Le virus de la dengue (DENV) et le virus Zika (ZIKV) sont deux virus transmis par le moustique et responsables de maladies importantes chez l’Homme. En absence de vaccin et de traitements antiviraux efficaces, ces pathogènes représentent des problèmes de santé publique majeurs. Les bases moléculaires des interactions qu’établissent le DENV et ZIKV et la cellule hôte lors de l’entrée virale sont peu connues. Notre laboratoire a récemment identifié, les protéines TIM (TIM-1 et TIM-4) et TAM (Tyro3 et Axl), deux familles de récepteurs à la phosphatidylsérine (PtdSer) impliqués dans la reconnaissance et l’élimination des cellules apoptotiques par phagocytose, comme de nouveaux facteurs d’entrée du DENV. Les récepteurs TIM et TAM permettent l’infection par le DENV en interagissant avec la PtdSer associée aux virions selon un mécanisme similaire à la reconnaissance des cellules apoptotiques (mimétisme apoptotique). L’objectif général de mon travail de thèse a été d’explorer les mécanismes moléculaires et cellulaires par lesquels TIM-1 et Axl médient l’entrée des flavivirus. A l’aide de techniques d’imagerie en temps réel nous avons montré que TIM-1 et DENV sont co-internalisés et que TIM-1 joue un rôle actif dans l’entrée du DENV. Notamment, nous avons montré que deux résidus lysine présentes dans le domaine cytoplasmique de TIM-1 sont importantes pour l’ubiquitination du récepteur et pour l’endocytose du virus. La recherche de partenaires de TIM-1 par des études de spectrométrie de masse a permis d’identifier STAM, un membre du complexe ESCRT-0 impliqué dans le trafic des récepteurs ubiquitinés, comme facteur important pour l’infection. Collectivement, nos résultats suggèrent très fortement que TIM-1 est le premier récepteur bona fide caractérisé pour le DENV.Identifier les facteurs d’entrée du ZIKV représente un enjeu majeur dans la compréhension du tropisme et de la pathogénèse associée à ce virus. Nous avons montré que le récepteur Axl est essentiel pour l’entrée du ZIKV dans les cellules microgliales, les astrocytes du cerveau humain en développement ainsi que dans les fibroblastes de la peau. Nos études ont démontré un double rôle du récepteur Axl dans l’infection par ZIKV. Axl lie et permet l’internalisation des particules virales, mais aussi, contribue à l’établissement d’un environnement favorable à la réplication virale en inhibant la réponse immunitaire innée. En conclusion, ce travail a contribué à améliorer notre compréhension des mécanismes d’entrée des virus DENV et ZIKV. Nos résultats indiquent que ces deux virus exploitent plusieurs récepteurs aux phospholipides pour initier leur cycle infectieux, ce qui pourrait contribuer à l’élargissement de leur tropisme. / Dengue virus (DENV) and ZIKA virus (ZIKV) are two mosquito-borne viruses responsible for important diseases in humans. Since there is currently no vaccine neither antiviral treatment available against these human pathogens, they are two major health concerns. The molecular basis of DENV and ZIKV host cell interactions leading to virus entry are poorly understood, hampering the discovery of new targets for antiviral intervention. Our laboratory recently discovered that TIM (TIM-1 and TIM-4) and TAM (Tyro3 and Axl) proteins, two receptor families that contribute to the phosphatidylserine (PtdSer)-dependent phagocytic removal of apoptotic cells, are DENV entry factors. TIM and TAM receptors mediate DENV infection by interacting with virion-associated PtdSer through a mechanism similar to the recognition and engulfment of apoptotic cells by phagocytes (viral apoptotic mimicry). The general objective of my PhD was to establish a detailed understanding of the molecular mechanisms by which TIM-1 and Axl mediated infection. Using live imaging, we demonstrated that TIM-1 and DENV are co-internalised and TIM-1 play an active role during DENV endocytosis. We showed that TIM-1 cytoplasmic domain is essential for DENV internalization, especially, we identified two lysine residues that are essential for TIM-1 ubiquitination and DENV endocytosis. Proteomic analysis of TIM-1 interacting partners identified STAM, a member of the ESCRT-0 complex involved in intracellular sorting of ubiquitinated cargos, as an essential host factor for DENV infection. Collectively our results establish TIM-1 as the first identified DENV bona fide receptor.Identifying ZIKV entry factors represents a major challenge in the understanding of ZIKV tropism and pathogenesis. We showed that Axl is responsible for ZIKV infection of microglial cells and astrocytes in the human developing brain and primary fibroblasts in human skin, suggesting an important role of this receptor during ZIKV life cycle. We also highlighted the dual role of the Axl receptor in ZIKV infection, which simultaneously promotes viral entry and dampens the innate immune response to facilitate a post entry step of the ZIKV life cycle. In conclusion, this work provided new insights in our understanding of the DENV and ZIKV entry program. Both viruses engage phospholipid receptors for their infectious entry, providing a rational to ascertain therapeutic strategies targeting virion-associated phospholipids.

