Le gliome infiltrant du tronc cérébral (en anglais “diffuse intrinsic pontine glioma”, DIPG) est une tumeur pédiatrique rare et très agressive. La durée moyenne de survie après diagnostic est inférieure à un an. Une caractéristique génétique majeure des DIPG est la mutation de l’histone H3 (H3K27M). L’évolution des connaissances en épigénétique a permis de concevoir des inhibiteurs de régulateurs épigénétiques capables de modifier, voire de contrebalancer, l’effet de cette mutation. Ainsi, le panobinostat (PS), un inhibiteur des histone-désacétylases, diminue la croissance cellulaire et conduit à la mort des cellules de DIPG in vitro et in vivo. Son efficacité est en cours d'évaluation dans des essais cliniques. Mon projet de thèse avait pour objectif de déterminer le rôle d’EBP50 et d’IRSp53, deux protéines spécifiquement dérégulées dans les lignées de DIPG après traitement des cellules par le PS. EBP50 est connue pour intervenir dans la progression tumorale mais sa dualité de fonction, à la fois oncogène et suppresseur de tumeur, nous a conduits à étudier plus précisément son rôle dans les cellules de DIPG. IRSp53 a été peu étudiée dans les cancers solides, bien qu'elle semble jouer un rôle important dans la motilité cellulaire et l’invasion. La diminution de l’expression d’IRSp53 et d’EBP50 par ARN interférence dans des lignées DIPG induit la mort des cellules par apoptose et bloque leur croissance ainsi que leur motilité cellulaire, ce qui suggérerait que ces deux protéines sont oncogéniques dans ce modèle. De plus, la localisation cytoplasmique et nucléaire d’EBP50 semble en accord avec son rôle pro-oncogénique dans les cellules de DIPG. En étudiant in vitro l’effet d’un traitement combinatoire du PS avec des inhibiteurs de l’expression d’EBP50 ou d’IRSp53, j’ai mis en évidence une augmentation de la sensibilité des cellules de DIPG au traitement par le PS. Enfin, j’ai validé le traitement ciblant EBP50 in vivo dans un modèle préclinique d’embryon de poulet. En conclusion, ces deux protéines constituent de nouvelles cibles thérapeutiques dans les DIPG et un moyen d’augmenter l’efficacité du PS. / Diffuse Intrinsic Pontine Glioma (DIPG), is a rare and highly aggressive pediatric tumor. The average survival time after diagnosis is less than one year. A major genetic characteristic of this disease is the mutation of histone H3 (H3K27M). The evolution of knowledge in epigenetics has made it possible to design epigenetic regulatory inhibitors able to modify, or even offset, the effect of this mutation. For example, panobinostat (PS), a histone deacetylase inhibitor, reduces cell growth and induces DIPG cell death, both in vitro and in vivo. Its effectiveness is currently being evaluated in clinical trials. My thesis project aimed at determining the role of two proteins, EBP50 and IRSp53, deregulated in different DIPG cell lines after treatment with PS. EBP50 has already been described as involved in tumor progression but its dual function, both oncogenic and tumor suppressor, has led us to further investigate its role in the DIPG cells. IRSp53 has been poorly studied in solid cancers, though it plays an important role in cell motility and invasion. Down-regulation by RNA silencing of these two proteins in DIPG cell lines induces apoptosis, decreases cell growth and motility, leading us to the hypothesis that these two proteins are oncogenic proteins. In addition, the cytoplasmic and nuclear localization of the EBP50 protein is consistent with its oncogenic role in DIPG cells. Then, I investigated the effect of combinatorial therapy that associates PS with EBP50 or IRSp53 expression inhibitors. My results show an increase in the antitumor effect in vitro for both proteins but also in vivo for EBP50, in a preclinical model, the chicken embryo. In conclusion, these two proteins could be the targets of new treatments for DIPG tumors in combination with PS to enhance its efficacy.

