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1
Lewis, Ian D. "Characterisation of normal and leukaemic stem cells in chronic myeloid leukaemia /." Title page, contents and abstract only, 1998. http://web4.library.adelaide.edu.au/theses/09PH/09phl6745.pdf.
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POTETI, MARTINA. "The metabolic profile of Chronic Myeloid Leukaemia: stem cells as a target to overcome resistance to therapy." Doctoral thesis, Università di Siena, 2019. http://hdl.handle.net/11365/1071850.
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Chronic Myeloid Leukaemia (CML) is a stem cell-driven disorder treated with Tyrosine Kinase inhibitors (TKi) with impressive efficacy. However, TKi are unable in most cases to prevent the relapse of disease, as even a very successful response to treatment results in the persistence of a state of Minimal Residual Disease (MRD). Our hypothesis predicts that MRD is sustained by the persistence of Leukaemic Stem Cells (LSC) capable to survive and cycle independently of BCR/Abl kinase activity within Bone Marrow (BM) stem cell niches where severe oxygen and glucose shortage would result in BCR/Ablprotein suppression. In this study, we addressed the role of the availability of glutamine, among a number of other metabolites possibly relevant in this context, in the control of BCR/Ablprotein expression in CML cell cultures where energy supply is markedly restricted, i.e. maintained under conditions likely mimicking those of stem cell niches in vivo.
We found that glutamine drives accelerated BCR/Ablprotein suppression and that this phenomenon is paralleled by the kinetics of glucose consumption from culture medium. The relationship between presence of glutamine and glucose consumption was deepened by investigating the effects of different metabolic inhibitors. We found that the inhibition of glycolysis via treatment with 2-DG or 3PO, as well as that of Pentose Phosphate Pathway (PPP) via treatment with 6-AN, prevented the effects of the presence of glutamine on BCR/Ablprotein expression, confirming that BCR/Ablprotein suppression requires the presence of glutamine and depends on glucose consumption irrespective of the pathway driving this consumption. On the contrary, the inhibition of OxPhos by means of metformin did not interfere with the effects of the presence or absence of glutamine on BCR/Ablpr
otein expression.
The effects of the presence or the absence of glutamine were also tested on the maintenance of stem cell potential in low oxygen. Using our in vitro LSC assay, Culture-Repopulation Ability (CRA) assay, we found that cells grown in the absence of glutamine exploited their stem cell potential promptly upon transfer to permissive conditions. The treatment of glutamine-free cultures with metformin did not interfere with the pattern of LC2 repopulation. On the contrary, BCR/Ablprotein-negative cells were affected by metformin treatment.
The treatment with BPTES, a GLS1 inhibitor, in either the presence or the absence of glutamine, favored the maintenance of BCR/Ablprotein expression in low oxygen, so that LSC transferred to permissive conditions were capable to exploit their stem cell potential rapidly, driving prompt LC2 repopulation. This result could represent the basis of an innovative CML treatment strategy using inhibitors of glutamine metabolism in combination with TKi to determine LSC eradication together with induction or maintenance of remission.
3
Volpe, Giacomo. "Regulation of flt3 gene expression in haematopoietic and leukaemic stem cells." Thesis, University of Birmingham, 2010. http://etheses.bham.ac.uk//id/eprint/822/.
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The interaction between the tyrosine kinase receptor Flt3 and its ligand leads to signalling during the commitment of haematopoietic stem cells (HSCs). Constitutive activation of the Flt3/FL pathway is a key factor in enhanced survival and expansion in acute myeloid leukaemia (AML). Although there is extensive knowledge regarding mutations leading to the constitutive activation of Flt3 receptor activity, the molecular mechanisms underlying the regulation of the \(flt3\) gene in HSCs, and how such mechanisms might be altered in leukaemia, are still poorly understood. Here, by using HSC and leukaemic cell lines, I locate several regulatory elements in the \(flt3\) locus by DNaseI mapping and have characterized their epigenetic environment. Analysis of the methylation and acetylation status of histones H3 and H4 around \(flt3 cis\)-regulatory regions highlights a distinct combination of epigenetic modifications specific to AML cells in a region that distinguishes the Flt3\(^-\) and Flt3\(^+\) stages of HSC differentiation. Moreover, I show the link between the \(in vivo\) binding of C/EBP and c-Myb on regulatory elements and epigenetic remodelling in the differential regulation of \(flt3\) in leukaemic cells. Finally, I identify the histone modifiers TIP60 and CBP as potential mediators of the epigenetic regulation of \(flt3\) in AML cells.
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Kuntz, Elodie Marie. "An investigation of metabolic vulnerabilities in chronic myeloid leukaemic stem cells." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/8615/.
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Chronic myeloid leukaemia (CML) is a myeloproliferative disorder that originates at the haematopoietic stem cell (HSC) level. CML is driven by BCR-ABL, a fusion oncoprotein with a constitutive tyrosine kinase activity. The discovery of imatinib, a c-Abl specific tyrosine kinase inhibitor (TKI), revolutionised the treatment of CML by inducing cytogenetic and molecular responses in the majority of CML patients in chronic phase. However, imatinib and second/third generation TKIs do not eradicate leukaemic stem cells (LSCs), leading to disease persistence with associated risk of toxicity, drug resistance and relapse. This suggests that effective eradication of CML LSCs requires identification of novel target(s) that can be exploited therapeutically in combination with TKI treatment. In recent years, a plethora of studies have demonstrated that cancer cells rewire their metabolism to fuel their high energy demands and targeting these metabolic alterations can be of therapeutic benefit. Thus far, investigation of CML LSCs metabolism has been restricted by technical limitations. In this study, we aimed to identify and target the metabolic dependencies in CML LSCs using stem cell-enriched (CD34+) primary cells isolated from CML patients and healthy donors. We initially investigated the metabolism of differentiated CD34- and primitive CD34+ cells and demonstrated that glucose and fatty acid oxidation was elevated in CD34+ CML cells. We as well demonstrated that CML CD34+ cells displayed an increase in their mitochondrial oxygen consumption rate (OCR). Next, we compared the metabolism of CD34+ and CD34+CD38- CML cells to their respective normal counterparts, which revealed that stem cell-enriched CML cells possess increased mitochondrial functions in comparison to normal cells. Of clinical significance, we show that the antibiotic tigecycline, an inhibitor of mitochondrial translation, reduced this aberrant oxidative metabolism. The combination of imatinib and tigecycline targeted primitive CML cells at a clinically achievable concentration while having minimal effect on colony formation potential of CD34+ cells derived from healthy donors. To validate these findings in vivo, human CML CD34+ cells were injected into irradiated immune-deficient mice. Remarkably, four-week combination treatment with tigecycline and imatinib in vivo eliminated the majority of CML LSCs, targeting 95% of the cells. Moreover, mice maintained low levels of CML LSCs upon discontinuation of the combination treatment whereas imatinib-treated mice showed signs of relapse. These results indicate that oxidative phosphorylation is crucial for the survival of CML LSCs and inhibition of mitochondrial metabolism with tigecycline, in combination with imatinib treatment, might be a suitable therapeutic strategy to selectively target these cells and improve cure rates.
5
Robinson, Simon N. "Proliferation regulation of haematopoietic stem cells in normal and leukaemic haematopoiesis." Thesis, University of St Andrews, 1992. http://hdl.handle.net/10023/14965.
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The cellular integrity of the blood is maintained by the cellular output of the haematopoietic stem cell population which produces the specialized precursors and differentiated cells which constitute the blood. The investigation of haematopoietic stem cell behaviour and regulation has been hampered by both the difficulty in their identification and the development of relevant assay systems. The purpose of this investigation was to study the behaviour and regulation of the haematopoietic stem cell population in normal and leukaemic haematopoiesis using an in vitro assay of a primitive haematopoietic precursor. The use of a combination of haematopoietic colony-stimulating factors [interleukin 3 (IL3)/multi-CSF and macrophage colony-stimulating factor (M-CSF/CSF-1)] in semi-solid agar culture of murine haematopoietic tissue, stimulated the proliferation of a haematopoietic colony-forming cell, defined as the "HPP-CFCIL3+CSF-1" population, which was characterized by a high proliferative potential, a multipotency and behavioural and regulatory properties consistent with its being a primitive haematopoietic precursor and possibly a component of the haematopoietic stem cell population. The proportion of the in vitro HPP-CFCIL3+csf-1 population in S-phase in normal murine marrow, was determined to be relatively low at approximately 10%, increasing to approximately 40% in sublethally X-irradiated, regenerating murine marrow and the respective presence of the haematopoietic stem cell proliferation inhibitor and stimulator was demonstrable by the induction of appropriate kinetic changes in the in vitro HPP-CFCIL3+CSF-1 population. In leukaemic haematopoiesis, leukaemic proliferation often occurs at the expense of apparently suppressed normal haematopoiesis. In vitro HPP-CFCIL3+CSF-1 assay of the haematopoietic stem cell proliferation regulators in a number of murine, myeloid leukaemic cell lines, failed to demonstrate either increased levels of the haematopoietic stem cell proliferation inhibitor, or evidence of a direct-acting, leukaemia- associated proliferation inhibitor, however, evidence of a leukaemia- associated impairment of inhibitor and stimulator production was observed and this may be a possible mechanism by which the leukaemic population develops a proliferative advantage over normal haematopoietic tissue. The identification of a possible mechanism of leukaemic progression and suppression of normal haematopoiesis may subsequently allow the development of potentially more effective disease treatment and management regimes. The endogenous haemoregulatory tetrapeptide: Acetyl-N-Ser- Asp-Lys-Pro [AcSDKP, Mr=487 amu] is reported to prevent the G0-G1 transition of haematopoietic stem cells into S-phase. The mechanism of action of AcSDKP and a number of related peptides, was investigated in relation to the stem cell proliferation stimulator and inhibitor. AcSDKP demonstrated no direct haemoregulatory role against the in vitro HPP-CFCIL3+CSF-1 population, which is consistent with reports that AcSDKP is not active against cells already in late G1, or S-phase, rather it appeared to act indirectly by impairing the capacity of the haematopoietic stem cell proliferation stimulator to increase the proportion of the in vitro HPP-CFCIL3+CSF-1 population in S-phase. An apparent impairment of stimulator action may explain the reported AcSDKP-associated 'block' of haematopoietic stem cell recruitment. A putative endogenous AcSDKP precursor and synthetic and degradative enzyme systems have been reported and the possible physiopathological role of AcSDKP in a number of myeloproliferative disorders has been implicated. The potential application of AcSDKP as a 'haemoprotective' agent administered prior to the use of S-phase- specific chemotherapy may be of clinical significance. The in vitro HPP-CFCIL3+CSF-1 assay of a primitive haematopoietic precursor cell population, which may be a component of the haematopoietic stem cell population, should play a significant role in the investigation of haematopoietic stem cell behaviour and regulation in both normal and aberrant haematopoiesis. With the characterization of the mechanism(s) of action of the haematopoietic stem cell proliferation inhibitor and stimulator and the haemoregulatory tetrapeptide AcSDKP, the manipulation of the haematopoietic system to clinical advantage can be envisaged, while the identification of the aberrant regulatory mechanism(s) in haematopoietic dysfunction may allow, the development of more effective disease treatment and management regimes.
