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Regulation of BCR-ABL Expression Via Its 3' Untranslated Region

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Regulation of BCR-ABL expression via its 3’untranslated region Bradley Chereda A thesis submitted for the degree of Doctor of Philosophy to the University of Adelaide December 2012 School of Medicine Leukaemia Biology Group Faculty of Health Sciences Centre for Cancer Biology University of Adelaide IMVS, SA Pathology ii Declaration Declaration This work contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution to Bradley Chereda and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. I give consent to this copy of my thesis, when deposited in the University Library, being made available for loan and photocopying, subject to the provisions of the Copyright Act 1968. I also give permission for the digital version of my thesis to be made available on the web, via the University‟s digital research repository, the Library catalogue, the Australasian Digital Theses Program (ADTP) and also through web search engines, unless permission has been granted by the University to restrict access for a period of time. Bradley Chereda December 2012 iii iv Table of Contents Table of contents Declaration.................................................................................................................................................... III Table of contents........................................................................................................................................... V Abstract....................................................................................................................................................... XII Acknowledgements................................................................................................................................... XIII Abbreviations.............................................................................................................................................. XV Chapter 1 .......................................................................................................................................................... 1 Chronic myeloid leukaemia .............................................................................................................................. 1 1.1 Preface ....................................................................................................................................................... 2 1.2 Chronic myeloid leukaemia ........................................................................................................................ 2 1.3 Disease pathogenesis ................................................................................................................................ 3 1.3.1 Chronic phase .................................................................................................................................. 4 1.3.2 Blast crisis ......................................................................................................................................... 4 1.3.3 Treatment ........................................................................................................................................ 4 1.4 BCR-ABL ..................................................................................................................................................... 5 1.4.1 BCR-ABL signalling in disease ........................................................................................................... 7 1.4.1.1 Proliferation and apoptosis .................................................................................................... 8 1.4.1.2 Genomic instability ................................................................................................................. 9 1.4.2 Expression patterns of BCR-ABL in CML ......................................................................................... 10 1.4.3 BCR-ABL dosage ............................................................................................................................. 12 1.4.3.1 Association with blast crisis .................................................................................................. 12 1.4.3.1.1 Dose-dependent downregulation of C/EBPα .................................................................. 12 1.4.3.1.2 Dose-dependent inhibition of DNA repair ....................................................................... 13 1.4.3.2 BCR-ABL levels and imatinib resistance ................................................................................ 13 1.4.3.3 BCR-ABL expression and TKI-sensitivity in leukaemic stem cells .......................................... 14 1.4.4 Control of BCR-ABL expression ...................................................................................................... 15 1.4.4.1 The BCR Promoter ................................................................................................................ 16 1.4.4.2 DNA/histone modifications relative to BCR-ABL .................................................................. 17 1.4.4.3 The BCR-ABL 3’UTR ............................................................................................................... 18 1.4.4.4 BCR-ABL is abnormal ............................................................................................................ 18 1.4.4.5 The key regulators of BCR-ABL expression are known, now what? ..................................... 19 1.5 Specific aims ............................................................................................................................................ 21 v Table of Contents Chapter 2 ....................................................................................................................................................... 22 Characterisation of the ABL1 3’UTR ............................................................................................................... 22 2.1 Introduction............................................................................................................................................. 23 2.1.1 The 3’UTR ...................................................................................................................................... 23 2.1.1.1 The PAS ................................................................................................................................ 24 2.1.1.2 MicroRNAs ........................................................................................................................... 24 2.1.1.3 RNA-binding proteins ........................................................................................................... 25 2.1.1.4 3’UTR avoidance .................................................................................................................. 25 2.1.2 The BCR-ABL 3’UTR ........................................................................................................................ 28 2.1.2.1 What is known about the BCR-ABL 3’UTR ........................................................................... 28 2.1.2.2 3’UTR shortening of ABL1 .................................................................................................... 31 2.1.2.3 Investigating the BCR-ABL 3’UTR ......................................................................................... 31 2.2 Methods .................................................................................................................................................. 33 2.2.1 Renilla reporter constructs ............................................................................................................ 33 2.2.1.1 ABL1 3’UTR reporters (PCR-based) ...................................................................................... 34 2.2.1.1.1 PCR .................................................................................................................................. 34 2.2.1.1.2 Restriction digest cloning ................................................................................................ 36 2.2.1.2 ABL1 3’UTR reporters (restriction site-based) ..................................................................... 36 2.2.1.3 ABL1 3’UTR reporters (restriction site insertion-based) ...................................................... 36 2.2.1.4 Blunt-end cloning: phRL-ABL1 3’UTR (1-840) ...................................................................... 39 2.2.1.5 Duplex insertion: phRL-FL_ABL1 (PAS) and phRL-FL_ABL1 (SAP) ........................................ 39 2.2.1.5.1 Oligo-duplex annealing ................................................................................................... 40 2.2.1.5.2 Restriction digest cloning ................................................................................................ 40 2.2.1.6 phRL-ABL1 (1116-1257) and phRL-ABL1 (1116-1257)ΔTTP ................................................. 41 2.2.1.7 Transformation .................................................................................................................... 42 2.2.1.8 DNA plasmid prep ................................................................................................................ 42 2.2.1.9 Plasmid amplification ..........................................................................................................
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