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Ddi1-like protein family has been recently raised into the spotlight by the scientific community due to its important roles in cellular homeostasis maintenance. It represents a specific group among shuttling proteins of the ubiquitin-proteasome system. When compared to other shuttles, Ddi1-like protein family members harbor a unique retroviral-protease like domain besides the conventional ubiquitin-like (UBL) domain and domains interacting with ubiquitin. In addition, a helical domain of Ddi (HDD) has been recently found in most of the orthologs. In this thesis, I focus on characterization of several members of Ddi1-like protein family, both on molecular level using NMR and in model mouse strains via a variety of biological methods. Solution structure of the UBL domain of Ddi1p of S. cerevisiae was solved and its characteristics were compared to those of the UBL domain of its human ortholog. Furthermore, we show that human DDI2 specifically binds to ubiquitin with its terminal domains, both the UBL and the UIM; however, with very low affinity in contrast to binding properties of its yeast counterpart. Our study also show that hDDI2 does not form a head-to-tail homodimer. Based on our structural studies, we hypothesize that human DDI2 might have evolved a different function compared to its yeast...

Les microtubules, composants clés du cytosquelette des cellules eucaryotes, sont des polymères de tubuline très dynamiques et impliqués dans une grande variété de processus cellulaires. Leur rôle essentiel dans le cycle cellulaire a fait d’eux une cible validée en thérapie anticancéreuse. Malgré l’efficacité clinique des agents ciblant les microtubules (ACM), les effets secondaires compliquent l’utilisation. Nous avons cherché à identifier des vulnérabilités génétiques qui peuvent être exploitées pour diminuer la dose requise tout en maintenant l'efficacité, et donc réduire les effets secondaires. En collaboration avec le laboratoire Tyers à l’IRIC, nous avons réalisé un criblage génétique basé sur la létalité synthétique avec des agents antiprolifératifs, dont les ACMs. Nous avons sélectionné les gènes dont l’extinction sensibilisait les cellules aux ACMs. J’ai confirmé que l’invalidation de chacun des gènes GNA13, SEPHS1, DLGAP5 et des gènes QRICH1, DLGAP5 sensibilisaient les cellules NALM6 au docétaxel et la vincristine respectivement. En revanche, aucune invalidation de ces gènes n'a augmenté la sensibilité au docétaxel dans les cellules U2OS. En plus de son effet avec le docétaxel, le gène GNA13 s’est distingué être une cible particulièrement intéressante. En effet, la perte complète de GNA13 augmente considérablement la fréquence et la gravité d’erreurs de ségrégation des chromosomes dans les cellules U2OS. Cette augmentation n’a pas été rectifiée à la suite d’un traitement avec la molécule UMK57, connue pour réduire le taux d’erreurs de ségrégation des chromosomes. De manière intéressante, la perte complète de GNA13 augmente également la fréquence des erreurs de ségrégation des chromosomes dans les cellules RPE1, cellules non-cancéreuses et stables au niveau chromosomique. Cela suggère que la perte complète de GNA13 ne nécessite pas de transformation ni d'instabilité chromosomique, comme conditions préalables pour exacerber l'instabilité chromosomique. L’ensemble de ces résultats ouvre une nouvelle voie de stratégies thérapeutiques anticancéreuses, à savoir, le traitement des cancers présentant une mutation des gènes QRICH1, DLGAP5, GNA13, et SEPHS1 avec de faibles doses d’ACMs. En particulier, GNA13 est fréquemment muté dans certains lymphomes. De plus, les résultats obtenus démontrent que la perte complète de GNA13 aggrave l’instabilité chromosomique et par conséquent, pourrait être impliquée dans la cancérogenèse. / Microtubules, key