Muscle invasive bladder cancer (MIBC) has a poor prognosis. Currently, therapy consists of radical radiotherapy or cystectomy with or without chemotherapy. The average age of patients with MIBC is high and older patients are less able to tolerate surgery or chemoradiation due to their impaired physical fitness and generally poor renal function. There is an urgent need to find new treatment regimes that are both tolerable and effective. The aims of this project were to investigate the radiosensitising effects of the histone deacetylase (HDAC) inhibitor panobinostat in bladder cancer cell lines, with the ultimate goal of proposing a novel radiosensitising therapy for MIBC, and to study the effects of panobinostat on the major DNA double strand break (DSB) repair pathways, homologous recombination (HR) and non-homologous end joining (NHEJ), to determine the predominant pathway targeted and to look further upstream at effects on the MRE11/RAD50/NBS1 (MRN) complex. The HDAC inhibitor panobinostat was found to be toxic in the low nanomolar range and significant radiosensitising effects were demonstrated at doses lower than IC50 in all the bladder cell lines studied. The radiosensitising effect of panobinostat was not influenced by TP53 status, which is generally regarded as an important determinant of bladder cancer response to radiotherapy. In the “synthetic lethality” bladder cancer cell model, panobinostat predominantly targets the HR pathway, reportedly the only proficient DNA repair pathway in MIBC. HR proteins RAD51 and CtIP were downregulated upon panobinostat treatment in a dose-dependent manner. Upstream, MRE11 and NBS1 proteins were also targeted by panobinostat, with levels slightly decreased in RT112 and T24 cells and in CAL29 cells post-ionising radiation. In summary, the HDAC inhibitor panobinostat was shown to be an efficient radiosensitiser in bladder cancer cells at low toxic doses and to predominantly target the HR pathway. These findings are promising and may contribute towards establishing a novel combination therapy of panobinostat with IR for MIBC patients.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Os mecanismos de ação citotóxica do inibidor de histona deacetilase LBH589 foram investigados em associação com o quimioterápico cisplatina (CDDP) em duas linhagens derivadas de câncer de pulmão de não pequenas células (CPNPC). Os resultados foram analisados em relação ao tipo de morte celular associada às alterações em enzimas relacionadas ao metabolismo energético e a via glicolítica. Para realização do trabalho, foram utilizadas as linhagens tumorais A549 (selvagem para o gene de p53) e Calu-1 (nulo para o gene de p53) tratadas com LBH589 em combinação ou não com CDDP. Foram realizadas curvas de tempo e dose-resposta com as drogas isoladamente pelo ensaio de viabilidade celular (MTT) nas duas linhagens para a escolha das melhores condições para o nosso estudo. As condições dos tratamentos isolados com redução da viabilida celular menores que o IC50 de cada fármaco foram selecionados para realização dos tratamentos combinados. As avaliações de apoptose foram realizadas por citometria de fluxo pelo ensaio de Anexina V/PI, e com a marcação de proteínas por Western Blotting. As proteínas relacionadas a via glicolítica foram avaliadas por Western Blotting e a expressão de RNAm por qPCR. Os resultados demonstraram que o LBH589 combinado a CDDP foi capaz de induzir apoptose em 70% das células (Calu-1) e 54,9% (A549) no tempo de 24 horas, e 90% (calu-1) e 62,1% (A549) em 48 horas, independendo, portanto, do status da p53. Os níveis de expressão de enzimas relacionadas com o metabolismos energético também sofreram alterações nos tratamentos estudados. O LBH589 induziu aumento de cerca de 4x dos níveis de RNAm de HK isoformas I e II em ambas as linhagens. Houve também um aumento na expressão proteica das isoformas de HK I e II. Outras enzimas relacionadas a via glicolítica como PFKP, PKM2 e LDHA foram analisadas e apresentaram redução da expressão proteica, principalmente na presença do LBH589. A combinação da CDDP com LBH589 parece ser promissora para o tratamento de CPNPC induzindo apoptose através de alterações no metabolismo energético tumoral. / The cytotoxic mechanisms of action of the histone deacetylase inhibitor LBH589 was investigated in association with cisplatin in two cell lines derived from non-small lung cancer (NSCLC). The results were analyzed based on the type of the cell death associated with alterations in enzymes related to energy metabolism and glycolytic pathway. To perform the study, we used two non-small lung tumor cell lines, A549 (wild type for the p53 gene) and Calu-1 (null for p53 gene) treated with LBH589 in combination with cisplatin or not. Assays for time and dose responses were performed through the cell viability analysis (MTT) for each drug separately in both cell lines to choose the best conditions for our study. The conditions of isolated treatments with reduced viability lower than the IC50 of each drug were selected for carrying out the combined treatments. Assays for apoptosis