Theses - Wellcome-MRC Cambridge Stem Cell Institute
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Item Embargo From Mechanisms of Leukaemogenic Dysregulation to New Candidate TherapeuticsKennedy, AlisonThe maintenance of a continuous flow of mature blood cells from a reservoir of specialised haematopoietic stem cells requires regulation from an intricate network of regulatory programmes. Gene regulatory networks are well evolved to control multi-lineage commitment and differentiation while maintaining self-renewal capacity in haematopoietic stem and progenitor cells. In leukaemia, mutations commonly hijack these regulatory programs causing both a block in normal differentiation and the misplacement of self-renewal capabilities. Rearrangements involving the *MLL1* gene (*Kmt2a*) produce powerful oncogenic proteins occurring predominantly in paediatric and infant acute myeloid and lymphoid leukaemias. Despite having more than 80 fusion partners, MLL-AF9, MLL-AF4 and MLL-ENL make up more than 60% of cases involving *MLL1* translocations. Conventional retroviral transduction models commonly use lineage depleted or c-Kit enriched mouse bone marrow haematopoietic stem and progenitor cells (HSPCs) that display distinct heterogeneity when profiled at the single-cell level which could explain inconsistencies across previously reported models. Conversely, our model, referred to as ME-Transformed cells, implements a conditionally blocked multipotent haematopoietic progenitor murine cell line (HoxB8-FL) allowing for the reproducible tracking of early leukaemic transformation and transcriptional changes due to MLL-ENL expression (Basillico., *et al* 2020). Using ME-Transformed cells, we knocked out 64 genes via CRISPR-Cas9 followed by small-bulk RNA-Sequencing. The 64 targets include transcription factors (TFs) and key transcriptional regulators implicated in the aetiology of MLL rearranged leukaemia. Bioinformatic analysis revealed a regulatory network connecting 19,437 links across 7,548 genes. Previous work in the Göttgens lab used a similar approach to establish the gene regulatory function of 39 TFs in the wild-type parental HoxB8-FL cells (Kucinski *et al*. 2020). The first aim of this thesis was to use ME-Transformed cells to establish a gene regulatory network underpinning the connection of 64 key TFs and transcriptional regulators in MLL rearranged leukaemogenesis. Thereby enabling the use of our newly established leukaemia transcription factor network to comparatively identify differences between the leukaemic and normal counterparts as a direct result of MLL-ENL expression. The second aim of this thesis was to investigate the molecular response to new candidate therapeutics. Using conventional cellular assays and RNA-Sequencing, this research project identified potential therapeutic vulnerabilities within several promising therapeutic avenues that need to be explored beyond the limits of this thesis. This experimental strategy makes the most of the nature of experimental system due to the presence of a known and accessible cell of origin (HoxB8-FL) allowing us to pinpoint candidates based on their sensitivity and specificity for the leukaemic model. The third aim was to ascertain whether prolonged exposure to AML therapeutics induces consistent molecular changes characteristic of resistant cells. Intratumoural heterogeneity underpins resistance to cancer therapies through both genetic and non-genetic means. Long term exposure of ME-Transformed cells to single small molecule inhibitory agents targeting the kinase activity of ATM recapitulated drug resistant mechanisms seen in patients. The fourth and final aim of this thesis was to determine potential combinations of small molecule therapeutics that could be combined to increase both the specificity and sensitivity of treatment while preventing the emerge of drug resistance populations. Subsequent single-cell RNA-Sequencing of ATM inhibitor resistant cells was able to identify crucial transcriptional changes between resistant and drug naive cells highlighting a new therapeutic vulnerability and a potential target for the eradication of drug resistant cells. In summary, the integration of experimental work and novel computational methods has allowed us to begin to decipher whether the perturbation of specific genes results in the identification of potential candidate therapeutic vulnerabilities as well offering a reference for which to project future perturbations, be it genetic or pharmacological.Item Open Access Role of ASCL1 and Its Interactors in NeuroblastomaMykhaylechko, LidiyaNeuroblastoma (NB) arises due to incomplete differentiation of sympathoadrenal cells during development, and is maintained by either an adrenergic or mesenchymal core transcriptional regulatory circuitry (CRC). Differentiation therapies are an attractive therapeutic approach for NB. However, it is still not fully understood how to modulate NB cell behaviour to limit proliferation and potentiate differentiation. Achaete-scute complex-like 1 (ASCL1) is a transcription