Identification and Functional Analysis of Gene Expression Changes in Acute Myeloid Leukaemia KOK Chung Hoow A thesis submitted in fulfilment of the requirement for the degree of Doctor of Philosophy in the School of Paediatrics and Reproductive Health at the University of Adelaide August 2010 Table of Contents List of Figures ……………………………………………………………………………..... i List of Tables ………………………………………………………………………………...v List of Appendix …………………………………………………………………………....vii Abbreviations ……………………………………………………………………………...viii Abstract …………………………………………………………………………………….xii Declaration …………………………………………………………………………………xiv Acknowledgement ………………………………………………………………………….xv Chapter 1: Introduction......................................................................................................1 1.1 Acute Myeloid Leukaemia ................................................................................................. 1 1.1.1 The classification and the prognostic outcome of AML .................................................. 1 1.1.2 Targeted therapies on AML ............................................................................................. 7 1.2 Haemopoiesis: interplay between growth factor signalling and lineage-specific transcription factors ...................................................................................................................... 12 1.2.1 The importance of growth factors in haemopoiesis ....................................................... 12 1.2.2 Transcription factors that determine haemopoietic cell fates......................................... 16 1.3 AML biology and pathogenesis ....................................................................................... 18 1.3.1 Genetic alteration of transcription factors in AML ........................................................ 21 1.4 Leukaemic stem cell.......................................................................................................... 22 1.5 Receptor signalling in haemopoiesis ............................................................................... 24 1.5.1 IL-3/IL-5/GM-CSF receptor........................................................................................... 24 1.5.2 FMS-like tyrosine kinase 3 receptor (FLT3).................................................................. 25 1.5.3 c-Kit receptor..................................................................................................................27 1.6 Activated receptor mutants in AML............................................................................... 28 1.6.1 Constitutive activation of GMR induces AML .............................................................. 28 1.6.1.1 GMR-V449E mutation in common beta chain................................................................... 28 1.6.1.2 A critical motif in hc regulates proliferation and survival................................................ 30 1.6.2 FLT3............................................................................................................................... 31 1.6.2.1 FLT3-ITD mutation............................................................................................................ 32 1.6.2.2 FLT3-TKD mutations......................................................................................................... 34 1.6.3 The c-Kit-TKD mutation................................................................................................ 35 1.7 Downstream signal transducers ...................................................................................... 36 1.7.1 The PI3K/AKT/mTOR pathway .................................................................................... 36 1.7.2 RAS/MAPK/ERK1/2 signalling..................................................................................... 37 A 1.7.3 JAK/STAT signalling..................................................................................................... 39 1.8 Application of gene expression profiling technology to AML ...................................... 40 1.8.1 Gene expression profiling for diagnostic and prognostic predictions ............................ 41 1.8.2 Gene expression profiling in target-based drug discovery ............................................. 43 1.9 Cell line models to study AML ........................................................................................ 44 1.9.1 A cell line model of granulocyte-monocyte growth and differentiation, FDB1............. 45 1.10 Aims of the project............................................................................................................ 46 1.10.1 Overall Aims:............................................................................................................. 46 1.10.1.1 Specific aims: ..................................................................................................................... 46 Chapter 2: Regulation of myeloid proliferation, differentiation and survival signals by the human GM-CSF/IL-3/IL-5 common beta chain .....................................................47 2.1 Introduction ...................................................................................................................... 47 2.2 Materials and methods..................................................................................................... 49 2.2.1 Reagents ......................................................................................................................... 49 2.2.2 Antibodies ......................................................................................................................49 2.2.3 Cell lines and culture conditions .................................................................................... 50 2.2.4 Flow cytometry .............................................................................................................. 50 2.2.5 Differentiation, cell viability, apoptosis and proliferation assays .................................. 51 2.2.6 Cell cycle analysis.......................................................................................................... 51 2.2.7 Western blotting ............................................................................................................. 52 2.2.8 Bioinformatics analysis .................................................................................................. 52 2.2.8.1 Gene-set enrichment analysis using the Wilcoxon rank sum test....................................... 52 2.2.8.2 Microarray dataset re-analysis............................................................................................ 53 2.2.8.3 Connectivity map, pathway and gene ontology analysis.................................................... 53 2.2.8.4 Transcription factor prediction using microarray significant gene-set ............................... 54 2.2.9 Statistical analysis .......................................................................................................... 54 2.3 Results................................................................................................................................ 54 2.3.1 V449E Y577F cells fail to proliferate but maintain viability......................................... 54 2.3.2 The V449E Y577F signature: A proliferation-associated signature .............................. 59 2.3.3 The Connectivity Map (CMAP) as a tool to explore the nature of the V449E proliferation signature ................................................................................................................. 68 2.3.4 Experimental validation of CMAP results ..................................................................... 72 2.3.5 Treatment of GMR-V449E cells with compounds identified from the CMAP analysis 74 2.3.5.1 Treatment with the PI3K-AKT-mTOR pathway inhibitors................................................ 74 2.3.5.2 Effects of the pathway inhibitors on survival of V449E FDB1.......................................... 75 2.3.5.3 Effect of pathway inhibitors on cell cycle status of V449E FDB1 cells............................. 76 2.3.5.4 Effects of pathway inhibitors on myeloid differentiation of V449E FDB1 cells................ 77 B 2.3.6 Gene-set enrichment analysis (GSEA) of the V449E proliferation signature in AML.. 80 2.3.7 Analysis of the hc Ser585 Signature – a signature associated with survival-only signalling ..................................................................................................................................... 83 2.3.8 Relevance of the survival-only signature to AML ......................................................... 87 2.4 Discussion .......................................................................................................................... 87 Chapter 3: Mechanisms associated with FLT3 mutations in AML..............................98 3.1 Introduction ...................................................................................................................... 98 3.2 Materials and Methods................................................................................................... 100 3.2.1 Reagents ....................................................................................................................... 100 3.2.2 Cell lines....................................................................................................................... 100 3.2.3 Cell viability................................................................................................................