Thèses : « AML acute myeloid leukaemia »

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Shah, Niraj Mayank. « The role of NRF2 in acute myeloid leukaemia (AML) ». Thesis, University of Liverpool, 2018. http://livrepository.liverpool.ac.uk/3022789/.

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Acute Myeloid Leukaemia refers to the excess proliferation of myeloid progenitor cells. Whilst the 5-year survival rate is 40% in younger patients, this falls to 5% in patients over 65 and resistance to front-line chemotherapy agents remain a problem. We previously identified NRF2, a regulator of anti-oxidant genes, to be constitutively activated in AML and this correlated with resistance to chemotherapy. Recent studies have also suggested NRF2 also plays a more oncogenic role. To further understand NRF2's role in both chemotherapy resistance and oncogenesis we looked at its ability to regulate miRNA in AML. Using a miRNA array, we identified several miRNAs, including miR-125b and miR-29b, whose expression correlated with that of NRF2 in both cell lines and AML patient samples. Both miRNAs exist as paralogs, in that they contain the same miRNA sequence but exist in different genomic locations and we used qPCR to identify miR-125b1 and miR-29b1 as paralogs regulated by NRF2. Using a reporter assay we confirmed the activity of putative NRF2-ARE binding sites in both miRNA promoters. To understand the function of both in AML we manipulated their expression using miRNA 'mimics' or antagomIRs. Individual manipulation of either miRNA resulted in a slight increase in apoptosis. However, the miRNAs appeared to act synergistically as when expressed simultaneously a significant increase in apoptosis was seen both in cell lines and patient blasts. Manipulation of both miRNA also resulted in the increased sensitivity of AML cells to chemotherapy agents. BAK1, STAT3 and AKT2 were shown to be targets of both miRNAs providing a novel mechanism by which NRF2 expression can affect AML cells. To further study the role of NRF2 in AML we used CRISPR-Cas9 to generate NRF2-deficient cells. CRISPR guide RNA were designed to target NRF2 Exons 1, 2 and genome editing validated in HEK293T cells. Leukaemic cell lines (K562 and THP-1) were virally transduced with guides targeting Exon 4 of NRF2. Once editing was verified clones were derived by growing selected cells in Methycellulose-containing medium. Putative clones were initially screened using MG-132 (to stabilise NRF2) and further confirmed by treatment with the NRF2 inducers CDDO-Me and Sulforaphane. Verified clones were characterised by Sanger Sequencing. In addition to CRISPR-Cas9 we also validated a number of other gene-editing methodologies including CRISPR-Cpf-1 and TALENs. Overall these methodologies represent powerful tools to further characterise the role of NRF2 in AML.

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Bajuaifer, Nada A. « Translational regulation of ABCB1 gene in acute myeloid leukaemia (AML) ». Thesis, University of Nottingham, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.594594.

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Introduction: Multidrug Resistance (MDR) was and remains a major obstacle in the way of successful chemotherapy for cancer patients and especially acute myeloid leukaemia (AML) patients. In AML, MOR mediated by ABCBl over-expression accounts for the majority of MOR cases. The ABCB1 gene encodes a surface protein known as P-glycoprotein (Pgp) which plays a major role in MOR by effluxing drugs out of cells leading to increased relapse rates in AML patients. The regulation of ABCBl and subsequently Pgp is multifactorial and can be altered at many levels. Common regulators of genes expression may play a role in the regulation of this gene such as genetic sequence variation in the form of single nucleotide polymorph isms and short noncoding RNAs known as microRNAs. Results and Discussion: Role of different regulators in the regulation of ABeBl gene was investigated; these were microRNAs and 3'UTR SNP (specifically rs3842). Microarray profiling of 11 AML cell lines and 45 NeRN AMLl4 and AMLl5 trial patients samples showed significant down-regulation of miR-34b and miR-503 in Pgp positive cell lines when compared with Pgp negative cell lines and miR-l48a, miR-45l, miR-659, miR-499-5p, miR-l97 and miR-642 in Pgp positive patient samples when compared with Pgp negative patient samples. This suggests a role of these miRs in MDR in AML. No significant up-regulation was found in the Pgp positive cell lines and patients samples when compared with the Pgp negative group. Functionally, no direct effect of miR-l48a and miR-34b on ASeSl3'UTR constructs and Pgp protein level (for miR-34b) was found but an indirect effect of these miRs on ASeS! still needs to be tested. Second, the role of rs3842 on regulation of ABeBl gene was investigated. rs3842 genotyping of 455 NCRN AMll4 and AML15 trial patients showed comparable distribution to the reported distribution of the genotypes of normal healthy European population. Furthermore, an effect of the rs3842 polymorphism on Pgp protein expression and function (but not at the mRNA level) was seen in the 35% of patients where ASeS! expression is high. Functionally, higher baseline activity of the ASeS! wild type 3'UTR construct was found when compared with the ABeBl variant 3'UTR construct and a further construct mutated by site-directed mutagenesis. On examination of the rs3842 polymorphism and surrounding sequence, it was predicted that miR-374a targets AseSl very close to the SNP. This went to show that miR-374a causes down-regulation of ASCSl 3'UTR activity but only when the wild type rs3842 allele A is expressed. In addition, an effect of miR-374a on Pgp protein expression was found at the protein level but not at the mRNA suggesting a role of miR-374a in the translational regulation of the ABCBl gene. To conclude, the role found of microRNAs (miR-374a) and rs3842 in ABCBl regulation in this work suggests a significant role of the 3'UTR of ABCBl gene in its regulation.

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Jawad, Mays Safa Hadi. « Genetic susceptibility to radiation-induced acute myeloid leukaemia (r-AML) ». Thesis, University of Leicester, 2006. http://hdl.handle.net/2381/30366.

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CBA/H inbred mice are susceptible to radiation-induced acute myeloid leukaemia (r-AML) while C57BL/6 mice are resistant to r-AML. CBA/H mice also have higher haemopoietic stem cell number (HSC) than C57BL/6 mice raising the possibility that HSC frequency is a risk factor in r-AML. F2 mice were therefore analysed to map QTL that determine the frequency of phenotypically defined bone marrow cells. The Lin-Sca-1+c-Kit+ cell QTL in F2 mice mapped to chromosomes 1 (65cM), 17 (6cM), and 18 (21cM), genetic evidence that they are HSC, while the more mature Lin-Sca-1+FLK-2+ cell QTL mapped to chromosome 9 (33cM), a novel progenitor cell frequency QTL. The same F2 mice were also scored for difference in peripheral blood and bone marrow red blood cell counts (RBC), and spleen weight, so QTL were mapped for these phenotypes. Different autosomal loci affect bone marrow (chromosome 5, 9, 11, and 19) and peripheral blood RBC counts (chromosome 4), and spleen weight (chromosomes 3, 15, and 17). This suggests that the relative contributions of spleen and bone marrow erythropoiesis to peripheral RBC counts are genetically determined in mouse. A human AML genetic association study was carried out to assess the contribution of variant alleles to risk of AML in patients with de novo or therapy-related AML (t-AML) compared to age matched controls. In humans, a variant HLX1 allele was associated with a 4-fold increased risk of t-AML. Together with a variant RAD51 DNA repair allele, the variant HLX1 allele increased the risk of t-AML by 14-fold. The variant HLX1 gene may determine stem cell frequency (target size) during chemo-/radiotherapy therapy and a reduced ability to repair therapy-induced DNA damage will increase the risk of malignant transformation.

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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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Gasiorowski, Robin. « CD300f As A Novel Therapeutic Antibody Target For Acute Myeloid Leukaemia ». Thesis, The University of Sydney, 2017. http://hdl.handle.net/2123/17190.

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Current outcomes for patient with acute myeloid leukaemia (AML) are unsatisfactory, particularly for older patients where the median overall survival is less than six months. Monoclonal antibodies (mAb) have been used with great success in a range of malignancies and the promising results seen with gemtuzumab ozogamicin suggest they may improve outcomes in AML. CD300f is an immunoregulatory leucocyte cell surface molecule with myeloid restricted expression, which we have investigated as a potential antibody target in AML. We found that CD300f is expressed on blasts and leukaemic stem cells (LSC) from the majority of AML patients and that AML cells express multiple isoforms of CD300f. We showed that there is a range CD300f epitopes expressed on healthy and malignant cells, and developed a novel CD300f mAb, DCR2, which has in vitro and in vivo activity against AML. However we showed that CD300f, like CD33, is expressed on haematopoietic stem cells suggesting that targeting this antigen is likely to result in myelosuppression. We also investigated the expression and regulation of other CD300 family members on myeloid cells, with the results suggesting that this family of molecules has the potential to regulate dendritic cell responses. We identified CD302 as another novel target for AML antibodies, again showing its expression on the majority of AML blasts and LSC. Finally, using the HLDA10 panel of mAbs, we investigated the expression of novel and more established AML targets on both healthy and malignant cells. We showed that all of the tested targets are expressed on both healthy and malignant cells. A key issue for products based on these murine mAbs will be establishing the therapeutic window where there is efficacy against AML without excessive toxicity. The binding patterns of DCR2, and the other mAbs investigated in this thesis, will help to drive the preclinical and clinical testing required to achieve this. Keywords: Therapeutic antibody, AML, CD300f

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Marsico, Paolo. « The effects of targeted therapy on cell viability and apoptosis on CML and AML cell lines ». Thesis, University of Chester, 2019. http://hdl.handle.net/10034/621798.

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Tyrosine kinase inhibitors (TKIs) are currently the first therapy option for chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) patients. However, many patients affected by CML and AML may develop resistance to TKIs or may not recover under this treatment regime. New potential and more effective treatments are recently emerging. Heat shock protein inhibitors (HSPIs) and the proteasome inhibitor Bortezomib are drugs which have been yet to be successfully tested on leukemic patients, despite being successful on other malignancies such as multiple myeloma (MM). The combination between HSPIs and Bortezomib could potentially be successful in killing leukemic cells, by enhancing their respective molecular mechanisms. Indeed, HSPIs would bind to HSP72 avoiding the protein to exert its ligase function to the proteasome, whilst Bortezomib could stop the ubiquitinated proteins to enter the proteasome and ultimately inducing apoptosis. To test the effects of such combination, cell viability was measured via MTS assay, apoptosis levels were tested through Annexin V\PI assays. Involvement of HSP72 and pro-survival protein Bcl-2 were measured via flow-cytometry. The cells were administered with HSPIs and Bortezomib first as single agents for 24 hours, to establish working minimal concentration. Also, the drugs were tested for a shorter time, to understand when the drugs start to be effective. It emerged that one hour is sufficient for the drugs to give an initial effect in terms of cell viability and apoptosis. Following, combination experiments of HSPIs and Bortezomib were performed; the first drug was administered for one hour, the second following one hour and the cells were incubated for 24 hours. This was repeated alternatively for both type of drugs on the different cell lines. MTS and Annexin V\PI showed that there is not a synergistic effect between drugs, but instead there is antagonism. No necrosis was found at any level of the study. The cells were then probed for HSP72 and Bcl-2, to investigate their involvement in apoptosis mechanisms. Following 6 hours of combined and single agent treatment, both type of drugs inhibit HSP72 but failed to reduce the expression of Bcl-2, particularly on AML cells. It is thus proposed that CML and AML cells may die by apoptosis following a short time of treatment with HSPIs and Bortezomib by an extrinsic pathway of apoptosis, independent from Bcl-2 involvement and from mitochondrial pathway of apoptosis. This study may be the first to indicate a potential use of HSPIs and Bortezomib on CML and AML patients for a short time of treatment, although not in combination. Future studies are needed to further investigate the mechanisms of action of these drugs, aiming to potentially give CML and AML patients another successful therapy option to overcome resistance to canonic chemotherapy.

