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<p> Proper gene expression programs cellular activities, while aberrant manipulation of transcription factors often leads to devastating consequences, such as cancer or cell death. The transcription factor family activator protein-1 (AP-1) plays an important role in many cellular activities including cell transformation, proliferation and survival (Shaulian and Karin 2002). However, little has been done to obtain a global view of the role of individual AP-1 members and how they cooperate in many cellular activities. We have discovered that blocking the AP-1 pathway by a c-Jun dominant negative mutant, TAM67, induced cell death in RSV-transformed primary chicken embryo fibroblasts (CEF), suggesting that AP-1 activity is vital for cell survival upon v-Src transformation. In addition, accumulation of cytoplasmic vesicles was observed in the cytoplasm of a proportion of RSV-transformed CEF expressing TAM67. Oil-red staining of these vesicles indicated the presence of lipid droplets in these cells, suggesting that the inhibition of AP-1 promotes the adipogenic conversion of v-Src transformed CEF. To understand the role of individual members of the AP-1 family, a retroviral-based shRNA expressing system was designed to stably downregulate individual AP-1 members. This retroviral-based RNAi system provided sustained gene downregulation of AP-1 family members. Reduction of the c-Jun protein level by shRNA induced senescence in normal CEF, while it modestly downregulated AP-1 activity in RSV -transformed CEF indicating that c-Jun is not the main component of the AP-1 complex in RSV-transformed CEF. Inhibition of JunD expression induced apoptosis and was deleterious to both normal and RSV-transformed CEF, suggesting that JunD is crucial for the survival of CEF. Transient express10n reporter-assays also showed that loss-of-function of JunD by shRNA dramatically repressed AP-1 activity. Hence JunD is the main component of the AP-1 complex that regulates the survival of CEF. Furthermore, we determined that loss of JunD expression resulted in an elevated level of tumour suppressor p53. Co-inhibition of p53 and JunD restored the transforming ability of v-Src transformed CEF, as indicated by foci formation in soft agar assays. Hence, repression of p53 induction was able to bypass the death signal released as a result of AP-1 inhibition in v-Src transformed CEF. Downregulation of Fra-2 (Fos-related antigen 2) level by shRNA did not affect the proliferation of normal CEF. However, RSV -transformed CEFs expressing fra -2 shRNA were transformation-defective with the presence of multiple vesicles in cytoplasm. Oil-red staining of these vesicles indicated the presence of lipid droplets, which resembles the effect of T AM67 in RSV -transformed CEF indicating that Fra-2 blocks differentiation. These findings help us to understand the role of individual members of the AP-1 transcription factor family in normal and RSV -transformed CEF. Importantly, global gene profiling of v-Src transformed CEF expressing shRNA for individual AP-1 members will improve our knowledge of the transformation process. Functional characterization of the cascade will rely on the use of retroviral-based shRNA expressing system as described above. </p> / Thesis / Doctor of Philosophy (PhD)

