Toxoplasmosis is an opportunistic disease caused by the protozoan parasite Toxoplasma gondii. The parasite is usually staved off by a healthy immune system and remains dormant in the body. In immunocompromised patients, the parasite can become active and spread throughout the body causing symptoms such as encephalitis, cognitive disorders, seizures, and death. Combination drug therapy is the usual treatment for toxoplasmosis; however, patients suffer from problems of intolerance, allergic reactions, and cytotoxicity. In an effort to identify new drug targets for toxoplasmosis, a series of compounds have been synthesized that can be used as tools to probe the unique pathways used by T. gondii to survive in the human host. One class of these compounds is pyridinyl imidazoles, which have been shown to be active against T. gondii MAP kinases. To set up a protein pull down assay, a biotinylated linker was synthesized. We have also synthesized a compound that’s being used to study the pathways involved in the most active and proliferative form of T. gondii.

Le cancer de la prostate est le plus fréquent chez l’homme et se place au 3ème rang des décès par cancer. Les formes métastatiques sont traitées par une castration centrale à base d’agonistes ou d’antagonistes de la LH-RH avec une efficacité limitée, puis avec une chimiothérapie à base de taxanes, ou une hormonothérapies de deuxième génération (abiratérone et enzalutamide) avec une espérance de vie qui ne dépasse pas 36 mois en phase de résistance à la castration. Dans le but d’identifier de nouvelles alternatives, de nombreuses classes thérapeutiques ont été testées, en particulier les inhibiteurs de kinases (IK) avec des résultats décevants. Or, de plus en plus d’IK arrivent sur le marché et il n’existe toujours pas de rationnel préclinique fort pour choisir les molécules à tester dans les futurs essais. Notre travail de Thèse vise à identifier des marqueurs prédictifs de la réponse aux IK dans des modèles de cancer de prostate pour orienter ce choix. Il repose sur une approche in silico utilisant les banques de données du panel des 60 lignées cancéreuses du NCI pour rechercher des corrélations entre l’expression de 92 gènes caractéristiques de la résistance à la castration et la sensibilité aux IK testés dans ce panel. Nous avons mis en évidence une signature de 20 gènes dont l’expression était corrélée à la réponse au vémurafénib et au sélumétinib ciblant respectivement RAF et MEK. Nous avons validé ces corrélations au niveau fonctionnel pour 11 d’entre eux dans les lignées DU145, 22RV1 et LNCaP, leur répression modulant la sensibilité des cellules à ces inhibiteurs. En particulier, la répression du gène ETV1, remanié dans 10% des cancers de prostate, confère une hypersensibilité au vémurafénib et au sélumétinib. En absence de traitement, la répression d’ETV1 induit un blocage en phase G2/M du cycle cellulaire qui est accompagné d’une diminution de la phosphorylation des kinases p38 (et d’une de ses cibles, HSP27) et AKT. En revanche, elle n’entraîne aucune altération de la voie des MAPK, suggérant que le phénotype d’hypersensibilité serait liée à un défaut d’autres voies de signalisation, notamment à une augmentation de l’apoptose que l’on observe dans les cellules 22RV1 traitées au vémurafénib. Parmi les gènes dont l’expression est régulée par ETV1 (identifiés par une approche in silico croisant des données de microarray, de Chip-Seq, et de corrélation dans le panel), nous avons recherché ceux qui pouvaient jouer un rôle dans l’hypersensibilité au vémurafénib des lignées où ETV1 est réprimé. Plusieurs candidats ont été identifiés notamment les gènes de la famille DUSP régulant la phosphorylation de p38. Ces gènes sont en cours de validation. Un volet plus « translationnel » de notre travail a consisté à évaluer, sur la base de nos résultats, des combinaisons de drogues entre inhibiteurs de kinase et inhibiteurs d’ETV1 (YK-279 et BRD32048) ou enzalutamide. Contrairement à nos attentes, aucune sensibilisation au vémurafénib n’a été observée en présence des inhibiteurs d’ETV1. En revanche le vémurafénib potentialise la réponse des cellules LNCaP à l’enzalutamide alors