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Bodini, M. « Genomics of treatment response in Acute Myeloid Leukemia ». Doctoral thesis, Università degli Studi di Milano, 2017. http://hdl.handle.net/2434/469570.

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Acute Myeloid Leukaemia (AML) is a cancer of the myeloid lineage of blood cells characterised by rapid growth of undifferentiated myeloid precursors that accumulate in the bone marrow and suppress normal haematopoiesis. It is the most common adult leukaemia with an estimated number of more than 60’000 new cases for the US in 2016. Despite the high rates of complete remissions achieved after treatment (60-80% in young adults), the number of patient that will result cured after induction and consolidation therapy is very low (~12%). The molecular basis of relapsing disease is still unclear and the small number of identified predictive factors has small predictive power. To date, chemotherapy induction treatment is similar for all patients and consists in the administration of mainly three drugs (fludarabine, cytarabine, and idarubicin). Prediction markers for the outcome of chemotherapy would instead reduce useless treatments and direct research through new possible therapeutic targets that would enhance AML treatment. In three successive studies, Ding et al., Corces-Zimmerman et al. and Krönke et al., described four possible behaviours for relapse patients: the return of the first leukaemia (dominant clone or a subclone), with or without additional evolution, or the emergence of ancestral clones, also in this case, with or without additional mutations. In this thesis, endowed of the NGS technologies advancement, we decided to delineate the possible process of relapse formation in order to be able in the future to predict which patients are more susceptible to relapse. Our experimental plan includes the whole exome analysis of 30 pairs of primary/relapsed AML samples using NGS to identify relapse-specific mutations, the bioinformatics analysis of the clonal evolution of the disease and the identification of pathways that correlate with the relapsing disease. The methods for the analysis of NGS data, at present, are still in a refinement phase, especially for the high level analysis (detection of variants and definition of their role in the pathogenesis). We broadly analysed the existing methods for the treatment of NGS data (aligners, mutation callers, CNV callers and methods to reconstruct clonal composition) in order to determine those better fitting to our cohort of patients and purposes: occasionally, we had the possibility to choose the best tool meeting our investigative needs, discovering that other methods were valuable as well, in other cases we verified that more improvements are needed to obtain reliable results. Our analysis shows that the genomic landscapes of primary and relapse AMLs are similar and in the majority of the patients (76%) some relapse clones were already present in the primary tumour and reappeared after chemotherapy at similar or augmented cellular frequencies. We also identified some functional gene categories (DNA methylation pathway, cohesin complex and chromatin modifiers) more prone to resistance and peculiar genes (e.g. ASXL1, TET2) presenting growing VAFs at relapse. In 4 out of 29 patients (14%) we were able to identify driver mutations in the blood sample of the complete remission at low frequency; we hypothesise that more sophisticated diagnostic tools, based on NGS analysis, would help in driving the treatment to obtain better outcomes for patients.

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MALLARDO, MARIA. « DISSECTING THE ROLE OF THE CYTOPLASMIC MUTANT NUCLEOPHOSMIN IN ACUTE MYELOID LEUKAEMIA DEVELOPMENT ». Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/234152.

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Acute myeloid leukaemia (AML) is a genetic heterogeneous group of diseases, with the largest subgroup showing a mutation in the Nucleophosmin gene (NPM1). Normally the NPM protein localizes mainly in the nucleolus, but in AML blasts it is aberrant localized to the cytoplasm (NPMc+AML). Notably, NPMc+AML patients show peculiar gene expression profiles, treatment response and prognosis. Hence, it has been proposed as an independent category for leukaemia classification according to WHO in 2008. In view of the relevance of NPMc+ mutation to AML pathogenesis and prognosis, understanding its role in leukaemia development represents a major issue in the field. The aim of this PhD project is to get further insight into the relevance of NPMc+ mutations to AML development. To this scope, here it is reported a characterization of a novel mouse model expressing the mutated protein. The hematopoietic restricted expression of the protein induces leukaemia in mice. This data definitively clarify that NPMc+ is an initiating mutation for leukaemia development. However, the long latency and low penetrance of disease onset strongly support the need of cooperating mutations. Since, the high frequency of FLT3-ITD mutations in NPMc+AML, we genetically tested the synergisms between the two abnormalities. To this scope, NPMc+ mice were crossed with FLT3-ITD mice (Lee, 2007). Double mutated mice developed leukaemia with sort latency and full penetrance indicating effective cooperation. Moreover, our data support the two hits model of tumourigenesis, where functional complementary mutations contribute to disease onset. Another major challenge of this project is to understand how NPMc+ affect the biology of normal HSPC and imposes the transition from normal to cancer stem cells. We found that NPMc+ expression perturbs the homeostasis of HSCP and expand the number of LT-HSC by increasing the proliferation rate. However, this enhanced proliferation is not associated to loss of quiescent and functional HSC, which may represent a reservoir of persistent pre-malignant cells available for the accumulation of additional genetic alteration. Further investigation into the biology of per-leukaemic stem cells may give insights into the molecular mechanisms imposed by the oncogene for malignancy transformation and finally may contribute for the development of new therapeutic strategies.

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GILIOLI, DIEGO. « Dissecting the role of cellular senescence in acute myeloid leukaemia immune-surveillance and response to therapy ». Doctoral thesis, Università Vita-Salute San Raffaele, 2022. http://hdl.handle.net/20.500.11768/133076.

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Tzelepis, Konstantinos. « Identification of novel genetic vulnerabilities and therapeutic targets in acute myeloid leukaemia using CRISPR dropout screens ». Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/271130.

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Acute myeloid leukaemia (AML) is an aggressive cancer with a poor prognosis, for which mainstream treatments have not changed for decades. To identify novel therapeutic targets in AML, I have optimized a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening platform and use it to identify genetic vulnerabilities in AML cells. This work led to the identification of 492 AML-specific cell-essential genes, including several established therapeutic targets such as that represent new clinically actionable candidates. I have validated selected genes using DOT1L, BCL2, MEN1 and many other genes genetic and pharmacological inhibition, and chose candidates for downstream studies. Both the epigenetic modifier KAT2A and SRPK1 as promising KAT2A and spliceosome kinase SRPK1 inhibition demonstrated anti-AML activity by inducing myeloid differentiation and apoptosis, and suppressed the growth of primary human AMLs while sparing normal hemopoietic stem-progenitor cells. My findings propose that KAT2A and SRPK1 inhibition should be investigated as new therapeutic strategies in AML and also provide a large number of novel genetic vulnerabilities of this leukaemia that can be pursued in downstream studies. As these screens were performed in immortalised AML cell lines, I then went on to develop a method for the performance of dropout screens in genotypically-defined primary murine AMLs developed in our lab and arising in Cas9-expressing mice. Through this work, I successfully carried out the first such screen in AML cells driven by mutant Npm1 (NPM1c) and Flt3-ITD, the commonest two-mutation combination in human AML. Downstream analysis of the results revealed the excellent potential of this type of screen and enabled me to investigate the molecular effects on mutant Npm1, which are currently poorly understood. Overall, my results demonstrate that unbiased CRISPR dropout screens can identify novel therapeutic targets in cancer while, in parallel, revealing novel biological insights.

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Mortera, Blanco Teresa. « Development of an ex vivo three dimensional (3-D) model of acute myeloid leukaemia (AML) ». Thesis, Imperial College London, 2008. http://hdl.handle.net/10044/1/4390.

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Acute Myeloid Leukaemia (AML) is a cancer of hematopoietic stem cells that develops in the three-dimensional (3-D) niches provided by the bone marrow microenvironment in vivo. The study of AML has been hampered by the lack of appropriate ex vivo models, which can mimic this microenvironment. It was hypothesised that the fabrication of scaffolds for the biomimetic growth of leukemic cells ex vivo could facilitate the study of the disease in its native 3-D niche. The growth of different leukemic cell lines was first evaluated, namely K- 562, HL-60 and Kasumi-6 on highly porous scaffolds fabricated from biodegradable and non-biodegradable polymeric materials: poly (L-lactic-co-glycolic acid) (PLGA), polyurethane (PU), poly (methyl-methacrylate) (PMMA), poly (D, L-lactade) (PDLLA), poly (caprolactone) (PCL), and polystyrene (PS). These results were compared with two commercially available scaffolds from BD™ Biosciences. Overall, out of all the scaffolds, PLGA and PU displayed the best seeding efficiency and leukemic cellular growth, assessed by MTS assay, scanning electron microscopy and immunohistochemistry. In order to improve the ex vivo 3-D leukemic cell culture, PLGA and PU scaffolds were coated with bone marrow extracellular matrix (ECM) proteins, collagen (62.5 or 125 μg/ml) and fibronectin (25 or 50 μg/ml) and a combination of both proteins: collagen + fibronectin (62.5 + 25 μg/ml) respectively. Once the abnormal hematopoietic 3-D model was established, a new model to culture normal hematopoietic cord blood mononuclear cells was studied and compared. All 3 leukemic cell lines and cord blood cells grew better in PU scaffolds coated with collagen type I using the low concentration and sustained growth in the absence of exogenous cytokines. As a result, it was concluded that PU-collagen scaffold could provide a practical model with which to study the biology and treatment of primary AML in an ex vivo mimicry without the use of animal models.

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Traikov, Sofia. « Loss of heterozygosity in acute myeloid leukaemia with normal karyotype ». Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2009. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-25082.

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Loss of heterozygosity (LOH) is detectable in many forms of cancer including leukaemia. It contributes to tumorigenesis through the loss of one of the two alleles of one tumor suppressor gene at a given locus, caused by deletion or uniparental disomy (UPD). UPD can only be the result of homologous recombination. Little is known about the mechanisms of UPD and what connection this aberration has with the outcome of this disease. In this study, 146 patients with primary AML were analysed using a novel technique based on single nucleotide polymorphisms (SNPs). Leukaemic cells and healthy T-cells from each patient were obtained using FACS-Vantage cell sorting. In cases with very few sorted cells whole genome preamplification was done. Genome-wide SNP analysis was carried out according to the standard GeneChip Mapping Assay protocol (Affymetrix, USA) using the Human Mapping 10K Arrays. Moreover, the impact of the FLT3-ITD mutation on the homologous recombination using pmHPRT-DRGFP /pCbASce vectors system and yHA2x assay was investigated. Of 146 patients with normal karyotype LOH was found in 30 cases. The potential LOH regions, were confirmed by microsatellite analysis of short tandem repeat (STR) markers. In 21 of these cases STR-analysis of T-cells, representing the corresponding tumor-free material, confirmed the regions of partial UPD. This aberration affected different chromosomes, but most commonly chr. 2, 6, 11, 21, 13, and 7, and covered between 11.5 and 88 Mb. Interestingly, in 6 LOH cases, long stretches of homozygosity present at the same positions as in the healthy cells and in the blasts were found. The impact of this phenomenon is unknown. Additionally, chromosome losses were detected in 3 patients classified with normal karyotype according to current methods. These 9 cases were not included in the UPD positive group. No differences were observed regarding any clinical factors including age, WBC-counts and sex. The FAB M1 subtype was observed in 47.6% of the UPD positive patients, compared to only 19.2% of the UPD negative patients (P=0.04, n=146). In addition, no correlation between FLT3-ITD, MLL-PTD and NPM1 mutations in the UPD patients was found, but the data indicate that patients with UPD have a higher rate of treatment failure. Moreover, in this study the relationship between UPD and gene aberrations was able to be confirmed. In some cases, UPD found on chromosomes 21, 19 and 11 was correlated with mutations in the RUNX1, CEBPA and WT genes, respectively. Furthermore, AML cases with and without UPD showed different but specific gene expression profiles, revealing different expression levels for genes involved in double strand break repairs. Furthermore, it was found that different mutations could be responsible for the increase in efficiency of HR, such as FLT3-ITD or BCR-ABL. Moreover, cells with a FLT3-ITD mutation (without wt expression) rapidly increased the HR efficiency compared with heterozygous (FLT3-ITD/wt) cells. Preliminary results showed that the high repair efficiency was mainly dependent on the translocation of RAD51. In conclusion, SNP array technology allow the identification and mapping of LOH in AML patients with normal karyotype. The obtained data also point out the necessity of analysing tumour-free material to confirm the somatic origin of the alteration. Furthermore, the available results indicate that compared to patients without UPD, patients with UPD have a higher relapse rate, which might be used as a prognostic marker in the future. Also, it could be hypothesized that downregulation of RAD51 (for example by FLT3 inhibition) might be beneficial DNA damage occurs through the genotoxic agent by reducing the relapse risk of AML.

