The Chronic Myeloid Leukemia (CML) is a clonal hematopoietic stem cell disorder characterized by the t(9:22) genetic translocation and expression of the oncogenic tyrosine kinase BCR-ABL . A first BCR-ABL Tyrosine Kinase Inhibitor (TKI), Imatinib (IM), was identified that inhibits proliferation of BCR-ABL expressing hematopoietic cells and leads to disease remission. However, BCR-ABL mRNA remains detectable in the most immature HSCs and discontinuation of IM results in clinical relapse. STAT5 factors play a crucial role in the CML pathogenesis of human primary CML cells. However, the contribution of the two related STAT5 genes, STAT5A and STAT5B, was unknown. We used an RNAinterference based strategy to analyze STAT5A or STAT5B roles in normal and CML cells. We showed that STAT5A/5B double knock-down (KD) triggers normal and CML cell apoptosis and suppressed long-term clonogenic potential of immature hematopoietic stem and progenitor cells known to be resistant to TKI treatment and responsible for residual disease. STAT5A loss alone was ineffective at impairing growth of both normal and CML cells under standard conditions. In contrast, STAT5A loss was sufficient to enhance Reactive Oxygen Species (ROS) which correlated with enhanced DNA damages in both normal and leukemic cells. We reported that STAT5A regulates oxidative stress through unconventional mechanisms, in a non-transcriptional-dependent manner. We further showed that, in contrast to primary cells at diagnosis, IM-resistant cells exhibited enhanced STAT5A dependence, by being sensitive to STAT5A single KD. To investigate the molecular basis of STAT5A activity in TKI-resistance and oxidative stress, we performed a transcriptomic analysis of STAT5 regulated genes. We identified Axl, which encodes a receptor tyrosine kinase, recently shown to be crucial in TKI-resistant CML cells. Specifically, Axl expression is enhanced by STAT5A. We investigated the role of Axl and we found that Axl KD did not affect survival of IM-sensitive CML cells. However, Axl KD decreased survival of IM-resistant cells, miming the activity of STAT5A. Moreover, Axl loss increased ROS levels in CML cells, promoting STAT5A anti-oxidant activity. We further sought to determine the expression of the Axl ligand, Gas6. Gas6 expression is dramatically reduced in CML primary cells at diagnosis compared to healthy cells. The strong and consistent down-regulation of Gas6 in CML cells suggested a possible role in the pathophysiology. Collectively, our findings highlight the pro-survival, stress protection and drug resistance roles of STAT5 factors, providing new understanding for medical treatment of CML patients. We suggest that STAT5A acts in synergy with Axl to face exogenous insults and propose a new mechanism by which CML cells increase their proliferation and reduce their motility by down-regulating Gas6 expression.

Pancreatic cancer is the fourth most lethal cancer and ranks as the eighth most commonly diagnosed cancer worldwide. This is due to its rapid proliferation, strong metastatic potential and its delayed detection. One major risk factor for developing pancreatic cancer is the aggressive inflammatory disease chronic pancreatitis. Chronic inflammation frequently precedes the development of certain pancreatic cancers. Inflammation is a protective and necessary process by which the body can alert the immune system of the existence of a wound or infection and mount an immune response to remove the harmful stimuli and start wound healing. The cross-talking of cells of the immune system and infected cells happens through cytokines, soluble proteins that activate and recruit other immune cells to increase the system’s response to the pathogen. Failure to resolve the injury can result in persistent cytokine production that in turn allows a cell that is damaged or altered to survive when in normal conditions it would be killed. Inflammation is thought to create a microenvironment that facilitates the initiation and/or growth of pancreatic cancer cells. Cytokines use two important kinases for