Les lymphomes anaplasiques à grandes cellules (LAGC) sont des lymphomes non-hodgkiniens dits de type T ou nul, représentant la majorité des lymphomes T pédiatriques (20 à 30%). Dans plus de 80% des cas, une translocation chromosomique réciproque aboutissant à l'expression anormale de protéines chimères de type X-ALK qui arborent constitutivement et de manière anormale l'activité tyrosine-kinase ALK (Anaplastic Lymphoma Kinase) est le moteur de la tumorigenèse (LAGC dits "ALK+ "). L'une des particularités de ces lymphomes, mise en évidence par mon équipe, est le fait que B-Cell Lymphoma-2 (BCL-2) demeure indétectable dans les cas ALK+ contrairement aux cas ALK-. Ce point est d'autant plus surprenant que BCL-2, oncogène largement établi comme prototype de protéines anti-apoptotiques ainsi que régulateur clé de l'autophagie, est fortement surexprimé dans la majorité des lymphomes. A l'inverse, Human Antigen R (HuR) est surexprimée dans les LAGC (comme dans la plupart des cancers). Il a été démontré que cette protéine de liaison aux ARN participait au maintien du phénotype tumoral, et que sa localisation subcellulaire et ses fonctions dépendaient étroitement de son statut de phosphorylation, lequel est régulé par ALK dans les LAGC ALK+. Au niveau du cytoplasme, HuR permet de stabiliser et d'augmenter la traduction d'ARNm possédant, dans leur région 3'-UTR, des séquences riches en adénine et uridine (AU-rich elements, "ARE"). De manière plus générale, HuR a la capacité de dialoguer avec les microARNs (miARN), soit en empêchant leur action par compétition, soit à l'inverse en coopérant avec ces derniers et en promouvant ainsi leur fonction de régulateur négatif sur certains transcrits cibles communs. Le transcrit Bcl-2, dont l'expression est réprimée dans les LAGC ALK+, fait partie des cibles potentielles de HuR. Au cours de ma thèse, j'ai ainsi cherché à comprendre les mécanismes moléculaires mis en jeu dans la répression de l'expression de Bcl-2, en me focalisant sur le rôle de HuR et de miARN "partenaires" dans ce processus. Mes données semblent indiquer que ce mécanisme implique le recrutement par HuR du miR-34a sur l'ARNm Bcl-2, conduisant à la mise en silence de ce dernier. A l'inverse, quand l'activité tyrosine- kinase de ALK est inhibée, l'interaction entre HuR et le transcrit Bcl-2 diminue, ce qui limite le recrutement de miR-34a et conduit à une restauration de l'expression de cette oncogène majeur dans les cellules lymphomateuses. Dans le contexte des essais cliniques d'inhibiteurs ciblant l'activité tyrosine kinase de ALK tels que le Crizotinib, la question de cette ré-expression de BCL-2 éclaire d'une lumière nouvelle certains échecs thérapeutiques subis par cette molécule pourtant prometteuse. Je me suis donc également consacré, pendant ma thèse, à l'étude des conséquences de cette ré-expression de BCL-2 sur les LAGC ALK+ traités par le Crizotinib. Les résultats que j'ai obtenus in vitro et in vivo montrent que contrecarrer, par interférence à l'ARN, l'élévation du taux de BCL-2 consécutive au traitement par le Crizotinib, permet de potentialiser les effets de la drogue : cela se traduit en particulier par une potentialisation de la mort par apoptose induite par le traitement mais aussi, de manière fascinante, par une conversion de la réponse autophagique initialement cytoprotectrice et pro-tumorale en une autophagie incontrôlée et délétère, qui participe alors à l'effet thérapeutique accru de la drogue. De manière globale, ce travail permet d'envisager de nouvelles combinaisons et alternatives thérapeutiques pour les patients souffrants de LAGC ALK+, et illustre la complexité des régulations croisées entre processus apoptotiques et autophagiques. / Anaplastic large cell lymphoma (ALCL) are T/-null non-hodgkin lymphoma representing most of childhood T-cell lymphoma (up to 30%). More than 80% of cases bear reciprocal chromosomic translocation responsible for abnormal expression and constitutive activation of X-ALK type (Anaplastic Lymphoma Kinase) chimeric proteins (ALK+ ALCL). A striking characteristic of this lymphoma is that B-Cell Lymphoma-2 (BCL-2) remains undetectable in ALK+ cases compared to ALK- cases. This is all the more surprising as the BCL-2 oncogene, which is firmly established as a prototypic anti-apoptotic factor as well as a key autophagy regulator, has been shown to be overexpressed in a majority of lymphomas. On the other hand, the RNA-binding protein HuR (Human Antigen R) is overexpressed in ALCL (as in most