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Saia, M. « GENOME-WIDE ANALYSIS OF TRANSCRIPTION FACTOR NETWORKS IN MYELOID DIFFERENTIATION AND ACUTE MYELOID LEUKAEMIA ». Doctoral thesis, Università degli Studi di Milano, 2013. http://hdl.handle.net/2434/219072.

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One of the most frequent genetic abnormalities underlying the pathogenesis of acute myeloid leukaemia (AML) is a reciprocal translocation between chromosomes 8 and 21 which results in the generation of a chimeric gene that encodes for the AML1/ETO fusion protein. AML1/ETO has the capacity to block myeloid differentiation, thus leading to the accumulation of immature precursor cells. The fusion protein functions as an aberrant DNA binding transcription factor, therefore altering the normal gene expression profile of the cell. Moreover, it interacts with a number of other transcription factors involved in myeloid differentiation. In this thesis, a murine haematopoietic stem/precursor cell line was exploited to study AML1/ETO functions. The binding pattern of AML1/ETO was investigated by ChIP-sequencing and correlated to the binding profiles of AML1, PU.1 and C/EBPα transcription factors. The distribution of three histone marks (H3K4me1, H3K4me3 and H3K27ac) upon expression of AML1/ETO was also analysed. The results showed that AML1/ETO expression is associated with modifications in the binding profile of the three transcription factors and in the distribution of chromatin marks, that changes in gene expression are associated with such modifications, and more rarely, with AML1/ETO binding. High-throughput profiling of miRNAs expression revealed the presence of two miRNAs up-regulated by AML1/ETO, miR-322* and miR-351, which are likely candidates for having a role in the block of differentiation, since their inhibition restores the ability of AML1/ETO-expressing cells to differentiate. Analysis of the AML1/ETO-associated gene expression profile predicted a modulation of the cell motility and cell localization. To further explore these functions, in vitro migration assays and in vivo homing experiments were performed. These assays confirmed that AML1/ETO endows the cell with an enhanced motility and an impaired homing of cells to haematopoietic organs. Bioinformatics analysis of AML1/ETO target genes and their regulatory regions revealed that a modification in the intensity of AML1, PU.1 and C/EBPα binding correlates with changes in gene expression. Sequence analysis showed that STAT6 and other STAT factors could synergize with AML1/ETO in the regulation of gene expression. Taken together, these results describe how AML1/ETO expression influences its target genes and how the three transcription factors included in the study, chromatin modifications, microRNAs, and putative co-regulators are involved in such regulation. In addition, alterations in cell motility and localization, functions not yet described for AML1/ETO, were identified.

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Woodward, Eleanor. « The use of gene expression profiling to identify novel minimal residual disease markers (MRD) in acute myeloid leukaemia (AML) ». Thesis, Cardiff University, 2010. http://orca.cf.ac.uk/55179/.

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Acute myeloid leukaemia (AML) is a heterogeneous disorder characterised by the accumulation of immature haematopoietic cells blocked at various stages of differentiation. Despite improved survival rates over the past decade, relapse occurs in approximately 70% patients undergoing chemotherapy. A potential reason for this is that current clinical protocols do not take account of the level of residual disease present at remission. Therefore, one strategy to reduce relapse rates is to monitor minimal residual disease and continue to treat until the patient is minimal residual disease negative. Current minimal residual disease markers are available for patients with characterised fusion genes but approximately 50% of patients have no detectable chromosomal aberration and therefore are without markers. Gene expression profiling is a powerful tool for disease classification, prognosis and therapeutic predictions. This study aimed to investigate the use gene expression profiling to identify novel minimal residual disease markers for specific AML sub-groups. Patient diagnostic samples were profiled to identify genes specific to AML patients with a favourable translocation in order to establish the "proof-of-principle". Several genes identified were followed in patient diagnostic and follow- up samples and compared to the markers currently used. Continuing with normal karyotype AML, genes were identified as specific to this sub-group. Several homeobox (HOX) genes and the Wilms' tumour (WT1) gene were identified and their MRD levels followed in diagnostic and follow-up samples. Only WT1 identified as specific to normal karyotype AML met the necessary criteria to be an MRD marker. Although the majority of genes selected from the GEP in this study proved unsuitable as markers, the identification and validation of a marker already used for MRD monitoring, WT1, demonstrates the ability of gene expression profiling to identify potential minimal residual disease markers in normal karyotype AML.

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Forster, Victoria Jane. « AML1/ETO promotes a mutator phenotype in t(8;21) acute myeloid leukaemia ». Thesis, University of Newcastle Upon Tyne, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.606731.

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The AM L1/ETO fusion protein in t(8;21) acute myeloid leukaemia is not sufficient for leukaemogenesis, but requires the acquisition of additional co-operating mutations. The "mutator phenotype" model of cancer suggests that the normal mutation rate in human somatic cells is not sufficient to mediate transformation, and that initiating mutations that increase the mutation rate are an essential "driver." As such, the major hypothesis of this thesis is that AML1/ETO confers a mutator phenotype and predisposes cells to the acquisition of co-operating mutations. Using cell lines transduced with the AML1/ETO fusion gene, investigations described within this thesis show that AML1/ETO confers a mutator phenotype, predisposing cells to acquisition of mutations at the TK and PIGA gene loci. This increase in mutation frequency in AML1/ETO-transduced cells is observed both spontaneously and after treatment with genotoxic agents, such as ionising radiation or the chemotherapy agent doxorubicin. Disruption of carefully controlled DNA repair mechanisms is well described to increase propensity to the acquisition of mutations. The effect of AML1/ETO on DNA repair in ectopic AML1/ETO-positive cell line models was investigated revealing dysregulated expression of several DNA repair-associated genes. The DNA base-excision repair pathway was notably affected, including downregulation of the OGGl DNA glycosylase, which is primarily responsible for the repair of 7,8-dihydro-8-oxoguanine lesions in DNA. The role of AML1/ETO in modulating angiogenic regulatory factors, was also investigated in ectopic AML1/ETO-transduced cell lines and t(8;21)-positive patient-derived cell lines. AML1/ETO was found to substantially upregulate the angiogenic regulatory factor ANGPTl. The study of dysregulated angiogenesis in leukaemia is a rapidly expanding field of research, and the investigations presented on AML1/ETO and ANGPT1, suggest that ANGPTl may be a possible therapeutic target in t(8;21) AML. The work presented in this thesis has provided new information as to how the AML1/ETO fusion protein promotes the acquisition of co-operating mutations leading to leukaemic transformation, and identified a 'possible role for AML1/ETO as a regulator of angiogenesis. This information could provide the basis for development of new methods of therapeutic intervention in t(8;21) AML.

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MICHELOZZI, ILARIA MARINA. « The role of the bone marrow microenvironment in aplastic anaemia and acute myeloid leukaemia : from pathogenesis to chemoresistance ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2019. http://hdl.handle.net/10281/241079.

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La nicchia staminale ematopoietica è formata da molteplici tipi cellulari che contribuiscono alla regolazione dell’ematopoiesi all’interno del midollo osseo. In alcuni disordini ematopoietici il microambiente midollare risulta alterato, sebbene il suo contributo alla patogenesi non sia ancora chiaro, fornisce supporto alle cellule staminali leucemiche (LSC), a spese delle normali cellule staminali ematopoietiche, e protezione contro i chemioterapici. Dunque, per eradicare le LSC, le strategie terapeutiche devono considerare l’effetto protettivo contro i chemioterapici esercitato dal microambiente midollare oltre alla refrattarietà intrinseca delle LSC alle convenzionali terapie. Nella prima parte di questo progetto di dottorato, abbiamo valutato il ruolo delle cellule stromali mesenchimali (MSC) nella patogenesi dell’anemia aplastica (AA). Le MSC derivate dai pazienti (AA-MSC) sono state caratterizzate in vitro e, soprattutto in vivo, utilizzando il modello di nicchia midollare in vivo recentemente sviluppato dal nostro gruppo di ricerca, basato sull’impianto sottocutaneo di pellet cartilaginei derivati dalle MSC (Serafini et al., Stem Cell Research, 2014). In vitro, le AA-MSC mostravano solo un ridotto potenziale clonogenico senza difetti morfologici, fenotipici, proliferativi e differenziativi. Inoltre, le AA-MSC erano in grado di ricreare una nicchia midollare funzionale in vivo dimostrando un’inalterata capacità di supportare l’ematopoiesi e l’assenza di un loro coinvolgimento nella patogenesi della AA. Nella seconda parte di questo dottorato, abbiamo analizzato gli effetti dell’asparaginasi (ASNase) sulle cellule derivate da pazienti affetti da leucemia mieloide acuta (AML), soprattutto sulle LSC, ed il contributo del microambiente alla resistenza ai chemioterapici. L’ASNase era egualmente efficace contro il bulk delle cellule AML e contro le LSC (CD34+CD38- e CD34+CD38+), mentre l’effetto nei confronti delle cellule ematopoietiche sane era trascurabile. L’azione del farmaco contro le cellule primitive di AML è stato ulteriormente confermato in saggi clonogenici ed in esperimenti effettuati in condizioni di coltura specifiche per il mantenimento delle LSC in vitro. Tuttavia, una nicchia midollare protettiva formata dalle MSC, che esprimono asparagina sintetasi, dai monociti/macrofagi e dai blasti leucemici stessi, che esprimono catepsina B, potrebbe ridurre il potenziale anti-neoplastico del farmaco. Infine, abbiamo testato un nuovo regime di condizionamento (somministrazione di fludarabina in aggiunta all’irraggiamento) in topi SCID-beige, un ceppo poco permissivo per l’engraftment umano. Tale strategia di condizionamento ha incrementato i livelli di engraftment della linea cellulare AML, KG-1, rispetto al solo irraggiamento, e ha permesso di ottenere l’engraftment nel 50% dei blasti AML primari trapiantati. In futuro, vorremmo combinare questo nuovo modello di xenograft così ottenuto con il trapianto di pellet cartilaginei per generare un modello di nicchia AML da poter usare per studiare il microambiente midollare leucemico e per testare promettenti agenti terapeutici contro l’AML, come l’ASNase.
The haematopoietic stem cell (HSC) niche is formed by several cell types which contribute to the regulation of the haematopoiesis within the bone marrow (BM). In pathological conditions, alterations of the BM microenvironment have been reported, but it is still debated whether they are cause or consequence of the disease. Support to leukaemic cells, especially to leukaemic stem cells (LSC), at the expense of normal HSC and protection against chemotherapeutic agents characterise the malignant BM niche. Thus, in order to eradicate LSC, promising therapeutic strategies may consider the chemoprotection exerted by the BM microenvironment in addition to LSC intrinsic features of refractoriness to conventional therapies. In the first part of this PhD project, we investigated the role of mesenchymal stromal cells (MSC) in the pathogenesis of aplastic anaemia (AA). Patient-derived MSC (AA-MSC) were characterised in vitro and, especially, in vivo taking advantage of our recently described in vivo BM niche model, based on the subcutaneous implantation of cartilaginous pellets generated from MSC (Serafini et al., Stem Cell Research, 2014). AA-MSC did not exhibit morphological, phenotypical, proliferative and differentiation defects in vitro. Only a reduced clonogenic potential was observed. Most importantly, they were able to recreate a functional and complete BM niche in vivo, proving their unaltered ability to support haematopoiesis and excluding their involvement in AA pathogenesis. In the second part of this PhD project, we analysed the effects of L-asparaginase (ASNase) on acute myeloid leukaemia (AML) cells, especially on LSC, and the contribution of the microenvironment to chemoresistance. ASNase was equally effective against unfractionated AML cells and against CD34+CD38- and CD34+CD38+ LSC, while slightly affecting healthy haematopoietic cells. The action of the drug against AML primitive cells was confirmed by clonogenic assays and by experiments performed in LSC supportive culture conditions. However, the anti-leukaemic potential of ASNase could be in part counteracted by MSC, expressing asparagine synthetase, and by monocytes/macrophages and blasts themselves, expressing cathepsin B, generating a protective niche. Lastly, we tested a new conditioning regimen based on the addition of fludarabine to irradiation in SCID-beige mice, a poorly permissive strain for human engraftment. The administration of fludarabine after irradiation increased the levels of engraftment of a AML cell line, KG-1, as compared to irradiation only, and it allowed the engraftment in 50% of primary AML blasts transplanted, reproducing human leukaemic infiltration. Thus, we generated a novel AML xenograft model in which we would like to test promising therapeutic agents, as ASNase, and to study different features of the malignant BM niche.

