RESUME Le proto-oncogène c-myc code pour un facteur de transcription qui est impliqué dans de multiples processus cellulaires tels que la prolifération, la différenciation et l’apoptose. Une dérégulation de son expression suite à des altérations génétiques (mutation, translocation, amplification) est retrouvée dans plusieurs tumeurs telles que le lymphome de Burkitt, des plasmacytomes murins ainsi que des tumeurs non-lymphoïdes. c-myc est un gène dont l’expression est régulée à différents niveaux. Chez l’homme, le gène c-myc est transcrit à partir de quatre promoteurs alternatifs appelés respectivement P0, P1, P2 et P3. P1 et P2 sont les deux promoteurs les plus utilisés. Ensemble, ils permettent de former 90% des transcrits c-myc dans des cellules normales. Les promoteurs P0, P1 et P2 permettent la transcription de trois ARNms qui comportent deux codons d’initiation de la traduction (un CUG et un AUG). L’utilisation alternative de ces deux codons d’initiation est à l’origine de la synthèse de deux protéines (c-Myc 1 et c-Myc 2) ayant à la fois des fonctions identiques et distinctes. La grande taille des parties 5’ non-traduites ainsi que la présence dans celles-ci de phases ouvertes de lecture sont des éléments défavorables à la traduction de l’ORF codant pour les protéines Myc par un mécanisme classique d’initiation de la traduction. Notre laboratoire avait précisément montré que les protéines c-Myc sont synthétisées par un processus d’initiation interne de la traduction. Les ARNms dont l’initiation de la traduction s’effectue par entrée interne des ribosomes présentent une structure spécifique appelée IRES (Internal Ribosome Entry Site). Cette structure permet la fixation du ribosome directement à proximité du codon d’initiation. Dans le cas des ARNms c-myc, on retrouve une IRES se situant en amont des codons CUG et AUG qui permet la synthèse des protéines c-Myc1 et 2 respectivement. Un tel mécanisme permet la synthèse des protéines c-Myc dans des conditions où toute traduction dépendante de la coiffe est inhibée (mitose, apoptose). Au cours de mon travail, tout d’abord j’ai montré qu’une séquence de 40 nt dans les transcrits P2 permet à elle seule une initiation interne efficace de la traduction. Nous avons déterminé aussi que cette séquence, appelée B4, est active dans quatre types cellulaires différents avec une efficacité variable et qu’elle active la traduction indépendamment de l’ORF placée en aval. D’autre part, il a été déterminé que la séquence B4 recrute le complexe de préinitiation 43S, qui ensuite scanne le messager jusqu’aux codons initiateurs comme c’est le cas de l’IRES du rhinovirus. Une analyse plus détaillée de la séquence B4 a permis d’identifier trois plus petites séquences de plus ou moins 14 nt (Ti1, Boucle, Ti2), qui indépendamment l’une de l’autre permettent une entrée interne des ribosomes. Il a été déterminé que la présence du motif A-N6-AC dans la séquence de Ti2 était importante pour l’activité IRES de celle-ci. Cependant, ce même motif également présent dans la séquence Ti1 n’est pas essentiel à l’activité IRES de Ti1. Par la suite, nous avons démontré que l’IRES de c-myc nécessite pour son activité un évènement nucléaire. Nous avons donc entrepris la recherche de facteurs cellulaires impliqués dans l’activité de l’IRES de c-myc. Dans un premier temps, nous avons exclu le rôle de certaines protéines connues pour activer d’autres IRES dont le mécanisme de recrutement du complexe de préinitiation est similaire. Ainsi, nous avons montré, par des expériences de complémentation d’un RRL, que les protéines PTB et unr connues pour activer l’IRES du rhinovirus ne contribuent pas à l’activité de l’IRES de c-myc. De plus, la complémentation de RRL avec des extraits S10 ou nucléaires de cellules HeLa n’a pas permis d’identifier des protéines impliquées dans l’activité IRES de c-myc. D’autre part, des méthodes alternatives d’interaction d’ARN et de protéine comme le triple hybride ou la chromatographie d’affinité d’ARN n’a pas permis dans un premier temps de détecter une interaction entre un facteur non canonique et l’IRES de c-myc. Dès lors, l’existence de facteurs cellulaires impliqués dans l’activité de l’IRES de c-myc reste à déterminer.

No description available.

