Les leucémies aiguës myéloblastiques (LAM) sont des maladies hématopoïétiques caractérisées par une prolifération incontrôlée de précurseurs myéloïdes bloqués à divers stades de différenciation. Le pronostic des LAM reste sombre à cause de la résistance aux traitements et des rechutes après rémission. En conséquence, des thérapies moins intensives et mieux tolérées doivent être développées ; ceci nécessite le développement de stratégies combinatoires associant des molécules avec des modes d’action différents pour augmenter l’efficacité des traitements. Plusieurs approches sont en cours d’étude préclinique et clinique [inhibiteurs des voies de signalisation PI3K/Akt/mTOR, anticorps monoclonaux couplés à une drogue (Mylotarg®), inhibiteurs du protéasome (bortezomib)…] Des travaux récents ont relancé l’intérêt de l’étude des molécules d’origine naturelle pour le traitement des cancers. Ainsi, l’acide flavone-8-acétique (FAA) a suscité de nombreux espoirs au vu de son action sur les tumeurs greffées chez la souris ; il s’est néanmoins révélé inactif chez l’homme du fait d’une métabolisation différente de celle de la souris. L’objectif de ma thèse a été d’étudier les effets d’anticorps monoclonaux dirigés contre l’antigène tumoral CD13 (aminopeptidase-N) et de deux dérivés de FAA, la 2’,3-Dinitroflavone-8-acétique (DNFAA ; inhibiteur de l’activité enzymatique de CD13) et la 3,3’-Diamino-4’-méthoxyflavone (DD1) dans les LAM. Mon étude a montré que DNFAA n’affecte ni la prolifération ni la survie des cellules de LAM (lignées et cellules primaires). Cependant, le traitement de ces cellules par les anticorps anti-CD13, (MY7, SJ1D1, WM15 ; reconnaissant ou non le site enzymatique) induit l’apoptose en activant les voies extrinsèque et intrinsèque. Dans la voie intrinsèque, les anti-CD13 régulent négativement l’expression des protéines anti-apoptotiques Bcl-2 et Mcl-1 et positivement l’expression de la protéine pro-apoptotique Bax. De plus, l’activation de la voie PI3K/Akt apparaît associée au processus apoptotique. Mon étude sur les effets du 3,3’-Diamino-4’-méthoxyflavone dans les cellules de LAM montre une induction d’apoptose résultant de la convergence de l’inhibition du protéasome et de l’activation des voies extrinsèque et intrinsèque. Les cibles de DD1 sont le protéasome, la kinase p70S6K (kinase en aval de mTOR), et les protéines pro-apoptotiques Bad et Bax. De plus, j’ai mis en évidence la dégradation de p70S6K sous l’action de la caspase 3, par le traitement avec DD1, nouvelle propriété partagée par DD1 et le bortezomib. En conclusion, mon travail a permis de mettre en évidence les capacités à induire in vitro des voies d’apoptose déficientes dans les cellules de LAM, d’anticorps monoclonaux anti-CD13 et de la flavone originale, 3,3’-Diamino-4’-methoxyflavone, en tant que nouvel inhibiteur du protéasome. Les propriétés de ces agents pro-apoptotiques méritent d’être analysées de façon plus approfondie. / Acute Myeloid Leukaemia (AML) is a deadly disease characterized by the clonal expansion and accumulation of hematopoietic stem cells arrested at various stages of development. Clinical research efforts are currently focusing on targeted therapies that induce apoptosis in AML cells such as PI3K/Akt/mTOR pathway inhibitors, monoclonal antibodies (Mylotarg®), proteasome inhibitor (bortezomib)… Natural products such as flavonoids have been reported as anticancer agents due to their antioxidant properties as well as to their possible interactions with signalling cascades. Therefore, flavone-8-acetic acid (FAA) has raised considerable attention since the discovery of its exceptional activity on several murine solid tumours. Unfortunately, these promising properties were not confirmed on human due to differential metabolization between human and mouse. The aim of my PhD was to study effects of monoclonal antibody against aminopeptide-N/CD13 and FAA derivatives, 2’,3-Dinitroflavone-8-acetic (DNFAA ; APN/CD13 inhibitor) and 3,3’-Diamino-4’-methoxyflavone (DD1) on acute myeloid leukaemia cells. My studies have shown that DNFAA does not modify proliferation or survival of LAM primary and cell lines. However, treatment of these cells by CD13 antibodies (MY7, SJ1D1 and WM15) induces apoptosis by triggering extrinsic and intrinsic apoptotic pathways. Regarding the intrinsic pathway, anti-CD13 down-regulate anti-apoptotic proteins Bcl-2 and Mcl-1 and up-regulate the pro-apoptotic protein Bax. Morever, PI3K/Akt signalling pathway seems to be associated with this apoptosis. My study about 3,3’-Diamino-4’-methoxyflavone effects on LAM cells has shown that DD1 induces apoptosis by proteasome inhibition and intrinsic and extrinsic pathways induction. DD1 targets p70S6 kinase (a downstream kinase of mTOR) and pro-apoptotic proteins Bad and Bax. Moreover, I have shown p70S6K degradation by caspase 3 during DD1 treatment, a new characteristic shared by DD1 and Bortezomib. As a conclusion, my works demonstrated that CD13 antibodies and a new synthetic flavone are able to induce apoptosis signalling pathway normally impaired on AML cells. Characteristics of these agents deserve to be more deeply analyzed.

