La topoisomérase I (Top1) élimine les surenroulements de l'ADN générés lors de la transcription en produisant transitoirement des complexes de clivage Top1-ADN (Top1cc). Ces Top1cc transitoires peuvent être stabilisés par les camptothécines, dont sont dérivés des agents anticancéreux, et par les fréquentes altérations de l'ADN. Bien que les Top1cc stabilisés soient des lésions qui bloquent efficacement la transcription, la compréhension des processus moléculaires qui résultent du blocage des complexes transcriptionnels par les Top1cc est encore limitée. Des travaux précédents ont montré que les Top1cc stabilisés produisent des cassures double-brin (DSBs) de l'ADN dépendantes de la transcription qui activent ATM. Dans ce projet, nous avons utilisé des cellules quiescentes traitées avec la camptothécine pour induire des Top1cc bloquant la transcription et nous avons étudié les mécanismes de la production et de la signalisation des DSBs. Nous montrons que les DSBs sont produites préférentiellement dans les régions sub-télomériques lors de la réparation des Top1cc bloquant la transcription par les cassures simple-brin de l'ADN générées après la protéolyse de la Top1 et avant l'action de Tdp1. L'analyse de la signalisation de ces DSBs révèle une nouvelle fonction de DNA-PK dans la promotion de l'ubiquitinylation conduisant (i) à l'activité complète d'ATM aux sites des DSBs en favorisant l'ubiquitination d'H2AX et H2A, et (ii) à l'augmentation de la réparation des Top1cc en favorisant la protéolyse de la Top1. Enfin, nous montrons que les DSBs co-transcriptionnelles induisent la mort des cellules quiescentes. L'ensemble de ces résultats apportent un nouvel aperçu des réponses cellulaires aux camptothécines, et suggèrent que les DSBs qui résultent des Top1cc bloquant la transcription puissent contribuer à la pathogénèse du syndrome neurodégénératif SCAN1, qui est causé par une déficience en Tdp1. / Topoisomerase I (Top1) removes DNA supercoiling generated during transcription by producing Top1-DNA cleavage complexes (Top1cc). These transient Top1cc can be stabilized by camptothecins, from which anticancer drugs are derived, and by common DNA alterations. Although stabilized Top1cc are potent transcription-blocking lesions, our understanding regarding the molecular processes resulting from the stalling of transcription complexes by Top1cc is currently limited. Previous work showed that stabilized Top1cc produce transcription-dependent DNA double-strand breaks (DSBs) that activate ATM signalling. In this project, we used camptothecin-treated quiescent cells to induce transcription-blocking Top1cc and study the mechanisms of DSB production and signalling. We show that DSBs form preferentially at subtelomeric regions during the repair of transcription-blocking Top1cc from DNA single-strand breaks generated after Top1 proteolysis and before Tdp1 action. Analysis of DSB signalling reveals a novel function of DNA-PK in promoting protein ubiquitination leading (i) to full ATM activity at DSB sites by promoting H2AX and H2A ubiquitination, and (ii) to enhancement of Top1cc repair by promoting Top1 proteolysis. Finally, we show that co-transcriptional DSBs kill quiescent cells. Together, these findings provide new insights into the cellular responses to camptothecins and further suggest that DSBs arising from transcription-blocking Top1cc may contribute to the pathogenesis of the neurodegenerative SCAN1 syndrome, which is caused by Tdp1 deficiency.

