Les voies de signalisation sont des moyens de communication intercellulaires très conservées au cours de l'évolution qui contrôlent tous les aspects du développement des organismes multicellulaires. La signalisation par les récepteurs Notch oriente les destins cellulaires au cours du développement embryonnaire et régule l'homéostasie des tissus. Dans les cellules normales, l'activation de la voie Notch est soumise à une régulation très stricte, ayant pour enjeu de maintenir un équilibre au sein des tissues entre prolifération et différenciation. Cependant des disfonctionnement de la voie Notch sont à l'origine de l'augmentation de la prolifération cellulaire, perturbant ainsi cet équilibre et aboutissant à la transformation maligne des cellules. Le rôle central de NOTCH1 dans l'oncogenèse humaine est soutenu par la présence de mutations activatrices de la voie dans plus de 50% des leucémies aigues lymphoblastiques T (LAL-T), ainsi qu'un large éventail de cancers hématopoïétiques et de tumeurs solides. Au cours des dernières décennies, d'importants progrès ont été réalisés dans la compréhension des mécanismes de transduction du signal Notch et l'identification des processus biologiques qui sont influencés par la voie Notch. Les fonctions physiologiques et pathologiques de NOTCH1 requièrent sa translocation dans le noyau, mais ses partenaires nucléaires et leurs rôles dans la tumorigénèse restent peu connus. De même, les mécanismes moléculaires par lesquelles Notch active la transcription des gènes cibles de la voie restent à élucider.Afin de comprendre les fonctions nucléaires de Notch dans un contexte tumoral, nous avons, par une approche protéomique, caractérisé les complexes protéiques associés à la forme oncogénique de NOTCH1 à partir d'extraits nucléaires de cellules leucémiques T humaines. Nos travaux, associant des approches biochimiques et fonctionnelles, mettent en évidence le rôle central de plusieurs enzymes et facteurs nucléaires dans le contrôle de l'activité transcriptionnelle et les fonctions oncogéniques de Notch.La perturbation de l'expression de plusieurs facteurs nouvellement identifiées induit un arrêt du cycle cellulaire et altère la prolifération in vitro des cellules cancéreuses portant des mutations de la voie Notch. De façon remarquable, l'inhibition des partenaires de NOTCH1 est capable de supprimer la croissance tumorale de cellules leucémiques humaines dans un modèle de xénogreffe murin. Ces travaux auront un impact important dans le développement des nouvelles stratégies capables d'interférer avec les fonctions oncogéniques de Notch et pourraient s'avérer utile dans le traitement des cancers humains. / Activating mutations in NOTCH1, an essential regulator of T-cell development, are frequently found in human T-cell acute lymphoblastic leukemia (T-ALL). However, the precise mechanisms by which Notch causes T-ALL are not fully understood. While several target genes regulated by Notch have been identified in thymocytes and T-ALLs, little is known about the identity of NOTCH1 interacting partners that are required for its oncogenic activity. Using an improved tandem affinity chromatography method, followed by mass spectrometry, we have purified the intracellular active form of NOTCH1 (ICN1) and its nuclear cofactors in human T-ALL cells. We identified and validated a large set of proteins associated with ICN1, including transcriptional activators, repressors and protein-modifying enzymes. Moreover, we found that NOTCH1 interacts with lineage-specifying transcription factors and components of other signaling pathways that could cooperate to confer a specific program of gene expression. This work represents the first comprehensive analysis of Notch partners in the nucleus and provides a framework to elucidate Notch functions and regulation.We subsequently used Notch nuclear interactome as resource to expand our understanding of how ICN1 orchestrates target gene activation. Biochemical and functional experiments demonstrated that Notch activation in T-ALL cells leads to the assembly of a large multisubunit complex in the nucleus containing ICN-CSL-MAML1 as a core subunit and several classes of transcriptional regulators that act at different steps of the transcriptional activation process. These include the transcriptional activator AF4p12, the histone demethylases LSD1 and PHF8, and the SWI/SNF nucleosome remodeling complex PBAF, all of which are required for expression of Notch-responsive genes. We found that PBAF recruitment to Notch-target enhancers facilitates transcription in a chromatin context, probably through the remodeling of nearby nucleosomal structures, while LSD1 and PHF8 act through their demethylase activity to promote local epigenetic modifications. In the presence of Notch, PHF8 demethylates the repressive dimethyl H3 lysine-27 (H3K27me2) mark and LSD1 removes the repressive H3K9me2 mark from Notch-responsive enhancers to promote gene expression. However, LSD1 is also associated with CSL corepressor complex in the absence of Notch and contributes to CSL-mediated repression of Notch targets by removing the activating H3K4me2 mark, suggesting that LSD1 plays a dual role in Notch signaling regulation. AF4p12 is a poorly-characterized protein that was first identified as one of the MLL translocation partners in leukemias. While its precise role awaits further investigation, our results indicate that AF4p12 is required for RNAPII recruitment at several Notch-target genes, therefore acting as a transcriptional coactivator