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Investigation of A Investigation of deoxythymidylate kinase (dTYMK) as an imaging and therapeutic target Alice Beckley A dissertation submitted for the degree of Doctor of Philosophy IMPERIAL COLLEGE LONDON Department of Surgery and Cancer Declaration of originality I declare that the contents of this dissertation are my original work and conducted by myself, except where otherwise stated and appropriately acknowledged. This thesis was conducted between March 2015 and March 2019 under the supervision of Prof. Eric Aboagye at Imperial College London UK. 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 and transmit the thesis on the condition that they do not change, transform nor build upon the document. It must not be used for commercial purposes. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. 1 Abstract The uncontrolled proliferative capacity of tumour cells, a hallmark of cancer, has been the main focal point for imaging modalities that provide non-invasive and quantitative estimates of tumour growth. Over the past few decades, several tracers have been developed for use with positron emission tomography (PET) to assess cell proliferation. More commonly, the exploitation of thymidine kinase-1 (TK1) substrate 18F-labeled-3-deoxy-3-fluorothymidine (18F-FLT) uptake for imaging of proliferation has been broadly accepted but, its limitation in accurately depicting the S-phase fraction has become more apparent over the past 10 years. This study explores the use of deoxythymidylate kinase (dTYMK) as a plausible imaging and therapeutic target since dTYMK participates in the only known pathway to synthesise deoxythymidine diphosphate and ultimately deoxythymidine triphosphate (dTTP). We introduce the first use of a novel squaramide-nucleotide radiotracer combined with the sensitivity of PET imaging to trace the proliferative tumour fraction with respect to the convergent enzyme, dTYMK. Initial in vitro 18F-SqFLT uptake in salvage proficient (HCT-116), de novo proficient (OST TK1) and CRISPR/Cas9 edited dTYMK knockdowns (B1 and B5) was found to be significantly low (~0.2 % ID/mg protein) when compared to 18F-FLT (~20 % ID/mg protein) suggesting that, 18F- SqFLT is not a substrate for dTYMK and, its rate-limiting step may be due to a low passive diffusion. As a pilot study, our observations were extended into an in vivo setting, which revealed non-significant tumour uptake in both wild-type and dTYMK knockdown models when compared to muscle. The highest accumulation of 18F- SqFLT occurred in the kidney, liver and bladder. A high uptake was also observed in 2 the gall bladder indicating partial excretion via the biliary pathway. While 18F-SqFLT was unsuccessful in tracing the tumour proliferative fraction, the study still provided pharmacodynamic information into the increasing interest of nucleoside analogues, presenting squaramide phosphate mimics, as potential biologically active cancer agents. Moreover, the CRISPR/Cas9 edited dTYMK knockdown models served as a good platform for understanding some of the mechanisms that may account for dTYMK targeted radiotracer accumulation and retention in cells. A key finding in this study was the disparity between in vitro and in vivo growth rate of dTYMK knockdown models. It was concluded that a dTYMK bypass mechanism that becomes more apparent in vitro than in vivo, may exist to sustain DNA synthesis and maintain genomic integrity. Given the increasing interest in targeting dTYMK as part of an adjunct therapy, these models present as a good system for future pharmaceutical application. To conclude, the exploitation of dTYMK from an imaging endpoint remains challenging; however, success will allow detailed evaluation of the cellular metrics of proliferation and overcome the key limitations associated with 18F-FLT imaging. 3 Acknowledgements I want to start by expressing my most profound appreciation to Professor Eric Aboagye for providing me with the opportunity to embark on the journey of research science. My personal experience left me with a passion for pursuing a career in cancer research, with the hope of having an impact on people’s lives, since my teenage years. I am forever grateful to you for giving me this opportunity and providing the platform I needed to fulfil my passion. You are an exceptional supervisor who has inspired me to be better and challenged my critical thinking. This project, our discussions along with the other collaborative work you have involved me in, has dramatically broadened my research skills and stimulated my drive for research. I am grateful to my siblings (Lola, Peter, Phillip and Paul), to Yeshua and my parents (Janet and Elijah Beckley), who have provided moral, emotional and practical support all through my life. I extend further thanks to Peter and Abi for their care and love during my write up and, to Paul for his outstanding presence in my life. Your patience, love and constant care have seen me through all aspects (from the joy of teaching me to ride a bike at 7 years old to soothing my pain during the most turbulent moments of my life). You are nothing short of a blessing to me. Thank you also to my all my nieces and nephews. You may all be to young to know right now but, your laughter, love and carefree nature fuelled my heart and gave me strength. I want to also express my gratitude to my best friends Marta, Lorraine and Willis for constantly pushing at me and getting the best out of me in your own ways. 4 Together we have laughed, cried our way from childhood to adulthood. I will always cherish this. I would like to extend a special thanks to Marta. Going through this PhD journey with you was possibly the best experience I could have. Your support for me both mentally and practically is invaluable. We grew together not just as scientists but as best friends. A very special thanks goes out to all the funders of Cancer Research UK for providing the funding for this and numerous research projects. It would not be possible without you all. Lastly, but certainly not the least, I would like to thank the whole of Aboagye lab for the warm atmosphere, the ability to bounce ideas and the fun we have had over the years. 5 Table of Contents TABLE OF CONTENTS ............................................................................................ 6 CHAPTER 1 ......................................................................................................... 18 INTRODUCTION ................................................................................................. 18 1.0. BACKGROUND ...................................................................................................... 19 1.1. NUCLEOTIDE SYNTHESIS AND DTYMK FUNCTIONALITY ............................................. 21 1.1.2. STRUCTURE AND CATALYTIC MACHINERY OF DTYMK ................................................ 24 1.1.3. CYTOPLASMIC AND MITOCHONDRIAL DTYMK .......................................................... 25 1.1.4. CURRENT RESEARCH ON TARGETING DTYMK ........................................................... 28 1.2. PET IN ONCOLOGY ................................................................................................ 31 1.2.1. PET IMAGING OF CELLULAR PROLIFERATION ............................................................ 34 i) - 2-[11C]Thymidine ............................................................................................ 35 ii) – 18F-FMAU ...................................................................................................... 36 iii) - 76Br-BrdU and 76Br-BFU ................................................................................ 37 1.2.2. 18F-FLT ........................................................................................................... 39 1.2.3. SUMMARY OF 18F-FLT UPTAKE CHARACTERISTICS – ITS ADVANTAGES AND LIMITATIONS ... 40 1.3. SELECTION OF A SUITABLE RADIOTRACER .................................................................... 44 1.3.1. BIOISOSTERES FOR PHOSPHATE MIMICRY ................................................................ 45 1.4. THESIS OBJECTIVES ................................................................................................ 47 CHAPTER 2 ......................................................................................................... 48 MATERIALS AND METHODS ............................................................................... 48 2.1. CELL CULTURE ...................................................................................................... 49 2.2 WESTERN BLOTTING ............................................................................................... 49 2.3 DNA CELL CYCLE ANALYSIS USING flOW CYTOMETRY ...................................................... 50 2.4. SYNTHESIS OF 18F-SQFLT, 18F-FLT, 18F-D4-FCH AND 18F-FDG .................................. 51 2.5. IN VITRO UPTAKE OF RADIOTRACERS .......................................................................... 52 2.6. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) ............................................ 53 2.7. LIPOSOME ENCAPSULATION ..................................................................................... 53 2.8. IN VITRO UPTAKE TO DETERMINE THE ACID INSOLUBLE FRACTION OF LABELLED NUCLEOTIDES 54 6 2.9. ENZYMATIC ASSAY OF DTYMK ...............................................................................
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