Mast cells are long-lived effector cells of importance for both acute and chronic inflammations. Mast cells can be activated in many different ways, leading to the release of inflammatory mediators. In contrast to most other inflammatory cells, activated mast cells have the capacity to recover, regranulate and thereby be activated again. In this thesis I have investigated the mechanisms involved in regulating activation-induced mast cell survival. We have found that cross-linking of FcεRI-bound IgE with an antigen (IgER-CL) induces a survival program in mast cells. Upon IgER-CL, mouse and human mast cells upregulate the pro-survival Bcl-2 family gene A1/Bfl-1. A1-/- mast cells degranulate upon FcεRI activation but they cannot recover most likely due to the lack of A1. Sensitized and provoked A1-/- mice exhibit lower amounts of mast cells compared to littermate controls. In contrast to mast cells, no Bfl-1 expression or survival promotion can be detected in basophils after IgER-CL. Another mast cell secretagogue, an adenosine receptor agonist, neither promoted upregulation of A1 nor survival. Although it is well established that mast cell survival is dependent on stem cell factor (SCF), it has not been described how this process is regulated. We have found that SCF promotes survival through Akt-mediated inhibition of the forkhead transcription factor FOXO3a and its transcriptional target Bim, a BH3-only pro-apoptotic protein. SCF-treatment prevents upregulation of Bim protein expression and leads to an upregulation of Bim phosphorylation through PI3-kinase and MEK-dependent pathways. Overexpression of FOXO3a causes an upregulation of Bim and induces mast cell apoptosis, even in the presence of SCF. Taken together, the work in this thesis demonstrates that A1/Bfl-1 and Bim play key roles in mast cell survival. These findings might be of importance in understanding the mechanisms of mast cell longevity and hence for possible new therapeutics used for mast cell-associated inflammations.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Les leucémies aiguës myéloblastiques (LAM) sont des maladies hématopoïétiques caractérisées par une prolifération incontrôlée de précurseurs myéloïdes bloqués à divers stades de différenciation. Le pronostic des LAM reste sombre à cause de la résistance aux traitements et des rechutes après rémission. En conséquence, des thérapies moins intensives et mieux tolérées doivent être développées ; ceci nécessite le développement de stratégies combinatoires associant des molécules avec des modes d'action différents pour augmenter l'efficacité des traitements. Plusieurs approches sont en cours d'étude préclinique et clinique [inhibiteurs des voies de signalisation PI3K/Akt/mTOR, anticorps monoclonaux couplés à une drogue (Mylotarg®), inhibiteurs du protéasome (bortezomib)...] Des travaux récents ont relancé l'intérêt de l'étude des molécules d'origine naturelle pour le traitement des cancers. Ainsi, l'acide flavone-8-acétique (FAA) a suscité de nombreux espoirs au vu de son action sur les tumeurs greffées chez la souris ; il s'est néanmoins révélé inactif chez l'homme du fait d'une métabolisation différente de celle de la souris. L'objectif de ma thèse a été d'étudier les effets d'anticorps monoclonaux dirigés contre l'antigène tumoral CD13 (aminopeptidase-N) et de deux dérivés de FAA, la 2',3-Dinitroflavone-8-acétique (DNFAA ; inhibiteur de l'activité enzymatique de CD13) et la 3,3'-Diamino-4'-méthoxyflavone (DD1) dans les LAM. Mon étude a montré que DNFAA n'affecte ni la prolifération ni la survie des cellules de LAM (lignées et cellules primaires). Cependant, le traitement de ces cellules par les anticorps anti-CD13, (MY7, SJ1D1, WM15 ; reconnaissant ou non le site enzymatique) induit l'apoptose en activant les voies extrinsèque et intrinsèque. Dans la voie intrinsèque, les anti-CD13 régulent négativement l'expression des protéines anti-apoptotiques Bcl-2 et Mcl-1 et positivement l'expression de la protéine pro-apoptotique Bax. De plus, l'activation de la voie PI3K/Akt apparaît associée au processus apoptotique. Mon étude sur les effets du 3,3'-Diamino-4'-méthoxyflavone dans les cellules de LAM montre une induction d'apoptose résultant de la convergence de l'inhibition du protéasome et de l'activation des voies extrinsèque et intrinsèque. Les cibles de DD1 sont le protéasome, la kinase p70S6K (kinase en aval de mTOR), et les protéines pro-apoptotiques Bad et Bax. De plus, j'ai mis en évidence la dégradation de p70S6K sous l'action de la caspase 3, par le traitement avec DD1, nouvelle propriété partagée par DD1 et le bortezomib. En conclusion, mon travail a permis de mettre en évidence les capacités à induire in vitro des voies d'apoptose déficientes dans les cellules de LAM, d'anticorps monoclonaux anti-CD13 et de la flavone originale, 3,3'-Diamino-4'-methoxyflavone, en tant que nouvel inhibiteur du protéasome. Les propriétés de ces agents pro-apoptotiques méritent d'être analysées de façon plus approfondie.