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Thomas, Karla Mari. « Interim analysis of Acute Myeloid Leukaemia treated on the Red Cross Children's Hospital Rx 2071 (adapted from the MRC AML 15 protocol) ». Master's thesis, University of Cape Town, 2017. http://hdl.handle.net/11427/27378.

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Background: Due to the poor outcomes achieved in acute myeloid leukaemia (AML) treatment, the Red Cross War Memorial Children's Hospital (RCWMCH) Oncology service changed from a BFM-87 based protocol to one based on MRC-AML15 in 2007. Rationale: This study was designed to assess the outcomes and treatment - related toxicity among children treated with RCWMCH protocol Rx 2071. Methods: This was a retrospective review of AML patients treated with Rx2071 between 2007 and 2012 at RCWMCH. Patients with acute promyelocytic leukaemia (APL) and Down Syndrome were excluded. Risk was assigned by cytogenetics. Good risk patients were those with t(8;21), t(16,16) and inv(16). Poor and standard risk included all other cytogenetics according to MRC-AML15. Data pertaining to toxicity was obtained from patient folders. Results: Thirty five children were treated on Rx 2071 during the study period. Males comprised 51.4% (18/35) and females 48.6% (17/35). Age at diagnosis ranged from 0.33 to 12.51 years with the median being 5.68 years. Follow-up from remission in the patients who survived ranged from 1 year 10 months to 9 years 1 month with a median of 62.5 months. Fifteen patients had favourable cytogenetics. Event free survival (EFS) for the good risk group was 85.6%. Twenty patients presented with standard/poor risk cytogenetics. Five patients were deemed poor risk with one having major karyotype abnormalities and four not achieving remission. The remaining fifteen were deemed standard risk by cytogenetics. EFS in this group was 32.4%. Two standard/poor risk patients were transplanted in first complete remission (CR1) and two patients were transplanted in second complete remission. (CR2) Patients had a median of four neutropaenic fevers, and required a median of eight packed cell and eleven platelet transfusions. There were 39 positive blood cultures. There were no chemotherapy related deaths. Discussion: The EFS for good risk patients is excellent but the EFS for standard/poor risk group is not on par with results being achieved in high income countries. The toxicity is not excessive on Rx2071. The results achieved on this protocol were superior to that of the previous BFM- based protocol. Conclusion: The results of this study support the continued use of Rx2071 at RCWMCH.

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Thomas, Karla Mari. « Interim analysis of Acute Myeloid Leukaemia treated on the Red Cross Children's Hospital Rx 2071 (adapted from the MRC AML 15 protocol) ». Master's thesis, University of Cape Town, 2002. http://hdl.handle.net/11427/27378.

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Background: Due to the poor outcomes achieved in acute myeloid leukaemia (AML) treatment, the Red Cross War Memorial Children's Hospital (RCWMCH) Oncology service changed from a BFM-87 based protocol to one based on MRC-AML15 in 2007. Rationale: This study was designed to assess the outcomes and treatment - related toxicity among children treated with RCWMCH protocol Rx 2071. Methods: This was a retrospective review of AML patients treated with Rx2071 between 2007 and 2012 at RCWMCH. Patients with acute promyelocytic leukaemia (APL) and Down Syndrome were excluded. Risk was assigned by cytogenetics. Good risk patients were those with t(8;21), t(16,16) and inv(16). Poor and standard risk included all other cytogenetics according to MRC-AML15. Data pertaining to toxicity was obtained from patient folders. Results: Thirty five children were treated on Rx 2071 during the study period. Males comprised 51.4% (18/35) and females 48.6% (17/35). Age at diagnosis ranged from 0.33 to 12.51 years with the median being 5.68 years. Follow-up from remission in the patients who survived ranged from 1 year 10 months to 9 years 1 month with a median of 62.5 months. Fifteen patients had favourable cytogenetics. Event free survival (EFS) for the good risk group was 85.6%. Twenty patients presented with standard/poor risk cytogenetics. Five patients were deemed poor risk with one having major karyotype abnormalities and four not achieving remission. The remaining fifteen were deemed standard risk by cytogenetics. EFS in this group was 32.4%. Two standard/poor risk patients were transplanted in first complete remission (CR1) and two patients were transplanted in second complete remission. (CR2) Patients had a median of four neutropaenic fevers, and required a median of eight packed cell and eleven platelet transfusions. There were 39 positive blood cultures. There were no chemotherapy related deaths. Discussion: The EFS for good risk patients is excellent but the EFS for standard/poor risk group is not on par with results being achieved in high income countries. The toxicity is not excessive on Rx2071. The results achieved on this protocol were superior to that of the previous BFM- based protocol. Conclusion: The results of this study support the continued use of Rx2071 at RCWMCH.

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Tinsley, S. P. « To establish the role of mutations in c-KIT tyrosine kinase in the pathogenesis and therapy of core-binding factor-related acute myeloid leukaemia (AML) ». Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1433249/.

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Haematopoiesis is controlled by complex signal transduction pathways and transcription regulators which may become dysregulated in acute myeloid leukaemia (AML). Activating mutations in FLT3 and c-KIT receptor tyrosine kinases (RTKs) are commonly found in AML and can impact on prognosis. Different types of FLT3 mutations are known to have distinct biological activities and prognostic implications. The presence of c-KIT mutations has been shown to increase the risk of relapse, but there has been no direct comparison of the biological activity of AML specific c-KIT mutations occurring in different receptor domains. In addition to prognostic information, RTKs are attractive potential targets for therapy using small molecule inhibitors. To evaluate their biological activity and response to targeted therapies, human UT-7 cells were transduced with wild-type and mutant c-KIT isoforms - c-KIT-Δ417-419>Y (extracellular domain [ECD]), c-KIT-L576P (juxtamembrane domain [JMD]), c-KIT-D816V and c-KIT-N822K (both in the activating loop domain [ALD). There were intrinsic differences in signal strength between the mutants examined - only c-KIT-Δ417-419>Y and c-KIT-D816V expressing cells had detectable constitutive c-KIT activation and showed ligand-independent growth. The response of transduced UT-7 cells to c-KIT inhibitors was assessed by treating the cells with dasatinib and masitinib. Cells expressing ALD c-KIT mutations were more resistant to dasatinib or masitinib-mediated cell killing in comparison to cells expressing c-KIT mutations in the ECD and JMD. Western blotting revealed that although c-KIT phosphorylation was potently inhibited, there was residual mTOR and/or PI3K/AKT activation in these cells. The resistance to dasatinib observed in c-KIT-D816V or c-KIT-N822K expressing cells could be overcome by co-blockade of c-KIT and PI3K/mTOR and blockade of c-KIT and PI3K/mTOR was synergistic in all c-KIT mutant cell lines at inducing cell death. Blockade of FLT3 and PI3K/mTOR in FLT3-ITD AML cell lines also showed similar results. During the screening of AML cell lines for c-KIT and FLT3 mutations a novel FLT3-T820N point mutation was identified in the ME-1 cell line. Expression of FLT3-T820N in 32D cells constitutively activated FLT3 and conferred ligand-independent growth. 32D FLT3-T820N cells were most sensitive to the FLT3 inhibitor AC220 with regard to growth inhibition, cell killing and decreased phosphorylation of FLT3 compared to FLT3-WT and FLT3-D835Y expressing cells. This work highlights the differences in biological outcomes and TK inhibitor-sensitivity between different RTK mutants found in AML and shows that simultaneous blockade of RTKs and PI3K/mTOR may provide a novel therapeutic approach for specific AML subtypes.

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Pyzer, Athalia Rachel. « Myeloid-derived suppressor cells in acute myeloid leukaemia ». Thesis, Queen Mary, University of London, 2017. http://qmro.qmul.ac.uk/xmlui/handle/123456789/36704.

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The tumour microenvironment consists of an immunosuppressive niche created by the complex interactions between cancer cells and surrounding stromal cells. A critical component of this environment are myeloid-derived suppressor cells (MDSCs), a heterogeneous group of immature myeloid cells arrested at different stages of differentiation and expanded in response to a variety of tumour factors. MDSCs exert diverse effects in modulating the interactions between immune effector cells and malignant cells. An increased presence of MDSCs is associated with tumour progression, poorer outcomes, and decreased effectiveness of immunotherapeutic strategies. In this project, we sought to quantify and characterise MDSC populations in patients with Acute Myeloid Leukaemia (AML) and delineate the mechanisms underlying their expansion. We have demonstrated that immune suppressive MDSCs are expanded in the peripheral blood and bone marrow of patients with AML. Furthermore, AML cells secrete extra-cellular vesicles (EVs) that skew the tumour microenvironment from antigen-presentation to a tumour tolerogenic environment, through the expansion of MDSCs. We then demonstrated that MDSC expansion is dependent on tumour and EV expression of the oncoproteins MUC1 and c-Myc. Furthermore, we determined that MUC1 signalling promotes c-MYC expression in a microRNA (miRNA) dependent mechanism. This observation lead us to elucidate the critical role of MUC1 in suppressing microRNA-genesis in AML, via the down-regulation of the DICER protein, a key component of miRNA processing machinery. Finally, exploiting this critical pathway, we showed that MDSCs can be targeted by MUC1 inhibition or by the use of a novel hypomethylating agent SGI-110.