Thesis (Ph.D.)--East Carolina University, 2009. / Presented to the faculty of the Department of Anatomy and Cell Biology. Advisor: Ann O. Sperry. Title from PDF t.p. (viewed June 12, 2010). Includes bibliographical references.

p53 is mutated in 50% of all human cancers, and up to 70% of lung cancer. Mutant p53 is usually expressed at elevated levels in cancer cells and has been correlated with a poor prognosis. Cancer cells that express mutant p53 show an increase in oncogenic phenotypes including an increase in growth rate, resistance to chemotherapeutic drugs, and an increase in motility and tumorigenicity to name a few. We have identified several genes involved in cell growth and survival that are upregulated by expression of common p53 mutants: NFκB2, Axl, and epidermal growth factor receptor (EGFR). The aim of this study was to determine the role NFκB2, Axl, and EGFR play in mutant p53’s gain of function (GOF) phenotype and to determine a mechanism for upregulation of mutant p53 target gene upregulation. Inhibition of mutant p53 in various cancer cell lines using RNAi in the form of transient siRNA transfection or stable shRNA cell line generation caused a decrease in the gain of function ability of those cells in the form of reduced chemoresistance, reduced cell growth and motility, and a reduction in tumor formation. Additionally, inhibition of NFκB2, Axl, and EGFR also showed similar effects. Promoter deletion analysis of the NFκB2 promoter did not show a specific mutant p53 response element needed for mutant p53 mediated transactivation. Similarly, deletion of the p53/p63 binding site on the Axl promoter did not inhibit mutant p53 transactivation. Sequence analysis of the NFκB2, Axl, and EGFR promoters revealed several transcription factor binding sites located throughout the promoters. ChIP analysis of mutant p53 and the promoter-specific transcription factor binding revealed that in the presence of mutant p53, individual transcription factor binding is increased to the NFκB2, Axl, and EGFR promoters as well as an increase in acetylated histone binding. This data suggests that mutant p53 promotes an increase in transcription by inducing acetylation of histones via recruitment of transcription factors to the promoters of mutant p53 target genes.