que cette association est antagoniste dans les cellules 22RV1, soulignant l’importance du contexte génétique pour l’utilisation de cette association (lignée 22RV1 : BRAF muté, expression du variant AR-V7, PTEN sauvage ; lignée LNCaP : PTEN muté).Notre travail a donc permis d’identifier une signature de réponse au vémurafénib pour prédire une réponse à cet IK actuellement en essai clinique dans les tumeurs de prostate BRAF mutées. Il a également permis pour la première fois d’établir le rôle fonctionnel d’ETV1 dans la réponse cellulaire au vémurafénib, données qui peuvent être transposées à d’autres types tumoraux où les inhibiteurs de RAF sont utilisés en routine clinique. / Prostate cancer is the most frequent in men and is the third leading cause of death by cancer. Metastatic form of the disease is treated by central castration using LH-RH agonist or antagonists with a limited efficacy, followed by taxane-based chemotherapy or second generation hormone therapies such as abiraterone acetate or enzalutamide. However, overall survival never exceeds 36 months when tumors become resistant to castration. In the search for new alternatives, numerous anticancer drugs from different classes have been tried, in particular kinase inhibitors (KI) but with desapointing results. Yet, increasing number of KI are being approved and there is still no strong preclinical rational to decide which molecule to test in further clinical trials. Our thesis was intended to identify predictive markers to the response to KI in prostate cancer cell models to orient this choice. It relies on an in silico approach based on the use of the NCI60 cell line panel to search for correlations between the expression of 92 genes that are characteristics of castration resistance and the sensitivity to KI that have been tested in the panel. We identified a 20 genes’ signature the expression of which is correlated to the sensitivity to vemurafenib and selumetinib targeting respectively RAF and MEK. We validated these correlations at the functional level for 11 of them in DU145, 22RV1 and LNCaP cell lines, their repression leading to altered sensitivity to these two inhibitors. In particular, repression of the ETV1 transcription factor that is rearranged in 10% of prostate tumors, sensitized cells to vemurafenib and selumetinib. In the absence of drug, ETV1 repression induced a G2/M blockage that was accompanied by a reduced phosphorylation of p38 (and its downstream substrate HSP27) and AKT kinases. Conversely, it did not altered the MAPK pathway, suggesting that hypersensitivity to vemurafenib and selumetinib could be linked to a deregulation of other signaling pathways, in particular to increased apoptosis that was observed in ETV1-repressed 22RV1 cells following treatment with vemurafenib. Among the genes that are regulated by ETV1 (that were identified by an in silico approach using microarray, Chip-Seq and correlations from the NCI60 panel), we identified several candidates that could be involved in the sensitization to vemurafenib when ETV1 is repressed, including genes from the DUSP family that are known to regulate p38 phosphorylation. These genes are currently being validated. A translational part of our thesis was to evaluate drug combinations between kinase inhibitors and either ETV1 inhibitors (YK-279 et BRD32048) or enzalutamide. Unexpectedly, we did not observed a sensitization of prostate cancer cells to vemurafenib by ETV1 inhibitors. Interestingly, we found that vemurafenib could potentiate enzalutamide cytotoxicty in LNCaP cells whereas this association had antagonistic effects in 22RV1 cells, pointing towards the importance of the genetic background to use such associations (BRAF mutated, AR-V7 expression and wild-type PTEN for 22RV1 ; mutated PTEN for LNCaP)Our thesis work has allowed the identification of a predictive signature of response to vemurafenib that is currently tested in BRAF mutated prostate tumors. It also allowed to establish for the first time a functional role of ETV1 in cell response to vemurafenib. These data could potentially being transposed to other cancer types where BRAF inhibitors are routinely used in the clinic.

