Medullary thyroid carcinoma (MTC) is a rare malignant tumor originating from thyroid parafollicular C cells. This tumor accounts for 3-4% of thyroid gland neoplasias. MTC may occur sporadically or inherited. The hereditary MTC is part of syndromes of multiple endocrine neoplasia (MEN) 2A and 2B, familial medullary thyroid carcinoma (FMTC). Germline mutations of the RET (REarranged during Transfection) protooncogene cause hereditary form of cancer, whereas somatic mutations can be present in sporadic form of the disease. The RET gene encodes a receptor tyrosine kinase involved in the activation of intracellular signaling pathways leading to proliferation, growth, differentiation, migration and survival. Nowadays, the only possibility of cure for MTC patients consists of total thyroidectomy associated with lymph node dissection. Based on the knowledge of the pathogenic mechanisms of MTC, new drugs have been developed in attempt to control metastatic disease. Of these, the small-molecule tyrosine kinase inhibitors (TKIs) represent one of the most promising agents for MTC treatment and clinical trials have shown encouraging results. Hopefully, the cumulative knowledge about the targets of action of these drugs as well as TKI-associated side effects will help on choosing the best therapeutic approach in order to enhance its benefits.

Whilst progress has been made in reducing mortality in some cancers, mortality rates remain high in many cancers and there is a need to develop novel therapeutic strategies. In this thesis, various pharmacological strategies designed to enhance the activity of existing therapeutic drugs were evaluated. Cancer cells are dependent upon aerobic glycolysis (the Warburg effect) and glutamine uptake. Using clinically approved tyrosine kinase inhibitors and Bortezomib, significant enhancement of chemosensitivity was observed when used in combination with inhibitors of lactate dehydrogenase (Gossypol) and pyruvate kinase dehydrogenase (Dichloroacetate). In contrast, depletion of glutamine from media had to be extensive in order to induce cell death and cell death only occurred after prolonged exposure to glutamine-deprived conditions. This suggests that glutamine depletion strategies alone are unlikely to be successful but may be useful in combination with other agents targeting glutamine addiction in cancer cells. Finally, Temozolomide (TMZ) is an important drug in the treatment of glioblastomas but its activity is reduced by resistance mechanisms including O6 methyl guanine methyltransferase (MGMT) and mismatch repair (MMR). This thesis has identified analogues of TMZ (EA02-45, EA02-59, EA02-64 and EA02-65) that are MGMT and MMR independent in terms of inducing cell kill in vitro. These compounds are promising leads for future development. In conclusion, this thesis has demonstrated that interfering with the metabolic phenotype of cancer can enhance the activity of existing drugs and identified novel analogues of TMZ that circumvent drug resistance mechanisms that hamper the efficacy of TMZ.

