Chez les eucaryotes supérieurs, la réplication de l'ADN est initiée à partir de plusieurs milliers d'origines. Mais la régulation spatio-temporelle de leur activation reste mal caractérisée. Une voie de contrôle (checkpoint) de la phase S est activée lorsque les fourches de réplication sont bloquées, inhibant ainsi l'activation d'origines tardives. L'objectif de ma thèse consistait à étudier deux facteurs essentiels dans le programme spatio-temporel de la réplication, dans le système du xénope : la protéine « checkpoint » Chk1, qui est un facteur inhibiteur de l'activation des origines, et les désoxyribonucléotides (dNTPs), précurseurs de la synthèse de l'ADN. Chez le xénope, après douze divisions embryonnaires, a lieu la transition mid-blastuléenne (MBT). A cette étape, une augmentation du ratio nucléo-cytosolique va entrainer la titration des facteurs de réplication, ce qui active le point de contrôle et ralentit la phase S. Il est possible de mimer in vitro les phases S rapides des embryons pendant le développement précoce en augmentant la concentration en noyaux dans l'extrait d'œufs.Nous avons pu voir par l'inhibition, la déplétion ou la surexpression de Chk1 que cette protéine régulait l'activation des origines lors d'un stress, mais également dans une phase S non perturbée, grâce à la technique du peignage moléculaire. Ce résultat montre que le niveau de Chk1 doit être finement régulé pour permettre une réplication correcte dans une phase S non perturbée, chez les eucaryotes supérieurs. Nous avons ensuite cherché à savoir si la concentration en dNTPs pouvait être limitante pendant le développement et comment elle modulait le programme de réplication. Nous avons comparé l'effet de l'ajout de dNTPs sur la réplication en mimant plusieurs stades précoces du développement pré-MBT. La variation de la concentration en dNTPs agit sur la réplication en augmentant à la fois l'activation des origines et, en fonction de la concentration en noyaux, aussi la vitesse des fourches. Cet effet est indépendant du checkpoint de la réplication dans ce système et d'autres études sont nécessaires pour comprendre les mécanismes moléculaires. / DNA replication in higher eukaryotes initiates at thousands of origins according to a spatio-temporal regulation program which is not well characterized. The S phase checkpoint is activated when replication forks are blocked which inhibits the firing of late origins. The aim of my thesis consisted to study two essentials factors in spatio-temporal replication program in Xenopus system: the checkpoint protein Chk1, inhibitor of origin activation, and the deoxyribonucleotides (dNTPs), DNA synthesis precursors. In Xenopus, the mid-blastula transition (MBT) occurs after twelve embryonic divisions. An increase of the nucleo-cytosolic ratio induces a titration of replication factors, that activates the checkpoint and slows down the S phase. It is possible to mimic in vitro the rapid S phases of early Xenopus development stages by increasing the nuclei concentration. By DNA combing combined with Chk1 inhibition, depletion and overexpression experiments, we show that Chk1 controls origins activation in perturbed but also unperturbed S phase. My results show that Chk1 levels needs to be tightly regulated in order to properly control the replication program during normal S phase in higher eukaryotes. In order to determine whether the concentration of dNTPs could be another limiting replication factor, we compared the effect of dNTPs addition on replication by mimicking in vitro several early stages of pre-MBT development. Addition of dNTPs affects DNA replication, by increasing origin activation and, dependent on nuclei concentration, also the fork speed. This effect is independent of the S phase checkpoint and further studies are needed in order to understand the molecular mechanisms behind.

