NEWCASTLE UNIVERSITY Control of Lineage Commitment in Acute Leukaemia Ricky Fong Tirtakusuma Northern Institute for Cancer Research Faculty of Medical Sciences Doctor of Philosophy September 2018 Abstract Acute leukaemia with the t(4;11) translocation is strongly associated with pro B-acute lymphoblastic phenotype. Here is described a lineage switch from acute lymphoblastic leukaemia (ALL) to acute myeloid leukaemia (AML) which carries identical t(4;11) breakpoints that provides insight into regulation of lineage commitment and the haematopoietic origin of leukaemia. Stable DNA microsatellite sequences argue against a therapy-related AML. Genome sequencing and RNAseq identified 12 novel and deleterious mutations unique to the AML. Immunoglobulin rearrangement analysis suggested the common cell of origin lied within a population prior to B cell differentiation. Sorting of haematopoietic stem/progenitor cell populations followed by multiplex PCR and next generation sequencing for the fusion and secondary mutations demonstrated the occurrence of the leukaemogenic MLL-AF4 fusion gene in cell populations as early as the multipotent progenitor, MPP, population in both ALL and AML. In this most primitive population, the AML carries mutations in chromatin modulating genes CHD4 and PHF3, suggesting their importance in lineage commitment. Knockdown CHD4 and PHF3 individually and in combination in the pro-B ALL t(4;11) SEM cell line resulted in ~3 fold higher expression of the myeloid cell surface marker CD33. Further analysis was performed using a recently described model of MLL-AF4 leukaemogenesis consisting of CD34+ cord blood cells transduced with a chimeric MLL-Af4 fusion gene. Knockdown of CHD4 and PHF3 resulted in loss of lymphoid differentiation potential in vitro. Analysis of different PHF3 splice variants revealed that only mutation-carrying PHF3 variants increased CD33 on SEM cells and that a balance between PHF3 variants was required for the lineage fidelity. This study suggests that the ALL and AML share a common primitive cell of origin and that mutations in CHD4 and PHF3 shift the lymphoid phenotype towards a myeloid lineage leukaemia. i ii Acknowledgment I would like to express my biggest thanks to my supervisor Olaf Heidenreich for his continuous teaching, ideas, patience, massive support and care, which not just limited to the project. To make it shorter, his guidance is much more prominent than an excellent supervisor. Thanks to Melanie, who was not only helping me a lot in the lab, but was also correcting my English in this thesis. Also thanks to Natalia for providing many valuables ideas in this project, in particular, the multiplex PCR candidate genes on the haematopoietic hierarchy and PHF3 isoforms. I am sure these are highly essential points in this study, and at least these parts are not from Olaf’s geniusness, but hers. I want to thank Alex, for firstly, providing the primograft samples (together with Helen), and also always explaining me the subjects that I didn’t even know where to start. His massive help was in particular at the beginning of the project, when I had the meetings with Olaf, didn’t understand what he meant, Alex would always be able to re-explain it to me. Massive thanks to Helen Blair for helping me transplant and harvest the primograft samples, also for her constant positive behaviour and support. Thanks to Natalie, Katie, Sarah Fordham, Dan Coleman, Hesta, and Helen Marr that introduced and taught me to the lab work at the beginning of the project, and for making the lab extraordinarily nice and exciting. Hesta helped me a lot with the cell sorting. Sarah Fordham and James Allan analysed the microsatellite instability data and explained it to me in great detail. Helen taught me the immunophenotyping of the patient samples in a very comprehensive manner. Thanks to Yuzhe, Hasan, Milene, Asmida, Eva, Peixun, Azira, Kasia, and Anja for not only being very nice, but also helping me with lab works, teaching some assays, and providing valuable discussion and ideas. Anja provided a highly valuable coffee automate. My huge thanks also to Lynne Minto, Marian, Liz, and Anne for being extremely helpful and keeping the lab running efficiently. Lynne, Marian, and Liz provided most of the patient samples. Also, Simon for reviewing this thesis. Thanks to Martyna, Claire, Richard, Judith, Bailey, and Gary for always answering my questions about the basic protein works. iii Thanks to Sarra Ryan and Paul Sinclair for teaching me the sequencing sample preparations, also still very helpful even though I ask plenty of questions! To Rin, Louise, and Matt for helping analysing bioinformatics data. Thanks to Paul Milne for helping the haematopoietic cell sorting and explaining me many things about the methods in flow cytometry. Thanks to Claus Meyer and Rolf Marschalek for helping to identify the sequences of the MLLr samples. This is a huge help in determining the lineage switch case also finding the pre-leukaemic population. Thanks to Ehud Shapiro’s team, especially to Rivka Adar, who was very kindly helped me with the single cell assay and the troubleshooting. Also to Mulloy lab for the cord blood cells MLL/Af4 cells, especially to Shan Lin who explained to me very clearly how to work with the model. Thanks to Gemma Llargues and Frederik as the source of ATP in the lab by always sharing the positivity and happy faces. I am sure they will still be positive even during torture. Thanks to everyone in the NICR, including the past members, since I am sure I have asked many of the colleagues here for many things, including questions, reagents, chemicals, etc. Lastly, thanks to the patients and the family. This study was coming from them and I hope I have not disappointed any of them and am grateful to them for giving their contributions and hopes. iv Table of Contents Abstract ....................................................................................................................... i Acknowledgment ...................................................................................................... iii Abbreviation ............................................................................................................ xii Chapter 1 Introduction ............................................................................................ 1 1.1. Haematopoiesis ............................................................................................................ 1 1.2. Acute leukaemia ........................................................................................................... 4 1.2.1. Infant ALL .............................................................................................................. 5 1.2.2. Paediatric acute myeloid leukaemia (AML) ........................................................... 8 1.2.3. MLL-rearranged leukaemia .................................................................................. 11 1.3. Leukaemia lineage switch ........................................................................................... 14 1.3.1. Introduction to leukaemia lineage switch ............................................................. 14 1.3.2. Study cases ......................................................................................................... 15 1.4. Patient L826 and preliminary data .............................................................................. 21 1.4.1. Immunophenotypes ............................................................................................. 22 1.4.2. Chromosome study .............................................................................................. 24 1.4.3. Fusion gene breakpoint sequences ..................................................................... 25 1.4.4. Microsatellite instability analysis .......................................................................... 25 1.4.5. Whole genome, whole exome, and RNA sequencing ......................................... 28 1.5. Candidate driver genes ............................................................................................... 30 1.5.1. ACAP1 ................................................................................................................. 31 1.5.2. CHD4 ................................................................................................................... 32 1.5.3. PHF3 .................................................................................................................... 35 1.5.4. PPP1R7 ............................................................................................................... 37 Chapter 2 Materials and Methods ........................................................................ 39 2.1. Materials ..................................................................................................................... 39 2.1.1. Laboratory equipment .......................................................................................... 39 2.1.2. Chemicals and reagents ...................................................................................... 40 2.1.3. Buffers and media ................................................................................................ 42 2.1.4. Bacterial strains ................................................................................................... 47 2.1.5. Antibodies ............................................................................................................ 48 2.1.6. Oligonucleotides