Avec près de 150 000 décès estimés en 2012 d’après l’Agence internationale pour la Recherche sur le Cancer, le cancer représente la première cause de mortalité en France. Cette maladie est caractérisée par la prolifération anarchique et incontrôlée de certaines cellules de l’organisme qui échappent aux mécanismes de contrôle. A l’heure actuelle, les thérapies anticancéreuses visent principalement ces cellules tumorales en agissant sur des protéines qu’elles surexpriment, telles que les récepteurs aux facteurs de croissance à activité tyrosine kinase.Nos travaux se sont essentiellement portés sur quatre de ces récepteurs : l’EGFR (Epidermal Growth Factor Receptor), le VEGFR (Vascular Endothelial Growth Factor Receptor), le PDGFR (Platelet-Derived Growth Factor Receptor) et le récepteur c-Kit. Plusieurs hétérocycles azotés (quinazoline, benzotriazine, thiénopyrimidine) se différenciant par leur motif anilino ou aryloxy en position 4 ont été conçus, synthétisés et évalués pharmacologiquement. Parmi ces produits, les 4-aryloxyquinazolines substituées en position 7 par des chaînes aminoalkoxy se sont révélées être de puissants inhibiteurs des récepteurs VEGFR, PDGFR et c-Kit présentant un fort pouvoir anti-angiogénique. En parallèle de ces travaux, des dérivés de type 2-aminoquinazoliniques ont été conçus. Ces composés substitués par différentes anilines en position 4 ont montré un pouvoir antiprolifératif intéressant grâce à leur intercalation entre les paires de bases de l’ADN. / According to the International Agency for Research on Cancer, cancer is the first cause of death in France with about 150 000 deaths estimated in 2012. This disease is characterized by anarchistic and uncontrolled proliferation of cells that escape control mechanisms. Currently, the anticancer drugs target mainly the cancerous cells that overexpress proteins, such as growth factor receptors with tyrosine kinase activity.Our work is mainly carried on four of these receptors: EGFR (Epidermal Growth Factor Receptor), VEGFR (Vascular Endothelial Growth Factor Receptor), PDGFR (Platelet-Derived Growth Factor Receptor) and c-Kit receptor. Several heterocycles (quinazoline, benzotriazine, thienopyrimidine) differing by their aniline or aryloxy moiety in C-4 position were designed, synthesized and evaluated. Among these products, the 4-aryloxyquinazolines substituted by aminoalkoxy chains in C-7 position have the characteristic to be potent inhibitors of VEGFR, PDGFR and c-kit receptor with high anti-angiogenic potency. Simultaneously, 2-aminoquinazoline derivatives were designed. These compounds substituted by various anilines in C-4 position showed interesting antiproliferative activity through their intercalation between the pairs of DNA bases.

O desenvolvimento de novas terapias para o câncer envolve um processo longo e custoso no qual apenas 5% dos candidatos a fármacos em ensaios clínicos são aprovados. Os ensaios celulares são um importante pilar neste processo, no entanto, eles são geralmente feitos em células cultivadas em monocamada, o que apresenta algumas limitações. Assim, o desenvolvimento e aplicação de modelos tridimensionais (3D) para ensaios pré-clínicos tem sido cada vez mais investigado, devido a suas maiores similaridades com tumores in vivo em relação a características físicas, espaciais e bioquímicas. A via PI3K-AKT-mTOR, que está frequentemente desregulada em diversos cânceres, é uma rota metabólica essencial por integrar fatores de crescimento e sinais internos com a expressão de proteínas relacionadas ao crescimento celular. Sendo assim, novos compostos inibitórios dessa via têm sido estudados pelo grupo NEQUIMED como alternativa terapêutica para essas doenças. Dessa forma, este trabalho teve o objetivo de avaliar o potencial citostático e citotóxico de novos inibidores da via da mTOR em culturas celulares bi e tridimensionais de câncer de próstata e hepatocarcinoma. Para isso, o modelo de cultura 3D foi padronizado para ambas linhagens usando duas técnicas diferentes, além da padronização da técnica de redução da resazurina para a determinação da viabilidade celular em 2D e 3D. No ensaio citotóxico, células em monocamada e esferoides foram tratadas com diferentes concentrações dos fármacos de referência e dos novos inibidores e tiveram sua viabilidade determinada pelo ensaio de redução da resazurina. Já para o ensaio citostático, baixas concentrações de células foram plaqueadas em monocamada