Evolutionarily conserved Pumilio (Pum) RNA-binding proteins act as translational repressors during embryo development and cell fate specification. Previous work in the lab has shown that over-expression of Pum2 (Pum2-EML) supports maintenance and suppresses mutilineage differentiation of murine multipotent HSC/MPP-like cell line EML. The subsequent analysis of HSC markers and functional analysis has revealed that wt EML cells share the LKS CD34 positive phenotype, whereas the majority Pum2-EML cells are similar to LKS CD34 negative. The CD34 positive wt EML cells can be divided into CD34low, CD34med and CD34high subpopulations, whereas Pum2-EML CD34 positive cells correspond to CD34low subpopulation. Colony forming assays have revealed that the overall multilineage differentiation of wt EML and Pum2-EML cells strongly correlates with the CD34 expression levels. Multiple experiments have revealed that purified CD34 negative and CD34 positive wt EML cells can generate each other and among CD34 positive wt EML cells the CD34low cells have the highest capacity to give rise to CD34 negative EML cells. We have proposed a model in which CD34 negative EML cells are more primitive cells in an "inactive" (differentiation inhibited) state, that give rise to CD3low "active" (differentiation ready) EML cells. The CD34low EML cells can revert back to the CD34 negative state or give rise to CD34med/high cells that can readily differentiate into multiple lineages. Based on that model, the over-expression of Pum2 leads to increased maintenance of cells in inactive CD34 negative state, and blocks development of CD34 positive cells past the CD34low stage. Cumulatively, these results support the notions that Pum2 could be involved in maintaining the balance between inactive and active state of multipotent hematopoietic cells. The c-kit receptor plays a vital role in self-renewal and differentiation of hematopoietic stem cells (HSC) and multipotent progenitors (MPPs). We have discovered that besides c-kit, the murine multipotent HSC/MPP-like cell line EML expresses the transcript and protein for a truncated form of c-kit, called tr-kit. Notably, the tr-kit transcript and protein levels were down-regulated during cytokine induced differentiation of HSC/MPP-like cell line EML into myelo-erythroid lineages. RT-PCR results show tr-kit is transcribed solely in cell populations enriched for LTR-HSC, STR-HSC and MPPs. The observation that tr-kit is co-expressed with c-kit only in more primitive, HSC and MPP-enriched cell populations raises an exciting possibility that tr-kit functions either as a new component of SCF/c-kit pathway, or is involved in a novel signaling pathway, present exclusively in HSC and MPPs. These findings necessitate functional characterization of tr-kit, and analysis of its potential role in the self-renewal, proliferation and/or differentiation of HSC and multipotent progenitors.

No description available.

Thesis (Ph. D.)--Cornell University, January, 1989. / Vita. Includes bibliographical references.

L’érythropoïèse est le processus aboutissant à la production des hématies à partir d’une cellule souche hématopoïétique. La différenciation érythroïde implique des changements morphologiques en partie liés à la perte d’expression membranaire du récepteur à activité tyrosine kinase de classe III, c-Kit. En réponse à son ligand, le SCF, c-Kit est activé puis internalisé et dégradé par la voie du protéasome, via l’ubiquitine E3-ligase c-Cbl, ou par la voie lysosomale suite à une endocytose. Dans la première partie de ce travail, nous avons pu mettre en évidence qu’en absence de SCF et en réponse à un inhibiteur de tyrosine kinase, l’imatinib, les érythroblastes cultivés ex vivo perdent l’expression membranaire de c-Kit et accélèrent leur entrée en différenciation terminale. Au vu de ces observations, nous avons cherché à comprendre les mécanismes impliqués. Sur un modèle de cellules érytholeucémiques dépendantes de l’érythropoïétine, mais exprimant de manière endogène c-Kit, nous avons montré que l’imatinib induit une internalisation du récepteur ainsi que sa dégradation par la voie lysosomale et de manière indépendant de c-Cbl. De plus, nous avons montré que cet effet est réversible et que l’imatinib ne bloque pas la réexpression de c-Kit après son internalisation en réponse au SCF. Des marquages métaboliques ont permis de montrer que l’imatinib ne modifie ni la synthèse ni la maturation de