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EGR-1 TUMOR SUPPRESSOR IN BCR-ABL DRIVEN LEUKEMIAMaifrede, Silvia January 2015 (has links)
Chronic Myelogenous Leukemia (CML) is a hematological disease originated with a chromosomal translocation t(9;22)(q34;q11) in a pluripotent hematopoietic stem cell. CML typically evolves in 3 different clinical phases: chronic and accelerated phases, and blast crisis. Disease progression is associated with the acquisition of secondary mutations that can be of very diverse origins, including inactivation of tumor suppressor genes, as well as inhibition of differentiation, DNA repair and telomere maintenance. While current therapies are very often successful, the remaining issues of resistance and the fact that therapy will not cure CML make it important that new therapy capable of effectively curing it be developed. The early growth response-1 (Egr-1) gene is a zinc-finger transcription factor localized to the human chromosome 5. Egr-1 belongs to a family of early response genes whose expression is rapidly stimulated by growth factors, hormones and neurotransmitters. In addition, Egr-1 is a myeloid differentiation primary response (MYD) gene, and is a positive regulator of terminal myeloid differentiation that potentiates macrophage differentiation. It also has been shown that Egr-1 plays a role in the development, growth control and survival of several cell types, such as T cells, B cells, and neuronal cells in addition to myeloid cells. There is a large amount of evidence consistent with Egr-1 behaving as a tumor suppressor in hematopoietic cells, both in vivo & in vitro, in both humans & mice, making it a prime candidate for a role in CML. In this study we asked if Egr-1 would behave as a tumor suppressor in CML. To answer that we investigated the function of Egr-1 in BCR-ABL driven leukemia using a mouse m bone marrow transplantation (BMT) model. We observe that loss of Egr-1 accelerates the onset of BCR-ABL driven CML. Furthermore, through Facs analysis we showed that most animals developed myeloid leukemia, determined by the observation that the majority of GFP+ cells in the BM were positive for Gr-1 and negative for B220. Interestingly a small cohort of mice developed B-cell acute lymphoid leukemia (B-ALL); this included both WT BCR-ABL and Egr-1 KO BCR-ABL BM-transplanted groups. In addition, we demonstrated that the loss of Egr-1 caused a more aggressive leukemia, which resulted not only in more rapid onset of disease but also greater enlargement of spleen and liver, as well as a tendency to more aggressive lung infiltration of leukemic cells. We also showed that decreased apoptosis, increased proliferation rates and resulting increased viability are consistent with, and probably contribute to, the increased leukemic potential of Egr-1 KO BCR-ABL BM. In addition, we demonstrated that Egr-1 expression was downregulated in BCR-ABL expressing BM cells in vitro, and in spleens of transplanted leukemic mice. Moreover, a very interesting observation, consistent with the rapid onset and aggression of disease, was that the bone marrow of leukemic mice caused by Egr-1 KO BCR-ABL BM transplantation, were enriched with lineage negative BCR-ABL-expressing cells, significantly more so than what was observed in WT BCR-ABL-transplanted mice. That this is also an enrichment of leukemia initiating cells was demonstrated using bone marrow from primary transplantation in a secondary bone marrow transplantation assay. Furthermore, using serial replating assays of colony forming units (CFUs), it was demonstrated that Egr-1 KO BCR-ABL-expressing BM had higher self-renewal ability than WT BCR-ABL-expressing BM, exhibiting an enrichment of primitive stem cells and fewer differentiated cells relative to WT counterparts. Finally, we also analyzed expression of Egr-1 in samples of CML human patients; the results are intriguing but due to small sample size inconclusive. Further inquiry on Egr-1 in CML, including expanding the study of human CML, signaling analysis, interaction of Egr gene family members in leukemia, and gain of function experiments should identify novel players that can impact on the aggressiveness of the disease, predict outcome for currently established therapies, as well identify targets for treatment regimens or adjunct therapy. In addition, these studies can provide a paradigm for understanding how Egr-1 functions as a tumor suppressor for other cancers and types of leukemia, and also delineate pathways that can be activated/inhibited by drugs, including reactivating Egr-1 expression. / Molecular Biology and Genetics
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Targeting the antagonism of AHR by MSI2 as a novel anti-leukemic strategy in human acute myeloid leukemiaLy, Michelle January 2017 (has links)
Acute myeloid leukemia (AML) is an aggressive malignancy of the hematopoietic system, characterized by the accumulation of abnormally differentiated blast cells that is driven by leukemic stem cells (LSCs). In murine AML, Musashi-2 (MSI2), an RNA-binding protein and positive regulator of stemness, has been implicated in the propagation of disease. While its enhanced expression correlated with poor disease outcome for human AML patients, no study has yet examined its actual functional role in human leukemia.
