The critical role of histone methylation in tumour progression and as anti-cancer targets in neuroblastoma Matthew Wong A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy Supervisor: Dr Tao Liu Co-supervisor: A. Prof Patsie Polly School of Women’s and Children’s Health Faculty of Medicine February 2016 PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: First name: Matthew Other name/s: Kwok Kei Wong Abbreviation for degree as given in the University calendar: PhD School: School of Women’s and Children’s Heath Faculty: Faculty of Medicine Title: The critical role of histone methylation in tumour progression and as anti-cancer targets in neuroblastoma N-Myc induces neuroblastoma by regulating the expression of target oncogenes. Histone H3 lysine 79 (H3K79) methylation at Myc-responsive elements of target gene promoters is a strict prerequisites for Myc-induced transcriptional activation. DOT1L is the only known histone methyltransferase that catalyses mono-methylation (me), di- methylation (me2) and tri-methylation (me3) at the histone H3K79 position, which have been linked to gene transcriptional activation. JMJD6 is a bi-functional arginine demethylase and lysyl-hydroxylase. The JMJD6 gene is located on the chromosome 17q25 position. 17q21-gter gain has been identified as the most frequent chromosome alternation in neuroblastoma and an indicator of poor patient prognostic. Here, I investigated the roles of DOT1L and JMJD6 in N-Myc over-expressing neuroblastoma. I found that N-Myc up-regulated DOT1L mRNA and protein expression, by binding to an E-box at the DOT1L gene promoter. Knocking-down DOT1L reduced the mRNA and protein expression of the N-Myc target genes, ODC1 and E2F2. DOT1L and N-Myc formed a protein complex, and knocking-down DOT1L reduced histone H3K79me2 and N-Myc protein binding at the promoters of the N-Myc target genes ODC1 and E2F2, and reduced neuroblastoma cell proliferation in vitro and tumour progression in neuroblastoma-bearing mice. In a publicly available microarray gene expression dataset, high levels of DOT1L gene expression in tumours correlated with high levels of MYCN gene expression and poor patient survival independent of MYCN amplification, age at diagnosis and disease stage. I have also demonstrated that JMJD6 up-regulated both N-Myc and c-Myc in neuroblastoma cell lines. Conversely N-Myc and c-Myc did not affect JMJD6 mRNA or protein expression. Knocking down JMJD6 reduced neuroblastoma cell proliferation in vitro and tumour progression in neuroblastoma-bearing mice. JMJD6 gene expression correlated with MYCN gene expression in human neuroblastoma tissue microarray gene expression datasets. High DOT1L gene expression was also a prognostic factor for poor neuroblastoma[Grab your reader’s patient attention outcome. with a great quote from the document or use this space to Inemphasize conclusion, a key thes point.e data To identify place this DOT1L text box as aanywhere novel co -onfactor the page,in N-Myc just dragoncogenesis, it.] and provide critical evidence for the potential utilization of DOT1L inhibitors for the therapy of MYCN amplified neuroblastoma. JMJD6 up-regulates N-Myc and c-Myc gene expression and JMJD6 gene gain is a potential mechanism for 17q21-qter gain driven neuroblastoma tumourigenesis. ii Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). Matthew Wong Pei Yan Liu 1/3/2016 ……………………………………… ……………………………..…………… ……………………………………… Signature Witness Signature Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS iii ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed …………………………………………….............. Date …………………………………………….............. COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed ……………………………………………........................... Date ……………………………………………........................... AUTHENTICITY STATEMENT ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed ……………………………………………........................... Date ……………………………………………........................... Table of Contents List of Figures ......................................................................................................................... vii List of Tables ........................................................................................................................... xi List of Abbreviations ............................................................................................................... x List of publications .................................................................................................................. xi Conference presentations ....................................................................................................... xi Acknowledgement .................................................................................................................. xii Abstract ................................................................................................................................. xiii Chapter 1: Literature review and project aims .................................................................... 1 1.1 Cancer ............................................................................................................................... 1 1.2 Neuroblastoma ................................................................................................................. 2 1.2.1 Neuroblastoma Staging.............................................................................................. 4 1.2.2 Hereditary Neuoblastoma ........................................................................................ 10 1.2.3 Neuroblastoma and anaplastic lymphoma kinase .................................................... 11 1.2.4 Drug resistance in neuroblastoma............................................................................ 12 1.3 C-Myc and N-Myc regulate gene expression ................................................................. 14 1.3.1 Myc mediates transcriptional activation .................................................................. 15 1.3.2 Myc mediates gene repression ................................................................................. 16 1.3.3 Myc regulation of miRNA expression ..................................................................... 18 1.4 N-Myc ............................................................................................................................ 20 1.4.1 N-Myc in embryonic development .......................................................................... 21 1.4.2 N-Myc induces neuroblastoma ................................................................................ 21 1.4.3 Strategies to target