components of the eukaryotic cytoskeleton, are highly dynamic polymers of tubulin implicated in a wide variety of cellular processes. Their essential roles in the cell cycle have made them a valid target in cancer therapy. Despite the clinical efficacy of microtubule targeting agents (MTA), their use is hampered by side effects. We sought to identify genetic vulnerabilities that can be exploited to decrease the required dose while maintaining efficacy, and therefore reduce side effects. In collaboration with the Tyers laboratory at IRIC, we carried out a genetic screen based on synthetic lethality with antiproliferative agents, including MTAs. We have selected genes whose knockout sensitized cells to MTAs. I have confirmed that the knockout of GNA13, SEPHS1, DLGAP5, and QRICH1, DLGAP5, sensitize NALM6 cells to docetaxel and vincristine respectively. However, no knockout of these genes increased the sensitivity to docetaxel in U2OS cells. In addition to its effect with docetaxel, GNA13 stood out as being a particularly exciting target. GNA13 knockout increased the frequency and severity of chromosome segregation errors in U2OS cells. This increase was not corrected following treatment with UMK57, a molecule known to reduce the rate of chromosome segregation errors. Interestingly, the GNA13 knockout also increased the frequency of chromosome segregation errors in non-cancerous and chromosomally stable RPE1 cells. This suggests that GNA13 does not require transformation nor chromosomal instability as prerequisites for exacerbating chromosomal instability. Overall, these results open up a new avenue of anticancer therapeutic strategies, namely, the treatment of cancers presenting mutations in QRICH1, DLGAP5, GNA13, and SEPHS1 with lower doses of MTAs. In particular, GNA13 is frequently mutated in certain lymphomas. In addition, the results obtained demonstrate that GNA13 knockout exacerbates chromosomal instability and, therefore, could be involved in carcinogenesis.

KRAS ist eines der am häufigsten mutierten Onkogene in Darmkrebspatienten. Dies macht es zu einem guten Ansatzpunkt für gezielte Krebstherapien. Trotz jahrzehntelanger Forschungsbemühungen hat sich jedoch keines der zur Inhibition des mutierten KRAS entwickelten Medikamente klinisch etablieren können. Um eventuelle Schwachstellen von KRAS mutierten Darmkrebszellen aufzudecken, wurde in der vorliegenden Studie ein shRNA basierter Screen in CaCo2 Zellen mit konditioneller KRAS(G12V) Expression ausgeführt. Die maßangefertigte shRNA-Bibliothek umfasste 121 ausgewählte Gene, die zuvor nach MEK Inhibition als stark hoch- oder herunterreguliert identifiziert wurden. Der Screen sowie die Screen-Validierung zeigten, dass KRAS(G12V) exprimierende CaCo2 Zellen besonders sensitiv für den Knockdown des DNA Replikationslizensierungsfaktors Minichromosome Maintenance Complex Component 7 (MCM7) waren, wohingegen sich KRAS(wt) CaCo2 Zellen als weitestgehend resistent gegenüber des MCM7 Knockdowns erwiesen. Ähnliche Ergebnisse wurden im isogenen DLD 1 Zellmodell erzielt. Des Weiteren hat der Knockdown von MCM7 spezifisch in KRAS mutierten Zellen zu erhöhtem Replikationsstress geführt, der durch gesteigerte nukleare RPA Fokalisierung nachgewiesen wurde. Weitere Untersuchungen haben außerdem eine signifikant erhöhte Anzahl an mitotischen Zellen nach gleichzeitigem MCM7 Knockdown und KRAS(G12V) Expression ergeben. Diese Zunahme an mitotischen Zellen wurde zusätzlich von einer stark angestiegenen Anzahl an DNS Schäden in der Mitose begleitet. Das hohe Maß an DNS Schäden in der Mitose kann auf den gesteigerten Replikationsstress zurückgeführt werden, der ungelöst zu einer gestörten Segregation der Chromosomen in der Mitose führt. Zusammenfassend zeigen die Ergebnisse, dass KRAS mutierte Darmkrebszellen sensitiv auf den Knockdown von MCM7 sind. Demzufolge könnte