Mutations in the RAS-RAF-MEK-ERK (MAPK) pathway give rise to a range of developmental disorders collectively referred to as the RASopathies. De novo germline mutations in patients suffering from these syndromes promote similar phenotypes, which include heart abnormalities, characteristic facial features, cutaneous malformations, gastrointestinal malfunctions, failure to thrive and a spectrum of mental retardation. Although many RASopathies patients show a propensity to develop early-onset benign and malignant tumours, Cardio-faciocutaneous (CFC) syndrome patients do not seem to share this predisposition, with the exception of an increased number of naevi. CFC syndrome is caused by mutations in BRAF, MEK1 or MEK2, with the majority of patients harbouring BRAF mutations. Intriguingly, both kinase-activating and kinase-impaired mutations have been identified in CFC patients. Here, I use the zebrafish system to address the activity of the CFC syndrome alleles and the MAPK pathway in a developmental context and test the potential of small molecule inhibitors to restore normal development. I established an assay for the activity of CFC, melanoma and engineered BRAF and MEK human mutated alleles in vivo. Using zebrafish as an animal model organism, a panel of 31 mutant and wild-type BRAF, MEK1 and MEK2 alleles were expressed in early zebrafish embryos to assess their role in development. Irrespective of the predicted kinase activity, all embryos expressing BRAF and MEK mutant alleles reproducibly manifested the cell movement phenotype during gastrulation. Consistent with aberrant fibroblast growth factor (FGF) signalling and defective gastrulation, in situ hybridisation against convergence-extension markers showed misregulated convergence-extension movement patterns in CFC zebrafish embryos. Finally, I performed whole embryo RNA expression microarrays to identify genes regulated downstream of the CFC mutations, and I discuss the potential for a possible link to some of the phenotypes associated with a CFC zebrafish model. I established that the CFC, BRAF and MEK mutant embryos are sensitive to inhibition of MEK signalling by small molecules. Importantly, a time-window of treatment was identified which was sufficient to restore normal gastrulation movements and to prevent the developmental side effects promoted by the inhibitors at later stages of development. In order to begin considering the therapeutic potential of small molecules in developmental disorders (at least in our model system), the effect of low concentrations of the inhibitors in the normal formation of diverse tissues was thoroughly examined during zebrafish development. From these studies, I identified a concentration of MEK inhibitor that could be administered in a continuous fashion to prevent CFC-associated cell movement defects during gastrulation, without additional later developmental defects. Finally, I addressed the role of MEK-ERK signalling in a specific behavioural phenotype in zebrafish. Many RASopathies patients suffer from mental retardation and experience learning and attention difficulties. Research in our laboratory has identified a novel zebrafish behaviour induced by enhanced cAMP signalling, where the zebrafish seek shaded areas in their environment and exhibit frequent defensive shoaling behaviour. I used western blotting to establish that enhanced cAMP signalling activates the MAPK signalling pathway and, in collaboration with members our laboratory, that this phenotype can be suppressed by administration of the PD325901 MEK inhibitor. While we do not yet know the effect of CFC syndrome mutations on this behaviour, we suggest that altered MEK-ERK signalling may underlie important features of vertebrate behaviour.

Thesis (doctoral)--Vrije Universiteit te Amsterdam, 1993. / Basic text in English; partial t.p. in Dutch; summaries in English, Dutch, and Una. "Stellingen" laid in. Includes bibliographical references (p. [157]-164) and index. Also issued online.