Introdução. O receptor epidérmico humano 2 (Her-2) e a topoisomerase-IIα (T2A) são dois marcadores biológicos importantes, ambos tendo um valor prognóstico e preditivo potencial em pacientes com tumores sólidos. A amplificação dos genes Her- 2 e T2A são eventos independentes, embora o último seja mais frequente em tumores com amplificação do Her-2 (34-90%), do que em tumores sem amplificação do Her-2 (5-10%). Existe uma melhor correlação entre amplificação e superexpressão do Her-2 no câncer de mama (CM) do que em outros tumores. No entanto, no CM, a correlação entre amplificação e superexpressão da T2A tem sido inconsistente, e existe uma carência de tais dados em outros tipos de tumores. A expressão da proteína T2A tem mostrado uma boa correlação com o índice de proliferação tumoral, particularmente no CM. Objetivos. Artigo 1: Sintetizar o conhecimento atual sobre a importância dos marcadores Her-2 e T2A nos tumores sólidos. Artigo 2: Investigar a prevalência de amplificação e superexpressão do Her-2 e da T2A, a correlação entre estas variáveis e a correlação entre as variáveis e estágio clínico, em amostras de câncer de ovário (CO) fixadas em parafina. Artigo 3: Investigar a prevalência de amplificação da T2A, assim como a correlação entre esta variável e a expressão da proteína T2A e do marcador de proliferação celular Ki-67, em amostras de CM fixadas em parafina, mostrando uma amplificação do Her-2. Métodos. Artigo 1: Os dados foram identificados através de busca em bases de dados eletrônicas (medline), livros de resumos de congressos e referências de artigos de revisão e originais. Artigo 2: 73 amostras de CO foram testadas para amplificação e superexpressão do Her-2 e T2A, por hibridização in situ fluorescente (FISH) e imuno-histoquímica (IHC), respectivamente. Artigo 3: 103 amostras de CM, com amplificação do Her-2, foram testadas para amplificação do gene T2A (por FISH) e superexpressão das proteínas T2A e Ki-67 (por IHC). Resultados. Artigo 2: Com base nos pontos de corte >1.5 e >2 (relação cópias/CEP17), as taxas de amplificação do Her-2 foram 15/64(23.4%) e 8/64(12.5%), versus 16/64(25%) e 5/64(7.8 %) para a T2A. Encontramos somente 3/72(4.2%) casos de superexpressão do Her-2(3+), contra 15/70(21.4%) para a T2A (marcagem em >10% das células). Foi observada uma modesta correlação entre amplificação e superexpressão da T2A (p= 0.01) e uma forte correlação entre amplificação da T2A e do Her-2, quando analisados como variáveis contínuas (p<0.001). A amplificação da T2A correlacionou-se com estágio FIGO avançado (p= 0.02). Artigo 3: Uma amplificação do gene T2A foi observada em 36.9%(38/103) dos casos. Os níveis de amplificação do Her-2 (número de cópias) não se correlacionaram com a amplificação da T2A. A porcentagem média de células positivas para a T2A (por IHC) foi de 5% e 10%, para casos T2A não-amplificados e amplificados, respectivamente. Uma correlação fraca, mas ainda significativa, foi observada entre amplificação do gene T2A e porcentagem de células T2A-positivas por IHC (Spearman=0.23, p=0.02); a correlação entre estas duas variáveis foi mais forte em tumores Ki-67 positivos. Conclusões. Artigo 2 : A avaliação da amplificação e da superexpressão do Her-2 e da T2A, por FISH e IHC, respectivamente, é realizável em amostras de CO. Foi observada uma boa correlação entre a amplificação dos genes Her-2 e T2A, mas a correlação entre amplificação do gene e superexpressão da proteína foi fraca para ambos marcadores. As taxas de amplificação dos genes Her-2 e T2A são mais elevadas quando não é realizada correção para o número de cópias do CEP17. Parece existir uma boa correlação entre amplificação da T2A e estágio clínico avançado. Estudos adicionais serão necessários para determinar o melhor ponto de corte para estes marcadores. Artigo 3: Contrariamente ao Her-2, a amplificação do gene T2A não parece necessariamente levar à superexpressão da proteína no CM. Outros fatores, como o índice de proliferação celular, podem interferir na síntese da proteína T2A. Embora a maioria dos casos de aberrações do gene T2A ocorram em tumores Her-2 positivos, os níveis de amplificação do Her-2 não se correlacionaram com a amplificação do gene T2A. / Background. The human epidermal receptor 2 (Her-2) and topoisomerase-IIα (T2A) are two important biomarkers, with potential prognostic and predictive value in patients with solid tumours. Her-2 and T2A gene amplification are separate events, although the latter is more frequently seen in Her-2 amplified (34-90%) than in Her-2 non-amplified (5-10%) tumours. There is a better correlation between Her-2 amplification and protein overexpression in breast cancer (BC) than in other tumour types. Nevertheless, there is a doubtful correlation between T2A amplification and overexpression in BC, with virtually no data available in other tumour types. In BC, the expression of the T2A protein has shown a good correlation with tumour proliferation rate. Objectives. Article 1: To summarise the available literature on Her-2 and T2A in solid tumours. Article 2: To investigate the prevalence of Her-2 and T2A amplification and overexpression, the correlation between these variables and with clinical stage, in paraffin-embedded samples of ovarian cancer (OC). Article 3: To investigate the prevalence of T2A amplification, as well as the correlation between this variable