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Platelets are central actors in haemostasis and thrombosis. Their stimulation by thrombin or collagen induces the phosphorylation and inhibition of the acetyl-CoA carboxylase (ACC). Since ACC is crucial for the synthesis of fatty acids, which are essential for platelet structure, signalling and metabolism, we hypothesized that this enzyme plays a role in platelet function. We showed that constitutive ACC activation increases thromboxane generation, leading to enhanced platelet reactivity and thrombus formation. Lipidomic analysis of intraplatelet content revealed an increase in arachidonic acid-containing phospholipids, which are major precursors of thromboxane. Interestingly, ACC is phosphorylated in high-risk coronary artery disease patients., (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2018

Deoxycytidine kinase (dCK) catalyses the first and rate limiting step of the deoxynucleoside salvage pathway that supplies cells with dNTPs for DNA synthesis. dCK is also required for the activation of several antiviral and anticancer nucleoside analogues and plays a crucial role in their therapeutic efficacy. Given these important functions, deciphering the mechanisms that control dCK activity is of major interest for both fundamental scientists and clinician researchers. Our group demonstrated that dCK is a phosphoprotein whose activity is increased by phosphorylation of the Ser-74 residue. Moreover, it was established that increase in dCK activity in response to several genotoxic treatments, including ionizing radiation (IR), UV-C, chemotherapeutic nucleoside analogues and DNA polymerase inhibitors, was related to an increase in Ser-74 phosphorylation. These observations suggested that Ser-74 could be phosphorylated by a DNA damage-activated protein kinase, possibly ATM, ATR or DNA-PK. Two different groups identified ATM, a transducer of the DNA damage response (DDR), as the protein kinase responsible for Ser-74 phosphorylation and dCK activation following ionizing radiation (IR), a DNA double-strand break (DSB) inducer. The first objective of this thesis consisted in investigating whether ATM was also involved in dCK activation in response to other types of DNA damage, such as induced by UV-C light that causes replication stress and induces single-stranded DNA (ssDNA). Using ATM-deficient lymphoblastoid cell lines, a selective ATM inhibitor or ATM siRNA, we demonstrated that ATM was not involved in dCK activation induced by UV-C, aphidicolin or cladribine. Surprisingly, we also found that ATM was not essential for dCK activation following IR, contrary to what was stated in the literature. On the other hand, a selective inhibitor of ATR, the kinase that is activated in response to ssDNA, as well as ATR siRNA prevented dCK activation by these agents, though not IR, (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2017

ATM, primarily activated by DNA double-strand breaks, and ATR, activated by single-stranded DNA, are master regulators of the cellular response to DNA damage. In primary chronic lymphocytic leukemia (CLL) cells, ATR signaling is considered to be switched off due to ATR downregulation. Here, we hypothesized that ATR, though expressed at low protein level, could play a role in primary resting CLL cells after genotoxic stress. By investigating the response of CLL cells to UV-C irradiation, a prototypical activator of ATR, we could detect phosphorylation of ATR at Thr-1989, a marker for ATR activation, and also observed that selective ATR inhibitors markedly decreased UV-C-induced phosphorylation of ATR targets, including H2AX and p53. Similar results were obtained with the purine analogs fludarabine and cladribine that were also shown to activate ATR and induce ATR-dependent phosphorylation of H2AX and p53. In addition, ATR inhibition was found to sensitize primary CLL cells to UV-C by decreasing DNA repair synthesis. Conversely, ATR inhibition rescued CLL cells against purine analogs by reducing expression of the pro-apoptotic genes PUMA and BAX. Collectively, our study indicates that ATR signaling can be activated in resting CLL cells and play a pro-survival or pro-apoptotic role, depending on the genotoxic context.

OBJECTIVES: Cardiac transplantation using hearts from donors after circulatory death (DCD) is critically limited by the unavoidable warm ischaemia and its related unpredictable graft function. Inasmuch as hypothermic machine perfusion (MP) has been shown to improve heart preservation, we hypothesized that MP could enable the use of DCD hearts for transplantation. METHODS: We recovered 16 pig hearts following anoxia-induced cardiac arrest and cardioplegia. Grafts were randomly assigned to two different groups of 4-h preservation using either static cold storage (CS) or MP (Modified LifePort© System, Organ Recovery Systems©, Itasca, Il). After preservation, the grafts were reperfused ex vivo using the Langendorff method for 60 min. Energetic charge was quantified at baseline, post-preservation and post-reperfusion by measuring lactate and high-energy phosphate levels. Left ventricular contractility parameters were assessed both in vivo prior to ischaemia and ex vivo during reperfusion. RESULTS: Following preservation, the hearts that were preserved using CS exhibited higher lactate levels (57.1 ± 23.7 vs 21.4 ± 12.2 µmol/g; P < 0.001), increased adenosine monophosphate/adenosine triphosphate ratio (0.53 ± 0.25 vs 0.11 ± 0.11; P < 0.001) and lower phosphocreatine/creatine ratio (9.7 ± 5.3 vs 25.2 ± 11; P < 0.001) in comparison with the MP hearts. Coronary flow was similar in both groups during reperfusion (107 ± 9 vs 125 ± 9 ml/100 g/min heart; P = ns). Contractility decreased in the CS group, yet remained well preserved in the MP group. CONCLUSION: MP preservation of DCD hearts results in improved preservation of the energy and improved functional recovery of heart grafts compared with CS.