die Inhibition von DNS Replikationslizensierung ein geeigneter Ansatz für die gezielte Therapie von KRAS mutierten Darmkrebs sein. / KRAS ist eines der am häufigsten mutierten Onkogene in Darmkrebspatienten. Dies macht es zu einem guten Ansatzpunkt für gezielte Krebstherapien. Trotz jahrzehntelanger Forschungsbemühungen hat sich jedoch keines der zur Inhibition des mutierten KRAS entwickelten Medikamente klinisch etablieren können. Um eventuelle Schwachstellen von KRAS mutierten Darmkrebszellen aufzudecken, wurde in der vorliegenden Studie ein shRNA basierter Screen in CaCo2 Zellen mit konditioneller KRAS(G12V) Expression ausgeführt. Die maßangefertigte shRNA-Bibliothek umfasste 121 ausgewählte Gene, die zuvor nach MEK Inhibition als stark hoch- oder herunterreguliert identifiziert wurden. Der Screen sowie die Screen-Validierung zeigten, dass KRAS(G12V) exprimierende CaCo2 Zellen besonders sensitiv für den Knockdown des DNA Replikationslizensierungsfaktors Minichromosome Maintenance Complex Component 7 (MCM7) waren, wohingegen sich KRAS(wt) CaCo2 Zellen als weitestgehend resistent gegenüber des MCM7 Knockdowns erwiesen. Ähnliche Ergebnisse wurden im isogenen DLD 1 Zellmodell erzielt. Des Weiteren hat der Knockdown von MCM7 spezifisch in KRAS mutierten Zellen zu erhöhtem Replikationsstress geführt, der durch gesteigerte nukleare RPA Fokalisierung nachgewiesen wurde. Weitere Untersuchungen haben außerdem eine signifikant erhöhte Anzahl an mitotischen Zellen nach gleichzeitigem MCM7 Knockdown und KRAS(G12V) Expression ergeben. Diese Zunahme an mitotischen Zellen wurde zusätzlich von einer stark angestiegenen Anzahl an DNS Schäden in der Mitose begleitet. Das hohe Maß an DNS Schäden in der Mitose kann auf den gesteigerten Replikationsstress zurückgeführt werden, der ungelöst zu einer gestörten Segregation der Chromosomen in der Mitose führt. Zusammenfassend zeigen die Ergebnisse, dass KRAS mutierte Darmkrebszellen sensitiv auf den Knockdown von MCM7 sind. Demzufolge könnte die Inhibition von DNS Replikationslizensierung ein geeigneter Ansatz für die gezielte Therapie von KRAS mutierten Darmkrebs sein. / With KRAS being one of the most frequently altered oncogenes in colorectal cancer (CRC), it is an obvious target for cancer therapy. However, despite enormous efforts over the past three decades to target mutated KRAS, not a single drug has made it to the clinic. To unravel vulnerabilities of KRAS-mutant CRC cells, a shRNA-based screen was performed in CaCo2 cells harboring conditional oncogenic KRAS(G12V). The custom-designed shRNA library comprised 121 selected genes, which were previously identified to be strongly up- or downregulated in response to MEK inhibition. The screen as well as the subsequent validations showed that CaCo2 cells expressing KRAS(G12V) were sensitive to the suppression of the DNA replication licensing factor Minichromosome Maintenance Complex Component 7 (MCM7), whereas KRAS(wt) CaCo2 cells were largely resistant to MCM7 suppression. Similar results were obtained in an isogenic DLD-1 cell culture model. Knockdown of MCM7 in a KRAS-mutant background led to replication stress as indicated by increased nuclear RPA focalization. Further investigation showed a significant increase in mitotic cells after simultaneous MCM7 knockdown and KRAS(G12V) expression. The increased percentage of mitotic cells coincided with strongly increased DNA damage in mitosis. Taken together, the accumulation of DNA damage in mitotic cells is due to replication stress that remained unresolved, which results in mitotic catastrophe and cell death. In summary, the data show a vulnerability of KRAS mutant cells towards suppression of MCM7 and suggest that inhibiting DNA replication licensing might be a viable strategy to target KRAS-mutant cancers.

No description available.

No description available.

