Les dérivés de la cytosine sont d’importantes modifications épigénétiques dont le rôle dans l’évolution de la leucémie lymphoïde chronique (LLC) n’est pas totalement exploré. Dans ce contexte, notre première étude vise à examiner le niveau global de la 5-methylcytosine (5-mCyt), 5-hydroxymethylcytosine (5-hmCyt), 5-carboxylcytosine (5-CaCyt) et 5-hydroxymethyluridine (5-hmU) dans des lymphocytes B purifiés de patients LLC (n=56) et d’individus sains (n=17). Les principaux acteurs de la régulation épigénétique (DNMT1/3A/3B, MBD2/4, TET1/2/3, SAT1) ont été évalués par PCR quantitative en temps réel. L’analyse a permis de mettre en exergue trois groupes de patients. En premier lieu, un groupe de patients stables (délai médian de progression [PFS] et délai au premier traitement [TFT] >120 mois), avec un profil épigénétique similaire au groupe contrôle. Deuxièmement, un groupe intermédiaire (PFS=84; TFT=120 mois) qui présente une augmentation de la déméthylation de l’ADN expliquée par l'induction SAT1 / TET2 pendant la progression de la maladie. Troisièmement, un groupe de patients avec une forme active de la maladie (PFS=52; TFT=112 mois) qui présentent une hyperlymphocytose, une réduction du temps de doublement des lymphocytes et des modifications épigénétiques majeures. Au sein de ce groupe, une réduction est observée pour la 5-mCyt, 5-hmCyt, 5-CaCyt et serait associée à une diminution des DNMTs, TETs et MBDs au cours de la progression de la maladie. Les profils épigénétiques mis en évidence sont indépendants du statut mutationnel IGHV mais sont associés avec les anomalies cytogénétiques. Nous nous sommes également intéressés à cette association et nous avons montré dans la deuxième étude que les modifications des dérivées de la cytosine peuvent affiner le pouvoir pronostic des anomalies cytogénétiques. En conclusion, nos résultats suggèrent que les variations de la méthylation ainsi que des intermédiaires de la déméthylation de l’ADN sont impliqués dans la progression de la LLC. / Cytosine derivatives are important epigenetic modifications whose role in the pathogenesis and evolution of chronic lymphocytic leukemia (CLL) is not fully explored. In this context, our first study aims to examine the global DNA level of 5-methylcytosine (5-mCyt), 5-hydroxymethylcytosine (5-hmCyt), 5-carboxylcytosine (5-CaCyt) and 5-hydroxymethyluridine (5-hmU) in purified B lymphocytes of CLL patients (n = 56) and healthy individuals (n = 17). The main actors in epigenetic regulation (DNMT1 / 3A / 3B, MBD2 / 4, TET1 / 2/3, SAT1) were evaluated by quantitative real time PCR. The analysis highlighted three groups of patients. First, a group of patients with stable disease (median time to progression [PFS] and time to first treatment [TFT]> 120 months), with an epigenetic profile similar to the control group. Secondly, an intermediate group (PFS = 84, TFT = 120 months) which shows an increase in DNA demethylation explained by SAT1 / TET2 induction during disease progression. Third, a group of patients with an active form of the disease (PFS = 52, TFT = 112 months) who have hyperlymphocytosis, a short lymphocyte doubling time, and major epigenetic changes. Within this group, a reduction is observed for 5-mCyt, 5-hmCyt, 5-CaCyt which is associated with a decrease in DNMTs, TETs and MBDs during disease progression. The identified epigenetic profiles are independent of IGHV mutational status but are associated with cytogenetic abnormalities. We were also interested in this association and we showed in the second study that modifications of cytosine derivatives levels can refine the prognostic power of cytogenetic abnormalities.In conclusion, our results suggest that methylation variations as well as DNA demethylation intermediates are involved in the progression of CLL.