An extract of the sponge <i>Rhabdastrella globostellifera</i> was active in an assay measuring stabilization of the binding of DNA with DNA polymerase β. From this extract, four isomalabaricane triterpenoids were isolated and characterized, three of which were active in the binding assay. All compounds were active in the A2780 ovarian cancer cell line assay. Bioassay-guided fractionation of an extract of a sponge of species <i>Dysidea</i> using the A2780 bioassay yielded the known scalarane sesterterpenoid heteronemin in good yield. Four derivatives of heteronemin were prepared semisynthetically from the natural product, tested for their bioactivity, and their structure-activity dependence was observed. Bioassay guided-fractionation of an extract of a <i>Tuemoya</i> sp. green alga, using an assay for inhibitors of the enzyme Tie2 kinase, afforded a two sulfated cycloartanol triterpenoids. Both the major and minor compounds were identified by spectroscopic methods. Bioassay-guided fractionation of an extract of <i>Petalonyx parryi</i> yielded three known oleanane triterpenoids which inhibited the lyase domain of DNA polymerase β. The structures were confirmed by NMR spectroscopic techniques. This is the first reported study of the chemical components of <i>Petalonyx parryi</i>. As part of our antitumor natural product drug discovery efforts, several extracts were selected for bioassay-guided fractionation based on their activity in initial in vitro screens. A new dereplication method using aminopropyl SPE cartridges was applied to six of these extracts, and four of the extracts were dropped due to the presence of long-chain fatty acids (LCFAs). We present results for the testing and application of this SPE-based method for LCFA dereplication. The cell cycle kinase Chk1 is an interesting target for the development of agents which might potentiate DNA damaging agents. Typical assays for Chk1 involve the use of expensive or radioactive reagents. To facilitate the development of new assays using shorter peptide substrates, small libraries of peptides have been synthesized and tested for their activity as Chk1 substrates. Several of the substrates synthesized displayed activity in the Chk1 assay. / Ph. D.

In the fission yeast, Schizosacchromyces pombe, blocks to DNA replication elongation trigger the intra-S phase checkpoint that leads to the activation of the Cds1 kinase. Cds1 is required to both stabilize stalled replication forks and to prevent premature entry into mitosis. Interestingly, although Cds1 is essential to maintain the viability of mutants defective in DNA replication elongation, my study shows that mutants defective in DNA replication initiation require the Chk1 kinase, rather than Cds1. This suggests that failed initiation events can lead to activation of the DNA damage checkpoint independent of the intra-S phase checkpoint. This might result from reduced origin firing that leads to an increase in replication fork stalling or replication fork collapse that activates the G2 DNA damage checkpoint. I refer to the Chk1-dependent, Cds1-independent phenotype as the rid phenotype (for replication initiation defective). The data shows that Chk1 is active in rid mutants when grown under semi-permissive conditions, and rid mutant viability is dependent on the DNA damage checkpoint, and surprisingly Mrc1, an adaptor protein required for activation of Cds1. Mutations in Mrc1 that prevent activation of Cds1 have no effect on its ability to support rid mutant viability, suggesting that Mrc1 has a checkpoint-independent role in maintaining the viability of mutants defective in DNA replication initiation. Like Mrc1, Swi1 and Swi3 have been hypothesized as a part of the replication fork protection complex (RFPC). They are required for maintaining the viability of rid mutants, but are not essential for activation of Chk1 in response to failed initiation events. This suggests that Mrc1 in conjunction with Swi1 and Swi3 function in a similar pathway to alleviate replicative stress resulting from defects in DNA replication initiation. Using flow cytometry, I demonstrate that inhibition of DNA replication initiation has no significant impact on the duration of S phase, suggesting dormant origins might be activated in response to defects in DNA replication initiation. Fission yeast Rad22 is implicated in forming nuclear foci in response to damaged DNA. By tracking YFP-labeled Rad22, I screened for potential DNA damage in rid mutants grown at semi-permissive temperatures, and the results show that DNA damage occurs as the result of defects in DNA replication initiation. I also identified camptothecin, a DNA topoisomerase I inhibitor that can at low dose (2 µM) induce the rid phenotype, suggesting our assay (Chk1-dependent, Cds1-independent) can be used to screen small molecule inhibitors that interfere with the initiation step of DNA replication.