The emergence of static warfare on the Western Front in late 1914, encouraged the reinvention of devices associated with siege warfare and the invention of hitherto unknown munitions. These munitions included hand and rifle grenades and trench mortars and their ammunition. At the outbreak of war, the British effectively possessed none of these devices and lacked an infrastructure by which they could be quickly designed, manufactured and supplied to the BEF. The British met this challenge with considerable success and the subsequent proliferation of trench warfare munitions had profound consequences for the evolution of British tactics on the Western Front. This thesis examines the processes by which these devices were invented, developed into manufacturable devices and supplied to the BEF. It considers their novelty in respect to similar devices from the American Civil War and the Russo-Japanese War. It looks at how their technical evolution affected tactical developments. The thesis discusses the relationship between the technical characteristics of these devices and the evolution of their tactical employment on the Western Front. It also considers how the characteristics of certain munitions, such as the Stokes mortar and the Mills grenade, directly effected tactics. It argues that the tactical employment of these munitions was dependent upon their functionality, utility and reliability. The present thesis provides a different model of trench warfare conducted by the British during the First World War and thereby demonstrates the significance of the novel munitions under discussion and the role they played in changing infantry warfare. This thesis also provides a different view of the Ministry of Munitions from that usually offered and argues that certain aspects of the Ministry’s role in providing the BEF with munitions has been overstated by virtue of its having underplayed the work of the War Office, while overlooking that conducted by the Royal Engineers in France.

Topoisomerase 2α (Topo2α) is an essential protein with DNA decatenating enzymatic properties, indispensable for chromosome decatenation and segregation. It is a target for a plethora of antitumour drugs and Topo2α protein levels have been associated with the success of treatment, but also drug resistance and secondary malignancies. Although unique in its ability to resolve catenated chromosomes, the role of Topo2α in other steps of DNA metabolism, such as DNA replication elongation and termination have been elusive. A thorough understanding of the role of Topo2α in the cell will not only allow for increased insight into the mechanisms it is involved in, but it will also shed light on proteins and pathways that can act as back-up in its absence, and therefore hopefully expand the basis on which to improve treatment options. Through a synthetic lethal interaction (SLI) screen with an siRNA library targeting 200 DNA repair and signalling genes, Topo2α emerged as being synthetic lethal to Werner protein (WRN), a RecQ helicase involved in maintaining genome integrity mainly in S phase, and the loss of which leads to Werner Syndrome (WS), a segmental progeroid syndrome. The screen was performed in WRN deficient cells, with the initial aim to find proteins that act to buffer against loss of viability, which is the central idea in the concept of synthetic lethality in the absence of WRN. The screen revealed an SLI between WRN and Topo2α and although we were unable to fully validate this, it spurred the question of Topo2α’s role in DNA replication. The findings in this thesis suggest that Topo2α is not required for DNA elongation and timely completion of S phase, and that simultaneous loss of the closely related isoform Topo2β does not affect replication, suggesting that these proteins do not act in parallel back-up pathways during replication. Interestingly, cells accumulate in the polyploid fraction after both depletion and inhibition of Topo2α, albeit with different kinetics. The mechanistic basis of this phenotype remains to be understood through further research, but it is highly interesting as aneuplidity and polyploidy are implicated in the initial stages of tumour development.

DNA is wrapped around proteins called histones, whose modification regulates numerous cellular processes. Therefore it is not surprising that mutations in the genes that modify the histones are frequently associated with human cancer. For example, mutations in SETD2, encoding the sole enzyme that catalyses histone H3 lysine 36 trimethylation (H3K36me3), occur frequently in multiple cancer types. This identifies H3K36me3 loss as an important event in cancer development, and also as a potential therapeutic target. This thesis investigates the following questions: (1) how does the loss of H3K36me3 contribute to cancer development; and (2) what therapy can be used to kill cancers that have already lost H3K36me3. To answer the first question, this thesis shows that H3K36me3 facilitates the accurate repair of DNA double-stranded breaks (DSBs) by homologous recombination (HR). H3K36me3 promotes HR by recruiting CtIP to the site of DSBs to carry out resection, allowing the binding of HR proteins (such as RPA and RAD51) to the damage sites. Thus it is proposed that error-free HR repair within H3K36me3-decorated transcriptionally active genomic regions suppresses genetic mutations which could promote tumourigenesis. To answer the second question, this thesis reveals a clinically relevant synthetic lethal interaction between H3K36me3 loss and WEE1 inhibition. WEE1 inhibition selectively kills H3K36me3-deficient cells by inhibiting DNA replication, and subsequent fork stalling results in MUS81 endonuclease-dependent DNA damage and cell death. The mechanism is found to be synergistic depletion of RRM2 (ribonucleotide reductase small subunit), the enzyme that generates deoxyribonucleotides (dNTPs). This work reveals two pathways that regulate RRM2: one involves transcriptional activation of RRM2 by H3K36me3, and the other involves RRM2 degradation regulated by Cyclin-Dependent Kinase, CDK1 (which is controlled by WEE1, CHK1 and ATR). Based on this mechanism, the synthetic lethal interaction is expanded, from between two genes, to between two pathways. Supported by in vivo experiments, the study suggests that patients with cancers that have lost H3K36me3 could benefit from treatment with the inhibitors of WEE1, CHK1 or ATR.

