Previous studies of genes associated with retroviral-induced neoplasia have provided the foundation for much of our current knowledge of both tumor suppressor and oncogenes, and have contributed to our understanding of both gene function and malignant transformation. The study of Friend virus-induced erythroleukemia, a well-studied example of multistage malignancy, has led to the identification of several oncogenes, including the Ets transcription factor, fli-1. Fli-1 plays a vital role in hematopoiesis, and vasculogenesis through the transcriptional regulation of its target genes, some of which are critical for the control of cellular proliferation, differentiation, and survival. The aberrant regulation of Fli-1 is associated with a number of cancers and human diseases, including erythroleukemia, Ewing’s sarcoma, lupus, and Jacobsen or Paris Trousseau syndrome. The essential goal set out to be achieved by the research presented herein is to establish a better understanding of both the oncogenic and developmental roles of Fli-1 by investigating the molecular basis by which its deregulated expression leads to fundamental aberration in the fine balance between proliferation and differentiation.

Previous studies of genes associated with retroviral-induced neoplasia have provided the foundation for much of our current knowledge of both tumor suppressor and oncogenes, and have contributed to our understanding of both gene function and malignant transformation. The study of Friend virus-induced erythroleukemia, a well-studied example of multistage malignancy, has led to the identification of several oncogenes, including the Ets transcription factor, fli-1. Fli-1 plays a vital role in hematopoiesis, and vasculogenesis through the transcriptional regulation of its target genes, some of which are critical for the control of cellular proliferation, differentiation, and survival. The aberrant regulation of Fli-1 is associated with a number of cancers and human diseases, including erythroleukemia, Ewing’s sarcoma, lupus, and Jacobsen or Paris Trousseau syndrome. The essential goal set out to be achieved by the research presented herein is to establish a better understanding of both the oncogenic and developmental roles of Fli-1 by investigating the molecular basis by which its deregulated expression leads to fundamental aberration in the fine balance between proliferation and differentiation.

La familia de tumores del sarcoma de Ewing (ESFT) incluye un grupo heterogéneo de neoplasias caracterizadas por la presencia de células redondas de pequeño tamaño con mínima evidencia morfológica de diferenciación. ESFT es el segundo tumor óseo más frecuente, afectando fundamentalmente a niños y adolescentes. A pesar del uso de terapias agresivas combinando quimioterapia, radioterapia y cirugía, la supervivencia de pacientes con metástasis es aproximadamente del 30%, mientras que en ausencia de enfermedad metastásica alcanza valores del 70 %. Debido a esto, son necesarias nuevas aproximaciones terapéuticas dirigidas a reducir la morbilidad relacionada con el tratamiento y a mejorar el índice de supervivencia. Afortunadamente, los ESFT presentan una diana molecular perfecta que resulta de la translocación cromosómica t(11;22)(q24;q12), que ocurre en aproximadamente un 95% de los casos, e involucra al gen EWS y a un miembro de la familia de factores de transcripción ETS, fundamentalmente FLI-1 o ERG. La translocación más común genera la formación de la oncoproteína de fusión EWS/FLI-1 que actúa como factor de transcripción y regula de forma aberrante la expresión de genes diana, favoreciendo el proceso tumorigénico. De esta forma la inactivación de la proteína de fusión EWS/FLI-1 se convierte en una estrategia atractiva, no sólo debido su papel fundamental en la tumorigénesis de ESFT, sino también por su especificidad en células transformadas. En este estudio se evaluaron diferentes estrategias dirigidas a reducir los niveles expresión de EWS/FLI-1 in vitro e in vivo. La primera estrategia utilizada para inhibir los niveles de expresión de la proteína de fusión EWS/FLI-1 se basó en el uso de la rapamicina, un antifúngico e inmunosupresor con propiedades anticancerígenas. La rapamicina inhibió la vía de señalización de mTOR/p70s6K y la proliferación de células de ESFT. Estos resultados sugirieron el uso de esta droga como agente citostático en el tratamiento de este tipo de tumores. La segunda aproximación terapéutica se basó en la inhibición simultánea de EWS/FLI-1 a nivel transcripcional y post-transcripcional. El tratamiento combinado de oligonucleótidos antisentido y rapamicina resultó en un incremento en la muerte de células de ESFT, a través de un proceso que involucra la restauración de la vía de señalización pro-apoptótica del TGF?1/TGF?-RII. In vivo, la administración del tratamiento combinado causó un retraso en el crecimiento de los tumores. Estos datos aportan la base para una mayor exploración del potencial del tratamiento combinado como una nueva estrategia en el tratamiento de este tipo de tumores. Los análisis moleculares mostraron que ESFT presentan alteraciones en proteínas reguladoras del ciclo celular, incluyendo la sobreexpresión de proteínas quinasas ciclina-dependientes (CDK2) y la pérdida o baja expresión de sus inhibidores. Basándonos en ésto, la tercera estrategia se basó en la reversión de alguna de estas alteraciones, mediante el uso de la roscovitina, un potente inhibidor de la actividad quinasa de las CDKs. El tratamiento con roscovitina resultó en un incremento de los niveles de apoptosis en células de ESFT in vitro e in vivo, por un mecanismo dependiente de la activación de caspasas. Estos resultados sugieren que la roscovitina puede ser un agente terapéutico efectivo en el tratamiento de ESFT, sola o en combinación con otras drogas potencialmente sinérgicas. Con el objetivo de identificar y evaluar nuevas proteínas que interactúan con EWS/FLI-1 y contribuir de esta forma a la comprensión de los mecanismos de transformación, identificamos como una diana transcripcional directa de EWS/FLI-1 a la caveolina-1, proteína involucrada en gran variedad de procesos celulares, tales como endocitosis, homeostasis del colesterol, transducción de señales y tumorigénesis. Los resultados de este trabajo mostraron que caveolina-1 juega un papel determinante en la tumorigénesis de células de ESFT y su inhibición podría permitir el desarrollo de nuevas estrategias terapéuticas moleculares dirigidas a mejorar el tratamiento de los pacientes de ESFT.