Early Xenopus laevis embryonic cell cycles exemplify rapid, non-pathological cell cycles without checkpoint pathways to arrest cell cycles in response to DNA assaults. There is no transcription or apoptosis during these cell cycles, and they continue unperturbed until the 12th cell cycle, marking a period called the midblastula transition (MBT). At the MBT, the embryo undergoes a period of developmental remodeling: gap phases are acquired, zygotic transcription is initiated, and the maternal mRNAs are degraded. After the MBT, checkpoint pathways can be activated in response to unreplicated DNA, and apoptosis initiates when continued embryonic survival is compromised. These studies examine how cell cycle regulation and apoptotic regulation are related. Specifically, the role of two cell cycle components, Chk1 and cyclin A1/Cdk2, during apoptosis was studied during early development of Xenopus embryos. Chk1 is a serine/threonine kinase that inhibits the activity of cyclin-dependent kinases (Cdks) in response to unreplicated DNA. In the pre-MBT embryo, Chk1 is present, but inactive. Injection of mRNA encoding dominant-negative Chk1 (DN-Chk1) into single-celled embryos results in the initiation of apoptosis after the MBT. The loss of Chk1 function also results in the initiation of additional rapid rounds of DNA replication after the MBT. These results suggest that Chk1 has a required function for the embryo after the MBT, possibly through the regulation of a cyclin/Cdk complex responsible for the apoptotic checkpoint. Cyclin A1 is a maternally provided mRNA that is degraded at the MBT. Prior to the MBT, cyclin A1 complexes exclusively with Cdc2 to regulate mitosis. When embryos are treated with ionizing radiation (IR), cyclin A1 activity and protein level persist after the MBT, and cyclin A1 complexes with Cdk2. When treated with aphidicolin, cyclin A1-associated activity and protein level persists. Injection of cyclin A1/Cdk2 into single-cell embryos results in apoptosis after the MBT; however, inhibition of cyclin A1 expression does not abrogate apoptosis. Therefore, cyclin A1/Cdk2 activity is sufficient, but not required, for the initiation of apoptosis in the early Xenopus embryo. These studies show that Chk1 and cyclin A1/Cdk2 have roles in regulating apoptosis in the post-MBT embryo. / Ph. D.

When unreplicated or damaged DNA is present, cell cycle checkpoint pathways cause cell cycle arrest by inhibiting cyclin-dependent kinases (Cdks). In Xenopus laevis, early embryonic development consists of twelve rapid cleavage cycles between DNA replication (S) and mitosis (M) without checkpoints or gap phases. However, checkpoints are engaged in Xenopus once the embryo reaches the midblastula transition (MBT). At this point, the embryo initiates transcription, acquires gap phases between S and M phases, and establishes a functional apoptotic program. During the cell cycle, there are two main checkpoints that regulate entrance into S and M phases. The focus of this study is the role of protein kinase Chk1 and the phosphatase Cdc25A in the DNA replication checkpoint. In the absence of active Chk1, Cdc25A activates cyclin dependent kinases (Cdks) allowing the cell to progress into S or M phase. Chk1 regulates cell cycle arrest in the presence of unreplicated DNA in somatic cells by phosphorylating Cdc25A and leading to its degradation. Chk1 is also transiently activated at the MBT in Xenopus laevis embryos, even when there is no block to DNA replication or damaged DNA. One goal of this work is to understand the developmental role and regulation of checkpoint signaling pathways due to its monitoring of DNA integrity within the cell. Chk1 plays a critical but not fully understood role in cell cycle remodeling and early embryonic development. In order to understand the function and regulation of Chk1 in checkpoints, the features of the MBT that activate Chk1 must be identified. The activation of Chk1 by two time-dependent events in the cell cycle, the critical nuclear to cytoplasmic (N/C) ratio and the cyclin E/Cdk2 maternal timer are explored in this study. Embryos treated with aphidicolin, resulting in a halted replication fork and therefore a reduced DNA concentration, were tested for Chk1 activation and Cdc25A degradation. Chk1 and Cdc25A were observed to undergo activation and degradation, respectively, in embryos with a reduced DNA concentration. In addition, embryos were injected with Δ34Xic cyclin E/Cdk2 inhibitor, in order to disturb the maternal timer and tested for Chk1 activation and Cdc25A degradation. Both Chk1 and Cdc25A were unaffected by the disruption of the cyclin E/Cdk2 maternal time in the embryo. Therefore, the N/C ratio and the cyclin E/Cdk2 maternal timer do not affect Chk1 activation and therefore Cdc25A degradation. Another