Le cancer colorectal est issu des cellules de l’épithélium et se définit comme une pathologie induite par une accumulation d’altérations génétiques. Des mutations de type gain-de-fonction dans les gènes KRAS, NRAS et BRAF sont retrouvées dans plus de 60% des cancers colorectaux entraînant potentiellement l’activation constitutive de la signalisation en aval, notamment le sentier MEK/ERK. Dans cette thèse, nous avons évalué les mécanismes moléculaires par lesquels l'activation du sentier MEK/ERK régularise la différenciation épithéliale et la tumorigénèse intestinale. Dans les cellules épithéliales intestinales (CEIs), nos résultats montrent que les kinases ERK1/2 doivent être inactivées pour permettre l’induction du processus de différenciation entérocytaire. En effet, l'expression d’une forme constitutive active de MEK1 (caMEK1) est suffisante pour bloquer la différenciation tant morphologique que fonctionnelle. Une augmentation de la phosphorylation du facteur de transcription Cdx2 sur la sérine 60, qui diminue son activité transcriptionnelle, constituerait un des mécanismes impliqués. Dans des cellules cryptales intestinales indifférenciées, l’expression du caMEK1 induit une transition épithélium-mésenchyme (EMT), un processus associé au cancer colorectal. Cette EMT confère aux CEIs des capacités invasives et métastatiques in vivo associées à la sécrétion de protéases extracellulaires (MMP2/9). Nous avons identifié plusieurs autres cibles moléculaires de l’activité MEK/ERK impliquées dans l’EMT et l’invasion tumorale. Nous avons démontré l’implication des cascades Fra-1/Snail2 et EGR-1/Snail1 dans la répression transcriptionnelle de la E-cadhérine, une étape clé de l’EMT. Ce mécanisme de répression est également présent dans les lignées cancéreuses colorectales humaines possédant la mutation oncogénique de KRAS. L’analyse comparative par micropuce d'ADN Affymetrix dans les CEIs transformées par caMEK1 a permis d’identifier le gène encodant pour la serpineE2, un inhibiteur de protéases, comme le gène le plus fortement induit. Nous avons démontré que la serpineE2 est une cible moléculaire directe de l'activité oncogénique de la voie KRAS/BRAF/MEK/ERK et démontré son importance dans la migration et l’invasion tumorale par l'utilisation d'ARN interférents. On observe d’ailleurs une très forte expression de la serpineE2 dans des tumeurs colorectales humaines en comparaison à la marge saine adjacente. Nous avons aussi démontré que la transformation des CEIs induite par l’expression des formes actives de KRAS ou de MEK1 stimule la voie signalisation Wnt/β-caténine, dont la fréquente dérégulation constitue un évènement majeur menant au développement du cancer du côlon. La phosphorylation MEK-dépendante de LRP6 (S1490/T1572) semble être responsable de l’augmentation de l’activité transcriptionnelle de la β-caténine, associée à une augmentation de sa présence au noyau. Cette interconnexion entre les voies KRAS/MAP kinase et Wnt/β-caténine a également été observée dans des cellules cancéreuses colorectales humaines mutées pour KRAS ou BRAF. Finalement, une augmentation du niveau de phosphorylation de LRP6 a été observée dans les adénomes et tumeurs colorectales humaines, supportant l’idée que la phosphorylation de LRP6 puisse être impliquée dans la progression tumorale.

No description available.

Neurofibromatosis type two (NF2) is a genetic disorder predisposing those affected to the development of multiple benign tumors in their central and peripheral nervous systems. This is due to the absence of the tumor suppressor protein merlin, which is encoded by the NF2 gene. In nearly all NF2 cases, patients present with bilateral schwannomas of the vestibulocochlear nerve, in addition to other schwannomas throughout the central and peripheral nervous systems, as well as meningiomas and ependymomas. Currently, no therapeutic alternatives to surgical removal and radiation therapy are available for NF2 patients. This study investigated cobimetinib, an inhibitor of the often-deregulated mitogen activated protein kinase (MAPK) pathway in NF2 tumors, and its in vitro mechanism of action in both mouse and human NF2 schwannoma model cell lines. It was demonstrated that the drug decreased 70% and 60% of the viability at 10μM in the mouse and human merlin-deficient cell lines, respectively. It was further demonstrated that this decrease in viability was due to cytostatic and cytotoxic effects of cobimetinib in the case of the mouse NF2 schwannoma model but only due to cytostatic effects of cobimetinib in the human NF2 schwannoma model. These results show promise in targeting the MAPK pathway in NF2 tumors, and the promise of cobimetinib specifically, supporting further cytometric flow and in vivo testing of the inhibitor.

Dans un village du Yucatán la majorité de la population s’est récemment convertie au protestantisme. Cette situation a contraint à une redéfinition de certains éléments de la culture locale et de ses pratiques rituels. Une rédefinition qui se réalise, d'une coté dans le refus du système d'orientement culturel, des pratiques rituelles et de l'organisation catholiques et, de l'autre coté, d'une maintenance selective et dans une remodulation de certains de ses termes. L'het'z mek, une pratique rituelle «traditionnelle», est aussi célébrée par les protestants sur la base de l'accent mis sur ses fonctionnalités par rapport au developpement physique et corporel associé à une intervention « orthopédiques ». Le parcour de l'initiation du chaman est par contre fortement contrasté à travers l'utilisation de la rhétorique des recits de conversion au protestantisme. / In a Yucatan village, the main population has recently converted to Protestantism. This situation brought to a redefinition of some elements of local culture and its practical rituals . This process of redefinition, which is realized partly by rejecting the cultural system, practices and rituals of the Catholic organization, and, partly, by a selective manteinance and a reshaping of some of its terms.The het'z mek ', a ritual "traditional" practice, is also celebrated by Protestants on the basis of the emphasis on its functionality over the physical body associated with an "orthopedic" intervention. The shaman's initiation path is rather strongly contrasted by the use of the rhetoric of conversions tale to Protestantism.

