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Les léiomyosarcomes (LMS) sont des tumeurs d’origine mésenchymateuse caractérisées par une différenciation musculaire lisse. La voie de signalisation PI3K/mTOR (qui contrôle la prolifération et la survie cellulaire) joue un rôle majeur dans le développement de ces tumeurs. De nos jours, cette voie est devenue une cible thérapeutique majeure en oncologie. Cette étude est la première qui évalue le bénéfice thérapeutique de l’inhibition de la voie PI3K/mTOR pour des patients atteint de LMS. Nous avons mis en évidence qu’une double inhibition de PI3K et mTOR est associée à une activité antitumorale supérieure à celle observée avec une inhibition de PI3K ou mTOR seule. Nous avons également montré que l’inhibition de la voie PI3K/mTOR est associée à une activation paradoxale de la voie MAPK et qu’un ciblage concomitant de cette voie est associé à une synergie antitumorale in vitro et in vivo. Afin de caractériser les mécanismes de résistance secondaire à l’inhibition de la voie PI3K/mTOR, nous avons développé in vitro et in vivo un modèle de résistance secondaire à l’inhibiteur double cible PI3K/mTOR. Nous avons notamment détecté une sous-population de cellules résistantes à l’inhibiteur et ayant des caractéristiques proches de celles des cellules souches. Nous avons mis en évidence que l’inhibition pharmacologique d’EZH2, une protéine cruciale du complexe Polycomb, permet de restaurer la sensibilité des modèles résistants. Ces résultats apportent de nouvelles perspectives thérapeutiques pour les patients atteints de LMS. / Leiomyosarcomas (LMS) are tumors of mesenchymal origin characterized by a smooth cell differentiation. The PI3K/mTOR pathway has been shown to play a crucial role in the tumorigenesis of LMS. Several agents targeting this pathway are under clinical development for the treatment of solid tumors and hematological malignancies. We report here the first study evaluating its potential therapeutic benefit for patients with LMS. We have demonstrated that dual inhibition of PI3K and mTOR is associated with more effective antitumor activity than agents targeting PI3K or mTOR only. We have also shown that PI3K and mTOR inhibition is associated with a paradoxal activation of the MAPK pathway and that combined treatment with MEK inhibitor resulted in synergistic antitumor activity in vitro and in vivo. Moreover, we developed in vitro and in vivo resistant model to dual PI3K/mTOR inhibitor. Interestingly, we have found that a cancer stem cell-like subpopulation may be involved in treatment resistance. We have shown that pharmacological inhibition of EZH2, a crucial protein of the Polycomb complex, is able to reverse dual PI3K/mTOR inhibitor resistance in vitro and in vivo. These results provide new therapeutic strategies for patients with LMS.

<p>Castration resistant prostate cancer (CRPC) causes significant morbidity and mortality around the world and improving treatment options for patients with CRPC is a major concern for biomedical research. Because of the importance of activating mutations in the PI3K/AKT/mTOR pathway in prostate cancer, several mTOR inhibitors have been tested for efficacy in CRPC but despite promising preclinical findings, the results of clinical trials have been disappointing. The findings of several groups, including a clinical trial of RAD001 conducted at Duke, suggest that feedback upregulation of PI3K and autophagy may be potential mechanisms for resistance of CRPC to mTOR inhibitor therapy. </p><p> The main goal of this dissertation was to explore these mechanisms in vitro and to determine if combinations of PI3K inhibitors and different classes of mTOR inhibitors can overcome resistance to mTOR inhibitor monotherapy. In particular, we used immunoblotting, reverse phase protein microarrays, polysome profile analysis, cell cycle analysis, and several techniques for determining cell survival and proliferation to explore the differences in survival, proliferation, autophagy, and activity of the AKT, translation initiation, and autophagy cell signaling networks between prostate cancer cell lines treated with different combinations of mTOR and PI3K inhibitors. Our findings revealed that the combination of PI3K and mTOR inhibition leads to a synergistic inhibition of prostate cancer cell survival and cytostasis that is correlated decreased translation rates, hypophosphorylation of 4E-BP1, autophagy, and an uncoupling of normal signaling between AKT and mTOR. We were able produce an effect on cell survival similar to treatment with high doses of mTOR/PI3K inhibitor combinations by inhibiting cap-dependent translation using a non-phosphorylatable mutant of 4E-BP1. In contrast, knocking down two major autophagy genes had little to no effect on the survival of prostate cancer cells treated with PI3K/mTOR inhibitors but did protect from cell death caused by the UPR activator tunicamycin. </p><p> We conclude that treatment strategies that target PI3K, mTORC1 and mTORC2 simultaneously have the potential to be clinically useful in CRPC, probably due to the increased inhibition of eIF4E activity and cap-dependent translation when compared to monotherapy with allosteric mTORC1 inhibitors. Although autophagic cell death can be induced in prostate cancer cells, the autophagy observed after inhibition of PI3K and mTOR does not appear to contribute to cell death and is not a major resistance mechanism under these conditions. Nevertheless, we did observe different roles for autophagy in the survival of cells exposed to different types of stressors, and further elucidation of autophagy signaling networks may yet provide useful clinical targets.</p> / Dissertation