Au sein des hémopathies myéloïdes malignes, les syndromes myélodysplasiques(SMD) et les leucémies aiguës myéloïdes (LAM) représentent des pathologies complexes ethétérogènes résultant d’anomalies clonales des cellules souches médullaires. Elles sontcaractérisées par une hématopoïèse inefficace provoquant des cytopénies sanguines graves.Les connaissances sur les anomalies moléculaires des SMD et des LAM, notammentà caryotype normal, sont globalement pauvres et leur physiopathologie encore mal connue.Une meilleure définition moléculaire est nécessaire pour une évaluation pronostique plusprécise de ces hémopathies et pour optimiser secondairement les stratégies thérapeutiques.Cette thèse présente un panorama des classifications cytogénétiques et moléculairesactuelles des SMD et LAM ainsi que l’étude de certaines altérations moléculairesrencontrées dans ces maladies.Grâce à l’apport des techniques d’analyse génomique à grande échelle, notamment laCGH-array, notre laboratoire a identifié de nouvelles altérations génétiques, parmi lesquellesles mutations du gène ASXL1, ainsi que des altérations des gènes codant les protéines de laCohésine et des régulateurs de la protéine CBL. Nous avons analysé une combinaison demutations de gène et émis l’hypothèse d’un modèle de leucémogenèse à 4 classes demutations, afin d’apporter des pistes dans la compréhension de la physiopathologie des SMDet LAM. / Among myeloid malignancies, myelodysplastic syndromes (MDSs) represent a groupof complex diseases characterized by clonal abnormalities of bone marrow hematopoieticprecursor cells. They are defined by an ineffective hematopoiesis leading to peripheralcytopenias. About 40% of MDSs secondarily evolve to acute myeloid leukemia (AML).This risk of transformation is evaluated by several international prognostic scoringsystems like IPSS and WPSS. The WHO classification recognizes several classes of MDSsessentially based on morphology and cytogenetics features, some with a high progressionrisk, like refractory anemia with excess of blasts type 2, others with a low risk, likerefractory anemia with ringed sideroblasts. However, the classification of MDSs is stillunsatisfactory and relevant prognostic markers allowing earlier treatments for patients with ahigh risk of transformation are still lacking. The physiopathology of SMDs and AMLs withnormal karyotype remains unclear. Currently, the only potentially curative treatment isallogenic stem cell transplant, which is feasible for a restricted number of patients and candisplay side effects and failures.A better knowledge of the molecular biology of MDSs and AMLs is necessary for abetter understanding of these diseases and may provide new early prognosis indicators andbetter strategies of treatments.

Acute myeloid leukemia (AML) develops following oncogenic alterations to hematopoietic stem (HSC) and progenitor cells (HSPCs) in the bone marrow, resulting in dysregulated proliferation of immature myeloid progenitors that interferes with normal hematopoiesis. Understanding the mechanisms of HSPC protection against damage and excessive division, and how these pathways are altered during leukemic progression, is vital for establishing effective therapies. Here, we show that autophagy, a lysosomal degradation pathway, is increased in HSPCs using a novel imaging flow cytometry autophagy assay. Loss of hematopoietic autophagy following deletion of key gene Atg5 resulted in increased HSC proliferation, leading to HSC exhaustion and bone marrow failure. Although erythrocyte and lymphocyte populations were negatively impacted by autophagy loss, myeloid cells showing immature characteristics were expanded. Deletion of Atg5 in an AML model resulted in increased proliferation under metabolic stress, dependent on the glycolytic pathway, and aberrant upstream mTOR signaling. Moreover, modulation of Atg5 altered leukemic response to culture with stromal cells. Finally, primary AML cells displayed multiple markers of decreased autophagy. These data suggest a role for autophagy in preserving HSC function, partially through suppression of HSPC proliferation, and indicate that decreased autophagy may benefit AML cells. We postulate that modulation of autophagy could help maintain stem cell function, for example during transplantation, and aid AML therapy in a setting-specific manner.

