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.

Acquired cellular resistance to traditional chemotherapeutics is a common obstacle in the treatment of most cancer cell types. This resistance occurs as a result of changes in the underlying molecular mechanisms of disease progression. The development of novel chemotherapeutic approaches designed to enhance the efficacy of protypical anti-cancer drugs is important in order to overcome this issue. Such approaches will aid in understanding the biomolecular phenomena responsible for drug resistance and disease progression. Combining signaling pathway inhibitors has become an effective strategy for enhancing tumor cell death by targeting multiple pathways known to regulate cell survival. Pemetrexed, an FDA-approved anti-folate drug, targets thymidylate synthase (TS) and a secondary folate-dependent enzyme, 5’ aminoimidazole-carboximide ribonucleotide formyltransferase (AICART); both important for DNA synthesis. Studies performed by our collaborator demonstrated that TS inhibition causes intracellular accumulation of ZMP+ and activation of AMPK which is known to induce autophagy in mammalian cells. Previous studies from our lab and others showed that sorafenib, a multi-kinase inhibitor of Raf-1 and class III receptor tyrosine kinases, was able to induce a cytotoxic form of autophagy in a variety of tumor cell types. Combination treatment using pemetrexed and sorafenib in these cancer cells resulted in an enhancement of autophagy and cell lethality beyond that of individual drugs alone. Inhibition of autophagy suppressed the toxic interactions of these drugs in all cell types examined. Pemetrexed/sorafenib cotherapy also proved to be an effective treatment for triple negative breast cancer cells having advanced to a stage of estrogen independence. Fulvestrant-resistant MCF7 cells were more sensitive to the drug combination than parental, estrogen-dependent MCF7 cells. Breast cancer cells cotreated with pemetrexed and sorafenib exhibited enhanced MEK/ERK signaling, Src activation that was dependent on platelet-derived growth factor β (PDGFRβ) downregulation, elevated protein phosphatase 2A (PP2A) activity, and increased de novo ceramide synthesis. Studies using a mouse model of experimentally-induced breast cancer validated drug combination effectiveness through inhibition of tumor growth, while no deleterious effects on normal tissues were observed. The data presented demonstrates that pemetrexed/sorafenib cotreatment augments chemosensitivity in both in vitro and in vivo systems. Based upon these findings, a Phase I clinical trial involving pemetrexed and sorafenib in breast cancer patients with solid, recurrent tumors was begun in 2011. In conclusion, this work strongly supports a promising therapeutic utility for the pemetrexed/sorafenib combination in treatment of various cancer cell types.