In normal human hematopoietic stem cells (HSCs), we have recently reported the inhibitory effects of MSI2 on the pro-differentiative aryl hydrocarbon receptor (AHR) signaling pathway as a mechanism for promoting self-renewal in HSCs. We hypothesized that elevated MSI2 is critical for maintenance of human AML and promotes unrestrained self-renewal of LSCs in part through constitutive repression of AHR signaling. Our work aimed to unravel the relationship between MSI2 and AHR in the human leukemic context and to determine if activation of AHR signaling can promote differentiation.
Results confirmed that MSI2 is preferentially expressed in primary patient LSCs and is negatively correlated with the expression of AHR gene targets. Upon lentiviral knockdown of MSI2 in-vitro and in-vivo, leukemic growth was compromised and increased AHR signaling was observed. Circumventing the inhibitory role of MSI2 in AML, activation of AHR with a potent agonist impaired leukemic progenitor activity and proliferation. In-vivo studies employing reconstitution of immunodeficient mice with primary AML samples showed impairment of AML engraftment for a significant proportion of tested samples upon treatment with an AHR agonist.
Overall, our findings from this project indicated that MSI2 is required for human AML propagation and that a decrease in MSI2 inhibitory effects on AHR signaling or direct activation of the AHR signaling pathway via a potent agonist can promote AML cell differentiation and loss. / Thesis / Master of Science (MSc) / The human blood system is sustained by a population of blood stem cells that are tightly regulated in their production of stem and differentiated cells. The Musashi-2 (MSI2) protein is a key regulator of blood stem cell identity through its inhibition of the aryl hydrocarbon receptor (AHR) signaling pathway. When there is dysregulation of blood cell homeostasis, blood malignancies such as acute myeloid leukemia (AML) may arise. In this work, the relationship between MSI2 and the AHR signaling pathway was explored within a myeloid leukemic context. It was shown that MSI2 imposes inhibitory effects on AHR to promote disease progression and that its reduction could help alleviate disease burden. Additionally, it was found that activation of the AHR signaling pathway could overcome the MSI2 differentiation block to create a therapeutic effect. Overall, the results of this project shed light on novel therapeutic strategies and targets for the treatment of AML.
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Role of S6K1 in regulating self-renewal of hematopoietic stem cells and propagatoin of leukemiaGhosh, Joydeep 15 December 2015 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / The development and function of hematopoietic stem cells (HSCs) is regulated by numerous signaling pathways including Akt-mechanistic target of rapamycin complex1 (mTORC1) pathway. Dysregulation of this pathway results in impaired HSC function and contributes to the development of hematologic malignancies. Activated mTORC1 phosphorylates and subsequently activates ribosomal protein S6 kinase 1 (S6K1). To study the role of S6K1 in hematopoiesis as well as leukemogenesis, we used a genetic model of S6K1 deficient mice (S6K1-/-). We found that loss of S6K1 expression in HSCs results in reduction of absolute HSC number in bone marrow (BM). Following chemotherapy, cycling HSCs undergo apoptosis and quiescent HSCs are required to cycle to regenerate the hematopoietic system. S6K1 regulates the quiescence of HSCs and in the absence of S6K1, mice are more susceptible to repeated myeloablative stress. We also observed that loss of expression as well as gain of expression of S6K1 affects the self-renewal ability of HSCs. Interestingly, when we overexpressed S6K1, it also resulted in reduced self-renewal of HSCs. Next, we assessed the role of S6K1 in the propagation of acute myeloid leukemia (AML). The mixed-lineage leukemia (MLL) gene is required for the maintenance of adult HSCs. Translocations in MLL are detected in approximately 5-10% of adult acute leukemia patients and in approximately 70% of acute leukemias in infants. We expressed MLL-AF9 fusion oncoprotein in WT and S6K1-/- hematopoietic stem and progenitor cells (HSC/Ps) and performed serial transplantation. Upon secondary transplantation, recipients of S6K1 deficient AML cells survived significantly longer compared to controls. In vitro, pharmacological inhibition of S6K1 activity resulted in reduced growth of primary human cells expressing MLL-AF9. Both human and murine HSC/Ps expressing MLL-AF9 showed reduced mTORC1 activity upon inhibition of S6K1 suggesting that loss of S6K1 activity results in reduced Akt-mTORC1 activation both upstream and downstream of mTORC1. Overall, our studies establish a critical role of S6K1 activity in the maintenance of HSC function and in the propagation of leukemia.
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The transformed consumer : collective practices and identity work in an emotional communityDunnett, Susan January 2009 (has links)
This interpretive consumer research study interrogates the idea that people turn to consumption as a means of self-determination. Proceeding from the understanding that the consumer enacts the development of their identity within the marketplace, it takes as its subject those in transition. Its context is a support group community of people brought together by an illness - multiple myeloma. Here, through a phenomenological approach designed to explore the lived experience of illness, the thesis discovers community to be the enabling context for the consumer’s negotiation of both selfhood and the market. Conclusions are drawn about the incremental, complex nature of identity work, and the collective practices that empower it. It is found that the marketplace requires significant mediation, but that the social resources of the community can equip the consumer to navigate its challenges. This transformation is manifested in the newly-diagnosed patient’s journey from dislocation and passivity to the empowered status of ‘skilled consumer’. The importance of the often-overlooked emotional texture of exchange within consumption communities is highlighted. In conclusion, it is offered that this study extends the concept of communities of practice into the field of consumption.