Le domaine de l'épigénétique couvre l'ensemble des phénomènes héritables et transmissibles qui interviennent dans l'expression du génome sans modifier la séquence nucléotidique. L'information épigénétique est régulée par les modifications de la chromatine impliquant les histones et l'ADN. La méthylation de l'ADN est un phénomène réversible jouant un rôle crucial dans l'expression des gènes puisque la méthylation des promoteurs de gènes empêche leur transcription. La modulation aberrante de cette marque épigénétique est associée à diverses pathologies telles que le cancer. Cette méthylation étant réversible, elle peut être ciblée afin de reprogrammer la cellule cancéreuse. Les méthyltransferases d'ADN (DNMT), étant les enzymes responsables de la méthylation, représentent la cible principale de notre stratégie de recherche. Leur inhibition par des petites molécules est au centre de nos recherches de thérapies anticancéreuses dont les bases sont représentées par deux catégories d'inhibiteurs de DNMT existant. Les premiers sont des analogues de cytosine qui est la cible de la méthylation. Ils sont connus pour s'intégrer dans l'ADN et former un complexe covalent irréversible avec l’enzyme (complexe suicide) mais ils souffrent d'un manque de stabilité et de certains effets indésirables dus à leur incorporation dans l’ADN. Les seconds sont les inhibiteurs non nucléosidiques qui sont divers et parfois connus pour cibler d’autres enzymes. Ils ont l’avantage de pouvoir être utilisés comme sondes pour comprendre plus précisément le mécanisme d'inhibition mais ils manquent de spécificité et de sélectivité. Au cours de cette thèse, une banque de molécules a été criblée à partir de la combinaison d'un test enzymatique et d'un test cellulaire visant à inhiber ces enzymes. Les synthèses de trois familles de molécules potentiellement inhibitrices de DNMT issus de ce criblage sont décrites en expliquant le chemin de drug design emprunté pour obtenir des informations mécanistiques d’inhibition de la méthylation d’ADN, notamment de réactivité avec la cible. Les découvertes ont été inspirées par des études de modélisation permettant de mettre en évidence une sélectivité de certains inhibiteurs. La synthèse chimique a également abouti à une nouvelle voie de synthèse d’accès aux diaminopyrimidines dont l’impact permet de faciliter les études chimiques de dérivés quinazolines comme inhibiteur non nucléosidiques utiles pour les thérapies anticancéreuses. / Epigenetic is defined as the study of heritable changes in the genes expression without altering the DNA sequence. Two main processes are implicated in this field, the histones modifications and the DNA methylation. By introducing an acetyl or a methyl group on the histone tails or by methylation of DNA, the chromatin state is modified and the gene expression is controlled. Aberrant epigenetic modifications are associated with several diseases, in particular with cancer. In cancer cells, the whole DNA is hypomethylated, thus promoting genome instability, while the promoter region is hypermethylated, inducing silencing of these genes. Overall, these observations indicate that DNA methylation is a central epigenetic process in cancerogenesis. Since DNA methylation is reversible, it is possible to target the methylation process in order to reactivate tumor suppressor genes. The DNA methyltransferases (DNMTs), the enzymes responsible for DNA methylation, use the SAM co-factor at specific CpG sites to product 5-methylcytosine. Three main isoforms (DNMT1, DNMT3A and DNMT3B) are described to ensure efficient methylation process during replication. Two families of DNMT inhibitors already exist, the nucleosidiques analogues are cytidine derivatives and are toxic molecules because of their incorporation into DNA, and the non-nucleosidic analogues are less toxic but also less potent. Our strategy of drug design is based on docking study and high throughput screening (HTS) information. First, novel potent derivatives of reference inhibitors are designed from molecular modelling. Then, three different families of compounds from HTS are described with appropriate structure-activity relationship studies. Mechanistic information on DNA methylation process are described through the discovery of a reactive inhibitor of DNMT3A. The study on a family of hydrazone derivatives of gallic acid is depicted and shows its selectivity for DNMT3A, compared to DNMT1, based on docking study. An alternative chemical pathway to diaminopyrimidines is described and extended to the synthesis of quinazolone in order to synthesize new quinazoline derivatives as potent inhibitors of DNMT. Promising informations are described in this thesis to enrich epigenetic knowledge of tumor genesis and to provide new molecules for anticancer therapy.