La maladie de Paget est une ostéopathie caractérisée par une augmentation multifocale du remodelage osseux, qui débute par un front de résorption osseuse, suivi d'une formation osseuse excessive, avec un remodelage anarchique et intense. Les ostéoclastes "OCs" impliqués dans la phase initiale sont les cellules responsables dans l'initiation du processus pagétique. Les OCs pagétiques sont caractérisés par une résistance à l'apoptose, et des anomalies du processus de l'autophagie "en particulier défaut d'induction"; afin de voir si ces deux caractéristiques étaient liées, nous avons émis l'hypothèse d’un rôle des complexes Bcl2-Beclin1. Beclin-1 est une protéine inductrice de l'autophagie qui peut lier les protéines anti-apoptotiques de la famille Bcl-2; Bcl-2 inhibe alors Beclin-1 "et donc l'induction de l'autophagie" en conservant ses fonctions anti-apoptotiques. Dans le but d'étudier l'impact de l'expression de Bcl2 sur l’autophagie dans les OCs humains, nous avons utilisé un modèle de différenciation in vitro à partir de monocytes dérivés de sang de cordon ombilical, cultivés en présence de RANKL et MCSF pendant 21 jours. Ces conditions permettent d'obtenir des cellules multinucléées au phénotype ostéoclastique. Pour augmenter l’expression de Bcl-2 dans les OCs et analyser son impact sur l’autophagie par interaction avec Beclin-1, les cultures ont été stimulées par TNFα ou RANKL dans le but d'induire une activation de NF-κB. L'expression de Beclin1 et Bcl2 a été confirmée par immunobuvardage dans les OCs. L’autophagie était induite dans les cultures réalisées en conditions stringentes "milieu pauvre en nutriments", sans variation de l'expression de Bcl2 ou Beclin 1 selon les conditions, et sans impact de TNFa ou RANKL. TNFa stimulait de manière significative l'activation de NF-kB dans les cellules HEK mais pas dans les OCs. Toutefois, et quelque soit les conditions, les immunoprécipitations ne permettaient pas de retrouver d'association entre Beclin1 et Bcl2. En revanche, le partenaire d'interaction classique de Beclin1, PI3K type III, était associé à Beclin1. En conclusion, notre travail n'a pas permis d'étudier la formation des complexes Beclin1/Bcl2 et les relations entre apoptose et autophagie, en partie du fait de la complexité du modèle "effets multiples de NF-kB et TNFa" ce qui n'exclut pas l'hypothèse initiale "à ré-évaluer par une méthodologie plus appropriée". En revanche les différentes techniques d'analyse sont maintenant au point pour la poursuite de l'étude. / Abstract : Paget's disease is an osteopathy characterized by a multifocal increase in bone remodeling, which begins with excessive bone resorption followed by increased bone formation. Osteoclasts "OCs" were incriminated in the initiation of the pagetic process. Pagetic OCs are characterized by a resistance to apoptosis, and abnormalities in the process of autophagy “in particular induction defect”. In order to define whether these two characteristics were linked, we hypothesized the role of Bcl2-Beclin1 complexes. Beclin-1 is an autophagy-inducing protein that can bind anti-apoptotic proteins of the Bcl-2 family; Bcl-2 then inhibits Beclin-1 "and thus the induction of autophagy" while retaining its anti-apoptotic functions. To study the impact of Bcl2 expression on autophagy in human OCs, we used an in vitro differentiation model that uses monocytes, which are derived from umbilical cord blood and grown in the presence of RANKL and MCSF for 21 days. These conditions make it possible to obtain multinucleated cells with an osteoclastic phenotype. To increase the expression of Bcl-2 in OCs and analyze its impact on autophagy due to its interaction with Beclin-1, cultures were stimulated with TNFα or RANKL in order to induce NF-κB activation. The expression of Beclin1 and Bcl2 was confirmed by immunoblotting of Ocs cell lysates. Autophagy was induced in cultures carried out under stringent conditions "nutriment-deprived mediun", but we did not observe any variation in the expression of Bcl2 or Beclin 1 according to the culture conditions or TNFα or RANKL stimulation. TNFα significantly stimulated the activation of NF-κB in HEK cells but not in OCs. However, whatever the conditions, results from immunoprecipitaion experiments did not reveal any association between Beclin1 and Bcl2. On the other hand, the classic interaction partner of Beclin1, PI3K type III, was associated with Beclin1. In conclusion, our work did not allow us to demonstrate the formation of Beclin1 / Bcl2 complexes and the relationship between apoptosis and autophagy, partly because of the complexity of the model "multiple effects of NF-κB and TNFα". Our initial hypothesis should thereby be re-evaluated using a more appropriate methodology. On the other hand, the different techniques are now ready for further study.

Melanoma is the deadliest form of skin cancer and one of the most difficult cancers to treat. Gene alterations identified in melanoma pointed to distinct molecular subsets of tumors with direct implications in therapeutic strategies. Activating mutations in NRAS, found in 20-30% of melanomas have been associated with aggressive clinical behavior and a poor prognosis. Nevertheless, there is lack of effective targeted therapies for NRAS mutant melanoma.Out of the few MEK inhibitors, pimasertib, a potent inhibitor of both MEK1 and MEK2 has showed promising results in NRAS mutant advanced melanoma. However, as a single agent and similar to other MEK inhibitors, it showed a limited clinical benefit due to its rather cytostatic effect and high toxicity. Our and other preliminary studies clearly indicated a stimulation of MITF (Microphthalmia associated transcription factor), the master transcription factor regulating cell growth and differentiation in the melanocyte, under MEK inhibition challenge. Thus, in a context where the tumor suppressor p53 is largely inactivated in melanoma, the stimulation of MITF may be the cause of the restraint cytotoxic effects of MEK inhibitors. Therefore, we aimed to further investigate the downstream MITF targets that can explain the resistance to the drugs.First, we showed that, MEK inhibition (by Pimasertib) led to a significant inhibition of cell proliferation but with a very limited effect on apoptosis that may be explained by the systematic MITF upregulation in all lines tested. Indeed, Mimicking MITF activation of expression by stimulating cAMP conferred resistance to MEK inhibition and interestingly up-regulated Bcl-2 expression. Further evidence was provided by the fact that, acquired resistance to MEK inhibition is associated with substantial upregulation of the anti-apoptotic signaling MITF/Bcl-2. More importantly, selective Bcl-2 inhibition by ABT-199 or Bcl-2 knock out using CRISPER/Cas9 system restores the sensitivity of NRAS mutant melanoma cells to MEK inhibition and breaks the acquired resistance.Given the known p53 regulating effect on Bcl-2, we evaluated p53 reactivation by PRIMA-1Met (APR-246) under MEK inhibition on the promotion of apoptosis in a panel of Q61NRAS mutant melanoma cells. Strikingly and similarly, this combination not only resulted in a synergistic effect to induce massive apoptosis but also broke resistance to MEK inhibitors both in cells with wild type or mutant p53 alike.In conclusion, we showed that the activation of cAMP/MITF/Bcl-2 pathway is a main anti-apoptotic mechanism associated with resistance to MEK inhibition in NRAS mutant melanoma. We propose drug combinations cotargeting MEK and other proteins regulating apoptosis -p53/Bcl-2- as a promising and clinically relevant therapeutic strategy to not only act in synergy to cause massive apoptosis but also to overcome resistance to MEK inhibitors in NRAS mutant melanoma / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