c-Kit et que le profil phospho-tyrosine des cellules traitées à l’imatinib est globalement inchangé. Enfin, nous avons montré que la fixation de l’imatinib à la poche catalytique de c-Kit est indispensable à son internalisation, et par conséquent à sa dégradation. Il apparait donc que l’imatinib lève l’auto-inhibition de c-Kit, qui semble nécessaire pour son maintien à la membrane. Dans la seconde partie de ce travail, nous nous sommes intéressés aux changements morphologiques subis par les nucléoles, lieu de la biogenèse des ribosomes, au cours de différenciation des érythroblastes. L’étude de la taille et du potentiel prolifératif des cellules, ainsi que l’analyse morphologique des nucléoles, nous a permis de confirmer que la réduction de taille des cellules est contemporaine d’un ralentissement de leur prolifération ainsi que de la réduction du volume et de la surface du composé granulaire (CG), « matrice » du nucléole. En microscopie électronique, nous montrons la persistance des CG en fin de maturation. Enfin, nous avons également étudié l’évolution des nucléoles dans un contexte pathologique de syndromes myélodysplasiques de faible risque, qui se caractérisent par une hématopoïèse inefficace. Nous observons que les cellules pathologiques immatures ont des CG plus volumineux que les cellules normales immatures, et qu’au cours de la différenciation, la morphologie des nucléoles est identique entre les cellules normales et pathologiques. En conclusion, ce travail a permis de décrire 1) le mécanisme d’internalisation d’un récepteur à activité tyrosine kinase de classe III, c-Kit par l’imatinib et 2) la morphologie du nucléole au cours de la différenciation érythroïde normale et pathologique des syndromes myélodysplasiques de faible risque. / Erythropoiesis is the process leading to the production of red blood cells from hematopoietic stem cell. The erythroid differentiation involves morphological cell changes, in part related to the loss of membrane expression of the type III receptor tyrosine kinase, c-Kit. In response to its ligand SCF, c-Kit is activated, then internalized and degraded by the proteasome pathway via the E3 ubiquitin ligase c-Cbl, or by the lysosomal pathway, after endocytosis. In the first part of this work, we demonstrated that in the absence of SCF and in response to tyrosine kinase inhibitor, imatinib, erythroblasts cultured ex vivo, lose membrane expression of c-Kit and accelerate their terminal differentiation. In view of these observations, we sought to understand the mechanisms involved. On an erythropoietin dependent cell line expressing c-Kit at the membrane, we showed that imatinib induces receptor internalization and degradation by the lysosomal pathway, independently of c -Cbl. Furthermore, we showed that this effect is reversible and that imatinib does not block the c-Kit re-expression after its internalization, in response to SCF. Metabolic labelling showed that imatinib does not alter synthesis or maturation of c -Kit and that the phospho-tyrosine profile of cells treated with imatinib is generally unchanged. Finally, we showed that the binding of imatinib to the catalytic pocket of c-Kit is essential for its internalization, and therefore its degradation. So, it appears that imatinib removes c-Kit self-inhibition, which seems necessary to its retention at the membrane. In the second part of this work, we studied the morphological changes of nucleoli, the site of ribosome biogenesis, during erythroid differentiation. We showed that the reduction of cell size takes place at the same time than reduction of cell proliferation and reduction of surface and volume of the Granular Compound (GC), the “matrix” of the nucleolus. Moreover, we showed by electronic microscopy, the persistence of GC at the end of maturation. Finally, we also studied the evolution of nucleoli in a pathological context of low risk myelodysplastic syndromes, which are characterized by ineffective hematopoiesis. We observed that immature pathological cells have larger GC than immature normal cells, but that during differentiation, the morphology of nucleoli is identical between normal and pathological cells. In conclusion, this work has allowed us to describe 1) the mechanisms of internalization of a class III receptor tyrosine kinase, c-Kit by imatinib and 2) the morphology of the nucleolus during normal and pathological low risk myelodysplastic syndromes of erythroid differentiation.