6
江卓庭 and Cheuk-ting Kong. "Understanding the function of the Mll-een leukaemic fusion gene by embryonic stem cell approaches." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2003. http://hub.hku.hk/bib/B31244312.
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Taylor, Alan. "The role of leukaemia inhibitory factor and a leukaemic associated inhibitor in the control of the proliferation of haematopoietic stem cells." Thesis, University of St Andrews, 1996. http://hdl.handle.net/10023/14962.
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Activities associated with, or interacting with, leukaemic cell populations were assayed for the ability to influence in vitro haematopoiesis. The first of these, the glycoprotein leukaemia inhibitory factor (LIF), has a role in aspects of murine, non human primate and human haematopoiesis. It is thought to be particularly important in the development of megakaryocytes and is also known to induce the terminal differentiation of certain leukaemic cell lines. LIF was assayed both for direct and indirect effects on the proliferation of haematopoietic precursor cell populations in vitro. As a direct acting agent in semi-solid agar culture of haematopoietic cell populations derived from normal bone marrow or 15 day foetal liver, LIF was unable to support colony formation. In cultures of cells derived from normal bone marrow stimulated with single, or combinations of, growth factors, the addition of LIF had no statistically significant effect on the level of colony formation. In cultures of cells derived from foetal liver, stimulated with particular growth factor combinations (medium conditioned by the Wehi3B leukaemic cell line + medium conditioned by the lung fibroblast cell line, L929); GM-CSF + M-CSF; IL-la + IL-3 + M-CSF), LIF, was shown to decrease the level of colony formation. LIF did not directly alter the proportion of the population in DNA synthesis in cell populations derived from normal femoral marrow, 15 day foetal liver or y- irradiated femoral marrow. As an indirect acting agent LIF failed to block the synthesis of a stem cell stimulator, or it's action, on a population of high proliferative potential colony forming cells derived from normal femoral marrow, cloned in the presence of Wehicm+L929cm. (HPP-CFC (Wehicm + L929cm)) LIF's actions on clones of a murine myeloid leukaemia (SA2JMB1) were also assessed. LIF had no statistically significant effect on colony formation or the level of DNA synthesis in populations of SA2JMB1 leukaemic cells. A second group of associated activities was produced by the X- irradiation induced murine myeloid leukaemia (SA2JMB1). Medium conditioned by the leukaemic cells was assayed in vitro both for direct and indirect effects on the proliferation of haematopoietic cells derived from femoral marrow. As a direct acting agent in 7 and 14-day semi-solid agar culture of femoral marrow, leukaemic conditioned medium alone stimulated limited colony formation. In 7 and 14 day cultures stimulated with single and combinations of specific colony stimulating factors: (rmGM-CSF, rhM-CSF, rhIL-1a) a significant increase in colony number was noted in all cases when cultures were supplemented with leukaemic conditioned medium. SA2JMBlcm was shown to support the proliferation of an IL-3 dependent cell line (FDCP-A4 cells). The colony enhancing ability of SA2JMBlcm was shown to be blocked by pretreatment with antibodies to IL-3. This suggested that SA2JMB1 conditioned medium contained IL-3 or an IL-3 like activity, as one of its components. The conditioned medium failed to directly alter the level of DNA synthesis in a population of HPP-CFC (Wehicm+L929cm) derived from normal bone marrow or y- irradiated bone marrow. As an indirect acting agent the conditioned medium did block the action of a stem cell proliferation stimulator on normal bone marrow derived HPP-CFC (Wehicm+L929cm). This leukaemia associated activity was shown to be larger than 50KD, sensitive to heat treatment and able to act in a different manner to the stem cell inhibitor MIP-1-a. Thus this novel activity may be important in blocking stimulator action in haematopoietic stem cells and thus contribute to the haematopoietic insufficiency seen in leukaemia.
8
Austin, Pamela M. "Defining biological properties of the leukaemic stem cell in Hoxa9Meis1-induced leukaemia." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=80222.
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A subset of the leukaemic clone, the leukaemic stem cell (L-HSC), is responsible for the maintenance and propagation of a leukaemia. Most current therapies, however, do not target this population. In this thesis, I show that a determinant of disease phenotype is the frequency of the leukaemic stem cell within the leukaemic population. Moreover, the frequency of L-HSC in a leukaemia is predetermined by the inherent properties of the cell that is transformed and can be attributed to the ontogenic origin of the cell. Ideal therapies would specifically target mechanistic defects in leukaemic stem cells; however, the pathways that are involved and what oncogenic defects can be targeted for therapy remain to be discovered. As it is not known if all oncogenes can be targeted or what the determinants of oncogenic dependency are, I have developed a system to further elucidate this as described herein.
9
Carlens, Stefan. "Leukaemic relapse after allogeneic haematopoietic stem cell transplantation and the use of the graft-versus-leukaemia effect /." Stockholm, 2000. http://diss.kib.ki.se/2000/91-628-4310-9/.
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10
Taussig, David. "Characterisation of acute myeloid leukaemia stem cells." Thesis, Queen Mary, University of London, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424766.
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Wilson, Kerrie Lauren. "Malignant stem cells in childhood acute lymphoblastic leukaemia." Thesis, University of Newcastle Upon Tyne, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.525030.
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Kvinlaug, Brynn Taryl. "Identification of leukaemic stem cell self-renewal pathways." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608840.
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Saleh, Lubaid. "Small molecule mediated targeting of haematopoietic stem/progenitor cell and leukaemic stem cell function." Thesis, Cardiff University, 2017. http://orca.cf.ac.uk/105172/.
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Haematopoietic stem cells (HSC) are a rare population of cells that have the ability to self-renew and differentiate giving rise to various blood lineages, thereby reconstituting the whole haematopoietic system. This is an essential characteristic, exploited in bone marrow transplantation therapy in response to myeloablative treatment. Due to their rarity, the lack of sufficient HSC numbers for transplantation has proved to be a major clinical issue. Separately, in the development of leukaemia, acquired mutations in HSCs give rise to malignant cells. These cells, like HSCs, have the ability to self-renew and differentiate forming immature blasts and are termed cancer (leukaemic) stem cells. They are thought to remain in a quiescent state and are therefore not targeted by standard chemotherapy, inducing relapse in haematopoietic malignancies. In this study, a cross species stem cell based screen was conducted on a 12,000 small molecule library across a range of adult and embryonic tissue types with a view to identifying compounds that would (i) expand HSCs ex vivo and in vivo for transplantation and (ii) eradicate cancer stem cells in leukaemia. A number of small molecules were identified as lead compounds and were assessed in our investigation. We found that Yohimbine, an alpha-2 adrenergic receptor (adra-2) antagonist, and Oxa-22, cis-2-Methyl-5-trimethylammoniummethyl-1,3-oxathiolane iodide (M3 Muscarinic acetylcholine receptor agonist) elicited a 2- and 1.5- fold increase in HSC frequency (respectively) in vivo. Further competitive transplantation studies showed that Yohimbine and Oxa-22 treated cells also enhanced the reconstitution of B cells and T cells respectively. In parallel, we also assessed Oxa-22 and a third compound, Phthalylsulfathiazole- an antibacterial sulphonamide, in the leukaemic setting to ascertain whether compounds could target leukaemic stem cells (LSCs). We found that these compounds promoted proliferation in acute myeloid leukaemia (AML) cell lines. Furthermore, when Oxa-22 and Phthalylsulfathiazole were administered in vivo models of AML, they accelerated disease progression by increasing the number of LSCs. Collectively, these results show that using small molecules we can target neuronal related pathways to enhance HSC number and function. Further investigation is required to elucidate the exact mechanisms of the compounds however, these data may prove to be influential in directing new methods of stem cell expansion for transplantation therapies. Small molecules targeting neuronal or antibacterial related pathways were also found to target malignant LSCs and alter their behaviour. By driving LSCs out of their dormant state, these small molecules may pave the way for potential targeting of LSCs in conjuction with standard current chemotherapies that incorporate and kill proliferating cancer cells.
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Anderson, Robert James. "Development of an affinity partitioning method for DNA/protein complexes and its application to interactions of topoisomerase II with DNA." Thesis, Open University, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386626.
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Rehe, Klaus. "Diversity of cancer stem cells in acute lymphoblastic leukaemia." Thesis, University of Newcastle Upon Tyne, 2012. http://hdl.handle.net/10443/1777.