Plus de 90% des érythroleucémies induites par le virus de Friend sont associées à l'activation récurrente de l’un ou l’autre des facteurs de transcription ETS Spi-1 ou Fli-1, ou du cluster miR-17-92. La contribution de ces trois oncogènes à la prolifération des clones érythroleucémiques correspondant a déjà été indépendamment démontrée. De plus, il a été montré dans l’équipe que Spi-1 active de façon directe l’expression de Fli-1 et que les deux facteurs activent des gènes cibles communs. Dans cette thèse, j’ai montré que Spi-1 et Fli-1 activent l’expression du cluster miR-17-92 en se liant sur un motif ETS conservé dans le promoteur de ce cluster. J’ai montré que la réexpression de miR-17 et miR-20a est suffisante pour contrebalancer partiellement la baisse de la prolifération et la survie cellulaire induite par l’inhibition de l’expression de Fli-1. De plus, j’ai identifié Hbp1 comme une cible de ces miARNs dans les cellules érythroleucémiques. Ces résultats montrent que les trois oncogènes activés de façon récurrente par le virus de Friend constituent un seul réseau oncogénique contrôlant la prolifération. Ces résultats suggèrent également un rôle potentiel de réseaux ETS-miR-17-92 dans d’autres contextes normaux ou pathologiques / Clonal erythroleudemia developing in susceptible mice infected by Friend virus complex are associated with higly recurrent orviral insertinons at one of three loci called Spi-1, Fli-1 or Fli-3, leading to deregulated expression of oncogenic Spi-1 or Fli-1 transcription factors or miR-17-92 miRNA cluster, respectively. Deregulated expression of each of these here ocongenes has been independently shown to contribute to cell profileration of erythroleukemic clones. Previous studies showed close relationship between Spi-1 and Fli-1, which belong te the seame ETS family, Spi-1 activating fli-1 gene and both Spi-1 and Fli-1 activating multiple common target genes involved in ribosome biogenesis. In this tehesis, we habe also demonstrated that physiological re-expression of exogenous miR-17 and MiR-20a are able to partially rescue proliferation arrest induced by Fli-1 knock down and we identified Hbp1 as a tarteg of these miRNAs in erythroleukemia cell line.These results establish that three of the most recurrently activated oncogenes in Friend erythroleukemia are arctually involved in the same oncogenic network controlling proliferation . The putative contribution of similar ETS-MiR-17-92 network in other normal or hyper

