Cancer cells exhibit genomic instability and are commonly defective in DNA damage signalling and/or DNA repair. There are many types of DNA damage inducing agents such as mechanical stress on chromosomes during recombination, chemotherapeutics, ionising and ultraviolet radiation and endogenously produced free radicals. These genetic lesions pose a serious threat to the cell and evoke a rapid and intricate DNA damage response signalling pathway involving many transducer and effector pathways including cell cycle arrest, DNA repair, chromatin remodelling, and apoptotic pathways. Genetic mutations within genes in this pathway often lead to genomic instability and cancer. The main effectors of the DNA damage response are the protein kinases ATM and ATR which are rapidly activated in response to DNA damage induction and phosphorylate a large and diverse number of targets including the checkpoint kinases Chk1, and Chk2, the tumour suppressors p53 and BRCA1 and chromatin associated proteins such as H2AX. BRCA1 is a key transducer molecule within the DNA damage response. This is evident from its loss, which leads to defects in many damage response processes such as cell cycle arrest and DNA repair. BRCA1s binding partner BARD1 has also been implicated in the DNA damage response and recent reports indicate that these proteins co-operate in this pathway. This study utilises a multifaceted approach to further characterise the function of the BRCA1/BARD1 complex within the DNA damage response. Firstly we have used shRNA to deplete the BRCA1/BARD1 complex and have shown that the BRCA1/BARD1 complex is required for ATM/ATR dependent phosphorylation of p53Ser-15 in response to IR and UV induced DNA damage. In contrast, we have shown that the phosphorylation of a number of other ATM/ATR dependent targets including H2AX, Chk2, and c-jun do not require the BRCA1/BARD1 complex. The study has also revealed that the prior phosphorylation of BRCA1 at Ser-1423 and Ser-1524 is required for the phosphorylation of p53 at Ser-15. Furthermore, we have shown that these phosphorylation events are required for IR induced G1/S cell cycle arrest via transcriptional induction of the cyclin dependent kinase inhibitor p21. The second part of this study involved the characterisation of a putative BRCA1 interacting protein – The KRAB associated protein 1 (Kap1). During this study we have been unable to confirm Kap1 as a bona fide BRCA1 interactor, however we have identified a clear role for Kap1 in the DNA damage response pathway. Using Mass spectrometric phospho amino acid mapping we have identified a novel Chk2 dependent phosphorylation site, Ser-473, within Kap1. Furthermore, we have shown that this phosphorylation event may regulate Histone H3-Lys-9 acetylation after DNA damage possibly regulating chromatin relaxation. This study has also identified a number of novel Kap1 interacting proteins, which appear to be regulated by Kap1 phosphorylation at Ser-473. These interactors may play an important role in the regulation of chromatin modification and/or structure after DNA damage. By studying the role of BRCA1 in the DNA damage response pathway we have not only uncovered a novel scaffolding function for BRCA1 in the G1/S checkpoint but have also identified a novel protein, Kap1, acting within the DNA damage response pathway. This study has identified a role for Kap-1 in the regulation of chromatin structure in response to DNA damage via the ATM – Chk2 pathway.

