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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.

Yes / The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor and member of the basic helix-loop-helix-PAS family. AHR is activated by numerous dietary and endogenous compounds that contribute to its regulation of genes in diverse signaling pathways including xenobiotic metabolism, vascular development, immune responses and cell cycle control. However, it is most widely studied for its role in mediating 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity. The AHR target gene and mono-ADP-ribosyltransferase, TCDD-inducible poly-ADP-ribose polymerase (TIPARP), was recently shown to be part of a novel negative feedback loop regulating AHR activity through mono-ADP-ribosylation. However, the molecular characterization of how AHR regulates TIPARP remains elusive. Here we show that activated AHR is recruited to the TIPARP promoter, through its binding to two genomic regions that each contain multiple AHR response elements (AHREs), AHR regulates the expression of both TIPARP but also TIPARP-AS1, a long non-coding RNA (lncRNA) which lies upstream of TIPARP exon 1 and is expressed in the opposite orientation. Reporter gene and deletion studies showed that the distal AHRE cluster predominantly regulated TIPARP expression while the proximal cluster regulated TIPARP-AS1. Moreover, time course and promoter activity assays suggest that TIPARP and TIPARP-AS1 work in concert to regulate AHR signaling. Collectively, these data show an added level of complexity in the AHR signaling cascade which involves lncRNAs, whose functions remain poorly understood. / This work was supported by Canadian Institutes of Health Research (CIHR) operating grants (MOP-494265 and MOP-125919), an unrestricted research grant from the Dow Chemical Company, and the Johan Throne Holst Foundation to J.M. G.G. was supported by European Union Seventh Framework Program (FP7-PEOPLE2013-COFUND) under the Grant Agreement n609020 - Scientia Fellows

Abstract Cytochrome P450 (CYP) enzymes are the major metabolizers of xenobiotics, e.g. drugs, and environmental toxins. Thus, changes in CYP expression have an important impact on drug metabolism and susceptibility to chemical toxicity. In the present study, the transcriptional mechanisms of both constitutive and inducible regulation of the Cyp2a5 gene in mouse liver were investigated. Mouse primary hepatocyte cultures were used as the main model system together with cell and molecular biology methods. The key activation regions of the Cyp2a5 5' promoter were determined using reporter gene assays. Two major transcription activation sites of the Cyp2a5 5' promoter, called the proximal and the distal, were found. Transcription factors hepatocyte nuclear factor-4 (HNF-4) and nuclear factor I were shown to bind to the proximal promoter. Aryl hydrocarbon receptor nuclear translocator (ARNT) and upstream stimulatory factor bound to a common palindromic E-box element in the distal promoter region. All three response elements were shown to be essential for constitutive expression of CYP2A5 in murine hepatocytes. ARNT appeared to control Cyp2a5 transcription without a heterodimerization partner suggesting active involvement of the ARNT homodimer in mammalian gene regulation. Aryl hydrocarbon receptor (AHR) ligands were shown to induce Cyp2a5 transcriptionally by an AHR-dependent mechanism, and established Cyp2a5 as a novel AHR-regulated gene. The AHR response element and the E-box, identified in these studies, were located near to each other and close to a separately defined nuclear factor (erythroid-derived 2)-like 2 binding site in the distal region of the Cyp2a5 promoter, suggesting cooperation between these elements. Peroxisome proliferator-activated receptor gamma coactivator-1α was shown to up-regulate Cyp2a5 transcription through coactivation of HNF-4α. This indicates that xenobiotic metabolism can be regulated by modification of co-activation. The present results show that CYP2A5 is regulated by several different cross-regulatory pathways. The regulatory mechanisms involved in the transcription of the Cyp2a5 gene may also control other CYP genes, especially the human ortholog CYP2A6, and may explain some of the individual variations in the metabolism of xenobiotics.