EIF4E, le facteur d’initiation de la traduction chez les eucaryotes est un oncogène puissant et qui se trouve induit dans plusieurs types de cancers, parmi lesquels les sous-types M4 et M5 de la leucémie aiguë myéloblastique (LAM). EIF4E est régulé à plusieurs niveaux cependant, la régulation transcriptionnelle de ce gène est peu connue. Mes résultats montrent que EIF4E est une cible transcriptionnelle directe du facteur nucléaire « kappa-light- chain- enhancer of activated B cells » (NF-κB).Dans les cellules hématopoïétiques primaires et les lignées cellulaires, les niveaux de EIF4E sont induits par des inducteurs de NF-κB. En effet, l’inactivation pharmaceutique ou génétique de NF-κB réprime l’activation de EIF4E. En effet, suite à l’activation de NF-κB chez l’humain, le promoteur endogène de EIF4E recrute p65 (RelA) et c-Rel aux sites évolutionnaires conservés κB in vitro et in vivo en même temps que p300 ainsi que la forme phosphorylée de Pol II. De plus, p65 est sélectivement associé au promoteur de EIF4E dans les sous-types LAM M4/M5 mais non pas dans les autres sous-types LAM ou dans les cellules hématopoïétiques primaires normales. Ceci indique que ce processus représente un facteur essentiel qui détermine l’expression différentielle de EIF4E dans la LAM. Les analyses de données d’expressions par séquençage de l’ARN provenant du « Cancer Genome Atlas » (TCGA) suggèrent que les niveaux d’ARNm de EIF4E et RELA se trouvent augmentés dans les cas LAM à pronostic intermédiaire ou faible mais non pas dans les groupes cytogénétiquement favorables. De plus, des niveaux élevés d’ARNm de EIF4E et RELA sont significativement associés avec un taux de survie relativement bas chez les patients. En effet, les sites uniques κB se trouvant dans le promoteur de EIF4E recrutent le régulateur de transcription NF-κB p65 dans 47 nouvelles cibles prévues. Finalement, 6 nouveaux facteurs de transcription potentiellement impliqués dans la régulation du gène EIF4E ont été prédits par des analyses de données ChIP-Seq provenant de l’encyclopédie des éléments d’ADN (ENCODE). Collectivement, ces résultats fournissent de nouveaux aperçus sur le control transcriptionnel de EIF4E et offrent une nouvelle base moléculaire pour sa dérégulation dans au moins un sous-groupe de spécimens de LAM. L’étude et la compréhension de ce niveau de régulation dans le contexte de spécimens de patients s’avère important pour le développement de nouvelles stratégies thérapeutiques ciblant l’expression du gène EIF4E moyennant des inhibiteurs de NF-κB en combinaison avec la ribavirine. / The eukaryotic translation initiation factor EIF4E is a powerful oncogene that is overexpressed in cancers, including the M4 and M5 subtypes of acute myeloid leukemia (AML). EIF4E is regulated at multiple levels; however not much is known about the transcriptional regulation of this gene. My findings show that the nuclear factor kappa-light- chain-enhancer of activated B cells (NF-κB) is a direct transcriptional regulator of EIF4E. EIF4E levels are induced in primary hematopoietic cells and in cell lines in response to NF-κB activating stimuli. Pharmacological and genetic inhibition of NF-κB suppresses EIF4E levels. NF-κB factors RelA (p65) and c-Rel are recruited to evolutionarily conserved κB sites in the EIF4E promoter in vitro and in vivo following NF-κB activation concurrent with the recruitment of p300 and phosphorylated Pol II. Furthermore, p65 is selectively associated with the EIF4E promoter in M4/M5 AML subtypes but not in other AML subtypes or normal primary hematopoietic cells and thus represents an underlying factor in determining the differential expression of EIF4E in AML. Analysis of gene expression RNA-Seq data from The Cancer Genome Atlas (TCGA) suggests that EIF4E and RELA mRNA levels are upregulated in intermediate and poor prognosis AML but not in the cytogenetically favorable group. Additionally, elevated EIF4E and RELA mRNA levels are significantly associated with worst patient survival outcome. Furthermore, 8 new putative NF-κB target genes that may be regulated with a pattern similar to EIF4E in poor prognosis AML were in silico predicted from Chip-Seq data. Finally, 6 new transcription factors that may be implicated in EIF4E gene regulation were predicted from the analysis of ChIP-Seq data from the encyclopedia of DNA elements (ENCODE). Collectively, these findings could offer novel insights into the transcriptional regulation of EIF4E and a novel molecular basis for its dysregulation in AML. Understanding this level of regulation within the context of patient specimens is important for the development of novel therapeutic strategies to target EIF4E gene expression with specific NF-κB inhibitors combined with ribavirin.