Cancer cells exhibit genomic instability and are commonly defective in DNA damage signalling and/or DNA repair. There are many types of DNA damage inducing agents such as mechanical stress on chromosomes during recombination, chemotherapeutics, ionising and ultraviolet radiation and endogenously produced free radicals. These genetic lesions pose a serious threat to the cell and evoke a rapid and intricate DNA damage response signalling pathway involving many transducer and effector pathways including cell cycle arrest, DNA repair, chromatin remodelling, and apoptotic pathways. Genetic mutations within genes in this pathway often lead to genomic instability and cancer. The main effectors of the DNA damage response are the protein kinases ATM and ATR which are rapidly activated in response to DNA damage induction and phosphorylate a large and diverse number of targets including the checkpoint kinases Chk1, and Chk2, the tumour suppressors p53 and BRCA1 and chromatin associated proteins such as H2AX. BRCA1 is a key transducer molecule within the DNA damage response. This is evident from its loss, which leads to defects in many damage response processes such as cell cycle arrest and DNA repair. BRCA1s binding partner BARD1 has also been implicated in the DNA damage response and recent reports indicate that these proteins co-operate in this pathway. This study utilises a multifaceted approach to further characterise the function of the BRCA1/BARD1 complex within the DNA damage response. Firstly we have used shRNA to deplete the BRCA1/BARD1 complex and have shown that the BRCA1/BARD1 complex is required for ATM/ATR dependent phosphorylation of p53Ser-15 in response to IR and UV induced DNA damage. In contrast, we have shown that the phosphorylation of a number of other ATM/ATR dependent targets including H2AX, Chk2, and c-jun do not require the BRCA1/BARD1 complex. The study has also revealed that the prior phosphorylation of BRCA1 at Ser-1423 and Ser-1524 is required for the phosphorylation of p53 at Ser-15. Furthermore, we have shown that these phosphorylation events are required for IR induced G1/S cell cycle arrest via transcriptional induction of the cyclin dependent kinase inhibitor p21. The second part of this study involved the characterisation of a putative BRCA1 interacting protein – The KRAB associated protein 1 (Kap1). During this study we have been unable to confirm Kap1 as a bona fide BRCA1 interactor, however we have identified a clear role for Kap1 in the DNA damage response pathway. Using Mass spectrometric phospho amino acid mapping we have identified a novel Chk2 dependent phosphorylation site, Ser-473, within Kap1. Furthermore, we have shown that this phosphorylation event may regulate Histone H3-Lys-9 acetylation after DNA damage possibly regulating chromatin relaxation. This study has also identified a number of novel Kap1 interacting proteins, which appear to be regulated by Kap1 phosphorylation at Ser-473. These interactors may play an important role in the regulation of chromatin modification and/or structure after DNA damage. By studying the role of BRCA1 in the DNA damage response pathway we have not only uncovered a novel scaffolding function for BRCA1 in the G1/S checkpoint but have also identified a novel protein, Kap1, acting within the DNA damage response pathway. This study has identified a role for Kap-1 in the regulation of chromatin structure in response to DNA damage via the ATM – Chk2 pathway.

Cancer cells exhibit genomic instability and are commonly defective in DNA damage signalling and/or DNA repair. There are many types of DNA damage inducing agents such as mechanical stress on chromosomes during recombination, chemotherapeutics, ionising and ultraviolet radiation and endogenously produced free radicals. These genetic lesions pose a serious threat to the cell and evoke a rapid and intricate DNA damage response signalling pathway involving many transducer and effector pathways including cell cycle arrest, DNA repair, chromatin remodelling, and apoptotic pathways. Genetic mutations within genes in this pathway often lead to genomic instability and cancer. The main effectors of the DNA damage response are the protein kinases ATM and ATR which are rapidly activated in response to DNA damage induction and phosphorylate a large and diverse number of targets including the checkpoint kinases Chk1, and Chk2, the tumour suppressors p53 and BRCA1 and chromatin associated proteins such as H2AX. BRCA1 is a key transducer molecule within the DNA damage response. This is evident from its loss, which leads to defects in many damage response processes such as cell cycle arrest and DNA repair. BRCA1s binding partner BARD1 has also been implicated in the DNA damage response and recent reports indicate that these proteins co-operate in this pathway. This study utilises a multifaceted approach to further characterise the function of the BRCA1/BARD1 complex within the DNA damage response. Firstly we have used shRNA to deplete the BRCA1/BARD1 complex and have shown that the BRCA1/BARD1 complex is required for ATM/ATR dependent phosphorylation of p53Ser-15 in response to IR and UV induced DNA damage. In contrast, we have shown that the phosphorylation of a number of other ATM/ATR dependent targets including H2AX, Chk2, and c-jun do not require the BRCA1/BARD1 complex. The study has also revealed that the prior phosphorylation of BRCA1 at Ser-1423 and Ser-1524 is required for the phosphorylation of p53 at Ser-15. Furthermore, we have shown that these phosphorylation events are required for IR induced G1/S cell cycle arrest via transcriptional induction of the cyclin dependent kinase inhibitor p21. The second part of this study involved the characterisation of a putative BRCA1 interacting protein – The KRAB associated protein 1 (Kap1). During this study we have been unable to confirm Kap1 as a bona fide BRCA1 interactor, however we have identified a clear role for Kap1 in the DNA damage response pathway. Using Mass spectrometric phospho amino acid mapping we have identified a novel Chk2 dependent phosphorylation site, Ser-473, within Kap1. Furthermore, we have shown that this phosphorylation event may regulate Histone H3-Lys-9 acetylation after DNA damage possibly regulating chromatin relaxation. This study has also identified a number of novel Kap1 interacting proteins, which appear to be regulated by Kap1 phosphorylation at Ser-473. These interactors may play an important role in the regulation of chromatin modification and/or structure after DNA damage. By studying the role of BRCA1 in the DNA damage response pathway we have not only uncovered a novel scaffolding function for BRCA1 in the G1/S checkpoint but have also identified a novel protein, Kap1, acting within the DNA damage response pathway. This study has identified a role for Kap-1 in the regulation of chromatin structure in response to DNA damage via the ATM – Chk2 pathway.