Introduction Le cancer bronchique est un problème de santé publique en étant la première cause de décès par cancer dans le monde. Il reste de mauvais pronostic avec une résistance au Cisplatine qui est inéluctable dans l’histoire naturelle de la maladie. Nous nous sommes intéressés à l’association du CDDP aux inhibiteurs de la Poly(ADP-ribose) polymérase. Les inhibiteurs pharmacologiques de PARP sont source d’optimisme en oncologie clinique en monothérapie pour des tumeurs déficientes pour une voie de réparation de l’ADN et en association aux cytotoxiques classiques.Matériel et méthodes Nous avons généré 9 clones résistants au CDDP après culture de la lignée A549 dans des faibles doses de CDDP. Deux inhibiteurs pharmacologiques de PARP, CEP8983 (CEP) et PJ34 (PJ), ainsi que des siRNA spécifiques de PARP1 sont utilisés pour l’inhibition de PARP. L’apoptose est mesurée en cytométrie de flux par l’intermédiaire du potentiel membranaire de la mitochondrie DiOC6(3) et la perméabilisation de la membrane plasmique est évaluée par l’iodide de propidium. Le test de clonogénicité permet d’évaluer la capacité des cellules à échapper à la mort et à former une colonie. L’activité métabolique des cellules est mesurée par la mesure de clivage du sel de tetrazolium WST-1. L’immunofluorescence sur cellules fixées a permis d’étudier les dommages de l’ADN (γH2AX), la voie intrinsèque de l’apoptose (l’activation de la caspase 3 et la libération du cytochrome c) et la recombinaison homologue (BRCA1, RAD51). En Western Blot nous avons mesuré l’expression et l’activité de PARP (PAR) ainsi que l’expression d’acteurs de la réparation par excision de base (BER) (XRCC1 and polymérase β). Nous avons développé une méthode de détection de PAR en immunohistochimie sur des tissus inclus en paraffine. Résultats Nous avons trouvé un effet synergique pour l’association du CDDP aux inhibiteurs de PARP in vitro. De façon inattendue nous avons observé que les clones résistants au CDDP développent une addiction à PARP et sont spécifiquement tués par l’inhibition de PARP contrairement à la lignée parentale. Ces clones exhibent une hyperexpression et une hyperactivité de PARP. La réponse aux inhibiteurs de PARP corrèle plus précisément avec l’activation plutôt qu’avec l’expression de PARP, pointant que PAR est un biomarqueur plus précis que PARP. Nous avons observé que l’hyperactivation de PARP accompagne une résistance induite au CDDP et prédispose à une sensibilité aux inhibiteurs de PARP dans d’autres lignées de cancer bronchique (H460 et H1650), de mésothéliome (P31), de cancer de l’ovaire (TOV112D) et de col (HeLa). Dans des expériences in vivo nous avons noté que dans les xénogreffes obtenues à partir de clones résistants au CDDP, l’expression de PAR est stablement retrouvée en immunohistochimie. Ces tumeurs répondaient à l’inhibition de PARP par le PJ en diminuant l’expression de PAR. Les clones résistants au CDDP sensibilisés aux I PARP ont une recombinaison homologue conservée, cependant ont un déficit dans les étapes terminales du BER.Conclusion Nous avons identifié un effet synergique pour l’association des inhibiteurs de PARP au CDDP de des lignées de cancer bronchique. Nous avons observé une dépendance à PARP dans des lignées de cancer bronchique résistantes au CDDP et déficientes pour l’élongation du BER. Nous postulant que PAR est un biomarqueur spécifique de la réponse aux inhibiteurs de PARP. / Introduction Driven by the facts that non small cell lung cancer (NSCLC) is the leading cause of cancer-related morbidity and mortality worldwide and that NSCLC