factor beta (TGFβ). I demonstrated that TGFβ stimulates the phosphorylation of THOC5. Via the expression of Y225F mutants of THOC5 and the chemical inhibition of TGFβ I show a role for this elevated TGFβ in the increased chemokinesis of MPL W515L expressing cells. TGFβ has been reported to upregulate sphingosine-1-phosphate (S1P) which contributes to fibrosis. Having previously published on the differential effects of S1P on the motility of HSC populations I investigated the potential role of S1P in the MPL W515L induced chemokinesis. Inhibition of sphingosine kinase reduced the increase in chemokinesis and THOC5 Y225 phosphorylation in MPL W515L expressing cells. Furthermore I demonstrated that MPL W515L expression led to an increase in the intracellular levels of S1P suggesting a role for S1P in MPN. To further understand the role of THOC5 phosphorylation in the increased chemokinesis I undertook a discovery proteomics screen of MPL W515L cells co-expressing either wild type or Y225F mutant THOC5. Enhancer zester homolog 2 (EZH2) was shown to increase in MPL W515L as compared to MPL W515L mutant THOC5 Y225F expressing and control cells and as such may be linked to the increases in chemokinesis observed. Present work is aimed at clarifying the role of EZH2 in chemokinesis. In conclusion I have identified a novel pathway disrupted in MPN and allow me to start to understand the mechanisms by which the phosphorylation of THOC5 may contribute to leukaemogenic transformation through links to TGFβ, MYC, and S1P biology.

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Original languageEnglish
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Award date31 Dec 2015