La dérégulation du système de surveillance qui bloque la prolifération lorsque l'intégrité du génome est compromise fait partie intégrante de la cancérogenèse. Nous cherchons à décortiquer les mécanismes qui, en phase G2, orchestrent l'arrêt du cycle cellulaire, irréversible, en présence des lésions de l'ADN (sénescence) ou réversible (quiescence), en absence de signaux mitogéniques ou confluence. L'objectif du premier volet fut d'élucider les rôles respectifs de l'inhibiteur de CDK (CKI) p21Waf1 et des kinases Chk1 et Chk2 dans l'arrêt en G2 dû au stress génotoxique menant à la sénescence. Nous avons montré que dans les cellules humaines normales cet arrêt nécessite l'action de p21 et Chk1 tandis que Chk2 n'est pas requise. Au contraire, dans plusieurs lignées cancéreuses, malgré la présence de p53, ce rôle de p21 est compromis à cause d'une activation inefficace de la kinase ATM. Par conséquent, en dépit d'une forte activation de Chk1 bloquant la mitose, ces cellules ne parviennent pas à initier la sénescence (Lossaint et al., soumis). L'objectif du deuxième volet fut de mettre en évidence le programme déclenchant la quiescence lors de confluence ou en absence de sérum. Les travaux antérieurs de l'équipe ont montré que cette décision pouvait être prise avant la mitose même si l'arrêt du cycle n'a lieu qu'en phase G1 suivante. En étudiant les fibroblastes synchronisés nous avons trouvé que la quiescence est précédée par l'inhibition pré-mitotique de la phosphorylation de pRb due à une diminution de cycline D1 et une stabilisation du CKI p27Kip1 (Chassot et al., 2008). De plus, nos résultats récents montrent que la présence de sérum entre le point R et la mitose est requise pour initier la réplication de l'ADN au cycle suivant. Les travaux futurs devraient élucider comment différentes voies de signalisation, via la voie cycline D-pRb, affectent divers composants de l'appareil de réplication de l'ADN pour inhiber la progression du cycle de façon réversible ou irréversible. / Cancer is a multi-step process resulting from abrogation of several barriers to uncontrolled proliferation. They include inhibitory pathways with appropriate checkpoints that lead to reversible (quiescence) or irreversible (senescence, apoptosis) block of cell proliferation. We are especially interested in pathways orchestrating cell cycle exit that operate in the G2 phase. The first objective of this thesis was to decipher mechanisms that prevent mitosis in response to DNA damage. We found that Cdk inhibitor p21Waf1 plays a crucial role in blocking mitotic onset in normal cells; acting in tandem with checkpoint kinase Chk1, p21 inactivates mitotic Cdks and inhibits pRb phosphorylation, thereby irreversibly blocking mitotic entry. In contrast, in p53-proficient transformed cells, the induction of p21 in G2 is impaired, most likely because of deficient ATM activation. While, in some cases, Chk1 hyper-activation prevents mitosis, the absence of p21 compromises the senescence program from G2. Finally, we showed that Chk2 is dispensable for G2 arrest in both non-transformed and transformed cells (Lossaint et al., submitted). Our second objective was to elucidate the pathways that induce quiescence (G0). This reversible cell cycle exit occurs in G1, requires pRb family members and p27Kip1-dependent Cdk inactivation. Based on observations obtained in our team and the data in the literature, we hypothesized that reversible cell cycle exit program might be launched before mitosis. By using an in vitro wounding model, we showed that confluence-driven quiescence is preceded by pre-mitotic CDK inhibition by p27, cyclin D1 downregulation and reduced pre-mitotic pRb phosphorylation (Chassot et al., 2008). Moreover, our results obtained in synchronized fibroblasts that were serum-starved after release from G1/S block suggest that cyclin D1 might stimulate proliferation by keeping pocket proteins phosphorylated during G2/M progression (Lossaint et al., in preparation).