cancers). It has been demonstrated that this protein was involved in the sustainability of the tumoral phenotype, and that its subcellular localization and functions were closely related to its phosphorylation status, which in turn heavily depends on ALK activity in ALK+ ALCL. In the cytoplasm, HuR has the ability to bind adenine and uridine-rich elements (ARE) located on the 3'-UTR of target mRNAs, and both protect them from degradation and increase their translation. From a general point of view, HuR is able to establish an interplay with microRNAs (miRNAs), either blocking them through competition, or actually cooperating with them and thus promote their function of negative regulators of gene expression on common target transcripts. The BCL-2 transcript, which expression seems to be silenced in ALK-expressing ALCL, has been described as a potential target of HuR. During my PhD work, I dedicated myself to understand the molecular mechanism at work in the silencing of BCL-2 expression with a focus on HuR and collaborating miRNA. The data I obtained point at a cooperation between HuR and miR-34a leading to the silencing of the BCL-2 transcript. However, when the ALK tyrosine kinase activity is inhibited, it appears the interaction between the BCL-2 mRNA diminishes, which limitates the miR-34a 's access to this transcript and ultimately results in a re-expression of the BCL-2 oncogene in these lymphoma cells. In the current context of clinical trials for ALK-targeting inhibitors, such as the Crizotinib, this BCL-2 re-expression observed upon ALK inhibition shed light on potential reasons behind some therapeutic failures that have recently been reported. Indeed, during my PhD work, I also studied the consequences of the BCL-2 re-expression observed in Crizotinib-treated cells. The data I obtained in vitro and in vivo show that, by blocking this re-expression using RNA interference, the Crizotinib anti-tumoral efficiency can be greatly potentiated. This potentiation took the form of an increase of apoptotic cell death induction and, interestingly, also affected the autophagic response triggered by the drug, making it switch from a cytoprotective- type, protumoral autophagic flux to an enhanced, deletary-type and tumor suppressive flux, adding to the therapeutic effect of the drug. This work in general provides insights for new therapeutic combinations that could potentially benefit to ALK+ ALCL patients, and illustrates the complex cross-regulations between apoptotic and autophagic pathway.

Severe Traumatic Brain Injury (sTBI) is a major cause of mortality and morbidity. At the Department of Neurosurgery Umeå University Hospital subjects with sTBI are treated with an intracranial pressure (ICP) guided therapy based on physiological principles, aiming to optimise the microcirculation of the brain so avoiding secondary brain injuries. The investigations in this thesis are unique in the sense that all patients with sTBI were treated according to the guidelines of an ICP targeted therapy based on the “Lund concept”. As the treatment is based on normalisation of the ICP, the accuracy and reliability of the measuring device is of outmost importance. Therefore the accuracy, drift, and complications related to the measuring device was prospectively studied (n=128). The drift was 0,9 ± 0,2 mmHg during a mean of 7,2 ± 0,4 days and the accuracy high. No clinical significant complications were noted. In 1997 uni- or bilateral decompressive hemi-craniectomy (DC) was introduced into the treatment guidelines. The effect of DC on the ICP and outcome was retrospectively analysed for subjects with sTBI treated 1998-2001. In the subjects who underwent DC the ICP was 36,4 mmHg immediately before and 12,6 mmHg immediately after the DC. The ICP then levelled out at just above 20 mmHg. The ICP was significant lower during the 72 hours following DC. The outcome did not differ between subjects who had undergone DC or not. Subclinical electroencephalographic seizures and status epilepticus have been reported to be common in subjects treated for traumatic brain injury (TBI). This can negatively influence the outcome giving rise to secondary brain injuries. The occurrence of seizures in subjects treated for TBI using continuous EEG monitoring was therefore prospectively studied. During 7334 hours of EEG recording in 47 patients no electroencephalographic seizures were observed. Theoretically, and based on animal studies, prostacyclin (PGI2) can improve the