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Knapper, Steven. « FLT3 inhibitors in acute myeloid leukaemia ». Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.432548.

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Smith, Matthew Liam Walker. « Mutation profiling in acute myeloid leukaemia ». Thesis, Queen Mary, University of London, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.416112.

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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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Dakik, Hassan. « Caractérisation des NADPH oxydases et effet de leur inhibition dans les leucémies aigues myéloïdes ». Thesis, Tours, 2017. http://www.theses.fr/2017TOUR3309.

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Dans le monde, 350 000 leucémies sont diagnostiquées chaque année. La rechute reste un problème majeur des leucémies aiguës myéloïdes (LAM) et le métabolisme oxydatif pourrait jouer un rôle essentiel dans la réponse au traitement. Un faible niveau des espèces réactives de l’oxygène (ROS) est associé à des propriétés des cellules souches leucémiques et la quiescence alors qu’un niveau plus élevé caractérise les leucoblastes proliférants. L’homéostasie des ROS repose sur un équilibre entre les systèmes oxydants et antioxydants. Les antioxydants sont bien documentés dans les LAM alors que les connaissances sur l’activité oxydante sont encore limitées. Dans ce travail nous avons choisi d’étudier les sept complexes NADPH oxydases (NOX) dans 25 lignées issues de LAM humaines et des LAM primaires. L’analyse des ARNm et des protéines montre des profils d’expression variables entre les lignées avec une expression plus forte des sous-unités du complexe NOX2 dans les lignées correspondant à des stades de différenciation myéloïde plus avancés. L’activité enzymatique des NOX est cependant équivalente entre les lignées. Deux inhibiteurs, DPI et VAS3947, ont été utilisés pour connaître la contribution des NOX à la production des ROS cellulaires. Alors qu’ils ont inhibé l’activité, ils ont aussi généré un stress oxydatif majeur conduisant à une diminution de la prolifération cellulaire et une forte apoptose, le DPI en augmentant les ROS mitochondriaux et VAS3047 les ROS cytoplasmiques. Afin de connaitre les sous-unités impliquées et de mieux comprendre les mécanismes, les sous-unités NOX2 et p22phox ont été inhibée par ARN interférence. Celle-ci n’ont pas affecté la prolifération mais ont montré des effets compensatoires. Nos data montrent qu’inhiber les NOX pourrait s’avérer une stratégie thérapeutique en augmentant le stress oxydatif dans les cellules leucémiques
350,000 leukaemia are diagnosed each year worldwide. In acute myeloid leukaemia (AML), relapse remains a major problem and the oxidative metabolism might play a crucial role in the therapeutic response. Low level of reactive oxygen species (ROS) is associated with properties of leukemic stem cells and quiescence whereas higher level promotes leukoblasts proliferation. ROS homeostasis relies on a tightly regulated balance between the oxidant and antioxidant systems. Although the antioxidant system is extensively studied in AML, the oxidant system remains poorly documented. In this work we aimed to study the seven NADPH oxidases (NOX) complexes in 25 AML human cell lines and primary samples. NOX transcriptional and protein profiles are variable with a higher expression of NOX2 in cell lines belonging to mature differentiation stages. An equivalent level of enzymatic activity was observed across all the cell lines. To reveal the contribution of NOX to global ROS production in the cells, two NOX inhibitors, DPI and VAS3947, were then used. Although both inhibitors efficiently blocked NOX activity they unexpectedly triggered strong oxidative stress leading to reduced cell proliferation and strong apoptosis, DPI by increasing mitochondrial ROS while VAS3947 by increasing cytoplasmic ROS production. To highlight which of the subunits were involved and to understand the mechanisms, NOX2 and p22phox subunits were inhibited using shRNA strategy. These did not affect cell proliferation but revealed a compensation effect. Our data suggest that NOX inhibition might be potential therapeutic strategy by increasing oxidative stress in leukemic cells

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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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Olwill, Shane. « Annexin II expression in acute myeloid leukaemia ». Thesis, University of Ulster, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274092.

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Armenteros-Monterroso, Elena. « Investigating reptin function in acute myeloid leukaemia ». Thesis, University College London (University of London), 2017. http://discovery.ucl.ac.uk/10038392/.

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Acute myeloid leukaemia (AML) is a disease characterized by the clonal expansion of immature white blood cells, which show increased proliferation, self-renewal and a block of differentiation. Despite recent advances in therapy, AML still causes over half of all leukaemia related paediatric deaths, as cytogenetically defined subgroups with poor prognosis are still prevalent. Chromosomal translocations, which encode abnormal fusion proteins, are common in patients with AML, and the MLL (Mixed Lineage Leukaemia) locus is the most frequently rearranged in paediatric AML. Previous studies in our laboratory used global gene expression analysis in conditionally immortalized MLL-rearranged mouse myeloid cells to demonstrate that Reptin was positively regulated by MLL-fusion genes. Reptin (also known as RUVBL2 or Tip48), functions as part of multi-protein complexes involved in chromatin remodelling, DNA repair, regulation of transcription and ribonucleoprotein assembly. Further work in our laboratory found Reptin to be essential for sustaining the hyperproliferative state and clonogenic potential, as well as suppressing apoptosis, of human AML cells, both MLL-rearranged and non-MLL rearranged. The aim of this study was to investigate the transcriptional pathways regulated by Reptin in human AML and to establish the efficacy of targeting Reptin in vivo. By using an inducible shRNA model to deplete Reptin expression we demonstrate that Reptin is essential for leukaemic progression in vivo, as Reptin knockdown in established human leukaemias resulted in increased survival and the cure of most xenotransplanted mice. Moreover, our analyses of global gene expression data in cells depleted for Reptin expression at different time points indicated that Reptin depletion is negatively correlated with the Leukaemic Stem Cell self-renewal signature. Furthermore, our gene expression results also indicated that Reptin modulates the expression signature of the transcription factors c-MYC and c-MYB, master regulators of survival and self-renewal pathways in AML. Additionally, immunoprecipitation assays identified a novel interaction between endogenous Reptin and c-MYB, and ChIP assays showed decreased binding of c-MYB and the epigenetic mark H3K27ac at the promoter region of the c-MYB target gene MPO after Reptin loss. Collectively, our data identify a new pathway modulated by Reptin and confirm that Reptin is a good therapeutic target for the treatment of AML.

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Stringaris, Katherine. « Natural killer cells and acute myeloid leukaemia ». Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/18014.

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Despite successful induction chemotherapy, most patients with acute myeloid leukemia (AML) will relapse. Immune surveillance by T cells or natural killer (NK) cells may play a role in preventing relapse. This thesis examines the potential of NK cells to control AML. Study 1 explored in 248 patients with haematological malignancies from the US National Institutes of Health, the genetic diversity of NK killer immunoglobulin receptor (KIR) genes in patients and their stem cell donors and their impact on outcome after stem cell transplantation (SCT) for haematological malignancy. Individuals with AML receiving SCT from donors inheriting 3 particular B-haplotype KIRs were 4 times less likely to relapse than those with donors without these favourable KIRs. Study 2 explored in samples obtained from 499 patients enrolled on UK MRC/NCRI AML trials, whether KIR genotype affects the risk of developing AML or the outcome of remission induction chemotherapy. While KIRs had no effect on the development or outcome of de novo AML, individuals with more activatory KIRs, in particular 2DS2, developed significantly less secondary AML, suggesting that activatory KIRs can protect against secondary AML. These studies support a role for NK-mediated immune surveillance in AML. Study 3 investigated the phenotype and function of AML NK cells. In 32 prospectively collected samples from AML patients undergoing remission induction chemotherapy at the Hammersmith Hospital, AML patients were found to have reduced NK activatory receptors, increased NK inhibitory receptors, and reduced cytotoxic function towards leukaemia, compared to healthy donors. These abnormalities corresponded with failure to achieve remission and can be induced in normal NK incubated with AML blasts. I conclude that KIR genetics have a limited influence on AML development and outcome but that AML itself can impair NK function, reducing the chance of achieving remission. These findings have implications for NK based immunotherapy for AML.

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Mannari, Deepak. « The genomics of acute myeloid leukaemia : an investigation into the molecular pathogenesis of acute myeloid leukaemia with t(8;21) ». Thesis, Queen Mary, University of London, 2012. http://qmro.qmul.ac.uk/xmlui/handle/123456789/8822.

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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.

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Nagura, Eiichi, Saburo Minami, Koichiro Nagata, Yoshihisa Morishita, Hideo Takeyama, Hiroshi Sao, Hisamitsu/ Suzuki et al. « Acute myeloid leukemia in the elderly : 159 Nagoya case studies ». Nagoya University School of Medicine, 1999. http://hdl.handle.net/2237/5348.

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Morgan, Rhys. « Role of γ-catenin in acute myeloid leukaemia ». Thesis, Cardiff University, 2011. http://orca.cf.ac.uk/55145/.

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Acute myeloid leukaemia (AML) is a heterogeneous clonal disorder of haematopoietic cells that primarily affects the elderly. Previously, our laboratory identified y-catenin as significantly overexpressed in AML. y-Catenin shares close structural and functional homology with the more intensively studied /3-catenin. Both catenins have dual roles in cell adhesion complexes and in transcription. Their transcriptional role is regulated by Wnt signalling which is critical for normal development and is one of the most frequently dysregulated processes in AML. In spite of this, little is known regarding the specific role of y-catenin in normal haematopoiesis or AML pathology. This study devised an intracellular flow cytometric staining assay to characterise the expression of y-catenin in normal haematopoietic subsets. y-Catenin exhibited a similar expression profile to /3-catenin. Expression was relatively high in haematopoietic stem/progenitor cells (HSC/HPC) and showed increased expression in myeloid differentiated cells (granulocytes and monocytes) while expression was lower in lymphoid cells and undetectable in red blood cells. Studies of subcellular distribution by confocal imaging showed reciprocal localisation of catenins in CD34+ cells, with /3-catenin predominantly nuclear translocated and y-catenin nuclear excluded. Conversely, in granulocytic and monocytic cells nuclear y-catenin levels were relatively high whilst nuclear /3-catenin levels were reduced. A small subset of the CD14+ monocyte population exhibited heavily nuclear translocated y-catenin. Subsequent knock-down studies of y-catenin showed this protein to be required for normal haematopoietic development in vitro, evidenced by the inhibition of macrophage differentiation and apparent reprogramming of committed monocyte progenitors for granulocytic development. In AML patients, y- catenin mRNA expression conferred a reduced complete remission rate arising from resistant disease, however discordance was found between mRNA and protein level, implying post-translational control of y-catenin expression in AML. In primary AML blasts (undifferentiated) y-catenin was aberrantly localised to the nucleus suggesting a transcriptional role in AML pathology. A correlation was identified between y- and (3-catenin protein expression in primary AML blasts and an association between nuclear levels of these proteins. To determine whether this association was causal, y-catenin was ectopically expressed in normal human CD34+ haematopoietic progenitor cells but had no significant influence on /3-catenin expression or localisation even following subsequent differentiation. In contrast, overexpression of y-catenin in leukaemic cell lines stabilised /3-catenin protein and promoted its translocation to the nucleus suggesting that the influence of y-catenin on /5-catenin is a feature of leukaemic cells but not normal cells. Phenotypically, overexpression of y-catenin had little effect on normal progenitor cells but was able to block agonist-induced differentiation of AML cell lines, probably via stabilisation of jS-catenin. In summary, this study indicates a role for y-catenin in the regulation of normal haematopoietic development and that its nuclear translocation is strictly regulated independently of /3-catenin in normal haematopoiesis. In leukaemic cells, however, this control is dysfunctional allowing y-catenin to promote the stabilisation and translocation of /3-catenin. This relationship may represent a pathological mechanism active in AML blasts to block myeloid differentiation and promote a leukaemic phenotype.