Malignant melanoma arises from the pigment producing melanocytes in epidermis and is the most aggressive type of skin cancer. The incidence of malignant melanoma is increasing faster than any other type of cancer in white population worldwide, with a doubling rate every 10-20 years. So far, the only identified external risk factor for malignant melanoma is UV exposure. Elimination of photodamaged cells by apoptosis (programmed cell death) is essential to prevent tumor formation. Melanocytes are considered relatively resistant to apoptosis, however, the regulation of apoptosis in melanocytes is still unknown. The aim of this thesis was to investigate the apoptotic process following ultraviolet (UV) irradiation in primary cultures of human melanocytes. Focus was on regulation of mitochondrial stability by Bcl-2 family proteins and the possible participation of lysosomal proteases, cathepsins. UV irradiation activated the mitochondrial pathway of apoptosis, leading to cytochrome c release, caspase activation, and nuclear fragmentation. No change in protein expression of Bax and Bcl-2 was observed in response to UV. Instead, translocation of the Bcl-2 family proteins from cytosol to mitochondia was important in the regulation of survival and death of melanocytes. The findings further demonstrated permeabilization of the lysosomal membrane to occur early in the apoptotic process, resulting in cathepsin release into the cytosol. The cathepsins were potent pro-apoptotic mediators and triggered apoptosis upstream of Bax translocation and mitochondrial membrane permeabilization. In response to both heat and UV irradiation, there was a marked increase in expression of stress-induced heat shock protein 70 (Hsp70), which inhibited apoptosis by binding lysosomal and mitochondrial membranes and counteracting the release of cathepsins and cytochrome c. Furthermore, UV irradiation activated c-jun N-terminal kinase (JNK), which triggered apoptosis upstream of cathepsins release from the lysosomes. In addition, JNK mediated apoptosis through phosphorylation of pro-apoptotic Bim, which was released from anti-apoptotic Mcl-1, by UV induced Mcl-1 depletion. This thesis illustrates that permeabilization of mitochondria and lysosomes and release of their constituents to the cytosol participates in UV induced apoptosis signaling in human melanocytes in vitro. The process is regulated by a complex network of pro- and anti-apoptotic proteins, exerting their effects through intracellular translocation and alteration of protein expression.

Of the thousands of dividing progenitor cells (PCs) generated daily in the adult brain only a very small proportion survive to become mature neurons through the process of neurogenesis. Identification of the mechanisms that regulate cell death associated with neurogenesis would aid in harnessing the potential therapeutic value of PCs. Apoptosis, or programmed cell death, is suggested to regulate death of PCs in the adult brain as overexpression of B-cell lymphoma 2 (Bcl-2), an anti-apoptotic protein, enhances the survival of new neurons. To directly assess if Bcl-2 is a regulator of apoptosis in PCs, this study examined the outcome of removal of Bcl-2 from the developing PCs in the adult mouse brain. Retroviral mediated gene transfer of Cre into adult floxed Bcl-2 mice eliminated Bcl-2 from developing PCs and resulted in the complete absence of new neurons at 30 days post viral injection. Similarly, Bcl-2 removal through the use of nestin-induced conditional knockout mice resulted in reduced number of mature neurons. The function of Bcl-2 in the PCs was also dependent on Bcl-2-associated X (BAX) protein, as demonstrated by an increase in new neurons formed following viral-mediated removal of Bcl-2 in BAX knockout mice. Together these findings demonstrate that Bcl-2 is an essential regulator of neurogenesis in the adult hippocampus.