microcirculation of the brain, decreasing the risk for secondary ischaemic brain injury. PGI2 was introduced to the treatment in a prospective randomised double blinded study (epoprostenol 0,5 ng/kg/min). The effect of PGI1 pkt was analysed using the lactate/pyruvate ratio (L/P) measured by cerebral microdialysis in order to study the energy metabolism in the brain. The outcome was measured as Glasgow Outcome Scale (GOS) at 3 months follow-up. Forty-eight subjects were included. The L/P was pathological high during the first day, thereafter decreasing. There was no significant difference in L/P or outcome between the treated and non-treated group. At 3 months the mortality was 12,5% (95,8% was discharged alive from the ICU), and favourable outcome (GOS 4-5) was 52%. In the same study the brain injury biomarkers S-100B and NSE were followed twice a day for five days to evaluate brain injury and investigate the possible use of these biomarkers for outcome prediction. Initially the biomarkers were elevated to pathological levels which decreased over time. The biomarkers were significant elevated in subjects with Glasgow Coma Scale 3 (GCS) and GOS 1 compared with subjects with GCS 4-8 and GOS 2–5, respectively. A correlation to outcome was found but this correlation could not be used to predict clinical outcome. It is concluded that the ICP measurements are valid and the treatment protocol is a safe and solid protocol, yielding among the best reported results in the world, in regard to favourable outcome as well as in regard to mortality. Epoprostenol in the given dose was not shown to have any effects on the microdialysis parameters nor the clinical outcome. In sTBI L/P and brain injury biomarkers can not be used to predict the final outcome.

EGFR is a transmembrane tyrosine kinase receptor from the HER family which, upon ligand stimulation, activates different signaling pathways involved in tumorogenesis. EGFR can be targeted by monoclonal antibodies, as cetuximab and panitumumab, which bind to EGFR preventing ligand stimulation of the receptor. Cetuximab and panitumumab are approved for colorectal cancer treatment. However, its clinical success is uniformily limited by the development of acquired drug resistance. We describe a new mechanism of acquired resistance to cetuximab in colorectal cancer that was due to a missense mutation in the EGFR ectodomain (S492R mutation). Upon chronic exposure to cetuximab, colorectal cancer cell lines acquired S492R mutation and became resistant to the treatment. We observed that cetuximab was not able to bind mutant EGFR. Notably, this amino acid change did not affect the ability of panitumumab to bind to EGFR, and panitumumab effectively suppressed growth of mutant cells. EGFRS492R mutation was detected in 2 out of 10 tumor specimens from patients following progression on cetuximab. One of these patients was subsequently treated with single agent panitumumab yielding a partial response. The S492R mutation defines a novel biomarker of resistance to cetuximab but not to panitumumab in colorectal cancer / EGFR és un receptor transmembrana tirosina cinasa de la família HER el qual, després de l’estimulació mitjançant lligands, activa vies de senyalització involucrades en processos tumorogènics. L’EGFR es pot inhibir amb anticossos monoclonals, com cetuximab i panitumumab, que s’uneixen al receptor prevenint-ne l’activació per part dels lligands. Cetuximab i panitumumab estan aprovats per al tractament del càncer colorectal, però el seu ús es veu limitat per el desenvolupament de resistència adquirida al tractament. Nosaltres describim un mecanisme de resistència adquirida a cetuximab en càncer colorectal degut a l’adquisió d’una mutació en el domini extracel•lular de l’EGFR, la mutació S492R. Durant l’exposició crònica a cetuximab, linies cel•lulars de càncer colorectal van adquirir la mutació S492R tornat-se resistents al tractament. Cetuximab no era capaç d’unir-se a l’EGFR mutat. Aquests canvi d’aminoàcid no afectava a l’habilitat que té panitumumab a unir-se al EGFR, pertant, panitumumab suprimia el creixement de les cèl•lules tumorals mutades. Vam detectar la mutació EGFRS492R en 2 de 10 mostres tumorals de pacients que havien recaigut al tractament amb cetuximab. Un d’aquest pacients va ser posteriorment tractat amb panitumumab obtenint-ne una resposta tumoral parcial. La mutació S492R defineix un nou mecanisme de resistència a cetuximab però no a panitumumab en el tractament del càncer colorectal.