Introdução: Os inibidores tirosino-quinase (ITQs) constituem uma nova terapia molecular para o carcinoma medular da tireoide (CMT). O vandetanibe, um ITQ que atua contra os receptores VEGFR, EGFR e RET, inibe a transformação e o crescimento do tumor no CMT. No entanto, os ITQs têm importantes efeitos adversos, incluindo o hipotireoidismo. O aumento da expressão da iodotironina desiodase do tipo 3 (D3/DIO3), uma enzima chave na inativação dos hormônios da tireoide, pode ser um possível mecanismo de indução do hipotireoidismo por estas drogas. Objetivo: Investigar os efeitos dos inibidores tirosino-quinase na expressão da D3 em células derivadas do CMT. Métodos: Estudo experimental in vitro, utilizando linhagem de células humanas oriundas de CMT (células TT). As células foram cultivadas em meio específico e tratadas com diferentes doses do ITQ vandetanibe (0,25; 0,5 e 1μM) ou com DMSO. A proliferação celular foi determinada por contagem em câmara de Neubauer. A expressão do mRNA foi avaliada por meio de PCR em tempo real, a expressão proteica por meio de Western Blot e a atividade da D3 foi avaliada por meio da técnica de cromatografia em colunas de Sephadex LH-20. Resultados: A adição do vandetanibe ao meio de cultura causou diminuição do número de células e seu efeito foi tempo e dose dependente, apresentando uma redução máxima (77%) após 6 dias de tratamento na dose de 1μM. Como esperado, o tratamento com vandetanibe inibiu a fosforilação do ERK. Não foram observadas alterações significativas dos níveis de mRNA da DIO3 após 3 (0,02 vs. 0,02 vs. 0,01 vs. 0,01; P = 0,34) ou 6 dias (0,02 vs. 0,02 vs. 0,03 vs. 0,02; P = 0,33) de tratamento. Consequentemente, a expressão proteica da D3 não aumentou nos grupos tratados. No entanto, observou-se um aumento de 2 a 5 vezes na atividade da D3 após 3 dias de tratamento e um aumento de 1,5 a 2,15 vezes em 6 dias de tratamento. Conclusões: O tratamento com vandetanibe não foi associado com níveis aumentados de expressão do mRNA e da proteína da D3 em células derivadas de CMT, embora tenha sido observado um aumento na sua atividade enzimática. / Background: Tyrosine kinase inhibitors (TKIs) constitute a novel molecular therapy for medullary thyroid carcinoma (MTC). Vandetanib, a TKI that acts against the VEGFR, EGFR and RET receptors, inhibits tumor transformation and growth in MTC. However, TKIs have important adverse effects, including hypothyroidism. Increases in the expression of type 3 iodothyronine deiodinase (D3/DIO3), a key enzyme in the inactivation of thyroid hormones, may be a possible mechanism of induction of hypothyroidism by these drugs. Objective: To investigate the effects of vandetanib on D3 expression in MTC-derived cells. Methods: In vitro experimental study using human MTC cell line (TT cells). Cells were cultured in specific medium and treated with different doses of vandetanib (0.25, 0.5 and 1μM) or DMSO. Cell proliferation was determined by counting in Neubauer's chamber. Expression of mRNA was evaluated by real-time PCR, protein expression by Western Blot and D3 activity was evaluated by Sephadex LH-20 column chromatography. Results: The addition of vandetanib to the culture medium caused a time and dose-dependent decrease in the number of cells, with a maximum reduction (77%) after 6 days of treatment at 1μM dose. As expected, vandetanib treatment inhibited ERK phosphorylation. No significant changes in DIO3 mRNA levels were observed after 3 (0.02 vs. 0.02 vs. 0.01 vs. 0.01; P = 0.34) or 6 days (0.02 vs. 0.02 vs. 0.03 vs. 0.02; P = 0.33) of treatment. Accordingly, D3 protein expression did not increase in treated groups. However, we observed a 2 to 5-fold increase in D3 activity after 3 days of treatment and a 1.5 to 2.15-fold increase in 6 days of treatment. Conclusions: Treatment with vandetanib was not associated with increased DIO3 mRNA and D3 protein expression levels in MTC-derived cells, although an increase in enzyme activity has been observed.