To enter cells, the filovirus, ebolavirus (EBOV), must bind to target cells and internalize into an endocytic vesicle. The properties surrounding filoviral entry into permissive cells remain poorly studied. To date, the kinetics associated with filoviral-glycoprotein (GP)-mediated entry have never been investigated past 6 hours. Our initial entry studies with filoviral-GP pseudotyped retrovirions at 37˚C indicated that virions entered permissive cells with a half time (T50) of ~8 hours. We found that 10 to 20% of retroviral based virions bound to cells in over a one hour period at 4˚C suggesting that virion binding was relatively inefficient. Surprisingly, we also observed that less than half of the retroviral based pseudovirions pre-bound to the cell surface were internalized by 7 hours at 37˚C indicating that virion internalization was a slow process. Consistent with slow internalization of retroviral particles, we observed that, while virus entry lost sensitivity to ammonium chloride treatment with time, 50% of the virions remained sensitive to low pH neutralization for at least 7 hours. These slow entry kinetics for filoviruses have not been appreciated thus far, and could have significant implications in the timing and types of treatments that could be administered to filoviral infected individuals. We also determined the impact of specific carbohydrate linkages on host cell plasma membrane proteins involved in filoviral entry, by using a series of Chinese hamster ovary (CHO) cell lines deficient in one or more enzymes required for N- and O- linked glycosylation. The LdlD CHO cell line that expresses normal surface N-linked glycans but has abbreviated O-linked surface glycans showed a 50% reduction in transduction by both Zaire (ZEBOV) and Lake Victoria MARV-GP pseudotyped particles as compared to the control wild type parental CHO cell line (Pro5). Use of the novel O-linked inhibitor drug 1-68A allowed us to confirm the necessity of O-linked glycans in efficient ZEBOV entry into additional permissive cells types. Interestingly, loss of terminal sialic acids (Lec2 cells) or galactose (Lec8 cells) on both N- and O- linked sugars resulted in a 2-fold enhancement of filoviral GP mediated entry compared to control. However, Lec1 cells that have wild type O-linked glycans but highly abbreviated N-linked glycans had similar levels of transduction to control Pro5 cells. Further studies indicated that binding of ZEBOV pseudovirions to Pro5 and all mutant CHO cells was equal, indicating that a post-binding defect or enhancement in ZEBOV internalization may be occurring. These data identify the importance of host cell O-linked glycosylation during the initial steps of filovirus infection. While the receptor(s) used by filoviruses for productive binding and entry into cells remains to be identified, several proteins have been shown to enhance filoviral entry into cells. Axl, a plasma membrane associated Tyro3/Axl/Mer (TAM) family member, is necessary for optimal ZEBOV-GP-dependent entry into some permissive cells, but not others. To date, the precise role of Axl in virion entry is unknown. Through the use of biochemical inhibitors, RNAi, and dominant-negative constructs, we set out to characterize entry pathways used for ZEBOV uptake in cells that require Axl for optimal transduction (Axl-dependent cells) and to define the role of Axl in these processes. We demonstrate that ZEBOV-GP-dependent entry into Axl-dependent cells occurs through multiple pathways including both clathrin-dependent and caveolae/lipid raft-mediated endocytosis. Surprisingly, both dynamin-dependent and -independent fluid-phase uptake (FPU) pathways mediated ZEBOV-GP entry into the Axl-dependent cells as well. Reduction of Axl expression by RNAi treatment resulted in abrogation of ZEBOV entry by FPU-dependent pathways, but had no effect on receptor-mediated endocytosis mechanisms. Our findings demonstrate for the first time that Axl enhances FPU, thereby increasing productive ZEBOV entry, and providing insight into the mechanisms surrounding filoviral entry.