Endocrine resistance is a significant clinical problem in the treatment of estrogen (E2) receptor positive breast cancers. There are two ER subtypes, ERα and ERβ, which promote and inhibit breast cancer cell proliferation respectively. While ER positive breast cancers typically express a high ratio of ERα to ERβ, the acquisition of antiestrogen resistance in vitro and in vivo is associated with increased relative expression of the ERβ. On some gene enhancers ERβ has been shown to function in opposition to the ERα in the presence of E2. Here we demonstrate that exposure to two different ERβ agonists results in decreased cell viability, and produced a marked reduction in G2/M phase in antiestrogen resistant breast cancer cell line in conjunction with altered cyclin D1, and cyclin E expression relative to E2. ERβ agonists also strongly downregulated Bcl-2 expression and recruited both ERs to the Bcl-2 and pS2 E2-response elements resulting in a reduction in mRNA transcripts from both of these genes. Bcl-2 reduction correlated with increased lipidation of LC3-I to LC3-II, indicative of increased autophagic flux. Although ERβ agonist treatment alone did not induce apoptosis, remarkably, the coaddition of ERβ agonist and the autophagy inhibitor, chloroquine, resulted in robust cell death. Lastly, in vivo studies demonstrate that preferential-ERβ agonists are not estrogenic in the uterus or mammary gland. Together, these observations suggest that combined therapies including an ERβ agonist and an autophagy inhibitor may provide the basis for a safe, novel approach to the treatment of antiestrogen-resistant breast cancers.

In the gut-associated lymphoid tissues, Th17 cells mediate mucosal homeostasis and inflammation. During HIV infection, Th17 cells become depleted and functionally impaired, which is implicated in the pathogenesis of chronic inflammation in patients treated with highly active antiretroviral therapy. IL-7 is a cytokine that mediates homeostatic responses in T lymphocytes, such as proliferation and survival, which are dysregulated during HIV infection. Whether similar dysregulation occurs in Th17 cells has yet to be reported. IL-7 receptor α (CD127) expression and IL-7 responses were therefore measured in blood-derived Th17 cells from uninfected individuals and effectively treated, HIV-infected individuals by flow cytometry. Th17 cells from uninfected individuals expressed CD127 and, in response to IL-7, exhibited phosphorylation of STAT5, upregulation of Bcl-2, and proliferation. During HIV infection, expression of CD127 and pSTAT5 in Th17 cells was comparable to that observed in cells from uninfected individuals. Interestingly, expression of Bcl-2 was upregulated while proliferation was dramatically impaired. These findings may provide further insight into the mechanisms by which Th17 cells fail to become restored during HIV infection.

<p>It has been long known that many types of cancers have high metabolic requirements and use reprogrammed metabolism to support cellular activities. The first identified metabolic alteration in cancer cells was elevated glucose uptake, glycolysis activity and lactate production even in the presence of oxygen. This metabolic program, termed aerobic glycolysis or the Warburg effect, provides cells with energy as well as biosynthetic substrates to sustain cell survival and rapid cell proliferation. Cancer metabolism is closely linked to genetic mutations and oncogenic signaling pathways, such as PI3K/Akt, cMyc and HIF pathways. These oncogenic signals can direct metabolic reprogramming while changes in metabolic status can regulate activities of these signaling pathways in turn. In addition to glucose, later studies also found utilization of alternate nutrients in cancer cells, including glutamine and lipids. Glutamine is the second major metabolic fuel and can be converted to various substrates to support cell bioenergetics needs and biosynthetic reactions. Usage of metabolic fuels in cancer cells, however, is variable. While certain cancers display addiction to one type of nutrient, others are capable of using multiple nutrients. </p><p>The unique metabolic features of cancer cells raise the possibility of targeting metabolism as a novel therapeutic approach for cancer treatment. Using pharmacological inhibitors, previous research has provided corroborating evidence that metabolic stress can impact survival and growth of proliferative cancer cells by regulating cell apoptotic machinery and cell cycle checkpoints. Due to lack of genetic tools and side effects from these inhibitors, however, mechanistic understanding of cell response to metabolic inhibition was limited in these studies. More importantly, how metabolic stress affects cancer progression in a physiological condition has not yet been well investigated. Lastly, current research has not examined metabolic program in indolent cancers and the metabolic requirements and activities in less proliferative cells also remain to be understood.