means of characterizing the unreplicated DNA checkpoint is through the use of mathematical modeling of the checkpoint-signaling cascade of the cell cycle. Mathematical modeling is the translating of biological pathways into mathematical equations that can simulate interactions without performing laboratory experiments. The Novák-Tyson checkpoint model made important predictions of hysteresis and bistability in the frog egg checkpoint model, predictions that were later confirmed experimentally. The model was updated with additional interactions, such as those including Myt1, a second inhibitor kinase, and lamin proteins, which become phosphorylated at the onset of nuclear envelope breakdown (NEB) at entry into mitosis. Also, experimental data was fit into the model while maintaining hysteresis and bistability. Therefore, the unreplicated DNA checkpoint model was updated with new interactions and experimental data while still preserving previously identified dynamic characteristics of the system. As described, Cdc25A regulation is dynamic in the embryo. The checkpoint original model represents the activity of Cdc25 phosphatase on the mitosis promoting factor (MPF) that leads the cell into mitosis. In the checkpoint model, Cdc25C is the phosphatase activating MPF. However, the model does not include Cdc25A, which is an integral part of the checkpoint-signaling pathway due to its role in activating the cyclin/Cdk complex allowing entry into S and possibly M phase. Experimental studies were performed in which Cdc25A levels were reduced in embryos and extracts using Cdc25A morpholinos. Embryos and extracts showed delayed cell cycle and mitotic entry, demonstrating the importance of Cdc25A plays in the cell cycle. Based upon experimental data, the mathematical model of the DNA replication checkpoint was expanded to include Cdc25A. The expanded model should more accurately demonstrate how checkpoints affect the core cell cycle machinery. Cdc25A was incorporated into the model by gathering experimental data and designing a signaling cascade, which was translated into differential equations. The updated model was then used to simulate the effect of synthesis and degradation rates of Cdc25A on the entry into mitosis dynamics. Therefore, using mathematical modeling and experimental design, we can further understand the role that Cdc25A plays in cell cycle progression during development. Understanding the regulation of Chk1 activity at the MBT and the role of Cdc25A in checkpoint signaling will help us further characterize the dynamics of early embryonic development. The use of mathematical modeling and experimental tools both contribute to further our understanding of controls of the checkpoint signaling pathway and therefore leading us one step closer to truly being able to model a pathway and make predictions as to the behavior of the cell during early embryonic development. / Ph. D.

Les sarcomes sont des cancers rares touchant toutes les zones du corps humain, caractérisés par une grande diversité de nature, de comportement clinique et de réponse aux thérapies existantes, certains étant de bon pronostic, d’autres très difficilement curables.Leur traitement de référence est la chirurgie ; la radiothérapie et les protocoles de chimiothérapie n’ayant que peu évolué lors des 30 dernières années.Récemment des caractéristiques génétiques leur étant propres ont été découvertes, prédictives de leur agressivité et contre lesquelles il est possible de diriger des drogues spécifiques pouvant améliorer le pronostic et diminuer les effets secondaires des thérapies conventionnelles.C’est sur l’inhibition d’Aurora Kinase A et B et CHK1 que s’est focalisé ce travail avec le test des effets de deux nouvelles drogues sur 9 types de cellules cancéreuses sarcomateuses avec des résultats très prometteurs, qu’il conviendra de conforter par d’autres expériences, notamment sur l’animal. / Sarcomas are rare cancers, which may arise in all parts of human body. They are characterized by great diversity in their nature, clinical behavior and response to existing therapeutics. Some are of good prognosis and others hard to cure.Their treatment essentially relies on surgery and radiotherapy or chemotherapy haven’t know major breakthrough over the last 3 decades.Recently new genetics abnormalities linked to sarcomas have been discovered. Their analysis can predict their aggressiveness and it is now possible to develop targeted therapies against them. This could help improving cancer prognosis and/or limiting conventional drugs adverse effects.Our work focused on Aurora Kinase A and B and CHK1 inhibition, testing the effects of 2 new drugs on 9 types of sarcoma cells with promising results, which we will confort by other experiments, including on the animal.