In recent years, there has been a major movement in the pharmaceutical industry towards the development of molecules that selectivity inhibit a previously-validated specific target. This is referred to as target-based drug discovery. It was hoped that adopting this approach would usher in a new golden age of drug discovery. However, this has not been the case, with issues arising such as the target’s mechanism of action being poorly understood, with it not playing the expected role in the disease progression, or feedback resistance mechanisms causing the target to lose its role in the disease. In contrast to this, in the past 20 years it has been argued that developing drugs in a target-agnostic way and screening them against an expressed phenotype i.e. phenotypic drug discovery, has been more successful, despite fewer programs being run in the manner. The AXL kinase is a receptor tyrosine kinase (RTK) and a member of the TAM family, along with MER and TYRO3. AXL has long been associated with numerous types of cancer. Having been first discovered in 1991 in acute myeloid leukaemia (AML), it has gone on to be more associated with advanced solid tumours such as brain, breast, and lung, with the trend being that increased AXL correlates with a poorer prognosis for the patient. Upon the activation of AXL by the vitamin K ligand GAS6, a series of downstream pathways are activated that go on to encourage cell survival, proliferation, and migration. In addition to this, AXL has been shown to be involved in crosstalk with other kinase pathways, resulting in AXL expression being associated with chemoresistance and survival mechanisms. Despite the promising outlook for AXL inhibitors, to date only one selective AXL inhibitor, BGB324 (formally R428) has entered clinical trials, with selective AXL inhibitors being difficult to develop due to a lack of a crystal structure or a reliable homology model. To address the aforementioned issues that target-based approaches can suffer from, and due to AXL lacking a crystal structure, the work in this thesis utilised a pragmatic drug design method that started from ligands/existing scaffolds known to inhibit the target from the literature (publications, clinical trials and patents). A series of small libraries were prepared and then tested against a selected phenotype e.g. cell viability, in at least two cell types: one that expressed the target (e.g. AXL) and one that did not. Hits were optimised for potency against the desired phenotype. The compounds then went through target deconvolution (kinase screening) to confirm the target of the inhibitors. Employing this approach, we initially synthesised two small libraries of potential AXL inhibitors. The potency of these compounds was tested using cell-based phenotypic assays, by evaluating cell viability in both native and chemo-resistant breast cancer cells. These libraries were optimised through focused combinatorial synthesis and phenotypic screening, to yield a small collection of antiproliferative hits. These hits were then profiled against a panel of twelve select kinases. The first library, while giving some important structural information, did not inhibit the kinases screened in a meaningful manner. However, the second library gave several potent compounds, inhibiting AXL, FLT3, and RET, with one compound being selective for AXL. The leads from this series were optimised further, through SAR studies, gaining important structural information in order to improve potency and selectivity of the compounds. The flexibility of the phenotypic cell-based approach allowed the pursuit of FLT3 inhibitors, resulting in the synthesis of one of the most potent FLT3 inhibitors synthesised to date.

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that localises to focal adhesions. FAK is crucial for many cellular processes that are disturbed in malignancy, including proliferation, cell cycle, cell survival, adhesion, and migration. Mouse models have shown that FAK is involved in tumour formation and progression. Other studies demonstrated a functional correlation between FAK expression, tumour progression and malignancy in human cancer, making FAK a potentially important therapeutic target. Several FAK inhibitors have been developed most of which target the FAK kinase function. However, FAK may predominantly act as a scaffolding molecule rather than as a kinase, therefore, disruption of FAK’s interaction with protein binding partners could be a good strategy to inhibit some cancer processes. The identification and characterisation of novel FAK interactions may help to uncover important molecular mechanisms that, in turn, regulate key cellular processes involved in tumour formation and/or progression. Disruption of their function, or inhibition of their binding to FAK, will define their roles and identify whether they are good anti-cancer targets. In this thesis work, I set out to identify novel binding partners of FAK, and study the role of a sub-set of these in tumour biology by impairing them in squamous cell carcinoma cancer cells in vitro. To do this I employed protein microarray and phage display methodologies using FAKΔ375 and FAK-FERM recombinant proteins as bait, respectively. I identified a number of novel proteins that interact directly with FAK. Then I set out to characterise some of these proteins. The first of these, Axl, is a protein receptor tyrosine kinase that has previously been linked with tumour progression and metastasis in number of human cancers. I confirmed the interaction between FAK and Axl in SCC cells and showed that the FAK-Axl interaction is predominantly a scaffolding function of FAK, which seems to be unregulated, at least by any of the major phosphorylation events characterised for FAK. I also found that Axl controls cell spreading, cell polarisation and invasive migration in this cancer cell lines. The second protein I characterise is the autophagy protein Ambra1. I found that Ambra1 is required for selective targeting of active Src to the autophagy pathway – a process that SCC cancer cells use when they are under adhesion stress, such as when FAK is deleted. Thus, Axl and Ambra1 are potentially important proteins in SCC biology. They bind to FAK and function at cell adhesions to promote cancer-associated cellular processes. Analysis of FAK binding proteins may be a useful strategy to discover proteins that function in various aspects of cancer cell behaviour.