Acute myeloid leukemia (AML) the most common acute leukemia in adults is characterized by various cytogenetic and molecular abnormalities. However, the genetic etiology of the disease is not yet fully understood. MicroRNAs (miRNAs) are small single- stranded noncoding RNAs that are negative regulators of gene expression. miRNAs influence processes of proliferation, differentiation and apoptosis. Deregulation of miRNAs expression can contribute to human disease. Circulating miRNAs are emerging biomarkers in many diseases and cancers such as breast cancer, colorectal cancer and lung cancer. However, defining a plasma miRNA signature in AML that could serve as a biomarker for diagnosis has been conducted only once. We studied miRNA expression in plasma of 8 AML patients in first detection of the disease and repeatedly after achieving remission using TaqMan miRNA microarray for 750 human miRNA. The plasma expression level of 25 miRNA was down-regulated whilst that of 20 miRNA was up-regulated in the AML group at diagnosis when compared to healthy controls. The plasma expression level of 21 miRNA was down-regulated whilst that of 13 miRNA was up-regulated in the AML group in remission compared to healthy controls. Keywords acute myeloid leukemia (AML), biomarker, microRNA (miRNA), plasma, TaqMan Low...

As células-tronco mesenquimais (MSCs) da medula óssea compreendem uma população de células multipotentes com propriedades imunorreguladoras e capacidade de secreção de fatores de crescimento, desempenhando um papel fundamental na regulação da hematopoiese. À luz dessas propriedades, alguns estudos fornecem uma análise das relações estabelecidas entre células-tronco hematopoiéticas normais (HSCs) e MSCs quando expostas à cocultura. Jing et al. (Haematologica, 2010) demonstram neste tipo de arranjos de cocultura a geração de três populações distintas de células: células não aderentes (Fração A), células aderidas à superfície de MSCs (Fração B) e células abaixo das MSCs (Fração C). Além disso, dados recentes apontam para a associação da progressão da doença com a evidência de transferência de mitocôndrias funcionais (mt) e espécies reativas de oxigênio (ROS) das MSCs para as células leucêmicas. É teorizado como um mecanismo de MSCs, a fim de reduzir as espécies reativas de oxigênio (ROS). No entanto, os desempenhos diferenciais nesses processos de transferência entre MSCs normais e leucêmicas em sistemas de cocultura em cada uma dessas populações de células distintas não foram estabelecidos. As células leucêmicas (CD45+) têm um aumento de quase três vezes na proliferação em todas as três populações após a cocultura com MSCs leucêmicas, mas não após a cocultura com MSCs saudáveis. As células CD45+ da fração A têm uma baixa taxa de proliferação em cocultura com MSCs normais comparadas com as células leucêmicas. Em 5d, as MSCs leucêmicas (CD73+) aumentam 20 vezes a coloração de mitotracker em comparação com 3d, implicando que os blastos AML estimulam MSCs a produzir mais mt, embora os MSCs normais apresentem os mesmos níveis de mitotracker em 3 / 5d. Além disso, os níveis de mtROS diminuem em 10 vezes em 5d em comparação com 3d em leucemia, mas não em MSCs normais, sugerindo uma recuperação mediada por mt em MSCs leucêmicas após a cocultura. Finalmente, o ROS total diminui 2 vezes nas células CD45+ após cocultura com MSCs leucêmicas por 5d, mas não em contrapartida normal. Em essência, esses achados sugerem diferentes mecanismos de doação mitocondrial de MSCs para blastos LMA. Além disso, o estudo fornece um passo importante nacompreensão da natureza complexa do metabolismo do tumor, não apenas na célula maligna, mas também dentro do microambiente que a suporta. / Bone marrow mesenchymal stromal cells (MSCs) comprise a population of multipotent cells with immunoregulatory properties and the capability of secreting growth factors, playing a key role in the regulation of hematopoiesis. In light of these properties, some studies provide analysis of the relations established between normal hematopoietic stem-cells (HSCs) and MSCs when exposed to coculture. Jing et al. (Haematologica, 2010) demonstrate in these kind of coculture arrangements the generation of three distinct cells populations: non-adherent cells (supernatant), phasebright cells (adhered to the surface of MSCs) and phase-dim cells (beneath the MSCs). Furthermore, recent data pointed to the association of disease progression in AML with the evidence of functional mitochondria (mt), and reactive oxygen species (ROS) transference from MSCs to the blasts cells. It is theorized as a mechanism of MSCs in order to reduce the reactive oxygen species (ROS). Nevertheless, the