Differential ammonium detoxification capacity influences mitochondrial anaplerotic pathways Aakriti Jain A dissertation submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy Division of Biosciences University College London and The Francis Crick Institute September 2019 1 Declaration I, Aakriti Jain, 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……. The tricarboxylic acid (TCA) cycle is a major pathway in central carbon metabolism that supports energy production and anabolic processes required for cancer cell survival and proliferation. To maintain flux through the TCA cycle, individual anaplerotic reactions need to coordinate with cataplerotic reactions and other anaplerotic pathways. This work uses in vitro reconstitution of mitochondrial metabolism to explore the mechanisms through which glutamine- and glucose-derived anaplerotic pathways are coordinated. In cell-free mitochondria, increased ammonium levels, as a consequence of glutamine deamidation, increased reductive amination through glutamate dehydrogenase (GDH) and flux through transaminases for anaplerosis. Ammonium- mediated changes to glutamine-derived anaplerosis were correlated with increased pyruvate-derived anaplerosis evidenced by increased flux through pyruvate carboxylase (PC). Addition of exogenous substrates to invoke flux through the urea cycle, as an alternative pathway to detoxify ammonium, rescued the effects of ammonium on GDH and PC. The studies in cell-free mitochondria revealed that acute changes in intramitochondrial ammonium levels coordinated glutamine- and glucose-derived anaplerosis without gene expression changes. To determine if chronic changes in gene expression could induce ammonium-mediated coordination of anaplerosis, enzymes that may affect intramitochondrial ammonium levels in whole cells were modulated. Overexpression of the urea cycle enzyme, carbamoyl-phosphate synthetase 1 (CPS1) was associated with decreased flux through PC and increased sensitivity to oxidative stress. Analysis of mRNA expression data from human cancers showed that CPS1 mRNA expression was down-regulated in hepatocellular carcinoma (HCC). A mouse model of HCC, which exhibited the same changes in gene expression on the protein level, was used to show that attenuation of CPS1 expression was associated with increased reductive amination through GDH and increased PC-derived anaplerosis. Altogether, this study provides evidence suggesting that down-regulation of the urea cycle and ammonium-mediated coordination of anaplerosis may promote survival and proliferation of HCC cells. 3 Impact Statement Cancers are often implicated with aberrant metabolism, which helps cancer cells survive and proliferate. In order to target cancer metabolism with increased efficacy, it is vital to understand both the impact of the changes to the metabolic network and the mechanisms through which such changes may take place. The tricarboxylic acid (TCA) cycle is a central metabolic pathway that can supply both the increased demands for energy and for biosynthetic macromolecules in cancer cells. Therefore, maintaining carbon flux through the TCA cycle is critical for cancer cell growth. Pathways that maintain TCA cycle flux are called anaplerotic pathways; these pathways can individually be regulated through several different mechanisms. However, lesser is known about how the different pathways may be coordinated with each other. This thesis provides evidence for ammonium-mediated coordination of two distinct anaplerotic pathways (glucose-derived and glutamine-derived). Furthermore, the study presented in this thesis examines how chronic changes in the expression of the canonical ammonium detoxifying pathway, the urea cycle, leads to increased ammonium-mediated coordination of anaplerosis in hepatocellular carcinoma. Altogether, this project increases our understanding of an important metabolic pathway and may, ultimately, lead to more efficient therapeutic strategies that target metabolism. 4 Acknowledgements It takes a village to make a doctor, and I am no exception. I have grown immensely, both personally and professionally, over the last few years and I have many (too many) people to thank for this; I will not get to all of them, but I am deeply grateful for all of the help and support I have received from all the colleagues, friends, and family in my life. First and foremost, I would like to thank my supervisor, Dimitrios Anastasiou, for giving me the opportunity to conduct my PhD studies in his lab. I am grateful for both the scientific direction and support that he has provided me over the years, but also thankful for the time and freedom he allowed me to explore and develop my own ideas. I would also like to thank my thesis committee members: Bart Vanhaesebroek, Erik Sahai, and Victoria Sanz-Moreno, for their guidance and scientific advice throughout my PhD. I would also like to thank Mariia Yuneva for the useful discussions about glutamine metabolism and more. My PhD was primarily funded by a fantastic EU program for early stage researchers (MetaRNA Marie-Curie ITN), and I am very thankful for the international and collaborative opportunities afforded by such programs. I am also fortunate to have to have experienced working in the collaborative environment of the Francis Crick Institute. One of the best aspects of the Crick are the helpful people and incredible wealth of knowledge they hold. My project would be nowhere without the Metabolomics STP: in particular, I would like to thank James MacRae, for being a great mentor and friend; Paul Driscoll, for all the hours spent solving NMR puzzles; Mariana Santos, for always finding ways to detect difficult metabolites on the LC-MS; and Jim Ellis for always helping fix the GC-MS, when I invariably break it. I would also like to thank Lucy Collinson from the EM STP, and, especially, Marie-Charlotte Domart, who painstakingly annotated hundreds of mitochondria for me. Probir Chakravarty from the Bioinformatics STP was also a great help in pointing me to the TCGA database and always answering my RNA-seq analysis- related questions. Mike Howell from the Crick High-throughput screening STP helped me with cell proliferation assays. I am also thankful for the Crick BRF staff, for all their help and patience with me over the years. Finally, I’d like to thank the Crick HR and PhD student admin team, and especially Caroline Ransom, for helping me sort out many complicated visa- and contract-related issues over the years. 5 Most integral in making me feel at home (in a place far away from “home”) are the colleagues that turned into great friends. I am thankful for all the current and former members of the Anastasiou and Yuneva labs for all the great scientific and non-scientific conversations during lunch, Friday drinks, and Phyzzmet. Particularly, I’d like to thank: Louise Fets, for teaching me how to be a beer-bourgeoisie and for all the pep-talks turned goss-sessions; Fiona Grimm, for opening my eyes to the world of R and for being a fellow podcast- and cat-lover; Patricia Nunes, for all the support, both as a scientist and as a friend, and for teaching me so much about metabolism; Steffi Gehrig, for being my old desk-buddy and catering to all my silly questions; Natalie Bevan, for being my new desk- buddy and pouring so many of my gels; Emma Still, for being a great friend and staying behind with me that one lunch after rhubarb-gate; and Joanna Segal, ditto re: rhubarb- gate and for being the best thesis-writing-buddy ever. Finally, thanks to Emma Powell, Ruta Ziukaite, and Luis Landas for great dinners, trips and board game nights; and to members of the LMCB ‘Secret Board Game Society’ for fun Friday evenings at the Square Tavern. Last, but not least, I want to thank my amazing family. Thanks Adish for being the best brother and for always questioning and challenging me. My mom and dad are my #1 fans and biggest supporters in life, and I would be nothing and nowhere without them. And finally, I’d like to thank my best friend and life partner – Henry de Belly (and our cute little kitten, Venus). You both have the patience and capacity to deal with my highs and my lows and I am more grateful for that than words can express! 6 Table of Contents Abstract…….3 Impact Statement .......................................................................................................... 4 Acknowledgements ...................................................................................................... 5 Table of Contents .......................................................................................................... 7 List of figures .............................................................................................................. 13 List of tables... ............................................................................................................. 17 Abbreviations .............................................................................................................. 18 Chapter 1.Introduction ............................................................................................... 23 1.1 The TCA cycle ................................................................................................... 24 1.1.1 Glucose-derived anaplerosis ................................................................................... 25 1.1.2 Amino