La formation de cancers est liée à des dérèglements de la progression du cycle cellulaire ou de l'apoptose. L'identification des acteurs cellulaires mis en jeu dans la maladie et l'élucidation des mécanismes responsables de ces dysfonctionnements sont à la base de nouveaux traitements anticancéreux. Ainsi, en vue du développement de thérapies ciblées, les kinases Pim et les protéines anti-apoptotiques de la famille des Bcl-2, surexprimées dans de nombreux types de cancers et associées à des phénomènes de chimiorésistance, constituent des cibles pertinentes. Les kinases Pim (Pim-1,-2 et -3) sont une famille de sérine / thréonine kinases qui jouent un rôle fondamental dans les processus de survie, de prolifération ou de différenciation cellulaire. Bien qu'elles possèdent un substrat commun avec les autres protéines kinases, l'ATP, des différences structurales permettent de les différencier et de les inhiber sélectivement. En tenant compte de ces spécificités, nous nous sommes intéressés à la synthèse de nouveaux inhibiteurs sélectifs des kinases Pim, compétitifs de l'ATP. Parmi les autres agents impliqués dans la formation de tumeurs, les protéines de la famille des Bcl-2, responsables du phénomène d'apoptose ou mort cellulaire programmée, font l'objet d'un domaine d'étude récent. Elles se classent en deux familles selon leur fonction : les protéines pro-apoptotiques et les protéines anti-apoptotiques dont la surexpression est observée dans de nombreux cancers. Nous avons poursuivi l'étude de relations structure-activité initiée au laboratoire à partir de trimères d'alkoxyquinoléines, inhibiteurs micromolaires des protéines anti-apoptotiques Bcl-2 et Bcl-xL, en préparant de nouveaux analogues.

<p>Hematopoiesis is the continual process of blood cell generation that primarily occurs in the bone marrow of adult animals. Hematologic neoplasms can also occur in the bone marrow and often result from dysregulation of signal transduction pathways. One example is the activation of the Ras oncogene, which has been linked to a variety of different cancers, including hematologic neoplasms. Ras is located proximal to the cell membrane and can activate many downstream effector pathways, thus it is difficult to determine which downstream pathway is mediating oncogenic Ras function. My thesis work focused on the effect of inappropriate activation of MEK/ERK, a downstream Ras effector pathway, in the hematopoietic system.</p><p>Using a retroviral transduction system, we expressed a constitutively active form of MEK1 in hematopoietic stem cells (HSCs). Mice transplanted with HSCs expressing active MEK developed a lethal myelodysplastic syndrome/myeloproliferative disease (MDS/MPN) characterized by the expansion of granulocytes/macrophages (GM) at the expense of lymphoid cell development. Transplantation of active MEK-induced MDS/MPNs into naïve mice did not result in further disease, suggesting that the MDS/MPN is not a frank leukemia.</p><p>Bcl-2 is an anti-apoptotic molecule that has been shown to play a role in leukemia development and maintenance. Coupling expression of active MEK and Bcl-2 resulted in MDS/MPNs that were phenotypically identical and had very similar disease onset compared to active MEK-induced MDS/MPNs. However, transplantation of Bcl-2/active MEK-induced MDS/MPNs did not result in a myeloid disease; rather, it resulted in the development of T-acute lymphoblastic leukemia (T-ALL) that was marked by activated Notch signaling. </p><p>These results led us to conclude that activation of MEK/ERK was sufficient to cause a pre-leukemic myeloid disease; however, additional oncogenic factors, such as Bcl-2 and Notch, were necessary for frank leukemia development. Moreover, additional oncogenic factors can alter the disease phenotype and disease course. Future analysis of the interplay between oncogenic factors will help shed light on disease development and aid in the development of more effective cancer treatments.</p> / Dissertation