</p><p>This work examines nutrients utilization in B cell derived acute and chronic leukemia (B-ALL and B-CLL). B-ALL is an aggressive form of leukemia. Using cell lines and primary patient samples, we found B-ALL cells primarily used glucose through aerobic glycolysis, similar to other proliferative cancer cells. B-ALL cells were also more sensitive to inhibition of glycolysis than normal B cells. Employing an untargeted metabolomics profiling in combination with isotope labeled glucose tracing approach, we show in a B-ALL model that genetic ablation of glucose transporter Glut1 partially reduced glucose uptake, sufficiently hindered anabolic pathways and promoted catabolic metabolism. This metabolic shift led to sharply curtailed B-ALL proliferation in vitro and reduced leukemic burden in vivo. Furthermore, this partial inhibition of glucose metabolism sensitized B-ALL cells to apoptotic stimuli and non-cytotoxic metabolic inhibition significantly enhanced efficacy of a tyrosine kinase inhibitor to eliminate B-ALL cells in vitro and in vivo. Thus, partial inhibition of glucose metabolism can provide a plausible adjuvant therapy to treat cancers that depend on glycolysis for survival and proliferation. </p><p>In contrast to B-ALL, B-CLL is an indolent form of cancer. Most B-CLL cells exhibited low glucose metabolic activities that were comparable with normal B cells at resting stage. Similar to chronically stimulated and anergic B cells, these B-CLL cells also failed to upregulate glucose metabolism in response to IgM stimulation. We also observed an altered amino acid and acyl-carnitine profile and increased glutaminase mRNA in B-CLL relative to normal B cells, suggesting the capability of using alternate nutrients such as glutamine in these cells. Finally, we explored the possibility of suppressing mitochondria metabolism to induce B-CLL cell death through inhibition of the nuclear hormone receptor and metabolic regulator ERRalpha. ERRalpha is known to regulate mitochondrial metabolism and was expressed higher in B-CLL than normal B cells. ERRalpha inhibition decreased viability of oncogene transformed pro-B cells, suggesting ERRalpha as a potential target for B-CLL treatment.</p><p> Collectively, this work investigates metabolic phenotype in two forms of leukemia derived from B cells. It reveals different metabolic requirements and activities in aggressive and indolent leukemia and explores different approaches to suppress metabolism in these cancers. Findings of this work shed light on how to potentially design metabolic approach to improve cancer treatment.</p> / Dissertation

The most common secondary structure of proteins is the alpha-helix. The alpha-helix can be involved in various protein-protein interactions (PPIs) through the recognition of three or more side chains along one face of the alpha-helix (Wells and McClendon, 2007). In recent years, there has been an increasing interest in the development of peptidic and non-peptidic compounds that bind to PPI surfaces. We focused on the design and synthesis of compounds that mimic the orientation of side chain residues of an alpha-helical protein domain. Although our scaffolds could potentially inhibit various PPIs, we focused mainly on the disruption of interactions among the Bcl-2-family of proteins and the Mdm-2-family of proteins to favor apoptosis in cancer cells. A summary of Bcl-2 and Mdm-2 structure and function relationships that focuses on the possibility of using peptidic and non-peptidic alpha-helical mimics as PPI inhibitors is described in Chapter One. Chapter Two discusses the design and synthesis of 3-substituted-2,6- and 2,5-piperazinedione oligomers as more hydrophilic scaffolds compared to previously reported alpha-helical mimetics (Yin, et al., 2005). A key feature of this design is the linkage of the units by a hydrazine bond. While we were able to prepare several monomers containing the hydrazine linkage, synthesis of the dimers and trimers is very challenging. Due to the difficulty of synthesizing oligomeric piperazine-diones in practical yields, we next focused on the design and synthesis of novel 2,5-terpyrimidinylene scaffolds as an alternative to obtain alpha-helical mimetics; this is discussed in Chapter Three. The main outcome of this project was the efficient preparation of a "first-generation" non-peptidic compound library via a facile iterative synthesis enabled by the key conversion of 5-cyanopyrimidine to 5-carboxamidine. Chapter Three also discusses our progress towards the synthesis of structurally similar substituted-2,5-terpyrimidinylenes, but with more drug-like properties as determined by QikProp calculations. Chapter Four describes an independent study on the synthesis of a guanidine derivative as an alkylating agent for the synthesis of cysteine peptide nucleic acids, CPNA, which is another current project in our lab.

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