Deoxycytidine kinase (dCK) (EC 2.7.1.74) is a key enzyme for salvage of deoxynucleosides and activation of numerous anticancer and antiviral nucleoside analogs. dCK activity is enhanced in response to several genotoxic treatments, which has been correlated with an increase of dCK phosphorylation at Ser-74. ATM was recently identified as the kinase responsible for Ser-74 phosphorylation and dCK activation after ionizing radiation (IR). Here, we investigated the role of ATM and the related kinase ATR in dCK activation induced by other types of DNA damage. Using ATM-deficient cells or the ATM inhibitor KU-60019, we found that ATM was not required for dCK activation caused by UV light, aphidicolin, cladribine, and unexpectedly also IR. On the other hand, the selective ATR inhibitor VE-821 significantly reduced up-regulation of dCK activity induced by these genotoxic agents, though not IR, and also down-regulated basal dCK activity. A role for ATR in the control of dCK activity was confirmed by using ATR siRNA and ATR-Seckel cells. ATR was also found to directly phosphorylate dCK at Ser-74 in vitro. Further studies revealed that ATR, which is also activated in response to IR, although later than ATM, was responsible for IR-induced dCK activation in ATM-deficient cells or in the presence of KU-60019. Overall, our results demonstrate that ATR controls basal dCK activity and dCK activation in response to replication stress and indicate that ATR can activate dCK after IR if ATM is lacking or inhibited.

Deoxycytidine kinase (dCK) catalyzes the phosphorylation of deoxycytidine, deoxyadenosine and deoxyguanosine, which constitutes the first and rate-limiting step in the synthesis of DNA precursors by the deoxynucleoside salvage pathway. In addition, dCK phosphorylates and activates numerous chemotherapeutic nucleoside analogs. As low dCK activity is often associated with reduced nucleoside analog efficacy, it is of particular interest to gain more insights into the mechanisms that regulate its activity. Prior to this thesis project, research conducted in our laboratory had demonstrated that dCK activity is regulated by post-translational modification, and more precisely that its activity can be increased in vivo through Ser-74 phosphorylation, a finding that could be exploited to improve therapeutic efficacy of nucleoside analogs. The first objective of this thesis was to investigate the impact of Ser-74 phosphorylation on nucleoside analog activation and efficacy in cancer cells. We started by a kinetic study using recombinant dCK in which Ser-74 phosphorylation was mimicked by a S74E mutation. Although it was well demonstrated that S74E mutation increases dCK activity toward deoxycytidine and pyrimidine analogs, such as gemcitabine, its effect toward purine analogs had not yet been investigated. We found that S74E mutation enhanced the catalytic activity (Kcat) of dCK toward cladribine and clofarabine, but not fludarabine, with ATP or UTP as phosphoryl donor. However, Km values were also increased so that the catalytic efficiencies (Kcat/Km) of dCK for cladribine and clofarabine were not, or only slightly increased, and even decreased for fludarabine. In addition, increase of endogenous dCK activity, which was easily detected with deoxycytidine or gemcitabine as substrates after in vivo-induced increase of Ser-74 phosphorylation, was not observed with deoxyadenosine or purine analogs. Accordingly, treatment of CLL (chronic lymphocytic leukemia) cells with aphidicol, (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2014

Deoxycytidine kinase (dCK) is a critical enzyme for activation of anticancer nucleoside analogs. Its activity is controlled via Ser-74 phosphorylation. Here, we investigated which Ser/Thr phosphatase dephosphorylates Ser-74. In cells, the PP1/PP2A inhibitor okadaic acid increased both dCK activity and Ser-74 phosphorylation at concentrations reported to specifically target PP2A. In line with this, purified PP2A, but not PP1, dephosphorylated recombinant pSer-74-dCK. In cell lysates, the Ser-74-dCK phosphatase activity was found to be latent, Mn2+-activated, responsive to PP2A inhibitors, and diminished after PP2A-immunodepletion. Use of siRNAs allowed concluding definitively that PP2A constitutively dephosphorylates dCK in cells and negatively regulates its activity.