factor required for both progenitor maintenance and neuronal differentiation in normal sympathoadrenal development. In NB, ASCL1 also has a dual role - as a component of the adrenergic CRC, ASCL1 supports cell proliferation, while promoting differentiation and cell-cycle exit on ASCL1 over expression. The mechanism behind these two functions is not fully understood but is likely to involve ASCL1 interactors regulating its transcriptional activity on chromatin. In this dissertation, the mechanisms by which ASCL1 induces differentiation are explored by characterising its genome-wide transcriptional targets, chromatin binding, whole proteome and protein-protein interactor differences between two pairs of NB cell lines overexpressing exogenous ASCL1. Each cell line was chosen to represent different genetic and epigenetic backgrounds of neuroblastoma disease. It was found that ASCL1 overexpression inhibited proliferation and induced morphological and transcriptional features of neuronal differentiation to a differing extent in each cell line. In the cell lines more responsive to ASCL1-induced differentiation, ASCL1 was found to associate more with proteins known to positively impact neuronal differentiation during development, such as BCL11B, PBX1, PHOX2B, SOX11, and others. On the other hand, in the cell lines less responsive to ASCL1-induced differentiation, ASCL1 associated more with proteins involved in cell-cycle regulation, such as CDKs and cyclins, or showed weaker association with most of its interactors, including E-protein TCF4, compared to a more responsive cell line. This work suggests understanding ASCL1 interactors could help explain whether this key transcriptional regulator promotes cell proliferation or differentiation of NB, allowing modulation of ASCL1-mediated differentiation to improve therapeutic options for this devastating disease.Item Embargo Alveolar stem cell dynamics and regulatory mechanisms in homeostasis and early oncogenesisEngland, Frances JaneLung adenocarcinoma (LUAD) is a leading cause of cancer-related mortality worldwide, yet the cellular dynamics and molecular mechanisms governing tumour initiation and progression remain elusive. Although alveolar type II (AT2) cells, which act as stem cells in the alveolar epithelium, have been identified as key cells of origin of LUAD, little progress has been made in understanding the precise sequence of events that transform single AT2 cells into complex malignant tumours. Here, I trace thousands of individual wildtype and oncogene-expressing mutant AT2-derived clones across multiple timepoints *in vivo* to shed insight into the early events driving neoplastic transformation. By integrating immunofluorescent analysis with 3D reconstruction and mathematical modelling of AT2- derived clones, I describe the presence of two independent AT2 subpopulations regulated by distinct dynamics. I show that these populations undergo neutral competition with their neighbours to maintain homeostasis, but that differentiation occurs more slowly at steady state than previously thought. Having established a physiological baseline for AT2 behaviour, I then utilised an oncogene-associated multi-colour reporter mouse model to simultaneously trace wildtype and *KrasG12D* mutant AT2 cells in the same mouse. Clonal analysis of the mutant compartment revealed a heterogeneous response to oncogenic activation where only one AT2 subset underwent significant clonal expansion. By leveraging single cell transcriptomics and 3D *in vitro* organoid systems, I show that these mutant cells hijack the regeneration pathway but fail to complete differentiation, resulting in aberrant expansion via stochastic fate transitions between reprogrammed cellular states. Further, I demonstrate that deletion of Interleukin 1 receptor 1 (*Il1r1*) acts as a roadblock to oncogenic expansion by preventing cells from entering the reprogramming trajectory. By performing comparative analyses between oncogenesis and homeostasis, I reveal that the presence of oncogenic clones induces a systemic injury response in wildtype AT2 cells, causing accelerated proliferation and differentiation through a similar state previously reported to be an injury-specific progenitor. These changes occur in conjunction with phenotypic changes in the surrounding niche, which, according to *in vitro* experiments, preferentially support oncogenic expansion over wildtype AT2 maintenance. Collectively, my results delineate key evolutionary trajectories driving neoplastic AT2 transformation and point towards a mechanism where mutant cells shape wildtype cellular dynamics to ultimately create a niche favoured towards tumour progression.Item Embargo Evolution of the genome in myeloproliferative neoplasms and the methylome in bloodLee, JoeThe increased accessibility of next generation sequencing (NGS) has unlocked the genomics of normal and diseased haematopoiesis, providing remarkable insights into the rates that mutations are acquired throughout life, mutations that drive disease and the growth rates