and the expression of T2A protein and the proliferation marker Ki-67, in paraffinembedded samples of Her-2 amplified BC. Methods. Article 1: The data were identified through search in electronic databases (medline), abstract books and references from review and original articles. Article 2: 73 samples of OC were tested for Her-2 and T2A amplification and overexpression, by fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC), respectively. Article 3: 103 samples of Her-2 amplified BC were tested for T2A amplification (by FISH) and overexpression (by IHC), and Ki-67 expression (by IHC). Results. Article 2: Based on cut-offs of ≥1.5 and ≥2 (ratio copies/CEP17), amplification rates for Her-2 were 15/64(23.4%) and 8/64(12.5%) versus 16/64(25%) and 5/64(7.8%) for T2A. We found only 3/72(4.2%) cases of Her-2 overexpression(3+) versus 15/70(21.4%) for T2A (staining in >10% of the cells). There was a modest correlation between T2A amplification and overexpression (p=0.01) and a strong correlation between T2A and Her-2 amplification when these markers were analysed as continuous variables (p<0.001). T2A amplification significantly correlated with advanced FIGO stage (p=0.02). Article 3: T2A gene amplification was observed in 36.9%(38/103) of the Her-2 amplified samples. Her-2 amplification level (i.e. copy number) was not predictive of T2A amplification. The median percentage of T2A positive cells for T2A non-amplified and amplified cases were 5% and 10%, respectively. A weak but still significant correlation was observed between T2A gene amplification level and percentage of positively stained cells (Spearman=0.23, p=0.02), the observed correlation being higher in patients with positive staining for Ki-67. Conclusions. Article 2: The assessment of Her-2 and T2A amplification and overexpression by FISH and IHC, respectively, is feasible in OC samples. There was a good correlation between Her-2 and T2A gene amplification, but the correlation between gene amplification and protein overexpression was poor for both markers. Amplification rates were higher in the absence of correction for the number of copies of the CEP17. Finally, we found a good correlation between T2A amplification and advanced disease stage. Further studies should aim to determine the optimal cut-offs for these markers. Article 3: Contrary to Her-2, T2A gene amplification does not always lead to protein overexpression in BC. Other factors, especially tumour proliferation rate, may interfere with the T2A protein status. Although the majority of the cases of T2A gene aberrations are seen in Her-2 positive tumours, the level of Her-2 amplification does not predict for T2A amplification.

Introdução. O receptor epidérmico humano 2 (Her-2) e a topoisomerase-IIα (T2A) são dois marcadores biológicos importantes, ambos tendo um valor prognóstico e preditivo potencial em pacientes com tumores sólidos. A amplificação dos genes Her- 2 e T2A são eventos independentes, embora o último seja mais frequente em tumores com amplificação do Her-2 (34-90%), do que em tumores sem amplificação do Her-2 (5-10%). Existe uma melhor correlação entre amplificação e superexpressão do Her-2 no câncer de mama (CM) do que em outros tumores. No entanto, no CM, a correlação entre amplificação e superexpressão da T2A tem sido inconsistente, e existe uma carência de tais dados em outros tipos de tumores. A expressão da proteína T2A tem mostrado uma boa correlação com o índice de proliferação tumoral, particularmente no CM. Objetivos. Artigo 1: Sintetizar o conhecimento atual sobre a importância dos marcadores Her-2 e T2A nos tumores sólidos. Artigo 2: Investigar a prevalência de amplificação e superexpressão do Her-2 e da T2A, a correlação entre estas variáveis e a correlação entre as variáveis e estágio clínico, em amostras de câncer de ovário (CO) fixadas em parafina. Artigo 3: Investigar a prevalência de amplificação da T2A, assim como a correlação entre esta variável e a expressão da proteína T2A e do marcador de proliferação celular Ki-67, em amostras de CM fixadas em parafina, mostrando uma amplificação do Her-2. Métodos. Artigo 1: Os dados foram identificados através de busca em bases de dados eletrônicas (medline), livros de resumos de congressos e referências de artigos de revisão e originais. Artigo 2: 73 amostras de CO foram testadas para amplificação e superexpressão do Her-2 e T2A, por hibridização in situ fluorescente (FISH) e imuno-histoquímica (IHC), respectivamente. Artigo 3: 103 amostras de CM, com amplificação do Her-2, foram testadas para amplificação do gene T2A (por FISH) e superexpressão das proteínas T2A e Ki-67 (por IHC). Resultados. Artigo 2: Com base nos pontos de corte >1.5 e >2 (relação cópias/CEP17), as taxas de amplificação do Her-2 foram 15/64(23.4%) e 8/64(12.5%), versus 16/64(25%) e 5/64(7.8 %) para a T2A. Encontramos somente 3/72(4.2%) casos de superexpressão do Her-2(3+), contra 15/70(21.4%) para a T2A (marcagem em >10% das células). Foi observada uma modesta correlação entre amplificação e superexpressão da T2A (p= 0.01) e uma forte correlação entre amplificação da T2A e do Her-2, quando analisados como variáveis contínuas (p<0.001). A amplificação da T2A correlacionou-se com estágio FIGO avançado (p= 0.02). Artigo 3: Uma amplificação do gene T2A foi observada em 36.9%(38/103) dos casos. Os níveis de amplificação do Her-2 (número de cópias) não se correlacionaram com a amplificação da T2A. A porcentagem média de células positivas para a T2A (por IHC) foi de 5% e 10%, para casos T2A não-amplificados e amplificados, respectivamente. Uma correlação fraca, mas ainda significativa, foi observada entre amplificação do gene T2A e porcentagem de células T2A-positivas por IHC (Spearman=0.23, p=0.02); a correlação entre estas duas variáveis foi mais forte em tumores Ki-67 positivos. Conclusões. Artigo 2 : A avaliação da amplificação e da superexpressão do Her-2 e da T2A, por FISH e IHC, respectivamente, é realizável em amostras de CO. Foi observada uma boa correlação entre a amplificação dos genes Her-2 e T2A, mas a correlação entre amplificação do gene e superexpressão da proteína foi fraca para ambos marcadores. As taxas de amplificação dos genes Her-2 e T2A são mais elevadas quando não é realizada correção para o número de cópias do CEP17. Parece existir uma boa correlação entre amplificação da T2A e estágio clínico avançado. Estudos adicionais serão necessários para determinar o melhor ponto de corte para estes marcadores. Artigo 3: Contrariamente ao Her-2, a amplificação do gene T2A não parece necessariamente levar à superexpressão da proteína no CM. Outros fatores, como o índice de proliferação celular, podem interferir na síntese da proteína T2A. Embora a maioria dos casos de aberrações do gene T2A ocorram em tumores Her-2 positivos, os níveis de amplificação do Her-2 não se correlacionaram com a amplificação do gene T2A. / Background. The human epidermal receptor 2 (Her-2) and topoisomerase-IIα (T2A) are two important biomarkers, with potential prognostic and predictive value in patients with solid tumours. Her-2 and T2A gene amplification are separate events, although the latter is more frequently seen in Her-2 amplified (34-90%) than in Her-2 non-amplified (5-10%) tumours. There is a better correlation between Her-2 amplification and protein overexpression in breast cancer (BC) than in other tumour types. Nevertheless, there is a doubtful correlation between T2A amplification and overexpression in BC, with virtually no data available in other tumour types. In BC, the expression of the T2A protein has shown a good correlation with tumour proliferation rate. Objectives. Article 1: To summarise the available literature on Her-2 and T2A in solid tumours. Article 2: To investigate the prevalence of Her-2 and T2A amplification and overexpression, the correlation between these variables and with clinical stage, in paraffin-embedded samples of ovarian cancer (OC). Article 3: To investigate the prevalence of T2A amplification, as well as the correlation between this variable and the expression of T2A protein and the proliferation marker Ki-67, in paraffinembedded samples of Her-2 amplified BC. Methods. Article 1: The data were identified through search in electronic databases (medline), abstract books and references from review and original articles. Article 2: 73 samples of OC were tested for Her-2 and T2A amplification and overexpression, by fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC), respectively. Article 3: 103 samples of Her-2 amplified BC were tested for T2A amplification (by FISH) and overexpression (by IHC), and Ki-67 expression (by IHC). Results. Article 2: Based on cut-offs of ≥1.5 and ≥2 (ratio copies/CEP17), amplification rates for Her-2 were 15/64(23.4%) and 8/64(12.5%) versus 16/64(25%) and 5/64(7.8%) for T2A. We found only 3/72(4.2%) cases of Her-2 overexpression(3+) versus 15/70(21.4%) for T2A (staining in >10% of the cells). There was a modest correlation between T2A amplification and overexpression (p=0.01) and a strong correlation between T2A and Her-2 amplification when these markers were analysed as continuous variables (p<0.001). T2A amplification significantly correlated with advanced FIGO stage (p=0.02). Article 3: T2A gene amplification was observed in 36.9%(38/103) of the Her-2 amplified samples. Her-2 amplification level (i.e. copy number) was not predictive of T2A amplification. The median percentage of T2A positive cells for T2A non-amplified and amplified cases were 5% and 10%, respectively. A weak but still significant correlation was observed between T2A gene amplification level and percentage of positively stained cells (Spearman=0.23, p=0.02), the observed correlation being higher in patients with positive staining for Ki-67. Conclusions. Article 2: The assessment of Her-2 and T2A amplification and overexpression by FISH and IHC, respectively, is feasible in OC samples. There was a good correlation between Her-2 and T2A gene amplification, but the correlation between gene amplification and protein overexpression was poor for both markers. Amplification rates were higher in the absence of correction for the number of copies of the CEP17. Finally, we found a good correlation between T2A amplification and advanced disease stage. Further studies should aim to determine the optimal cut-offs for these markers. Article 3: Contrary to Her-2, T2A gene amplification does not always lead to protein overexpression in BC. Other factors, especially tumour proliferation rate, may interfere with the T2A protein status. Although the majority of the cases of T2A gene aberrations are seen in Her-2 positive tumours, the level of Her-2 amplification does not predict for T2A amplification.