Interactions between DNA and ligands are important in the rational design of drugs and in research into DNA function. In particular, the interaction of DMZ with DNA structures named “G-quadruplexes” was considered. G-quadruplexes are structures present in telomeres and several oncogenes. The main purpose of this project was to provide a computational tool to study DNA ligand interactions using a variety of molecular modeling techniques that include molecular docking, molecular dynamics simulations (MD) and MM/PBSA (Molecular Mechanics/Poisson Boltzmann Surface Area). We investigated the binding modes and binding affinities of DMZ with c-MYC G-quadruplexes (G4s). We found that the conformation and structural design of the quadruplex can dramatically influence the binding profiles of the ligand. The binding free energies for each site were estimated by the MM/PBSA method. The binding of small molecules to DNA can result in the disruption of oncogene transcription, making it an effective anticancer strategy.

The transcription factor c-Myc plays a central role in cell growth and proliferation owing to the large number of genes it transactivates or transrepresses and to the fact that these genes are in turn implicated in these cellular processes. Also, c-Myc's deregulation and/or overexpression contribute to most aspects of tumoral cellular biology. As a heterodimer with Max, c-Myc activates the transcription of genes leading to cell proliferation and represses the transcription of cytostatic genes such as p15[superscript ink4b] and p21[superscript CiP1]. In contrast to the transactivation mechanism, our current understanding of the transrépression by c-Myc is still incomplete, aside from the fact that an interaction with Miz-1 is essential. Coupling preliminary results from a collaboration with Martin Eilers' group to data obtained following a bioinformatics' approach to predict Miz-1 DNA binding, we were able to elaborate a now transrepression mechanism for c-Myc/Miz-1. In this mechanism, the c-Myc/Max heterodimer directly binds the noncanonical E-box sequences present in the promoters and provoke the supercoiling of DNA assisted by the interaction between c-Myc and Miz-1. This supercoiling impairs accessibility to the initiation site to the transcriptional machinery. This thesis aims at the study on a structural and biophysics viewpoint of the determinants for the specific heterodimerization and DNA binding by c-Myc and Max and the interaction between c-Myc and Miz-1 in order to validate our mechanistic model for the transrépression by c-Myc. In chapter 1, we present an overview of the actual knowledge on Myc and the repression model along with some of the results that led to its elaboration. Chapters 2 and 3 report the study of the structural determinants for the heterodimerization and E-box binding by the b-HLH-LZ domains of c-Myc and Max. Our model allows to predict that b-HLH-LZ peptides able to bind the E-box present in the repressed promoters without interaction with Miz-1 could reverse the inaccessibility and reactivate p15[superscript ink4b] and p21[superscript Cip1] expression in cancer cells where c-Myc is overexpressed.The results presented in this thesis will find application in the development of new inhibitors of c-Myc eventually leading to novel therapies to fight cancer.

Murine plasmacytoma models human cancers that involve deregulation of MYC. Overexpression and duplication of the immature colon carcinoma transcript 1 gene, Ict1, along with MYC deregulation may contribute to the aggressive mechanism for disease development in fast-onset mouse plasmacytomas. This study looks at Ict1 and c-MYC overexpression in mouse PreBmycER cells that serve as a cell culture model for MYC-dependent plasmacytomagenesis. An Ict1 inducible vector was transfected into the mouse PreBmycER cell line that contains inducible c-MYC. This allowed us to examine the effect of overexpression of ICT1 and c-MYC proteins simultaneously or each separately, on selected hallmark cancer cell traits such as increased proliferation, evasion of apoptosis and increased genomic instability. An increase in the number of cells in the S-phase was observed by 15 % and up to 20 % at 24 and 36 hours respectively, and cell doubling time shortened by almost 2 hours at 24 hours during peak ICT1 and c-MYC overexpression. Although, no noticeable change in apoptosis levels, or large scale genomic alterations were detected up to 96 hours post-ICT1 and c-MYC peak-overexpression, genomic instability was observed when MYC protein was overexpressed with or without ICT1 protein overexpression. Extrachromosomal elements increased in number and size during conditional MYC deregulation, and most of these elements (25 %) classified as Chromosome 11. These findings support Ict1 as a candidate gene that is selected for by MYC-deregulation during plasmacytomagenesis, and show promise that the experimental model of induced MYC and ICT1 overexpression in mouse PreB cells, deserves further investigation, specifically with in vivo studies. / October 2016

