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published_or_final_version / Medicine / Master / Master of Philosophy

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Telomeres are regions of tandem repeats at the ends of chromosomes ensuring chromosome stability or inducing replicative senescence when critically short. Telomerase extends telomeres and its catalytic subunit, telomerase reverse transcriptase is tightly regulated at multiple levels. Cancerous cells prevent telomere-mediated senescence to attain unlimited proliferation, in most cases by enhancing telomerase activity. Chronic myeloid leukaemia is characterised by the translocation, t(9;22), in haematopoietic stem cells. The resulting fusion protein exhibits constitutive tyrosine kinase activity in the cytoplasm, promoting cellular proliferation, inhibiting apoptosis and impeding cell adhesion. Changes in telomere biology have been observed in chronic myeloid leukaemic cells. The current study aimed to investigate telomere biology in 18 chronic myeloid leukaemia patients at various time intervals from date of diagnosis. Although telomeres were significantly shorter in patients compared to controls, results point to complex telomere dynamics in the malignancy. Increased telomerase activity did not necessarily accompany telomere lengthening and increased transcription of the telomerase catalytic subunit was not necessarily indicative of telomerase activity. Ultimately the current study could not detect any trends between telomere length, telomerase activity and telomerase catalytic subunit expression in chronic myeloid leukaemia patients. Together with inherent patient-to-patient variation and the high cost per assay, measurement of telomere biology does not appear to hold prognostic value in chronic myeloid leukaemia and does not warrant inclusion into a routine test repertoire.

by Chan Shuk Chong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 135-142). / Statement --- p.i / Acknowledgment --- p.ii / Abbreviations --- p.iii / Abstract --- p.iv / Chapter Chapter 1: --- General Introduction / Chapter 1.1 --- Haematopoiesis --- p.1 / Chapter 1.1.1 --- Sites of haematopoiesis / Chapter 1.1.1.1 --- Bone marrow stroma / Chapter 1.1.1.2 --- Thymus / Chapter 1.1.1.3 --- Spleen and lymph node / Chapter 1.1.1.3.1 --- Spleen / Chapter 1.1.1.3.2 --- Lymph Nodes / Chapter 1.1.2 --- Blood Cell / Chapter 1.1.2.1 --- Development of T and B cells / Chapter 1.1.2.1.1 --- T cells / Chapter 1.1.2.1.2 --- B cells / Chapter 1.1.2.2 --- Development of Granulocytes and monocytes / Chapter 1.2 --- White Cell Disorder -Leukemia --- p.13 / Chapter 1.2.1 --- Leukemia - general concept / Chapter 1.2.1.1 --- Classification of leukemia / Chapter 1.2.1.2 --- Pathophysiology and Clinical features / Chapter 1.2.1.3 --- Etiology of myeloid leukemia / Chapter 1.2.2 --- Genetic basis of leukemia / Chapter 1.3 --- Acute myeloid leukemia (AML) cell model --- p.19 / Chapter 1.3.1 --- Cell Model for human acute myeloid leukemia / Chapter 1.3.2 --- Murine leukemia cell lines / Chapter 1.4 --- Induction of leukemia cell differentiation --- p.21 / Chapter 1.4.1 --- Overview of different inducers / Chapter 1.4.2 --- Cytokines as Inducers / Chapter 1.5 --- Objectives and Research Strategy --- p.26 / Chapter 1.5.1 --- Objectives / Chapter 1.5.2 --- Research strategy / Chapter Chapter 2 : --- Materials and Methods / Chapter 2.1 --- Materials --- p.29 / Chapter 2.1.1 --- Cell line / Chapter 2.1.2 --- Tissue culture medium / Chapter 2.1.3 --- Tumor necrosis Factor - alpha (TNF-α) / Chapter 2.1.4 --- Interleukin 1- alpha (IL-lα)and Interleukin 1- beta (IL-1β) / Chapter 2.1.5 --- "Monoclonal hamster anti-mouse IL-lα monoclonal hamster anti-mouse IL-1β, and Polyclonal rabbit anti-mouse TNF-α antibodies" / Chapter 2.1.6 --- Lipopolysaccharides (LPS) / Chapter 2.1.7 --- Buffers and solutions / Chapter 2.2 --- Methods : --- p.33 / Chapter 2.2.1 --- Cell culture / Chapter 2.2.2 --- Cytotoxicity assay / Chapter 2.2.3 --- Proliferation assay / Chapter 2.2.4 --- Cell morphology / Chapter 2.2.5 --- Phagocytosis assay / Chapter 2.2.6 --- Preparation of undifferentiated and differentiated murine leukemia WEHI3B (JCS) cells for cell lysate / Chapter 2.2.7 --- Isolation of total cellular RNA / Chapter 2.2.8 --- Extraction of the total RNA / Chapter 2.2.9 --- Spectrophotometry / Chapter 2.2.10 --- Electrophoresis of RNA in agarose gel containing formaldehyde / Chapter 2 2.11 --- First strand cDNA synthesis / Chapter 2.2.12 --- Cytokines phenotyping of the uninduced and induced WEHI 3B (JCS) by The Reverse Trancription Polymerase Chain Reaction method / Chapter 2.2.13 --- Gel electrophoresis of PCR- product / Chapter 2.2.14 --- Southern blot / Chapter 2.2.15 --- Dot blot / Chapter 2.2.16 --- Hybridization with oligonucleotides / Chapter 2.2.17 --- Chemiluminescent detection / Chapter Chapter 3 : --- Growth Inhibitory and Differentiation Effects of Lipopolysaccharides ( LPS ) on WEHI 3B (JCS) cells / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.1.1 --- Chemical structure of LPS / Chapter 3.1.2 --- Biological activity of LPS / Chapter 3.2 --- Results --- p.55 / Chapter 3.2.1 --- Anti-proliferative effects of LPS / Chapter 3.2.2 --- Differentiation inducing effect of LPS on WEHI 3B (JCS) cells / Chapter 3.2.3 --- Phagocytic activity LPS treated WEHI 3B (JCS) cells / Chapter 3.2.4 --- Anti-proliferative effect of TNF-α / Chapter 3.2.5 --- Differentiation inducing effect of TNF-α / Chapter 3.2.6 --- Phagocytic activity of TNF-α treated WEHI3B (JCS) cells / Chapter 3.3 --- Discussion --- p.67 / Chapter 3.4 --- Summary --- p.69 / Chapter Chapter 4 : --- The Cytokine Genes Expression of the TNF-α and LPS Treated WEHI 3B (JCS) cells / Chapter 4.1 --- Introduction --- p.70 / Chapter 4.1.1 --- Differentiation of leukemia cell line / Chapter 4.1.2 --- Study of the cytokine genes expression of WEHI 3B (JCS) cells / Chapter 4.2 --- Results --- p.72 / Chapter 4.2.1 --- Isolation of total RNA from uniduced and induced WEHI 3B (JCS) cells / Chapter 4.2.2 --- The cytokine genes expression during differentiation / Chapter 4.2.2.1 --- "Up-regulation of IL-lα, IL-1β,TNF-α and IFN-γ in both TNF-α induced and LPS induced WEHI 3B (JCS) cells" / Chapter 4.2.2.1.1 --- Southern blot / Chapter 4.2.2.1.2 --- Semi-quantitation of PCR-products by gel electrophoresis and dot-blot hybridization / Chapter 4.2.2.2 --- up-regulation of GM-CSF and G-CSF in LPS induced WEHI 3B (JCS) cells / Chapter 4.3 --- Discussion --- p.92 / Chapter 4.4 --- Summary --- p.95 / Chapter Chapter 5 : --- Growth inhibitory and Differentiation Inducing Effect of IL-l( IL-1α and IL-1β) on WEHI 3B (JCS) cells / Chapter 5.1 --- Introduction --- p.96 / Chapter 5.1.1 --- The interleukin 1 (IL-1) family / Chapter 5.1.1.1 --- Structure of IL-1 / Chapter 5.1.1.2 --- The biological function of IL-1 / Chapter 5.1.2 --- Tumor necrosis factor - alpha ( TNF-α) / Chapter 5.1.2.1 --- Structure of TNF-α / Chapter 5.1.2.2 --- Biological functions of TNF-α / Chapter 5.1.3 --- The similarity between TNF and IL-1 / Chapter 5.2 --- Results --- p.102 / Chapter 5.2.1 --- Anti-proliferative effect of IL-1 / Chapter 5.2.2 --- Differentiation inducing effect of IL-1 / Chapter 5.2.3 --- Phagocytic activity of IL-1 treated JCS cells / Chapter 5.2.4 --- "Role of endogenously produced IL-lα, IL-1β and TNF-α in LPS cytokines differentiation of WEHI 3B (JCS) cells" / Chapter 5.2.4.1. --- "Effect of neutralizing anti- ILl-α,anti - IL-l-β, and anti-TNF-α antibodies on the growth inihbition of the treated WEHI 3B (JCS) cells" / Chapter 5.2.4.2 --- "Effects of neutralizing anti-IL-lα, anti- IL-1β, and anti-TNF-α antibodies on differentiation of the treated WEHI 3B (JCS) cells" / Chapter 5.3 --- Discussion --- p.124 / Chapter 5.4 --- Summary --- p.127 / Chapter Chapter 6 --- : Concluding Discussion --- p.128 / References --- p.135

Acute myeloid leukaemia is a clonal disorder characterised by recurrent chromosomal translocations. One of the commonest, is the t(8;21) which results in part of the AML1 gene being juxtaposed to most of the ETO gene with the resultant chimeric protein, AML1-ETO, acting predominantly as a transcriptional repressor. Despite the extensive literature available, the exact mechanism by which the chimeric protein results in AML has not been fully elucidated. By using exon arrays and high throughput sequencing as tools it was hoped to gain further insights into the molecular basis of this disease. Gene expression profiling using the exon arrays highlighted molecular pathways and specific genes that play a key role in the pathogenesis in t(8;21). Exon arrays were also used to profile individual exon expression of the ETO gene. This demonstrated that the genomic breakpoint of ETO in the t(8;21) is variable between different patients. This technique also resulted in the discovery of a new exon in the ETO gene. This novel exon results in formation of alternative transcripts of AML1-ETO and was shown in mouse models to play a key role in leukaemogenesis. Chromatin immunoprecipitation followed by high throughput sequencing revealed novel aspects of AML1-ETO binding. A number of novel genes that bind AML1-ETO were recognized and in conjunction with the expression data, a number of hypothesis on how AML1-ETO binding effects gene expression are made.

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.

Phosphoinositides (PIs) are pivotal lipid molecules with both scaffolding and signalling functions regulating key aspects of cellular physiology. For example, phosphatidylinositol (3,4,5)-trisphosphate, generated by phosphoinositide 3-kinase (PI3K), is an essential mediator of the PI3K/AKT signalling pathway, which is crucial for cell proliferation, survival and apoptosis. Constitutive activation of this signalling cascade has been identified in acute myeloid leukaemia (AML), the most common haematopoietic malignancy in adults, and experimental deletion of the PI3K antagonists PTEN and SHIP cause leukaemia in mice. However, little is known regarding the role of other PI modulator proteins in AML. Thus, in this thesis, a lentivirally delivered small hairpin RNA (shRNA) library targeting 103 genes (345 pLKO knockdown constructs) with presumed or established roles in PI metabolism was utilised to screen for genes required for AML blast cell viability/proliferation and differentiation. First, knockdown constructs were tested for their impact on proliferation/viability in seven human AML cell lines by measuring fold change in fluorescence of the cell viability dye alamarBlue relative to controls (cells transduced with a non-targeting control hairpin) over three days. This identified 13 candidate genes selected with the criterion that two or more knockdown constructs per gene reduce cell viability/proliferation relative to control by greater than or equal to50 % across all cell lines. From these candidate genes, PIP4K2A, INPP5B and IMPAD1 were selected for downstream validation experiments, which reproduced the observation from the primary screen. For INPP5B and IMPAD1, knockdown constructs also reduced clonogenic potential of primary human AML samples but only showed a modest effect on normal CD34+ haematopoietic stem or progenitor cells (HSPCs) in a methylcellulose based assay. This could be recapitulated in a murine setting where knockdown constructs targeting both genes reduced clonogenic potential of murine MLL- AF9 AML cells with little effect on normal KIT+ HSPCs. In line with this, Inpp5b knockout KIT+ BM cells either failed to immortalise or weakly immortalised, following forced expression of the powerful MLL-AF9 oncogene. A further screen was performed to identify regulators of THP-1 blast cell differentiation, by seeding knockdown construct transduced cells into methylcellulose based semisolid media. After ten days of incubation the degree of macrophage differentiation was evaluated by light microscopy and an arbitrary differentiation score was given. With the criterion that greater than or equal to2 knockdown constructs per gene received the highest differentiation score, reflecting terminal macrophage differentiation of all seeded cells, SBF2 was identified as the top-scoring hit. Validation experiments have confirmed macrophage differentiation based on cytospin preparations of SBF2 knockdown THP-1 cells. Moreover, xenograft assays have shown that knockdown constructs targeting PIP4K2A and SBF2 delayed or abrogated in vivo leukaemogenesis. Thus this work has identified novel roles for PI modulator genes in human AML with possible therapeutic potential.

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.

The finely tuned regulation of haematopoiesis is crucial in order to maintain life-long haematopoiesis. The disruption of the balance among cell fates, can lead to malignant transformation. It has become increasingly evident that the metabolic regulation haematopoietic stem cells is critical for stem cell fate decisions. Haematopoietic stem cells reside in a hypoxic microenvironment within the bone marrow and are thought to mainly utilize glycolysis rather than oxidative phosphorylation in order to maintain their pool. However recent evidence suggests that oxidative phosphorylation is critical for quiescent HSCs and in several cases, for leukaemic stem cells (LSCs). One of the key parts of mitochondrial respiration is the tricarboxylic cycle (TCA), providing co-factors for its efficient activity. The TCA functions by catalysing the oxidation of pyruvate via key enzymatic activities. A key component of the TCA cycle is fumarate hydratase (Fh1) which catalyses the hydration of fumarate into malate within the mitochondria, but also catalyses the same reaction in the cytoplasm. FH is a tumour suppressor in human lyomeioma and renal kidney cancer (HLRCC). Previous work conducted by our team has shown that Fh1 is essential for foetal and adult haematopoiesis, as Fh1 deletion within the haematopoietic system is embryonic lethal. Furthermore, conditional deletion of Fh1 in donor cells of the Mx1-Cre system that were injected in lethally irradiated recipients, resulted in the complete reduction of their chimerism in the