e tratadas por até 6 dias, tendo sua viabilidade medida a cada 48 h pelo ensaio de MTT. Os esferoides foram tratados por 9 dias com as mesmas substâncias, tendo seu volume medido a cada 3 dias. No geral, os novos compostos não apresentaram efeitos citotóxicos relevantes, contudo, os compostos Neq0438 e seu análogo, Neq0679, tiveram maior efeito citostático que a Rapamicina em culturas 2D e 3D de HepG2. Além disso, os compostos tiveram maior efeito citostático em esferoides do que em células cultivadas em monocamada para as duas linhagens. Isso pode ser justificado pela alteração na expressão de proteínas da via da mTOR em relação ao modelo de cultura, já que foi demonstrado sua maior ativação nos modelos tridimensionais. Futuramente esses compostos poderão ser testados sozinhos, ou em terapia combinada em modelos animais para melhor avaliação de sua segurança e sua evolução no estudo pré-clínico. / The development of new cancer therapies involves a long and expensive process in which only 5% of the clinical trial candidates for new drugs get approval. Cell assays are an essential pillar in this process; however, they are usually carried out in cells grown as a monolayer, which have some limitations. Thus, the development and application of new tridimensional (3D) models for preclinical trials has been deeply investigated, due to their higher similarities with in vivo tumors, regarding physical, spatial and biochemical features. The PI3K-AKT-mTOR pathway, which integrates growth factors and the expressions of proteins for the cellular growth, is often upregulated in several types of cancer. Hence, the NEQUIMED group is studying new pathway inhibitors as a therapeutic alternative for cancer. Here, the cytotoxic and cytostatic effects of the new PI3K-AKT-mTOR inhibitors were assessed in two and three-dimensional cells models of prostate cancer and hepatocarcinoma. To that end, the 3D culture model was standardized for both cell lines using two different techniques. The resazurin reduction assay was also standardized to determine cell viability in 2D and 3D models. For the cytotoxic assay, cells grown in a monolayer and 3D spheroids were treated with a range of concentrations of the reference drugs and new mTOR inhibitors and had their viability assessed by the resazurin assay. For the cytostatic assay, low cell density was plated on 2D and treated for 6 days, having their viability determined every 48 h using the MTT assay. Spheroids were treated for 9 days with the same substances and had their volume measured every 3 days. Overall, the new mTOR inhibitors did not show relevant cytotoxic effects, however, Neq0438 and its analog Neq0679, had a higher cytostatic effect than Rapamycin for both 2D and 3D cultures of HepG2. Besides, all mTOR inhibitors had a higher cytostatic effect on 3D cultures when compared to monolayers, which can be related to the overexpression of the mTOR pathway in this culture system, already reported on the literature. Based on these results, Neq0438 and Neq0679 should now be used alone or in combination using in vivo models to investigate their antitumoral effects.

In recent years, tyrosine kinase inhibitors have been more and more used for the targeted cancer therapy, due to their ability to disrupt intracellular signalling pathways associated with the development of tumours. Cabozantinib is the tyrosine kinase inhibitor which has been approved for the treatment of thyroid cancer and it is also effective against several other types of cancer. However, multiple drugs combination is often used in anticancer therapy, which may result in their cytochrome P450-mediated interactions. Although this may affect the therapeutic effect of the drugs and cause adverse effects on the organism, very little is known about the effect of cabozantinib on biotransformation enzymes. Therefore, the effect of cabozantinib not only alone but also in combination with the known cytostatic ellipticine on the expression and the activity of cytochromes P450 1A1, 1A2 and 1B1 in rat liver and kidney in vivo was studied in this work. The gene expression was determined by quantitative PCR, the amount of protein was studied by Western blotting and consecutive immunodetection. The enzyme activity was studied using specific marker reactions, 7-ethoxyresorufin O-deethylation for CYP1A1, 7-methoxyresorufin O-demethylation for CYP1A2 and 17β-estradiol 4-hydroxylation for CYP1B1. Our results...