detection by flow cytometry were performed by Annexin V / PI, the expression of proteins related to apoptosis by Western Blotting. The expression of glycolytic related proteins were performed by Western blotting and their mRNA expressions by qPCR. The results demonstrated that the combined LBH589 plus CDDP was able to induce apoptosis in 70% of cells -Calu-1- and 54.9% -A549- in 24 hours, and 90% (Calu-1) and 62.1 % (A549) in 48 hours regardless of the p53 status. The expression levels of enzymes related to energy metabolism also presented changes in the studied treatments. The LBH589 increase of about 4x the HK isoforms I and II mRNA levels in both cell lines. There was also an increase in the expression isoforms I and II of HK protein. Other enzymes related to the glycolytic pathway as PFKP, PKM2 and LDHA were analyzed and we observed reduced protein expression, especially in the presence of LBH589. The combination of CDDP with LBH589 appears promising for the treatment of NSCLC inducing apoptosis via alterations in energy metabolism tumor.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Os mecanismos de ação citotóxica do inibidor de histona deacetilase LBH589 foram investigados em associação com o quimioterápico cisplatina (CDDP) em duas linhagens derivadas de câncer de pulmão de não pequenas células (CPNPC). Os resultados foram analisados em relação ao tipo de morte celular associada às alterações em enzimas relacionadas ao metabolismo energético e a via glicolítica. Para realização do trabalho, foram utilizadas as linhagens tumorais A549 (selvagem para o gene de p53) e Calu-1 (nulo para o gene de p53) tratadas com LBH589 em combinação ou não com CDDP. Foram realizadas curvas de tempo e dose-resposta com as drogas isoladamente pelo ensaio de viabilidade celular (MTT) nas duas linhagens para a escolha das melhores condições para o nosso estudo. As condições dos tratamentos isolados com redução da viabilida celular menores que o IC50 de cada fármaco foram selecionados para realização dos tratamentos combinados. As avaliações de apoptose foram realizadas por citometria de fluxo pelo ensaio de Anexina V/PI, e com a marcação de proteínas por Western Blotting. As proteínas relacionadas a via glicolítica foram avaliadas por Western Blotting e a expressão de RNAm por qPCR. Os resultados demonstraram que o LBH589 combinado a CDDP foi capaz de induzir apoptose em 70% das células (Calu-1) e 54,9% (A549) no tempo de 24 horas, e 90% (calu-1) e 62,1% (A549) em 48 horas, independendo, portanto, do status da p53. Os níveis de expressão de enzimas relacionadas com o metabolismos energético também sofreram alterações nos tratamentos estudados. O LBH589 induziu aumento de cerca de 4x dos níveis de RNAm de HK isoformas I e II em ambas as linhagens. Houve também um aumento na expressão proteica das isoformas de HK I e II. Outras enzimas relacionadas a via glicolítica como PFKP, PKM2 e LDHA foram analisadas e apresentaram redução da expressão proteica, principalmente na presença do LBH589. A combinação da CDDP com LBH589 parece ser promissora para o tratamento de CPNPC induzindo apoptose através de alterações no metabolismo energético tumoral. / The cytotoxic mechanisms of action of the histone deacetylase inhibitor LBH589 was investigated in association with cisplatin in two cell lines derived from non-small lung cancer (NSCLC). The results were analyzed based on the type of the cell death associated with alterations in enzymes related to energy metabolism and glycolytic pathway. To perform the study, we used two non-small lung tumor cell lines, A549 (wild type for the p53 gene) and Calu-1 (null for p53 gene) treated with LBH589 in combination with cisplatin or not. Assays for time and dose responses were performed through the cell viability analysis (MTT) for each drug separately in both cell lines to choose the best conditions for our study. The conditions of isolated treatments with reduced viability lower than the IC50 of each drug were selected for carrying out the combined treatments. Assays for apoptosis detection by flow cytometry were performed by Annexin V / PI, the expression of proteins related to apoptosis by Western Blotting. The expression of glycolytic related proteins were performed by Western blotting and their mRNA expressions by qPCR. The results demonstrated that the combined LBH589 plus CDDP was able to induce apoptosis in 70% of cells -Calu-1- and 54.9% -A549- in 24 hours, and 90% (Calu-1) and 62.1 % (A549) in 48 hours regardless of the p53 status. The expression levels of enzymes related to energy metabolism also presented changes in the studied treatments. The LBH589 increase of about 4x the HK isoforms I and II mRNA levels in both cell lines. There was also an increase in the expression isoforms I and II of HK protein. Other enzymes related to the glycolytic pathway as PFKP, PKM2 and LDHA were analyzed and we observed reduced protein expression, especially in the presence of LBH589. The combination of CDDP with LBH589 appears promising for the treatment of NSCLC inducing apoptosis via alterations in energy metabolism tumor.