The AMP-activated protein kinase (AMPK) acts as a sensor of cellular and whole body energy homeostasis. Once activated, it inhibits energy consuming anabolic pathways while at the same time stimulating energy producing catabolic pathways. AMPK activation in skeletal muscle stimulates glucose uptake, which contributes towards the beneficial effects of exercise in counteracting the symptoms of type 2 diabetes. AMP is mainly degraded by AMP-deaminase (AMPD) or soluble 5’-nucleotidases. Three separate genes code for AMPD isozymes: AMPD1 (expressed in skeletal muscle), AMPD2 (expressed in liver) and AMPD3 (expressed in erythrocytes). The soluble 5’-nucleotidase IA (cN-IA) is mainly expressed in skeletal muscle and heart, whereas the other soluble 5’-nucleotidase (cN-II) is ubiquitous. In this study, it was first investigated whether overexpression of AMPD1, AMPD2 and cN-IA would antagonize oligomycin-induced AMPK activation in HEK293T cells. In this model, cN-IA rather than AMPDs, exerted most control over AMP and ADP concentrations. Overexpression of cN-IA markedly reduced oligomycin-induced AMPK activation, whereas overexpression of AMPD1 and AMPD2 effects on AMPK activation were much less. The design of cN-IA inhibitors could be an alternative strategy to direct kinase activators for achieving AMPK activation. The data also support the idea that ADP as well as AMP are important for AMPK activation and that AMPK is regulated by the AEC. As most of the circulating glucose is taken up by muscle, the second part of the thesis focuses on the consequences of AMPD inhibition for AMPK activation in this tissue. Pharmacological AMPD inhibition as well as AMPD1 deletion in mice led to a substantial enhancement of contraction-induced AMP and ADP accumulation in incubated skeletal muscles. However, this did not systematically lead to potentiation of AMPK phosphorylation and activation in response to electrical stimulation. Also, pharmacological AMPD inhibition did not enhance con, (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2013

The AMP-activated protein kinase (AMPK) is an important protein kinase that controls cellular and whole body energy homeostasis. During energy stress, AMPK is activated by an increase in AMP/ATP and/or ADP/ATP ratio. In order to restore energy balance, AMPK activation stimulates catabolic pathways to produce ATP and while at the same time inhibiting ATP-consuming anabolic pathways. The search for new AMPK targets is thus fundamental to understanding the mechanisms by which organisms can adapt their metabolism to deal with physiological and pathophysiological stress conditions. This thesis was devoted to the study of two potential AMPK targets, myosin regulatory light chains (MLCs) and the liver isoenzyme of glycogen synthase (GYS2). Firstly, MLCs have been proposed to be directly phosphorylated by AMPK (Lee et al., 2007). This observation contradicted results previously obtained in our laboratory. The first publication compared the kinetics of MLC phosphorylation by recombinant AMPK with that catalyzed by a commercial purified preparation of AMPK from rat liver (used by Lee et al., 2007) and by MLC kinase (MLCK), the dedicated kinase well-known to phosphorylate MLCs. Our results show that i) the commercial AMPK preparation was contaminated by kinase(s) able to phosphorylate MLC, explaining the result obtained by Lee et al. (2007) ii) MLCs are not a physiological substrate for AMPK. In this publication, it was also demonstrated that AMPK activation in epithelial cells by A769662, a pharmacological activator of AMPK, led to MLC phosphorylation. Moreover, this phosphorylation was reduced by H1152, a Rho kinase inhibitor, suggesting that Rho kinase could be involved in the control of MLC phosphorylation by AMPK in intact cells. In the second publication, we found that both recombinant GYS1, the muscle isoenzyme already known to be an AMPK substrate, and recombinant GYS2 were phosphorylated to a similar extent by recombinant AMPK. We also identified Ser7 as the AMPK phos, (BIOL 3) -- UCL, 2012

