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Novel Modification of Human Myeloma Proteasomes and Development of Non-Active Site Directed Inhibitors

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Novel Modification of Human Myeloma Proteasomes and Development of Non-Active Site Directed Inhibitors Novel modification of Human Myeloma proteasomes and development of non-active site directed inhibitors. By David S. Pitcher A thesis submitted for the degree of Doctor of Philosophy October 2016 Centre of Haematology Imperial College London COPYRIGHT The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. Figures 1.2, 1.3, 1.4 and 1.5 are from the sources stated in the legends of each figure and permission has been obtained for reproduction and republishing in an online open access format by the licences stated below: Figure 1.2 is licenced by ‘American Society of Hematology’ number #3973070918521 Figure 1.3 is licenced by ‘Nature Publishing Group’ number #3960180094287 Figures 1.4 & 1.5 are licenced by ‘Nature Publishing Group’ #3960180835983 I DECLARATION All the experiments described in this thesis were designed and performed by myself unless otherwise stated. Contributions from other people are acknowledged in the appropriate sections. I performed the experiments, analysed the data, produced the graphs and figures, and wrote the text; all with the guidance of my supervisor, Dr Maurits Kleijnen. David Pitcher October 2016 II ABSTRACT Multiple Myeloma (MM) is a plasma cell malignancy that is characterised by bone lesions and production of excessive amounts of monoclonal protein. Treatment involves the use of chemotherapy agents, immunomodulatory agents and proteasome inhibitors (PI). The 26S proteasome is a 2.5 MDa molecular machine that is integral to the viability of all eukaryote cell. Its main function is to hydrolyse proteins that are marked for degradation by a poly-ubiquitin chain. Although MM is treatable, it is as yet incurable with mean survival of ~6 years. In this thesis, I show that human proteasomes contain a charged polymeric posttranslational modification (PTM), one which has some similarity to poly-ADP- ribose. This modification is not normally resolvable via normal SDS-PAGE electrophoresis, but can be resolved by the lesser used CTAB-PAGE or after separation of the proteasome from other cellular components. This modification appears to be present predominantly in the nucleus of the cell, and may provide a mechanism for how nuclear proteasomes interact with chromatin, DNA and other nuclear components. The use of proteasome inhibitors as a valid therapy for MM has been hypothesised to be due to a high proteasome load in MM; therefore, a small amount of inhibition would suffice to perturb proteostasis. However, I show that myeloma cells experience severe proteasome inhibition upon treatment with compounds such as Bortezomib, to a degree that far exceeds the levels of inhibition observed with purified proteasomes. This suggests that, when PIs engage with proteasomal active-sites, they trigger a cellular mechanism which exacerbates this inhibition to a far greater degree than would otherwise be achieved. I excluded trivial explanations including additional binding of the inhibitor, caspase mediated proteasome inhibition or cell death initiation. Intriguingly, I found that early changes to CTAB-PAGE detectable PTMs coincided with PIs’ ability to achieve an excessive degree of cellular proteasome inhibition. In addition to this work I continue the development of an allosteric proteasome inhibitor identified by a phage display technique. Through a number of rounds of chemical optimisation, I show the ability of these compounds to inhibit proteasome degradation of an ubiquinated substrate and a lethality in myeloma cells at 25 nM. III ACKNOWLEDGEMENTS I am greatly indebted to my funding body, Bloodwise (Registered Charity #216032). Without their input to my work over the last four years I would not have been able to fulfil my love of investigative science and hopefully have provided my small input to their overall aims. I also would like to thank all those who have raised money for this hugely great charity. I am so grateful to have been able to contribute to the important work funded by this organisation in the fight against all blood cancers. I would also like to thank the many other people that as part of the chain that made my PhD possible. It is four years ago that I was invited for an interview at Imperial College London. I would like to sincerely thank my supervisor Dr Maurits Kleijnen for having the faith in me, and offering me the opportunity to study in his lab. As well as constantly motivating me and pushing me to exceed my own personal expectation, he enabled me to expand my thinking and develop my ability. I am exceedingly grateful for his patience, assistance and most importantly his faith in me enabling me to achieve so many goals and achievements. I also have to thank my second supervisor Prof. Tassos Karadimitris for giving me the additional support as well as conveying his knowledge into my work. A big thank you goes to my parents who have given me the freedom in my education to develop and pursue my love of science. They have always supported me in my, pathway through life and my love and interest in science, from the very early age of watching ‘The Open University’ in the early hours on TV whilst they were asleep. I would also like to acknowledge the efforts, support, training and interest of my previous supervisor Dr Helen Dawe at Exeter University. She gave me the basic experience and taught me the need for strict laboratory skills so necessary for accurate experimentation. I have on many occasions been able to overcome difficulties down to her strict training and advice which I will carry with me forever. During my time in the lab I have been very privileged to work alongside some great lab members. Katerina, Mai, Kate P, Kate MS and Kostas deserve a special mention IV for working alongside and teaching me protocols and sharing reagents as well as the other members of Lab 211 in Exeter, especially Kate McI, Kim, Dan, Natalie. All of these people showed me that science is not just a job and lab members are not just colleagues. I would also like to thank my housemates Pete and Mo for keeping me sane throughout my four years. They were kind enough to feign excitement upon my discoveries, made to watch scientific documentaries and advise me practicing presentations before Game of Thrones, I sincerely hope they have suffered no lasting damage!! Lastly my thanks go to Louise and Pete for reading sections of my thesis and giving me invaluable feedback from a laypersons point of view. V CONTENTS COPYRIGHT ............................................................................................................................................ I DECLARATION ...................................................................................................................................... II ABSTRACT............................................................................................................................................ III ACKNOWLEDGEMENTS ......................................................................................................................IV ABBREVIATIONS, UNITS AND PREFIXES .......................................................................................... 1 LIST OF FIGURES ................................................................................................................................. 3 LIST OF TABLES ................................................................................................................................... 6 CHAPTER 1 - INTRODUCTION ............................................................................................................. 7 1.1 MULTIPLE MYELOMA (MM) .............................................................................................................. 8 1.2. PROGRESSION OF THE DISEASE ....................................................................................................... 8 1.3. DIAGNOSIS ..................................................................................................................................... 9 1.3.1. Clinical features .................................................................................................................. 9 1.4. EPIDEMIOLOGY ............................................................................................................................. 13 1.5. CYTOGENETIC ABERRATION AND MUTATIONS ................................................................................. 14 1.5.1. Common Translocations .................................................................................................. 14 1.5.2. Copy number variations ................................................................................................... 16 1.6. CURRENT TREATMENT OF MULTIPLE MYELOMA ............................................................................. 18 1.6.1. Prednisone ......................................................................................................................... 18 1.6.2. Thalidomide ....................................................................................................................... 19 1.6.3. Proteasome Inhibitors .....................................................................................................
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