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Characterisation and Functional Validation of ERG Phosphorylation in Normal Haematopoietic and Leukaemic Cells

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Characterisation and Functional Validation of ERG Phosphorylation in Normal Haematopoietic and Leukaemic Cells Characterisation and Functional Validation of ERG Phosphorylation in Normal Haematopoietic and Leukaemic Cells Yizhou Huang A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy Prince of Wales Clinical School Faculty of Medicine September 2015 Huang: ERG Phosphorylation and Leukaemogenesis 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 ii Huang: ERG Phosphorylation and Leukaemogenesis 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. iii Huang: ERG Phosphorylation and Leukaemogenesis Acknowledgements First and foremost my heart-felt thanks go to my parents, Wei Huang and Yingwei Liu, whose unconditional support and patience made my overseas study possible ever since 2007. Heart-warming love also goes to my husband Han Shen, who is without doubt one of the greatest treasures I found in the Lowy Cancer Research Centre, and has always been holding my hands during the ups and downs throughout my PhD. My gratitude goes to my supervisors John Pimanda, Jason Wong and Julie Thoms for their advice and guidance, and for their constant encouragement and help in preparing this thesis. Julie Thoms offered enormous help in revising the literature review, and more importantly, she has always had faith in me during my candidature. My friends and co-workers in the Pimanda lab have been particularly wonderful. They were always ready to help, and provided a truly collegial environment to work in. Their support, funny stories and laughter brought a smile to my face even in the most arduous moments. “Thank you” cannot be said enough to Julie Thoms, Melinda Tursky and Kathy Knezevic for keeping me company during my major emotional breakdowns, whose comforting hugs I will always remember. Special thanks also go to my dear friend and my lovely desk neighbour Kate Yifang Guan, who has always been there to share the joyful and upset moments in our “nerdy” lives and has made my PhD a truly colourful journey. iv Huang: ERG Phosphorylation and Leukaemogenesis Thank you to Julie Thoms, Melinda Tursky and Kathy Knezevic for teaching me experimental techniques and data analysis, to Dominik Beck and Julie Thoms for the bioinformatic analysis, to Santi Suryani for the protocols and tricks in handling primary leukaemic cells, and to Jake Olivier for his help and advice with statistical analysis. Additional thanks go to the people mentioned in each research chapter and the Contributions chapter. My gratitude goes to the University of New South Wales (UNSW) Australia for the University International Postgraduate Scholarship and the Translational Cancer Research Network for the top-up scholarship. Thank you also to UNSW Australia Postgraduate Research Support Scheme and my supervisors for the funding which allowed me to present at conferences. v Huang: ERG Phosphorylation and Leukaemogenesis Publications Wong JJL, Ritchie W, Ebner OA, Selbach M, Wong JWH, Huang Y, Gao D, Pinello N, Gonzalez M, Baidya K, Thoeng A, Khoo TL, Bailey CG, Jolst J, Rasko JEJ, “Orchestrated intron retention regulates normal granulocyte differentiation.” Cell, 2013; doi:10.1016/j.cell.2013.06.052 Tursky ML, Beck D, Thoms JAI, Huang Y, Kumari A, Unnikrishnan A, Knezevic K, Evans K, Richards LA, Lee E, Morris J, Goldberg L, Izraeli S, Wong JWH, Olivier J, Lock RB, MacKenzie KL, Pimanda JE, “Over-expression of ERG in cord blood progenitors promotes expansion and recapitulates molecular signatures of high ERG leukaemias.” Leukaemia, 2014; doi: 