Le cancer colorectal est responsable de plus de 17500 décès par an et se situe à la 3ème place des cancers les plus fréquents en France. En altérant le processus de réplication de l’ADN des cellules cancéreuses, certaines des molécules utilisées en chimiothérapie induisent du stress réplicatif. Au niveau cellulaire, ce stress est géré par la voie ATR-CHK1. Quand elle est activée par des régions d’ADN simple brin protégées par RPA au niveau de fourches de réplication ralenties/bloquées, ATR déclenche l’activation du checkpoint intra-S via la phosphorylation de son effecteur CHK1. Ce checkpoint permet alors aux cellules de gérer ce stress réplicatif, via différents processus (arrêt du cycle cellulaire, régulation de l’allumage d’origines de réplication, stabilisation des fourches…). Depuis quelques années, l’intérêt thérapeutique de cibler cette voie est clairement établi dans la littérature. Ce rationnel thérapeutique repose sur une inhibition pharmacologique de la voie ATR-CHK1, éventuellement couplée à des traitements par des molécules génotoxiques. Par ailleurs, certaines tumeurs présentent fréquemment des mutations hétérozygotes des gènes ATR et CHK1. Nous avons émis l’hypothèse que ces déficiences puissent représenter leur talon d’Achille. L’équipe a généré un modèle cellulaire permettant d’étudier spécifiquement ces mutations hétérozygotes d’ATR et CHK1. Nous avons commencé notre étude par la caractérisation des impacts de ces mutations en conditions normales de culture. Nos données montrent que les mutations d’ATR et CHK1 altèrent l’activation basale du checkpoint intra-S, provoquent un stress réplicatif endogène, et aboutissent à une induction de dommages de l’ADN. Par ailleurs, ces mutations sensibilisent les cellules à certaines drogues. Entre autres, les cellules mutantes présentent des sensibilités cytotoxiques accrues au SN-38 (principe actif de l’Irinotécan, inhibiteur de topoisomérase 1) et au VE-822 (inhibiteur d’ATR). De plus, nous avons montré que ces deux composés ont un effet synergique important, et nous avons par la suite étudié les mécanismes moléculaires sous-jacents à ces phénotypes de sensibilisation et de synergie. Nos résultats démontrent que la combinaison SN-38+VE-822 entraîne une apoptose dépendante de la caspase-3, exacerbée chez les cellules mutantes ATR ou CHK1. Ces altérations génétiques limitent le potentiel d’activation du checkpoint intra-S et aboutissent à une accumulation de dommages de l’ADN. L’inhibition d’ATR par le VE-822 permet aux cellules de court-circuiter l’arrêt du cycle cellulaire en S-précoce normalement induit par le SN-38. Nos analyses démontrent que ce phénotype entraine un épuisement de RPA et une catastrophe réplicative subséquente, la mutation d’ATR prédisposant les cellules à ces phénotypes. Les cellules survivant à la combinaison SN-38+VE-822 complètent la réplication et s’accumulent en G2 de façon DNA-PK-dépendante. Ce checkpoint post-réplicatif protège les cellules de la catastrophe mitotique. Ensemble, ces observations suggèrent que RPA et DNA-PK représentent des cibles thérapeutiques prometteuses pour optimiser les effets de l’inhibition de la voie ATR-CHK1. En définitive, les mutations d’ATR et CHK1 retrouvées chez les patients pourraient représenter des facteurs pronostiques importants de la réponse à ces stratégies thérapeutiques. De plus, certains de nos résultats suggèrent également une implication de la voie ATR-CHK1 dans la régulation du remodelage des fourches de réplication, notamment dans la résection de l’ADN néo-synthétisé. En affinant la compréhension des processus moléculaires impliqués dans la réponse au stress réplicatif, notre étude pourrait contribuer à l’amélioration de la prise en charge thérapeutique du cancer colorectal. / Colorectal cancer is responsible for more than 17,500 deaths per year and ranks third among the most frequent cancers in France. By interfering with the DNA replication process of cancer cells, several chemotherapeutic molecules induce replication stress. At the cellular level, this stress is managed by the ATR-CHK1 pathway. When activated by RPA-protected single-stranded DNA regions at slowed/blocked replication forks, ATR triggers the activation of the intra-S checkpoint via the phosphorylation of its CHK1 effector. This checkpoint then allows the cells to manage this replicative stress, via different processes (stopping the cell cycle, regulating the ignition of replication origins, stabilizing the forks...). In recent years, the therapeutic value of targeting this pathway has been clearly established in the literature. This therapeutic rationale is founded on pharmacological