differential performances in these transference process among normal and leukemic-MSCs in coculture systems in each of those distinct cells populations were not established. AML cells (CD45+) have an increase of almost 2.5-fold in proliferation in all of 03 populations after coculture with leukemic-MSCs but not after coculture with a normalMSCs. The CD45+ cells in phase-bright/dim have a low proliferation rate in coculture with normal-MSCs compared with the leukemic cells. In 5d, the leukemic-MSCs (CD73+) increase 20-fold the mitotracker staining compared with 3d, implying that AML blasts stimulate MSCs to produce more mt, albeit the normal-MSCs present the same mitotracker levels in 3/5d. Additionally, the mtROS levels decrease by 10-fold in 5d compared with 3d in leukemic, but not in normal-MSCs, suggesting mt mediated recover in leukemic-MSCs after coculture. Finally, total ROS decrease 2-fold in CD45+ cells after coculture with leukemic-MSCs for 5d, but not in normal counterpart. In essence, these findings suggest different mechanisms of mitochondrial donation from MSCs to AML blats. Moreover, the study provides an important step in the understanding of the complex nature of tumor metabolism, not only in the malignant cell, but also within the microenvironment which supports it.

Les Leucémies Aiguës Myéloïdes (LAM) représentent un groupe hétérogène d’hémopathies malignes, caractérisées par une accumulation de progéniteurs myéloïdes indifférenciés. Cette accumulation proviendrait de l’existence de cellules souches leucémiques responsables de la résistance aux traitements et de la rechute de la maladie. Les Cellules Souches Leucémiques (CSL) se comportent comme les cellules souches embryonnaires, lesquelles expriment des marqueurs embryonnaires leur procurant des capacités de prolifération, d’autorenouvellement et d’absence de différenciation. Plusieurs études ont démontré le rôle des marqueurs embryonnaires (OCT4, NANOG, SOX2, SSEA1 et SSEA3) dans la cancérogénèse mais peu de données concernent les LAM. Dans le but d’identifier le rôle fonctionnel des marqueurs embryonnaires dans la LAM, une évaluation de leur expression dans les compartiments CD34+ de cellules souches hématopoïétiques et leucémiques a été réalisée. Leur sur-expression et leur implication dans les propriétés des cellules leucémiques (notamment OCT4), nous laisse penser que ces antigènes embryonnaires peuvent être utilisés comme marqueurs discriminants de la maladie résiduelle mais aussi comme cible thérapeutique potentielle. Cependant, les mécanismes de leucémogénèse par lesquels les antigènes embryonnaires seraient impliqués restent encore à être élucidés / Acute Myeloid Leukemias (AMLs) represent a heterogeneous group of malignant haemopathies, characterized by an accumulation of undifferentiated myeloid progenitors. This accumulation comes from the presence of Leukemic Stem Cells (LSCs) responsible for the resistance to treatment and relapse of the disease. LSCs behave as embryonic stem cells, which express embryonic markers giving them proliferation, self-renewal and lack of differentiation. Several studies have demonstrated the role of embryonic markers (OCT4, NANOG, SOX2, SSEA1 and SSEA3) in carcinogenesis, but there is few data in AML. In order to identify the functional role of the embryonic markers in AML, an evaluation of their expression in haematopoietic and leukemic stem cells CD34+ compartments was carried out. Their overexpression and involvement in the properties of leukemic cells (especially OCT4), suggest that these embryonic antigens can be used as discriminating markers of residual disease as well as a potential therapeutic target. However, the mechanisms of leukemogenesis by which embryonic antigens are involved remain to be elucidated

The hematopoietic stem cell (HSC) is characterized by its ability to self-renew and produce all mature blood cells throughout the life of an organism. This is tightly regulated to maintain a balance between survival, proliferation, and differentiation. The HSCs are located in specialized niches in the bone marrow thought to be low in oxygen, which is suggested to be involved in the regulation of HSC maintenance, proliferation, and migration. However, the importance of hypoxia in the stem cell niche and the molecular mechanisms involved remain fairly undefined. Another important regulator of human HSCs maintenance is the tyrosine kinase receptor FLT3, which triggers survival of HSCs and progenitor cells. Mutations in FLT3 cause constitutively active signaling. This leads to uncontrolled survival and proliferation, which can result in development of acute myeloid leukemia (AML). One of the purposes with this thesis is to investigate how survival, proliferation and self-renewal in normal HSCs are affected by hypoxia. To study this, we used both in vitro and in vivo models with isolated Lineage-Sca-1+Kit+ (LSK) and CD34-Flt3-LSK cells from mouse bone marrow. We found that hypoxia maintained an immature phenotype. In addition, hypoxia decreased proliferation and induced cell cycle arrest, which is the signature of HSCs with long term multipotential capacity. A dormant state of HSCs is suggested to be critical for protecting and preventing depletion of the stem cell pool. Furthermore, we observed that hypoxia rescues HSCs from oxidative stress-induced cell death, implicating that hypoxia is important in the bone marrow niche to limit reactive oxidative species (ROS) production and give life-long protection of HSCs. Another focus in this thesis is to investigate downstream pathways involved in tyrosine kinase inhibitor-induced cell death of primary AML cells and cell lines expressing mutated FLT3. Our results demonstrate an important role of the PI3K/AKT pathway to mediate survival signals from FLT3. We found FoxO3a and its target gene Bim to be key players of apoptosis in cells carrying oncogenic FLT3 after treatment with tyrosine kinase inhibitors. In conclusion, this thesis highlights hypoxic-mediated regulation of normal HSCs maintenance and critical effectors of apoptosis in leukemic cells expressing mutated FLT3. / <p>On the day of the defence date the title of article II was "Hypoxia, via hypoxia-inducible factor (HIF)-1, mediates low cell cycle activity and preserves the engraftment potential of mouse hematopoietic stem cells" and one of the authors is no longer included in the article.</p>

Ces travaux de thèse ont porté sur le développement de formulations innovantes et nanoparticulaire, destinées à améliorer la prise en charge des patients atteints de leucémie aigüe myéloïdes (LAM). Cette amélioration de la qualité de vie peut passer par le développement d’une formulation orale de décitabine.Trois stratégies de formulations différentes ont été développées : deux formulations de nanocapsules lipides (LNCs) avec encapsulation ou de décitabine, ou d’une prodrogue de décitabine (décitabine(C12)2) . La troisième stratégie a été le développement de particules de type liposomal, dans lesquelles la décitabine a été encapsulée. Après avoir été caractérisée sur des critères physicochimiques, chacune des stratégies basées sur les LNCs a été évaluée par des essais in vitro pour évaluer la perméabilité intestinale de la décitabine lorsqu’elle a été encapsulée. Une des stratégies a permis d’accroitre la perméabilité, in vitro, de la décitabine. L’activité sur la prolifération cellulaire a ensuite été évaluée sur des cellules humaines de LAM. Il a été démontré que l’encapsulation dans les LNCs améliore l’activité de la décitabine et de la décitabine (C12)2. Après l’ensemble de ces essais, en vue d’évaluer le potentiel avantages de ces formulations pour augmenter la demi-vie plasmatique de la décitabine, leurs stabilités dans du plasma humain a été évaluée. La décitabine (C12)2 libre et encapsulée permettent de limiter la dégradation rapide de la décitabine. Finalement, une étude de pharmacocinétique a été mise en place. L’encapsulation de la décitabine, en synthétisant au préalable une prodrogue permet d’augmenter les concentrations maximales atteintes. / The aim of this phD work was to develop nanoparticle formulations to improve patients’quality of life in case of acute myeloid leukemia (AML). These formulations could, for example, allow an oral administration of decitabine. Three different formulations were developed: two were based on lipid nanocapsules (LNCs) with an encapsulation of decitabine or a decitabine prodrug (decitabine(C12)2). The third strategy was aliposomal formulation with a decitabine encapsulation. After being characterized on physico-chemical parameters, in vitro intestinal permeability studies were performed on LNCs strategies. One strategy was able to enhance decitabine permeability. Cell proliferation studies performed on human AMLcell lines showed that encapsulations into LNCs improve decitabine and decitabine(C12)2 activities. In order to evaluate the potential of these formulations to enhance decitabine plasma half-life, their stabilities in human plasma were then assayed. Free decitabine(C12)2 or encapsulated into LNCs has been shown to limit the rapid decitabine degradation. Finally, pharmacokinetic studies were performed. Decitabine encapsulation into LNCs with a previous decitabine prodrug synthesis was able to increase maximal plasma concentrations.