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For many cancers research led to controversial results regarding frequency and identity of cancer stem cells, cells that self-renew and are able to reconstitute the full phenotype of the original malignancy. In some malignancies such as acute myeloid leukaemia the hierarchical stem cell model that suggests that only rare and immunophenotypically immature blasts exhibit stem cell characteristics, resembling the normal physiological haematopoietic hierarchy, has been well established. In contrast to that, the stochastic model states that all or at least a substantial proportion of malignant cells has stem cell potential whereby this is supported by extrinsic stimuli. Initially, several studies suggested that acute lymphoblastic leukaemia (ALL) also is organised in a hierarchy. However, using more immunocompromised mouse strains and refined transplantation techniques for in vivo xenotransplantation models, previous findings regarding frequency and restriction of stem cells to very early B-precursor cell stages have been challenged in more recent studies. In order to address the questions of identity and frequency of stem cells in ALL, a robust orthotopic mouse model with the most immunocompromised mouse strain currently available (NOD/scid IL2Rγnull; NSG) was established. Primary diagnostic patient ALLs or blasts harvested from engrafted mouse bone marrow were sorted for B-cell lineage differentiation markers CD10, CD19, CD20 or CD34 to purify candidate stem cell populations of different maturity. All transplanted cell populations, from the most immature CD34+CD19low stage to the already more differentiated stage of CD19+CD20high cells were able to reconstitute the original ALL in mouse bone marrow and followed a typical dissemination pattern with infiltration of the spleen. Furthermore, this more mature CD19+CD20high subpopulation proved self-renewal ability in serial transplantation experiments. To investigate, whether stem cells in ALL are a rare entity or more abundant, unsorted bulk leukaemia blasts were transplanted in limiting dilutions from 1 x 104 to 10 cells per mouse. Cell numbers required for engraftment varied between leukaemias but the leukaemia engrafted in mice when only 10 to 1,000 cells were transplanted. The limiting dilution experiments were repeated with sorted blast populations according to the surface antigens CD10, CD20 and CD34. Stem cell frequencies in all sorted populations were comparable and as few as 10 to 100 cells were sufficient to reconstitute the leukaemia in mice. Stem cells do not seem to be restricted to immature blast populations as in the hierarchical model but a broad spectrum of different blast immunophenotypes display stem cell capabilities. Furthermore, the frequency of stemness among unsorted bulk ALL cells as well as subpopulations of different maturity according to the blast immunophenotype is high and similar. The results from this thesis provide strong evidence for the stochastic cancer stem cell model in B precursor ALL.
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O'Brien, Stephen Gerard. "Haemopoietic stem cell purging in chronic myeloid leukaemia." Thesis, Imperial College London, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.300928.
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McLaughlin, Fiona. "Transcriptional regulation of the stem cell leukaemia gene." Thesis, University of Cambridge, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285447.
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Zhai, Xiao Qun. "Biology of adult human normal and leukaemia CD133+ stem cells." Thesis, University of Central Lancashire, 2010. http://clok.uclan.ac.uk/21488/.
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Recently, CD133 has been used extensively as a marker for identification of stem cells from human normal and malignant tissues. Human normal CD 133+ stem cells are capable of multilineage in vitro and in vivo differentiation providing a novel source of stem cells for regenerative therapy, whereas, malignant CD133+ stem cells are a potential therapeutic target for anti-cancer treatment. This PhD thesis investigated the neural differentiation of human adult bone marrow and foetal liver CD133+ stem cells; explored the presence of CD133 and embryonic stem cell marker Oct-4 positive stem cells in adult human normal and malignant tissues, including normal brain tissues, normal and malignant bone marrow stem cells; and evaluated the anti-acute myeloid leukaemia (AML) effect of 5 novel synthetic ajoene compounds using drug-resistant AML cell line for overcoming drug resistance in elderly AML patients. A novel serum-free culture system for inducing neural differentiation of human adult bone marrow and foetal liver CD 133+ stem cells in vitro was demonstrated. Following only two weeks' culture of selected bone marrow CD 133+ cells in serum-free medium supplemented by 50% human astrocyte conditioned medium and other cytokines, 25.5% of bone marrow CD 133+ stem cells differentiated into neural (17.8%) and glial (7.7%) cells. After three weeks' culture of selected foetal liver CD133+ cells in serum-free medium supplemented by several cytokines without astrocyte conditioned medium, only 10.8% of these stem cells differentiated into neural and glial cells. These neural differentiated cells expressed mature neural and glial markers including NF-h, NF-m, NSF and GFAP, and exhibited mature neural morphologies. The astrocyte conditioned medium was the essential ingredient in all effective serum-free culture conditions suggesting it may contain other more potent neural/glial inducing factors. The serumfree culture system is clinically relevant and provides a vehicle for generating neural cells from adult human bone marrow CD 133+ stem cells for the treatment of patients with neurodegenerative diseases. CD 1 33-isoform 2 (CD 133-2) and embryonic stem cell marker Oct-4 expression was revealed in three adult human normal and malignant tissues and cells, including normal brain (substantia nigra and striatum), normal and malignant CD34+ marrow stem cells. There was no expression of CD133-isoform I (CD133-1) in these tissues. The very small population of CD133-2 and Oct-4 positive stem cells in adult human normal substantia nigra and striatum may represent the embryonic remnants and provide a potential source of adult neural stem cells that may be induced to undergo neural diffeentiation for the treatment of neurodegenerative conditions, such as Parkinson's disease. The substantial expression of Oct-4 in adult human normal and drug-resistant myeloid leukaemia KG1a marrow stem cells demonstrates that, alongside CD133, Oct-4 is a novel cancer stem cell marker suggesting the CD 133-2 and Oct-4 positive KGIa cells are the most likely targets in disease development and are novel therapeutic targets for effective treatment of AML. Novel synthetic ajoene compounds were shown to significantly inhibit growth, induce apoptosis plus necrosis, and reduce Bcl-2 expression in human drug-resistant CD34+ AML KG 1 a cells, both alone and in combination with low dose (1 jxM) cytarabine. E/Zbenzyl was the most potent growth inhibition compound,. whereas, Z-ajoene was the most cytotoxic compound. Low dose cytarabine-induced cytotoxicity was significantly enhanced by the five novel compounds in the following order: Z-ajoene > E-ajoene > Ephthalimide> E/Z-benzyl > Z-pthalimide. The combination of Z-ajoene and low dose cytarabine provides a promising combination therapy for elderly AML patients to overcome drug resistance. This PhD thesis provides the basis for appropriate identification and clinical application of stem cells from adult human normal tissues (brain and bone marrow) and foetal liver for autologous transplantation for the treatment of neurodegenerative diseases, such as Parkinson's disease, and also the identification of cancer stem cells in human AML marrow stem cells indicating novel therapeutic targets for anti-cancer treatment. Moreover, it demonstrates significant anti-cancer effect of 5 novel synthetic ajoene compounds in drug-resistant AML cells highlighting their potential for overcoming drug resistance in elderly patients with AML.
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Barton, L. M. "Transcriptional regulation of the stem cell leukaemia (SCL) gene." Thesis, University of Cambridge, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596444.
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To facilitate the characterisation of SCL regulatory elements, 33 kb of sequence encompassing the SCL genomic locus of the pufferfish, Fugu rubripes and 84 kb from the locus of the zebrafish, Danio rerio were sequenced and analysed. Sequence comparisons with the SCL genes of human, mouse and chicken identified four conserved non-coding regions. The most striking of these was a 150 bp stretch corresponding to the mouse SCL promoter 1a. Five conserved blocks of sequence were identified including two new putative transcription factor binding motifs, CS1 and CS2. In vitro analysis of mouse promoter constructs showed that mutation of CS1 and CS2 reduced promoter activity to 67% and 63% respectively within the erythroid cell line, J2E. Two protein complexes were shown to bind CS1 but the nature of the proteins within these complexes remains unclear. Expression studies in transgenic mice demonstrated that two conserved GATA motifs are necessary for full midbrain expression of SCL, the first demonstration that GATA acts upstream of SCL in neural development. Although the pattern of SCL expression is highly conserved from mammals to teleost fish, the genes flanking the pufferfish SCL gene were unrelated to those known to flank both avian and mammalian SCL genes. These data suggest that SCL regulatory elements are confined to the region between the upstream and downstream flanking genes, a region of 65 kb in moue and 8.5 kb in pufferfish. Consistent with this hypothesis, the entire 33 kb pufferfish SCL locus directed appropriate haemopoietic and neural expression in transgenic zebrafish embryos as did a 10.4 kb fragment containing the SCL gene and extending to the 5' and 3' flanking genes.
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Wickham, Caroline Louise. "Molecular detection and monitoring of leukaemia and lymphoma." Thesis, University of Exeter, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341343.
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Hong, Dengli. "Clonal evolution of stem cells in TEL-AML1-associated childhood leukaemia." Thesis, Oxford Brookes University, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.543817.
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Abadir, Edward. "Novel Targets in Acute Myeloid Leukaemia." Thesis, University of Sydney, 2020. https://hdl.handle.net/2123/23677.
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Background: Antibody based immunotherapies have revolutionised the treatment of haematological malignancies. Despite recent advances in Acute Myeloid Leukaemia (AML) most patients still have poor outcomes. Current surface targets in AML are not ideal and ongoing work is required to examine new antigens for meaningful clinical outcomes. Hypothesis: CD302 and CD300f have inherent properties that make them promising potential targets in AML. Preclinical work will establish these antigens as suitable targets in AML for further study. Methods: We looked at the distribution of CD302 on AML and Haematopoietic Stem and Progenitor Cells (HSPC) using flow cytometry from local patient cohorts and compared this data with AML gene expression profiling from public databases. The expression of CD302 from healthy organs was examined using PCR. The rate of internalisation of anti-CD302 antibodies was assessed using flow cytometry and fluorescent microscopy. The ability of unmodified antibodies to perform Antibody Dependant Cellular Cytotoxicity (ADCC) was examined. The impact of CD302 on AML cell migration was assessed using a transwell system. The influence of an anti-CD302 antibody upon AML cell line engraftment was tested in vivo. CD300f on AML and HSPC was analysed using flow cytometry from local patient cohorts and we compared the data to gene expression profiling from public databases. The distribution of CD300f isoforms across AML and HSPC was assessed by PCR and confirmed with TCGA RNA sequencing data. Alterations in antibody binding epitopes using multiple CD300f antibodies was assessed in AML cell lines as well as primary AML and HSPC. The cytotoxicity of an anti-CD300f based ADC was examined in vitro using cell lines. An anti-CD300f ADC was examined in vitro and in vivo against cell lines as well as primary AML and healthy HSPC. The rate of cytotoxicity and synergy of the ADC with Fludarabine was assessed in vitro with cell lines. Results: In a cohort of 33 AML patients, 88% were found to express CD302 on the surface of blasts and 80% on the surface of CD34+ CD38- population. Expression of CD302 was found on the surface of HSPC. A monoclonal antibody (mAb) targeting human CD302 was effective in mediating ADCC and was internalised, making it amenable to toxin conjugation. Targeting CD302 with this antibody limited in vivo engraftment of the leukaemic cell line HL-60 in NOD/SCID mice. While CD302 was expressed in a hepatic cell line, HepG2, this molecule was not detected on the surface of HepG2, nor could HepG2 be killed using a CD302 antibody-drug conjugate. CD300f antibodies bind to AML from 85% of patient samples. Transcriptomic analysis found that CD300f transcripts are expressed by healthy HSPC. Several CD300f protein isoforms exist as a result of alternative splicing. The extracellular region of CD300f can be present with or without the exon 4‐encoded sequence. This results in CD300f isoforms that are differentially bound by CD300f‐specific antibodies. Analysis of publicly available transcriptomic data indicated that CD34+ HSPC expressed fewer CD300f transcripts that expressed exon four compared to AML with monocytic differentiation. An anti-CD300f antibody, DCR‐2, to CD300f exposes a structural epitope recognized by a second CD300f mAb, UP‐D2. Analysis of a small cohort of AML cells revealed that this UP‐D2 conformational binding site could be induced in cells from AML patients with monocytic differentiation but not those from other AML or HSPC. CD300f is expressed evenly across HSPC subtypes. CD300f has equivalent transcription and protein expression as CD33 on AML. We have developed an anti-CD300f antibody which efficiently internalises into target cells and conjugated it with a PBD warhead that selectively depletes AML cell lines and AML Colony Forming Units (CFU) in vitro. CFU derived from healthy HSPC are depleted by the ADC. The ADC synergises with Fludarabine, which is often used in allogeneic Haematopoietic Stem Cell Transplant (allo-HSCT) conditioning. The ADC prolongs the survival of mice engrafted with human cell lines and depletes primary human AML engrafted with a single injection. In a humanised mouse model, a single injection of the ADC depletes CD34+ HSPC and CD34+ CD38- CD90+ HSC. Conclusions: CD302 is a potential target in AML. The hepatic expression may limit potential therapeutics but this could be mitigated as hepatocytes appear to express CD302 predominantly intracellularly, further work is required in this field. CD302 is expressed on HSPC and any future therapeutics would likely need to be part of a conditioning strategy. CD300f is a more promising target given the lack of non-haematopoietic expression. Certain isoforms or epitopes may be targeted to generate selective binding to AML with monocytic differentiation and avoid HSPC. An anti-CD300f ADC has promise as a targeted conditioning agent that may deplete residual AML and facilitate allo-HSCT.
