The non-receptor tyrosine kinase activity of fusion gene BCR-ABL derived oncoproteins is the key factor responsible for development and progress of Philadelphia positive (Ph+) chronic myeloid leukemia (CML). In the search for a superior and novel peptide-based inhibitor of Bcr-Abl, here I investigated a naturally occurring molecule, called apoptin. Apoptin is a 13.6 kDa protein derived from chicken anemia virus (CAV) and known to induce apoptosis in a wide range of transformed but not in primary cells. Apoptin is a protein without any reported structural and/or functional homolog and is an interesting candidate to initiate protein-protein interactions and subsequent downstream effects. Initially by an array-based analysis I found that apoptin interacts with the SH3 domain of Abl. By high stringency pull-down and co-immunoprecipitation assays the apoptin and Bcr-Abl interaction was further confirmed. Subsequently, a set of apoptin and Bcr-Abl deletion mutants were used to map this interaction precisely that mainly occurred between a proline rich domain of apoptin and the SH3 domain of Bcr-Abl. I further investigated the role of apoptin on Bcr-Abl. Apoptin was able to modify the phosphorylation of a series of targets (e.g. CrkL, STAT5, c-Myc) downstream of Bcr-Abl kinase. In addition, I used computational algorhythms for protein modeling to study the 3D structure of apoptin and it’s docking with Bcr-Abl at the molecular level. In controlled studies using the 2-pheny-laminopyrimidine derived specific tyrosine kinase inhibitor Imatinib® I found that apoptin has comparable effects on CML cells, suggesting that the interacting segment of the apoptin molecule acts as an adaptor and negatively regulates the Bcr-Abl kinase by deactivating many cell proliferation and anti-apoptotic pathways in CML cells. Briefly, this work provides important insights towards the development of peptide based tyrosine kinase inhibitors as new anti-cancer agents.

The non-receptor tyrosine kinase activity of fusion gene BCR-ABL derived oncoproteins is the key factor responsible for development and progress of Philadelphia positive (Ph+) chronic myeloid leukemia (CML). In the search for a superior and novel peptide-based inhibitor of Bcr-Abl, here I investigated a naturally occurring molecule, called apoptin. Apoptin is a 13.6 kDa protein derived from chicken anemia virus (CAV) and known to induce apoptosis in a wide range of transformed but not in primary cells. Apoptin is a protein without any reported structural and/or functional homolog and is an interesting candidate to initiate protein-protein interactions and subsequent downstream effects. Initially by an array-based analysis I found that apoptin interacts with the SH3 domain of Abl. By high stringency pull-down and co-immunoprecipitation assays the apoptin and Bcr-Abl interaction was further confirmed. Subsequently, a set of apoptin and Bcr-Abl deletion mutants were used to map this interaction precisely that mainly occurred between a proline rich domain of apoptin and the SH3 domain of Bcr-Abl. I further investigated the role of apoptin on Bcr-Abl. Apoptin was able to modify the phosphorylation of a series of targets (e.g. CrkL, STAT5, c-Myc) downstream of Bcr-Abl kinase. In addition, I used computational algorhythms for protein modeling to study the 3D structure of apoptin and it’s docking with Bcr-Abl at the molecular level. In controlled studies using the 2-pheny-laminopyrimidine derived specific tyrosine kinase inhibitor Imatinib® I found that apoptin has comparable effects on CML cells, suggesting that the interacting segment of the apoptin molecule acts as an adaptor and negatively regulates the Bcr-Abl kinase by deactivating many cell proliferation and anti-apoptotic pathways in CML cells. Briefly, this work provides important insights towards the development of peptide based tyrosine kinase inhibitors as new anti-cancer agents.

Yes / Vandetanib is an orally available tyrosine kinase inhibitor used in the treatment of cancer. The current synthesis proceeds via an unstable 4-chloroquinazoline, using harsh reagents, in addition to requiring sequential protection and deprotection steps. In the present work, use of the Dimroth rearrangement in the key quinazoline forming step enabled the synthesis of Vandetanib in nine steps (compared to the previously reported 12–14). / This work was supported by the Cancer Research UK-Cancer Imaging Centre (grant: C1060/ A16464), the Institute of Cancer Research and the University of Hull.

New approaches are needed for the treatment of patients with T-cell acute lymphoblastic leukemia (T-ALL) who fail to achieve remission with chemotherapy. Analysis of the effects of pan-PIM protein kinase inhibitors on human T-ALL cell lines demonstrated that the sensitive cell lines expressed higher PIM1 protein kinase levels, whereas T-ALL cell lines with NOTCH mutations tended to have lower levels of PIM1 kinase and were insensitive to these inhibitors. NOTCH-mutant cells selected for resistance to gamma secretase inhibitors developed elevated PIM1 kinase levels and increased sensitivity to PIM inhibitors. Gene profiling using a publically available T-ALL dataset demonstrated overexpression of PIM1 in the majority of early T-cell precursor (ETP)-ALLs and a small subset of non-ETP ALL. While the PIM inhibitors blocked growth, they also stimulated ERK and STAT5 phosphorylation, demonstrating that activation of additional signaling pathways occurs with PIM inhibitor treatment. To block these pathways, Ponatinib, a broadly active tyrosine kinase inhibitor (TKI) used to treat chronic myelogenous leukemia, was added to this PIM-inhibitor regimen. The combination of Ponatinib with a PIM inhibitor resulted in synergistic T-ALL growth inhibition and marked apoptotic cell death. Treatment of mice engrafted with human T-ALL with these two agents significantly decreased the tumor burden and improved the survival of treated mice. This dual therapy has the potential to be developed as a novel approach to treat T-ALL with high PIM expression.