Despite major advances in our understanding of the biology of childhood acute myeloid leukemia (AML) and the development of new cytotoxic drugs, the prognosis of long-term survival is still only 60-65 %. In the present research, we studied the pharmacokinetics of drugs used in the induction therapy of childhood AML and performed in vitro drug sensitivity testing of leukemic cells from children with AML. The aims of the studies were to correlate the results of the analysis to biological and clinical parameters and to identify subgroups of AML with specific drug sensitivity profiles in order to better understand why treatment fails in some patients and how therapy may be improved. Blood samples were analysed to study the pharmacokinetics of doxorubicin (n=41), etoposide (n=45) and 6-thioguanine (n=50). Doxorubicin plasma concentration and total body clearance were correlated to the effect of induction therapy, and doxorubicin plasma concentration was an independent factor for complete remission, both in univariate and multivariate analysis including sex, age, and white blood cell count at diagnosis. For etoposide and 6-thioguanine no correlation was found between pharmacokinetics and clinical effect. Children with Down syndrome (DS) tended to reach higher blood concentrations of etoposide and thioguanine nucleotides, indicating that dose reduction may be reasonable to reach the same drug exposure as in children without DS. Leukemic cells from 201 children with newly diagnosed AML, 15 of whom had DS, were successfully analysed for in vitro drug sensitivity by the fluorometric microculture cytotoxicity assay (FMCA). We found that samples from children with DS were highly sensitive to most drugs used in AML treatment. In non-DS children, the t(9;11) samples were significantly more sensitive to cytarabine (p=0.03) and doxorubicin (p=0.035) than other samples. The findings might explain the very favorable outcome reported in children with DS and t(9;11)-positive AML. A specific drug resistance profile was found for several other genetic subgroups as well. A detailed study of MLL-rearranged leukemia showed that cellular drug sensitivity is correlated both to partner genes and cell lineage, findings that support the strategy of contemporary protocols to include high-dose cytarabine in the treatment of patients with MLL-rearrangement, both in AML and acute lymphoblastic leukemia (ALL). Our results indicate that drug resistance and pharmacokinetic studies may yield important information regarding drug response in different sub-groups of childhood AML, helping us to optimize future chemotherapy in childhood AML.

The diagnosis and monitoring of AML relies predominantly on the identification of blast cells in the bone marrow and peripheral blood. While at the time of diagnosis the identification of leukemic cells is relatively easy, during remission the identification of small numbers of blasts is problematic. This is most evident by the fact that patients who achieve complete remission frequently relapse, despite pathologic examination indicating a marked reduction in leukemic cell burden. In this thesis I have explored the potential of using serum proteins secreted by leukemic cells as a means of monitoring disease in patients. To identify proteins that might be useful for monitoring, I took advantage of published gene expression arrays and looked into online bioinformatics databases. Using specific characteristics, I was able to identify approximately 107 candidate proteins secreted by AML cells. RT-PCR analysis and ELISA assays were performed to evaluate the variability of expressions and serum level differences of twelve different proteins in the list.