Characterisation of functional validation of ERG phosphorylation in normal haematopoietic and leukaemic cells

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Embargoed until 2017-12-31
Copyright: Huang, Yizhou
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Abstract
Direct modulation of oncogenic transcriptional programs by targeting aberrantly regulated transcription factors is a promising area of research for cancer treatment. The ETS factor ERG (ETS-related gene) plays an important role in haematopoiesis and is also a potent oncoprotein with leukaemogenic activity in mouse models. In humans, high ERG expression is associated with poor patient outcomes in acute myeloid leukaemia (AML) and T-cell acute lymphoblastic leukaemia (T-ALL). However, gaps still exist in our knowledge regarding the post-translational regulation of ERG. Protein phosphorylation is known to regulate the transcriptional activity of many ETS factors. Yet, as a known phosphoprotein, how ERG activity responds to phosphorylating signalling pathways that are associated with the onset and progression of leukaemia is largely unknown. To unravel the post-translational regulation of ERG, I used liquid chromatography coupled tandem mass spectrometry to identify five phosphorylated serines (S) (S55, S88, S103, S222, S283) on endogenous ERG immunoprecipitated from MOLT-4 T-ALL cells with S283 phosphorylation (pS283) strongly enriched in leukaemic cells compared with healthy haematopoietic stem/progenitor cells (HSPCs). Generation of a customised anti-ERG pS283 antibody to probe upstream signalling pathways in primary ALL and AML xenografts identifies early T-cell precursor ALL cells with poor prognosis to exhibit particularly high levels of pS283, and there was a direct association between levels of pS283 and active extracellular signal-regulated kinase (ERK). Over-expression of phosphomimetic ERG mutant (S283D) enhanced the expansion and clonogenicity of transduced primary HSPCs more than wild-type (WT) ERG. This phenotype was associated with induction of genes involved in mitogen-activated protein kinase (MAPK)/ERK signalling. Further experiments showed ERG pS283 was directly modulated by ERK, consistent with the existence of a positive feedback loop that stabilised this modification in leukaemic cells. There were no substantial differences between WT and mutant ERG with regards to protein stability, nuclear transfer or direct DNA binding, however, there was increased enrichment of S283D ERG at specific leukaemia-associated enhancers. This work significantly expands existing knowledge of ERG phosphorylation in leukaemic cells and identifies a specific modification that could be targeted to modulate ERG-driven leukaemic transcriptional programs.
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Author(s)
Huang, Yizhou
Supervisor(s)
Pimanda, John
Wong, Jason
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Publication Year
2015
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Thesis
Degree Type
PhD Doctorate
UNSW Faculty
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