Epigenetic Regulation of Acute Myeloid Leukaemic Stem Cells Halina Hoi Laam Leung Bachelor of Medicinal Chemistry Advanced (Honours Class 1) A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Children‘s Cancer Institute Australia for Medical Research School of Women‘s and Children‘s Health Faculty of Medicine University of New South Wales November 2015 PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Leung First name: Halina Other name/s: Hoi Laam Abbreviation for degree as given in the University calendar: PhD School: Women’s and Children’s Health Faculty: Medicine Title: Epigenetic Regulation of Acute Myeloid Leukaemic Stem Cells Abstract 350 words maximum: (PLEASE TYPE) Acute myeloid leukaemia (AML) is a heterogeneous blood disease with high relapse rates. A small population of highly drug resistant leukaemic stem cells (LSC) persists after chemotherapy, causing disease relapse. This emphasises the need to develop novel LSC-targeted therapies. While increasing evidence has emerged in recent years highlighting the role of epigenetic regulators in cancer development and maintenance, knowledge in epigenetic regulation of LSC is limited. This study investigated the role of two novel histone demethylases, Jmjd1c and Jmjd5, in regulating LSC, as well as the effectiveness of currently available epigenetic agents on leukaemogenesis. This study has shown that Jmjd1c is required for AML development and maintenance. Experiments involving enforced expression of Jmjd1c in haematopoietic stem cells (HSC) suggested that Jmjd1c has the capacity to enhance stem cell proliferation. Jmjd1c overexpression also promoted proliferation of HSC transduced with oncogenes, Hoxa9/Meis1a, and accelerated leukaemia development in mice. Gene expression profiling identified Jmjd1c to be a critical regulator of metabolic pathways such as glycolysis. These novel findings support the importance of targeting Jmjd1c. This study also provided the first evidence of Jmjd5 as a potential tumour suppressor in AML. Overexpression of Jmjd5 significantly impaired LSC proliferation and prolonged mouse survival. Gene expression profiling and functional studies suggested that Jmjd5 negatively regulated a LSC self-renewal pathway driven by G protein-coupled receptor 84 (Gpr84). Given the partially shared downstream signalling of Jmjd5 and Gpr84, it is likely that Jmjd5 negatively regulates LSC function, at least in part, by inhibiting Gpr84 signalling. Jmjd1c and Jmjd5 have previously been shown to regulate histone methylation. Although global H3K9me2 levels were not altered by Jmjd1c, Jmjd5 reduced global H3K36me2 and H3K27me3 levels. Reduction in H3K27me3 could also be achieved by DZNep-induced Ezh2 inhibition at low doses, which altered the gene expression profile of DZNep-treated AML cells. In summary, both Jmjd1c and Jmjd5 were found to be crucial in the proliferation and maintenance of AML cells, suggesting that targeting aberrant methylation mediated by these epigenetic regulators may provide a promising approach in AML therapy. 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). …………………………………………………………… ……………………………………..……………… ……….…....11/11/2015..…….… Signature Witness 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. FOR OFFICE USE ONLY Date of completion of requirements for Award: THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS 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 ……11/11/2015..…………………… i 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 ………11/11/2015………………… ii 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 ………11/11/2015..………………… iii ABSTRACT Acute myeloid leukaemia (AML) is a heterogeneous blood disease with high relapse rates. A small population of highly drug resistant leukaemic stem cells (LSC) persists after chemotherapy, causing disease relapse. This emphasises the need to develop novel LSC-targeted therapies. While increasing evidence has emerged in recent years highlighting the role of epigenetic regulators in cancer development and maintenance, knowledge in epigenetic regulation of LSC is limited. This study investigated the role of two novel histone demethylases, Jmjd1c and Jmjd5, in regulating LSC, as well as the effectiveness of currently available epigenetic agents on leukaemogenesis. This study has shown that Jmjd1c is required for AML development and maintenance. Experiments involving enforced expression of Jmjd1c in haematopoietic stem cells (HSC) suggested that Jmjd1c has the capacity to enhance stem cell proliferation. Jmjd1c overexpression also promoted proliferation of HSC transduced with oncogenes, Hoxa9/Meis1a, and accelerated leukaemia development in mice. Gene expression profiling also identified Jmjd1c to be a critical regulator of metabolic pathways such as glycolysis. These novel findings support the importance of targeting Jmjd1c. This study also provided the first evidence of Jmjd5 as a potential tumour suppressor in AML. Overexpression of Jmjd5 significantly impaired LSC proliferation and prolonged mouse survival. Gene expression profiling and functional studies suggested that Jmjd5 negatively regulated a LSC self-renewal pathway driven by G protein-coupled receptor 84 (Gpr84). Given the partially shared downstream signalling of Jmjd5 and Gpr84, it is likely that Jmjd5 negatively regulates LSC function, at least in part, by inhibiting Gpr84 signalling. Jmjd1c and Jmjd5 have previously been shown to regulate histone methylation. Although global H3K9me2 levels were not altered by Jmjd1c, Jmjd5 reduced global H3K36me2 and H3K27me3 levels. Reduction in H3K27me3 could also be achieved by DZNep-induced Ezh2 inhibition at low doses, which altered the gene expression profile of DZNep-treated AML cells. iv In summary, both Jmjd1c and Jmjd5 were found to be crucial in the proliferation and maintenance of AML cells, suggesting that targeting aberrant methylation mediated by these epigenetic regulators may provide a promising approach in AML therapy. v ACKNOWLEDGEMENTS I would like to thank a number of people for their support and patience during the last four challenging years. I would not have made it without you all. Firstly, I would like to thank my supervisor, Dr Jenny Wang, for giving me the opportunity to join her research group. I have learnt so much and appreciate the supervision and advice she has given me throughout the time spent in her lab. I also would like to thank my co-supervisor, Professor Murray Norris, for the time taken out of his busy schedule for his constructive feedback and support in getting me through my PhD. I also appreciate the support and guidance given to me by my