Les ectéinascidines (la trabectédine, la lurbinectédine) sont de nouveaux dérivés de produits naturels marins qui se lient de façon covalente à l'ADN, actifs contre les cancers chimio-résistants. L'objectif de ma thèse est 1) d'identifier les principales voies de transduction activées en réponse à l'apparition des lésions de l'ADN induites par les ETs 2) d'établir si l'abrogation pharmacologique de la réponse cellulaire induite par l'endommagement de l'ADN (ATM, ATR, Chk1, Chk2) peut moduler l'activité thérapeutique des ETs. Dans un premier temps, nous avons montré que la voie ATR/Chk1 activée principalement en réponse à l'apparition d'un stress réplicatif et la voie ATM/Chk2 qui initie la réponse cellulaire suite à la formation de lésions double-brins, sont activées en réponse aux adduits créés par les ETs. Dans un second temps, nous avons montré que les combinaisons des ETs avec les inhibiteurs Chk1/Chk2 ou les inhibiteurs ATR ou ATM seuls s'accompagnent d'une modeste potentialisation. Inversement, la combinaison simultanée des ETs avec les inhibiteurs ATR et ATM entraine une forte synergie dans les modèles du cancer de l'utérus et de l'ovaire sensibles ou résistants au cisplatine. Enfin, nous avons montré que cette potentialisation passe par l'inhibition du recrutement des protéines impliquées dans l'initiation et la réalisation des mécanismes de réparation par recombinaison homologue. Ces résultats suggèrent qu'en inhibant simultanément les vois initiés par ATR et ATM, l'activité thérapeutique des ETs pourrait être potentialisée en clinique. / Ecteinascidins (Trabectedin, Lurbinectedin) are novel marine derived natural products, DNA minor groove binders and active against chemo-resistant cancers. The purpose of my thesis was to 1) characterize the DNA damage response (DDR) to both trabectedin and lurbinectedin 2) to establish whether the pharmacological abrogation of cell response induced by DNA damage (ATM, ATR, Chk1, Chk2) can modulate the therapeutic activity of ETs. Our results show that both compounds activate the ATM/Chk2 (ataxia-telangiectasia mutated/checkpoint kinase 2) and ATR/Chk1 (ATM and RAD3-related/checkpoint kinase 1) pathways. Interestingly, the pharmacological inhibition of either Chk1/2, ATR or ATM kinases is not accompanied by a significant improvement of either trabectedin or lurbinectedin cytotoxic activity. However, the simultaneous inhibition of both ATM and ATR strongly potentiates the activity of both ETs. Importantly, these results are not restricted to HeLa cells but can also be extended to cisplatin-sensitive or -resistant ovarian carcinoma cell lines. Finally, we showed that the concomitant inhibition of both ATR and ATM is an absolute requirement to efficiently block the initiation and realization of homologous recombination repair mechanisms. Together, our data identify ATR and ATM as central coordinators of the DDR to trabectedin and lurbinectedin and provide a mechanistic rational for combinations of these compounds with both ATR and ATM inhibitors.