of clones that acquire driver mutations. Such metrics for DNA methylation, another heritable marker, are less well understood. In this thesis, I use two NGS approaches to answer questions pertaining to genomic evolution in myeloproliferative neoplasms (MPNs) and identify changes in the methylome of blood with age and in the context of driver mutations. In the first study, serial sequencing of bulk samples from 30 patients with MPN coupled with clinical data show that clonal evolution is associated with disease transformation. Moreover, by robustly identifying all SNV mutations in major clones, we can ascribe estimates of timing over which the driver mutations may have occurred. Our timing estimates are in line with recent work by our group using single cell derived haematopoietic colonies (scHCs) and generating phylogenetic trees to time mutation acquisition. In addition, analysis of mutational signatures highlights mutagenic signals associated with therapy. In the second and more substantial body of work, a novel method to interrogate genome- wide methylation is developed and optimised and I show the steps taken to achieve this. I used this method to sequence > 700 scHCs from individuals with normal, aged and MPN haematopoiesis that had already undergone whole genome sequencing. This unique dataset provides fascinating insights into the mechanisms behind epigenetic clocks and how driver mutations may impact upon this. I also demonstrate how we discovered unexpected heritable signals from early life that are retained in scHCs of individuals up to 77 years of age. I conclude by highlighting the additional work we are undergoing to explore such insights further.Item Open Access Targeting the B-Cell Receptor in Diffuse Large B-Cell LymphomaCorcoran, SeanDiffuse Large B-Cell Lymphoma (DLBCL) is an aggressive malignancy of which one-third of diagnosed patients ultimately die. New treatment paradigms for relapsedrefractory patients are needed. Almost all DLBCL tumours express a B-cell receptor (BCR), which is a cell surface antigen receptor of immunoglobulin in tandem with the signalling adapters CD79A and CD79B. Over the last 2 decades, the importance of signals emanating from the BCR have gained prominence in how we understand oncogenic signalling in DLBCL. Multiple agents targeting BCR signalling have been tried in large clinical trials, but none have yet succeeded in altering front-line therapy. A greater understanding of BCR biology is needed. Here, I studied regulators of the BCR using whole genome CRISPR screens. First, I created an assay for internalisation of the BCR and combined it with a sorted CRISPR screen to try to understand how the BCR transits to intracellular signalling platforms. Next, I used the anti-CD79B antibody drug conjugate Polatuzumab-Vedotin (POLA-V) as a tool to study regulators of surface BCR. By combining drug CRISPR screens with surface-CD79B sorted screens, I uncovered both regulators of synergy and resistance to an important therapeutic and discovered regulators of BCR protein levels. Mechanistically, I found specific glycosylated residues on CD79A and CD79B that block Polatuzumab-Vedotin from binding its target, and I found that KLHL6 targets CD79B for degradation via CD79B K219. Based on these findings, I identified strategies for enhancing POLA-V killing by removing cell surface sialic acid. In a second line of work, I revealed a novel role for the E3 ligase KLHL6 in suppressing BCR complex cell surface expression in the germinal centre. These findings have clinical implications for patients receiving POLA-V and further our understanding of a regulator of the immune response.Item Embargo Neural and Physical Niches Orchestrate Skeletal Remodelling and Promote Cartilage RegenerationGadomski, StephenThe skeleton is densely innervated by sympathetic noradrenergic and cholinergic nerve fibres. While several studies suggest the bone-catabolic nature of noradrenergic innervation in decreasing osteoblast activity and promoting osteoclastogenesis, there is little known on the development and function of skeletal cholinergic innervation. Therefore, the first goal of my dissertation research was to elucidate potential secreted factors that promote cholinergic differentiation during postnatal skeletal development, and to examine the function of the cholinergic system in bone using genetic and pharmacological depletion of cholinergic neurons. Treatment of superior cervical ganglion cultures with interleukin-6 (IL-6) for 14 days induced cholinergic, and reduced noradrenergic, gene and protein expression. In vivo studies showed IL-6 expression in developing skeletal muscle cells, and depletion of IL-6 using targeted antibodies and IL-6-deficient mice led to reduced cholinergic innervation in developing long bones. Further, genetic ablation of GDNF family receptor-α2 (Gfra2) induced expression of tumour necrosis factor-alpha converting enzyme (TACE) in neurons, which promotes cleavage of IL-6 receptor leading to enhanced trans-IL-6 signalling, reducing cholinergic