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Hodby, Katharine Ailsa. "Investigating the mechanism of bone marrow failure observed in patients with acute myeloid leukaemia." Thesis, Queen Mary, University of London, 2018. http://qmro.qmul.ac.uk/xmlui/handle/123456789/36673.
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Patients with Acute Myeloid Leukaemia (AML) present with the signs and symptoms of bone marrow failure. This finding spans the genetic and phenotypic diversity of the disease. The mechanism which underlies it is poorly understood. This thesis explores the effect of AML on the normal haematopoietic stem cell (HSC) population, using primary human diagnostic bone marrow samples. Previous work from our group suggested that AML induces a state of quiescence in HSCs, producing a differentiation block responsible for the observed cytopenias1. Reversal of this process might offer an alternative to the current treatment of patients with palliative transfusions. I have developed a flow cytometry-based technique to differentiate normal HSCs from leukaemia cells, selecting cells with the CD34+38-ALDHhighCLL1- expression signature. Validation of this technique by assessment of sorted cells by FISH and PCR, suggests it is successful in 73% of AML samples. In a further 25% of samples, it selects for a population significantly enriched for normal HSCs. We used this panel to investigate the concentration of HSCs at AML diagnosis, compared to controls. We show that there is no significant difference between HSC concentration at AML diagnosis (n=38, median [HSC] 2.5 cells/μl) and controls (n=24, median [HSC] 2.4 cells/μl). HSC concentration was not significantly affected by AML karyotype, patient age or gender. However, those patients presenting with a low HSC concentration at diagnosis (< 0.1 HSC/μl) were found to have a significantly worse outcome both in terms of overall and relapse-free survival, an effect apparently independent of age, gender and underlying karyotype. HSC concentration at diagnosis with AML may therefore represent a new independent prognostic marker. We then studied CD33 expression patterns on HSCs within Core Binding Factor mutated AML (n=37) at diagnosis, and found its expression to be significantly lower than on HSCs within controls (n=9) (17% versus 58%, p=0.005). CD33 expression on HSCs from AML samples rose significantly from diagnosis to remission (n=16) (17% to 58%, p=0.0001). This mirrors previous findings from our group using CD34low AML samples, and is, we believe, the first time that the antigenic signature of normal HSCs has been shown to be modified. 6 by the presence of AML. However, an in vitro assay to test the significance of these changes in terms of the cytotoxicity of GO towards normal HSCs did not demonstrate a significant difference between HSC subgroups. Finally, we attempted to investigate the mechanism by which AML might induce HSC quiescence by studying the comparative transcriptomes of HSCs from CD34low AML (n=6) and controls (n=6) by RNA-Seq, using direct cell to cDNA synthesis, followed by amplification. A first attempt resulted in poor quality data, with a significant proportion of reads mapping to non-coding DNA regions. A repeat approach, using utilising immediate RNA extraction post sorting resulted in significantly better quality data Bioinformatics analysis revealed differential expression of 6 genes between the 2 datasets (GNPDA1, ADGRG3, MIAT, WDR31, RP11-244H3.1 and RXFP1). GO enrichment studies using David highlighted a number of pathways including the TNF signalling pathway (p=0.003; after Benjamini-Hochberg correction p=0.51). Validation of these findings by independent qPCR, and functional exploration of enriched signalling pathways remains outstanding.
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Harris, William. "The histone demethylase KDM1A sustains the oncogenic potential of MLL-AF9 leukaemia stem cells." Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/the-histone-demethylase-kdm1a-sustains-the-oncogenic-potential-of-mllaf9-leukaemia-stem-cells(347b6f68-1a3d-453f-9eab-c35ed895ba64).html.
Full textAbstract:
Rearrangements involving the mixed lineage leukaemia (MLL) gene are found in 5-10% of human leukaemias and are likely propagated by a deregulated self renewing pool of leukaemia stem cells (LSCs). Targeting of the LSC pool represents a key novel strategy for the treatment of AML. In recent years epigenetic dysfunction has been identified as a key driving factor in a range of solid tumours and haematological malignancies. Evidence for this includes identification of mutations in the genes coding for critical epigenetic modifiers, characterisation of localised regions of abnormal chromatin at oncogene or tumour suppressor genes and the efficacious use of epigenetic-targeted therapies already present in the clinic. The data submitted in this thesis identify the histone demethylase KDM1A as a critical regulator of LSC potential in MLL-AF9 acute myeloid leukaemia (AML). Of all the histone demethylases, we found that only Kdm1a expression correlated positively and significantly with LSC frequency in murine models of human MLL fusion AML. Genetic knockdown or Cre-mediated excision of Kdm1a resulted in loss of LSC potential, reduced expression of LSC maintenance transcriptional programs and induction of macrophage differentiation in MLL-AF9 cells. These effects were phenocopied by chemical inhibition of KDM1A using the monoamine oxidase inhibitor tranylcypromine (TCP), as well as novel TCP analogues which inhibit KDM1A with greater potency and selectivity. These results were seen in murine, human cell line and primary patient cells harbouring MLL rearrangements. Global transcriptome and epigenome analyses revealed a key role for KDM1A in maintaining the histone three lysine four (H3K4) methylation status at highly expressed MLL-AF9-bound genes. In vivo transplantation of Kdm1a knockdown MLL-AF9 cells conferred a significant survival advantage compared with control littermates. Similarly, TCP analogue treatment of mice transplanted with MLL-AF9 cells revealed a reduction in LSC potential of the donor-derived AML cells but little impact on normal recipient haematopoietic stem and progenitor cells (HSPCs). Critically the clonogenic and repopulating potential of normal HSPCS, of both murine and human origin, was spared following either knockdown or chemical inhibition of KDM1A. Taken together, the data presented establish KDM1A as a potential therapeutic target in MLL fusion leukaemia.
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Richardson, S. E. "Modeling TEL-AML1 childhood acute lymphoblastic leukaemia using human pluripotent stem cells." Thesis, University College London (University of London), 2016. http://discovery.ucl.ac.uk/1496130/.
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Childhood acute lymphoblastic leukaemia (cALL) is distinct from that in adults with higher incidence, better prognosis and a distinct mutational spectrum. One hypothesis for this difference is that cALL arises in transient cells unique to early human development. I explored this in ETV6-RUNX1 cALL where evidence from twins and neonatal heel prick testing has shown that this mutation arises in utero and is an initiating event. I hypothesised that human pluripotent stem cells (hPSCs) could model the developmental features of in utero B cell development. In vitro B cell differentiation of hPSCs produced both pre and proB cells, and a CD19-IL7R+ progenitor that switched from myeloid to lympho-myeloid priming during culture, akin to that identified in parallel studies of human fetal liver (FL). At the global transcriptional level the hPSC lymphoid hierarchy mapped closely with FL, with both separating from adult suggesting that hPSCs provide a developmentally relevant model of early FL B lymphopoiesis. I used CRISPR-directed homologous recombination to engineer the expression of ETV6-RUNX1 under the endogenous ETV6 promoter. ETV6-RUNX1 hPSCs displayed a partial block in B cell differentiation at the level of the CD19-IL7R+ progenitor. ETV6-RUNX1 expressing proB cells co-expressed an abnormal B-myeloid gene expression signature similar to that seen in the CD19-IL7R+ progenitor. These data support a model where expression of ETV6-RUNX1 inhibits lymphoid specification in an early FL CD19-IL7R+ lymphomyeloid progenitor, arresting B lineage differentiation and resulting in the production of myeloid-primed B cells. This may explain the propensity for aberrant myeloid antigen expression seen in cALL. ETV6-RUNX1 hPSCs provide a platform for the systematic evaluation of the contribution of additional mutations seen in cALL and may offer a tractable platform for drug screening. In conclusion I propose the human fetal CD19-IL7R+ lymphomyeloid progenitor as a candidate target cell for in utero pre-leukemic initiation in cALL.
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Siriboonpiputtana, Teerapong. "The role of beta-catenin in development of origin-specific leukaemia stem cells." Thesis, King's College London (University of London), 2015. http://kclpure.kcl.ac.uk/portal/en/theses/the-role-of-betacatenin-in-development-of-originspecific-leukaemia-stem-cells(49ca7704-4fdc-4b31-8bfa-ce6fdb68be2d).html.