O sarcoma de Ewing (SE) é um dos mais agressivos tipos de câncer pediátrico. Apesar dos significativos avanços no tratamento dessa doença, ainda há uma grande necessidade no aumento das taxas de cura, redução da toxicidade quimioterápica e redução da resistência ao tratamento. Tem sido proposto que SE provém de precursores neuronais, podendo ter sua fisiologia afetada, pois, por neurotrofinas (NTs). Examinamos a influência de receptores de NTs (Trks) em SE. Foram avaliadas a expressão proteica de NTs (NGF e BDNF) e seus receptores (TrkA e TrkB, respectivamente) em amostras de tumores de pacientes com SE, e a expressão de mRNA nas linhagens celulares RD-ES e SK-ES-1. O tratamento das linhagens com o pan-inibidor de Trks (K252a) modificou a morfologia celular e diminuiu a expressão de mRNA de NGF, TrkA, BDNF e TrkB. Ainda, a inibição de Trks diminuiu drasticamente a proliferação e capacidade clonogênica celular. Efeitos sinérgicos foram observados quando as células foram tratadas em conjunto com baixas doses de quimioterápicos, tanto em células selvagens de SE, quanto nas quais induzimos quimiorresistência. Esse estudo sugere, pela primeira vez, que a inibição de Trks reduz a proliferação e sobrevivência celular em SE, além de aumentar a sensibilidade ao tratamento quimioterápico. / Ewing's sarcoma (ES) is one of the most aggressive types of pediatric cancer. Despite significant advances in the treatment of this disease, there is still a great need in increasing cure rates, reducing chemotherapy toxicity and treatment resistance. It has been proposed that ES might derive from neuronal precursors and may be influenced, therefore, by neurotrophins (NTs). We have examined the influence of Trk neurotrophin receptors in ES. Protein expression of NTs (NGF and BDNF) and their receptors (TrkA, and TrkB, respectively) was detected in tumor samples from patients with ES, and mRNA expression was analyzed in the RD-ES, SK-ES-1 cell lines. Treating cells with a Trk Pan-inhibitor (K252a) altered cell morphology and decreased the mRNA expression of NGF, TrkA, BDNF, and TrkB. In addition, Trk inhibition dramatically decreased cell proliferation and clonogenic capacity. Synergistic effects were observed when cells were treated in combination with low doses of cytotoxic chemotherapeutics, both in normal ES cells and cells in which chemoresistance was induced. The results suggest for the first time that Trk inhibition can reduce the proliferation and survival of ES cells and sensitize them to cytotoxic chemotherapy.

O sarcoma de Ewing (SE) é um dos mais agressivos tipos de câncer pediátrico. Apesar dos significativos avanços no tratamento dessa doença, ainda há uma grande necessidade no aumento das taxas de cura, redução da toxicidade quimioterápica e redução da resistência ao tratamento. Tem sido proposto que SE provém de precursores neuronais, podendo ter sua fisiologia afetada, pois, por neurotrofinas (NTs). Examinamos a influência de receptores de NTs (Trks) em SE. Foram avaliadas a expressão proteica de NTs (NGF e BDNF) e seus receptores (TrkA e TrkB, respectivamente) em amostras de tumores de pacientes com SE, e a expressão de mRNA nas linhagens celulares RD-ES e SK-ES-1. O tratamento das linhagens com o pan-inibidor de Trks (K252a) modificou a morfologia celular e diminuiu a expressão de mRNA de NGF, TrkA, BDNF e TrkB. Ainda, a inibição de Trks diminuiu drasticamente a proliferação e capacidade clonogênica celular. Efeitos sinérgicos foram observados quando as células foram tratadas em conjunto com baixas doses de quimioterápicos, tanto em células selvagens de SE, quanto nas quais induzimos quimiorresistência. Esse estudo sugere, pela primeira vez, que a inibição de Trks reduz a proliferação e sobrevivência celular em SE, além de aumentar a sensibilidade ao tratamento quimioterápico. / Ewing's sarcoma (ES) is one of the most aggressive types of pediatric cancer. Despite significant advances in the treatment of this disease, there is still a great need in increasing cure rates, reducing chemotherapy toxicity and treatment resistance. It has been proposed that ES might derive from neuronal precursors and may be influenced, therefore, by neurotrophins (NTs). We have examined the influence of Trk neurotrophin receptors in ES. Protein expression of NTs (NGF and BDNF) and their receptors (TrkA, and TrkB, respectively) was detected in tumor samples from patients with ES, and mRNA expression was analyzed in the RD-ES, SK-ES-1 cell lines. Treating cells with a Trk Pan-inhibitor (K252a) altered cell morphology and decreased the mRNA expression of NGF, TrkA, BDNF, and TrkB. In addition, Trk inhibition dramatically decreased cell proliferation and clonogenic capacity. Synergistic effects were observed when cells were treated in combination with low doses of cytotoxic chemotherapeutics, both in normal ES cells and cells in which chemoresistance was induced. The results suggest for the first time that Trk inhibition can reduce the proliferation and survival of ES cells and sensitize them to cytotoxic chemotherapy.