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Green, C. L. « Studies on CEBPA mutations in acute myeloid leukaemia ». Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1379542/.

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Acute myeloid leukaemia (AML) is a highly heterogeneous disease with regard to clinical outcome, and molecular markers with prognostic impact can be used to stratify patients for risk-adapted therapy. CEBPA mutations have been associated with a favourable prognosis, however several questions remained, in particular whether one (CEBPA-single) or two (CEBPA-double) mutations were necessary for this benefit, and their interaction with other molecular markers. A method of detecting CEBPA mutations in patient samples using denaturing HPLC was developed and the CEBPA status of 1427 young adult AML patients (median age 43 years, range 15-68 years) determined. Overall, 107 (7%) were CEBPA-mutant: 48 (45%) CEBPA-single and 59 (55%) CEBPA-double. The majority of CEBPA-double patients (83%) had an out-of-frame insertion/deletion in the N-terminus and a mutation in the C-terminal DNA-binding/leucine zipper domains (DBD/LZD) that were on different alleles as determined by cloning. By contrast, mutations in CEBPA-single cases were distributed across the gene. CEBPA-double patients were less likely to have a FLT3/ITD (P=.04) and highly unlikely to have an NPM1 mutation (P<.0001) compared to CEBPA-WT/CEBPA-single cases. Eight year overall survival (OS) was higher in CEBPA-double patients compared to CEBPA-WT and CEBPA-single cases (54%, 34%, 31%, respectively, P=.004). In multivariate analyses, CEBPA-double, but not CEBPA-single, was an independent favourable factor for OS (P=.004) and relapse (P=.02). However, this benefit was completely lost in the presence of a FLT3/ITD. The mutant level of 101 mutations was determined by fragment analysis and the majority were of a level consistent with a heterozygous mutation present in most cells. The impact of ten atypical CEBPA mutations on C/EBPα transactivation activity was explored by a luciferase reporter assay. Only mutations affecting the DBD or LZD functional domains had an impact on transactivation activity. This work provides insight into the biology of CEBPA mutations and their use as clinical markers.

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Putwain, Sarah Lucy. « The role of Sox4 in acute myeloid leukaemia ». Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648624.

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Grandage, V. L. « Investigation of aberrant signal transduction in acute myeloid leukaemia ». Thesis, University College London (University of London), 2007. http://discovery.ucl.ac.uk/1445527/.

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Haematopoiesis is the result of tightly regulated signal transduction pathways mediated by cytokines and their receptors. Aberrations in these pathways are an underlying cause for diseases such as leukaemia and other myeloproliferative and lymphoproliferative disorders. The PI3-Kinase/Akt pathway is central to regulation of cell survival and proliferation. This study found that the PI3-Kinase pathway is activated in AML cells. This activation was reduced or abolished when the cells were incubated with the PI3-Kinase inhibitor, LY294002. Leukaemic cell survival appeared to be dependent on PI3-Kinase activation as incubation with LY294002 resulted in a reduction in cell number and an increase in apoptosis. This was also true for the CD34+/38- leukaemic stem cell population. Further work indicated that activation of Akt alone was sufficient to protect factor dependent cells from cytokine withdrawal induced apoptosis and also from the cytotoxic effects of Ara-C and Etoposide. The JAK/ STAT Pathway is important for many biological responses including differentiation and proliferation and its dysregulation has also been reported in many malignancies. It was shown that G66976, a selective PKC inhibitor, is a potent inhibitor of JAK 2 in in vitro kinase assays and in whole cell systems and inhibits signaling downstream of multiple JAK2 coupled cytokines including IL-3, GM-CSF and EPO. G66976 was also found to inhibit signalling downstream of disease-associated forms of JAK2 such as the TEL-JAK2 fusion and mutant JAK2 V617F. The majority of primary AML cells investigated had constitutive STAT activation which was reduced by incubation with G66976 in the majority of cases. This reduction was accompanied by a decrease in cell survival and proliferation. This work indicates that both the PI3-Kinase/Akt and the JAK/STAT pathways would be appropriate targets for the development of small molecule inhibitors for use in the treatment of AML.

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Ivey, Adam Stuart. « Molecular characterisation and tracking disease in acute myeloid leukaemia ». Thesis, King's College London (University of London), 2016. https://kclpure.kcl.ac.uk/portal/en/theses/molecular-characterisation-and-tracking-disease-in-acute-myeloid-leukaemia(a3aac920-4bbe-4360-afbd-2bd1dbe5e738).html.

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Acute myeloid leukaemia (AML) is a highly heterogeneous disease in terms of genetics and at the clonal level. Despite improved understanding of the genetic landscape of AML, pathogenesis and cause of disease relapse remain poorly understood. To address these issues exome sequencing was carried out on 25 diagnostic and 12 relapse samples from 28 cytogenetic standard risk AML patients. More than 500 somatic mutations were identified, including recognised recurrent mutations, and novel variants with driver potential. Copy number variations were also investigated, identifying loss of heterozygosity of chromosome 13 involving the FLT3 locus as a common event acquired at disease relapse. The heterogeneity of disease relapse was also highlighted with the analysis of paired diagnostic and relapse samples. To further develop an understanding of AML clonal architecture and disease evolution, targeted deep sequencing was then carried out using a custom gene panel informed by exome sequencing results. Two cohorts were analysed including 223 diagnostic and 49 relapse samples from NPM1 mutant AML patients, followed by 43 unselected AML patients. In the NPM1 mutant cohort co-operating mutations were identified in 99% of patients, while at least one mutation was detected in 91% of the unselected cohort. Furthermore, quantification of variant allele frequencies allowed detailed analysis of mutational hierarchy and clonal evolution of relapse. The prevalence of mutations in candidate genes was also assessed, notably identifying mutations of MYC as recurring events in NPM1 mutant AML. Knowledge of AML genetics can also be applied post-treatment to track minimal residual disease (MRD) using molecular markers. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assays were designed and optimised for rare fusion genes and NPM1 mutations. As a model, the NPM1 mutant RT-qPCR assays were used to track MRD in follow up samples from a large cohort of NPM1 mutant AML patients. These data identified a single MRD measurement in the blood following the second course of chemotherapy was the most informative outcome predictor, independent of mutational profile. It was also established that analysis of sequential samples could identify patients destined for relapse providing an opportunity for pre-emptive treatment. In the future a more personalised treatment approach, incorporating both mutational profiling and MRD monitoring, may improve patient outcome.

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Lim, Seah-Hooi. « Immunotherapy and recombinant interleukin-2 in acute myeloid leukaemia ». Thesis, University of Aberdeen, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.484307.

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In this study 12 acute myeloid leukaemia (AML) patients (3 in 1^st complete remission (CR), 3 in 2^nd CR, 3 in early relapse or partial remission and 3 in frank relapse) were treated with continuous infusion of recombinant Interleukin-2 (rIL-2). Adverse reactions among these patients were common. In all patients, there were evidence of lymphocyte activation with subsequent upregulation of the cellular cytotoxic functions following the infusion of rIL-2. Despite this, clinical response among patients treated with active disease remains disappointing, with only 1 patient achieving a 3^rd complete remission after being treated in early 2^nd relapse (marrow blast counts of 10%). The other patients had brief periods of stable disease but died eventually of disease progression. No conclusion however can be drawn from patients treated in complete remission due to the small number of patients entered into the study. In vitro studies were performed in a different cohort of AML patients, at presentation and during complete remission. In all the patients, both the Natural Killer (NK) and Lectin-Dependent Cellular Cytotoxicity (LDCC) activities were significantly reduced when compared to normal healthy controls. Patients in complete remission however had higher values than those studied during active diseases. These findings would suggest a strong rationale for the use of immunotherapy capable of upregulating the NK and LDCC activities, e.g. rIL-2. Further rationale for the use of immunotherapy has been drawn from the findings that leukaemia blast cells of AML are immunogenic, as evidenced by data of T cell activation in these patients and the presence of complement-fixing antibodies for autologous myeloblasts. More importantly no stimulation of the myeloblast proliferation by IL-2 was observed in any of the myeloblasts studied. All these findings would point to a good and safe rationale for the use of rIL-2 in AML patients.

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Le, Dieu Helen Rifca. « Characterisation of T cell defects in acute myeloid leukaemia ». Thesis, Queen Mary, University of London, 2009. http://qmro.qmul.ac.uk/xmlui/handle/123456789/561.

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Understanding the immune system in patients with cancer and how it interacts with malignant cells is critical for the development of successful immunotherapeutic strategies at a time when novel cancer treatment approaches are required. Acute myeloid leukaemia (AML) results in widespread interaction between the malignant cells and T cells and as such, offers an opportunity to study these interactions. A flow cytometric analysis of T cells in the peripheral blood of patients presenting with AML illustrated that the absolute number of T cells is increased in AML compared with healthy controls. Furthermore, a large population of CD3+56+ cells was identified. These cells are not natural killer T cells but effector T cells that may represent a failing immunosurveillance mechanism. Two technical issues were explored: how to separate T cells from the peripheral blood of newly diagnosed AML patients and the impact of the method of immunomagnetic cell separation on the gene expression profile of healthy T cells. Gene expression profiling was subsequently performed on T cells from AML patients compared with healthy controls. Global differences in transcription were observed suggesting aberrant T cell activation patterns in AML. As differentially regulated genes involved in actin cytoskeletal formation were noted, a functional assessment of the ability of T cells from AML patients to form immunological synapses was performed. This illustrated that although T cells from AML patients can form conjugates with autologous blasts, their ability to form immune synapses and recruit phosphotyrosine signalling molecules to that signalling interface is impaired. Taken together, these findings demonstrate that numerically T cells are plentiful in AML however they are abnormal in terms of the genes they are transcribing and in their interactions with tumour cells. Targeting immunological synapse formation may represent an important means of improving T cell recognition of tumour cells across a range of cancers.

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El-Sharkawi, D. « Studies to investigate epigenetic factors in acute myeloid leukaemia ». Thesis, University College London (University of London), 2016. http://discovery.ucl.ac.uk/1528735/.

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Acute myeloid leukaemia (AML) is a heterogeneous disease with numerous recurrent cytogenetic and molecular abnormalities. This heterogeneity is reflected in the variation in clinical outcome seen in patients. This disparity in outcome is also seen within groups of patients who have the same mutation or no known molecular abnormalities. To investigate whether the DNA methylation profile of samples can provide prognostic information, the methylome of forty cytogenetically normal AML samples that were wild-type for NPM1 and FLT3 was analysed, 20 were from patients with chemosensitive disease and 20 with chemoresistant disease. Unsupervised cluster analysis revealed the DNA methylation profile to be most associated with underlying CEBPA genotype hence a CEBPA signature was created using the 25 CpG sites that differed the most between wild-type (n=30) and classic CEBPADM (double mutant) samples (n=10). Two follow-up cohorts were analysed, validating the initial signature in differentiating classic CEBPADM samples from wild-type. CEBPASM (single mutant) samples had profiles more similar to the CEBPAWT (wild-type) signature. Non-classic CEBPADM samples with at least one mutation leading to loss of function of the C terminal were associated with a CEBPA mutant methylation profile. Methylation of the CEBPA promoter was not associated with a classic CEBPADM methylation profile in eight of the nine cases exhibiting hypermethylation. The ASXL1 gene, known to have a role in histone regulation, was screened in 371 patients using denaturing HPLC. The overall mutation rate was 9%. Overall survival was significantly lower in patients with an ASXL1 mutation, however the mutation was associated with secondary disease and older age, and thus in multivariate analysis mutations in ASX L1 lost significance. These studies indicate that epigenetic factors are closely linked to other prognostic traits such as age or underlying molecular status of the AML. Given this association, DNA methylation could play an important role in assessing the significance of different types of mutations.