Atherosclerosis is an inflammatory disease of the arterial walls and is the major cause of heart attack and stroke. Atherosclerosis is localized to curves or branches in the vasculature where disturbed blood flow alters endothelial cell (EC) gene expression and induces EC dysfunction. Epigenetics controls aberrant gene expression in many diseases, but the mechanism of flow-induced epigenetic gene regulation in ECs via DNA methylation has not been well studied until very recently. The goal of this project was to determine how the DNA methylome responds to flow, causes altered gene expression, and regulates atherosclerosis development. Here, we found that d-flow increases DNA Methyltransferase 1 (DNMT1) expression in ECs, and we hypothesized that this causes a shift in the EC methylome and transcriptome towards a pro-inflammatory, pro-atherosclerotic gene expression program, and further that this leads to atherosclerosis development. To test this hypothesis, we employed both in vitro and in vivo experimental approaches combined with genome-wide studies of the transcriptome and DNA methylome according to the following three specific aims: 1) to elucidate the role of DNA Methyltransferase 1 in EC function, 2) to uncover the DNA methylation-dependent EC gene expression response to flow, and 3) to discover and examine master regulators of EC function that are controlled by DNA methylation. The work presented here has resulted in new knowledge about the epigenetic EC shear response, details the previously unstudied EC methylome, and implicates specific loci within the genome for additional studies on their role in EC biology and atherosclerosis. This work provides a foundation for novel and more targeted therapeutic strategies for CVD.

Folic acid is a B vitamin involved in DNA CpG methylation. Mandated dietary fortification has led to a subsequent increase in blood folate concentration which has been correlated to a simultaneous spike in colorectal cancer incidence in Canada and the US. Several human colorectal cancer cell lines were cultivated under low (0 mg/L), standard (4 mg/L), and high (16 mg/L) folate conditions for seven days, then assessed for DNA methyltransferase1 protein expression, changes in DNA methylation, and ability to generate colonospheres in culture. Low folic acid levels generally led to reduced DNMT1 protein expression, CpG hypomethylation, and reduced colonosphere yield. High folic acid levels led to increased DNMT1 protein expression, CpG hypermethylation, and maintained colonosphere yield. This data demonstrates that varying levels of folic acid in vitro can influence the methylation status and cancer stem cell self-renewal ability of human colorectal cancer cells. / Canadian Cancer Society

La méthylation de l’ADN est un phénomène épigénétique qui joue un rôle important dans le développement des mammifères et qui est associé à une répression transcriptionnelle. La méthylation de loci CpG de l’ADN est médiée par les méthyltransférases de l’ADN – les Dnmts. La méthylation joue également un rôle clef dès les stades précoces de la cancérogenèse dans une grande partie des tumeurs où on observe une méthylation, notamment la répression des gènes suppresseurs de tumeurs et une déméthylation, notamment l’expression de séquences d’ADN parasites. <p>\ / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished

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The rationale behind targeted molecular therapy in cancer, oncogene addiction, is that tumors rely on driver oncogenes to control their proliferation and survival. Therefore, an efficacious targeted therapy should induce a dual, detrimental response to the tumor. While there have been clinical success stories using targeted therapies, even tumors that are initially sensitive invariably develop resistance. In the case of triple negative breast cancer (TNBC), despite extensive evidence pointing to its driver oncogene status, inhibitors of the Epidermal Growth Factor Receptor (EGFR) are considered clinically inefficacious. Resistance to EGFR inhibition has been predominantly described as due to genetic alterations. Yet it remains unclear why patients exhibiting the same dysregulated status of a driver oncogene react to targeted therapy, as in the case of EGFR-mutant non-small cell lung cancer, while others do not at all (i.e., TNBC). Furthermore, not all of resistance can be described by genetic alterations to EGFR, to its pathway effectors, or to compensatory pathways. Emerging data reveals that drugs can induce resistance by rewiring epigenomic, transcriptional, and translational regulatory mechanisms. Unfortunately, a major limitation in designing efficacious treatments is our inability to predict whether cell types can rewire in response to drug exposure. Therefore, it is necessary to elucidate mechanisms of growth and survival in cells that have undergone rewiring. This study characterized intrinsic resistance to EGFR inhibition in TNBC. We found that EGFR inhibition induces rewiring, which results in a resistant growth state that bypasses the EGFR-MAPK pathway as a whole. Additionally, we found that a tRNA-modifying complex masks the oncogene addiction status of EGFR in TNBC by stabilizing the protein abundance of a pro-survival protein. Importantly, this happens solely in the context of EGFR inhibition. Taken together, this study highlights potential therapeutic strategies for TNBC and strategies that can be used to improve our understanding of targeted therapy resistance, especially intrinsic resistance.