Hohe Telomeraseaktivität bedingt durch genomische TERT-Rearrangements definiert eine Gruppe an Hochrisiko-Neuroblastompatienten mit ungünstiger Prognose. Das Abzielen auf Telomerase ist ein hochpriorisierter Ansatzpunkt in der Therapie, für die es bislang keine klinisch erfolgreichen Inhibitoren gibt. Der Einsatz von epigenetisch wirksamen Histondeacetylase Inhibitoren (HDACi) stellt dabei eine interessante Therapieoption dar. In TERT-rearrangierten Neuroblastomzellen erzielte die Behandlung mit verschiedenen pan-, Klasse I oder spezifischen HDAC1/2 Inhibitoren eine Supprimierung der TERT mRNA Expression und der Telomeraseaktivität. RNA-Interferenz Studien bestätigten, dass HDAC1 und HDAC2 die TERT Expression positiv regulieren. Die transiente Überexpression von TERT zeigte einen partiellen Rescue des HDACi-bedingten anti-proliferativen Effekts. Der präventive und therapeutische Einsatz von HDACi Panobinostat verlangsamte das Xenografttumorwachstum, die TERT-Expression und Telomeraseaktivität in subkutanen NMRI-Foxn1nu/nu Mausmodellen des TERT-rearrangierten Neuroblastoms bei klinisch relevanten Dosen. Dies zeigt das translationale Potential und die klinische Durchführbarkeit der Panobinostat-Behandlung. ChIP Sequenzierung und Methylierungsanalyse zeigten keine bedeutenden Unterschiede der Histonmodifikationen und der Methylierung von CpG Dinukleotiden am TERT Lokus nach Panobinostatbehandlung. Die Inhibierung der de novo RNA Synthese zeigte, dass die Stabilität des TERT mRNA Transkripts nach Panobinostatbehandlung verringert war. Dies deutet darauf hin, dass die reduzierte Transkriptstabilität der zugrundeliegende molekulare Mechanismus ist. Zusammenfassend konnte gezeigt werden, dass die hohe Telomeraseaktivität in TERT-rearrangierten Neuroblastommodellen durch den Einsatz zugelassener HDACi supprimiert werden kann. / Telomerase activation by genomic TERT-rearrangements defines a subgroup of high-risk neuroblastomas with adverse outcome. Accordingly, telomerase activity presents a high-priority drug target with no currently available clinical inhibitors. It was assessed whether telomerase activity could be inhibited through histone deacetylase (HDAC) inhibition in models of TERT-rearranged neuroblastoma. Treatment with a panel of seven pan-, class I- or specific HDAC1/2 inhibitors suppressed TERT mRNA expression and telomerase activity in TERT-rearranged neuroblastoma cells at clinically achievable concentrations. RNA interference-based studies confirmed that HDAC1 and HDAC2 positively regulate TERT transcript levels. Enforced TERT expression partly rescued the anti-proliferative effect of HDAC inhibition indicating a causal role of TERT suppression in the HDAC inhibitormediated tumor-suppressive phenotype. Panobinostat treatment, in preventive and therapeutic settings, considerably attenuated tumor growth in subcutaneous TERT-rearranged neuroblastoma xenograft models in NMRI-Foxn1nu/nu mice and suppressed TERT transcript levels and telomerase activity at clinically relevant doses, thus demonstrating translational potential and clinical feasibility. ChIP sequencing detected no major differences in the chromatin context of the TERT locus between HDAC inhibitor-treated and control cells. Likewise, HDAC inhibition did not substantially alter the methylation profile in the TERT region. Blocking de novo RNA synthesis, however, reduced TERT mRNA transcript levels in HDAC inhibitor-treated cells, suggesting reduced TERT transcript stability as the underlying molecular mechanism. In summary, high-level telomerase activity caused by genomic rearrangements in neuroblastoma models is suppressed by treatment with clinically approved HDAC inhibitors, suggesting indirect druggability and a potential molecular rationale for therapeutic intervention.