Tyrosine kinases regulate fundamental signal pathways in cells including cell proliferation, motility, and differentiation. The kinase activity is tightly controlled in normal cells but is usually excessive activated in cancers. Several tyrosine kinase inhibitors are used in cancer therapies nowadays. Our novel tyrosine kinase inhibitor, 1J-309, is a multiple kinase inhibitor that targets several receptors including vascular endothelial growth factor receptors (VEGFRs). We find 1J-309 dramatically reduces cell proliferation of VEGFR3+/VEGF-C+ A549 human lung cancer cells by decreasing the expression of CDK1 and cyclin B1 following growth arrest at G2/M phase. After long term drug treatment, 1J-309 causes cell death. Moreover, 1J-309 represses CDK1 expression at early stage but it does not change CDK1 RNA expression and protein stability. Additionally, 1J-309 significantly decreases the migration ability of A549 cells. 1J-309 also reduces gelatin-related invasion potency. The AKT and p38 MAPK activity are significantly repressed by 1J-309 and it dramatically drives the expression of tumor suppressor, p53, at low-dose treatment. Our results demonstrate that 1J-309 significantly attenuates cell proliferation by inducing G2/M growth arrest, reduces the invasion and migration potency, and promotes a dramatic increase of p53 in A549 cells.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21960号 / 医博第4502号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 平井 豊博, 教授 松原 和夫, 教授 湊谷 謙司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13158号 / 論医博第2145号 / 新制||医||1029(附属図書館) / (主査)教授 川上 浩司, 教授 戸井 雅和, 教授 松田 秀一 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Des mutations faux-sens au niveau du récepteur à activité tyrosine kinase FGFR3 (Fibroblast Growth Factor Receptor 3) entrainent sa suractivation qui apporte des dysfonctions biologiques dans plusieurs maladies. L’achondroplasie, la forme la plus commune de chondrodysplasie liée à Fgfr3, est une maladie génétique rare, touchant 1 nouveau-né sur 20 000, caractérisée par des signes cliniques spécifiques : nanisme rhizomélique, membres courts, macrocéphalie, hypoplasie de l’étage moyen de la face, compression cervico-médullaire. L’activité anormale du récepteur induit des défauts de l’ossification endochondrale responsables du phénotype pathologique. Pendant longtemps le seul traitement pour cette maladie a été l’allongement chirurgical des membres, cependant au cours des dernières années de nombreux chercheurs ont développé des potentielles stratégies thérapeutiques basées sur des études moléculaires. L’objectif de ma thèse était d’évaluer une nouvelle approche thérapeutique pour l’achondroplasie. Une stratégie thérapeutique prometteuse prévoit l’utilisation de petits inhibiteurs chimiques, connus sous le nom d’inhibiteurs de tyrosine kinases, qui sont capables d’arrêter l’activité de FGFR3. J’ai estimé les effets d’un de ces composés, NVP-BGJ398, dans un modèle murin mimant le nanisme achondroplase (Fgfr3Y367C/+). Des expérimentations effectuées ont montré une amélioration des caractéristiques pathologiques dans les souris traitées avec NVP-BGJ398. Nous avons également examiné l’impact de la mutation activatrice de FGFR3 sur le développement mandibulaire. L’étude a reconnu un défaut dans la croissance mandibulaire chez l’homme et la souris atteints. En outre nous avons pu investiguer la croissance osseuse de la mandibule et corriger le défaut pathologique avec NVP-BGJ398. Enfin j’ai participé à des analyses moléculaires pour décrire comment trois mutations de FGFR3 localisées à la même position (Lys650) peuvent induire trois différents nanismes avec sévérité croissante. Les résultats ont fourni une meilleure compréhension des mécanismes moléculaires pathologiques et pourront mener à des nouvelles cibles pour des approches thérapeutiques. / Missense mutations in the tyrosine kinase receptor FGFR3 (Fibroblast Growth Factor Receptor 3) lead to its overactivation causing biological dysfunctions in several diseases. Achondroplasia, the most common Fgfr3-related chondrodysplasia, is a rare genetic disorder, affecting 1 in 20000 live births, characterized by particular clinical features: rhizomelic dwarfism, short limbs, macrocephaly, midface hypoplasia, cervicomedullary compression. The abnormal activity of the receptor induces endochondral ossification defects that are responsible for the pathological phenotype. For a long time the only treatment for this disease was the limb lengthening surgery, however in recent years several researchers have developed potential therapeutic strategies based on molecular studies. The objective of my thesis was to evaluate a novel therapeutic approach for achondroplasia. A promising therapeutic strategy involved the use of small chemical inhibitors, known as tyrosine kinase inhibitors, that are able to arrest the FGFR3 activity. I have assessed the effects of one of these compounds, NVP-BGJ398, in a mouse model mimicking the acondroplastic dwarfism (Fgfr3Y367C/+). The experiments performed showed an improvement of all pathological hallmarks in NVP-BGJ398 treated mice. We have also inspected the impact of the activating FGFR3 mutation on the mandibular development. The study established a defect in mandibular growth in both affected patients and mice. Furthermore we could investigate the mandibular bone growth and correct the pathological defect with NVP-BGJ398. Finally I have participated in molecular analyses to describe how three FGFR3 mutations at the same position could lead to three different dwarfisms with increasing severity. The results provided a better understanding of FGFR3 pathological molecular mechanisms and could lead to new targets for therapeutic approaches.