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Naiel, Abdulbasit. « Study of acute myeloid leukaemia with known chromosomal translocations ». Thesis, Brunel University, 2014. http://bura.brunel.ac.uk/handle/2438/9303.

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Acute myeloid leukaemia (“AML”) is a clonal disease characterised by increased, uncontrolled abnormal white blood cells and the accumulation of leukaemia immature cells in the bone marrow and bloodstream. Chromosomal rearrangements have been detected in almost half of AML cases. It has been proven that the chromosomal rearrangements constitute a marker for the diagnosis and prognosis of AML and have therapeutic consequences. The discovery of these rearrangements has led to a new World Health Organization (“WHO”) classification system. However, small regions of cryptic chromosomal rearrangements have been identified among these cases. Such cryptic rearrangements can be explained by the identification of small regions which cannot be found by conventional chromosome banding techniques. Moreover, approximately 50% of AML cases have been found with normal karyotypes. The improvement of cytogenetic techniques, including fluorescence in situ hybridization (“FISH”) and single nucleotide polymorphism (“SNP”) platforms, have allowed the detection of small rearranged regions (such as copy number changes) both in normal and abnormal karyotype AML. This study identifies: (i) cryptic chromosomal translocations in leukaemia cells of AML patients; (ii) DNA copy number changes in patients with known chromosomal translocations; and (iii) the proliferating state of leukemic cells harbouring chromosomal abnormalities within a series of patients. In the initial study, the FISH technique was performed on 7 AML patient samples to validate a novel three colour probe for the detection of t(7; 12). The results demonstrated that the new three-colour FISH approach used in this study has enabled the detection of a cryptic t(7;12) translocation as part of a complex rearrangement in one patient previously been described as having t(7;16) and ETV6-HLXB9 fusion transcript at the molecular level. To date there are only two cases of a cryptic t(7; 12) translocation reported in the literature. Additionally, the new three-colour FISH approach also enabled identification of t(7; 12) in a new seven year-old AML patient (the first case of childhood leukaemia with an onset after infancy to be found positive for t(7; 12)). In the second study the FISH technique was used to validate three colour probe sets for the detection of 7(q22-q31) and 7(q22-q36.1) regions on several myeloid cell lines. The results indicate that the probes found chromosome 7 rearrangements in myeloid cell lines with complex rearrangements. The three colour probe sets enabled detection of a new rearrangement in the k562 cell line, described as a duplication of 7q36 region, followed by intrachromosomal insertion of long arm material into the short arm of chromosome 7. The intrachromosomal insertion identified in k562 cell line is an uncommon form of chromosomal rearrangement in myeloid leukaemia which has not been previously reported. In the third study, the Illumina BeadArray approach was used to assess copy number alterations (“CNAs”) and copy number loss of hertrozygosity (“CN-LOH”) regions in 22 AML patients samples with inv(16)(p13;q22) and t(8;21)(q22;q22) rearrangements. In order to distinguish between true CNAs and false-positive findings as well as to verify whether CNAs are present in the same clone harbouring inv (16), FISH was used on fixed chromosome and cell suspensions from the same patients. The results showed a low number of copy number losses and copy number gains in 17 (77.27%) out of 22 cases, with an average of 1.86 CNAs per case as well as copy neutral-LOH with an average of 6.7% per patient. Furthermore, interphase FISH was carried out on four cases showing a 7q36.1 deletion, 4q35.1 deletion, 16.13.11 deletion and 8q24.21-q24.3 gain identified by array. The FISH results confirmed CNAs in most cases while CNA was not confirmed in one patient. Moreover, the FISH data analysis showed that the CNAs were found in both cells without inv (16) and cells harbouring the inv (16) rearrangement. In the final study, indirect immunofluorescence (IF) was used to determine the ki67 staining patterns in 8 stimulated and unstimulated peripheral blood samples and k562 cell lines. The results showed a high percentage of ki67 positive staining in the stimulated samples in comparison with unstimulated samples, which showed a low percentage of ki67 positive staining. In addition, a high percentage of proliferating cells were detected in the k562 cell line. ImmunoFISH was performed on five different patient samples and leukaemia cell lines using specific probes in the regions of interest to detect the chromosomal abnormalities and using the ki67 antibody to assess the proliferation state of the cells. The results showed that the proliferation state of the cells carrying chromosomal abnormalities in two patients was higher than the proliferation state of the cells carrying abnormalities in three patients; in other words, most of the cells carrying abnormalities were proliferating in two cases and non-proliferating in three cases.

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RIBEIRO, Ana Rita Matias Rosa de Almeida. « Insights into Acute Myeloid Leukaemia metabolism using NMR Spectroscopy ». Master's thesis, Instituto de Higiene e Medicina Tropical, 2017. http://hdl.handle.net/10362/22443.

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A Leucemia Mielóide Aguda (LMA) é o segundo tipo de leucemia mais diagnosticado e o mais comum entre adultos. É uma condição hematológica caracterizada pela infiltração da medula óssea e do sangue por populações clonais de células mielóides imaturas, anormalmente diferenciadas e altamente proliferativas. É uma doença muito heterogénea, originando-se a partir de uma grande variedade de linhagens hematopoiéticas, com perfis genéticos distintos.(2,3) Uma das apresentações menos comuns desta doença é a acidose láctica (acumulação de lactato no sangue), relacionada com um mau prognóstico de sobrevivência.(4) Esta condição está relacionada com elevadas taxas de glicólise aeróbica, o “efeito de Warburg”, resultando na produção de lactato que é excretado para o microambiente. Vários estudos demonstram que alguns tipos de células tumorais incorporam lactato e utilizam-no como base para o metabolismo oxidativo. (5,6) Estudou-se o metabolismo de lactato e glucose de três linhagens de LMA (HL60, THP1 e HEL) através de RMN. Investigou-se também a influência do VEGF (importante no microambiente tumoral, estando envolvido no processo de angiogénese em LMA) nestas linhas celulares.(7) Os resultados demonstraram que as linhas promielocítica e monocítica conseguem utilizar o lactato como base para a manutenção das suas necessidades energéticas e de biomassa, utilizando-o como substrato para o ciclo de Krebs e para a fosforilação oxidativa. Observámos que a linha monocítica metaboliza a glucose através da glicólise e da via das pentoses fosfato. A linha eritroleucémica depende da presença de glucose, sendo incapaz de suster o seu metabolismo usando o lactato como substrato. O VEGF parece ter um efeito positivo nas linhas HL60 e THP1, potenciando as taxas de glicólise e promovendo a produção de nucleótidos, o que sugere que o VEGF pode ter influência neste padrão metabólico alterado. Assim, as três linhagens de LMA apresentam diferentes graus de plasticidade metabólicas, tendo algumas se adaptado para tirar partido do microambiente, enquanto que outras parecem incapazes de se ajustar. Também investigámos como a exposição a diferentes microambientes influencia o metabolismo da LMA. O microambiente natural dos blastos de LMA encontra-se na medula óssea mas as células podem espalhar-se e acumular-se noutras zonas do corpo onde, para sobreviver, têm de se adaptar a um microambiente diferente. Em alguns casos, a LMA resulta na infiltração do Sistema Nervoso Central, normalmente afectando as meninges. (1) Células HEL foram inoculados na medula e cérebro de ratinhos e, através de análise multivariada de espectros de RMN, foi possível fazer a distinção entre os perfis metabólicos das células expostas a cada um dos microambientes. Os resultados mostraram que esta linhagem está melhor adaptada para sobreviver no microambiente da medula do que no microambiente cerebral. Em ambos os microambientes observaram-se alterações graduais no metabolismo resultantes de mais tempo de exposição, demonstrando que o metabolismo celular não é estático, sendo influenciado pelas características do meio envolvente. Em conclusão, foi demonstrado que a elucidação do metabolismo de leucemia e da sua interacção com o microambiente é indispensável à caracterização desta doença e pode contribuir para esclarecer os mecanismos biológicos responsáveis pelo seu desenvolvimento e progressão.
Acute Myeloid Leukemia (AML) is the second most diagnosed type of leukemia and the most common in adults. It is a haematological disorder characterized by the bone marrow and peripheral blood infiltration of clonal populations of abnormally differentiated and highly proliferative blasts. It is a very heterogenous disease, originating from a wide variety of haematopoietic lineages with a diverse genetic landscape.(2,3) One of the less typical presentations of AML involves lactic acidosis (the accumulation of lactate in the blood) which has been correlated with poor survival prognosis. (4) This condition is correlated with high rates of aerobic glycolysis, the “Warburg effect”, resulting in the production of lactate that is excreted to the microenvironment. Several studies have found that some cancer cells have can take up lactate and utilize it as a source for oxidative metabolism.(5,6) We studied the glucose and lactate metabolism of three different AML lineages (HL60, THP1 and HEL) through NMR spectroscopy. We also investigated the influence of VEGF (another key player in tumour microenvironment, known to be involved in tumour-related angiogenesis in AML) in these cell lines.(7) Our results showed that the promyelocytic and monocytic lines can rely on lactate to sustain their energy and biomass demands, utilizing it as substrate for the TCA cycle and oxidative phosphorylation. On the monocytic line we confirmed that glucose is mainly metabolized through glycolysis and pentose phosphate pathway. The erythroleukemic line was unable to sustain its metabolism on lactate consumption, being dependent on glucose to sustain the cells’ metabolism. VEGF seem to have a positive effect on the HL60 and THP1 lines, increasing glycolytic rates and promoting nucleotide production, which suggest that it may have a role in supporting the alternative metabolism exhibited by these cells. So, the three lineages of AML display very different metabolic plasticity, with some having adapted to take advantage of their environment, while others seeming unable to adjust. We also investigated how exposure to different microenvironments influences AML cell metabolism. The normal environment of AML is the bone marrow (BM), but the cells can spread and accumulate in other organs of the body, where they are required to adapt to a completely different environment. In some cases, AML can involve infiltration of the Central Nervous System, usually in the form of leptomeningeal disease.(1) We inoculated HEL cells into the brain and BM of mice and, through multivariate analysis of NMR data, were able to distinguish between the metabolic profiles of the cells exposed to the two environments. Our results showed that the HEL cell line is much better suited to survive in the BM microenvironment than in the brain microenvironment. In both microenvironments, there were gradual metabolic modifications as the result of increased exposure, indicating that cell metabolism is not static and is influenced by the characteristics of the surrounding microenvironment. In conclusion, we showed that understanding leukaemia metabolism and its interaction with the microenvironment is indispensable for disease characterization and can provide valuable insights into the biologic mechanisms that govern AML progression and survival.

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Yu, N. « Identifying and targeting dormant cells in acute myeloid leukaemia ». Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/33679/.