trans-differentiation. Therefore, during postnatal life, sprouting sympathetic neurons contact the periosteum and respond to IL-6 secreted by skeletal muscle, resulting in cholinergic differentiation when intrinsic TACE levels are low. Genetic lineage tracing of choline acetyltransferase (ChAT) confirmed dense cholinergic innervation in periosteum and cortical bone, with sparse branches reaching trabecular bone, and labelled bone-lining osteoprogenitors shown to amplify the cholinergic signal to the bone marrow parenchyma. Selective depletion of cholinergic neurons using Gfra2 knockout (Gfra2-/-) mice reduced cortical and trabecular bone mass, bone strength, and bone formation rate. While osteoclast numbers were unaffected in Gfra2-/- mice, osteocytes showed reduced dendritic connectivity and survival. Mechanistically, cholinergic fibres provide a rich source of the neurotrophic factor, Neurturin (NRTN), which supports osteocyte survival and growth. Loss of cholinergic innervation also led to increased expression of the Wnt inhibitor, sclerostin, in osteocytes, and inhibition of sclerostin in Gfra2-/- mice rescued deficits in bone formation. Overall, skeletal cholinergic innervation provides neurotrophic signals and suppresses sclerostin in osteocytes, promoting bone formation and osteocyte survival. Differentiated chondrocytes from human bone marrow stromal cells including skeletal stem cells (hBMSCs/SSCs) and induced pluripotent stem cells (hiPSCs) have the potential to permanently restore damaged cartilage in arthritic joints, yet chondrocyte hypertrophy is a major barrier for translational therapy. With chondrogenic differentiation, hBMSCs/SSCs undergo hypertrophy in vitro and mineralisation in vivo, leading to inferior fibrocartilage and bone formation. However, hBMSCs/SSCs attached to a fibrin microbead scaffold coated with hyaluronic acid (HyA-FMBs) produce hyaline-like cartilage for up to 28 weeks in vivo. Therefore, the second goal of my dissertation research was to examine the signalling pathways that govern the development of hypertrophic-resistant chondrocytes using the HyA-FMB model system, and to guide hiPSCs to stable chondrocyte-like cells using this mechanistic knowledge. After one day of chondrogenic differentiation in vitro, hBMSCs/SSCs attached to HyA-FMBs exhibited higher expression of extracellular matrix proteins—including Insulin-like Growth Factor Binding Protein-5 (IGFBP5) and Matrix Gla Protein (MGP)—and decreased Bone Morphogenic Protein (BMP) signalling evidenced by pathway analysis. However, BMP signalling was restored by day 5, and increased by day 10, in a chondrogenic subpopulation enriched for IGFBP5 and MGP, accompanied by diminished expression of hypertrophic and osteogenic markers (COL10A1, ALPL, IBSP, and SPP1) exclusively in hBMSCs/SSCs attached to HyA-FMBs. Transcriptomic measurements confirmed increased BMP signalling in stable hyaline-like cartilage produced by ectopic transplantation of hBMSCs/SSCs attached to HyA-FMBs. Using this knowledge, I developed a serum-free hiPSC differentiation strategy that inhibited, then activated BMP signalling in a purified SOX9+ subpopulation that naturally detaches from monolayer cultures (termed “chondrospheroids”). Treatment of SOX9+ chondrospheroids with BMP-2 and GDF-5 produced uniform and stable expression of COL2A1, ACAN, and PRG4 and minimal expression of COL10A1 in vitro and in vivo. Osteochondral transplantation of day 35 chondrospheroids, which mimicked the transcriptional identity of a foetal chondrocyte, produced stable hyaline-like cartilage for up to 5 months in NSG mice and SRG rats when attached to HyA-FMBs. Therefore, BMP signalling activates and maintains a hypertrophic-resistant chondrogenic cell enriched for SOX9, MGP and IGFBP5 during chondrogenic differentiation.Item Embargo Functional and Transcriptional Heterogeneity of the Human Haematopoietic Stem Cell Pool at Steady-State and Under Inflammation(2021-02-10) Calderbank, Emily; Calderbank, Emily [0000-0002-9559-6593]Blood production is coordinated by a functionally heterogeneous pool of multipotent haematopoietic stem cells (HSCs), downstream of which lineage-restricted progenitors are generated. The advent of single cell technologies has changed our view of the haematopoiesis to a dynamic continuum. Understanding the early differentiation trajectories of HSCs, and how environment and molecular factors can modify them, is vital in furthering our insight into human haematopoiesis in health and disease. Here I combined index sorting, single cell functional assays in vitro, RNA-sequencing (RNAseq) and in vivo assays to i) study lineage commitment heterogeneity within the HSC compartment of cord blood (CB) and foetal liver (FL) ii) to explore the role of inflammatory signals in HSC differentiation, using an in vitro model of human early HSC differentiation I developed. Using in vitro functional assays, I uncovered that at single cell resolution, the CB HSC/Multipotent progenitor (MPP) compartment is polarised based on lineage output. I established novel