La mise en évidence de la variabilité pharmacocinétique et/ou pharmacodynamique permet l’optimisation de l’utilisation des cytotoxiques. L’association des thérapies ciblées à la chimiothérapie conventionnelle peut apporter des avantages supplémentaires en termes de bénéfice thérapeutique, mais aussi peut provoquer des interactions médicamenteuses et augmenter les variabilités interindividuelles. Les interactions médicamenteuses sont maintenant connues pour avoir un impact significatif sur l'élimination des médicaments.Le premier volet de ces travaux consiste à mesurer les concentrations sanguines de deux molécules (le lapatinib et la vinorelbine) chez les patientes et à les utiliser pour la modélisation pharmacocinétique. Cette modélisation a clairement montré l’influence du poids et du nombre des plaquettes sur la pharmacocinétique de la vinorelbine permettant ainsi de diminuer les variabilités pharmacocinétiques. Elle a également mis en évidence l’influence du lapatinib sur la pharmacocinétique de la vinorelbine. Cependant, compte tenu de l’absence de groupe témoin, nous n’avons pas réussi à obtenir une significativité statistique pour ces résultats. En parallèle, aucune influence de la vinorelbine sur le comportement pharmacocinétique du lapatinib n’a été mise en évidence.Le second volet concerne la modélisation de la réponse et de la tolérance des patientes pour cette association médicamenteuse (modélisation pharmacodynamique). La neutropénie est la toxicité dose-limitante et comme le lapatinib n’est pas connu pour être myélotoxique, nous avons modélisé cette toxicité par rapport au comportement pharmacocinétique de la vinorelbine. Là encore, nous avons observé une très forte tendance quant à l’influence du lapatinib sur la myélotoxicité de la vinorelbine. Concernant l’efficacité de cette association, la modélisation de l’évolution de la croissance tumorale a mis en évidence une synergie d’action entre ces deux molécules. A notre connaissance c’est la première fois qu’une telle modélisation tant du point de vue de la tolérance que de celui de la réponse, est réalisée lors d’une association de chimiothérapie classique et d’une thérapeutique ciblée.La modélisation PK-PD de population des médicaments anticancéreux peut apporter une aide précieuse aux cliniciens dans ce domaine. Elle peut également être essentielle dans la prise de décision clinique précoce. / The identification of sources of pharmacokinetic and / or pharmacodynamic variabilities, optimizes the use of cytotoxic agents. The combination of targeted therapies with conventional drugs may provide additional benefits in terms of therapeutic benefit, but also can cause drug interactions and increased variability. Drug interactions are known to have a significant impact on drug disposition.The first part of this work is to measure blood concentrations of two molecules (lapatinib and vinorelbine) in patients and to perform pharmacokinetic modeling. This modeling clearly showed the influence of weight and the platelet number on the pharmacokinetics of vinorelbine reducing thereby the pharmacokinetic variabilities. It also highlighted the influence of lapatinib on the pharmacokinetics of vinorelbine. However, due to the lack of control group, we failed to obtain statistical significance for these results. In parallel, no effect of vinorelbine on the pharmacokinetics of lapatinib has been detected.The second part concerns the modeling of the response and tolerance of patients for this drug combination (pharmacodynamic modeling). Neutropenia was detected as the dose-limiting toxicity of the combination and the fact that lapatinib is not known to be myelotoxic, we performed toxicity modeling based on the pharmacokinetics of vinorelbine. Again, we observed a strong tendency on the influence of lapatinib on the myelotoxicity of vinorelbine. Regarding the efficacy of this combination, tumor growth modeling showed a synergistic action between the two molecules. To our knowledge this is the first time that such a model, both in terms of tolerance and response, is implemented in a combination of conventional chemotherapy and targeted therapy.Population PK-PD modeling of cancer drugs can provide valuable assistance to clinicians in this field. It can also be essential in the early clinical decision making.

