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.

Le cycle cellulaire est l'ensemble des étapes qui conduisent une cellule mère à se diviser en deux cellules filles. La protéine Checkpoint kinase 1 (Chk1) est importante pour sa progression. Nous avons d'une part cherché à savoir si Chk1 intervenait lors des mécanismes de production des plaquettes, car ces cellules permettant la coagulation du sang sont issues d'un cycle cellulaire particulier. Par ailleurs, nous avons étudié le rôle de Chk1 dans la Leucémie Aiguë Myéloïde (LAM), cancer des cellules sanguines. Les patients atteints de LAM sont traités par une chimiothérapie visant à endommager l'ADN afin d'entrainer la mort des cellules cancéreuses. Chk1 est garante du contrôle de la réparation des dommages de l'ADN, ce qui contrecarre l'effet de la chimiothérapie. Elle pourrait donc favoriser l'apparition de résistance. Son rôle dans les LAM étant peu connu, l'objectif de ce projet est donc de vérifier si Chk1 favorise la résistance des cellules leucémiques aux chimiothérapies. / The cell cycle is a series of events that takes place in a mother cell, leading to its division into two daughter cells. The protein Checkpoint kinase 1 (Chk1) is mandatory for its coordinated progression. In this PhD projet, we wondered on the one hand whether Chk1 could be involved in the platelets production process, because these componants of blood that enables coagulation are produced due to a particular cell cycle dedicated to this end. On the other hand, we studied the role of Chk1 in Acute Myeloid Leukemia (LAM) physiopathology. LAM is a cancer of blood cells, in which patients are treated with drugs that create DNA damages, causing the death of tumoral cells. The role of Chk1 in the drug response in LAM is not well studied, but, as it enables DNA repair, it may render theses medicines less efficient, leading to relapses to therapies. So the goal of this project is to check wether Chk1 favors the resistance of some LAM cells to chemotherapeutic treatments.

Mes études doctorales ont permis de démontrer que les souris déficientes en VIP présentent une microcéphalie ayant principalement une origine maternelle qui affecte secondairement le développement de la substance blanche. Cette production placentaire par les lymphocytes T pourrait être affectée dans des pathologies du système immunitaire. De plus, nos données indiquent qu’une déficience en VIP prédispose à l'apparition de troubles sensoriels, en particulier de la nociception. Il est donc possible que les déficits précoces de développement du cerveau murin et l'apparition de l'hypersensibilité cutanée mécanique et thermique froide soient deux facettes d'une même pathologie. Des mesures d'activité de décharge spontanée des neurones dans le thalamus sensoriel chez des mâles adultes anesthésiés ont montré que les neurones des animaux KO sont hyper-excités, ce qui suggère un traitement aberrant des informations, notamment nociceptives, ou que l'activité inhibitrice des interneurones des réseaux locaux est réduite. / The studies carried out during my PhD demonstrate that VIP-deficient mice suffer from microcephaly and as well as white matter deficits mainly due to the absence of maternal VIP during embryogenesis, Placental secretion of VIP is dependent on T lymphocytes and could be altered in pathologies of the immune system. Moreover, our data links VIP deficiency to sensory alterations, specifically, the nociceptive system. Thus, it is possible that early developmental defects and hypersensitivity to mechanical and cold stimuli are two manifestations of the same pathology. This hypothesis was reinforced following analysis of spontaneous firing patterns of neurons in the sensory thalamus of anesthetized adult males. Neurons from VIP-KO mice are hyperactive, which suggests aberrant local processing of nociceptive input or that the inhibitory inputs from local interneuron networks is reduced.

Hypoxia (low physiological O₂ levels) is a characteristic of solid tumours. It not only alters the chemical microenvironment of a tumour but initiates a number of mechanisms which enable cells to cope and thrive under these conditions, resulting in therapy-resistant and aggressive tumours. The replication stress induced by severe hypoxia activates a DNA damage response which involves the kinases ATR and Chk1. Moreover, periods of hypoxia are often followed by reoxygenation, which induces DNA damage. Chk1 inhibitors have been used to potentiate chemotherapy with cytotoxic agents and have recently been proposed as single agents in tumours with high levels of replication stress. However, inhibition of Chk1 also affects normal DNA replication, cell cycle progression and DNA repair. The herein presented study chose known inhibitors of Chk1 and, with methods of synthetic organic chemistry, modified them into agents to selectively target hypoxic cells. Three different Chk1 inhibitors were selected and bioreductive analogues synthesised which were evaluated in chemical, biochemical and cellular assays. We found a convenient route to access a precursor of the bioreductive 2-nitroimidazole group and established a three-step protocol for the testing of bioreductive drugs. This protocol allows us to determine whether a bioreductive drug undergoes reduction and prodrug activation. In addition, bioreductive Chk1 inhibitors were shown to induce DNA damage and cellular toxicity in a hypoxia-selective fashion. While reduction of the prodrugs occurred in all three cases, fragmentation was always the rate-limiting step. We propose that the use of bioreductive Chk1 inhibitors is a promising strategy to target the most therapy-resistant tumour fraction while sparing normal tissue.