L’administration de Facteur VIII thérapeutique (FVIII) chez les patients hémophiles A entraine l’apparition d’anticorps anti-FVIII appelés « inhibiteurs » chez 30% des hémophiles A sévères. Ceci constitue alors une impasse thérapeutique. Si de nombreuses investigations ont permis de caractériser les effecteurs lymphocytaires T et B impliqués dans cette réponse immunitaire, elles n’ont toutefois pas permis de proposer aux patients des stratégies thérapeutiques pour prévenir l’apparition des inhibiteurs du FVIII. La première partie de ma thèse explore la possibilité de prévenir la réponse immunitaire anti-FVIII en inhibant la capture et l’apprêtement antigénique du FVIII par les cellules présentatrices de l’antigène (CPA). Il avait été montré précédemment que les deux structures hautement mannosylées du FVIII, sur les asparagines 239 et 2118, étaient reconnues par le CD206 exprimé par les cellules dendritiques humaines dérivées de monocytes, et que cette voie d’endocytose menait à l’apprêtement antigénique du FVIII. Je me suis donc intéressée à la possibilité de réduire l’immunogénicité du FVIII en éliminant ces deux glycosylations. La seconde partie de ma thèse porte sur la possibilité de prévenir ou d’éradiquer la réponse immunitaire anti-FVIII en inhibant une molécule de la signalisation du récepteur des lymphocytes B (LB) : la tyrosine kinase de Bruton (BTK). La BTK jouant un rôle central dans la signalisation des LB, l’inhibition de celle-ci a montré un intérêt thérapeutique dans le cas de certaines pathologies malignes et auto-immunes. J’ai donc exploré le potentiel thérapeutique d’un inhibiteur de la BTK dans la réponse immunitaire anti-FVIII. / Administration of therapeutic factor VIII (FVIII) to hemophilia A patients leads to the development of anti-FVIII antibodies called “inhibitors” in 30% of severe hemophilia A patients. Despite a well characterization of T and B effectors cells involved in this immune response, there is still no therapeutic strategy proposed to the patients to prevent the occurrence of FVIII inhibitors. The first part of my thesis explores the possibility to prevent the anti-FVIII immune response by blocking FVIII capture and processing by antigen presenting cells (APC). It has been previously demonstrated that the two highly mannosylated structures on FVIII, on asparagines 239 and 2118, were recognized by the CD206 expressed on human monocyte-derived dendritic cells. This endocytic pathway led to FVIII processing and presentation to T cells. Therefore, I have investigated the possibility to reduce FVIII immunogenicity by eliminating those two glycosylations. The second part of my thesis focuses on the possibility to prevent or eradicate the anti-FVIII immune response by inhibiting a molecule involved in B cell receptor signaling: the Bruton’s tyrosine kinase (BTK). BTK plays a key role in B cells signaling and inhibition of BTK has shown a great interest in B cell malignancies, but also in some auto-immune diseases. Therefore, I have investigated the therapeutic potential of a new BTK inhibitor against the development of the anti-FVIII immune response.

Multidrug resistance (MDR), a phenomenon in which tumors that were initially sensitive, recur and start showing resistance not only to the initial chemotherapeutic agent but also to various anticancer drugs that are structurally and functionally different from the initial drug, constitutes one of the main reasons for the failure of chemotherapy. An important mechanism of MDR is the enhanced cellular efflux of anticancer agents due to an overexpression of ATP-binding cassette (ABC) transporters (i.e. efflux transporters), especially P-glycoprotein (Pgp), Multidrug Resistance-associated Protein 1 (MRP1) and Breast Cancer Resistance Protein (BCRP), in cancer cells. In order to reverse this resistance, there has been a lot of emphasis on the development of Pgp, MRP1 and BCRP inhibitors. Although this search has been ongoing for three decades, there are still no clinically available efflux transporter modulators. Tyrosine kinase inhibitors (TKIs) are a novel, rapidly growing class of anticancer agents that have a target-based mechanism of action, and their use transformed cancer chemotherapy due to higher specificity and enhanced safety profiles compared to conventional chemotherapeutic agents. Despite their tremendous success in treating various types of tumors, patients develop resistance to TKIs over time. Most of the FDA- approved TKIs are substrates of Pgp and/or BCRP, and as a result, these efflux transporters are also an important cause of conferred resistance against TKIs in cancer cells. Additionally, none of the 31 approved TKIs have an indication for use in brain tumors and interestingly, this may also due to the presence of Pgp and BCRP at