10.1038/leu.2014.299. Thoms JAI, Knezevic K, Liu J, Glaros E, Thai T, Qiao Q, Huang Y, Papathanasiou P, Tunningley R, Whittle B, Yeung A, Chandrakanthan V, Wong JWH, Ward R, Thomas S, Pimanda JE. “Arrested Haematopoiesis and Vascular Relaxation Defects in Mice with a Mutation in Dhfr.” Blood, 2015. (Submitted manuscript) Unnikrishnan A, Guan Y, Thoms JAI, Huang Y, Knezevic K, Beck D, Wong JWH, Pimanda JE, “A quantitative proteomics approach identifies new transcriptional regulators of ERG.” Nucleic Acid Research, 2015. (Submitted manuscript) vi Huang: ERG Phosphorylation and Leukaemogenesis Presentations Huang Y, Thoms JAI, Tursky ML, Knezevic K, Chandrakanthan V, Suryani S, Lock R, MacKenzie K, Wong JWH, Pimanda JE. “ERG phosphorylation in normal haematopoietic and leukaemic cells”. Prince of Wales Clinical School Postgraduate Research Seminar, 2013. Oral presentation. Huang Y, Thoms JAI, Tursky ML, Knezevic K, Chandrakanthan V, Suryani S, Lock R, MacKenzie K, Wong JWH, Pimanda JE. “Leukaemogenic potential of ERG is mediated by phosphorylation via the MAPK/ERK pathway”. New Directions in Leukaemia Research (NDLR), 2014. Poster presentation. Huang Y, Thoms JAI, Tursky ML, Knezevic K, Chandrakanthan V, Suryani S, Lock R, MacKenzie K, Wong JWH, Pimanda JE. “Leukaemogenic potential of ERG is mediated by phosphorylation via the MAPK/ERK pathway”. Lorne Genome, 2014. Poster presentation. Huang Y, Thoms JAI, Tursky ML, Knezevic K, Chandrakanthan V, Suryani S, Lock R, MacKenzie K, Wong JWH, Pimanda JE. “Leukaemogenic potential of ERG is mediated by phosphorylation via the MAPK/ERK pathway”. Prince of Wales Clinical School Postgraduate Research Seminar, 2013. Poster presentation. vii Huang: ERG Phosphorylation and Leukaemogenesis Abbreviations ALL Acute lymphoblastic leukaemia AML Acute myeloid leukaemia AMKL Acute megakaryocytic leukaemia APML Acute promyelocytic leukaemia ATP Adenosine triphosphate BM Bone marrow BMIF Biomedical imaging facility BRIL Biological resources imaging laboratory BSA Bovine serum albumin BTK Bruton’s tyrosine kinase CB Cord blood CDK Cyclin-dependent kinase CFU Colony forming unit ChIP Chromatin immunoprecipitation CK Casein kinase CMP Common myeloid progenitors CLP Common lymphoid progenitor Ct Threshold cycle DDA Data dependent acquisition DMEM Dulbecco's modified Eagle’s medium DMSO Dimethyl sulfoxide dNTP Deoxynucleoside triphosphate DP Distal promoter DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid EMSA Electromobility shift assay EPO Erythropoietin ERG ETS-related gene ERK Extracellular signal-regulated kinase ES Enrichment score ETP Early T-cell precursor ETS E-twenty six EWS Ewing sarcoma EWSBR Ewing sarcoma breakpoint region FACS Fluorescence activated cell sorting FBS Foetal bovine serum FLI1 Friend Leukaemia virus Integration site 1 Flt3L FMS-like tyrosine kinase 3 ligand FSC Forward scatter viii Huang: ERG Phosphorylation and Leukaemogenesis FP Fluorescence polarisation FT Fourier Transform GATA Globin transcription factor G-CSF Granulocyte-colony stimulating factor GFOLD Generalised fold change GFP Green fluorescence protein GMP Granulocyte-macrophage progenitors GRB2 Growth factor receptor-bound protein 2 GREAT Genomic regions enrichment of annotations tool GSEA Gene set enrichment analysis GST Glutathione S-transferase HBSS Hanks’ balanced salt solution HEK Human embryonic kidney HF High fidelity HPC Haematopoietic progenitor cell/s HREC Human Research Ethics Committee HRP Horseradish peroxidase HSC Haematopoietic stem cell HSPC Haematopoietic stem and progenitor cell IL Interleukin IMDM Iscove's modified Dulbecco's medium IPTG Isopropyl β-D-1-thiogalactopyranoside IRES Internal ribosomal entry site ITC Isothermal titration
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