their signaling: Janus Kinases (JAKs) and Signal Transducers and Activators of Transcription (STATs). The JAKs are activated upon the binding of cytokines to their corresponding receptors. When activated, the JAKs activate STATs through tyrosine phosphorylation. The STATs transduce signals to the nucleus of the cells to induce expression of critical genes essential in normal physiological cellular events such as differentiation, proliferation, cell survival, apoptosis and angiogenesis. STAT3 (a member of the STAT family) is constitutively activated in some pancreatic cancers, promoting cell cycle progression, cellular transformations and preventing apoptosis. Therefore, STAT3 is a promising target for cancer treatment. Novel therapies that inhibit STAT3 activity in cancers are urgently needed. Natural products are a very good resource for the discovery of new drugs against pancreatic cancer. Covering more than 70% of the Earths surface, The Ocean is an excellent source of bioactive natural products. Harbor Branch Oceanographic Institute’s Center for Marine Biomedical and Biotechnology Research (HBOI-CMBBR) situated in Florida, aims to find new marine natural products useful in disease prevention and drug therapy. Their current focus is to look for novel treatments for preventing both the formation of new pancreatic tumors and the metastasis of existing tumors. The hypothesis of this degree project was that novel inhibitors of STAT3 useful in the treatment of pancreatitis and/or pancreatic cancer could be found from marine-natural products. The first specific aim of this degree project was to set up an assay to identify bioactive marine natural products as inhibitors of inflammation. Furthermore the assay was validated using a commercially available inhibitor of inflammation (Cucurbitacin I). The last aim was to further validate the assay by screening pure compounds and peak library material from the HBOI marine specimen collection. At the end of the experimentation time, the assay still was not set-up as there were difficulties in proper cell culture techniques and the cell line did not respond as advertised. While the results were not as expected, the work performed resulted in familiarization with research laboratory practices and increased laboratory skills. Moreover, the results from the assays point to future directions to accomplish this project. / Development of a screening assay for inhibitors of inflammation useful against pancreatic cancer

La leucémie myéloïde chronique (LMC) est une pathologie de la cellule souche hématopoïétique caractérisée par la présence de la translocation chromosomique t(9 :22) conduisant à l’expression de la kinase BCR-ABL responsable de la maladie. Un inhibiteur de l’activité de BCR-ABL a été identifié, l’Imatinib (IM). L’IM a révolutionné la prise en charge de la LMC en bloquant sélectivement la croissance des cellules tumorales, conduisant à la rémission des patients. Cependant, une majorité d’entre eux subissent des récidives en cas d’arrêt du traitement, et environ 15% développent des résistances à l’inhibiteur. BCR-ABL active de multiples voies de signalisation parmi lesquelles figurent les facteurs de signalisation STAT5. Nous avons analysé les rôles respectives des deux facteurs STAT5, STAT5A et STAT5B, dans les cellules souches hématopoïétiques normales et de LMC, par une approche d’ARN interférence. Nos observations indiquent que l’activité des deux facteurs STAT5 permet la survie et le maintien à long terme des cellules souches de patients LMC au diagnostic. Nous avons de plus montré qu’indépendamment de son activité transcriptionnelle, STAT5A aide les cellules normales et leucémiques à limiter leur stress oxydatif. Nous avons aussi pu observer que les cellules de patients présentant des résistances secondaires à l’IM, sans mutations ni surexpression de BCR-ABL, manifestent une dépendance caractéristique vis-à-vis de l’activité STAT5A. Pour mieux comprendre les mécanismes d’action des facteurs STAT5, nous avons recherché les gènes cibles de STAT5 par une approche transcriptomique