the blood-brain barrier (BBB) and in the tumor cells, which prevent the TKI from crossing the BBB and reaching its target tumor site. Since Pgp- and BCRP- mediated TKI efflux has been shown to be involved in TKI resistance, the inhibition of these transporters could represent a potential TKI resistance reversal strategy. Over the last three decades, a large number of Pgp and/or BCRP inhibitors have been identified, but none of them have successfully made it to the clinic. It was observed that most drugs identified as inhibitors were either unable to achieve Pgp and BCRP inhibitory concentrations in-vivo without imparting severe toxicity, or did not possess adequate bioavailability and tissue distribution profiles in order to reach the tumor site. From these identified candidate inhibitors, after much thought and consideration, we chose to investigate TKIs and methylated flavones as modulators of efflux transporter-mediated TKI resistance. The overall goal of this project was to investigate the promising chemosensitizing potential of TKIs and methylated flavones in efflux transporter-mediated TKI resistance, both in-vitro and in-vivo. To identify potent efflux transporter inhibitor TKIs, we evaluated the effect of various TKIs on the accumulation of afatinib, the model TKI substrate, in Pgp- and BCRP- overexpressing cell lines. Afatinib was chosen as the model TKI substrate for our study because it undergoes very minimal metabolism in several species. Afatinib is a substrate of both Pgp and BCRP, but is not a substrate of uptake transporters. Therefore, it was anticipated that an in-vivo efflux transporter-mediated interaction with afatinib would most likely not be confounded or masked by other factors influencing its disposition. From the in-vitro cell uptake studies, we found that nilotinib is a potent inhibitor of both Pgp and BCRP, and it reversed Pgp- and BCRP- mediated afatinib efflux. Subsequently, an in-vivo study was carried out in mice to investigate the interaction between afatinib and nilotinib; and also the impact of nilotinib on the pharmacokinetics and tissue distribution of afatinib. Afatinib exposure in the plasma and in most tissues, namely liver, lung, kidney, heart, muscle, fat, and skin, was found to be significantly increased when nilotinib was coadministered with afatinib. Further, the nilotinib concentrations in most mice tissues was above that needed for Pgp and BCRP inhibition. These results showed that nilotinib could be a potent chemosensitizing agent for Pgp- and BCRP- mediated TKI resistance. Additionally, a significant increase in afatinib brain exposure was observed in the mice which were administered afatinib in combination with nilotinib. This is an interesting and important finding that could potentially be very useful in the treatment of primary and metastasized brain tumors. We also developed a physiologically based pharmacokinetic model of afatinib to characterize its tissue disposition in mice organs, and this model was then scaled up to humans. The developed model accurately predicted afatinib plasma exposure in healthy volunteers and patients with solid malignant tumors, renal impairment, and hepatic impairment. To investigate the chemosensitizing potential of methylated flavones in efflux transporter-mediated TKI resistance, the Bcrp1 inhibitory effect of 5,7-DMF and its effect on sorafenib accumulation was evaluated in-vitro. 5,7- DMF was found to be a potent inhibitor of Bcrp1 and consequently, its impact on the pharmacokinetics and tissue distribution of sorafenib was evaluated in mice. Results showed that co-administration with 5,7-DMF led to significantly greater sorafenib exposure in plasma and in most tissues collected. This indicated that 5,7-DMF may represent a promising chemosensitizing agent for Bcrp1-mediated TKI resistance due to its low toxicity and potent Bcrp1 inhibition. Our results may have important clinical implications as TKIs are currently the most widely used anticancer agents. 5,7-DMF may show great potential in reversing MDR in tumors expressing BCRP. On the other hand, TKI-TKI combination therapy, especially with nilotinib as the perpetrator, is an attractive strategy to combat both Pgp- and BCRP-mediated TKI resistance. Additionally, since nilotinib has a wide volume of distribution and can reach various tissues at concentrations sufficient enough to inhibit Pgp and BCRP; it could potentially be used as a chemosensitizer in the treatment of numerous types of cancers. Furthermore, its chemosensitizing potential could particularly be useful in the treatment of primary and metastatic brain tumors. Further studies are warranted to assess the chemosensitizing effect of nilotinib in tumor xenograft models.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 199-227).

