Molecular Interactions Between Childhood Acute Lymphoblastic Leukaemia Cells And The Bone Marrow Microenvironment Ana Markovic 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 THE UNIVERSITY OF NEW SOUTH WALES 2009 THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: MARKOVIC First name: ANA Other name/s: Abbreviation for degree as given in the University calendar: PhD School: Women’s & Children’s Health Faculty: MEDICINE Title: Molecular Interactions Between Childhood Acute Lymphoblastic Leukaemia Cells And The Bone Marrow Microenvironment. Abstract 350 words maximum Acute lymphoblastic leukaemia (ALL) is the most common cause of death from disease in children. Whilst cure rates over the last 30 years have drastically improved, children that do go on and relapse have a very poor prognosis. Additionally, the ones that do survive can have significant long term side effects from existing treatments. Understanding the molecular mechanisms of the relationship between leukaemia and its microenvironment is essential for the identification of novel targets for treatment and/or the manipulation of existing treatments. Our previous work has established a panel of childhood ALL xenografts from patient biopsies in NOD/SCID mice. Several samples secrete vascular endothelial growth factor (VEGF), an integral component of neovascularisation and normal haematopoiesis, and express FMS-like tyrosine kinase-3 (FLT-3), a receptor tyrosine kinase, which plays an essential role in regulating normal haematopoiesis. This thesis builds on previous work by examining the relationship between VEGF and FLT-3, two widely, yet independently studied molecules in leukaemia, with the aberrant expression of either having adverse outcomes for patients. The results show that the high expression and activation of FLT-3, significantly increases secretion of VEGF. To assess whether VEGF secretion is triggered by FLT-3 signalling, we measured VEGF in the absence and presence of an inhibitor (SU11657), humanised anti-FLT-3 blocking antibodies as well as decreasing the receptors with siRNA. All of these manipulations were able to decrease the secretion of VEGF in leukaemia cells. To further investigate this relationship, we examined the phosphorylation status of FLT-3 and the downstream signalling pathway. Our results indicate that FLT-3 signalling may be an important factor in the induction of VEGF secretion in a sub-type of leukaemia cells, and in turn, VEGF secretion can be attenuated by an FLT-3 specific inhibitor. Two separate microarray studies were also used to assess simultaneous gene expressions between the leukaemia and bone marrow microenvironment, and to examine the effects of FLT-3 ligand on ALL xenograft cells. The results of the microarray studies confirm the previously observed results regarding the manipulation of the microenvironment by the leukaemic cells. Inhibition of the FLT-3/VEGF pathway may disrupt paracrine signalling between leukaemia cells and the bone marrow microenvironment, and future studies into how this disruption may influence leukaemia cell responses to conventional chemotherapy are warranted. 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: Declaration 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 …………………………………………………… Declaration iii Acknowledgements This thesis would not be possible without the support, belief and guidance of many people. I thank my supervisor A/Prof. Richard Lock for the opportunity he provided to undertake this work, along with Dr. Karen Mackenzie for her support. I have learned a great deal during my time at the Children's Cancer Institute Australia. I would also like to acknowledge the Berry family together with the Australian Academy of Science and the Foundation of the National Institutes of Health for establishing the fellowship which enabled me to experience working at the NIH. This includes, in particular, Dr. Javed Khan and his lab for accepting me for the short time I was there and teaching me about microarrays. I thank the staff at the faculty of Science at the University of NSW, for giving me the opportunity to experience the joy of teaching. Also, I want to thank the friends that have stayed by me in this madness, otherwise known as a PhD; especially Dr. Marina Pajic, Dr. Scott Melville and Laura Veas. Thank you to Dr. Andrew Kinsela, who has learned more about leukaemia, more than he ever thought he would or even wanted to know. Thank you, I am very grateful for all your love and support. Looks like I too have made it to the end. A thank you also needs to go to my grandparents. I am just sorry my grandfather is not here to see the completion of this thesis. Finally my mum and dad and my brother Ivan – thank you, I love you. Acknowledgements v Conferences, Publications and Awards Conferences Markovic A, MacKenzie KL, Lock RB. (2007) Induction of vascular endothelial growth factor secretion in acute leukemia cells via the FLT-3 signaling pathway, AACR- NIH-EORTC Conference “Molecular Targets and Cancer Therapeutics: Discovery, Biology, and Clinical Applications, San Francisco, USA. Markovic A, MacKenzie KL, Lock RB. (2007) Induction of VEGF Secretion in Acute Leukaemia Cells Through the FLT-3 Signalling Pathway, 19th Lorne Cancer Conference, Lorne, Victoria, Australia. Markovic A, MacKenzie KL, Lock RB. (2006) Novel Function of FLT-3: Induction of the Vascular Endothelial Growth Factor in Acute Leukaemia, Australian Society for Medical Research annual NSW Meeting, Sydney, Australia. Markovic A. (2005) Experience at the NIH as the Inaugural Recipient of the Adam J. Berry Memorial Fellowship, Annual Meeting of the Australian Academy of Science, Canberra, Australia. Conferences, Publications and Awards vii Publications Markovic A, MacKenzie KL, Lock RB. FLT-3: a new focus in the understanding of acute leukemia. Int J Biochem Cell Biol. 37(6):1168-72. Markovic A, MacKenzie KL, Lock RB. Induction of VEGF in leukaemia cells (manuscript in preparation) Awards 2007 - AACR Eli-Lilly Travel Award, for the conference AACR-NIH-EORTC Conference “Molecular Targets and Cancer Therapeutics: Discovery, Biology, and Clinical Applications, San Francisco, USA. 2005 - Australian Academy of Science & the Foundation for the National Institutes of Health USA - Inaugural Recipient of the Adam J. Berry Memorial Fellowship Conferences, Publications and Awards viii Abstract Acute lymphoblastic leukaemia (ALL) is the most common cause from death of disease in children. Whilst cure rates over the last 30 years have drastically improved, the children that do go on and relapse have a very poor prognosis. Additionally, the ones that do survive can have significant long term side effects from existing treatments. Understanding the molecular mechanisms of the relationship between leukaemia and its microenvironment is essential for the identification of novel targets for treatment and/or the manipulation of existing treatments. The role that vascular endothelial growth factor (VEGF), an integral component of both neovascularisation and normal haematopoiesis, plays in the progression and invasiveness of solid tumours is well established. However, its function in haematological malignancies has been a more recent and thus less considered observation. Human leukaemia cells secrete VEGF, which may act in a paracrine manner with the bone marrow microenvironment to promote the survival and proliferation of leukaemia cells. In addition to VEGF being produced by leukaemias, it also increases