Extensive efforts have shed light on the identity and biology of cancer stem cells, required and sufficient for the propagation of hematological malignancies and solid tumours. Much less is understood about the closely related issue as to the identity and properties of the normal stem and progenitor cells targeted by oncogenic lesions, and how the nature of the targeted cell might impact on the biology and clinical picture of the resulting cancer. To address this, we developed a mouse model allowing targeted inactivation of Ezh2 and Runx1 to different haematopoietic compartments. Inactivating mutations of EZH2 and RUNX1 frequently co-occur in haematological malignancies with markedly different phenotypes including myelodysplastic syndrome (MDS) and early thymic progenitor (ETP) leukaemia. Inactivation of Ezh2 and Runx1 in adult haematopoietic stem cells (HSCs) resulted in perturbed haematopoiesis leading to development of an MDS-like disease. Unexpectedly, this MDS phenotype could be fully reproduced when Ezh2 and Runx1 inactivation was targeted to multipotent progenitors (MPPs) using Flt3-Cre. Furthermore, the disease was transplantable by MPPs, but not more committed progenitor populations, demonstrating that MDS tumour propagating potential is not exclusive to intrinsically self-renewing HSCs. Targeting Ezh2 and Runx1 inactivation to early lympho-myeloid progenitors did not result in an MDS phenotype. These mice showed a marked expansion of ETPs within the thymus, combined with a block in thymocyte differentiation. These expanded ETPs displayed transcriptional features characteristic of ETP leukaemia, a treatment-resistant acute leukaemia subtype hypothesised to originate from ETPs. Combination of inactivation of Ezh2 and Runx1 in ETPs with the constitutively activating Flt3-ITD signalling mutation resulted in an aggressive lympho-myeloid acute leukaemia, which could be propagated by the expanded ETP population. These findings demonstrate the potential of lympho-myeloid progenitors such as ETPs to become leukaemia stem cells which propagate a disease retaining lympho-myeloid features. We used this novel ETP leukaemia model to explore therapeutic targeting of Ezh2-inactivated ETP leukaemias using inhibitors of the bromodomain and extra terminal (BET) proteins. Aberrant transcription resulting from epigenetic changes induced by Ezh2 loss could be reversed by BET inhibitors, and these compounds showed therapeutic efficacy against both mouse and human ETP leukaemias in vitro and in vivo.

Acute Myeloid Leukemia (AML) is a type of cancer that affects the process of hematopoiesis. In individuals affected with AML, normal blood cells do not develop into red blood cells, white blood cells, and platelets, leading to symptoms such as anemia, neutropenia, and thrombocytopenia. The prognosis of AML is affected by multiple factors including: the genetic make-up of the leukemic cells, age of the affected individual, and underlying blood disorders such as myelodysplastic syndrome (MDS). MDS affects the development of stem cells into red blood cells, white blood cells, and platelets. Due to their clinical heterogeneity, AML and MDS continue to be a challenge that should be investigated in the community hospital setting. Remission rates between patients diagnosed with de-novo AML and patients diagnosed with AML with MDS were compared in a community hospital setting following induction therapy using a retrospective study design. Length of stay between patients diagnosed with de-novo AML and patients diagnosed with AML with MDS was compared during induction therapy. The association of age at diagnosis and number of chromosomal abnormalities to remission status was evaluated in each disease group. The association of blood transfusion requirements and neutropenic fever to length of stay was evaluated in each disease group. There were no statistically significant differences found between disease groups with respect to remission rates and length of stay. There were no statistically significant associations found between blood transfusion requirements and neutropenic fever in each disease group. There was an association found between age at diagnosis and remission status in patients diagnosed with AML with MDS. This indicates that older patients with AML with MDS are less likely to benefit from therapy and achieve complete remission. It is important to consider the small sample size, rare nature of the disease, and other variables that could have contributed to trends seen in the study population. The impact of predictors such as growth factor use and incidence of fungal infections should be investigated in future studies with AML patients. Considering these factors will allow for the development of targeted therapies and mechanisms against drug resistance for affected individuals.