Les facteurs de transcription inducteurs de la transition épithélio-mésenchymateuse (EMT) sont fréquemment réactivés de manière aberrante dans de nombreux cancers. Dans les carcinomes, ils favorisent dans les étapes précoces le développement tumoral en inhibant les systèmes de sauvegarde tels que la sénescence ou l'apoptose et à terme dans les étapes finales, promeuvent la formation de métastases. Dans le cadre du mélanome nous avons mis en évidence des fonctions antagonistes de ces facteurs : il était connu que ZEB2 et SNAIL2 sont impliqués dans la délamination de la crête neurale et la détermination mélanocytaire mais de manière inattendue ZEB2 et SNAIL2 sont aussi exprimés dans les mélanocytes adultes normaux et leur expression est diminuée au cours de la progression maligne, au profit des facteurs ZEB1 et TWIST1. Ce changement de profil d'expression est un facteur de mauvais pronostic pour les patients. Mes travaux ont permis de montrer que cet effet antagoniste passe par la régulation de MITF, le facteur clef du développement mélanocytaire. ZEB2 et SNAIL2 exercent une fonction oncosuppressive en activant l'expression de MITF et en promouvant la différenciation mélanocytaire. Au contraire, ZEB1 et TWIST1 jouent un rôle oncogénique en inhibant MITF et favorisent l'acquisition de propriétés de cellules souches. J'ai poursuivi l'étude de la fonction oncogénique de Twist1 in vivo dans un modèle murin de mélanome induit par l'oncogène BRAFV600. J'ai ainsi mis en évidence que l'expression conjointe de Twist1 et de BRAFV600 entraîne la formation de mélanomes agressifs, dédifférenciés et invasifs. Enfin, j'ai analysé et caractérisé l'implication de ces facteurs dans le processus de résistance aux thérapies ciblées anti-BRAF dans les mélanomes BRAFV600E. J'ai démontré que ZEB1 peut contribuer à la résistance aux inhibiteurs de BRAF. En effet, l'expression de ZEB1 est augmentée dans des cellules de mélanomes résistantes (innée ou acquise), par rapport aux cellules sensibles. Tandis que l'expression de ZEB1 favorise l'émergence de cellules résistantes, cibler ZEB1 augmente la sensibilité des cellules à l'inhibiteur de BRAF et resensibilise les cellules résistantes, mettant en évidence l'intérêt de cette combinaison d'un point de vue thérapeutique / Embryonic transcription factors inducers of Epithelial to Mesenchymal Transition (EMT-TFs) are frequently and aberrantly reactivated in many cancers. In carcinomas they promote, in early stage, tumor development by inhibiting the failsafe programs of the cell like senescence and apoptosis, and in final stages, they promote metastases formation. In melanoma we highlighted antagonist’s functions of these factors: it was known that ZEB2 and SNAIL2 were involved in delamination of neural crest cells and melanocytes determination but unexpectedly, ZEB2 and SNAIL2 are also expressed in normal adult melanocytes and their expression is decreased during malignant progression, for the benefit of ZEB1 and TWIST1 factors. This change in expression profiling is a factor of poor prognosis for patients. The results I provide show that this antagonistic effect might go through the regulation of MITF, the key factor in the development of melanocytic. ZEB2 and SNAIL2 exert an oncosuppressive function by activating the expression of MITF and promoting melanocytic differentiation. On the contrary, ZEB1 TWIST1 plays an oncogenic role in inhibiting MITF and promotes the acquisition of stem cells properties. In order to go deeper in the study of TWIST1 oncogenic functions, I used an in vivo Melanoma murine model induced by the BRAFV600 oncogene. I thus put in evidence that the combined expression of TWIST1 and BRAFV600 leads to the formation of aggressive, dedifferentiated and invasive melanoma. Finally, I analyzed and characterized the involvement of these factors in the process of resistance to targeted therapies against BRAFV600E melanomas. I demonstrated that ZEB1 may contribute to BRAF inhibitors resistance. Indeed, ZEB1 expression is increased in resistant (innate or acquired) melanomas cells, compared to sensitive cells. While ZEB1 expression promotes the emergence of resistant cells, targeting ZEB1 increases the sensitivity to BRAF inhibitor and sensitizes resistants cells, highlighting the interest of this combination from a therapeutic point of view