Introdução: A artrite reumatóide (AR) é uma artropatia autoimune, de caráter inflamatório, com prevalência em torno de 1% da população. O tabagismo é considerado o principal fator de risco para o desenvolvimento da AR. O receptor de hidrocarbonetos de arila (AhR), um fator de transcrição intracelular ativado por hidrocarbonetos aromáticos componentes da fumaça do cigarro, foi identificado como alvo de regulação da diferenciação de células Th17. Objetivos: Avaliar se os polimorfismos genéticos do AhR estariam associados ao desenvolvimento da AR , e se este receptor estaria mais expresso em pacientes com AR. Pacientes e Métodos: Nós analisamos sete polimorfismos genéticos por mudança de única base (SNP) por PCR em tempo real utilizando sondas TaqMan em 138 pacientes com AR e 129 indivíduos saudáveis. A quantificação da expressão do mRNA do AhR em células mononucleares isoladas de pacientes com AR e indivíduos saudáveis foi realizada por PCR em tempo real. Resultados: Identificamos que haplótipos formados por SNPs no AhR estariam associados com desenvolvimento da AR, podendo ser fator protetor ou de risco para a doença. Em adição, os pacientes com haplótipos de risco apresentavam doença com índice de atividade elevado, principalmente quando o tabagismo estava presente. De fato, pacientes com AR apresentaram aumento na expressão de AhR (mRNA) em relação a indivíduos saudáveis. Conclusões: Em conjunto estes resultados sugerem que o AhR possui um papel importante para o desenvolvimento da artrite reumatóide. Possivelmente mutações neste receptor podem estar relacionadas com alterações na sua atividade e conseqüentemente na diferenciação de células Th17 e a susceptibilidade a AR. / Introduction: Rheumatoid arthritis (RA) is a chronic autoimmune arthropaty with inflammatory characteristics and prevalence around 1% in the population. Tabagism is the main risk factor to RA development. The aryl hydrocarbon receptor (AhR) is an intracellular transcription factor activated by aromatic hydrocarbons present in smoking, whichwas identified to be a target of regulation of Th17 differentiation. Purpose: Study the relationship of genetic polymorphisms in AhR with RA development, and if this receptor expression is upregulated in RA patients. Patients and Methods: We analyzed seven genetic single nucleotide polymorphisms by Real-Time PCR using TaqMan probes in 138 patients with Rheumatoid Arthritis and 129 healthy controls. The AhR mRNA quantization in mononuclear cells isolated from AR patients and healthy controls has been done by Realt-Time PCR. Results: We identified that AhR haplotypes were associated with RA development and that they could be protector or risk factors to disease. In addition, patients with risk haplotypes showed higher disease activity index, mainly when smoking was present. Indeed, patients with RA showed upregulation in the AhR expression (mRNA) when compared with healthy controls. Conclusions: These results suggest that AhR has an important role in AR development. Probably, mutations in this receptor could be related with alterations in its activity and consequently in the differentiation of Th17 cells and RA susceptibility.

Le récepteur aux hydrocarbures aromatiques (AhR) est un facteur de transcription activé par de nombreux xénobiotiques (molécules étrangères à l’organisme) qui régule l’expression d’enzymes et transporteurs permettant le métabolisme et l’élimination de ces ligands. Cette protéine exprimée dans toutes les cellules chez les vertébrés, joue un rôle majeur dans la détoxication et la protection des organismes vis à vis des molécules toxiques. Des orthologues de celle-ci ont été identifiés chez les invertébrés mais ne semblent pas jouer le même rôle; ils sont exprimés principalement dans des neurones et ne sont pas activés par des polluants. L’absence du AhR chez ces organismes entraîne au niveau cellulaire, des défauts de morphologie dendritique et sur le plan comportemental, des anomalies dans le comportement de nutrition. Malgré ces découvertes, peu de recherches ont été entreprises sur les conséquences d’une invalidation du AhR sur le fonctionnement du système nerveux central chez les vertébrés. Au cours de ma thèse, j’ai étudié ces conséquences au niveau moléculaire, cellulaire et comportemental: les souris AhR KO développent un nystagmus pendulaire horizontal dont l’origine est en partie liée à des défauts structuraux de la gaine de myéline. Au niveau moléculaire, nous avons mis en évidence un changement de la composition lipidique, de l’expression des gènes de la myéline et de l’inflammation, défauts qui sont retrouvés en partie chez des souris dont le AhR a été invalidé spécifiquement dans l’oligodendrocyte, la cellule responsable de la formation de la gaine de myéline. J’ai donc réalisé des études en parallèle sur la lignée murine d’oligodendrocyte, 158N, et montré que l’invalidation du AhR dans cette lignée ainsi que in vivo, modifiait l’expression du gène MAG (Myelin Associated Glycoprotein). Compte tenu du rôle du AhR en tant que récepteur de polluants, nous avons également exposé ou traité nos modèles avec de la TCDD (dioxine de Seveso) et montré que celle-ci modifiait également l’expression du gène MAG. Mes travaux démontrent donc que le AhR joue un rôle au niveau oligodendrocytaire dans la formation de la gaine de myéline. Son rôle connu en tant que récepteur de polluants laisse supposer que certaines contaminations environnementales pourraient jouer un rôle dans l’incidence de pathologies au niveau du système nerveux central, ce qui soulève de nombreuses questions en terme de santé publique. / The aryl hydrocarbon receptor (AhR) is a transcription factor activated by many xenobiotics (foreign molecules) that regulates the expression of enzymes and transporters which allow the metabolism and elimination of these ligands. This protein expressed in all cells in vertebrates, plays a major role in detoxication and protection of the organisms against toxic molecules. Some orthologs have been identified in invertebrates but do not seem to play the same role; they are expressed mainly in neurons and are not activated by pollutants. The absence of the AhR in these organisms causes at the cellular level, defects of the dendritic morphology and behaviourally, abnormalities in the feeding behavior. Despite these findings, little research has been conducted on the consequences of the AhR invalidation in the central nervous system of vertebrates. During my PhD, I studied these consequences at the molecular, cellular and behavioral : the AhR knockout mice develop a horizontal pendular nystagmus whose origin is partly related to structural defects in the myelin sheath. At the molecular level, we have shown modifications in the lipid composition, myelin and inflammation gene expression, defects that are found partly in mice whose AhR was invalidated specifically in the oligodendrocytes, the cells involved in myelin sheath formation. I therefore made parallel studies on the murine oligodendrocyte lineage, 158N, and showed that the invalidation of the AhR in this cell line and in vivo, altered MAG (Myelin Associated Glycoprotein) gene expression. Given the role of the AhR as a receptor of pollutants, we have also exposed or treated our models with TCDD (dioxin of Seveso) and showed that it also changed the expression of MAG gene. My works show that the AhR is involved in oligodendrocyte level in the formation of the myelin sheath. As the AhR is also a receptor of pollutants, some environmental contaminants may play a role in the incidence of diseases in the central nervous system, which raises many issues in terms of public health.