Some myelodysplastic syndrome (MDS) patients present with macrothrombocytopenia due to impaired megakaryocyte (MK) differentiation. Transgenic mice that express the NUP98-HOXD13 (NHD13) fusion gene is a model for MDS and recapitulates the key features of MDS. The study investigated the hypothesis that expression of NHD13 disrupts actin architecture during MK differentiation leading to macrothrombocytopenia. To test the hypothesis, sternums were stained with hematoxylin and eosin, and evaluated by light microscopy to analyze MK morphology in vivo. NHD13 bone marrow (BM) contained many dysplastic MK. BM from wild type (WT) and NHD13 mice were also flushed, cultured in media supplemented with thrombopoietin only or with estrogen to induce proplatelet formation, and MK harvested after 5 days. Harvested MK and BM cores were processed and analyzed by transmission electron microscopy (TEM) to detail the ultrastructural features. TEM of MK revealed that NHD13 leads to formation of an irregular demarcation membrane system and fewer proplatelets. Cultured WT and NHD13 MK were also cytospun onto glass slides, labeled with fluorescent-tagged F-actin, α/β-tubulin and myosin IIa, and their cytoskeleton compared. Interestingly WT MK had actin either distributed evenly or predominantly in the periphery of the cytoplasm, NHD13 MK displayed only the former phenotype. Additionally, proplatelets lacked actin cytoplasmic extensions. The results from the present thesis demonstrate actin expression and architecture are impaired in dysplastic MK expressing the NHD13 leukemic fusion gene and leads to macrothromcytopenia. Understanding the molecular mechanisms of abnormal MK differentiation in MDS is important as many MDS patients die of hemorrhagic complications. / Master of Science

Myeloid Leukemia Factor 1 (Mlf1) is the murine homolog of MLF1, which was identified as a fusion gene with Nucleophosmin (NPM) resulting from the (3;5)(q25.1;q34) translocation associated with acute myeloid leukemia and myelodysplastic syndrome (Yoneda-Kato et al., 1996). Mlf1 was independently isolated using cDNA representational difference to identify genes up-regulated when an erythroleukemic cell line underwent a lineage switch to display a monoblastoid phenotype (Williams et al., 1999). Mlf1 has been shown to enhance myeloid differentiation and suppress erythroid differentiation; however, its mechanism of action is unknown. A yeast two hybrid screen was employed to identify Mlf1-interacting proteins. This screen isolated a number of known protein, as well as several novel molecules, that bound Mlf1. One of these was 14-3-3ξ, a member of a family of molecules that bind phosphoserine motifs and regulate the subcellular localization of partner proteins. Mlf1 contains a classic RSXSXP sequence for 14-3-3 binding and associated with 14-3-3ξ; via this phosphorylated motif (Lim et al., 2002). The aim of this thesis was to characterise a novel Mlf1-interacting protein that had some homology to protein kinases and was named Mlf1 Adaptor Molecule (Madm). Adaptor proteins are molecules that possess no enzymatic or transcriptional activity, but instead mediate protein-protein interactions. Madm is encoded by a gene consisting of 18 exons and promoter analysis suggested Madm expression might be widespread; indeed Northern blotting of adult tissues and in situ hybridization of embryos demonstrated ubiquitous Madm expression. Significantly, the Madm protein sequence is highly conserved across diverse species. / Madm formed dimers and although it contains a kinase-like domain, the protein lacks several critical residues required for catalytic activity, including an ATP-binding site. Purification of recombinant Madm revealed that the protein was not a kinase; however, studies in mammalian cells showed that Madm associated with a kinase and that Madm was phosphorylated on serine residues in vivo and in vitro. Madm also contains a nuclear localization sequence and nuclear export sequence and was shown to localise to both cytoplasm and nucleus by subcellular fractionation and confocal microscopy. The presence of two nuclear receptor binding motifs (consensus MILL) suggests that Madm may have a functional role in the nucleus. Madm co-immunoprecipitated with Mlf1 and co-localized in the cytoplasm. In addition, the Madm-associated kinase phosphorylated Mlf1 on serine residues, including the RSXSXP motif. In contrast to wild-type Mlf1, the oncogenic fusion protein NPM-MLF1 did not bind 14-3-3i; and localized exclusively in the nucleus. Although Madm co-immunoprecipitated with NPM-MLF1 the binding mechanism was altered. As Mlf1 is able to reprogram erythroleukemic cells to display a monoblastoid phenotype and potentiate myeloid maturation (Williams et al., 1999), the effects of Madm on myeloid differentiation was investigated. However, unlike Mlf1, ectopic expression of Madm in M1 myeloid cells suppressed cytokine-induced differentiation. / In summary, the data presented in this thesis reports on the cloning and characterization of a novel adaptor protein that is involved in the phosphorylation of the proto-oncoprotein MIM. Phosphorylation of Mlf1 is likely to affect its interaction with other proteins, such as 14-3-3~. Complex formation, therefore, may well alter the localization of Mlf1 and Madm, and influence hematopoietic differentiation.

