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Investigation of the Role of Cbl-B in Leukemogenesis and Migration

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Investigation of the Role of Cbl-B in Leukemogenesis and Migration INVESTIGATION OF THE ROLE OF CBL-B IN LEUKEMOGENESIS AND MIGRATION by Karla Michelle Badger-Brown A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Medical Biophysics University of Toronto © Copyright by Karla Michelle Badger-Brown (2009) Investigation of the Role of CBL-B in Leukemogenesis and Migration Doctor of Philosophy, 2009 Karla Michelle Badger-Brown Department of Medical Biophysics University of Toronto ABSTRACT CBL proteins are E3 ubiquitin ligases and adaptor proteins. The mammalian homologs – CBL, CBL-B and CBL-3 show broad tissue expression; accordingly, the CBL proteins play roles in multiple cell types. We have investigated the function of the CBL-B protein in hematopoietic cells and fibroblasts. The causative agent of chronic myeloid leukemia (CML) is BCR-ABL. This oncogenic fusion down-modulates CBL-B protein levels, suggesting that CBL-B regulates either the development or progression of CML. To assess the involvement of CBL-B in CML, bone marrow transduction and transplantation (BMT) studies were performed. Recipients of BCR-ABL-infected CBL-B(-/-) cells succumbed to a CML-like myeloproliferative disease with a longer latency than the wild-type recipients. Peripheral blood white blood cell numbers were reduced, as were splenic weights. Yet despite the reduced leukemic burden, granulocyte numbers were amplified throughout the animals. As well, CBL- B(-/-) bone marrow (BM) cells possessed defective BM homing capabilities. From these results we concluded that CBL-B negatively regulates granulopoiesis and that prolonged latency in our CBL-B(-/-) BMT animals was a function of perturbed homing. To develop an in vitro model to study CBL-B function we established mouse embryonic fibroblasts (MEFs) deficient in CBL-B expression. Transduction of the wild-type and CBL-B-deficient MEFs with BCR-ABL did not confer transformation; nevertheless, the role of CBL-B in fibroblasts was evaluated. The CBL-B(-/-) MEFs showed enhanced chemotactic migration toward serum in both Transwell migration and time-lapse video microscopy studies. The biochemical response to serum was extensively evaluated leading to the development of a model. We predict that CBL-B deficiency either: (a) augments GRB2-associated binding protein 2 (GAB2) phosphorylation leading to enhanced extracellular signal-regulated kinase (ERK) and protein kinase B (PKB / Akt) signaling, or (b) alleviates negative control of Vav3 resulting in stimulation of Rho effectors. In either case, our results reveal a negative regulatory role for CBL-B in fibroblast migration. The two studies detailed herein expand our knowledge of CBL-B function. They strongly suggest that CBL-B can modulate granulocyte proliferation and point toward a role for CBL-B in the motility of numerous cell types. ii ACKNOWLEDGMENTS My PhD endeavor was driven by a desire for independent scientific- and self- discovery. I was searching for an opportunity to steer my own course and follow a road less traveled. While the experiences I take from this journey are my own, I cannot pretend that I was not significantly aided along the way. This road has been pebbled by many friends, colleagues and family that have provided useful experimental and personal guidance. As a newcomer from Manitoba I would have expected Toronto to be a difficult city to navigate, and yet I felt immediately welcomed into the Department of Medical Biophysics. From the “2001 Freshmen” to the Drowned Rats and the editorial board of Hypothesis, each experience granted me new friendships. Many a merry meeting was imbibed with not only beverage but scientific discourse. There is no doubt that I will happily cultivate these friendships for many years to come. As a PhD student I had the fortunate opportunity to exist as a true member of not only one, but two great labs. Thanks so much to the Dick lab members for sharing your input, reagents and a cozy Isolab corner; you guys all deserve a huge thanks for helping me evaluate my science from a stem cell point of view and entertaining me in the process. And to the “Barber Babes” and “Boys” - I can’t thank you enough for useful advice, help in the rough patches, and again, a healthy dose of fun. I particularly want to thank Hani and Terri. It is with great regard that I value your scientific intellect and drive. As two strong women, there is no doubt in my mind that you will find great success in your future. Which brings be to the Barber boss – Dwayne. Thanks for being an excellent supervisor. From the moment I stepped into your office for my initial interview I knew that our relationship would work. You have always shown confidence in me, providing the independence that I so desperately craved. As well, you have continuously challenged me to strive for greater success. From my experience under your tutelage I really feel that if I work hard enough, no task is insurmountable. Which brings me to “family”. Thanks first to my family – Mum, Ron, Holly and Julie. It’s been difficult being so far away from home, but I thank you for your support of my dreams. A special thank- you goes out to my role model - Mum. You were “single-Mum-extrodinaire”! I can’t tell you how much I appreciated your encouragement then, and now, as I near graduation. To my other family, Covell and Mary, you have grown to be not only my “in-laws”, but also my second set of parents. Thanks goes to Mary for recognizing those moments when a warm hug and delicious Sunday dinner would be appreciated. And thanks to Covell - my “great self-development over these years” is definitely partly a function of your useful guidance and advice. Membership in the Brown family would not have existed without my dearest Nicholas. You have been an integral part of this PhD process. Thanks so much for taking the time to truly appreciate my science and for your unwavering support in trying times. Moreover, it is from you Nicholas, that I have learned compassion and love, and for that, I am truly blessed. iii TABLE OF CONTENTS ABSTRACT................................................................................................................................................ii ACKNOWLEDGMENTS ........................................................................................................................iii TABLE OF CONTENTS..........................................................................................................................iv LIST OF TABLES ....................................................................................................................................vi LIST OF FIGURES .................................................................................................................................vii LIST OF ABBREVIATIONS...................................................................................................................ix LIST OF APPENDICES.........................................................................................................................xvi CHAPTER 1. INTRODUCTION .............................................................................................................1 1.1. Hematopoiesis................................................................................................................................... 2 1.1.1. Hematopoiesis during ontogenesis............................................................................................2 1.1.2. Adult hematopoiesis ..................................................................................................................2 1.1.3. Differentiation of the HSC into mature blood cells .................................................................. 2 1.1.4. Hematopoietic Growth Factors ................................................................................................. 6 1.1.5. Methods to identify hematopoietic cells ...................................................................................6 1.1.6. Summary ................................................................................................................................... 8 1.2. Chronic Myeloid Leukemia............................................................................................................10 1.2.1. BCR-ABL is the causative agent of CML...............................................................................10 1.2.2. Molecular mechanisms of BCR-ABL-mediated transformation.............................................12 1.2.3. Biological mechanisms of BCR-ABL-mediated in vitro transformation................................20 1.2.4. Modeling CML in animals ...................................................................................................... 30 1.2.5. Using the BMT assay to identify key pathways of BCR-ABL-mediated leukemogenesis..... 33 1.2.6. Therapies for CML..................................................................................................................37 1.2.7. Summary ................................................................................................................................43 1.3. The CBL family of proteins............................................................................................................44 1.3.1. The domain structure of CBL proteins....................................................................................44 1.3.2. CBL proteins mediate ubiquitination ......................................................................................47
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