et avons identifié le récepteur tyrosine kinase Axl dont l’expression est augmentée par STAT5A. L’inhibition d’Axl dans les cellules LMC sensibles à l’IM n’a aucun effet sur leur survie, alors qu’elle diminue fortement la survie des cellules LMC résistantes à l’IM. De plus, Axl contrôle le niveau des réactifs oxygénés dans les cellules de patients LMC. Nous avons analysé l’expression d’un des activateurs d’Axl, le ligand Gas6, et avons observé que son expression diminue fortement dans les cellules primaires de LMC par rapport aux contrôles sains. Ces résultats suggèrent que le tandem Gas6/Axl pourrait participer au processus leucémique de la LMC à différents niveaux. De manière globale, nos travaux montrent que les facteurs STAT5 favorisent le maintien des cellules souches de LMC, leur résistance au stress oxydatif et aux traitements thérapeutiques Ces deux dernières activités sont au moins en partie liées à l’activité d’une nouvelle cible de STAT5, le récepteur Axl, par ailleurs déjà impliqué dans la résistance aux traitements thérapeutiques. Les facteurs STAT5 représentent donc des nouvelles cibles thérapeutiques potentielles dans l’éradication de la maladie résiduelle. / The Chronic Myeloid Leukemia (CML) is a clonal hematopoietic stem cell disorder characterized by the t(9:22) genetic translocation and expression of the oncogenic tyrosine kinase BCR-ABL . A first BCR-ABL Tyrosine Kinase Inhibitor (TKI), Imatinib (IM), was identified that inhibits proliferation of BCR-ABL expressing hematopoietic cells and leads to disease remission. However, BCR-ABL mRNA remains detectable in the most immature HSCs and discontinuation of IM results in clinical relapse. STAT5 factors play a crucial role in the CML pathogenesis of human primary CML cells. However, the contribution of the two related STAT5 genes, STAT5A and STAT5B, was unknown. We used an RNAinterference based strategy to analyze STAT5A or STAT5B roles in normal and CML cells. We showed that STAT5A/5B double knock-down (KD) triggers normal and CML cell apoptosis and suppressed long-term clonogenic potential of immature hematopoietic stem and progenitor cells known to be resistant to TKI treatment and responsible for residual disease. STAT5A loss alone was ineffective at impairing growth of both normal and CML cells under standard conditions. In contrast, STAT5A loss was sufficient to enhance Reactive Oxygen Species (ROS) which correlated with enhanced DNA damages in both normal and leukemic cells. We reported that STAT5A regulates oxidative stress through unconventional mechanisms, in a non-transcriptional-dependent manner. We further showed that, in contrast to primary cells at diagnosis, IM-resistant cells exhibited enhanced STAT5A dependence, by being sensitive to STAT5A single KD. To investigate the molecular basis of STAT5A activity in TKI-resistance and oxidative stress, we performed a transcriptomic analysis of STAT5 regulated genes. We identified Axl, which encodes a receptor tyrosine kinase, recently shown to be crucial in TKI-resistant CML cells. Specifically, Axl expression is enhanced by STAT5A. We investigated the role of Axl and we found that Axl KD did not affect survival of IM-sensitive CML cells. However, Axl KD decreased survival of IM-resistant cells, miming the activity of STAT5A. Moreover, Axl loss increased ROS levels in CML cells, promoting STAT5A anti-oxidant activity. We further sought to determine the expression of the Axl ligand, Gas6. Gas6 expression is dramatically reduced in CML primary cells at diagnosis compared to healthy cells. The strong and consistent down-regulation of Gas6 in CML cells suggested a possible role in the pathophysiology. Collectively, our findings highlight the pro-survival, stress protection and drug resistance roles of STAT5 factors, providing new understanding for medical treatment of CML patients. We suggest that STAT5A acts in synergy with Axl to face exogenous insults and propose a new mechanism by which CML cells increase their proliferation and reduce their motility by down-regulating Gas6 expression.