京都大学 / 0048 / 新制・課程博士 / 博士(社会健康医学) / 甲第22152号 / 社医博第100号 / 新制||社医||10(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 川上 浩司, 教授 武藤 学, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Public Health / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / Myelodysplastic syndrome (MDS) is a clonal disease arising from mutated hematopoietic stem cells (HSCs). MDS stem cells originate from pre-leukemic HSCs, which have enhanced competitive advantage over wild-type (WT) HSCs but normal differentiation capacity. Recently, acquired somatic gain-of-function (GOF) TP53 mutations were identified in the blood of aged healthy individuals as well as in patients with MDS. However, the role of GOF TP53 mutations in clonal hematopoiesis and the pathogenesis of MDS is largely unknown. Based upon our previous studies and clinical findings, I hypothesized that GOF mutant p53 drives the development of pre-leukemic HSCs with enhanced competitive advantage, leading to clonal expansion and the pathogenesis of MDS. To test my hypothesis, I examined HSC behaviors in young p53+/+ and p53R248W/+ mice. I discovered that p53R248W enhances the repopulating potential of HSCs without affecting terminal differentiation. I also found that GOF mutant p53 protects HSCs from genotoxic stress and promotes their expansion. To investigate the role of mutant p53 in the pathogenesis of hematological malignancies, I monitored disease development in p53+/+ and p53R248W/+ mice and observed that some mutant p53 mice develop MDS during aging. Therefore, I demonstrated that GOF mutant p53 enhances the repopulating potential of HSCs and drives the development of pre-leukemic HSCs, predisposing aged mutant p53 mice to MDS development. Mechanistically, I found that mutant p53 increases the chromatin accessibility to genes important for HSC maintenance, including pluripotent gene Sox2 and chemokine gene Cxcl9. By performing biochemical experiments, I discovered that GOF mutant p53, but not WT p53, interacts with histone methyltransferase EZH2 and enhances histone H3 lysine 27 trimethylation (H3K27me3) at genes, including Mef/Elf4 and Gadd45g, that negatively regulate HSC self-renewal. Collectively, these findings demonstrated that GOF mutant p53 drives pre-leukemic HSC development through modulating epigenetic pathways. Thus, our studies have uncovered novel mechanistic and functional links between GOF mutant p53 and epigenetic regulators in pre-leukemic HSCs. This research may identify epigenetic regulator EZH2 as a novel target for the prevention and treatment of MDS patients with TP53 mutations.

No description available.

Ce projet de thèse portait sur le développement d’outils microfluidiques pour la toxicologie et la recherche contre le cancer. En permettant l’analyse simultanée d’un très grand nombre de réactions biologiques ou chimiques réalisés dans des compartiments indépendants (ie. gouttelettes), la microfluidique de gouttes offre une sensibilité de détection et une précision sans précédent pour l’analyse de molécules biologiques, telles que l’ADN ou les Anticorps, en comparaison des expériences réalisées conventionnellement en tubes ou en microplaques (essais en « bulk » ou volume). Ce format permet également de réaliser des expériences à très haut débit et est particulièrement pertinent pour la toxicologie, où des analyses robustes de l’effet des médicaments sont nécessaires. De même, ces procédures sont également très adaptées à l’analyse de cellules uniques pour le séquençage ADN ou ARN et l’épigénomique. Tout cela fait de la microfluidique en goutte un outil puissant pour la toxicologie et la recherche sur le cancer. En premier temps, une méthode du comptage précise des cellules encapsulée dans des microgouttelettes, nommée « hémocytométrie microfluidique », a été développée. Un nouvel algorithme de comptage a été proposé. Des cellules bactériennes (Escherichia Coli) et des cellules de 2 lignées humaines différentes (HL60 and H1975) ont été testées. Le nombre de chaque type de cellules a été déterminé avec une haute corrélation entre la théorie (basée sur la distribution de Poisson) et les résultats expérimentaux. Avec ces résultats robustes, un