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While we and others have prospectively identified potential origins of leukaemic stem cells (LSCs) resulting in phenotypically identical myeloid leukaemia, the functional differences among these origin-specific LSCs have not been vigorously investigated. Recently, our lab has shown that β-catenin, which is dispensable for normal HSCs, is essential for MLL LSCs, highlighting it as a potential therapeutic target for selective eradication of LSCs while sparing normal HSCs. Here, I investigated the role of β-catenin in LSCs of distinctive cellular origins using both RTTA (in vitro) and bone marrow transplantation (in vivo) assays. I demonstrated that conditional deletion of β-catenin abolished the leukaemogenic potential of LSK-Meis1-Hoxa9 pre-LSCs whereas CMP-Meis1-Hoxa9 pre-LSCs were able to develop into LSCs in vivo regardless of β-catenin status. Interestingly, conditional inactivation of β-catenin abolished the in vivo leukaemogenic property of GMP-MLL pre-LSCs whereas LSK-MLL pre-LSCs were still able to induce leukaemia in vivo in the absence of β-catenin, revealing functional differences in origin-specific LSCs in spite of their ability to induce phenotypically identical leukaemia. Since a major function of β-catenin is to mediate stem cell self-renewal, I hypothesized the presence of multiple alternative self-renewal pathways in normal HSCs that may allow the LSK/HSC-origin specific LSCs overcoming inactivation of β-catenin. Hox genes have been previously shown by others and us as key players in mediating self-renewal in normal and malignant haematopoiesis. Consistently, Hoxa9 exhibited functions as β-catenin in mediating developing of MLL pre-LSCs originated from GMP but not HSCs. To test if Hoxa9 may compensate the loss of β-catenin in LSK-MLL pre-LSCs, I have created a conditional β-catenin/Hoxa9 knockout model where a combination of these two molecules can be inactivated in normal HSCs and various myeloid progenitors. As a result, I demonstrated that specific inactivation of β-catenin or Hoxa9 abolished the oncogenic potentials of GMP-MLL pre-LSCs, but not LSK-MLL pre-LSCs. Strikingly, inactivation of both pathways suppressed oncogenic transformation by LSKMLL pre-LSCs and resulted in down-regulation of several Meis1-Hoxa9 target genes. Among them is Prmt1, which has been identified as a critical epigenetic modifying enzyme associated with an oncogenic MLL fusion complex. Prmt1 knockdown inhibited clonogenic activity of LSKMLL-ENL LSC with Hoxa9 or β-catenin knockout, suggesting Prmt1 as a key modulator for transformation of LSK-MLL-ENL LSC. Together, I have demonstrated, for the first time, specific molecular and functional differences among origin-specific LSCs, in which crosstalk between multiple self-renewal pathways inherited from the cell of origins may determine the biology of the disease and mediates resistance to potential targeted therapies.
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Vlachou, T. "FUNCTIONAL AND GENETIC HETEROGENEITY IN ACUTE MYELOID LEUKAEMIA." Doctoral thesis, Università degli Studi di Milano, 2017. http://hdl.handle.net/2434/471776.
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Acute myeloid leukaemia (AML) is the most frequent leukaemia in adults, and still represents a disease with an unmet medical need, with 50-60% of patients relapsing within 3 years after diagnosis. AMLs are characterised by a high degree of intra-tumour heterogeneity, both at the biological and the genetic level, which is critical for tumour maintenance and response to treatments. Biologically, AMLs are organised hierarchically, with rare stem-like cells (leukaemia stem cells, LSCs) endowed with the unique properties of self-renewal and differentiation. Genetically, AMLs harbour patient-specific combinations of different driver mutations, which are organised within individual cases in sub-clones with distinct growth properties. We hypothesized that tumour maintenance and relapse in AMLs are driven by the selective expansion of quiescent sub-clones within the LSC population, which serve as the genomic and functional reservoir of the tumour. The experimental strategy we employed to test this hypothesis is based on the xenotransplantation of human leukaemias, the implementation of an in vivo clonal tracking approach, the functional isolation of leukaemic subpopulations with diverse proliferation histories and whole-exome sequencing (WES) of bulk and isolated leukaemic subpopulations. Our aims were to assess the proliferative hierarchy of LSCs and to examine their intrinsic genetic heterogeneity. We identified two functional LSC classes, quiescent and cycling, that are in equilibrium in the tumour and largely share the same clonal architecture. We further observed that genetic leukaemic clones appear to consist of a high number of individual LSCs, the majority of which exhaust upon serial transplantation. Finally, by genetic analyses of isolated leukaemic subsets, we were able to detect a specific enrichment for rare mutations in the quiescent compartment of two patient xenografts. Our data indicate that tumour evolution is sustained by the quiescent LSC pool and suggest that their highly proliferating counterpart has a finite lifespan. We expect that the results of our studies will provide new insights into the mechanisms of disease progression and treatment response in AML, and potentially reveal novel therapeutic approaches.
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Muthukumarana, Poorni Apsara de Silva. "Stem cell factors, axotrophin and leukaemia inhibitory factor in immune regulation." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611884.
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Latif, Elda Surhaida. "Understanding the proliferative and self-renewal potential of different leukaemic stem cell populations." Thesis, University of Newcastle Upon Tyne, 2013. http://hdl.handle.net/10443/1828.
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Although contemporary treatment cures most children with ALL, with survival rates in the region of 90%, treatment outcomes in certain cytogenetic subgroups remain poor. Patients with such abnormalities have a greatly increased risk of relapse and re-treatment responses are poor. Understanding the mechanisms of resistance in these patients is therefore a priority in the search for improvements in chemotherapy. The translocation t(17;19)(q22;p13) is a rare cytogenetic abnormality, which occurs in less than 1% of childhood ALL and is associated with a very poor prognosis and chemotherapy resistance. The translocation results in the fusion of E2A on chromosome 17 with HLF on chromosome 19. It has been commonly assumed that, following relapse, ALL cells will divide more rapidly than at presentation. To test this hypothesis, competitive transplantation studies were performed using paired presentation and relapse samples from a case of t(17;19)-positive ALL in a NSG xenograft mouse model. Surprisingly mice engrafted with the presentation cells survived significantly shorter (p<0.05, logrank test) than those with relapse cells, indicating the aggressiveness and rapid proliferation of presentation cells, while the survival curves of mice engrafted with various proportions of presentation and relapse cells had intermediate levels of survival. Administration of dexamethasone prolonged the survival of mice engrafted with the presentation cells and those with various proportions of cells, with no effect on survival of mice with relapse cells. Flow cytometry analysis showed that a high percentage (70%-95%) of human leukaemic cells engrafted in bone marrow and spleen across all groups tested. RT-PCR amplification identified this t(17;19) case to be a Type 1 E2A/HLF fusion. In order to determine the genes responsible for chemo-insensitivity, whole genome SNP array analysis was performed on the matched presentation and relapse samples which demonstrated deletion of glucocorticoid receptor (NR3C1) gene in relapse cells which is not seen in presentation cells, confirmed by fluorescence in situ hybridisation. This deletion was used to identify the relapse cells (R) in mice engrafted with mixtures of presentation and relapse cells. FISH analysis showed the percentages of presentation cells (P) were higher than the ratios of the cell initially transplanted. In concordance, real-time PCR analysis showed high levels of NR3C1 in all mix cells ratios. These results indicated that presentation cells proliferate more rapidly and outgrow relapse cells in competitive clonal repopulation experiments. Dexamethasone treatment reduced the percentages of presentation cells in all mix populations with high significance (p<0.01, t-test) in the 30%-P+70%-R ratio. Levels of NR3C1 in all mix cell populations were significantly depleted (p<0.05, t-test) by dexamethasone. To be able to physically track the proliferation of leukemic cells populations in vivo and to follow progression of the disease, two lentiviruses pSLIEW (expressing firefly luciferase and green fluorescent protein) and pSRLICW (expressing Renilla luciferase and mCherry fluorescent protein) were generated. pSRLICW was just successfully generated but has not yet been tested. Lentivirus pSLIEW functionality was validated by transducing various leukaemic cell lines. By assessing the percentage of green fluorescence (gfp) cells, transduction efficiencies in SEM and RS4;11 (ALL cell lines) were 45% and 5% respectively, whereas transduction efficiencies in Kasumi-1 and SKNO-1 (AML cell lines) were 72% and 98% respectively, higher than the ALL cell lines. Transduced SEM and Kasumi-1 lines were sorted for higher gfp populations for xenograft purposes. Real time bioluminescence imaging on mice xenografted with sorted transduced SEM cells showed rapid progression of the disease in the systemic while Kasumi-1 cells xenografted in mice produced localised tumours and progressed much slower. These lentiviral-transduced cell lines xenografts have proven the in vivo monitoring capability by real time luminescence imaging. The information gained from this project study provides novel insights into the mechanisms of relapse in childhood ALL. The growth characteristics of the leukaemic blasts should be considered in assigning patients to different therapeutic options.
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Zhou, Yan. "Transcriptional regulation of the stem cell leukaemia gene (SCL/TAL1) via chromatin looping." Thesis, University of Glasgow, 2012. http://theses.gla.ac.uk/4004/.
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The bHLH protein TAL1 (SCL) is a critical regulator of vertebrate hematopoiesis and is misregulated in T-cell acute lymphoblastic leukemia (T-ALL). This thesis studied chromatin looping interactions at the TAL1 locus – defining the first structural model which accounts for a number of phenomena associated with TAL1, its flanking genes and its relationship with its functional paralogue LYL1. The chromosome conformation capture (3C) and its high-throughput variant 4C-array technologies have been applied to characterise the chromatin interactions. Intriguing chromatin organisations have been identified at the TAL1 and LYL1 loci, which are closely associated with transcriptional regulation, chromosomal abnormality and regulatory remodelling through evolution. Firstly, in TAL1 expressing cells, the locus adopts a “cruciform” configuration – forming an active chromatin hub which brings together the TAL1 promoters, its stem cell and erythroid enhancers, and two CTCF/Rad21-bound insulators. Secondly, loss of a GATA1-containing complex bound by the TAL1 erythroid enhancer and its promoter is sufficient to disrupt the formation of the hub and the entire cruciform structure and results in decreased TAL1 expression. Thirdly, it demonstrates that genes flanking TAL1 are also dependent on this hub and that TAL1 promoters interact directly with intron 1 of the neighbouring STIL gene. This TAL1/STIL interaction also provides a structural link between the DNA sequences which mediate micro-deletions in 25% of cases of T-ALL. Finally, it demonstrates that a GATA1-dependent chromatin looping mechanism also exists at the LYL1 locus which is strikingly similar to that mediating contact between the TAL1 promoter and its erythroid enhancer. Conservation of core chromatin looping at the TAL1 and LYL1 loci may account for some aspects of their functional relationships. It also suggests that looping mechanisms at both loci could also facilitate cis-regulatory maintenance and/or remodelling during vertebrate evolution.