Le rétinoblastome est une tumeur maligne de la rétine qui touche essentiellement les nourrissons et jeunes enfants. Sa prise en charge est associée à la survenue d’effets secondaires sévères, certains traitements induisant le développement de tumeurs secondaires. Dans ce contexte, la thérapie photodynamique (PDT) apparaît comme une alternative prometteuse, car elle est non mutagène et génère des effets secondaires moins importants. Elle consiste à injecter un agent photosensibilisateur (PS) - une porphyrine par exemple – puis à illuminer la zone tumorale avec un laser. L'efficacité de la PDT nécessite l'accumulation de PS dans la tumeur. Cependant, la plupart des porphyrines sont hydrophobes et s'agrègent en milieu aqueux. Leur incorporation dans un nano-vecteur peut améliorer leur distribution au cytoplasme. Malheureusement, lorsqu'elles sont encapsulées dans le cœur des nanoparticules, les molécules de PS perdent leur phototoxicité en raison de leur auto-extinction. Dans ce travail, nous avons conçu des lipo-nanoparticules biodégradables (LNP) constituées d'une nanoparticule (NP) de poly (D,L)-lactide (PLA) recouverte d'une bicouche de phospholipides (POPC-DOTAP). Un principe actif anticancéreux, la bêta-lapachone et un agent photosensibilisateur ont ensuite été co-encapsulés dans notre système en vue de favoriser un effet synergique sur le rétinoblastome. Nous avons démontré la formation effective des LNPs et leur internalisation dans les cellules de rétinoblastome en quelques heures.Enfin, nous avons démontré une amélioration de l'activité antitumorale en combinant les deux traitement dans notre système par rapport au traitement simple par PDT ou chimiothérapie. / Retinoblastoma is a malignant tumor of the retina in infants. Conventional therapies are associated to severe side effects and some of them induce secondary tumors. Therefore, photodynamic therapy (PDT) appears as a promising alternative as it is non-mutagenic and generates minimal side effects. It consists in injection of a photosensitizer (PS) like a porphyrin, and then illumination of the tumor area with a laser. The effectiveness of PDT requires the accumulation of the PS in the tumor. However, most porphyrins are hydrophobic and aggregate in aqueous medium. Their incorporation into a nanocarrier may improve their delivery to the cytoplasm. Unfortunately, when incorporated into a nanoparticle core, PS molecules lose their phototoxicity due to self-quenching. In this work, we have designed biodegradable liponanoparticles (LNPs) consisting of a poly(D,L)-lactide (PLA) nanoparticle (NP) coated with a phospholipid (POPC/DOTAP) bilayer. An anticancer drug, beta-lapachone (β-Lap), and a photosensitizer were then co-encapsulated in these LNPs for achieving synergistic effect on retinoblastoma. We have first demonstrated the effective formation of the LNPs and their internalization in retinoblastoma cells within few hours. Then we studied the cyto/phototoxicity of the system.The hybrid nanoparticles showed an improved antitumor activity when the PS and β-Lap were combined, compared to the single treatment by PDT or chemotherapy.

L’histiocytose langerhansienne (HL) est la plus fréquente des histiocytoses, liée à l’accumulation de cellules pathologiques de phénotype langerhansien. La découverte de la mutation somatique BRAFV600E dans environ 50% des cas à ouvert un nouveau champ d’investigation pour tirer bénéfice de ce statut moléculaire pour la prise en charge des patients.Tout d’abord, nous avons montré l’efficacité des inhibiteurs de BRAF sans rapporter de résistance dans les formes actives d’HL, en particulier dans les formes multisystémiques avec atteinte des organes à risque (MS OR+) du nourrissons, confirmant le rôle driver de cette mutation dans l’HL. Toutefois, après l’arrêt du traitement administré durant 2 à 6 mois, de nombreuses récidives ont été constatées.Ensuite, nous avons montré que pour les enfants atteints d’HL, la mutation BRAFV600E était significativement associée aux formes MS OR+, retrouvée dans 87,8% de ces cas. Comparés aux patients non mutés BRAF, les patients avec la mutation BRAFV600E présentaient un taux de résistance plus élevé à la chimiothérapie de première ligne velbé - corticoïde (21,9% contre 3,3%), un taux plus élevé de réactivation à 5 ans (42,8% contre 28,1%) et un taux de séquelles supérieur (27,9% contre 12,6%).Par ailleurs, nous avons montré que, pour les HL BRAFV600E mutées, la quantification de BRAFV600E dans l’ADN libre circulant par PCR digitale en gouttelette était un biomarqueur