La voie JAK / STAT est une voie majeure de signalisation intracellulaire, activée par plusieurs cytokines, y compris par les interférons (IFNs). Mon laboratoire a précédemment établi que l'endocytose et le trafic du récepteur de l’IFN alpha/beta (IFNAR) jusqu’à l'endosome précoce, joue un rôle clé dans l’activation de la voie de signalisation JAK/STAT et dans les activités antivirales et antiprolifératives induites par les IFNs de type I. Le récepteur de l’IFN de type I se compose de deux sous-unités: IFNAR1 et IFNAR2. Durant le trafic du récepteur vers l'endosome précoce, les deux sous-unités prennent des itinéraires différents - IFNAR1 est ubiquitiné et dégradé au lysosome tandis que IFNAR2 est recyclé vers la membrane plasmique. Les mécanismes moléculaires permettant un trafic différent des IFNAR1 et IFNAR2 restent mal compris.J’ai tout d’abord étudié le trafic intracellulaire des sous-unités IFNAR1 et IFNAR2 et j’ai pu montrer que les protéines Rab4 et Rab11 sont nécessaires au recyclage d’IFNAR2 à la membrane plasmique. Par la suite, j’ai identifié le complexe rétromère endosomal - VPS26A/VPS29/VPS35 comme un partenaire d’interaction avec IFNAR2 nécessaire pour son recyclage. En outre, j’ai montré que le complexe rétromère contrôle la séparation spatiotemporelle des sous-unités IFNAR1 et IFNAR2 au niveau de l'endosome précoce. En effet, la déplétion du complexe rétromère entraine une prolongation de l’association endosomale de deux sous-unités et une prolongation de la signalisation JAK/STAT induite par l’IFN tant pour la réponse précoce (phosphorylation de STAT1) que la réponse à plus long terme (expression des gènes stimulés par l'IFN). Des résultats en cours de publication par l’équipe montrent un rôle d’ESCRT-0 dans l’initiation de la signalisation JAK/STAT. J’ai montré que le rôle de la machinerie ESCRT dans ce processus est limité au complexe ESCRT-0. Les sous-complexes en aval d’ESCRT-0, bien que nécessaires à la dégradation d’IFNAR1, ne sont pas impliqués dans l’activation de la voie JAK/STAT.En conclusion, ces résultats nous ont permis d’établir un modèle dans lequel le retromère joue un rôle clef dans la régulation spatio-temporelle du tri endosomal d’IFNAR et de la signalisation JAK/STAT au niveau de l’endosome précoce. Après la séparation rétromère-dépendante des sous-unités du récepteur et la terminaison de la signalisation JAK/STAT, la dégradation lysosomale d’IFNAR1 est assurée par la machinerie ESCRT en aval d’ESCRT-0. / The JAK/STAT pathway is a major intracellular signaling pathway that is activated by several cytokines including interferons (IFNs). My laboratory has previously established that endocytosis and trafficking of the IFN alpha/beta receptor (IFNAR) to the early endosome is key for the activation of JAK/STAT signaling and for the antiviral and antiproliferative activities induced by type I IFNs. The functional type I IFN receptor - IFNAR - consists of two subunits: IFNAR1 and IFNAR2. Upon endocytosis to the early endosome, both subunits take different trafficking routes – IFNAR1 is ubiquitinated and degraded in the lysosome, whereas IFNAR2 recycles back to the plasma membrane. The molecular mechanisms behind the separation of IFNAR1 and IFNAR2, as well as their distinct trafficking routes remain poorly understood.First, I studied the intracellular trafficking of IFNAR1 and IFNAR2 subunits and showed the requirement for Rab4 and Rab11 in IFNAR2 recycling to the plasma membrane. Next, I identified the endosomal retromer complex – VPS26A/VPS29/VPS35 as an IFNAR2 interacting partner, required for IFNAR2 recycling. Additionally, I was able to show that retromer controls the spatiotemporal separation of IFNAR1 and IFNAR2 subunits at the early endosome. Indeed, retromer depletion resulted in prolonged endosomal association of both subunits and prolonged activation of IFN-induced JAK/STAT signaling, at both early (STAT1 phosphorylation) and longer time response (IFN-stimulated gene expression). Unpublished results of our team indicate the role of ESCRT-0 in initiation of the IFN-mediated JAK/STAT signaling. I found that role of the ESCRT machinery in this process is limited to the ESCRT-0. ESCRT subcomplexes downstream of ESCRT-0, although required for IFNAR1 degradation, are not involved in activation of the JAK/STAT pathway.In conclusion, these results permit us to draw a model in which the retromer is a key spatiotemporal regulator of IFNAR endosomal sorting and the JAK/STAT signaling at level of the early endosome. Once retromer-mediated subunits separation is accomplished and JAK/STAT signaling is terminated, ESRCT machinery downstream to ESCRT-0 mediates IFNAR1 lysosomal degradation.