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Neylon, Annette J. "Cytokine gene polymorphisms and their involvement in stem cell transplantation in chronic myeloid leukaemia." Thesis, University of Newcastle upon Tyne, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.275583.
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Bakker, E. Y. "Analysis of drug resistance and the role of the stem cell niche in leukaemia." Thesis, University of Salford, 2017. http://usir.salford.ac.uk/43810/.
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Glucocorticoids and etoposide are used to treat acute lymphoblastic leukaemia (ALL) as they induce death in lymphoblasts through the glucocorticoid receptor (GR) and p53. However, glucocorticoid resistance, cell death mechanisms and the contribution of the bone marrow microenvironment to drug response/resistance all require investigation. Using microenvironment-mimicking conditioned media (CM), dexamethasone (a synthetic glucocorticoid) and etoposide to treat glucocorticoid-sensitive (C7-14) and glucocorticoid-resistant (C1-15) cells, pathways by which the microenvironment exerts its chemoprotective effect have been investigated. CM reduced caspase-3/8 activation, downregulated RIPK1 (necroptotic marker), and limited chemotherapy-induced BECN1 downregulation, suggesting protective effects of CM. Glucocorticoids upregulated BIRC3 (which ubiquitinates RIPK1), whilst CM altered GR phosphorylation. GR occupancy was observed on the RIPK1, BECN1 and BIRC3 promoters and changed depending on its phosphorylation. High-molecular weight proteins reacting with the RIPK1 antibody increased with CM, and reduced following AT406 BIRC3 inhibitor treatment suggesting they represent ubiquitinated RIPK1. These results suggest mechanisms by which CM promotes survival, as well as indicating novel glucocorticoid-regulated pathways. Complementing laboratory investigation is the construction of a Boolean model of the GR interaction network (GEB052, GR “interactome”) containing 52 nodes (proteins, inputs/outputs) connected by 241 interactions. In silico mutations and analyses have generated predictions that were subsequently validated on a genome-wide scale via comparison to microarray data. GEB052 demonstrated high prediction accuracy, consistently achieving a better prediction rate than a randomised model. Quantitative algorithmic analysis via microarray superimposition has also been performed, and lastly the model has been preliminarily validated as a clinical tool via superimposition of patient microarray data and comparing model predictions to clinical data. In summary, this thesis provides novel insight into the effects of the microenvironment, and identifies new glucocorticoid-regulated pathways. The GEB052 model of GR signalling represents the novel application of this modelling approach to GR research, and generates accurate predictions.
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Green, Ann Frances. "Immune reconstitution in patients who have undergone stem cell transplantation for acute lymphoblastic leukaemia." Thesis, University of the West of England, Bristol, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429536.
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Holyoake, Tessa Laurie. "Investigation of the effects of in vitro cytokine exposure on short and long term reconstituting haemopoietic stem and progenitor cells in a murine model." Thesis, University of Glasgow, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.320310.
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Irvine, David Arthur. "Defining novel therapeutic targets in chronic myeloid leukaemia stem cells : targeting self-renewal through hedgehog pathway inhibition." Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4584/.
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Verbiest, Tom. "Identification and characterisation of the haematopoietic stem/progenitor cells at risk for leukaemia development following radiation exposure." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:0a9fa5f8-c882-4d29-a502-9445a0cc224a.
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A variety of epidemiological studies have provided support for an increased leukaemia incidence following exposure to both high and low dose radiation. There is a close histopathological similarity between human and murine acute myeloid leukaemia with mouse models of the disease being most often used. We report here, for the first time, that allogeneic haematopoietic stem cells can compete for niches in the nonmyeloablated NSG bone marrow compartment and that the nonmyeloablated NSG bone marrow microenvironment is capable of supporting allogeneic long-term HSC engraftment and differentiation. Using this NSG transplantation model, we then provided evidence of a reduced contribution of low dose irradiated HSCs towards longterm haematopoiesis. We further report on the generation and characterisation of a novel Chr2MDRmCh mouse model where a construct positioned in the minimal deleted region following radiation exposure carries the fluorescent protein mCherry, which is expressed under control of the ubiquitous Rosa26 promoter. Crossing these mice with Sfpi1GFP mice allowed the early detection of an expanding haematopoietic clone which had lost mCherry fluorescence following radiation exposure (and so also the chromosome 2 homologue carrying the fluorescent construct and the Sfpi1 gene). Finally, we report on a gender-dependent leukaemic progression and characterisation where 3 Gy irradiated male mice only present with acute myeloid leukemia and irradiated female mice mainly develop leukaemia with a lymphoid phenotype. In conclusion, the work in this thesis postulates data obtained from two novel mouse models and which could help to further identify key molecular mechanisms involved in leukaemia initiation and development.
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Hamilton, Ashley. "Investigation into the relevance of BCR-ABL for the survival of cancer stem cells in chronic myeloid leukaemia." Thesis, University of Glasgow, 2010. http://theses.gla.ac.uk/2208/.
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Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder of the haemopoietic stem cell, defined by the Philadelphia chromosome (Ph) - the outcome of a balanced, reciprocal translocation between the long arms of chromosomes 9 and 22. The novel fusion oncogene generated on chromosome 22 as a result of this translocation is called BCR-ABL. In the majority of patients, this oncogene transcribes a 210-kDa constitutively active protein tyrosine kinase, often referred to as p210BCR-ABL, which is necessary for the transformation of the disease. The introduction of the orally available, tyrosine kinase inhibitor (TKI) - imatinib mesylate (IM) - marked a major advance in CML treatment in terms of efficacy and tolerability and has now become the first line of therapy. IM acts by competing with ATP to block ABL-kinase activity, resulting in the selective apoptosis induction of BCR-ABL+ cells. However, despite the success of IM as standard therapy for CML, only a small proportion of patients obtain a complete molecular response, where they become negative for BCR-ABL transcripts by RTPCR. It is hypothesised that this minimal residual disease is the result of a primitive quiescent subpopulation of leukaemic cells, which may be the cause for relapse at a later date. Another major clinical concern is the observation of molecular resistance in IM-treated patients. Proposed mechanisms of resistance include BCR-ABL amplification, decreased intracellular IM concentrations caused by drug efflux proteins such as multi drug resistance-1 and the development of point mutations within the ABL-kinase domain. In an attempt to overcome IMresistance, a second generation of BCR-ABL inhibitors has been developed. Dasatinib (BMS-354825, Sprycel) is a TKI developed for the treatment of IM resistant or -intolerant patients with Ph+ leukaemias, which has a 325-fold greater potency than IM against cells expressing wild-type BCR-ABL, and is effective against all IM-resistant BCR-ABL mutants tested in vitro, except T315I. Previously, we showed that dasatinib induced durable inhibition of BCR-ABL and impressive clearance of Ph+ cells, however, the primitive quiescent cell population did not appear to undergo apoptosis even after several days TKI exposure. Therefore, it was still not clear whether early CML progenitor cells depend on BCR-ABL for their growth and survival. In this study we have attempted to determine whether CML stem cells are dependent on BCR-ABL TK activity for their survival and/or proliferation using dasatinib treatment and aimed to characterise the cells which survived drug exposure. We found that 10% of the CML cells were able to survive the dasatinib treatment. We also showed that maximal BCR-ABL TK inhibition was achieved in the surviving CML cells, both in the bulk population of cells and the more problematic primitive stem cell population. Those cells which survived the dasatinib treatment were found to be primitive, residing mainly in the undivided cell fraction and the very early cell divisions. Since these BCR-ABL TK-inhibited, resistant cells were also able to grow when re-cultured in cytokines and form longterm culture-initiating cell (LTC-IC) colonies; these data suggested that ~10% of primitive CD34+ CML cells are not addicted to BCR-ABL TK activity for their survival. This also suggested that these primitive, resistant CML cells appeared to survive and proliferate by BCR-ABL-independent mechanisms. Therefore, the next experiments were then designed to investigate the cellular process of autophagy as a potential means of primitive CML cell survival. Analysis of the properties of CD34+ CML cells which remained viable following dasatinib treatment, revealed the existence of cytoplasmic autophagic structures determined by electron microscopy and significantly increased autophagosome-asociated LC3-II, particularly in the cells cultured without growth factors (GF)s. This suggested that autophagy is induced following GF deprivation of CML cells and is significantly increased within these cells, upon BCR-ABL inhibition following dasatinib treatment. Furthermore, we also found that the inhibition of autophagy greatly potentiated the effect of TKI treatment on the reduction of primitive CML progenitor cells, in terms of the effective eradication of functionally defined colony forming cells and LTC-ICs. Overall, this thesis has shown for the first time that the most TKI-resistant primitive CML cells are likely to be independent of BCR-ABL TK activity for their proliferation and/or survival. Furthermore, we have shown that these resistant CML stem cells rely on the BCR-ABL independent autophagy process for survival in response to stressful conditions, such as, GF deprivation and TKI treatment.
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Korfi, Koorosh. "Epigenetic programming defines stem cell identity and entry into the proliferative compartment in chronic myeloid leukaemia (CML)." Thesis, University of Glasgow, 2012. http://theses.gla.ac.uk/3929/.