Skeletal muscle regeneration is a critical process that replaces damaged muscle fibers with new fibers. The regenerative process can be segmented into four main phases: necrosis, inflammation, regeneration, and maturation. While many of the key signaling molecules are known and characterized, there are still gaps in our understanding of how this process is regulated. While it is reported that growth arrest specific 6 (Gas6) and its receptor Axl are expressed in mature muscle tissue, nothing is known about the effect that Gas6 and Axl have on regulating skeletal muscle regeneration. In this study we investigated the regenerative process in a Gas6/Axl double knockout (dKO) mouse model. The tibialis anterior (TA) muscle was chemically injured with BaCl2 and allowed to recover for 3, 7, or 14 days. We investigated satellite cell (SC) activation and muscle growth. We found that the dKO injured muscle has fewer SCs at 3-days post-injury, but the percentage of mitotically active SCs were no different between WT and dKO injured muscle. Interestingly, basal and injured dKO muscle has an increased cross-sectional area compared to wild type in male mice. Together this may suggest that in the absence of Gas6/Axl signaling may lead to impaired regeneration and compensatory fiber hypertrophy. The mechanism behind the hypertrophy remains unknown, but ultimately our findings suggest that Gas6/Axl signaling has an effect on skeletal muscle regeneration.

Current research spans a wide range of objectives whose diversity includes the understanding of global epidemiology and the detailing of molecular interactions leading to specific pathologies. This work aligns more closely with the goal of mechanistic clarity by elucidating several aspects of signaling pathways involved in inflammatory and obstetric pathologies. Prior research has confirmed the role of Receptors for Advanced Glycation End-Products (RAGE) activation in signaling leading to chronic inflammation such as that observed in chronic obstructive pulmonary disease (COPD). RAGE activation has also been identified in other disease states including diabetes, Alzheimer’s disease, osteoarthritis, and cancers. We examined the role of RAGE in the obstetric complication intrauterine growth restriction (IUGR) wherein fetal development is delayed and infants are born at low birthweight. Exposure to tobacco smoke is known to activate RAGE, and smoke exposure also increases risk for IUGR. We confirm a role for RAGE signaling in development of IUGR. RAGE inhibition by semi-synthetic glycosaminoglycan ethers (SAGEs) significantly improved fetal and placental weights and reduced inflammatory signaling molecules. Interactions between RAGE and other signaling pathways have been noted in several research endeavors, and we sought to further understand signaling interactions specifically in obstetric pathologies by examining relationships between RAGE and Gas6/AXL signaling. We confirm that RAGE and Gas6/AXL signaling are not independent. Using tobacco smoke as a means of inducing RAGE, we determined that total AXL is inhibited when RAGE is active, but that phosphorylated AXL is increased. Inhibition of RAGE also increased Gas6 expression. These interactions require further clarification, but provide a foundation to expand upon. We further studied interactions within the Gas6/AXL pathway independent of RAGE. High levels of Gas6 have been noted in the serum of some women with preeclampsia, and early diagnosis and treatment of preeclampsia are currently limited. We demonstrate that, in a rat model, administration of Gas6 during pregnancy is sufficient to induce symptoms of preeclampsia including high blood pressure, increased proteinuria, and decreased trophoblast invasion. This provides a novel model which will further both diagnosis and treatment of preeclampsia. We also demonstrated that trophoblast invasion is influenced in a cell-type dependent manner by Gas6 and mTOR signaling, with decreased trophoblast invasion when Gas6 is high in trophoblast cells, but increased invasion with high Gas6 in a pulmonary adenocarcinoma cell type and in oral squamous cell carcinoma cells. Our work has clarified details of both RAGE and Gas6/AXL signaling that are crucial to further study of the pathways in which they are active, and the pathologies resulting from signaling misregulation.