pertinent pour les cas d’HL MS OR+ et les présentations résistantes au traitement de première ligne.Enfin, après un criblage de points chauds mutationnels d’une série d’échantillons tissulaires d’HL ayant permis de mettre en évidence un cas avec la mutation somatique PIK3CAE542K, 9 couples d’échantillons tumeur/constitutionnel ont été étudiés par séquençage d’exome. Cela nous a permis de mettre en évidence une mutation récurrente (n=2) de BRAF au niveau du site d’épissage 5’ (donneur) de l’intron 12. Selon l’analyse de l’ARN, cette mutation conduirait à l’insertion de 3 acides aminés (LLR) dans le domaine kinase de la protéine mutée, dont l’analyse fonctionnelle est en cours. / Langerhans cell histiocytosis (LCH) is the most common type of histiocytosis owing to accumulation of pathologic CD1a+ CD207+ histiocytes. The identification of BRAFV600E in more than half of patients with LCH has launched a new field of investigation to study potential patient’s management benefits and implications from this molecular status.First, in BRAFV600E mutated LCH, we reported the effectiveness of BRAF inhibitors. Efficacy with no resistance to vemurafenib was reported in all cases with active LCH disease, especially for multi-system LCH with risk organ (MS RO+) involvement, confirming the driver status of this mutation in LCH. However, after discontinuation of this treatment administered during 2-6 months, many recurrences were observed.Then, we showed that children with BRAFV600E mutated LCH manifested more severe disease, comprised 87.8% of patients with MS RO+ involvement. Compared to patients with wild-type BRAF, patients with BRAFV600E more commonly displayed resistance to combined vinblastine and corticosteroid therapy (21.9% vs. 3.3%), showed a higher 5-year reactivation rate (42.8% vs. 28.1%) and had more long-term permanent consequences (27.9% vs. 12.6%).Moreover, we showed that BRAFV600E quantification in circulating cell-free DNA by droplet digital PCR is a relevant biomarker to monitor response to therapy for MS RO+ LCH and RO- LCH children who failed to respond to first line chemotherapy.Finally, after the screening of LCH biopsy (n=86) for the BRAF, KRAS, NRAS and PI3KCA most common mutations, leading to highlight one case with the PIK3CAE542K somatic mutation, 9 paired tumor-normal samples from children with LCH were analyzed by whole exome sequencing. Data showed a new BRAF recurrent mutation (n=2) in the 5′ splice sites of the intron 12. According to RNA analysis, this mutation would lead to the insertion of 3 amino acids (LLR) in the smaller N-terminal lobe of the BRAF kinase domain. Functional analysis is ongoing.

Background: Aberrant MET tyrosine kinase signaling is known to cause cancer initiation and progression. While MET inhibitors are in clinical trials against several cancer types, the clinical efficacies are controversial and the molecular mechanisms toward sensitivity remain elusive. Methods: With the goal to investigate the molecular basis of MET amplification (MET amp ) and hepatocyte growth factor (HGF) autocrine-driven tumors in response to MET tyrosine kinase inhibitors (TKI) and neutralizing antibodies, we compared cancer cells harboring MET amp (MKN45 and MHCCH97H) or HGF-autocrine (JHH5 and U87) for their sensitivity and downstream biological responses to a MET-TKI (INC280) and an anti-MET monoclonal antibody (MetMab) in vitro, and for tumor inhibition in vivo. Results: We find that cancer cells driven by MET amp are more sensitive to INC280 than are those driven by HGF-autocrine activation. In MET amp cells, INC280 induced a DNA damage response with activation of repair through the p53BP1/ATM signaling pathway. Although MetMab failed to inhibit MET amp cell proliferation and tumor growth, both INC280 and MetMab reduced HGF-autocrine tumor growth. In addition, we also show that HGF stimulation promoted human HUVEC cell tube formation via the Src pathway, which was inhibited by either INC280 or MetMab. These observations suggest that in HGF-autocrine tumors, the endothelial cells are the secondary targets MET inhibitors. Conclusions: Our results demonstrate that MET amp and HGF-autocrine activation favor different molecular mechanisms. While combining MET TKIs and ATM inhibitors may enhance the efficacy for treating tumors harboring MET amp , a combined inhibition of MET and angiogenesis pathways may improve the therapeutic efficacy against HGF-autocrine tumors.