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Relapse in AML is thought to arise from dormant leukaemic cells that are characterised by low RNA synthesis activity, protected by the bone marrow (BM) niche, and may evade the effects of chemotherapeutic drugs. Our aim was to investigate agents which might be able to overcome chemoprotection by targeting the intrinsic apoptosis pathway. We developed in vitro assays to identify and characterise the dormant AML cells using combinations of markers, including the cell-division marker PKH26, leukaemia-associated phenotypes (LAPs), and dormancy markers. In a dormancy model based on 12-day AML/stroma co-culture, we have shown that the expression of some aberrant phenotypes can persist for several days. Also, after 12 days, some of the CD34+, PKH26high (dormant), and LAP+ (leukaemic) cells maintained their primitiveness and were still clonogenic. Furthermore, our chemosensitivity data showed that novel agents TG02, and BH3 mimetics ABT-737 and ABT-199, which inhibit the B-cell lymphoma 2 (BCL-2) family of anti-apoptotic molecules, could efficiently target BM niche-mediated chemoresistance, which is thought to be one of the main obstacles to traditional chemotherapy. We explored various candidate dormancy markers based on the low RNA, non-proliferative profile of dormant cells. Among those tested, the RNA synthesis marker Pyronin Y (PY), and an antibody to the transferrin receptor CD71 were the most reproducible in terms of marker expression and stability. We endeavoured to characterise cell dormancy on the molecular level by investigating gene expression in the PYlow (dormant) and PYhigh (proliferating) subsets and have obtained limited results. In summary, this study has identified and partly characterised dormant AML cells by development of in vitro assays, and has shown chemosensitivity to novel agents TG02, ABT-737 and ABT-199 in dormant leukaemia cells.

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Krishnamurthy, Pramila. « Cellular and vaccination-based immunotherapy of acute myeloid leukaemia ». Thesis, King's College London (University of London), 2014. http://kclpure.kcl.ac.uk/portal/en/theses/cellular-and-vaccinationbased-immunotherapy-of-acute-myeloid-leukaemia(ea5188b6-c807-4a99-8473-e99f55f19559).html.

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Approximately 50% of Acute Myeloid Leukaemia (AML) patients relapse within 5 years of diagnosis. Allogeneic haematopoietic stem cell transplantation (HSCT) has curative potential, partly mediated by a Graft-versus-Leukaemia (GvL) effect. GvL activity may be boosted by donor lymphocyte infusions (DLI) after HSCT, given pre-emptively (pDLI), to prevent relapse in mixed donor chimeric recipients, or therapeutically (tDLI) following disease recurrence. Few publications report efficacy of these approaches, therefore a retrospective analysis of outcomes after DLI at our institution, following lymphodepleted, reduced intensity conditioned HSCT for AML/myelodysplastic syndromes (MDS), was performed. Encouraging estimated 5-year overall survival rates following DLI of 80% for pDLI recipients and 40% for tDLI recipients were observed. Incidence of GvHD was only moderate, suggesting delayed add-back of immune cells can boost GvL reactions in AML/MDS patients without excessive toxicity. However, despite association of DLI with reduced relapse, leukaemia recurrence in a proportion of patients highlights that GvL activity is neither guaranteed nor universally sustained. Two forms of vaccination are described in this thesis, aiming to enhance priming and activity of leukaemia-reactive T-cells. Both offer broad applicability across all human leucocyte antigen (HLA)-types. T-cell responses to peptide vaccinations targeting the leukaemia-associated antigen Wilms’ Tumour protein (WT1) were enhanced by exploration of novel adjuvants for induction of cell-mediated immunity. Vaccinations comprising these adjuvants and single peptides or overlapping peptides spanning the whole WT1 protein, induced functional T-cell responses (antigen-specific in vivo cytolytic activity and interferon-gamma production) in C57BL/6 mice. Secondly, autologous AML blasts, genetically modified to express the immunostimulatory molecules CD80 and IL-2, were co-administered as a vaccine with tDLI in a Phase I clinical trial. Preliminary data supporting safety of this approach and possible induction of immune responses to vaccination, evidenced by development of a delayed-type hypersensitivity reaction in a subject, are presented. This thesis describes broad-based immunotherapeutic strategies in AML patients for prevention of disease recurrence by enhancement of anti-leukaemic immune responses.

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Gilby, Daniel C. « Analysis of myeloid tumour suppressor genes in acute myeloid leukaemia and idiopathic myelofibrosis ». Thesis, University of Sheffield, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.505559.

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Chim, Chor-sang James. « Study of gene promoter methylation in acute promyelocytic leukaemia ». Hong Kong : University of Hong Kong, 2002. http://sunzi.lib.hku.hk/hkuto/record.jsp?B25256725.

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Walsby, Elisabeth Jane. « Role of FUS (TLS) in differentiation in acute myeloid leukaemia ». Thesis, Cardiff University, 2004. http://orca.cf.ac.uk/55540/.

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Leukaemia is the result of molecular abnormalities which lead to a block in differentiation that is the defining characteristic of myeloid leukaemia cells. Work to identify the genes that are associated with this defective differentiation led to the identification of the involvement of the FUS gene. FUS (TLS) is a housekeeping gene that is capable of binding DNA and RNA. FUS has roles in coupling mRNA transcription and processing and in the repair of double stranded DNA breaks. Previous work within this department has shown that FUS is down-regulated in human leukaemia cell lines in response to induction of differentiation with ATRA and that FUS is up-regulated in acute myeloid leukaemia (AML) patients. The aim of this study was to identify whether this is a causative or correlative relationship and also to identify target genes associated with FUS dysregulation. Transduction of human (HL- 60, NB4, NB4R2) and murine (32D, 32D AML-ETO, 32D B2A2) leukaemia cell lines with retroviral constructs expressing FUS or antisense FUS resulted in some over-expression or down-regulation of FUS in these cells. The effect of FUS modulation in the transduced cell lines was assessed through cell growth and viability. No effect on the growth and viability of transduced cells was observed as a result of FUS modulation. To determine whether FUS had a role in differentiation, the transduced cell lines were induced to differentiate using ATRA and G-CSF. Differentiation was assessed by measurement of cell growth, viability and the expression of cell surface markers by flow cytometry. The result of expression of FUS antisense in the ATRA sensitive NB4 and 32D cell lines was to generate resistance to differentiation induction using ATRA. Conversely in the ATRA resistant NB4R2 and 32D B2A2 cells, expression of FUS antisense reinstated the ability to differentiate in these cells. In response to treatment with G-CSF, the 32D cells expressing FUS antisense developed a resistance to differentiation while the previously G-CSF resistant 32D B2A2 cells became capable of differentiation when FUS antisense was expressed in these cells. Following the demonstration of an altered phenotype in response to treatment with different differentiation inducers in cells containing FUS antisense constructs, genes acting as target genes of FUS were identified using the Affymetrix gene expression system. The effect of FUS over-expression and down- regulation were studied in all the murine 32D derived cell lines and in the human NB4 cell line. In addition to this, gene expression changes resulting from FUS modulation in the NB4 cells during treatment with ATRA over 96 hours was investigated in this manner. Expression levels of genes associated with FUS dysregulation were verified using quantitative RT-PCR. Genes identified as having altered expression as a result of the expression of the FUS antisense construct included transcription factors, genes involved in apoptosis and differentiation and genes that have previously been shown to interact with, or have homology to, FUS itself. Further analysis of these candidate genes suggested that they were not likely to have a dominant effect in the altered phenotype seen in the transduced cells but were more likely to play a participatory role in the effects observed. This study has concluded that FUS may have a role in haematopoietic differentiation induced by both ATRA and G-CSF but this role appears to be context dependent making it important to study the effects of its modulation in primary AML blasts. The mechanism through which FUS affects the ability of the cells to differentiate remains unresolved.

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Owen, Carolyn Jane. « The Molecular Basis of Familial Myelodysplasia and Acute Myeloid Leukaemia ». Thesis, University of London, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.517976.

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Darkhshan, Fatemeh. « Mapping of murine radiation-induced acute myeloid leukaemia susceptibility loci ». Thesis, University of Reading, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367693.

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Zhao, L. « Identify critical signalling pathways in MLL-rearranged acute myeloid leukaemia ». Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1432505/.

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Chromosome translocations that disrupt the Mixed Lineage Leukaemia (MLL) gene are associated with a unique subset of acute myelogenous and lymphoblastic leukaemias. MLL translocations are most prevalent in infant leukaemia, where they comprise 80% of cases of acute lymphoblastic leukaemia and 60% of cases in acute myeloid leukaemia. To identify transcriptional target genes required for the immortalisation, previous work in our laboratory involved generating constitutively and conditionally immortalised primary mouse haematopoietic progenitor cells. Global gene expression analysis, upon loss of MLL-fusion protein, identified a number of genes that were differentially expressed. This project aims to investigate the importance of two differentially expressed genes, Msi2 and Ruvbl2, in MLL-fusion mediated leukaemogenesis. To examine the efficacy of targeting Ruvbl2 and Msi2 in leukaemia elimination in vivo, shRNA inducible models were generated for the human leukaemic cell line, THP1 and murine MLL-ENL leukaemic cells. While targeting Msi2 had no significant impact on leukaemia progression in vivo, inhibiting RUVBL2 function significantly delayed leukaemia onset. RUVBL2 is a member of the AAA+ family of DNA helicases and plays an important role in diverse cellular processes. RUVBL2 was inhibited either via shRNA-mediated knockdown or through the expression of a dominant-negative mutant form of RUVBL2, and its inhibition resulted in a marked increase in apoptosis, reduction in colony formation and a block in cell cycle. In parallel, while expression of dominant-negative RUVBL2 induced apoptosis in mouse MLL-ENL leukaemic cells, it had a relatively mild impact on normal haematopoietic progenitor colony formation in vitro. Further experiments indicated that RUVBL2 functioned mainly through influencing the transcriptional activity of c-MYC. Taken together, our data suggest that a potential therapeutic window exists for targeting RUVBL2 function in MLL-rearranged leukaemia.

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MILOVANOVIC, SARA. « NON-GENETIC MECHANISMS OF CHEMORESISTANCE IN ACUTE MYELOID LEUKEMIA ». Doctoral thesis, Università degli Studi di Milano, 2022. https://hdl.handle.net/2434/946408.

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Acute Myeloid Leukemia (AML) is the most common type of acute hematological malignancy in adults. 40–60% of patients relapse due to the emergence of cellular resistance to anti- leukemic drugs. Drug resistance in leukemic cells has been associated with intratumoral heterogeneity, among which quiescence, specifically, is considered a key factor for cell survival. Experimental evidence collected both from patients and model systems suggests that relapse is due to rare persistent AML cells which survive chemotherapy. Chemotherapy persistent cells are not yet biologically and molecularly defined. Open questions are whether persistent cells are drug-resistant, what are their cellular and molecular features, as well as their content in leukemic stem cells. My working hypothesis is that chemoresistance in AMLs is associated with specific phenotypic states that characterize rare cell populations found within the pool of quiescent leukemic cells. These cells are selected by chemotherapy and represent the cellular basis of the relapse. To test my hypothesis, I explored transcriptional and functional characteristics of quiescent leukemic cells and tested the effect of chemotherapy. Here, I present two newly established xeno-models of chemoresistant human AMLs that closely recapitulate clinical data. My data show that quiescent cells accumulate over time and quiescent and proliferating cells can switch from one state to another. Notably, quiescent cells are selectively spared by chemotherapy and show resistance to additional rounds of treatment. Finally, quiescent cells appear as the only carrier of tumorigenic capacity in AMLs and are, therefore, deemed essential for leukemia development and, possibly, relapse.

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Maher, John. « The role of ras in myeloid leukaemogenesis ». Thesis, Imperial College London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.283668.

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