La duplication interne en tandem au sein du gène du Fms-like tyrosine kinase 3 (FLT3) est l’une des mutations les plus fréquemment observées dans les leucémies aiguës myéloblastiques (LAM). Elle est corrélée à un mauvais pronostic. Des inhibiteurs de tyrosine kinase anti-FLT3 (FLT3-ITK) sont en cours de développement mais les premiers essais cliniques ont été décevants. Les rémissions sont de courte durée, et si une clairance leucémique sanguine est observée, la LAM persiste au sein de la moelle osseuse. Dans ce travail, nous avons démontré que les cytokines activatrices de STAT5, telles que l’interleukine-3 et la thrombopoïétine, et les basses pressions en oxygène, telles que celles observées au sein de la niche hématopoïétique augmentent l’expression et l’activité du récepteur tyrosine kinase AXL qui protège les cellules de LAM FLT3-ITD de l’apoptose induite par le FLT3-ITK quizartinib (AC220). Nous avons démontré dans un modèle murin que les cellules de LAM FLT3-ITD « knock-down » pour AXL sont plus sensibles au quizartinib, et que cette différence se révèle spécifiquement dans un modèle de prise de greffe hématopoïétique. La combinaison de stratégies inhibitrices du FLT3-ITD et d’AXL permettra d’améliorer l’efficacité des FLT3-ITK en atteignant la fraction de cellules responsable des rechutes, nichée dans son microenvironnement. A l’issue, nous avons démontré que le gilteritinib (ASP2215), double FLT3/AXL-ITK est plus efficace que le quizartinib pour atteindre ces cellules leucémiques médullaires. Enfin, nous avons démontré que la combinaison d’un anticorps monoclonal anti-AXL avec un FLT3-ITK ou de la cytarabine était une stratégie thérapeutique prometteuse dans les LAM FLT3-ITD ou sauvage. / Internal tandem duplication in Fms-like tyrosine kinase 3 gene (FLT3-ITD) is the most frequent mutation observed in acute myeloid leukemia (AML), and correlates with poor prognosis. FLT3 tyrosine kinase inhibitors (FLT3-TKI) have been promising for therapeutic strategies but clinical trials have revealed rarely long-lasting remission with persistent leukemic cells present in the bone marrow. In this work, we show that the hematopoietic niche microenvironment protects FLT3-ITD AML cells from FLT3-TKI quizartinib (AC220) through convergent up-regulation of AXL expression and activity. Cytokine-dependent activation of STAT5 enhances AXL gene transcription and expression, while low O2 concentration up-regulates AXL protein levels. Moreover, cytokines such as thrombopoietin or interleukin-3 directly activate AXL. RNA interference-based inhibition of AXL expression in FLT3-ITD AML cells allowed a selective purge of leukemic cells within their microenvironment when combined with FLT3-TKI in immuno-compromised mice. Altogether, our data support a strategy combining FLT3-TKI and anti-AXL therapy to eradicate FLT3-ITD AML cells, including those protected by the hematopoietic niche. In such a setting, we performed a study to test the efficacy of gilteritinib (ASP2215) and we showed in vitro and in vivo that this dual FLT3/AXL-TKI is more efficient to eradicate leukemic cells in their microenvironment than quizartinib which is a more specific FLT3-TKI. Finally, we also studied an anti-AXL monoclonal antibody on primary AML cells and showed that its efficacy could be interesting with FLT3-TKI and cytarabine in both FLT3-wild type and FLT3-ITD AML.