inhibition of the ATR-CHK1 pathway, possibly coupled with genotoxic molecules treatments. In addition, some tumours frequently have heterozygous mutations of the ATR and CHK1 genes. We have hypothesized that these deficiencies may represent their Achilles' heel. Our team generated a cellular model to specifically study these heterozygous mutations of ATR and CHK1. We began our study by characterizing the impacts of these mutations under normal growing conditions. Our data show that the ATR and CHK1 mutations alter the basal activity of the intra-S checkpoint, cause endogenous replicative stress, and lead to spontaneous DNA damage. In addition, these mutations sensitize the cells to certain drugs. Amongst other things, mutant cells show increased cytotoxic sensitivities to SN-38 (active ingredient of Irinotecan, topoisomerase inhibitor 1) and to VE-822 (ATR inhibitor). Furthermore, we showed that these two compounds have a strong synergistic. We then studied the underlying molecular mechanisms to these sensitization and synergy phenotypes. Our results show that the combination SN-38+VE-822 causes caspase-3-dependent apoptosis, exacerbated in mutant ATR or CHK1 cells. These genetic alterations limit the activation potential of the intra-S checkpoint and lead to extensive DNA damages. Inhibition of ATR by VE-822 allows cells to bypass the S- early cell cycle arrest normally induced by SN-38. Our analyses show that this phenotype leads to RPA depletion and subsequent replicative catastrophe, with ATR mutation predisposing cells to these phenotypes. Cells surviving the SN-38+VE-822 combination complete the replication and accumulate to G2 in a DNA-PK-dependent manner. This post-replicative checkpoint protects the cells from mitotic catastrophe. Together, these data suggest that RPA and DNA-PK represent promising therapeutic targets to optimize the effects of inhibition of the ATR-CHK1 pathway. Moreover, some of our results also suggest that the ATR-CHK1 pathway could be involved in the regulation of replication forks' remodeling, particularly in the resection of newly-synthetized DNA. Ultimately, the mutations of ATR and CHK1 found in patients may represent important prognostic factors in the response to these therapeutic strategies. By achieving a better understanding of the molecular processes involved in the response to chemically-induced replication stress, our study could contribute to the improvement of colorectal cancer’s therapeutic management.

High Mobility Group AT-hook protein 2 (HMGA2) is a non-histone chromatin binding protein expressed in stem cells, cancer cells but not in normal human somatic cells. The presence of HMGA2 in cancer correlates with advanced neoplastic disease and poor prognosis. HMGA2 plays important roles in Base Excision Repair (BER) and at replication forks. HMGA2 is present at mammalian metaphase telomeres and its loss induces chromosomal aberrations. However, the functional role of HMGA2 at telomeres remains elusive. We hypothesized a protective role of HMGA2 that guards telomeres and modulates DNA damage repair signaling pathways. Employing different HMGA2+ human tumor cell models, we investigated the HMGA2-mediated functions that contribute to chemoresistance in glioblastoma (GB). This study presents a novel interaction of HMGA2 with telomeric protein TRF2 (Telomere Repeat-Binding Factor 2). This interaction retains TRF2 at telomeres, thus capping the telomeres and reducing telomere-dysfunction induced foci despite induced telomere stress. Loss of HMGA2 coincides with increased phosphorylation of TRF2, decreased TRF2 retention at telomeres and increased formation of telomeric aggregates, anaphase bridges and micronuclei. These findings provide new evidence for a unique role of HMGA2 at telomeres as a novel contributor of telomeric integrity. We show that upon DNA damage, HMGA2 causes increased and sustained phosphorylation of Ataxia Telangiectasia and Rad3-related kinase (ATR) and checkpoint kinase 1 (CHK1). Prolonged presence of pCHK1Ser296 coincides with prolonged G2/M block and increased tumor cell survival. The relationship between (ATR)-CHK1 DNA damage response pathway and HMGA2 identifies a novel mechanism by which HMGA2 can alter DNA repair function in cancer cells. We identified HMGA2 as a novel factor contributing to temozolomide (TMZ) resistance in GB. HMGA2 knockdown sensitizes the GB cells to TMZ. We propose a specific combination of FDA-approved drugs, TMZ and Dovitinib (DOV), to increase GB cell death. We show that DOV downregulates key BER proteins, attenuates pSTAT3-coordinated Lin28A and HMGA2 expression. Our results suggest that a sequential therapeutic strategy of pretreating GB cells with DOV followed by a sequence of TMZ and DOV diminishes TMZ resistance and enhances the ability of TMZ to induce GB cell death. Overall, we identified HMGA2 as a multifunctional survival factor in human cancer cells and showed that targeting HMGA2 is a valid strategy to combat HMGA2+ cancer cells. / February 2016