of ICN1. Importantly, we found that Notch cofactors are recruited to Notch-responsive genes in primary developing T cells and are required for Notch-mediated tumor growth in a xenograft T-ALL model. Thus, this newly characterized Notch-activation complex controls both Notch developmental and oncogenic functions in immature T-cells.In addition, our preliminary data provide insights into regulation of ICN1 stability and identify several protein-modifying enzymes as potential regulators of Notch signaling. Moreover, we propose that non-canonical Notch activities, as well as extensive crosstalk with lineage-specifying transcription factors, might shape the repertoire of Notch regulated genes, therefore contribute to Notch oncogenic functions in T-ALL cells.The identification of Notch-associated proteins in T-ALL uncovered novel aspects of Notch signaling and provided a powerful tool for dissecting mechanisms of Notch function and regulation that could be translated into new therapeutic approaches.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, the 5-year survival rate being less than 5%. At the time of diagnosis, 90% of PDACs extend beyond the pancreas and distant metastases are often present. Due to aggressive growth, local expansion and early appearance of metastasis, primary PDAC tumours are local enough for curative surgical resection in only 10–20% of the cases. Adjuvant chemotherapy is indicated in these curative-treated cases, with slight improvement in survival. PDAC is considered to represent a heterogeneous group of biologically and prognostically different malignancies. Characterization of these subgroups is essential and there is an urgent need for more accurate biomarkers and targeted treatments in PDAC. In the current work, we immunohistochemically investigated the expression levels and prognostic values of oxidative stress markers (8-OHdG, Keap1, Prx I, II, III, V and VI), epigenetic histone modifiers (KDM4A, KDM4B, KDM4D and SIRT1–4), and cell-cycle regulators (p16, Rb, CDK4) and DNA-repair enzymes (FEN1 and MGMT) in the cohort of surgically treated PDAC patients. We found that Keap1 expression was associated with better pancreatic cancer-specific survival. Expression of antioxidative peroxiredoxins I, III, V and VI was also connected with a more favourable tumour characteristics and Prx I and VI showed prognostic value. When considering the biology of PDAC, we noticed that pivotal epigenetic regulation also occurred in exocrine pancreatic tissue adjacent to resection margins. Overexpression of the cell-cycle regulator CDK4 and the DNA-repair enzyme FEN1 in the whole population, and elevated expression level of MGMT in the most high-risk patients were connected with worse prognosis. The results of the study can be utilized in the future when individualized therapies are being designed for PDAC patients. Due to occurrence of the epigenetic regulation also in exocrine pancreatic tissue adjacent to resection margins, it could be evaluated in future for routine diagnostics and treatment optimization. The potential role of MGMT in the development of PDAC chemoresistance should be studied in the future. / Tiivistelmä Haiman duktaalinen adenokarsinooma (PDAC) on yksi aggressiivisimmista syöpäsairauksista. Viiden vuoden elossaoloennuste on vain lähellä 5 prosenttia. Diagnoosihetkellä 90% haiman adenokarsinoomista yltää haiman ulkopuolelle ja usein kasvain on jo lähettänyt etäpesäkkeitä. Kasvutaipumuksen sekä metastasoinnin takia kuratiivinen kirurginen hoito on mahdollista vain 10–20% tapauksista. Liitännäissolunsalpaajahoito on aiheellista näissä kuratiivistavoitteisesti hoidetuissa tapauksissa. Kuitenkin vaikutus kokonaiselossaoloaikaan on melko vähäinen. Uusimman tutkimustiedon valossa PDAC:aa pidetäänkin heterogeenisenä ryhmänä biologisesti ja ennusteellisesti erilaisia tautiryhmiä. Näiden tautiryhmien tunteminen ja tunnistaminen riittävän tarkkojen merkkiaineiden avulla olisi ensiarvoisen tärkeää, jotta hoitoja voitaisiin kohdentaa niistä hyötyville potilaille. Väitöskirjatutkimuksessa selvitimme immunohistokemiallisin menetelmin oksidatiivisen stressin merkkiaineiden (8-OHdG, Keap1, Prx I, II, III, V ja VI), epigeneettisten histonimodifikaattorien (KDM4A, KDM4B, KDM4D ja SIRT1–4) sekä solusyklin säätelijöiden (p16, Rb, CDK4) ja DNA-korjausentsyymien (FEN1 ja MGMT) ilmentymistä ja ennusteellista arvoa kirurgisesti hoidetuilla PDAC-potilailla. Tutkimuksessamme totesimme, että kasvainkudoksen Keap1-ilmentymä yhdistyi parempiennusteiseen taudinkuvaan. Antioksidatiivisten peroksiredoksiinien I, III, V ja VI ilmentyminen yhdistyi niin ikään suotuisampaan kasvaimen fenotyyppiin ja Prx I ja VI osoittivat ennusteellista arvoa. Havaitsimme lisäksi, että PDAC:n biologiaan keskeistesti vaikuttavaa epigeneettistä säätelyä tapahtuu myös malignin haimakudoksen viereisessä eksokriinisessä haimakudoksessa. Solusyklin säätelijä CDK4:n ja DNA-korjausentsyymi FEN1:n voimakas ilmentyminen koko tutkimuspopulaatiossa sekä kohonnut MGMT:n ilmentyminen korkeimman riskin potilailla yhdistyivät huonompaan taudin ennusteeseen. Väitöskirjatyön tutkimustuloksia voidaan tulevaisuudessa hyödyntää, kun tutkitaan yksilöllisiä hoitomuotoja PDAC-potilailla. Koska epigeneettistä säätelyä tapahtuu myös syövän viereisessä eksokriinisessa haimakudoksessa, voidaan tulevaisuudessa tämän kudoksen arviointia mahdollisesti käyttää rutiinisti diagnostiikassa sekä hoidon optimoinnissa. MGMT:n mahdollinen rooli PDAC:n kemoresistenssin kehittymisessä tulisi tulevaisuudessa selvittää.