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western world, but its etiology has not yet been completely elucidated. Purine nucleoside analogs (PNAs) are among the most effective drugs used in CLL treatment. However, resistance remains a major challenge in clinical management of CLL. The major objectives of this thesis were to better understand the mechanisms of resistance to PNAs in CLL as well as the pathogenesis of the disease, and to find new strategies to predict chemoresistance. In order to identify new mechanisms of chemoresistance, we used genome-wide expression profiling to compare the response to PNAs of chemosensitive and chemoresistant CLL samples. In chemosensitive cells, PNAs predominantly increased the expression of p53-dependent genes, among which PLK2 was the most highly up-regulated at an early time point. Conversely, in chemoresistant samples, p53-dependent and PLK2 responses were abolished. Using quantitative real-time PCR, we confirmed that PNAs significantly increased PLK2 mRNA expression in chemosensitive, but not in chemoresistant CLL samples. The analysis of a larger cohort of CLL patients showed that in vitro cytotoxicity induced by PNAs correlated well with PLK2 mRNA induction. Therefore, we proposed that testing PLK2 induction in response to PNAs might be a new strategy to investigate the integrity of the p53/DNA damage pathway in CLL and to predict clinical sensitivity to these drugs. This study was completed by a review of the current knowledge of p53 functional analysis in CLL. The next step was to investigate the potential role of PLK2 induction in the mechanism of action of PNAs. Unfortunately, we could not reliably detect Plk2 protein in CLL cells, either because of insufficient sensitivity and specificity of the antibodies used for its detection or because of its low expression level. Comparison with other cancer cell lines suggested that PLK2 mRNA and protein expression levels would be rather low in, (SBIM 3) -- UCL, 2012

We investigated whether overexpression of AMP-metabolizing enzymes in intact cells would modulate oligomycin-induced AMPK activation. Human embryonic kidney (HEK) 293T cells were transiently transfected with increasing amounts of plasmid vectors to obtain a graded increase in overexpression of AMP-deaminase (AMPD) 1, AMPD2, and soluble 5'-nucleotidase IA (cN-IA) for measurements of AMPK activation and total intracellular adenine nucleotide levels induced by oligomycin treatment. Overexpression of AMPD1 and AMPD2 slightly decreased AMP levels and oligomycin-induced AMPK activation. Increased overexpression of cN-IA led to reductions in the oligomycin-induced increases in AMP and ADP concentrations by ∼70 and 50%, respectively, concomitant with a 50% decrease in AMPK activation. The results support the view that a rise in ADP as well as AMP is important for activation of AMPK, which can thus be regulated by the adenylate energy charge. The control coefficient of cN-IA on AMP was 0.3-0.7, whereas the values for AMPD1 and AMPD2 were <0.1, suggesting that in this model cN-IA exerts a large proportion of control over intracellular AMP. Therefore, small molecule inhibition of cN-IA could be a strategy for AMPK activation.

Aberrations of TP53 (mutations and/or deletions) are associated with a dismal prognosis in chronic lymphocytic leukemia (CLL). Complete loss of ATM is another mechanism of failed DNA damage response and also associated with poorer prognosis in CLL. However, p53 dysfunction may arise through alternative mechanisms unrelated to structural aberrations (deletion and/or mutation) of TP53 or ATM, and thus be undetectable by traditional DNA-directed approaches (fluorescence in situ hybridization [FISH], sequencing, karyotyping). In order to address the latter changes, and also to better understand the consequences of TP53/ATM aberrations, p53 functional assays have recently been developed. The purpose of dynamic assessment of p53 response in CLL is to carry out a comprehensive analysis of all mechanisms causing p53-deficient phenotype, including those unrelated to genomic aberrations of TP53 and ATM. The present review focuses on the current knowledge of p53 function assays in CLL, including important features such as technical issues, correlation with structural aberrations and clinical value.

Deoxycytidine kinase (dCK) (EC 2.7.1.74) is a key enzyme in the activation of several therapeutic nucleoside analogs (NA). Its activity can be increased in vivo by Ser-74 phosphorylation, a property that could be used for enhancing NA activation and clinical efficacy. In line with this, studies with recombinant dCK showed that mimicking Ser-74 phosphorylation by a S74E mutation increases its activity toward pyrimidine analogs. However, purine analogs had not been investigated. Here, we show that the S74E mutation increased the k(cat) for cladribine (CdA) by 8- or 3-fold, depending on whether the phosphoryl donor was ATP or UTP, for clofarabine (CAFdA) by about 2-fold with both ATP and UTP, and for fludarabine (F-Ara-A) by 2-fold, but only with UTP. However, the catalytic efficiencies (k(cat)/Km) were not, or slightly, increased. The S74E mutation also sensitized dCK to feed-back inhibition by dCTP, regardless of the phosphoryl donor. Importantly, we did not observe an increase of endogenous dCK activity toward purine analogs after in vivo-induced increase of Ser-74 phosphorylation. Accordingly, treatment of CLL cells with aphidicolin, which enhances dCK activity through Ser-74 phosphorylation, did not modify the conversion of CdA or F-Ara-A into their active triphosphate form. Nevertheless, the same treatment enhanced activation of gemcitabine (dFdC) into dFdCTP in CLL as well as in HCT-116 cells and produced synergistic cytotoxicity. We conclude that increasing phosphorylation of dCK on Ser-74 might constitute a valuable strategy to enhance the clinical efficacy of some NA, like dFdC, but not of CdA or F-Ara-A.