Neutrophils form a first line of defence against infections. These short-lived, terminally differentiated cells perform many important functions, including chemotaxis, degranulation, reactive oxygen species (ROS) release and cytokine production. Whilst neutrophils are essential for host immunity, their inappropriate recruitment, activation and/or removal can contribute to excessive inflammation and host damage, as exemplified in autoimmune diseases such as rheumatoid arthritis. It is therefore essential that neutrophil function is tightly regulated. Neutrophils are activated by a range of stimuli, including immune complexes. Neutrophil functions are tightly regulated by intracellular signalling events that are induced by the ligation of cell surface receptors, for example, the binding of immune complexes to Fc receptors. Phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (Erk) are key signalling intermediates that act downstream of many cell surface receptors. They are involved in the regulation of numerous biological processes in the neutrophil. Using pharmacological interventions, I analysed PI3K signalling in immune complex-stimulated human neutrophils and uncovered a previously uncharacterised, noncanonical signalling pathway, PI3K-Cdc42-Pak-Mek-Erk. This represents an unusual situation where Pak acts as the MAP3K downstream of Cdc42 in a PI3K-dependent fashion. By performing a range of functional experiments, I showed that this unconventional signalling pathway promotes apoptosis in human neutrophils by regulating the ratio between anti- and pro-apoptotic members of the Bcl-2 family proteins. No other immune complex-induced, PI3K-dependent neutrophil function tested depended on PI3K-Cdc42-Pak-Mek-Erk signalling. Mouse knock-outs of all components of this signalling pathway have been described. Immune complex-induced apoptosis was also PI3K-dependent in mouse neutrophils, but experiments performed with inhibitors showed that, in contrast to human neutrophils, this was not dependent on PI3K-Cdc42-Pak-Mek-Erk signalling. The myeloid leukaemia cell line, PLB-985 is amenable to knock-down and can be differentiated to become neutrophil-like. These cells are not notably activated by immune complexes, perhaps because they do not express the major Fcγ receptor, CD16. Since retroviral expression of CD16 in PLB985 cells did not improve their response to activation by immune complexes, I was not able to confirm my observations with human neutrophils genetically. Collectively, I showed that a novel, pro-apoptotic signalling pathway operates downstream of Fcγ receptors in the human neutrophil. The fact that this signalling pathway appears to regulate apoptosis specifically suggests uncoupling pro- and anti-inflammatory effects induced by immune complexes might be possible. This may be helpful in the design of improved therapies of autoimmune diseases such as rheumatoid arthritis, in which immune complex-driven neutrophilic inflammation contributes to disease pathogenesis and where neutrophil apoptosis is disturbed.