Ce travail a consisté en la synthèse d’outils chimiques et au développement de leurs applications biologiques. Dans un premier temps, des sondes pour l’étude de la Topoisomérase IIA humaine ont été synthétisées. Ces sondes ont alors été testées sur lysat cellulaire pour la capture des protéines présentant une affinité pour ces médicaments. Dans un second temps, un nouveau lien clivable en conditions non dénaturantes pour des applications en protéomique chimique a été developpé. Ainsi, après optimisation de la structure, il a été intégré au sein d’une sonde d’affinité pour évaluer sa capacité de capture et libération de la PARP 1. Enfin, la réaction de click entre un azoture et un cyclooctyne a été appliquée à l’élimination d’une drogue circulante dans le sang.Après l’étude cinétique de la réaction, l’activité biologique et la pharmacocinétique des différents composés ont été évaluées pour optimiser la réaction de click in vivo. / The main goal of this work was to synthesize chemical tools and to developp their biological applications. In the first part, probes for the study of Topoisomerase II via chemical proteomic were synthesized. They were then used for pulldown experiments on cell lysats. In a second part, a new cleavable linker in non denaturing conditions was developped for chemical proteomic applications. After optimisation of the structure, it was incorporated in an affinity probe and tested for the pulldown of PARP 1. Finally, a click chemistry reaction, the SPAAC, was used to provok the elimination of a circulating drug. After the study of the kinetic of the reaction, the biological activity and the pharmacokinetic of the different compounds were evaluated to optimise the click reaction in vivo.

Des liens de caténation sont progressivement crées lors de la réplication de l’ADN et sont responsables de la cohésion des chromatides sœurs. La topoisomérase IV est une topoisomérase de type II impliquée dans la résolution de ces liens de caténation accumulés derrière la fourche de réplication, et lors de la dernière étape de séparation des chromatides sœurs à la fin de la réplication. Nous avons étudié la liaison de la topoIV à l’ADN ainsi que son activité catalytique à l’aide de méthodes de biologie moléculaire et de génomique. Une expérience de ChIPseq a révélé que l’interaction de la topoIV de chez E.coli avec l’ADN est contrôlée par la réplication. Durant la réplication, la topoIV a accès à des centaines de sites sur l’ADN mais ne se lie qu’à quelques sites où elle exerce son activité catalytique. La conformation locale de la chromatine et l’expression des gènes influencent la sélection de certains sites. De plus, une forte liaison et une activité catalytique renforcée a été trouvée au site de résolution des dimers, dif. Le site dif est situé à l’opposé de l’origine de réplication dans le macrodomaine ter. Nous avons montré qu’il existe une interaction physique et fonctionnelle entre la topoIV et la recombinase XerCD, qui agit au site dif. Cette interaction est médiée par MatP, une protéine essentielle dans l’organisation du macrodomaine ter. L’ensemble de ces résultats montre que la topoIV, XerCD/dif et MatP œuvrent ensemble pour permettre l’étape finale de ségrégation des chromosomes lors du cycle cellulaire. / Catenation links between sister chromatids are formed progressively during DNA replication and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chromatin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle.

La résistance acquise aux traitements anticancéreux représente un problème clinique majeur. Les voies de réparation de l'ADN fournissent un mécanisme de résistance, mais celle-ci peut aussi résulter de mutations ou d'une expression réduite de la protéine ciblée. La surexpression ou la localisation nucléaire de la Y-Box binding (YB-1) protéine est considérée comme un marqueur pronostique de chimiorésistance de la tumeur. YB-1 interagit avec plusieurs partenaires ; dans cette étude, nous nous sommes concentrés sur son interaction avec l'enzyme de réparation de l'ADN NTH1 (hNTH1) et l'ADN topoisomérase I (hTopoI), deux enzymes stimulées par YB-1. L'abondance du complexe hNTH1/YB-1 est accrue dans les cellules tumorales résistantes au cisplatine. La TopoI humaine est une enzyme essentielle impliquée dans la régulation cellulaire du surenroulement de l'ADN et est la cible de plusieurs agents anticancéreux. YB-1 augmente la sensibilité à l'inhibiteur de TopoI, la camptothécine, dans les tumeurs. Nous avons caractérisé les complexes YB-1/hNTH1 et YB-1/hTopoI in vitro et in vivo en utilisant des mesures de transfert d'énergie par résonance en fluorescence (ou FRET) pour identifier et développer de nouvelles stratégies pour le traitement de tumeurs chimio-résistantes. Nous avons développé et optimisé un biosenseur original basé sur le FRET pour cribler deux chimiothèques de taille moyenne afin d’identifier des inhibiteurs potentiels du complexe hNTH1/YB-1. Plusieurs «hits» ont été identifiés qui réduisent de façon significative le niveau de FRET de notre biosenseur. Pour certains de ces composés, nous avons reproduit ces résultats à partir de poudres, effectué des courbes dose-réponse et validé leurs actions en tant qu'inhibiteurs de l'interface hNTH1/YB-1 en utilisant d'autres tests d’interactions. Ensemble nos résultats démontrent que YB-1 interagit directement et stimule des enzymes de la réparation de l'ADN et du relaxation de l’ADN, et que cibler l’interface YB-1/hNTH1 représente une nouvelle stratégie intéressante pour le développement de traitements anticancéreux. / Acquired resistance to anti-cancer therapy is common and is a major clinical issue. Functional DNA repair pathways provide a common mechanism for drug resistance, but it can also result from mutations or reduced expression of the targeted protein. The overexpression or nuclear localisation of the multifunctional Y-box binding protein (YB-1) is considered as a prognostic marker for drug resistance in tumours. YB-1 has several interaction partners in cells; in this study, we have focused on its interaction with the human DNA repair enzyme NTH1 (hNTH1) and human DNA topoisomerase I (hTopoI), two enzymes that have been shown to be stimulated by YB-1. The abundance of the hNTH1/YB-1 complex was shown to increase in cisplatin-resistant tumour cells. Human TopoI is an essential enzyme involved in cellular regulation of DNA supercoiling and is the target of several anti-cancer agents. YB-1 enhances the activity of hTopoI and its sensitivity to hTopoI inhibitor, camptothecin in tumour cells. We have characterised the YB-1/hNTH1 and YB-1/hTopoI complexes in vitro and in vivo using Fluorescence Resonance Energy Transfer (FRET) measurements to identify and develop new strategies for the treatment of drug-resistant tumours. We also designed and optimised an original FRET-based biosensor to screen two medium-sized chemical libraries in order to find potential inhibitors of the hNTH1/YB-1 complex. Several “hits” were identified that significantly reduced the FRET level of our biosensor. For some of these compounds, we have reproduced these results starting from powders, have performed dose-response curves and have validated their actions as inhibitors of the hNTH1/YB-1 interface using alternative binding assays. Taken together, our results demonstrate that YB-1 directly interacts and stimulates a DNA repair and a DNA relaxing enzyme and targeting the YB-1/hNTH1 interface represents an interesting new strategy for the development of anti-cancer drugs.

Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis, has been a global health problem for years. The emergence of drug resistance in this organism generates the necessity of exploring novel targets and developing new drugs. Topoisomerases are enzymes found in all kingdoms of life responsible for overcoming the topological barriers encountered during essential cellular processes. The genomes of mycobacteria encode only one type IA topoisomerase (MtopI), which has been validated as a novel TB drug target. The goal of this study is to obtain new information on the mechanism and resistance of endogenous and synthetic inhibitors of MtopI. Rv1495 is a M. tuberculosis toxin that belongs to the MazEF family (MazE is the antitoxin and MazF is the toxin), with endoribonuclease activity. Rv1495 (MazF homolog in M. tuberculosis) toxin has been shown to interact directly with the C-terminal domain of MtopI for mutual inhibition. In this study the interaction of Rv1495 with the positively charged C-terminal tail in Mtop I is reported. This new information is useful for rational design and discovery of antibiotics for mycobacteria. Ethacridine, an FDA approved drug has shown activity against MtopI. In this project we studied the mechanisms of resistance associated with this drug as well the use of Ethacridine in combination with Moxifloxacin, to potentiate the bactericidal effect of this current second line drug for TB treatment. Results from sequencing of the genomic DNA isolated from the resistant mutants suggested the involvement of the Holliday-junction Ruv resolvase. Further studies showed that co-treatment with Ethacridine can enhance the moxifloxacin-mediated killing of M. smegmatis. FP-11g, a novel fluoroquinophenoxazine inhibitor of bacterial topoisomerase I, has shown promising activity against M, tuberculosis. We explored the bactericidal activity and resistance mechanisms associated to FP-11g using M. smegmatis as model organism. Additionally, the inhibitory effect of FP-11g was also evaluated on M. abscessus. FP-11g at concentration 4X MIC showed complete bactericidal activity against M. smegmatis after 24 hours. Inhibitory activity against M. abscessus was also observed. Results from sequencing of the genomic DNA isolated from the M. smegmatis resistant mutants revealed mutations in genes associated with general drug resistance.

Le protozoaire Leishmania est à l’origine d’une maladie tropicale négligée qui peut s’avérer mortelle si elle n’est pas traitée adéquatement. Dans les dernières années, l’efficacité des médicaments utilisés dans le combat de la leishmaniose a grandement diminué en raison de la résistance du parasite à ceux-ci. Bien que le Topotecan (TPT), présentement utilisé comme antitumoral, ait démontré une activité antileishmaniale puissante (EC50 de l’ordre des nanomolaires), son efficacité pourrait être compromise par l’émergence de la résistance du parasite d’autant plus que des résistances tumorales cliniques au TPT ont déjà été rapportées. Dans cette étude, les mécanismes de résistance au TPT par Leishmania infantum ont été caractérisés d’un point de vue moléculaire. Le génome complet des parasites sélectionnés résistants au TPT (16 x EC50) (TPT700.1, TPT700.2, TPT700.3) a été séquencé. Le rôle dans la résistance des différents éléments génétiques identifiés a été confirmé à l’aide d’une complémentation par nucléofection épisomale dans le parasite sauvage et a été étudié avec des simulations computationnelles. Aucune amplification ni délétion n’a été identifiée et seulement une variation mineure du nombre de chromosomes a été observée. Cependant, un polymorphisme d’un seul nucléotide non synonyme a été identifié dans le gène de la grande sous-unité de la topoisomérase IB (TOP IB), la cible du TPT, chez chacun des parasites mutants résistants conférant des niveaux de résistance variables (TPT700.1 F187Y > TPT700.3 W232R > TPT700.2 G191A > sauvage). De plus, des modélisations in sillico ont permis d’illustrer la proximité de ces substitutions d’acides aminés au site catalytique de TOP IB ainsi qu’au site de liaison du TPT. En conclusion, ces résultats suggèrent qu’une mutation ponctuelle dans la grande sous-unité TOP IB est suffisante pour engendrer des hauts niveaux de résistance (environ 24 x EC50) chez TPT700.1F187Y, TPT700.2G191A et TPT700.3 W232R. TPT pourrait être considéré comme un modèle pharmacologique pour l’étude de la résistance chez Leishmania. / Leishmania, a protozoan parasite, causes a neglected tropical disease that is fatal if left untreated. In recent years, the effectiveness of the drugs used to tackle leishmaniasis has decreased dramatically due to the emergence of drug resistant parasites. Even though Topetecan (TPT), currently employed as an anti-tumoral drug, has shown strong anti-leishmanial activity (its EC50 being measured in nanomoles), its efficiency may be compromised by the resistance developped by the parasites, similarly to the resistance already recorded by tumoral cells to the drug. In this study, the mecanisms of resistance to TPT by Leishmania infantum were caracterised at the molecular level. The whole genome of parasites resistant to TPT (16 x EC50) (TPT700.1, TPT700.2, TPT700.3) was sequenced. The role of various genetic elements in the resistance mecanisms was confirmed via a complementation by episomal nucleofection in the wild type and was studied with the help of computational models. Neither amplications nor deletions were identified and only a minor variation in the chromosome number was observed. However, a non-synonymous single nucleotide polymorphism was identified in the gene coding the large subunit of topoisomerase IB, TPT’s target, within each of the resistant mutant parasites confering variable levels of resistance (TPT700.1 F187Y > TPT700.3 W232R > TPT700.2 G191A > wild type). Furthermore, in sillico models highlighted the proximity of these amino acid substitutions to the catalytic site of topoisomerase IB and also to the binding pockets of TPT. In conclusion, these results suggest that a point mutation in the large subunit of TOP IB is sufficient to confer high levels of resistance (about 24 x EC50) to TPT700.1F187Y, TPT700.2G191A and TPT700.3 W232R. Therefore, TPT can be considered a pharmacological tool to study resistance in Leishmania.

No description available.

