SUPPRESSION OF NEUROBLASTOMA TUMORIGENESIS USING ENU MUTAGENESIS IN THE TH-MYCN MOUSE MODEL OF NEUROBLASTOMA A thesis submitted to the University of New South Wales in fulfilment of the requirements for the degree of Doctor of Philosophy Jayne Murray Children’s Cancer Institute and School of Women’s and Children’s Health Faculty of Medicine University of New South Wales December 2017 i THESIS/DISSERTION SHEET PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Murray First name: Jayne Other name/s: Elizabeth Abbreviation for degree as given in the University calendar: PhD School: School of Women’s and Children’s Health Faculty: Faculty of Medicine Title: Suppression of neuroblastoma tumorigenesis using ENU mutagenesis in the Th-MYCN mouse model of neuroblastoma Abstract 350 words maximum: (PLEASE TYPE) Neuroblastoma, a disease of the neural crest and the most common solid tumour of infancy, accounts for 7-10% of all childhood cancer. The disease arises from primitive cells within the neural crest, and can develop at any point along the sympathetic nervous system, including sites within the abdomen, pelvis, neck and thoracic cavity. Neuroblastoma tumours with MYCN oncogene amplification are highly aggressive, leading to a dismal outcome in most patients. A mouse model of neuroblastoma previously generated by targeting the human MYCN oncogene to neural crest cells (Th-MYCN), leads to 100% of MYCN homozygous mice developing tumours at an early age. This thesis describes a forward genetic screen involving the use of N-ethyl-N-nitrosourea (ENU) to generate heritable mutations causing delayed tumorigenesis in this model, as well as genomic sequencing to identify the mutated gene(s) responsible for the delayed tumorigenesis observed. Of 1716 ENU progeny screened, two mice demonstrated an unprecedented delay in tumour development. Subsequent breeding generated lines from these founders in which the delayed phenotypes were inherited in a dominant Mendelian fashion. Mapping, and whole exome and genome sequencing, identified a single gene in each of the lines as strong candidates responsible for delayed tumour formation, namely Runt-related transcription factor-1; translocated-to-1 (Runx1t1) and RING finger protein 121 (Rnf121). Runx1t1 was investigated further. Confirming the ENU mutagenesis findings, crossing of Runx1t1 knock-out mice with Th-MYCN mice, showed that loss of one allele of Runx1t1 abrogated tumour formation. The mechanism by which mutant Runx1t1 prevents neuroblastoma tumorigenesis was found to involve inhibition of neuroblast hyperplasia in mouse ganglia, a known prerequisite for neuroblastoma initiation. There was little evidence of MYCN directly regulating RUNX1T1 transcription, but rather increased RUNX1T1 protein levels were observed in human MYCN-amplified tumours. Knock-down of RUNX1T1 using shRNA resulted in decreased neuroblastoma colony formation in vitro, suggesting that this gene may also have a role in neuroblastoma maintenance. Collectively this study has employed ENU mutagenesis in an in vivo forward genetic screen to identify RUNX1T1 and RNF121 as novel neuroblastoma-associated genes. These genes represent potential new therapeutic targets for the treatment and ultimately prevention of this disease. 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). ……………………………………………………… ……………………………………..………… ……….……………………...… 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: 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 ........................................04052018 iv 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 ..........................................04052018 v 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 ..................................04052018 vi ACKNOWLEDGEMENTS Firstly I would like to thank my supervisors Professor Murray Norris and Professor Michelle Haber for entertaining the crazy idea of doing a part-time PhD while also working at Children’s Cancer Institute full-time as Program Officer for Molecular Diagnostics. I could not have done this without your supervision and guidance and allowing me time to write and do part of my PhD overseas in the laboratory of Professor Giovanni Perini. I cannot say thank you enough. Also, to my co-supervisor, Professor Douglas Hilton for suggesting the idea of doing an ENU mutagenesis study in the first place, and for performing the ENU injections. A project of this scope and longevity could not have been done without the support of a number of people. To the ‘mouse team’; Ashleigh Clark, Hannah Webber, Michelle Ruhle, Tony Huynh, Georgina Eden, Sophie Allan, Aaminah Khan, Sara Sarraf and new recruits Steph Alfred and Jenn Brand, that have battled through the huge number of experiments that were part of my full-time job and supported me in this endeavour, allowing me time to do my PhD. Whilst some of you have come and gone through this journey, we remain in contact and I could not have gotten to this point without each, and every one of you. Dr Emanuele Valli, not only for your expertise in creating any construct you could ask for and help with experiments, but also for welcoming me in Bologna when I came to do part of my PhD there. I value your friendship enormously. Also to Professor Giovanni Perini and Dr Daniela Erriquez, for allowing me to come to your laboratory and for working with me on the fractionation portion of this thesis, thank you. Thanks must also go to the collaborators that this project has collected over the years. To Dr Benjamin Kile and Dr Janelle Collinge, for your valuable insight into ENU mutagenesis, and for performing the exome and whole genome sequencing. To Dr Jamie Fletcher, for the protein modelling and for insightful scientific advice. To Amanda Russell and Professor Joel Mackay, for the NMR work and to Dr Andrew Gifford and Dr Laura Gamble for the histology analysis. To all the members of Experimental Therapeutics/Molecular Diagnostics, both past and present, thank you for providing a great working environment. To my panel members Professors Richard Lock and Maria Kavallaris and Dr Belamy Cheung, thank you for all your support. vii To Jessica Koach, Claudia Flemming, Joanna Keating, Leanna Cheung, Kathleen Kimpton and Tanya Dwarte: I could not have done this without your friendship, help, training, ideas and the occasional tap takeover. You all helped keep me sane during this period, whether it was just a chat over coffee or travelling to a great destination together, believe me, you helped. Finally, to my family, that have put up with the constant weekend work or late nights. I promise, life will get back to some sense of normalcy from now on. viii CONFERENCES, PUBLICATIONS AND AWARDS Conferences: Oral Presentations Murray, J., Kile, B.T., Collinge, J.E., Marshall, G.M., Hilton, D.J., Haber, M. and Norris, M.D. “Suppression of neuroblastoma tumorigenesis using ENU mutagenesis in the Th-MYCN mouse model of neuroblastoma.” Oral Presentation at the Advances in Neuroblastoma Research meeting, Toronto, Canada, June 2012. Murray, J., Haber, M., Carnegie-Clark, A., Webber, H., Marshall, G.M., Gurova, K., Burkhart, C., Purmal, A., Gudkov, A.