protocole de microfluidique en goutte a été mis en place pour interroger la viabilité cellulaire et la prolifération des 2 lignées humaines. Ces résultats sont en concordance avec ceux de la littérature. Pour la toxicologie, 3 différents modèles, y compris des microsomes (extrait de cellules d’insectes infectées par un baculovirus exprimant le cytochrome P450 3A4 humain, CYP3A4), HepG2-CYP3A4 (modifiée génétiquement pour exprimer le gène CYP3A4 humain), et HepaRG, une lignée hépatique, ont été évaluées pour l’activité enzymatique du CYP3A4, une enzyme largement utilisée en routine pour le criblage de médicament candidat. Les microsomes ont permis de développer un essai fluorogénique permettant de mesurer l’inhibition du CYP3A4. Cependant, ni l’utilisation des microsomes ni des cellules HepG2 exprimant CYP3A4 n’a donné de résultats satisfaisants en microgouttelettes. L’utilisation des cellules HepaRG, une lignée cellulaire qui conserve la majorité de l’expression des cytochromes P450 et des récepteurs nucléaires nécessaire à leur expression, a montré des résultats encourageant à la fois sur les tests de mesure de l’activité enzymatique et d’analyse de l’induction du CYP3A4. Pour la recherche sur le cancer, 4 essais originaux de PCR digitale en gouttes ont été mis en place pour la détection et la quantification de mutations (NRAS, DNMT3A, SF3B1 and JAK2) importante pour les syndromes myélodysplasiques, un groupe hétérogène de maladies touchant les cellules souches hématopoïétiques caractérisées par une hématopoïèse inefficace et des cytopénies périphériques. Finalement, un essai de PCR sur cellule unique encapsulées au sein de billes agarose a été proposé. / This thesis project consists in developing droplet-based microfluidic tools for toxicology and cancer research. Owing to its large numbers of discretized volumes, sensitivity of detection of droplet-based microfluidics for biological molecules such as DNA and antibody is much higher than bulk assays. This high throughput format is particularly suitable for experiments where a robust dose-response curve is needed, as well as for single cell analysis with applications in genomic or sequencing and epigenetics. All above makes droplet-based microfluidics a powerful tool for toxicology and cancer research. In a first part of the work, an accurate cell counting method, named “microfluidics hemocytometry”, has been developed. A new counting algorithm was proposed to count the cells within each droplet. Escherichia Coli and two different human cell lines (HL60 and H1975) were used to validate our strategy. The number of each type of cells in droplets was determined with a high consistency between theory (Poisson distribution) and experimental results. With these robust results, a droplet-based microfluidic protocol has then been established to inquiry both cell viability and proliferation for the two human cell lines. The results are in good agreement with the one of the literature. For the toxicology, 3 different biological models, including microsomes (extracted from baculovirus-infected insect cell expressing human CYP3A4), HepG2-CYP3A4 (genetically modified to express the human CYP3A4 gene) and HepaRG liver cells lines were evaluated for enzymatic activity of cytochromes P450 (CYP3A4), a routinely used enzyme for drug candidate screening. Microsome-based assays were used to validate a fluorogenic inhibition assay. However neither microsome-based assay nor the assay using CYP3A4 expressing HepG2 gave satisfying results in droplet-based format. However, HepaRG cells, a hepatic function-conserved cell line with most cytochrome and related nuclear receptors, demonstrated high relevance both for enzymatic activity testing and CYP3A4 expression induction study. For cancer research, 4 different picoliter droplet-based PCR assays were developed for the detection and quantification of mutations (NRAS, DNMT3A, SF3B1 and JAK2) present in Myelodysplastic syndromes, a heterogeneous group of clonal bone marrow hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral cytopenias. Furthermore, a single cell multistep PCR assay using encapsulation of target DNA in agarose droplets was proposed.