Immunotherapy for cancer is a young research field progressing at high speed. The first chimera of an antibody and a signaling chain was designed by Zelig Eshhar and was later further developed to enhance existing T cell therapy by combining a single-chain fragment of an antibody with the CD3 zeta chain of the TCR complex. T cells expressing these chimeric antigen receptors (CARs) could recognize and specifically kill tumor cells. However the T cells, lacked in persistence and tumor rejection did not occur. Thus, the CAR constructs have been improved by providing the T cell with costimulatory signals promoting activation. The focus of this thesis has been to evaluate second and third generation αCD19-CAR T cells for the treatment of B cell leukemia and lymphoma. B cell tumors commonly upregulate anti-apoptotic proteins such as Bcl-2, which generates therapy resistance. In the first paper a second generation (2G) αCD19-CD28-CAR T cell was combined with the Bcl-2 family inhibitor ABT-737. ABT-737 sensitized tumor cells to CAR T cell therapy and may be an interesting clinical combination treatment. In paper II, the phenotype and function of a third generation (3G) αCD19-CD28-4-1BB-CAR T cell were evaluated. B cell-stimulated CAR T cells showed increased proliferation and an antigen-driven accumulation of CAR+ T cells. 3G CAR T cells had equal cytotoxic capacity, similar lineage, memory and exhaustion profile phenotype compared to 2G CARs. However, 3G CAR T cells proliferated better and had increased activation of intracellular signaling pathways compared to 2G CAR T cells. In paper III, αCD19-CD28-4-1BB-CAR T cells were used to stimulate immature dendritic cells leading to an upregulation of maturation markers on co-cultured dendritic cells. Hence, CAR T cells may not only directly kill the tumor cells, but may induce bystander immunity that indirectly aids tumor control. This thesis also include supplementary information about the development and implementation of protocols for GMP production of CAR T cell batches for a phase I/IIa clinical trial currently ongoing for patients with refractory B cell leukemia and lymphoma. So far, two patients have safely been treated on the lowest dose.

La apoptosis es un proceso de muerte celular programada que está involucrada en la selección del repertorio de linfocitos T y en el mantenimiento de la tolerancia inmunológica, ya que es el mecanismo por el que se eliminan las células que podrían dar lugar a respuestas autoinmunes. Existen evidencias de que la alteración en los mecanismos apoptóticos están implicados en la patogenia y la actividad de las enfermedades autoinmunes sistémicas, de las cuales, el lupus eritematoso sistémico (LES) es la más representativa. Se han identificado en el ser humano algunos genes que codifican oncoproteínas y citocinas cuya transcripción parece ser crucial en este proceso: unos pro-apoptóticos (fas, p53 y TNF-alfa) y otros anti-apoptóticos (bcl-2 y IL-10). Se sospecha que cambios en la expresión de los mismos podrían desempeñar algún papel en la patogenia del LES, al favorecer la proliferación de determinadas poblaciones celulares de efecto autorreactivo.Con los trabajos de la presente tesis doctoral nos propusimos: determinar la implicación de los oncogenes (bcl-2, fas y p53) y las citocinas (IL-10 y TNF-alfa) en la disregulación apoptótica que presentan los pacientes con LES; estudiar la interrelación existente entre ellos; y analizar la relación de las posibles disregulaciones de los elementos que participan en la apoptosis con la actividad de la enfermedad lúpica.Los resultados obtenidos muestran cómo los oncogenes fas, bcl-2 y p53, las citocinas IL-10 y TNF-alfa, y la fracción proteica soluble del Fas (sFas) tienen una notable importancia en la patogenia y la actividad de la enfermedad lúpìca. Las vías apoptóticas del Fas y p53 son independientes entre sí. Sin embargo, diversas citocinas (IL-10, TNF-alfa), oncoproteínas (Bcl-2) y fracciones proteicas solubles (sFas) pueden ser las encargadas de relacionarlas entre sí. La interferencia de estas vías apoptóticas produciría una eliminación deficiente de los linfocitos autorreactivos. Ello favorecería su supervivencia, lo que provocaría las alteraciones de disregulación inmunológica propias del LES.

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 104 pages. Includes vita. Includes bibliographical references.

Thesis--University of Oklahoma. / Bibliography: leaves 71-78.