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La leucémie myéloïde aiguë (LMA) est l’une des formes de cancer le plus génétiquement hétérogène avec un faible taux de survie globale sur 5 ans de 21 % 1. En effet, les traitements standards sont peu efficaces pour les patients plus âgés, ceux présentant des comorbidités, ceux en rechutes ou pour les cas résistants. Bien que notre compréhension génétique de la LMA ait progressé ces dernières années, les traitements ont peu évolué et le taux de survie reste toujours faible chez les patients. À la suite d’un criblage de plus de 10 000 composés sur 56 échantillons primaires de LMA, nous avons regroupé des composés actifs contre la LMA à l’aide d’une nouvelle approche développée par notre groupe, nommée Compound Correlation Cluster (CCC) 2. L’hypothèse à l’origine de cette méthode de regroupement est que les composés d’un même CCC agissent sur les mêmes cibles moléculaires. Dans le présent mémoire, nous caractérisons une nouvelle petite molécule issue d’un de ces CCC, le BMS-249 du CCC88, un potentiel agent thérapeutique prometteur ciblant les sous-types de LMA à mauvais pronostic clinique. En effet, nous avons démontré que les spécimens de LMA TP53 mutés, à caryotype complexe, ou à risque défavorable, sont plus sensibles au BMS-249. Grâce à un criblage CRISPR/Cas9 sur l’ensemble du génome humain, nous avons déterminé que les gènes importants de la voie moléculaire du mévalonate et du cholestérol étaient impliqués dans son mécanisme d’action. Par des études de synergie et de quantification des lipides, nos résultats montrent que le BMS-249 impacte la voie métabolique du cholestérol dans des modèles de cellules leucémiques. De manière intéressante, des études récentes sur les statines ont montré que le métabolisme du cholestérol est une cible thérapeutique d’intérêt en LMA 3, et l’effet du BMS-249 sur cette voie démontre effectivement qu’elle est cruciale pour la survie des cellules cancéreuses. Dans l’avenir, de plus amples études sur la relation entre la structure et l’activité du BMS-249, à des fins d’optimisation et d’identification directe de la cible moléculaire, seront grandement pertinentes. Globalement, nos résultats ont démontré que l’approche par CCC permet de rapidement trouver des voies moléculaires importantes pouvant être ciblées pour le développement de nouveaux agents thérapeutiques contre la LMA. / Acute myeloid leukemia (AML) is one of the most genetically heterogeneous forms of cancer with a low 5-year overall survival rate of 21% (1). Indeed, standard treatments are not very effective for older patients, those with comorbidities, those in relapse or for drug resistant cases. Although our genetic understanding of AML has progressed in recent years, treatments have slowly evolved, and the survival rate remains low among patients. Following a screening of more than 10,000 compounds on 56 primary AML samples, we clustered compounds active against AML using a novel approach developed by our group, named Compound Correlation Cluster (CCC) (2). The assumption behind this clustering method is that compounds of the same CCC act on the same molecular targets. In this thesis, we characterize a new small molecule derived from one of these CCCs, the BMS-249 of CCC88, a potential promising therapeutic agent targeting AML subtypes with poor clinical outcomes. Indeed, we demonstrated that specimens of AML TP53 mutated, with a complex karyotype, or at unfavorable risk are more sensitive to BMS-249. Through a human genome-wide CRISPR/Cas9 screen, we determined that important genes of the mevalonate and cholesterol molecular pathway are involved in its mechanism of action. Through synergy and lipid quantification studies, our results show that BMS-249 impacts the cholesterol metabolic pathway in leukemic cell models. Interestingly, recent studies on statins have shown that cholesterol metabolism is a therapeutic target of interest in AML (3), and the effect of BMS- 249 on this pathway effectively demonstrates that it is crucial for cancer cell survival. In the future, further studies on the relationship between the structure and activity of BMS-249, for the purpose of optimization and direct identification of the molecular target, will be highly relevant. Overall, our results demonstrated that the CCC approach allows to quickly find molecular pathways that can be targeted for new therapeutic agents against AML.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

We have identified a novel Cyclin, called Cyclin O, which is able to bind and activate Cdk2 in response to intrinsic apoptotic stimuli. We have focused on the study of Cyclin O&#945; and Cyclin O&#946;, alternatively spliced products of the gene. Upon treatment with different stress stimuli, transfected Cyclin O&#945; accumulates in dense aggregations in the cytoplasm compatible with being Stress Granules (SGs). Furthermore, we have seen that Cyclin O&#946; and a point mutant of the N-terminal part of the protein constitutively localize to the SGs. Although both alpha and beta isoforms are proapoptotic, only Cyclin O&#945; can bind and activate Cdk2. On the other hand, we have demonstrated that Cyclin O is upregulated by Endoplasmic Reticulum (ER) stress and is necessary for ER stress-induced apoptosis. Cyclin O activates specifically the PERK pathway and interacts with the PERK inhibitor protein p58IPK. Moreover, Cyclin O participates in the activation of other eIF2&#945; kinases. We have also observed that a pool of Cyclin O is located in active mitochondria, suggesting a function of the protein linked to oxidative metabolism.Hemos identificado una nueva Ciclina, llamada Ciclina O, que es capaz de unirse y activar Cdk2 en respuesta a estímulos apoptóticos intrínsecos. Nos hemos centrado en el estudio de la Ciclina O&#945; y la Ciclina O&#946;, productos de splicing alternativo del gen. En respuesta a diferentes tipos de estrés, la Ciclina O&#945; se acumula en agregaciones citoplásmicas densas que podrían corresponder a Gránulos de Estrés (SGs). Además, hemos visto que la Ciclina O&#946; y un mutante puntual de la parte N-terminal de la proteína se localizan constitutivamente en los SGs. Aunque las dos isoformas alfa y beta son proapoptóticas, solo la Ciclina O&#945; es capaz de unirse y activar Cdk2. Por otro lado, hemos demostrado que los niveles de Ciclina O se incrementan en respuesta al estrés de Retículo Endoplásmico (RE) y que esta proteína es necesaria para la inducción de apoptosis dependiente de estrés de RE. La Ciclina O activa específicamente la vía de PERK e interacciona con la proteína inhibidora de PERK p58IPK. Además, la Ciclina O participa en la activación de otras quinasas de eIF2&#945;. La Ciclina O se localiza en mitocondrias activas, lo que sugiere una función de la proteína ligada al metabolismo oxidativo.

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