Made available in DSpace on 2016-08-10T10:39:27Z (GMT). No. of bitstreams: 1 CRISTIANO LUIZ RIBEIRO.pdf: 575229 bytes, checksum: 94f5c01fc439ef40126c533a4e1c43c8 (MD5) Previous issue date: 2009-03-16 / This was a study of a low incidenced disease in the population that is myelodysplastic syndrome (MDS). This disease comprises a group of heterogeneous hematopoietic disorders of clonal nature that have in common, varying degrees of bone marrow failure and distinct levels of peripheral blood cytopenias and dysplasia in cell differentiation and may provide various forms of cytogenetic changes, sometimes progressing to leukemia. For MDS, can be identified through the changes with the aid of the karyotype, determine prognosis and to classify them into risk groups, which each patient will be subjected to a more individualized treatment.The objective of this study was to evaluate the cytogenetic and correlated the cytogenetic findings found with the clinical data of patientes in an attempt to improve prognostic information in cases of MDS. This study was conducted at NPR Núcleo de Pesquisas Replicon of Universidade Católica de Goiás e LaGene - de Citogenética Humana e Genética Molecular / SuLeide Superintendência Leide das Neves Ferreira, SES-GO in partnership with the Hospital of the Universidade Federal de Goiás and Hospital Araújo Jorge. We evaluated samples of peripheral blood and bone marrow with the aid of conventional cytogenetics, of 25 patients with clinical indication of MDS, with only 15 confirmed cases with MDS were included in this study. The results of cytogenetic tests showed a wide range of chromosomal abnormalities, including trisomy of chromosomes 21 and 22, monosomy of chromosomes 17 and Y translocation between chromosomes 3q, 8q and 10q, 16q and a duplication on chromosome 1. The deletions appeared more frequently, including deletions on chromosomes 7p, 5q 10q, 11q and 12p. We also observed an isochromosome 17, a ring chromosome and a marker chromosome of unknown origin in addition to the normal karyotypes. This diversity of findings is related to genomic instability, and the monosomy and deletions may be related to inactivation of tumor suppressor genes (TSG) and translocations can activate oncogenes, so these changes are related to the ineffective hematopoiesis in MDS. Also associated with prognosis, the clinical course and progression of the disease, and providing information to assist the diagnosis, classification, monitoring, treatment choice and a more adequate understanding to the broad diversity of the disease. This research will be important to stimulate new research in this area and may establish new partnerships between research centers and treatment, primarily to provide a better quality of life for patients with MDS and their families. / Tratou-se de um estudo sobre uma doença de baixa incidência na população que é a síndrome mielodisplásica (SMD). Esta doença compreende um grupo de desordens hematopoiéticas heterogêneas de natureza clonal que tem em comum, graus variados de insuficiência medular e níveis distintos de citopenias no sangue periférico e displasias na diferenciação celular podendo apresentar várias formas de alterações citogenéticas, evoluindo algumas vezes para leucemia. Para SMD, pode-se através das alterações identificadas com auxílio do cariótipo, determinar o prognóstico e classificá-las em grupos de risco, cujo, cada paciente será submetido a um tratamento mais individualizado. O objetivo deste estudo foi avaliar as alterações citogenéticas nas síndromes mielodisplásicas, utilizando a citogenética convencional, correlacionando os achados citogenéticos encontrados com os dados clínicos dos pacientes, na tentativa de incrementar informações de valor prognóstico para os casos de SMD. O presente estudo foi conduzido no NPR Núcleo de Pesquisa Replicon da Universidade Católica de Goiás e no LaGene Laboratório de Citogenética Humana e Genética Molecular / SuLeide Superintendência Leide das Neves Ferreira, SES-GO em parceria com o Hospital das Clínicas, da Universidade Federal de Goiás e com Hospital Araújo Jorge. Foram avaliadas amostras de sangue periférico e medula óssea com auxílio da citogenética convencional de 25 pacientes com indicação clínica de SMD, sendo que somente os 15 casos confirmados com SMD foram incluídos neste estudo. Os resultados dos testes citogenéticos demonstraram uma grande diversidade de alterações cromossômicas, entre elas trissomias nos cromossomos 21 e 22, monossomias dos cromossomos 17 e Y, translocações entre o os cromossomos 3q;10q e 8q;16q e uma duplicação no cromossomo 1. As deleções apareceram com mais freqüência, entre elas citamos deleções nos cromossomos 7p, 5q 10q, 11q e 12p. Foi observado também um isocromossomo de 17, um cromossomo em anel e um cromossomo marcador de origem indeterminada além dos cariótipos normais. Esta diversidade de achados está relacionada com uma instabilidade gênomica, sendo que as monossomias e as deleções podem estar relacionadas com inativação de genes supressores tumorais (GST) e as translocações podem ativar os oncogenes, logo estas alterações estão relacionadas com a hematopoiese ineficaz em SMD. Também apresenta relação com o prognóstico, o curso clínico e a evolução da doença, oferecendo ainda informações para auxiliar o diagnóstico, a classificação, o acompanhamento, a escolha terapêutica e um entendimento mais adequado em função da grande diversidade biológica da doença. Esta pesquisa será importante para estimular novos estudos nesta área podendo estabelecer novas parcerias entre centros de pesquisas e de tratamento, principalmente para oferecer uma melhor qualidade de vida para os pacientes com SMD e seus familiares.

名古屋大学博士学位論文 学位の種類:博士(医療技術学) (課程) 学位授与年月日:平成20年9月30日