La mastocytose est une maladie hétérogène, caractérisée par une accumulation de mastocytes dans l’organisme. Les enfants et les adultes ont des mutations différentes de c-Kit. Dans un premier travail, nous avons montré que seules les formes adultes sont associées à la réactivation de la télomérase, alors que les formes pédiatriques ne sont pas. Cela semble être lié aux différences de mutations de c-Kit observées entre adultes et enfants et pourrait expliquer pourquoi seules les formes pédiatriques de mastocytose régressent spontanément et non les formes adultes. Ces résultats aident à mieux comprendre la physiopathologie de la mastocytose. Dans un second travail, nous a étudié le lien entre la longueur des télomères et les troubles psychologiques des adultes atteints de mastocytose. Nous avons montré que réactions émotionnelles négatives sont corrélées au raccourcissement de la longueur des télomères des leucocytes et que l’érosion télomérique est fortement prédite par les défauts de régulation des émotions. Nous émettons l'hypothèse qu’au cours des troubles neuropsychologiques, le mastocyte pourrait être impliqué dans le raccourcissement de la longueur des télomères en périphérie. / Mastocytosis is a heterogeneous disease characterized by an accumulation of mast cells. Children and adults hold different c-Kit mutations. In a first work, we showed that only adult forms are associated with reactivation of telomerase whereas pediatric forms are not. This seems to be linked to the differential c-Kit mutations observed between adults and children and could explain why only pediatric mastocytosis spontaneously regress in comparison with adult forms. These results help to better elucidate the pathophysiology of mastocytosis. In a second work, we studied the link between the telomere length and the psychological features of adults with mastocytosis and showed that negative emotionality correlated negatively to telomere length and that telomere shortening was strongly predicted by emotion regulation deficits. We hypothesize that in psychological disorders, mast cell may represent the link between brain and periphery and induce telomere shortening.

C-Kit est généralement considéré comme un récepteur à tyrosine kinase et comme un proto-oncogène, dont la surexpression et la mutation conduisent à une progression tumorale médiée par son activité kinase. En clinique, les traitements ciblant l’activité kinase de c-Kit, comme l’Imatinib (Gleevec), ont été largement utilisés pour traiter les patients atteints de maladies liées à c-Kit. Alors que le rôle de c-Kit comme proto-oncogène ne fait aucun doute, certaines études et analyses de bases de données diffèrent avec l’idée d’un rôle pro-tumoral de c-Kit, laissant penser à un rôle différent de c-Kit dans le cancer. Ici, nous montrons que c-Kit appartient à la famille des récepteurs à dépendance, de la même façon que d’autres récepteurs de la famille des tyrosine kinases tel que MET, RET et TrkC. En absence de son ligand Stem Cell Factor (SCF), au lieu de rester inactif, c-Kit déclenche l’apoptose, qui peut être renforcée par l’invalidation de son activité kinase. En parallèle, nous montrons que c-Kit est capable de se lier à la caspase-9 et de l’activer. De plus, à la manière d’autres récepteurs à dépendance, c-Kit est aussi clivé par des protéases de type caspase sur son résidu acide aspartique D816, qui est nécessaire à son activité pro-apoptotique. La mutation du site D816 inhibe l’interaction entre c-kit et la caspase-9 et invalide l’activité pro-apoptotique de c-Kit. De façon intéressante, la mutation D816 est l’une des mutations les plus communes de ce récepteur dans la plupart des cancers liés à c-Kit, et cette mutation favorise la résistance au traitement Gleevec. Nous montrons aussi que la surexpression de c-Kit invalidé pour son activité kinase est capable d’inhiber la croissance tumorale dans des modèles