AXL est un récepteur tyrosine kinase (RTK) impliqué dans de nombreux mécanismes cellulaires tels que la migration, l'invasion, l'angiogenèse et la prolifération des cellules. Sa surexpression a été observée dans de nombreux cancers et est souvent liée à un mauvais pronostic vital pour le patient. De plus, ce récepteur semble agir dans un mécanisme important dans la formation de métastases et la résistance aux thérapies anticancéreuses : la transition épithélio-mésenchymateuse (EMT). Nous avons dans un premier temps généré des anticorps monoclonaux murins spécifiques du récepteur AXL. Deux de ces anticorps ont ensuite été sélectionnés pour leurs propriétés inhibitrices de l'expression d'AXL à la surface et de l'activation de ce récepteur par son ligand GAS6. En effet ces deux anticorps, le 20G7D9 et le 3E3E8, entraine l'internalisation et la dégradation lysosomale d'AXL.Nous avons dans un deuxième temps étudié l'expression et le rôle de ce récepteur dans le cancer du pancréas qui possède un manque cruel de solutions thérapeutiques aujourd'hui et dont le taux de survie reste très faible (moins 5% des patients survivent 5 ans après son diagnostic). Nous avons ainsi observé une expression d'AXL dans une majorité des tumeurs de patients (76%), notamment au niveau du front invasif de ces tumeurs. Le ciblage d'AXL par nos deux anticorps inhibe sa signalisation et permet une réduction in vitro et in vivo de la croissance tumorale.Enfin, l'importante expression d'AXL dans le front invasif des tumeurs nous a incité à étudier le rôle d'AXL au cours de la transition épithélio-mésenchymateuse. Nous avons ainsi démontré que le couple AXL/GAS6 induit l'EMT dans des modèles invasifs de cancer du sein triple négatifs. De plus, l'expression du récepteur dans des tumeurs de cancer du sein de type basal-like est corrélée à celle de différents marqueurs importants dans l'EMT. L'application de nos anticorps anti-AXL dans ce type de cancer permet d'inhiber l'induction de l'EMT par le récepteur ainsi que l'invasion cellulaire in vitro et in vivo.Cette thèse a ainsi permis de démontrer l'importance du récepteur tyrosine kinase AXL dans des mécanismes oncogéniques clés et l'efficacité de son ciblage par des anticorps monoclonaux dans des modèles précliniques de cancer. / The Tyrosine Kinase Receptor (TKR) AXL is implicated in various cellular mechanisms (migration, invasion, angiogenesis and cell proliferation). Its overexpression has been observed in many cancers and is often correlated with poor prognosis. Moreover, this receptor seems to be important in Epithelial to Mesenchymal Transition (EMT), a mechanism related to metastasis formation and resistance to anticancer therapies.We have generated several AXL specific murine monoclonal antibodies. Two of them, 20G7D9 and 3E3E8, have been selected for their inhibition properties in AXL expression and activation by its ligand GAS6. In fact, both antibodies induce internalization and lysosomal degradation of AXL.Then we decided to study AXL expression and role in pancreatic cancer, which is characterized by a dramatic overall survival (<5%, 5 years after diagnosis) and a lack of efficient therapeutic solutions. We observed an ectopic expression of AXL in a majority of patient' pancreatic tumors (76%), notably in the invasive front of the tumor. Targeting AXL with both 20G7D9 and 3E3E8 inhibits its signaling and decreases tumor growth in vitro and in vivo.As AXL is mainly expressed in the invasive front of tumors, we analyzed its role during EMT. We observed that AXL/GAS6 signaling induces EMT in triple negative breast cancer cell lines. Furthermore, its expression is correlated with well-defined EMT markers in basal-like breast cancer tumors. In vitro and in vivo application of our antibodies inhibits AXL-dependant EMT signaling and cellular migration and invasion.In conclusion, this thesis demonstrates the importance of AXL Tyrosine Kinase Receptor in oncogenic processes and the efficacy of targeting this receptor with monoclonal antibodies in cancer preclinical models.