Introdução: A artrite reumatóide (AR) é uma artropatia autoimune, de caráter inflamatório, com prevalência em torno de 1% da população. O tabagismo é considerado o principal fator de risco para o desenvolvimento da AR. O receptor de hidrocarbonetos de arila (AhR), um fator de transcrição intracelular ativado por hidrocarbonetos aromáticos componentes da fumaça do cigarro, foi identificado como alvo de regulação da diferenciação de células Th17. Objetivos: Avaliar se os polimorfismos genéticos do AhR estariam associados ao desenvolvimento da AR , e se este receptor estaria mais expresso em pacientes com AR. Pacientes e Métodos: Nós analisamos sete polimorfismos genéticos por mudança de única base (SNP) por PCR em tempo real utilizando sondas TaqMan em 138 pacientes com AR e 129 indivíduos saudáveis. A quantificação da expressão do mRNA do AhR em células mononucleares isoladas de pacientes com AR e indivíduos saudáveis foi realizada por PCR em tempo real. Resultados: Identificamos que haplótipos formados por SNPs no AhR estariam associados com desenvolvimento da AR, podendo ser fator protetor ou de risco para a doença. Em adição, os pacientes com haplótipos de risco apresentavam doença com índice de atividade elevado, principalmente quando o tabagismo estava presente. De fato, pacientes com AR apresentaram aumento na expressão de AhR (mRNA) em relação a indivíduos saudáveis. Conclusões: Em conjunto estes resultados sugerem que o AhR possui um papel importante para o desenvolvimento da artrite reumatóide. Possivelmente mutações neste receptor podem estar relacionadas com alterações na sua atividade e conseqüentemente na diferenciação de células Th17 e a susceptibilidade a AR. / Introduction: Rheumatoid arthritis (RA) is a chronic autoimmune arthropaty with inflammatory characteristics and prevalence around 1% in the population. Tabagism is the main risk factor to RA development. The aryl hydrocarbon receptor (AhR) is an intracellular transcription factor activated by aromatic hydrocarbons present in smoking, whichwas identified to be a target of regulation of Th17 differentiation. Purpose: Study the relationship of genetic polymorphisms in AhR with RA development, and if this receptor expression is upregulated in RA patients. Patients and Methods: We analyzed seven genetic single nucleotide polymorphisms by Real-Time PCR using TaqMan probes in 138 patients with Rheumatoid Arthritis and 129 healthy controls. The AhR mRNA quantization in mononuclear cells isolated from AR patients and healthy controls has been done by Realt-Time PCR. Results: We identified that AhR haplotypes were associated with RA development and that they could be protector or risk factors to disease. In addition, patients with risk haplotypes showed higher disease activity index, mainly when smoking was present. Indeed, patients with RA showed upregulation in the AhR expression (mRNA) when compared with healthy controls. Conclusions: These results suggest that AhR has an important role in AR development. Probably, mutations in this receptor could be related with alterations in its activity and consequently in the differentiation of Th17 cells and RA susceptibility.