Base teórica: Nos Estados Unidos da América (EUA) e no Norte da Europa o carcinoma de ovário é a principal causa de morte por câncer ginecológico. A maioria dos casos é diagnosticada em estágios avançados – III ou IV FIGO . A sobrevida em 5 anos neste grupo é de 30% das pacientes. O tratamento envolve cirurgia oncológica para citorredução tumoral e quimioterapia adjuvante, na maioria dos casos. A via do apoptose está envolvida no desenvolvimento tumoral e resistência ao tratamento. As proteínas da família Bcl2 são ativas na via apoptótica e sua atividade se divide em anti e próapoptose. Estudos em pacientes com câncer de ovário foram realizados nos EUA, Europa e Canadá e os resultados observados foram controversos com relação ao papel dessas proteínas no câncer de ovário. Objetivos: Determinar a prevalência de Bcl2, Bcl-xL, Bax, Bad e p53 em uma amostra de pacientes do HCPA. Estudar a possível correlação entre a expressão dessas proteínas e aspectos de desfecho clínico (resposta ao tratamento, sobrevida total e livre de doença). Pacientes e métodos: Foram avaliadas, retrospectivamente, 45 pacientes com diagnóstico de carcinoma epitelial de ovário, tratadas de forma padronizada no HCPA no período de 1996 a 2004. Tecido tumoral foi avaliado através da técnica de imunohistoquímica com relação à positividade para as proteínas Bcl2, Bcl-xL, Bax, Bad e p53. Resultados: A prevalência das proteínas encontrada nessa amostra de pacientes foi: Bcl2 49%, Bcl-xL 80%, Bax 98%, Bad 87% e p53 58%; apresentando similaridade, em geral , com a literatura mundial revisada. A expressão positiva da proteína p53 correlacionou-se com pior sobrevida livre de doença (16 versus 58 meses – p=0.04) na análise univariada. As demais proteínas não apresentaram correlações com resposta ao tratamento ou sobrevida, nesta amostra. Conclusão: O padrão de expressão das proteínas da família Bcl-2 e da proteína p53 encontrado nesta coorte foi similar ao descrito na literatura mundial disponível. A expressão positiva de p53 demonstrou correlação com menor sobrevida livre de doença na análise univariada, o que aponta para as investigações, já em andamento, acerca desta proteína, suas mutações e potencial alvo terapêutico. As proteínas da família Bcl-2 não se correlacionaram com resposta ao tratamento ou sobrevida nesta amostra e requerem investigações em estudos prospectivos, utilizando novas tecnologias de detecção. / Background: In the USA, Europe and Canada epithelial ovarian cancer is the principal cause of death from gynecological cancer and most patients are diagnosed in advanced stages of the disease – Stages III or IV FIGO. The 5 years survival for these patients is around 30%. Treatment is based in laparoscopy to stage and debulk the tumor volume and most cases will need chemotherapic treatment, based on platinum compounds. Apoptosis route is involved in the tumor development and, probably in treatment resistance also. Bcl2 family proteins are active in the apoptosis route and its activity is divided in pro and anti-apoptosis pathways. Studies in ovarian cancer and Bcl2 proteins family activity were done in USA, Europe and Canada. However, the results remain controversial concerning the role of these proteins in ovarian cancer. Objective: Determine Bcl2, Bcl-xL, Bax, Bad and p53 prevalence in a sample of patients from HCPA. Evaluate a possible correlation between these proteins expression and the clinical response, as well as the survival. Methods and patients: Forty-five patients were retrospectively analyzed. They had epithelial ovarian carcinoma diagnosed and treated in a standardized way on HCPA, between the years 1996 to 2004. Tumor tissue samples were evaluated through immunohistochemical analysis to detect positive expression of the proteins Bcl2, Bcl-xL, Bax, Bad and p53. Results: The prevalence found for the studied proteins in this sample was: Bcl2 49%, Bcl-xL 80%, Bax 98%, Bad 87% and p53 58%; similar to the levels described in the literature. Positive expression of p53 correlated with worst disease free survival (16 versus 58 months – p=0.04) in the univariate analysis. The other proteins did not show correlation with treatment response or with survival in this sample. Conclusion: The expression pattern of Bcl-2 protein family and of the p53 protein in this cohort was similar to the one usually described in the literature. The positive expression of p53 was correlated with a smaller time of disease free survival in the univariate analysis. In fact, there are currently investigations about this protein and its mutations as a potential therapeutic goal. Bcl-2 family proteins did not correlate with response to treatment or survival in this sample. Prospective studies are required, as well as the use of new technologies, to know better the role of these proteins in ovarian cancer.