Exploring the therapeutic potential of protein tyrosine phosphatase inhibition in neuroblastoma Elsa Irving University College London Great Ormond Street Institute of Child Health A thesis submitted for the degree of Doctor of Philosophy 1 I, Elsa Irving, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract Neuroblastoma accounts for 15% of paediatric cancer deaths and there is an urgent need for improved therapeutic strategies. Phosphotyrosine signalling, regulated by the opposing actions of protein tyrosine kinases and protein tyrosine phosphatases (PTPs), is critical for virtually all aspects of cell behaviour, and is commonly perturbed in cancer. We have previously shown that pan-inhibition of PTPs using oxidovanadium induces cytotoxicity in a panel of neuroblastoma cell lines. We therefore hypothesise that there exist specific PTPs that promote tumour cell survival, and that their specific or pan-inhibition may be beneficial for the treatment of neuroblastoma. Whilst promising preclinical data using vanadium-derived compounds in in vitro and in vivo models of cancer has been reported, clinical trials have been prevented in part due to concerns surrounding off-target tissue toxicity. I have taken several approaches to harness the cytotoxic properties of oxidovanadium, and PTP inhibition, with the aim to develop new therapeutic strategies for neuroblastoma. The tumour-promoting roles of specific PTPs were investigated using loss-of-function approaches including RNAi and CRISPR/Cas9 gene knockout. The dual specificity phosphatase CDC14B was identified as a potential candidate, although further validation studies need to be considered for this enzyme to be taken forward as a potential therapeutic target. In a parallel study, the first genome-wide transcriptomic analysis in neuroblastoma cells treated with oxidovanadium has revealed a potential role for cAMP signalling in bismaltolato oxidovanadium (BMOV)-induced cytotoxicity. This pathway and others that are affected by oxidovanadium may be a source of useful therapeutic targets for neuroblastoma in the future. Finally, I have shown for the first time that hydrophobic oxidovanadium can be packaged into liposomes and maintains its cytotoxicity when delivered to neuroblastoma cells. This presents a novel opportunity to deliver vanadium with potentially fewer safety concerns, whilst retaining its broad activity and high levels of anti-cancer efficacy. 3 Impact Statement The aim of this project was to explore the potential of targeting protein tyrosine phosphatase (PTP) signalling for the treatment of neuroblastoma and to deliver new conceptual approaches and opportunities for future therapies. We believe that there is enormous therapeutic potential in targeting PTPs. These enzymes, along with their functional partners, protein tyrosine kinases, are responsible for the regulation of tyrosine phosphorylation, which is vital for proper cell function, and its deregulation is implicated in a broad spectrum of human diseases including cancer. PTPs are increasingly being positively implicated in tumourigenesis and cancer progression, and several have now been labelled as bona fide oncogenes in several tumour types including neuroblastoma. We therefore suggest that therapeutic inhibition of these, and other yet to be identified PTPs, will be useful in developing much needed improved therapeutic strategies for high-risk neuroblastoma. Here I present data indicating that targeting of specific pro-tumour PTPs, as well as pan-PTP inhibition using vanadium-derived compounds may be of benefit for neuroblastoma treatment. I utilised genomic technologies including siRNA and shRNA-mediated gene knockdown, and CRISPR/Cas9-mediated gene deletion, to identify specific PTPs that are required for growth and/or survival of neuroblastoma cell lines. If confirmed in vivo, therapeutic targeting of these enzymes may have significant clinical impact. Vanadium-derived compounds are considered broad-spectrum inhibitors of the entire PTP superfamily and have been of interest in disease research including cancer for many decades. Despite considerable research efforts reporting anti-cancer activity in in vitro and in vivo tumour models, and even human clinical trials in the diabetes setting, vanadium compounds have yet to achieve clinical approval, due in part to concerns regarding the safety of their use in humans. Here I attempted to deconvolute the mechanisms driving oxidovanadium cytotoxicity, using genome-wide transcriptomics. These data revealed a broad range of transcriptional changes, suggesting that several effector pathways likely contribute to the observed cytotoxicity. One such 4 pathway was identified as cAMP signalling, and early validation suggests that cAMP signalling is activated by oxidovanadium and may be involved in cytotoxicity. Targeting of this pathway and other oxidovanadium effectors may be useful in the treatment of neuroblastoma. Both the targeting of specific pro-tumour PTPs and BMOV effectors are reductionist approaches to harness the cytotoxicity associated with oxidovanadium-mediated pan-PTP inhibition. An alternative approach is the therapeutic use of oxidovanadium itself. Here I present a novel hydrophobic oxidovanadium liposomal formulation that can be delivered to neuroblastoma cells in culture. This nanoparticle formulation may allow safer delivery of oxidovanadium to tumour cells in vivo, both by enhancing the efficiency of delivery and reducing off-target tissue toxicities. These formulations may be useful not only in the treatment of neuroblastoma but also in other cancers, as well as other diseases related to PTP signalling, namely diabetes, where vanadium-derived compounds have previously been trialled. My research will therefore raise the profile of PTP signalling as a therapeutic target in the cancer field and will also promote new thinking about how to utilize the cytotoxic potential of oxidovanadium for cancer and other disease treatment. 5 Acknowledgements I would first like to thank Andy Stoker. Your calming influence, patience, enthusiasm, and willingness to help is inspirational, and I truly couldn’t have hoped for a better supervisor. I also owe a huge thank you to Vruti Patel. I am so glad that you joined the lab, your advice, and let’s face it counselling, really has helped me through to the end! I am also very grateful for the help of many other scientists based at ICH. In particular, thank you to JP for his support and advice, Aris and Ruhina for synthesising my liposomes, and Simon and Mike for their help with HPLC. I have made great friends during my time at ICH, who have supported me enormously. I’d especially like to thank all ‘residents’ of office W2.04 past and present. Amy, Vruti, Aara, Lisa, and Elliot, you are all fantastic people and you deserve medals for putting up with me during writing this thesis! Lucy, we’ve been in it together since day one, and we’ve done it! (And Amy you will too!). I hope I will be lucky enough to work with a group of people just as fun, driven, kind, and coffee and cake dependent again. Most importantly, thank you to my amazing family and friends in London and further afield, who have supported me unconditionally. Catherine, you’ve been my cheer leader! Thank you for your pep talks over coffee and breakfast, you’ve kept me sane (ish!). I will be forever grateful to the Flahertys for welcoming me into their wonderful home and providing me with endless fun and distraction. I have been so lucky to come home to supportive family, lovely food and a comforting home every day. Owen, thank you for always telling me I can do this and for reminding me that ‘No stress is best!’. 2018 has been a very challenging year but, thanks to you, also one of my best! Alice, you know me better than I know myself, and never fail to make me smile during meltdowns, however ridiculous they may seem! You and Dicken have been inspiring and encouraging me for my whole life, and this PhD has been no different. Finally, thank you to my parents. Mum and John, you have supported me in everything I have achieved, and have always told me I can do anything I put my mind to. I love you both, and if there is one thing I know, it is that this PhD would not have been possible without your love and support. 6 Table of contents Abstract ........................................................................................................ 3 Impact Statement ......................................................................................... 4 Acknowledgements ..................................................................................... 6 List of figures ............................................................................................. 12 List of tables ............................................................................................... 12 Abbreviations ............................................................................................. 17 Chapter 1. Introduction.............................................................................. 21 1.1. Neuroblastoma .................................................................................. 22 1.1.1. Incidence and clinical presentation ............................................. 22 1.1.2. Disease classification and treatment ........................................... 24 1.1.3. MYCN amplification ...................................................................