Les analogues de nucléosides sont de petites molécules cytotoxiques chimiquement similaires aux nucléosides physiologiques, utilisées dans le traitement d’hémopathies malignes et de certaines tumeurs solides. Deux analogues puriques, la 2-chloroadénosine (2-CAdo), toujours à l’étude fondamentale, et la 2-chloro-2’-désoxyadénosine (CdA), utilisée en clinique, ont fait principalement l’objet de cette thèse. La 2-CAdo est un analogue de l’adénosine utilisé initialement comme agoniste des récepteurs de l’adénosine. Cette molécule est capable d’induire l’apoptose via l’activation des récepteurs de l’adénosine ou via son métabolisme intracellulaire. Cependant, les mécanismes d’induction de l’apoptose n’étaient pas bien connus. La première partie de cette thèse a été consacrée à l’étude de ces mécanismes dans un modèle de leucémie lymphoïde chronique (LLC), la lignée cellulaire EHEB. Nous avons d’abord confirmé, par plusieurs approches, que la 2-CAdo induit effectivement l’apoptose dans ces cellules leucémiques et que celle-ci résulte de son métabolisme. En effet, l’inhibition de la conversion intracellulaire de la 2-CAdo en son dérivé triphosphate, le 2-CATP, supprime sa cytotoxicité. L’accumulation de 2-CATP est accompagnée d’une chute de l’ATP intracellulaire, d’une inhibition de la synthèse d’ADN, d’ARN et des protéines, ainsi que d’une libération de cytochrome c dans le cytosol, indiquant que la 2-CAdo active la voie intrinsèque de l’apoptose. L’activation de cette voie ne fait pas intervenir le facteur de transcription p53, mais a pu être corrélée à une diminution du taux de Mcl-1, une protéine anti-apoptotique, à courte demi-vie. Enfin, il est apparu que la chute d’ATP qui accompagne la métabolisation de la 2-CAdo joue un rôle important dans le processus apoptotique déclenché par cet analogue de nucléoside. Dans la seconde partie de cette thèse, nous nous sommes intéressés à la CdA, un analogue de la 2’-désoxyadénosine. La CdA, après sa conversion en composé triphos, (SBIM 3) -- UCL, 2011

IMPORTANCE OF THE FIELD: Despite considerable advances, B-cell chronic lymphocytic leukemia (CLL) is incurable with standard approaches. Thus, there remains a need for new therapies, particularly for patients who develop chemoresistance to DNA-targeting treatments. AICA-riboside (acadesine) is a nucleoside with a wide range of metabolic effects, including release of adenosine and activation of AMP-activated protein kinase (AMPK), which was initially developed as a cardioprotective agent. More recently, it has been shown that AICA-riboside induces apoptosis in various models of leukemia, including CLL. AREAS COVERED IN THIS REVIEW: The literature data show that apoptosis induced by AICA-riboside in CLL is not dependent on a functionally normal p53 pathway. Moreover, AICA-riboside is active towards resting and proliferative models of leukemia cells, including resistant phenotypes. Finally, studies in healthy subjects and during coronary artery bypass graft surgery show that AICA-riboside is devoid of serious toxicity. WHAT THE READER WILL GAIN: This paper reviews the mechanisms of action of AICA-riboside in normal and malignant cells and discusses how AICA-riboside could impact CLL treatment. TAKE HOME MESSAGE: We propose that AICA-riboside, which displays a relative selectivity and a favorable toxicity profile, may offer a new treatment option for CLL.

Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxyribonucleosides and in the activation of several anticancer and antiviral nucleoside analogues. We have recently shown that dCK is a phosphoprotein. Four in vivo phosphorylation sites were identified: Thr-3, Ser-11, Ser-15, and Ser-74. Site-directed mutagenesis demonstrated that phosphorylation of Ser-74, the major phosphorylated residue, strongly influences dCK activity in eucaryotic cells. Here, we show that phosphorylation of the three other sites, located in the N-terminal extremity of the protein, does not significantly modify dCK activity, but phosphorylation of Thr-3 could promote dCK stability.