prospective purification strategies, that maximise enrichment of cells with myeloid (My)-erythroid (Ery) (CD34lo CLEC9Ahi; Subset1) or My-lymphoid (Ly) (CD34hi CLEC9Alo; Subset2) potential in vitro. In vivo, I used an optimised NSG xenograft model for detection of erythroid potential, to show that Subset2 cells were restricted to My-Ly differentiation and displayed infrequent long-term repopulation capacity. In conclusion, I demonstrated that the first lineage restriction step in human haematopoiesis occurs within the human HSC/MPP pool and generates My-Ly committed cells with no erythroid differentiation capacity. Using similar methodologies as above, I report 2 main findings in the human FL HSC/MPP compartment: i) there is a decrease in multipotency and Ery potential with gestational age but an increase in Ly potential and ii) there is an increased percentage of cells in G0 of the cell cycle with gestational age, indicating a progressive shift to quiescence. Finally, I developed an in vitro model of early haematopoiesis by culturing long-term (LT-) HSCs for 5-days then performing single cell RNAseq and single cell functional assays. In this model most lineage types were produced: My, Ly, Ery, megakaryocyte (Meg) and mast cells (MC). Studying the differentiation trajectories observed in this model, I identified IL1RL1, the gene encoding the IL-33 receptor, ST2, as a potential modulator of the Ery, Meg and MC branch. When exposed to IL-33, LT-HSCs showed increased differentiation towards the Meg, MC (in vitro) and Ery lineages (in vitro and in vivo) but maintained long-term engraftment potential. This demonstrates a novel role of the pro-inflammatory cytokine IL-33 as a regulator of early LT-HSC differentiation.Item Open Access MYC transcriptional functions controlling epidermal stem cell self-renewal and differentiation(2011-09) Nascimento, ElisabeteThe oncoprotein MYC has long been recognized as an important stem cell regulator, yet its direct biological contributions have been difficult to determine. MYC activation can induce pleiotropic phenotypes and mediates cellular functions as opposing as cell growth and proliferation, metabolism, differentiation and apoptosis. In addition, functional redundancy with MYCL and MYCN proteins as well as dose dependency, complicates the identification of the most relevant biological functions. Studies in tissues with high proliferative capacity and rapid turnover have shown that MYC is a key regulator of homeostasis by balancing stem cell self-renewal, proliferation and differentiation processes. In skin, MYC induces the exit of epidermal stem cells from their niche, increases proliferation of progenitor cells and subsequently stimulates lineage specific differentiation into interfollicular epidermis and sebaceous glands; yet the direct transcriptional roles of MYC in these processes remained elusive. To gain insight into the transcriptional roles of MYC in epidermal stem cell homeostasis, I performed chromatin immunoprecipitation on microarrays (ChIP-on-Chip) using mouse proximal promoter arrays combined with mRNA expression data that was generated using epidermal cells from wild-type and transgenic K14MycER mice, treated in a time-course from zero to six days with tamoxifen, to induce the ‘Myc’ transgene expression in the basal undifferentiated layers of the epidermis. Data analysis revealed that 2187 genes, which corresponds to 15% of the promoter regions covered, were directly regulated by MYC. To identify genes uniquely regulated by MYC in skin, I performed gene expression studies on mouse skin in which MYC was conditionally deleted in the basal layer of the epidermis. Remarkably, I found that 45% of all repressed genes were related to epidermal maintenance and differentiation. To better understand the mechanism of how MYC induces keratinocytes to differentiate specifically into lineages of sebaceous glands and interfollicular epidermis, I analyzed whether MYC might have directly regulated genes involved in skin differentiation. Here, I focused my studies on a single 2.2 Mb locus located on mouse chromosome 3 designated as the epidermal differentiation complex (EDC). To assess how activation of MYC could influence the expression of genes localized to the EDC, I performed ChIP-on-Chip for MYC, H3K4me3, H3K27me3, as well as transcription factors, which have been described to regulate terminal differentiation in skin, such as CEBPα, OVOL-1, KLF4, TCFAP2-γ and SIN3A, among others. I demonstrated that MYC recruits a specific set of tissue-specific transcription factors to the EDC, (e.g. KLF4 and OVOL-1) and thereby prevents binding of a different and distinct set of genomic regulators, (e.g. CEBPα , MXI1 and SIN3A). Using a combination of mouse models and systems biology tools, I then identified SIN3A as a key regulator in this MYC-dependent transcriptional network. I found that MYC and SIN3A form a negative feedback loop, which is required to balance proliferation and differentiation in epidermis, and both factors are essential to maintain skin homeostasis.