Le stroma inflammatoire joue un rôle primordial lors de la carcinogénèse. Dans ce contexte, nous montrons que la cytokine LIF est à l'origine d'une population de fibroblastes capable de remodeler la matrice extracellulaire de manière à la rendre permissive à l'invasion collective des cellules tumorales. En effet, nous montrons que la production de LIF par les cellules tumorales et fibroblastiques, après une stimulation au TGFβ, va réguler les capacités contractiles et pro-invasives de ces dernières via la régulation du cytosquelette d'acto-myosine et de manière indépendante de l'expression de α-SMA. En effet, l'inhibition pharmacologique des kinases JAKs permet de bloquer l'environnement fibrotique des tumeurs et d'ainsi bloquer l'invasion des cellules tumorales in vitro et in vivo. Nous montrons ensuite que LIF est à l'origine d'un switch épigénétique responsable de l'activation constitutive de la voie de signalisation JAK1/STAT3. Ce processus, régulé par la forme acétylée de STAT3, et son interaction avec l'ADN methyltransférase DNMT3b permet l'hypermethylation du promoter de la phosphatase SHP1 et donc la phosphorylation constitutive de JAK1. Une fois mis en place, ce nouveau profil de méthylation est maintenu par DNMT1. La surexpression de LIF dans les carcinomes humains corréle avec un environnement fibrotique, la présence de nodules invasifs et un mauvais pronostic clinique. De même, il existe une forte corrélation négative entre l'acétylation de STAT3 et l'expression de SHP1 dans le stroma tumoral. Nos résultats montrent qu'inhiber l'activité des DNMT et des kinases JAK permet de reprogrammer les capacités pro-invasive des fibroblastes associés aux carcinomes. / Signaling crosstalk between tumor cells and fibroblasts confers proinvasive properties to the tumor microenvironment. We identify LIF as a tumor promoter that mediates proinvasive activation of stromal fibroblasts independent of alpha-smooth muscle actin expression. We demonstrate that a pulse of transforming growth factor β (TGF-β) establishes stable proinvasive fibroblast activation by inducing LIF production in both fibroblasts and tumor cells. In fibroblasts, LIF mediates TGF-β-dependent actomyosin contractility and extracellular matrix remodeling, which results in collective carcinoma cell invasion. Indeed, pharmacological inhibition of JAK activity by counteracts fibroblast-dependent carcinoma cell invasion in vitro and in vivo. We next unveil that LIF initiates an epigenetic switch leading to the constitutive activation of JAK1/STAT3 signaling, which results in sustained pro-invasive activity of fibroblasts. The process is mediated by p300-histone acetyltransferase acetylation of STAT3, and DNA methyltransferase DNMT3b, which induce the hypermethylation of SHP1 phosphatase promoter and results in constitutive phosphorylation of JAK1. Sustained JAK1/STAT3 signaling is maintained by DNMT1. Accordingly, carcinomas display strong LIF upregulation, which correlates with dense collagen fiber organization, cancer cell collective invasion, and poor clinical outcome. Moreover, we show that STAT3 acetylation and phosphorylation are inversely correlated with SHP1 expression in tumors stroma. Combined inhibition of DNMT activities and JAK signaling results in long-term reversion of CAF-associated pro-invasive activity and restoration of the wild-type fibroblast phenotype.

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