Currently there is no curative therapy for Chronic Myelogenous Leukemia (CML), and patients must remain on the current prescribed treatment, tyrosine kinase inhibitors (TKI), indefinitely. Although many patients can survive in the chronic phase of the disease under TKI treatment, some patients do progress to the terminal blast crisis phase of the disease. Patients in this terminal phase do not respond to TKI treatment. We evaluated the therapeutic benefit of targeting the oncogene c-MYC in CML, using the CML cell line K562. This was achieved by inhibiting the enzyme O-linked β-N-acetylglucosamine Transferase (OGT), using two indirect inhibitors 2-deoxyglucose and Azaserine, and the direct inhibitor ST078925. Treatment with these inhibitors resulted in decreased half-life of c-MYC protein in K562, reduced c-MYC protein in K562 cells, and reduced K562 cell growth. Together these results suggest that targeting c-MYC through OGT may be a potential therapeutic option for patients with CML.

No description available.

8q24.21 is a highly amplified region in cancer and associated with many epithelial cancer such as bladder, breast, colorectal and prostate cancer. The proto-oncogene c-myc is located in this region and surrounded by many lncRNAs genes such as PCAT family, CCAT family, PRNCR1. In this study, we used CRISPR-Cas9 constructs to knock out PCAT1, PRNCR1, CASC8, CASC11 and also the sequences between PCAT1-CASC11 and CASC8-CASC11in the prostate cancer cell PC3. The transfected cells with CRISPR-Cas9 targeting CASC11 gene had less proliferation ability comparing with the transfected cells with CRISPR-Cas9 targeting PCAT1, PRNCR1 or CASC8. The role of CASC11 in cancer progression and development is obscure. In our study, The CASC11 Knockout efficiency was 90% compare to the control cell. Furthermore, the study showed the importance of CASC11 in cell proliferation by significantly decreasing in the forming colonies and the growth rate comparing to the control. Also, MMP2, MMP3 and MMP9 expression levels were detected in the transfected cell by using real time PCR and the result revealed the crucial role for CASC11 in metastasis and migration. The slug and vimentin expression levels were reduced in the transfected and the double transfected clones which indicate the possible role of CASC11 in epithelial mesenchymal transition and cell motility. Taken together, our study revealed that the lncRNA CASC11 plays important roles in prostate cancer progression and metastasis by promoting the cell proliferation and migration.

Breast cancer (BC) is a complex disease with over 25,000 new diagnoses made in Canadian women every year. The disease can be caused by inactivation of the ataxia telangiectasia mutated (ATM) pathway, a major anti-tumor mechanism that protects against the abnormal cell division and growth that occurs in breast cancer, but how the pathway is inactivated has yet to be completely elucidated. BMI1 is an established oncogene that is overexpressed in BC and is associated with poor disease prognosis. BMI1 is a component of the polycomb repressive complex 1 (PRC1) that acts to repress transcription of the ARF/INK4A locus encoding two important tumor suppressor genes. We have recently shown a novel property of BMI1 in attenuation of ATM function independent of this locus. We thus hypothesize a role of BMI1 in promoting BC formation through inhibiting oncogene-induced ATM activation, allowing cancer-promoting genes to induce abnormal cellular growth. To examine this hypothesis, we transiently expressed oncogene c-Myc with or without BMI1 co-expression. As expected, ectopic c-Myc expression upregulated γH2AX, a demonstrated target of ATM; concurrent BMI1 expression reduced the γH2AX levels. Similar observations were also obtained using a BMI1 mutant deficient in promoting PRC1-mediated repression of the ARF/INK4A locus. These observations support the concept that BMI1 contributes to ATM inactivation during BC tumorigenesis through mechanisms independent of PRC1. To further examine this concept, we investigated the association of γH2AX and BMI1 in vivo. In MCF7 cell-produced xenograft tumors, the presence of γH2AX nuclear foci was clearly observed, indicative of ATM activation during BC tumorigenesis. In xenografts generated by MCF7 cells stably expressing BMI1, a trend of reduction in γH2AX nuclear foci was observed. To further model BMI1’s pathological relevance in c-Myc induced BC under a more physiological setting, we are developing transgenic mouse models (GEM) with breast-specific c-Myc expression with or without a breast-specific BMI1 knockout. The goal of these experiments is to recapitulate the above in vitro and in vivo observations. The expectation, should it be achieved, will significantly strengthen the connection between BMI1 and ATM during breast cancer tumorigenesis. / Thesis / Master of Science (MSc) / Breast cancer (BC) is a complex disease with over 25,000 new diagnoses made in Canadian women every year. Normally there are anti-tumor mechanisms in place to protect against the abnormal cell division and growth that is associated with breast cancer. We propose a novel function of protein BMI1 to explain how breast cancer cells override these protective pathways. BMI1 is known to contribute to BC through inhibiting production of key tumor suppressing proteins and has recently been shown to decrease activity of the ataxia telangiectasia mutated (ATM)- mediated tumor inhibiting pathway. We propose a novel role of BMI1 in promoting breast tumor formation through inhibiting the ATM-mediated anti-tumor barrier, allowing cancer-promoting genes (oncogenes) to induce abnormal cellular growth. BMI1 was shown to be able to reduce oncogene induced ATM activity, in an action independent of established mechanisms. Additionally in MCF7 BC tumors, the presence of BMI resulted in a trend of reduction in ATM activity. Continued work to develop a transgenic mouse model with a breast specific BMI1 knockout will help further our understanding of BMI1’s role in BC tumorigenesis.