Full textAbstract:
Chronic myeloid leukaemia (CML) is a haematological malignancy that is identified by the presence of a fusion oncogene, BCR-ABL1, which is a constitutive tyrosine kinase. The discovery of tyrosine kinase inhibitors (TKIs) over that past decade has resulted in significantly improved survival rates and disease management in CML patients. However, a subpopulation of BCR-ABL1+ cells in the niche are found which exhibit stem cell-like features, such as self-renewal and quiescence. These CML stem cells (LSCs) are shown to be insensitive to TKI treatment and are capable of deriving the disease during the relapse. Consequently, the elimination of LSCs is a primary goal of current research. Therefore, the aim of this thesis was to obtain a global view of the cellular processes that maintain stem cell identity in CD34+ CD38- LSCs as well as identify those processes which initiate the transition to proliferative CD34+ CD38+ CML progenitor cells (LPCs). A combined approach was exploited to investigate genome-wide gene expression profiles and histone modification signatures of normal HSCs and committed progenitors (HPCs), and their LSC and LPC counterparts. Despite having increased activity in pathways involved in cell division and proliferation, expression levels of the pathways involved in stem cell identity were not significantly different in LSCs to those found in HSCs. These pathways included Wnt, TGF-β signalling, and several novel neurotransmitter signalling pathways. By examining genome-wide histone modification patterns using ChIP-sequencing it was shown that the stem cell identities of HSCs and LSCs are programmed at the epigenetic level. All of the pathways which confer stem cell identity to both HSCs and LSCs are significantly enriched for bivalent gene promoters having both the H3K4me3 and H3K27me3 marks. These similarities were most evident in neurotransmitter signalling and it was demonstrated that these pathways are capable of promoting LSC maintenance in vitro. Intriguingly, although the stem cell entry into the proliferative state occurs through the repression of many of the same stem cell identity pathways in both HSCs and LSCs, it was shown that epigenetic reprogramming in CML mediates this repression via a different mechanism than in normal HSCs. Furthermore, abnormalities in levels of several chromatin enzymes were identified that are likely to be responsible for the epigenetic reprogramming of CML cells. The work presented in this thesis defines the chromatin landscape of a cancer stem cell for the first time and provides new therapeutic targets for the eradication of TKI resistant CML stem cells.
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Hallböök, Helene. "Acute lymphoblastic leukaemia in adult patients : studies of prognostic factors, treatment results and in vitro cellular drug resistance /." Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-5768.
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Monteiro, C. J. "Function of MYST3/MOZ, a histone acetyltransferase and chromatin regulator implicated in haematopoietic stem cell development and leukaemia." Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/52018/.
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MOZ (also known as KAT6A and MYST3) is a histone acetyltransferase that is implicated in the development of hematopoietic, heart and craniofacial tissues. Reciprocal translocations generating MOZ fusion proteins are associated with acute myeloid leukaemia (AML), a heterogeneous hematopoietic stem cell disorder where the myeloid cells do not enter into differentiation process, resulting in accumulation of blast cells and absence of a fully functional granulocyte lineage. Also, mutations that truncate the C-terminus of MOZ are associated with global developmental delay. Mechanistically, it is known that MOZ acts as a cofactor for certain transcription factors, particularly those with haematopoietic or neuronal specificity, such as acute myeloid leukaemia/Runt-related transcription factor 1 (AML1/RUNX1) and PU.1. However, at the outset of this project, it was relatively unknown which genes that are regulated by MOZ in leukaemia. Thus, this work investigates MOZ target genes in a leukemic cell line to fully understand its importance in cell function and disease conditions. Firstly, we performed siRNA silencing in K562 cell line, coupled with transcription profiling by RNA sequencing. We identified 406 deregulated genes under MOZ knockdown, of which 76 were downregulated. Many of the MOZ-target genes identified are implicated in haematopoietic development and cell proliferation. Secondly, we generated MOZ/KAT6A knockout K562 cells using CRISPR CAS9n nickase technology. It allowed further analysis of the function of MOZ in leukaemia. Our results confirmed the role of MOZ in maintaining levels of H3K14ac and H3K9ac and an apparent inverse relationship with the levels of H3K14 crotonylation. Furthermore, the results indicated that MOZ is important for leukemic cell proliferation and the gene dosage influences on the cell proliferation rate. Finally, we found that genes requiring MOZ for their expression are sensitive to a novel class of bromodomain inhibitors called iBETs. Furthermore, we found evidence for the existence of MOZ in complex with BRD4 protein. This provides a hitherto unknown link between MOZ, MLL and BRD2-4 complexes and insight into how these epigenetic regulators cooperate to control gene expression.
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Kaveri, Deepika. "Study of the role of Wnt pathway in a murine model of T-ALL." Phd thesis, Université de Strasbourg, 2012. http://tel.archives-ouvertes.fr/tel-00912330.
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We report a murine model, R26-βcat, expressing a stable form of β-catenin in T cells. R26-βcat pre-leukemic mice show a developmental block in T-cell differentiation and exhibit increased resistance to apoptosis. Interestingly, the mice develop T cell lymphomas independent of the Notch pathway. Furthermore, we showed that loss of the tumour suppressor Pten and over-expression of Myc was favoured; and may constitute the secondary events contributing to this leukemogenesis. We also demonstrated that R26-βcat tumours are malignant, heterogeneous and that leukaemia stem cells (LSC) were enriched in DP cells. Furthermore, the self-renewal capapcity of R26-βcat LSCs can to be exhausted.We propose that the R26-βcat model defines a new sub-group of Notch-independent T-ALL and the β-catenin may serve as a potential therapeutic target for these tumours.
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Mony, Ullas. "Establishment of a defined bonr marrow niche for testing in vitro chemosensitivity of acute myeloid leukaemic stwm and progenitor cells." Thesis, University of Nottingham, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.517769.
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Kassouf, Mira. "In vivo study of the DNA-binding requirements of the haematopoietic transcription factor SCL (stem cell leukaemia) in erythropoiesis." Thesis, University of Oxford, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.492059.
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The bHLH (basic helix-loop-helix) transcription factor SCL (Stem Cell Leukaemia) is required for HSC (haematopoietic stem cell) specification and for differentiation of the megakaryocytic and erythroid lineages. bHLH proteins bind DNA as dimers and this has been thought to be a prerequisite for their function. However, this concept has been recently challenged as SCL DNAbinding activity was shown to be dispensable for some of its functions, in in vitro haematopoietic differentiation assays. In my thesis, I studied the in vivo requirements for SCL DNA-binding activity in mice with a germ line DNA-binding (SCL-RER) mutation. In contrast to SCL knock-out embryos that die at E9.5 from absence of haematopoietic development, specification of haematopoietic progenitors was observed in SCLRERIRER yolk sacs. Lethality was first observed at E14.5. However 5% of homozygous mice were born from heterozygous crosses. A specific defect in erythropoiesis was documented. Anaemia was observed throughout development in homozygous embryos and adult mice. A combination of complementary phenotypic and cellular analyses of primitive and definitive erythropoiesis showed that specification of erythroid progenitors occurs but revealed defective erythroid maturation. To understand the molecular defects underlying the phenotype, expression levels of candidate target genes were assessed in fetal liver cells enriched for early erythroid progenitors or late normoblasts. We observed decreased or increased mRNA expression of some genes tested in SCLRERIRER erythroid populations when compared to controls, suggesting that SCL DNA-binding activities are required for both activation and repression of target genes. We then pursued the analysis of SCL-mediated transcriptional regulation by studying the recruitment of SCL to known cl.s-regu Iatory regI.Ons 0 f genes W.ith perturbed expressI.On pattern I.n SCLRER/RER erythro'dl populations by ChIP assay. Our data showed that SCL recruitment to specific hypersensitive sites was compromised on chosen targets such as a-globin, the transcription factor GATAl, and the red cell membrane protein band 4.2. In conclusion, this in vivo model confirms the dispensability of SCL DNA-binding activity for specification of primitive erythroid progenitors and definitive HSCs. As for erythroid development, it is the first in vivo proof in mammals that SCL DNA-binding activity is not required for specification of progenitors but is crucial for their differentiation into mature red blood cells. Finally, through our molecular studies, SCL appears as a multifunctional transcriptional regulator of erythropoiesis that can act as either an activator or a repressor of gene expression depending on the target and the maturation stage.
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Court, Emma Louise. "Profiling of differential gene expression in acute myeloid leukaemia and the effects of stem cell factor on expression levels." Thesis, University of the West of England, Bristol, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.409461.
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Toofan, Parto. "Using induced pluripotent stem cells (IPSCS) as a replacement for in vivo models to screen novel therapies in chronic myeloid leukaemia (CML)." Thesis, University of Glasgow, 2016. http://theses.gla.ac.uk/7729/.
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Tyrpsine kinase inhibitors (TKIs) effectively target progenitors and mature leukaemic cells but prove less effective at eliminating leukaemic stem cells (LSCs) in patients with chronic myeloid leukaemia (CML). Several reports indicate that the TGFβ superfamily pathway is important for LSC survival and quiescence. We conducted extensive microarray analyses to compare expression patterns in normal haemopoietic stem cells (HSC) and progenitors with CML LSC and progenitor populations in chronic phase (CP), accelerated phase (AP) and blast crisis (BC) CML. The BMP/SMAD pathway and downstream signalling molecules were identified as significantly deregulated in all three phases of CML. The changes observed could potentiate altered autocrine signalling, as BMP2, BMP4 (p<0.05), and ACTIVIN A (p<0.001) were all down regulated, whereas BMP7, BMP10 and TGFβ (p<0.05) were up regulated in CP. This was accompanied by up regulation of BMPRI (p<0.05) and downstream SMADs (p<0.005). Interestingly, as CML progressed, the profile altered, with BC patients showing significant over-expression of ACTIVIN A and its receptor ACVR1C. To further characterise the BMP pathway and identify potential candidate biomarkers within a larger cohort, expression analysis of 42 genes in 60 newly diagnosed CP CML patient samples, enrolled on a phase III clinical trial (www.spirit-cml.org) with greater than 12 months follow-up data on their response to TKI was performed. Analysis revealed that the pathway was highly deregulated, with no clear distinction when patients were stratified into good, intermediate and poor response to treatment. One of the major issues in developing new treatments to target LSCs is the ability to test small molecule inhibitors effectively as it is difficult to obtain sufficient LSCs from primary patient material. Using reprogramming technologies, we generated induced pluripotent stem cells (iPSCs) from CP CML patients and normal donors. CML- and normal-derived iPSCs were differentiated along the mesodermal axis to generate haemopoietic and endothelial precursors (haemangioblasts). IPSC-derived haemangioblasts exhibited sensitivity to TKI treatment with increased apoptosis and reduction in the phosphorylation of downstream target proteins. 4 Dual inhibition studies were performed using BMP pathway inhibitors in combination with TKI on CML cell lines, primary cells and patient derived iPSCs. Results indicate that they act synergistically to target CML cells both in the presence and absence of BMP4 ligand. Inhibition resulted in decreased proliferation, irreversible cell cycle arrest, increased apoptosis, reduced haemopoietic colony formation, altered gene expression pattern, reduction in self-renewal and a significant reduction in the phosphorylation of downstream target proteins. These changes offer a therapeutic window in CML, with intervention using BMP inhibitors in combination with TKI having the potential to prevent LSC self-renewal and improve outcome for patients. By successfully developing and validating iPSCs for CML drug screening we hope to substantially reduce the reliance on animal models for early preclinical drug screening in leukaemia.