patients often develop resistance against Cisplatin (CDDP)-based therapies, we addressed the question of the combination therapy of CDDP with poly(ADP-ribose) polymerases (PARP) inhibitors. Inhibitors of PARP have raised great expectations for the treatment of a variety of cancers, either as monotherapeutic agent against DNA repair-deficient tumours or combined to DNA-damaging compounds.Material and methods We generated nine CDDP-resistant clones by prolonged exposure to low dose CDDP of the A549 NSCLC parental cell line. Two distinct PARP inhibitors, CEP8983 (CEP) and PJ34 (PJ) as well as PARP1 knockdown with small interfering RNAs (siRNAs) were used for PARP inhibition. Apoptosis was measured by the simultaneous assessment for the loss of the mitochondrial transmembrane potential (m) and the breakdown of the plasma membrane using the m-sensitive fluorochrome DiOC6(3) and the vital dye propidium iodide, respectively. Moreover clonogenic survival was assessed. In vitro assessments of the enzymatic activity of cells were based on the reduction of the colorless tetrazolium salt. Immunofluorescence microscopy determinations were performed with antibodies specific for DNA damage (γH2AX), intrinsic apoptosis (cleaved Caspase-3 and cytochrome c), and homologous recombination (RAD51 and BRCA1). Immunoblotting was assed for PARP1 expression and activity (PAR) and base excision repair (BER) effectors (XRCC1 and polymerase β). We developed an immunohistochemical staining method that specifically detects PAR on paraffin-embedded cell pellets and tissue sections.Results We found that PARP inhibitors and PARP1 siRNAs synergized with CDDP in the killing of NSCLC cells in vitro. Unexpectedly, CDDP-resistant NSCLC cell clones developed addiction to PARP hyperactivation, thereby becoming susceptible to apoptosis induction by PARP inhibition. We showed that these cisplatin-resistant clones, exhibited high PARP protein levels and increased PARP activity, leading to an increased poly-ADP ribosylation of cellular proteins, as compared to their parental, cisplatin-sensitive counterparts. These cisplatin-resistant cells become susceptible to cell death as induced by PARP inhibition, correlating with the hyperactivity of PARP (elevated PAR levels) more accuratly than with the overexpression of PARP. Suggesting that PAR levels may constitute a more accurate biomarker than PARP to predict the sensitivity of cells to PARP inhibition. We expanded the observation that cisplatin resistance causes PARP upregulation and hyperactivation and subsequent sensitization to PARP inhibition to additional five human cancer cell lines including two NSCLC (H1650 and H460), one mesothelioma (P31), one ovarian (TOV112D) and one cervical cancer (HeLa) cell line. To get further insight into this issue, we generated in vivo experiments. Tumors derived from CDDP-resistant cells were characterized by elevated levels of PAR suggesting that PAR levels are preserved during tumor formation. Those PAR-overexpressing tumors responded to the administration of PJ in vivo with a consistent reduction in PAR immunoreactivity. CDDP resistant clones that are specifically killed by PARP inhibitors assessed efficient homologous recombination repair however deficient BER elongation.Conclusion We showed a beneficial effect for the association therapy of PARP inhibitors with CDDP in several NSCLC cell lines. We have identified an addiction to PARP in CDDP resistant cell lines with deficient BER elongation. We postulate that PAR is a specific predictive biomarker for the response to PARP inhibitors.