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Cancer is a global health catastrophe, with neuroblastoma, the most common solid childhood tumor, and glioblastoma, a deadly brain tumor, being aggressive and unresponsive to current treatment modalities. These tumors are known to utilize uncontrollable cell proliferative capabilities as a mechanism for tumor survival. Therefore, malignant cell growth can be mitigated by targeting the essential proteins that regulate cell growth, such as receptor tyrosine kinases (RTKs). Under normal physiological conditions, RTKs bind with varying affinity to mitogenic stimuli such as growth factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) which, in turn, leads to receptor phosphorylation and activation.

Non-small cell lung cancer (NSCLC) makes up the majority of lung cancers, which remains the leading cause of cancer mortality worldwide. NSCLC with mutant epidermal growth factor receptor (EGFR) is currently treated with tyrosine kinase inhibitors (TKIs). TKIs have proven effective in improving survival until resistance is conferred, mostly by way of the exon 20, threonine 790 to methionine (T790M) point mutation in EGFR. The mechanism by which this point mutation arises is poorly understood. Herein we report a possible pathway by which the C to T transition that leads to T790M comes about. We show that activation-induced cytidine deaminase (AID) mRNA expression is induced upon treatment with EGFR TKIs in mutant-EGFR human lung cancer cell lines but not in control cell lines. We also show that stable expression of AID is sufficient to produce resistance to one such TKI, erlotinib, and is sufficient to produce T790M itself. We also report that B-cell lymphoma 6 (BCL6) may precede AID in this pathway. Our results show that BCL6 is upregulated in these cell lines treated with EGFR TKIs but not in normal bronchial cells. We then treated human lung cancer cell lines with EGFR TKIs in combination with BCL6 inhibitors. Our results show that AID is dependent upon BCL6 expression. Finally, we report on results from a transient BCL6 overexpression which lead us to believe that AID mRNA receives input from at least one alternate pathway in addition to BCL6. We also performed these experiments on a family of apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases, that show they may be involved in this pathway downstream of AID. Taken together, our results suggest a potential pathway involving BCL6, AID, and APOBEC cytidine deaminases that lead to the C to T transition that produces T790M, thereby conferring resistance to EGFR TKIs in mutant-EGFR NSCLC. They also provide potential new targets for treatment should further study confirm our results. / 2021-10-07T00:00:00Z

Chronic myeloid leukemia is characterized by a unique reciprocal translocation between chromosomes 9 and 22 resulting in deregulated tyrosine kinase activity. Tyrosine kinase inhibitors, such as imatinib, dasatinib, and nilotinib have revolutionized treatment of Chronic myeloid leukemia. However, tyrosine kinase inhibitors? use has presented new challenges in managing both acute and chronic toxicities, particularly ?off-target? toxicities like pleural effusion. Pleural effusions are seen less often with imatinib and very rarely with nilotinib. A 66-year-old male presented to emergency department with complaints of mild chest pain and dyspnea of 3 days duration with progressive worsening, including dyspnea at rest. Patient was currently taking nilotinib after failing imatinib for chronic myeloid leukemia. Nilotinib was put on hold. After exclusion of cardiac and pulmonary etiologies patient was treated for community acquired pneumonia with minimal improvement. Despite the very low incidence of pleural effusion with nilotinib (<1%), he was started on 20?mg of prednisone PO for 3 days. Patient had a dramatic improvement within 48?h after beginning prednisone. This treatment approach suggests that pleural effusions associated with nilotinib can be successfully treated in the same way as pleural effusions associated with dasatinib.

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