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Gallipoli, Paolo. "Investigation of the role of autocrine and paracrine growth factors in the survival and proliferation of chronic myeloid leukaemia stem and progenitor cells." Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4172/.
Full textAbstract:
Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder arising in a haemopoietic stem cell (HSC) as a result of the reciprocal translocation between the long arms of chromosomes 9 and 22 (t9;22), leading to the formation of the fusion oncogene BCR-ABL. BCR-ABL has constitutive tyrosine kinase (TK) activity which drives, at least during the chronic phase (CP) of the disease, myeloid progenitor cells expansion through terminally differentiated cells and is necessary for the transformed phenotype. The introduction at the end of the last century of BCR-ABL TK inhibitors (TKI) has dramatically changed the management of newly diagnosed CP CML patients as the vast majority achieve deep molecular responses while enjoying good quality of life when treated with TKI. However about 20% of patients still show various degree of resistance to all currently available TKI while in those achieving deep responses, there is compelling evidence of persistent minimal residual disease demanding lifelong treatment which has obvious implications in terms of compliance, adverse events and costs. It is now known that the main reason for disease persistence in CML patients treated with TKI lies in the insensitivity of the most primitive CML leukaemia stem cell (LSC). More recent evidence has demonstrated that, in contrast to more mature leukaemic progenitor cells, CML LSC are not addicted to BCR-ABL kinase activity but rather rely on other stem cell intrinsic pathways for their survival. The main focus in the CML field is therefore to identify these pathways while also trying to exploit them therapeutically to achieve CML LSC eradication and as a result disease cure. Growth factor (GF) signals are known to provide survival cues to CML stem and progenitor cells (SPC) and potentially support their survival even in the presence of TKI. Moreover CML SPC are also known to produce higher levels of some GFs via an autocrine loop and support their survival and proliferation through this mechanism. In this thesis, the characterisation of the autocrine GF production by CML SPC was extended while also investigating the role of several GFs and downstream signals in survival, proliferation and self-renewal of CML SPC. Whenever possible, the consequences of therapeutic targeting of these signals on CML SPC survival and proliferation were also assessed in vitro. In particular the role of the intracellular janus kinase (JAK) 2, which relays several myeloid GF signals, such as those from interleukin (IL)-3 and granulocyte macrophage colony-stimulating factor (GM-CSF), in CML SPC survival and proliferation was investigated mainly because higher levels of autocrine expression of GM-CSF by CML SPC relative to normal were demonstrated, while autocrine IL-3 production by CML SPC had already been shown. Moreover the cognate receptor of both GM-CSF and IL-3 (CSF2RB) was also shown to be expressed at higher levels in CML SPC relative to their normal counterparts, further supporting investigations on the role of JAK2 in CML SPC biology. Indeed targeting JAK2 with small molecule inhibitors in CML SPC in vitro, particularly in the presence of maximal BCR-ABL TK inhibition, resulted in increased apoptosis, reduced proliferation and colony output of CML SPC. The JAK2 inhibitor plus TKI combination treatment, compared to either single agent, further reduced survival of the more primitive quiescent LSCs in vitro, while also reducing engraftment of primary CML CD34+ cells in vivo in immunocompromised hosts. Although a degree of toxicity to normal haemopoietic stem and progenitor cells (HSPC) was observed, this was not as great as seen in CML SPC, thus suggesting that a therapeutic window for using JAK2 inhibitors in CML patients might be present when a carefully selected concentration of these compounds is chosen. Tumour necrosis factor (TNF)-α was another GF shown to be produced in an autocrine fashion at higher levels by CML SPC relative to normal HSPC. Moreover its levels of production by CML SPC were not modulated by BCR-ABL TK activity. Using a small molecule TNF-α inhibitor and exogenous TNF-α, it was shown that autocrine TNF-α acts as a survival and proliferative signal in CML SPC. Moreover its role became even more important in the presence of TKI, as combining TNF-α inhibition with TKI led to high levels of apoptosis in CML CD34+ cells, including the more primitive quiescent population, while also causing increased apoptosis in a population enriched for CML LSCs based on its surface marker expression (CD34+ CD38-). Finally given the known importance of quiescence and self-renewal pathways in CML LSC persistence following TKI treatment, the role of transforming growth factor (TGF)-β1 and novel neurotransmitter mediated pathways in CML LSC quiescence and self-renewal was investigated based on the findings of a genome and epigenome-wide screen of primary CML LSCs and normal HSCs carried out in our laboratory. Using in vitro assays the putative role of the neuromediators norepinephrine and acethylcoline in CML LSC self-renewal was demonstrated. Moreover the role of TGF-β1 in inducing primary CML LSC quiescence mainly by modulating the AKT pathway was also demonstrated. Overall the work presented in this thesis has furthered our understanding of the role of both autocrine and paracrine known and novel regulators of haemopoiesis in several aspects of CML SPC biology such as their survival, proliferation and self-renewal. Furthermore the efficacy in eradicating CML SPC of therapeutic strategies targeting some of these GF signals has been explored in vitro, thus providing evidence supporting their subsequent testing in in vivo assays and in due course in clinical studies. It is hoped therefore that the work presented will contribute to devise novel therapeutic strategies to eradicate CML LSC and in turn lead to a cure for CML patients.
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Chang, Chao-Hui. "Haematopoietic stem/progenitor cell interactions with the bone marrow vascular niche." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:452da334-bd4e-45c7-a7bd-fc8767d1239c.
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Umbilical cord blood (UCB) is used as a source of haematopoietic stem cells (HSCs) for transplantation but shows defective homing to the bone marrow niche and delayed haematological reconstitution. Following transplantation, HSCs will home to the bone marrow in response to the CXCL12 chemokine, adhere to the bone marrow sinusoidal endothelial cells and then migrate into and lodge in bone marrow niches. In addition to CXCR4, a variety of molecules have been described as being important in these processes. In this laboratory, junctional adhesion molecule-A (JAM-A) was shown to be expressed on human UCB CD133⁺/CD34⁺ cells and regulated by hypoxia. In this thesis, further phenotypic studies show that this molecule is most highly expressed on human CD41a⁺ megakaryocytes and CD14⁺ monocytes/macrophages in UCB. JAM-A was also found to be expressed on all human UCB CD133⁺ cells, which have been shown by others to encompass the HSCs and early myeloid-lymphoid precursors and on the majority of CD34⁺ haematopoietic progenitor cells (HPCs). While it is also present on bone marrow sinusoidal endothelium (BMEC), JAM-A is not detected on cultured bone marrow mesenchymal stromal cells (MSCs). JAM-A blockade, silencing and overexpression experiments showed that JAM-A contributes to, but is not solely responsible for, the adhesion of CD34⁺ haematopoietic progenitor cells to IL-1β activated BMEC-60 cells and fibronectin. Lack of significance in cell migration suggested that JAM-A is more likely to act as an adhesion molecule or a regulator of adhesion rather than as a migratory molecule in such cells. Further functional studies using the proximity ligation assay highlight a potential association of JAM-A with CXCR4 and the adhesion molecules, tetraspanin CD82 and integrin β1. Mechanistic studies were commenced to establish if JAMA could modulate CXCR4 signalling following CXCL12 stimulation, but time constraints prevented these from being completed. These preliminary experiments which were carried out first in the Jurkat cell line lacking JAM-A or transduced to express JAM-A, however, suggest that JAM-A may modulate CXCL12-induced Rap1 phosphorylation and ERK1/2 phosphorylation. The former pathway is important for integrin function and the latter pathway is important in cell adhesion. The results described here, although requiring finalisation, support the hypothesis that JAM-A acts as an adhesion molecule and also may fine tune CXCR4 and integrin mediated functions on human CD34⁺ cells, thereby potentially regulating engraftment of these cells to the bone marrow niche.
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Hanke, Maximilian Paul [Verfasser], and Axel [Akademischer Betreuer] Rosenhahn. "How Stem Cells Roll : a microfluidic characterisation of the CD44-hyaluronic acid interaction and its role in leukaemia / Maximilian Paul Hanke ; Betreuer: Axel Rosenhahn." Heidelberg : Universitätsbibliothek Heidelberg, 2014. http://d-nb.info/1180547837/34.
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Hanke, Maximilian [Verfasser], and Axel [Akademischer Betreuer] Rosenhahn. "How Stem Cells Roll : a microfluidic characterisation of the CD44-hyaluronic acid interaction and its role in leukaemia / Maximilian Paul Hanke ; Betreuer: Axel Rosenhahn." Heidelberg : Universitätsbibliothek Heidelberg, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:16-heidok-177021.
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Lim, Chee Yee. "Understanding transcriptional regulation through computational analysis of single-cell transcriptomics." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/267786.
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Gene expression is tightly regulated by complex transcriptional regulatory mechanisms to achieve specific expression patterns, which are essential to facilitate important biological processes such as embryonic development. Dysregulation of gene expression can lead to diseases such as cancers. A better understanding of the transcriptional regulation will therefore not only advance the understanding of fundamental biological processes, but also provide mechanistic insights into diseases. The earlier versions of high-throughput expression profiling techniques were limited to measuring average gene expression across large pools of cells. In contrast, recent technological improvements have made it possible to perform expression profiling in single cells. Single-cell expression profiling is able to capture heterogeneity among single cells, which is not possible in conventional bulk expression profiling. In my PhD, I focus on developing new algorithms, as well as benchmarking and utilising existing algorithms to study the transcriptomes of various biological systems using single-cell expression data. I have developed two different single-cell specific network inference algorithms, BTR and SPVAR, which are based on two different formalisms, Boolean and autoregression frameworks respectively. BTR was shown to be useful for improving existing Boolean models with single-cell expression data, while SPVAR was shown to be a conservative predictor of gene interactions using pseudotime-ordered single-cell expression data. In addition, I have obtained novel biological insights by analysing single-cell RNAseq data from the epiblast stem cells reprogramming and the leukaemia systems. Three different driver genes, namely Esrrb, Klf2 and GY118F, were shown to drive reprogramming of epiblast stem cells via different reprogramming routes. As for the leukaemia system, FLT3-ITD and IDH1-R132H mutations were shown to interact with each other and potentially predispose some cells for developing acute myeloid leukaemia.