peripheral blood of recipient mice. Mechanistically, these phenotypes were mostly associated with supra-physiological levels of fumarate as a result of Fh1 deletion. Interestingly, by employing mice that ubiquitously express the human cytosolic isoform of FH (FHCyt, which lacks the mitochondrial targeting sequence and therefore is excluded from the mitochondria), we rescued the embryonic lethality that Fh1 causes, and reduced the levels of fumarate. Importantly, although FHCyt expression restored fumarate-associated lethality, it did not restore the mitochondrial defects, allowing us to study the importance of genetically intact TCA in the context of haematopoiesis. Here I investigated the impact that a genetic truncation of the TCA cycle (as a result of the lack of the mitochondrial isoform of Fh1) has on leukaemic transformation and on aged haematopoiesis. Fh1fl/fl; FHCyt; Vav-iCre mice of approximately 60 weeks old displayed and expansion in the pool of early stem and progenitor compartment (Lin- Sca-1+ c-Kit+), as well as in the early progenitors HPC-1 (LSK CD48+ CD150-) and HPC-2 (LSK CD48+ CD150+). Furthermore, the mice exhibited a drastic depletion of B cells (CD19+ B220+) and an expansion in the frequency of the myeloid compartment (Mac-1+ Gr1+). In order to assess the importance of the TCA cycle in malignant transformation, I isolated stem and progenitor cells from Fh1fl/fl; FHCyt; Vav-iCre (and control (Fh1fl/fl; FHCyt Vav-iCre negative or Fh1fl/fl Vav-iCre negative)) E 14.5 day old embryos and infected them with retroviruses expressing Meis1 and Hoxa9, and generated pre-leukaemic cells (pre-LCs). Genetically intact TCA was required for the efficient generation of leukaemia-initiating cells (LICs), as injection of pre-LCs lacking mitochondrial Fh1 into sub-lethally irradiated recipient mice, resulted in 76 % of leukaemia-free mice while injection of control pre-LCs resulted in 25 % of leukaemia-free mice. However, the genetic perturbation of the TCA did not exert and effect on the long-term self-renewal capacity of LICs. Inducible deletion of mitochondrial Fh1 in established LICs of the Mx1-Cre background using poly (I:C) did not affect their ability to generate AML in primary and secondary recipient mice. These data indicate that genetically intact TCA is required for the efficient generation of LICs in vivo but is dispensable for their long-term self-renewal capacity, highlighting the metabolic rewiring that occurs at different stages of leukaemic transformation. In an effort to understand whether, similarly to HLRCC, Fh1 plays a tumour-suppressive role in malignant haematopoiesis, I isolated LSK cells from the foetal liver of E 14.5 old embryos lacking both isoforms of Fh1. Fh1fl/fl; Vav-iCre cells transduced with Meis1/Hoxa9 or MLL-AF9, MLL-ENL, AML-ETO (chromosomal translocations involved in AML development) -expressing retroviruses, failed to generate colonies in methylcellulose, indicating that stem and progenitor cells require Fh1 to undergo in vitro transformation by these oncogenes. Furthermore, acute deletion of Fh1 (via the use of lentivirally-expressed Cre) in pre-LCs generated using the Meis1/Hoxa9 retroviruses, rendered them unable to generate colonies in methylcellulose, indicating that Fh1 is required for the self-renewal capacity of pre- LCs in vitro. Similarly, when LICs (Fh1fl/fl; Vav-iCre negative) isolated from primary recipient mice were infected with Cre to induce deletion of Fh1, they were unable to generate colonies indicating that Fh1 is required for the self-renewal capacity of LICs in vitro. Finally, in order to identify whether Fh1 is important for LIC self-renewal in vivo I generated Fh1fl/fl; Mx1-Cre pre-LCs by infecting stem and progenitor cells of E 14.5 embryos with Meis1/Hoxa9 retroviruses, and injected them into sub-lethally irradiated mice. After the mice developed AML, I induced the deletion of Fh1, by injecting the mice with poly (I:C). Interestingly, the percentage of LICs in the peripheral blood of recipient mice was drastically decreased, leaving recipient mice leukaemia-free for the remaining time they were monitored. Surprisingly however, approximately 50 % of the recipient mice exhibited a drastic increase in LIC chimerism after two weeks post poly (I:C). Assessment of LICs isolated from recipient mice indicated that Fh1 was fully deleted. These data indicate that while in some cases Fh1 is required for LIC self-renewal in vivo, in other cases it is dispensable. Therefore, the tumour-suppressive roles of Fh1 are likely tissue-specific and do not extend to haematopoietic cells. Overall, this study agrees with published work supporting the notion that intact mitochondrial respiration is important (in varying degrees), in both the contexts of normal and malignant haematopoiesis.