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Optimizing Chemotherapy in Childhood Acute Myeloid LeukemiaPalle, Josefine January 2008 (has links)
<p>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 %.</p><p>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.</p><p>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.</p><p>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.</p><p>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).</p><p>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.</p>
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Sensibilité des cellules leucémiques aux immunoconjugués anti-CD33Savoie Rondeau, Isabelle 08 1900 (has links)
La leucémie myéloïde aigue (LMA), cancer du sang causé par une prolifération excessive des précurseurs myéloïdes à un stade précoce de maturation, est associée à une survie variant entre 20 et 30% à cinq ans en dépit des traitements de chimiothérapie les plus intensifs. L’antigène CD33 est exprimé chez les cellules malignes dans 90% des LMA ce qui en fait une cible de choix pour le développement d’immunoconjugué (IC). Trois IC composés d’un anticorps monoclonal anti-CD33 couplé à la maytansine, une toxine s’attaquant aux fuseaux mitotiques, ont été créés. Nous avons étudié l’effet de ces IC sur des cellules primaires et des lignées cellulaires LMA et étudier les mécanismes pouvant expliquer différents niveaux de sensibilité. Les études effectuées ont permis de déterminer que le niveau d’expression du CD33 n’explique pas la variation de sensibilité face aux IC. Il a été démontré que les IC anti-CD33 sont internalisés rapidement par la cellule et que le conjugué est retrouvé au niveau de l’endosome en premier lieu. Il a été confirmé que le lysosome est essentiel à l’effet anti-mitotique induit par le conjugué. Aussi, il est proposé que la protéine SOCS3 pourrait jouer un rôle dans la résistance aux IC anti-CD33 en dirigeant le complexe IC-CD33-SOCS3 vers le protéasome et ainsi empêcher la libération du composé toxique par le lysosome. Nous avons aussi conclu que les variations d’agent de liaison et l’augmentation du nombre de molécules toxiques entre les 3 IC n’ont pas été suffisantes pour augmenter leur efficacité à éliminer les cellules LMA. L’évaluation de ces IC ainsi que l’identification des mécanismes de résistance permettra de cibler les patients les plus susceptibles de bénéficier de ce type de traitement et potentiellement d’identifier de nouvelles voies pour améliorer l’efficacité des traitements. / Acute myeloid leukemia (AML), a cancer where hematopoietic precursors are arrested in an early stage of development, is associated with a poor survival rate of 20 to 30% over five years, despite intensive chemotherapy treatments. In approximately 90% of AML cases the malignant cells express CD33 antigen, which makes it a target of choice for development of immunotoxin based therapy. Three immunoconjugates (IC) composed of anti-CD33 monoclonal antibody coupled with maytansine derivative, which prevent tubulin polymerization and thus formation of mitotic spindle, were designed. These three IC were tested for their activity against several AML cell lines and primary AML patient cells and we investigate mechanisms responsible for variation in sensitivity to IC treatment. In this report, we show that differences in number of CD33 molecules on AML cell surface does not explain the observed differences in IC sensitivity. We demonstrate that binding of huMy9-6 antibody to CD33 induces rapid internalization and that it is first process through endosome. We confirm that lysosomal processing is essential for the antimitotic effect induced by IC treatment. Also, we provide evidence that SOCS3 protein may play a role in resistance of AML cells to anti-CD33 therapy by directing IC-CD33-SOCS3 complex to the proteasome and therefore affecting lysosomal decoupling of IC-CD33 and intracellular release of maytansine derivatives. Finally, the linkers and maytansine derivative modifications were not sufficient to increase sufficiently the efficacy of conjugates to eliminate higher numbers of AML cells. The identification of mechanisms responsible for increased resistance of AML cell lines and primary AMLs may allow us to identify IC responsive AML cells and also identify strategies to improve the efficacy of IC treatment.
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Recherche de nouvelles cibles moléculaires dans les syndromes myélodysplasiques et leucémies aiguës myéloïdes / Identification of new molecular targets in myelodysplastic syndromes and acute myeloid leukemiasRocquain, Julien 29 November 2010 (has links)
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.
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Autophagy in hematopoiesis and acute myeloid leukemiaWatson, Alexander Scarth January 2014 (has links)
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.
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Understanding the transcriptional control of EIF4E and its dysregulation in acute myeloid leukemia: role of NF-κBHariri, Fadi 08 1900 (has links)
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.
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Caractérisation de l'oncogène MLF1 (Myeloid Leukemia Factor 1)Bourgoin, Vincent January 2005 (has links)
Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.
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