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Investigating HK2 As a Potential Therapeutic Target in Glioblastoma

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Investigating HK2 As a Potential Therapeutic Target in Glioblastoma Investigating HK2 as a potential therapeutic target in glioblastoma Daniel Blakeway, MSc A thesis submitted in fulfilment of the requirements of the University of Wolverhampton for the degree of Doctor of Philosophy Brain Tumour Research Centre Research Institute in Healthcare Sciences Faculty of Science and Engineering University of Wolverhampton September 2019 I Authors Declaration This work or any part thereof has not previously been presented in any form to the University or to any other body whether for the purposes of assessment, publication or for any other purpose (unless otherwise indicated). Save for any express acknowledgments, references and/or bibliographies cited in the work, I confirm that the intellectual content of the work is the result of my own efforts and of no other person. The right of Daniel Blakeway to be identified as author of this work is asserted in accordance with ss.77 and 78 of the Copyright, Designs and Patents Act 1988. At this date copyright is owned by the author. Daniel Blakeway Signature ….…………………………………………………….. Date …………27/02/2020……………………………………. II ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my supervisor, Prof. Tracy Warr for providing me with the privilege and opportunity to pursue my PhD. Her tremendous support and guidance throughout this project have been a major factor in its successful completion and has helped shape the scientist I am today. I am very grateful to my co-supervisor’s Dr Katherine Karakoula and Dr Mark Morris, for their valuable support, suggestions and constant encouragement that have always been inspiring. I also have great appreciation to Prof. John Darling, Dr Farzana Rowther and Dr Angel Armesilla for all their valuable support. I would like to thank my colleagues at the university, of which there are too many to thank, but I do want to note my key appreciation of Dr Lawrence Eagles and Dr Maggie Trela for their help, encouragement and support over the last few years, it has been a pleasure to work alongside them. I am grateful to the technical staff at the University of Wolverhampton for all the support over my time here. I especially want to thank the efforts of Dr Angie Williams, Henrik Townsend and Clare Murcott for the technical support. I would like to thank the University for its philosophy, without which I would not have been able to undertake this PhD project. I am grateful to the Colin Oliphant Charitable Trust for funding this project and for the Sydney Driscoll Trust for their support. I also extend my thanks to the collaborators in this project, brain tumour north west and Mr Andrew Beggs from the University of Birmingham, for providing support. I am grateful to all my friends outside the University, for their love and encouragement. On a personal note, I would love to thank my parents, family and partner Jessica, for their constant source of encouragement, love and support, and for always being there pushing me onward and upward to greater things. Lastly, I would like to thank my brother Chris, who’s influence, and nurture has impacted my life beyond measure and shaped the person I am today and will continue to aspire to emulate. This PhD would not have been possible without all your help. Thank you! III DEDICATION To my parents and brother IV Abstract Glioblastoma is the most common high-grade primary brain tumour in adults. Current treatments have limited success with average survival comprehensively low. Effective drug treatments are hindered predominantly by the complex genetic background of glioblastoma heterogeneity, as such there is a compelling need for the development of effective therapeutics. An approach is to target abnormal metabolic pathways that are universally dysregulated in glioblastoma. The identification of potential targets and the development of therapeutic agents is crucial for the advancement of treatment. Hexokinase 2 (HK2) has a prominent role in glycolysis, acting as the rate-limiting step in the pathway. HK2 is highly expressed in many cancers and its role as the rate-limiting step of glycolysis may potentially contribute to tumour growth. Overexpression of HK2 has also been associated with drug resistant phenotypes, in parallel, its inhibition has improved the effectiveness of anticancer agents, suggesting HK2 as a potential therapeutic target. The role of methylation and its association with expression levels of HK2 was determined in glioblastoma fresh frozen biopsies and patient-derived cultures, through pyrosequencing and quantitative PCR. CRISPR knockout was utilised to investigate the effect of inhibiting HK2 on proliferation and to determine the role of HK2 in chemoresistance. The anti-proliferative effects of HK2 inhibitors 3-bromopyruvic-acid (3-BPA) and metformin were investigated via cytotoxic assays and FACS analysis was used to determine their mechanism of action. Additionally, downstream expression changes were investigated via expression profiler arrays, across 84 key genes involved in the regulation and enzymatic pathways of glucose metabolism. Hypomethylation was demonstrated in all biopsies (n=100) and cultures (n=15) compared to normal brain tissue; with average methylation of 4.6% compared to 26% respectively (p<0.0001) determined across 15-CpGs. A significant increase (p<0.0001) in HK2 expression was discovered, ranging between 6 to >1000- fold change in all biopsies and cultures compared to normal brain tissue and a strong correlation between hypomethylation and increased level of expression (p<0.0001) was established. Furthermore, elevated levels of HK2 revealed notably poorer survival outcomes. Sensitivity to 3-BPA in glioblastoma cultures was associated with V elevated expression of HK2. Significantly different levels of apoptosis were observed with average levels 38% greater in high HK2 expressing cultures (p=0.0014). A significant growth rate reduction (p<0.007) was demonstrated in HK2-KO cultures compared to parent cultures. Importantly sensitivity to both metformin and TMZ was also significantly increased (p<0.0001) in response to HK2-KO, with substantial reduction in both ID50 values and cell survival. Notably, HK2-KO cultures yielded greater synergistic effects with metformin and TMZ combination treatment. Array data revealed significant downstream gene expression alterations (p<0.005) with HK2-KO and with the supplementation of both 3-BPA and metformin, where >50% genes demonstrated reduced levels of expression compared to the corresponding parent/non-treated cultures. This study demonstrates the predominant role of HK2 within the glycolytic pathway, with overexpression potentially key in driving the genetic alterations downstream. This study also verifies a strong correlation between increased expression of HK2 and hypomethylation, additionally highlighting the impact HK2 has on inferior patient prognosis. HK2-KO revealed considerable ubiquitous reductions in downstream gene expression compared to glioblastoma biopsy tissue and parent cultures. Additionally, an increase in drug sensitivity was depicted with the loss of HK2 signifying the potential of targeting HK2 as a novel therapy in a significant subset of glioblastoma. VI TABLE OF CONTENTS Abstract ................................................................................................... V List of tables ............................................................................................ XIII List of figures ........................................................................................... XVI Abbreviations ........................................................................................... XXII Chapter 1 Introduction .......................................................................................... 1 1.1 Cancer ...................................................................................................2 1.2 Brain tumours ........................................................................................2 1.2.1 Glioblastoma ...................................................................................... 3 1.2.2 Glioblastoma classification ....................................................................4 1.3 Treatment of GBM ................................................................................ 6 1.4 Molecular biology of glioblastoma............................................................ 10 1.4.1 PI3K/Akt pathway alterations.............................................................. 12 1.4.2 EGFR/RTK alterations.......................................................................... 12 1.4.3 p53 pathway alterations...................................................................... 13 1.5 Role of epigenetics in cancer.................................................................. 15 1.5.1 DNA methylation ............................................................................... 15 1.5.1.2 DNA methylation in cancer .............................................................. 17 1.5.1.3 MGMT methylation status ................................................................ 19 1.5.2 Non-coding RNAs/ Micro RNAs ............................................................ 20 1.5.2.2 miRNA in cancer ............................................................................. 20 1.6 Cancer as a metabolic disease ............................................................... 21 1.6.1 Glucose pathway ............................................................................... 21 1.6.2 Hexokinase ......................................................................................
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