Megakaryocytes (Mks) are unique bone marrow cells, which produce platelets. Dysregulated Mk development can lead to abnormal platelet number and the production of functionally defective platelets, causing bleeding, thrombotic events, and leukaemia. Understanding the molecular mechanisms driving megakaryopoiesis may yield insights into the molecular genetics and cellular pathophysiology of a diversity of disorders. The primary aim of this thesis was to gain insight into the molecular events required for normal Mk development. As transcription factors and cytokines play a central role in driving Mk development, both of these processes were investigated. Fli-1 and GATA-1 are key transcription factors regulating Mk-gene expression, alone and co-operatively. To understand the mechanism of transcriptional synergy exerted by Fli-1 and GATA-1, in vitro assays were carried out investigating the interactions between Fli-1, GATA-1 and DNA that mediate synergy. A novel mechanism of synergy was identified, where Fli-1 DNA binding is not required, although an interaction between Fli-1 and GATA-1, and GATA-1 DNA binding is required. Importantly, the results demonstrate that Fli-1 DNA binding is not essential for promoting Mk-gene expression in primary murine bone marrow cells. Thrombopoietin (TPO) is the primary cytokine responsible for Mk and platelet development. Identifying novel TPO gene-targets may provide invaluable information to aid the understanding of the complex and unique processes required for Mk development. Using microarray technology, IFI16 was identified as a TPO-responsive gene that has not previously been studied in the Mk lineage. This work demonstrated that IFI16 is expressed in CD34+ HSC-derived Mks, and that the Jak/STAT pathway is essential for the activation of IFI16 by both TPO and IFN-??. Of biological significance, IFI16 was found to regulate both the proliferation and differentiation of primary Mks, suggesting that IFI16 may control the balance between these two essential processes. In conclusion, the data in this thesis presents a novel mechanism through which Fli-1 and GATA-1 regulate the synergistic activation of Mk genes. The identification and functional characterisation of a novel TPO-inducible gene, IFI16, involved in regulating the proliferation and differentiation of Mks is also described. These findings have implications for several congenital and malignant conditions affecting Mk and platelet development, and possibly a mechanism for IFN-induced thrombocytopaenia.

Megakaryocytes (Mks) are unique bone marrow cells, which produce platelets. Dysregulated Mk development can lead to abnormal platelet number and the production of functionally defective platelets, causing bleeding, thrombotic events, and leukaemia. Understanding the molecular mechanisms driving megakaryopoiesis may yield insights into the molecular genetics and cellular pathophysiology of a diversity of disorders. The primary aim of this thesis was to gain insight into the molecular events required for normal Mk development. As transcription factors and cytokines play a central role in driving Mk development, both of these processes were investigated. Fli-1 and GATA-1 are key transcription factors regulating Mk-gene expression, alone and co-operatively. To understand the mechanism of transcriptional synergy exerted by Fli-1 and GATA-1, in vitro assays were carried out investigating the interactions between Fli-1, GATA-1 and DNA that mediate synergy. A novel mechanism of synergy was identified, where Fli-1 DNA binding is not required, although an interaction between Fli-1 and GATA-1, and GATA-1 DNA binding is required. Importantly, the results demonstrate that Fli-1 DNA binding is not essential for promoting Mk-gene expression in primary murine bone marrow cells. Thrombopoietin (TPO) is the primary cytokine responsible for Mk and platelet development. Identifying novel TPO gene-targets may provide invaluable information to aid the understanding of the complex and unique processes required for Mk development. Using microarray technology, IFI16 was identified as a TPO-responsive gene that has not previously been studied in the Mk lineage. This work demonstrated that IFI16 is expressed in CD34+ HSC-derived Mks, and that the Jak/STAT pathway is essential for the activation of IFI16 by both TPO and IFN-??. Of biological significance, IFI16 was found to regulate both the proliferation and differentiation of primary Mks, suggesting that IFI16 may control the balance between these two essential processes. In conclusion, the data in this thesis presents a novel mechanism through which Fli-1 and GATA-1 regulate the synergistic activation of Mk genes. The identification and functional characterisation of a novel TPO-inducible gene, IFI16, involved in regulating the proliferation and differentiation of Mks is also described. These findings have implications for several congenital and malignant conditions affecting Mk and platelet development, and possibly a mechanism for IFN-induced thrombocytopaenia.