A l’instar du lymphome de Burkitt (LB) avec la translocation de MYC, les lymphomes diffus à grandes cellules B (DLBCL) par d'autres mécanismes (mutation, amplification, dérégulation du promoteur) sont associés à une dérégulation de c-Myc, facteur de transcription maitre de la prolifération. Les DLBCL sont classés en deux sous-groupes: « centre germinatif » (GCB) et « cellule B activée » (ABC) avec activation constitutive de NF-κB. Cette activation constitutive de NF-κB peut être le résultat d'altérations génétiques (MYD88, A20, TRAF2 et TRAF5) ou de l'activation du BCR ou CD40. Ces caractéristiques soulèvent la question de la synergie d’action entre NF-κB et c-Myc dans les ABC-DLBCL. Nous avons analysé l’effet d'une activation continue de c-Myc dans un contexte de sur-activation de NF-κB par plusieurs inducteurs. Nos résultats montrent que la surexpression de c-Myc dans un contexte d'induction de NF-κB, i) par le programme EBV latence III, apporte un avantage sélectif à ces cellules (expression génique en faveur d'un métabolisme élevé, prolifération intense et protection contre apoptose), ii) par le TLR9 (modèle in vivo et in vitro), augmente la survie et la prolifération des lymphocytes B des souris λc-Myc (augmentation des cellules B activées, splénomégalie, augmentation de la prolifération des lymphocytes B, modification du microenvironnement tumoral), et iii) par CD40, induit une lymphomagenèse B très agressive dans les souris doubles transgéniques CD40/Myc, les tumeurs ont un phénotype proche des ABC-DLBCL. Ces résultats suggèrent que c-Myc est un événement co-transformant dans les lymphomes agressifs avec un phénotype activé par NF-κB, tel que les ABC-DLBCL. / Not only Burkitt lymphoma (BL) with the translocation of MYC, but also diffuse large B-cell lymphoma (DLBCL) by other mechanisms (mutation, amplification, promoter dysregulation…) are associated with dysregulation of c-Myc, the master transcription factor for proliferation. DLBCL’s are classified in two subgroups: “Germinal center B-cell” (GCB) without and “activated B-cell” (ABC) with constitutive NF-κB activation. This constitutive activation of NF-κB can be the result of genetic alterations (MYD88, A20, TRAF2, and TRAF5) or the activation of B-cell receptor or CD40. These features raise the question of the synergy of action between NF-κB and c-Myc in ABC-DLBCL. We analyzed the effect of a continuous activation of c-Myc in a context of over-activation of NF-κB by several inductors. Our results show that overexpression of c-Myc in the context of induction of NF-κB, i) by EBV latency III program, provides a selective advantage to those cells (gene expression in favor of a high metabolism, intense proliferation and protection against apoptosis), ii) by TLR9 (in vivo and in vitro model) increases the survival and proliferation of B lymphocytes of λc-Myc mice (increase of activated B cells, splenomegaly, increased B cells proliferation, modification of tumor microenvironment), and iii) by CD40, induces a very aggressive B lymphomagenesis in CD40/Myc double transgenic mice, the tumors have a phenotype close to ABC-DLBCL. These results suggest that c-Myc is an NF-κB co-transforming event in aggressive lymphomas with an activated phenotype by NF-κB, such as ABC-DLBCL.