La sclérose systémique (ScS) est une maladie auto-immune d’origine inconnue qui est caractérisée par des atteintes vasculaires, des dérèglements cellulaire et immunitaire. La majorité des patients atteints de ScS possède des auto-anticorps dirigés contre des protéines nucléaires. Ces auto-anticorps sont associés à des manifestations cliniques spécifiques favorisant la classification et le diagnostic de la ScS. Les anti-ADN topoisomérase I (antitopo) sont l’un des principaux auto-anticorps retrouvés dans la ScS. Ils sont associés à la forme la plus grave de la maladie, soit la forme diffuse. Celle-ci se caractérise par une importante fibrose progressant vers une atteinte viscérale. La fibrose résulte d’une production excessive et dérégulée de matrice extracellulaire par les fibroblastes. Bien que les anti-topo soient associés à un très mauvais pronostic et qu’ils corrèlent avec l’activité et la sévérité de la maladie, leur rôle dans la pathogenèse de la ScS n’est pas élucidé. Toutefois, depuis que certains auto-antigènes ont démontré des fonctions additionnelles lorsque retrouvés dans le milieu extracellulaire, leur contribution suscite un intérêt marqué. En effet, ces auto-antigènes, dits bifonctionnels, influencent la physiologie de certaines cellules en se liant à leur surface. Ainsi, la détermination du rôle de ces autoantigènes ouvre la voie pour l’exploration du rôle potentiellement pathogène de leurs autoanticorps. Tout d’abord, nous avons démontré que l’auto-antigène topo, ciblée par les antitopo, pouvait influencer la physiologie du fibroblaste suite à l’activation de voies de signalisations intracellulaires stimulant la migration cellulaire. Nos résultats suggèrent fortement que la topo stimule le fibroblaste suite à son interaction avec le CCR7, un récepteur de chimiokine, présent à sa surface. Nous avons également démontré que la topo utilisait les protéoglycans à chaînes d’héparanes sulfates (HSPG) à titre de corécepteurs. Il avait été démontré que la topo liée à la surface des fibroblastes entraînait le recrutement d’anti-topo, l’adhésion et l’activation monocytaires. Nous avons ici démontré que la présence d’anticorps anti-topo entraîne l’amplification de la liaison de la topo au niveau des HSPG. De ce fait, le complexe immun à la surface des fibroblastes pourrait contribuer à l’initiation d’une cascade inflammatoire propice au développement d’une fibrose, caractéristique de la ScS. En dernier lieu, nos résultats nous ont permis de suggérer l’utilisation de l’héparine et des héparines de bas poids moléculaires comme approche thérapeutique pour la ScS puisqu’elles permettent autant de prévenir la liaison du complexe immun topo/anti-topo au niveau des HSPG que de le dissocier une fois lié. En résumé, notre étude soutient d’abord le rôle actif de l’auto-antigène dans la physiologie des fibroblastes mais également le rôle pathogène des anti-topo en présence de la topo dans la ScS. Finalement, les résultats de notre étude permettent de proposer une approche thérapeutique potentielle pour inhiber le développement d’une cascade inflammatoire et pro-fibrotique. / Systemic sclerosis (SSc) is an autoimmune disease of unknown etiology characterized by vascular damage, cellular and immunological disorders. The vast majority of patient sera are characterized by the presence of autoantibodies directed against nuclear proteins. The autoantibodies are associated with specific clinical manifestations and thus useful for diagnostic and classification of the disease. One of the major autoantibody groups are the anti-DNA topoisomerase I (anti-topo). They are associated with the diffuse form of the disease which is characterized by extensive cutaneous and visceral fibrosis. Increased extracellular matrix synthesis and deposition by fibroblast result in the development of fibrosis. Although anti-topo are associated with the worst form of the disease, correlated with the activity and the severity of SSc, their exact role in the pathogenesis of SSc is controversial and still unravelled. On the other hand, there is now strong evidence for active contribution of autoantigens, targeted by autoantibodies, in autoimmune diseases. Indeed, numerous cells have been shown to be influenced by the interaction of autoantigens with their cognate receptors present on their surface. These autoantigens display cytokine-like effects toward their target cell and are called bifunctional autoantigen. Hence, determination of the exact role of these autoantigens and characterization of their interaction with their target cell may open up research perspectives for the elucidation of the potential pathogenic role of their autoantibodies. In our study, we demonstrated that topo activates intracellular signaling pathways leading to the stimulation of fibroblast migration. We undertook experiments to characterize the interaction of the autoantigen topo with fibroblasts responsible of these cellular effects. Our results strongly suggest a direct interaction of topo with CCR7, a chemokine receptor, present on the surface of fibroblasts. Heparan sulfate proteoglycans (HSPG), abundantly present on fibroblast surfaces, were found to act as coreceptors for topo binding. Previous work has demonstrated that once bound to fibroblast surfaces, topo recruits anti-topo autoantibodies, which subsequently lead to adhesion and activation of monocytes. Here, we demonstrated that anti-topo autoantibodies from SSc sera lead to the amplification of topo binding to HSPG on fibroblast surfaces. The binding of topo/anti-topo IC could mediate the initiation and maintenance of an inflammatory cascade and further fibrosis development. Hence, perturbing the binding of topo/anti-topo immune complexes to HSPG became an interesting therapeutic approach. Heparin and low molecular weight heparins were found to prevent the binding of topo and topo/anti-topo immune complexes to the fibroblast surfaces. Moreover, topo/anti-topo immune complexes could be dissociated from fibroblast surfaces by these molecules. Hence, the prevention of topo/anti-topo immune complexes binding to HS chains could result in the absence of the inflammatory cascade initiation. Overall, our results support an active role for topo as a bifunctional autoantigen toward fibroblasts and a pathogenic role for anti-topo autoantibodies in SSc. Finally, a potential therapeutic approach is proposed which could target inflammatory and fibrotic development characteristic of SSc.