Checkpoint kinase 1 (Chk1) is a key player in the DNA damage response signalling pathway and the mode of Chk1 activation whereby it undergoes ATRdependent phosphorylation at Ser317 and Ser345 is well characterised. It has been suggested that phosphorylation at the ATR sites relieves the auto-inhibitory action conferred by the C-terminal negative regulatory domain on the catalytic core of Chk1. In this study, we show that Chk1 activity can also be stimulated by docking to an N-terminal region of the growth regulator p21waf1 and this docking domain is necessary for efficient Chk1-dependent phosphorylation of p21 at Ser146. In addition, Chk1 and p21 are shown to form a transient interaction by immunoprecipitation. Interestingly, although the isolated p21 docking domain can activate Chk1 in trans, a mutant where the C-terminal 70 amino acids are truncated is refractory to stimulation whereas mutation of the ATR phosphoacceptor sites does not affect docking dependent activation. Furthermore, when the amino acid sequence of the p21 docking domain was aligned with the sequence of Chk1, homology to the F region on the kinase domain was identified. Mutation of two conserved tryptophan residues within the homology region appears to release the C-terminus from intramolecular interactions rendering it susceptible to cleavage and refractory to allosteric stimulation. Furthermore, small peptides based on this region of Chk1, like the p21 docking domain, are able to activate Chk1 in trans and disrupt interaction between the N-terminal and Cterminal domains. Interestingly, peptide microarray showed that Chk1 stimulated by activating peptide is able to phosphorylate novel peptide substrates which are not observed with unstimulated Chk1. The data suggest that the last C-terminal 70 amino acids of Chk1 play an important role in auto-inhibition through interaction with the F region of the core catalytic domain. Binding to p21 is able to activate Chk1 by inhibiting the auto-inhibitory interaction independent of phosphorylation at the Ser317 and Ser345 sites. Furthermore, activating peptide is able to modulate Chk1 specificity towards other substrates.

Myc is a transcription factor frequently found deregulated in human cancer. Cells with deregulated expression of Myc carry a selective advantage against its neighbours due to the fact that Myc-mediated transcription governs crucial cellular events such as proliferation and growth. In addition, Myc has been implicated in several other aspects of tumour biology like cellular immortality, the formation of new blood vessels and the colonization of distant tissues through the process of metastasis. Therapy aimed at disrupting essential pathways regulated by Myc is important because of the many different types of cancers that depend on continued signalling along these pathways.  This thesis describes new treatment opportunities for cancers with a high Myc signature. In Paper Ι, we describe a new role for the DNA methyltransferase inhibitor Decitabine in the treatment of Myc transformed tumours cells. We show that the therapeutic potential of Decitabine in the treatment of Burkitt Lymphoma relies not only on its ability to cause reactivation of silenced genes such as pro-apoptotic PUMA, but also on the DNA damage that this drug induces. In vivo, Decitabine delays disease progression of transplanted lymphoma cells. In Paper ΙΙ, we identify the DNA damage checkpoint kinase Chk1 as a therapeutic target in Myc overexpressing cancers. We show that targeting Chk1 with shRNA or with a novel small molecule inhibitor cause a delay in disease progression of transplanted lymphoma cells in vivo. In Paper ΙΙΙ, the Chk1-related kinase Chk2 is evaluated as a therapeutic target in Myc overexpressing cancers. Myc overexpressing cells are not dependent on Chk2 but we show that Chk2 abrogation using shRNA causes polyploidization and protection against DNA damage. However, Chk2-targeted therapy elicits a synergistic lethal response in combination with inhibition of the DNA repair associated protein PARP. In conclusion, this thesis shows the potential of targeting the DNA damage machinery and the functional hubs important for maintenance of genomic stability in tumours with a deregulated expression of Myc.