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Defining and exploiting the developmental origin of MLL-AF4-driven infant Acute Lymphoblastic Leukaemia Vasiliki Symeonidou Doctor of Philosophy The University of Edinburgh 2019 ii Declaration I declare that this thesis was composed by myself that the work contained herein is my own except where otherwise stated in the text, and that this work has not been submitted for any other degree or professional qualification except as specified. Vasiliki Symeonidou Edinburgh, December 2019 iii Acknowledgements I would like to thank my supervisor Prof. Katrin Ottersbach for giving me the opportunity to do a Ph.D. in her lab and for her support; what a great journey this has been! I would also like to thank my Ph.D. committee, Profs Kamil Kranc, Lesley Forrester and Keisuke Kaji. Dr. Simon Tomlinson and his group as well as Dr. Stephen Clegg for their support during my first steps in the Bioinformatics arena. I would like to thank Dr. Andrew Finch for performing the lipidomics experiments, analysing the accompanied data and helpful discussions. I would also like to thank the SCRM flow cytometry team - Fiona Rossi, Clare Cryer, Andrea Corsinotti and Bindy Heer - many times they have gone out of their way to help and support me and for this I am most grateful. I would also to thank Dr. Linh My Huynh for help with the cloning aspects of my work and Benedetta Carbone for her support for the CRISPR-Cas9 work. I would also like to thank Prof. Pablo Menendez for giving me the opportunity to spend time in his lab and learning from their experience, following from this I would like to thank Drs Talia Velasco and Clara Bueno for their help with the human foetal liver transduction experiments and MLL-AF4 lentiviral particles production. iv Abstract Infant MLL-AF4-driven pro-B Acute Lymphoblastic Leukaemia (ALL) is the most common leukaemia in infants. This devastating disease, which arises in utero, renders the infant patients with an aggressive disease and with a 5-year survival rate of less than 50%. It has long been speculated that along with the fusion protein, the foetal origin of the disease is one of the main contributing factors to its aggressive nature. The first aim of this work was to identify if and how this was the case. Towards this end, multiple RNA sequencing experiments were performed comparing foetal and neonatal/adult populations in both humans and mice. This allowed for the identification of the transcriptional differences between foetal and neonatal/adult cells. The results showed that the foetal derived cells were characterised by a proliferative and oncogenic nature whereas neonatal/adult cells had a mature and immune cell- like profile. From this it can be concluded that the foetal nature of the leukaemia-initiating cell could support the aggressive nature of the infant disease. To address the question of whether the foetal characteristics were maintained in the transcriptome of the blasts, the transcriptional profile of the foetal cells was compared to that of blasts derived from infant patients. Interestingly, there was a large commonality between the two. To further investigate whether the common genes were critical for the disease, 21 were selected and functional assays performed using the SEM cell line. With this approach, several genes were identified deletion of which had a tremendous impact on the survival of the SEM cells. The genes that were shown to be critical for the SEM cells included PLK1, BUB1B, HSPD1, ELOVL1, CCNB1, NUTF2 and TPX2. Of particular interest was PLK1 because there is an inhibitor (Volasertib) available that is currently in phase III clinical trials. Inhibition of PLK1 using Volasertib in the SEM cells resulted in cell cycle arrest, which led to apoptosis. Another gene of interest was ELOVL1, because its knockout effect appears to be unique to the infant disease. Knockout of ELOVL1 in SEM resulted in apoptosis and v investigation into the lipidome of the knockout cells identified a dramatic decrease in lipids that contain very long fatty acid chains. Additionally, using overexpression assays, DACH1 was shown to decrease the proliferation potential of the SEM cells. From this data it can be concluded that the foetal origin of the disease could be used as a means to identify novel therapeutic targets. A further aim of this work was to investigate and understand the early disease stages. For this, an additional RNA sequencing experiment was performed. This experiment used an Mll-AF4 expressing mouse model to characterise the transcriptional profile of a pre-leukemic population. Of particular interest was Skida1 which was shown to be upregulated in the Mll-AF4 expressing cells. Intriguingly, SKIDA1 was also upregulated in the blasts of infants with MLL- AF4 driven ALL compared to blasts derived from paediatric patients with the same disease and healthy controls. Interestingly, Skida1 belongs to the same family of proteins as DACH1. Intriguingly, while SKIDA1 was upregulated in the infant patients, DACH1 was not expressed at all. These findings suggest that this family of proteins could play an important role for the infant disease. This has been a proof of concept study where it was shown that by defining the transcriptome of the cell of origin of the disease and by identifying early molecular aberration caused by MLL-AF4 it was possible to identify novel disease targets. vi Lay Summary Leukaemia is a cancer of the blood and like the majority of cancers it is caused by mutations. Typically, an accumulation of multiple mutations is required for the leukaemia to develop which leads to the production of abnormal cancer cells called blasts. In general, it takes time for an individual cancer or leukaemia to develop and therefore these diseases are predominantly associated with aging. Unfortunately, there is a unique set of leukaemias that run a different course and these type of leukaemias affect infants. These leukaemias are caused by a single very potent mutation where one part of one gene (called MLL) breaks and fuses with part of another gene (AF4). The leukaemias that arise by such events are called MLL-AF4-driven and occur while the infant is still in their mother’s womb (in utero). Unfortunately, infants with this disease have an extremely poor prognosis because the disease is very aggressive and, owing to the fact that we do not completely understand the underling biology of this disease, there are few therapeutic targets. It has long been speculated that the foetal origin of these types of leukaemias is what makes them unique and very different from adult leukaemias. We know that the cells of the developing embryo (foetal cells) are very different from adult cells, as they need to support the needs of a rapidly growing organism. They have acquired specific features, for example, we know that these cells divide a lot more than other cells. Although, we do have some knowledge of how these cells are different, we still do not have the complete picture. The first aim of this work was to investigate how foetal cells are different from adult ones. Interestingly, it was observed that the foetal cells not only divide a lot more than adult cells but also that they have a very oncogenic nature. This oncogenic nature can form a supportive environment for the initial mutation to flourish and lead to this aggressive infant disease. vii Having identified these unique features of the foetal cells, the next step was to ensure that they are present in the blasts of the infant patients. Interestingly, some of them were, indeed, present suggesting that this environment seems to play a role not only in the disease initiation but also in its maintenance. To ensure that this was true, experiments were performed where the specific features were removed from the cells. This was achieved by removing a gene from a model of the infant disease. With this approach, it was observed that the removal of some of these features had a tremendous impact on the disease, as it dramatically decreased the survival of the disease model cells. Intriguingly, none of these features have been associated with the infant disease prior to this study. In summary, this study was able to define the foetal nature of the leukaemia initiating cells thereby adding to our comprehension of the underlying biology of this unique disease. Additionally, features were identified in the blasts of infant patients that were residue of the foetal cell of origin of the disease and removal of these features in a disease model resulted in a tremendous decrease in the survival of those cells. Therefore, this study serves to present a new approach for the identification of novel therapeutic targets for infant patients