共著者あり。共著者名:Munakata Mitsuru, Sato Atsuko, Amishima Masaru, Homma Yukihiko, Kawakami Yoshikazu. / Hokkaido University (北海道大学) / 博士 / 医学

Les proteines ErbBjHER appartiennent a la famille des recepteurs a activite tyrosine kinase (RrK). Ces recepteurs participent a un reseau complexe d'jnteractions a la surface de la cellule et controlent la proliferation et la differenciation cellulaire. Cette famille de 4 membres (ErbBljEGFR, ErbB2, ErbB3, ErbB4) forme des homodimeres etjou des heterodimeres apres fixation du ligand. La deregulation de ce reseau d'interactions est associee a de nombreux cancers humains.<br />Le domaine tr~smembranaire joue un role dans la fonction de ces recepteurs et l'implication des motifs de type GxxxG dans les processus d'association suscite un interet particulier. Des etudes experimentales montrent une correlation entre la hierarchie des interactions des recepteurs entiers et celIe des domaines transmembranaires.<br />Une methode theorique de recherche de modeles d'association des heterodimeres a ete develoHpee permettant de quantifier les interactions entre les domaines membranaires et de definir Ie role des motifs dans l'association. Des simulations de dynamique moleculaire effectuees sur l'ensemble des modeles montrent une preference d'association gauche des helices. Les petits residus appartenant aux motifs de dimerisation sont presents a l'interface des deux helices. Nos etudes montrent que les domaines transmembranaires participent a la specificite et a la selectivite des recepteurs dans les processus de dimerisation.

La schistosomiase constitue un problème majeur de santé publique dans de nombreux pays émergents d'Afrique, d'Amérique Latine et d'Asie du Sud Est, causant près de 300 000 décès par an. Cette maladie est due au schistosome qui est un ver parasite possédant un cycle de vie complexe. A ce jour une seule drogue est utilisée en monothérapie, le praziquantel ou PZQ, pour lutter contre cette maladie. En raison d'apparitions de résistances au PZQ, il devient nécessaire de rechercher de nouvelles cibles thérapeutiques contre le ver. Au cours de ma thèse je me suis penché sur l'utilisation potentielle des Récepteurs Tyrosine Kinase (RTK) de schistosomes comme cibles thérapeutiques contre le parasite. En effet, les kinases du schistosome semblent présenter un fort degré de spécificité, ce qui les rend attractives pour le l'élaboration d'inhibiteurs potentiels. Dans ce cadre, nous avons poursuivi l'étude de SmIR-1, un récepteur de l'insuline de Schistosoma mansoni découvert au laboratoire, et montré qu'il pouvait être impliqué dans la prise de glucose chez le parasite. Dans un second temps, à partir d'un RTK totalement atypique décrit au laboratoire chez S. mansoni, nous avons découvert une nouvelle famille de RTK nommés les VKR pour Venus Kinase Recepteur, qui semblerait étendue à l'ensemble des invertébrés et dont nous avons entrepris l'étude fonctionnelle

La dimérisation induite par la fixation de ligand est nécessaire à l'activation des récepteurs tyrosine kinase. Le domaine transmembranaire unique de ces protéines joue un rôle crucial dans ces mécanismes. Le récepteur Neu chez le rat et son homologue ErbB2 chez l'humain sont à l'origine de nombreux cancers provoqués par la mutation ponctuelle Val/Glu dans le segment transmembranaire ou la surexpression du récepteur. La structure du domaine transmembranaire dans le récepteur dimère semble déterminante dans les processus d'activation. Les simulations de dynamique moléculaire montrent que, dans une membrane modèle constituée de DMPC ou de POPC hydratés, les hélices transmembranaires de Neu sauvage et oncogénique s'associent préférentiellement en enroulement gauche formant une interface symétrique. Dans le cas de Neu oncogénique le résidu polaire Glu, enfoui dans la membrane, stabilise les interactions entre hélices en formant de fortes liaisons hydrogène