animaux, alors que la mutation du site D816 empêche son effet suppresseur de tumeur. En outre, nous avons développé un outil permettant de bloquer l’interaction entre SCF et c-Kit, déclenchant l’activité pro-apoptotique de c-Kit dans les cancers positifs pour ce récepteur. En utilisant l’activité pro-apoptotique de c-Kit, en combinaison avec des inhibiteurs de kinases comme le Gleevec, nous proposons une nouvelle stratégie thérapeutique. En conclusion, nous démontrons que c-Kit est un membre de la famille des récepteurs à dépendance, présentant une activité pro-apoptotique, et pouvant être utilisé comme un outil alternatif dans le cadre d’un traitement contre le cancer / C-Kit has been generally considered as a receptor tyrosine kinase and a proto-oncogene, whose upregulation and mutation lead to tumor progression through its kinase activity. Clinically, drugs targeting the kinase activity of c-Kit, such as Imatinib (Gleevec), have been wildly used to treat patients with c-Kit related diseases. While the role of c-Kit as a proto-oncogene is of no doubt, some research reports and database analysis do not fit well the tumor promoting role of c-Kit, indicating a possible different role of c-Kit in cancer. Here, we show that c-Kit belongs to the dependence receptor family, similarly to other receptor tyrosine kinases such as MET, RET and TrkC. In the absent of its ligand SCF (stem cell factor), instead of staying inactive, c-Kit triggers apoptosis, which can be enhanced by silencing its kinase activity. Besides, we have shown that c-Kit is able to bind and activate caspase-9. Moreover, similarly to other dependence receptors, c-Kit is also cleaved by caspases-like protease at aspartic acid residue D816, which is crucial for its pro-apoptotic activity. The mutation of D816 site inhibits the c-Kit/caspase-9 binding and silences the pro-apoptotic activity of c-Kit. Of interest, c-Kit D816 mutation is one of the most common mutation of this receptor in many c-Kit related cancers and it promotes resistance against Gleevec treatment. We also show that overexpression of kinase mutated c-Kit is able to inhibit tumor growth in animal models, while the mutation of D816 site impairs the tumor suppressing activity. Furthermore, we develop a tool to block the SCF/c-Kit interaction, which unleashes the pro-apoptotic activity of c-Kit in cancers expressing this receptor. By using the pro-apoptotic activity of c-Kit, in combination with kinase inhibitors like Gleevec, we propose a novel therapeutic strategy. In conclusion, we demonstrate that c-Kit is a member of dependence receptor family, harboring intrinsic pro-apoptotic activity, which can be used as an alternative tool in cancer treatment

La mastocytose est une maladie hétérogène, caractérisée par une accumulation de mastocytes dans l'organisme. Les enfants et les adultes ont des mutations différentes de c-Kit. Dans un premier travail, nous avons montré que seules les formes adultes sont associées à la réactivation de la télomérase, alors que les formes pédiatriques ne sont pas. Cela semble être lié aux différences de mutations de c-Kit observées entre adultes et enfants et pourrait expliquer pourquoi seules les formes pédiatriques de mastocytose régressent spontanément et non les formes adultes. Ces résultats aident à mieux comprendre la physiopathologie de la mastocytose. Dans un second travail, nous a étudié le lien entre la longueur des télomères et les troubles psychologiques des adultes atteints de mastocytose. Nous avons montré que réactions émotionnelles négatives sont corrélées au raccourcissement de la longueur des télomères des leucocytes et que l'érosion télomérique est fortement prédite par les défauts de régulation des émotions. Nous émettons l'hypothèse qu'au cours des troubles neuropsychologiques, le mastocyte pourrait être impliqué dans le raccourcissement de la longueur des télomères en périphérie.

This work explored features of canine mast cell tumours (MCT) to improve prognosis and to discover potential therapeutic targets. Subcutaneous MCT - a subset of these tumours arising in the subcutis - are usually grouped with cutaneous MCT, but there is evidence that they may be clinically different. The first objective was to develop a grading scheme for subcutaneous MCT. Over 300 canine subcutaneous MCT were evaluated retrospectively and parameters were correlated with clinical outcomes, making this the largest retrospective survival study of these tumours to date. The results of the study showed that the majority of subcutaneous MCT had excellent outcomes and key prognostic markers were identified (mitotic index, surgical margins and degree of infiltration). A subset of the subcutaneous MCT from the retrospective study was further evaluated to assess the cellular localization of KIT - a receptor tyrosine kinase (RTK) which is dysregulated and constitutively activated in some cutaneous MCT - as well as Ki67, a proliferation marker. In addition, evaluation of mutations of c-KIT, the gene for KIT, was determined for each MCT. Cytoplasmic KIT localization and high Ki67 values were predictive of decreased survival time and time to local reoccurrence, but no c-KIT mutations were detected. The majority of canine MCT do not appear to depend solely upon KIT for tumour progression and few other RTK targets have been studied in canine MCT. Based on evidence that vascular endothelial growth factor receptors (VEGFR) and platelet-derived growth factor receptors (PDGFR) - may play a role in the progression of canine MCT; the expression and distribution of these RTK were evaluated. The results showed that canine MCT have unique expression profiles and activity of KIT, VEGFR2 and PDGFR. Two novel mast cell tumour cell lines were generated and used to assess signalling of KIT and VEGFR2 in vitro. Stimulatory and inhibitory responses were assessed and found to be different in both cell lines. Both had autophosphorylated VEGFR2 and an autocrine VEGF/VEGFR2 signalling pathway existed for both cell lines. These findings are unique and the first that identify autocrine VEGF signalling as a possible survival mechanism for canine MCT. / Pet Trust Foundation, Ontario Veterinary College

In der vorliegenden Arbeit wurden die Effekte einer frühzeitigen Zelltherapie im Langzeit in-vivo Infarktmodell studiert. Erstmals wurden dabei auch Veränderungen der kardialen -Adrenozeptoren untersucht und Zelltherapie mit einer reversiblen präinfarziösen Ischämie kombiniert. Initial wurden dafür bei 38 männlichen weißen Neuseeländer Kaninchen Knochenmarkspunktionen durchgeführt, MSC durch Kultur isoliert und 60 Minuten nach induziertem Infarkt und ohne Reperfusion in den Randbereich des Infarktgebietes injiziert. Zur Untersuchung möglicher Interaktionen zwischen Zelltherapie und Präinfarktgeschehen wurde bei einigen Tieren das Myokard durch eine kurzzeitige Präinfarktischämie präkonditioniert. Die Ergebnisse der vorliegenden Arbeit zeigen, dass auch die frühzeitige Zellinjektion ohne Reperfusion mit signifikanten Effekten auf die Kontraktilität und spezifischen sympathoadrenergen Veränderungen verbunden ist.

Research Doctorate - Doctor of Philosophy (PhD) / Reversible protein phosphorylation plays a central role in the regulation of intracellular signalling, and is controlled by the opposing activities of protein kinases and phosphatases. Deregulation of these mechanisms can result in increased proliferation and enhanced survival, which is a hallmark feature of malignant transformation. For example, over 90% of chronic myeloid leukaemia (CML) patients express the BCR/ABL oncoprotein, which exhibits unrestrained tyrosine kinase activity. In addition, activating mutations within the receptor tyrosine kinase, c-KIT, contribute to the pathogenesis of gastrointestinal stromal tumours (GIST), systemic mastocytosis, acute myeloid leukaemia (AML), testicular seminoma and melanoma. The advent of small molecule tyrosine kinase inhibitors, such as imatinib, has revolutionised the treatment of malignancies driven by these oncogenic kinases. However, a proportion of patients are either unresponsive or develop resistance, and as such, relapse and disease progression is a major clinical problem. In order to improve the treatment outcome for these patients, a greater understanding of the signalling pathways regulated downstream of BCR/ABL and c-KIT is required. The data presented in this thesis indicates that oncogenic BCR/ABL and mutant c-KIT both require inhibition of the tumour suppressor, protein phosphatase 2A (PP2A), to induce tumourigenesis. PP2A is a large family of serine/threonine phosphatases that provide the fine control on signalling pathways by governing the rate and duration of phosphorylation. The heterotrimeric PP2A enzyme is comprised of a structural subunit (PP2A Aα and Aβ), a catalytic subunit (PP2Acα and cβ) and a regulatory subunit, which consists of three unrelated families: B55 (α, β, γ, δ), B56 (α, β, γ, δ, ε) and B" (PR72/130 / PR70/48). Binding of the regulatory subunit to the core PP2A AC dimer directs both the substrate specificity and cellular localisation of the enzyme. The combinatorial assembly of these individual components permits the formation of distinct complexes which have been implicated in numerous cellular functions such as proliferation, survival and mitosis. In particular, important roles for PP2A in various aspects of malignant transformation are beginning to emerge. Recent work demonstrates that PP2A is functionally inactivated by BCR/ABL in myeloid progenitor cells. Using the mouse myeloid progenitor cell line, FDC-P1, these observations were confirmed in the current study. Detailed investigation into the underlying mechanisms have demonstrated for the first time that active BCR/ABL increases the expression of the PP2A structural and certain regulatory subunits. This alters the PP2A holoenzyme composition and results in the abundance of complexes containing B55α and B56α. Consequently, B56γ, a known tumour suppressive subunit, appears to be simultaneously displaced. To investigate which subunits are functionally important for BCR/ABL-mediated leukaemogenesis, individual PP2A subunits were targeted with shRNA sequences in WT BCR/ABL FDC-P1 cells. Subsequent evaluation identified B56α as a key player which facilitates the leukaemic phenotype. In accordance with an increase in PP2A activity, knockdown of B56α significantly inhibited the cellular growth and reduced the clonogenic potential of BCR/ABL⁺ myeloid progenitors. Furthermore, suppression of the B56δ subunit in WT BCR/ABL FDC-P1 cells appears to delay progression through the cell cycle. Together, these findings provide new insights into the biology of PP2A and begin to define the precise mechanisms by which BCR/ABL induces leukaemogenesis via PP2A in CML. Investigation of the regulation of PP2A was also extended to the oncogenic tyrosine kinase, c-KIT. Using FDC-P1 cells expressing imatinib-sensitive (V560G) or –resistant (D816V) mutant c-KIT, this work demonstrates for the first time that constitutive activation of c-KIT impairs the activity of PP2A, and this is essential for tumourigenesis. Pharmacological reactivation of PP2A with FTY720 significantly reduced the proliferation, impaired the clonogenic potential and induced apoptosis of oncogenic c-KIT cells, whilst having no effect on empty vector controls or WT c-KIT cells stimulated with stem cell factor (SCF). These cytotoxic effects of FTY720 are mediated, in part, by the rapid dephosphorylation, and hence inactivation, of oncogenic c-KIT receptors. These promising in vitro findings were translated into an in vivo model, where the daily administration of FTY720 significantly delayed the growth of mutant c-KIT⁺ tumours. Furthermore, FTY720 markedly prevented the infiltration of D816V c-KIT tumour cells into secondary lymphoid organs, such as the spleen and bone marrow. As a result, the survival of FTY720-treated mice was significantly prolonged compared to saline-treated controls. Overall, this body of work greatly enhances our understanding of PP2A function and identifies the complex mechanisms of PP2A regulation by the oncogenic tyrosine kinases, BCR/ABL and c-KIT. Taken together, the data suggests that inhibition of PP2A may represent a general mechanism employed by constitutively active kinases to facilitate tumour growth. As such, this work supports the future application of PP2A-activating agents in a broad range of human malignancies.