Melanoma manifests itself from the malignant transformation of melanocytes and represents the deadliest form of skin cancer, being responsible for the disproportionate majority of all skin cancer deaths. The 2002 discovery that 50% of all melanoma patients possess activating BRAF mutations ignited a significant paradigm shift in the way the melanoma field approached research and how patients were treated [1]. The era of targeted therapy had begun and with it came successful targeted BRAF inhibitor therapy regimens, which have accomplished improved clinical benefit (response rate, progression free survival, and overall survival) compared with treatment with chemotherapy in three phase III clinical trials [2]. Although there has been much success in the subgroup of patients whose melanomas harbor activating BRAF mutations, approximately 50% of all melanoma patients do not harbor BRAF mutations. This subgroup of melanoma is composed of ~15-20% of all patients with NRAS mutations and another ~25-30% of patients with neither BRAF nor NRAS mutations. Successful targeted treatment strategies are currently lacking for this subgroup of BRAF-wild type melanomas and therefore novel targeted therapeutic modalities are urgently needed. The work described in this dissertation sheds light on potential approaches for the treatment of BRAF wild type melanoma and will be split into three separate strategies. The first will focus upon the treatment of melanomas without BRAF or NRAS mutations (BRAF/NRAS wild type melanoma) and will expand upon a clinical observation where two melanoma patients were treated with an experimental combination of carboplatin and paclitaxel, with the addition of the AKT inhibitor MK-2206. We demonstrate that the inhibition of AKT significantly enhances the efficacy of chemotherapy in a reactive oxygen species (ROS) mediated fashion, and an induction of autophagy plays a cyto-protective role. The second story focuses upon the treatment of NRAS mutant melanomas by investigating resistance mechanisms to MEK inhibitor treatment. We discovered a MEKi-mediated induction of receptor tyrosine kinase (RTK) signaling to serve as a significant mechanism of escape for NRAS mutant melanomas treated chronically with the MEK inhibitor AZD6244, as well as the recently U.S. Food and Drug Administration (FDA) approved MEK inhibitor trametinib. Novel targeted therapy combinations were then added to overcome the escape from MEK inhibitor therapy. Co-targeting of the receptor tyrosine kinases AXL, PDGFR-β and c-MET with a pan-RTK inhibitor, as well as the mitogen-activated protein kinase (MAPK) pathway with a MEK inhibitor greatly enhanced treatment-induced apoptosis and inhibition of proliferation. The final strategy builds upon the observation that single agent MEK-inhibition is largely ineffective in the treatment of NRAS mutant melanomas. A recovery of MAPK pathway activity in response to MEK inhibition was established to play a significant role in escape of NRAS mutant cells from cell cycle arrest and apoptosis. The combination of a MEK inhibitor with the novel ERK inhibitor VTX-11e prevents the onset of resistant clones and enhances cytotoxicity of the NRAS mutant melanoma cells. This body of work establishes original targeted therapy combinations for the treatment of both NRAS mutant melanomas and BRAF/NRAS wild type melanomas. We propose future clinical investigation with these strategies in the treatment of BRAF wild type melanoma patients in hopes to further extend overall survival.

Le neutrophile humain est une composante essentielle du système immunitaire inné. Il joue un rôle-clé comme phagocyte professionnel pour la défense contre les agents externes. De plus, il a la capacité de libérer un large éventail de produits antimicrobiens et de produire également diverses protéines immunorégulatrices, dont une vaste gamme de cytokines (IL-8, MIP-1[alpha]/[beta], IP-10, I-TAC, TNF-[alpha], etc.). La génération de ces dernières permet le recrutement massif de neutrophiles et d'autres populations leucocytaires au site inflammatoire, contribuant ainsi au bon déroulement de la réponse inflammatoire. La génération de cytokines par le neutrophile humain est induite par différents agonistes, dont des molécules bactériennes (LPS, peptides N-formylés) ou les médiateurs inflammatoires (cytokines, chimiokines, facteurs de croissance). Ces molécules vont activer des récepteurs à la surface du neutrophile et déclancher ainsi plusieurs voies de signalisation et des facteurs transcriptionnels. Dans la présente étude, nous avons déterminé l'impact de la voie de signalisation MEK/ERK dans l'induction de l'expression des cytokines chez le neutrophile humain isolé du sang périphérique. Nous avons noté un découplage du module MEK/ERK suite à une stimulation avec certains agonistes pro-inflammatoires (LPS, TNF-[alpha]), mais par pour d'autres (fMLP, GM-CSF). L'utilisation de différentes classes d'agonistes et d'inhibiteurs pharmacologiques des voies de signalisation nous a permis de mettre en évidence les rôles différents de MEK et de ERK en ce qui concerne la sécrétion et la transcription de cytokines. Les kinases ERK et MEK sont toutes deux impliquées dans la sécrétion de cytokines, mais ERK est la seule des deux qui est associée à la transcription. Par contre, nous n'avons toujours pas identifié la kinase responsable de l'activation de ERK lorsque le module MEK/ERK est découplé. Enfin, à défaut d'identifier la kinase qui phosphoryle ERK, nous montrons que la MAP3K, TAK1, agit en amont de ERK et de MEK chez les neutrophiles. Nos résultats suggèrent que les thérapies basées sur l'inhibition de MEK devront être complémentées d'une inhibition de ERK, en particulier dans des maladies inflammatoires chroniques à forte prédominance neutrophilique.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23079号 / 医博第4706号 / 新制||医||1049(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 稲垣 暢也, 教授 妹尾 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM