Die Synthese von Glycopeptiden und Glycopeptid-Protein-Konjugaten mit einer Partialstruktur des tumorassoziierten Mucins MUC1 zur Entwicklung von Tumorvakzinen

„Synthese von Glycopeptiden und Glycopeptid-Protein-Konjugaten mit einer Partialstruktur des tumorassoziierten Mucins MUC1 zur Entwicklung von Tumorvakzinen“ Das Glycoprotein MUC1 ist in Tumorepithelzellen sonderlich stark überexprimiert und wegen der vorzeitig einsetzenden Sialylierung sind die Saccharid-Epitope der O-Glycanketten stark verkürzt (sog. tumorassoziierte Antigene). Dadurch werden auch bisher verborgene Peptidepitope des Glycoprotein-Rückgrates auf der Zelloberfläche der Epithelzellen zugänglich, die als fremd von den Zellen des Immunsystems erkannt werden können. Dies macht das MUC1-Zelloberfächenmolekül zu einem Zielmolekül in der Entwicklung von Tumorvakzinen. Diese beiden strukturellen Besonderheiten wurden in der Synthese von Glycohexadecapeptiden verbunden, indem die veränderten tumorassoziierten Saccharidstrukturen TN-, STN- und T-Antigen als Glycosylaminosäure-Festphasenbausteine synthetisiert wurden und in das Peptidepitop der Wiederholungseinheit des MUC1 durch Glycopeptid-Festphasensynthese eingebaut wurden. Wegen der inhärenten schwachen Immunogenität der kurzen Glycopeptide müssen die synthetisierten Glycopeptidstrukturen an ein Trägerprotein, welches das Immunsystem stimuliert, gebunden werden. Zur Anbindung der Glycopeptide ist ein selektives Kupplungsverfahren nötig, um definierte und strukturell einheitliche Glycopeptid-Protein-Konjugate zu erhalten. Es konnte eine neue Methode entwickelt werden, bei der die Konjugation durch eine radikalische Additionsreaktion von als Allylamide funktionalisierten Glycopeptiden an ein Thiol-modifiziertes Trägerprotein erfolgte. Dazu wurde anhand von synthetisierten, als Allylamide modifizierten Modellaminosäuren untersucht, ob diese Reaktion generell für eine Biokonjugation geeignet ist und etwaige Nebenreaktionen auftreten können. Mit dieser Methode konnten verschiedene MUC1-Glycopeptid-Trägerprotein-Konjugate hergestellt werden, deren immunologische Untersuchung noch bevorsteht. Das tumorassoziierte MUC1 nimmt in der immundominanten Region seiner Wiederholungseinheit eine knaufartige Struktur ein. Für die Entwicklung von selektiven Tumorvakzinen ist es von großer Bedeutung möglichst genau die Struktur der veränderten Zelloberflächenmoleküle nachzubilden. Durch die Synthese von cyclischen (Glyco)Peptiden wurde dieses Strukturelement fixiert. Dazu wurden olefinische Aminosäure Festphasenbausteine hergestellt, die zusammen mit den oben genannten Glycosylaminosäuren mittels einer Glycopeptid-Festphasensynthese in acyclische Glycopeptide eingebaut wurden. Diese wurden dann durch Ringschlussmetathese zyklisiert und im Anschluss reduziert und vollständig deblockiert. In einem dritten Projekt wurde der Syntheseweg zur Herstellung einer C-Glycosylaminosäure mit einer N-Acetylgalactosamin-Einheit entwickelt. Wichtige Schritte bei der von Glucosamin ausgehenden Synthese sind die Keck-Allylierung, eine Epimerisierung, die Herstellung eines Brom-Dehydroalanin-Derivates und eine B-Alkyl-Suzuki-Miyaura-Kreuzkupplung sowie Schutzgruppenoperationen. Der racemische Baustein konnte dann in der Peptid-Festphasensynthese eines komplexen MUC1-Tetanustoxin-Konjugates eingesetzt werden.

Untersuchung der DNA-bindenden Eigenschaften neuer Nukleobasen-gekoppelter Pyrrolcarboxamide, Combilexine und Hetaren[a]carbazol-Derivate: Validierung und Etablierung von in-vitro- und in-silico-Methoden

Da maligne Neoplasien durch Mutationen in Proto-Onko- und/oder Tumorsuppressorgenen ausgelöst werden, stellt die DNA eines der wichtigsten Targets für die Entwicklung neuer Zytostatika dar. Auch bei den im Arbeitskreis Pindur designten und synthetisier-ten Verbindungen der Nukleobasen-gekoppelten Pyrrolcarboxamid-, der Hetaren[a]carbazol- und der Combilexin-Reihe handelt es sich um DNA-Liganden mit potentiell antitumoraktiven Eigenschaf-ten. Die einen dualen Bindemodus aufweisenden Combilexine bestehen aus einem Interkalator (u. a. Naphthalimid, Acridon), der über einen Linker variabler Kettenlänge mit einer rinnenbin-denden, von Netropsin abgeleiteten Bispyrrol-, oder einer bioisosteren Imidazol-, Thiazol- oder Thiophen-pyrrolcarboxamid-struktur verknüpft ist. Das N-terminale Ende der Combilexine wird von einer N,N-Dimethylaminopropyl- oder -ethyl-Seitenkette gebildet. Die DNA-Affinitäten der Liganden wurden mittels Tm-Wert-Messung-en bestimmt. Diese Denaturierungsexperimente wurden sowohl mit poly(dAdT)2- als auch mit Thymus-DNA (~42% GC-Anteil) durchge-führt, um Aussagen zur Stärke und zur Sequenzselektivität der DNA-Bindung machen zu können. Des Weiteren wurden die Bindekon-stanten einiger ausgewählter Vertreter mit Hilfe des Ethidium-bromid-Verdrängungsassays ermittelt; einige Testverbindungen wurden zudem auf potentiell vorhandene, TOPO I-inhibierende Eigenschaften untersucht. Diese biochemischen und biophysika-lischen Tests wurden durch Molecular Modelling-Studien ergänzt, die die Berechnung von molekularen Eigenschaften, die Durch-führung von Konformerenanalysen und die Simulation von DNA-Ligand-Komplexen (Docking) umfassten. Durch Korrelation der in vitro-Befunde mit den in silico-Daten gelang es, vor allem für die Substanzklasse der Combilexine einige richtungweisende Struktur-Wirkungsbeziehungen aufzustellen. So konnte gezeigt werden, dass die Einführung eines Imidazol-Rings in die rinnen-bindende Hetaren-pyrrolcarboxamid-Struktur der Combilexine aufgrund der H-Brücken-Akzeptor-Funktion des sp2-hybridisierten N-Atoms eine Verschiebung der Sequenzselektivität der DNA-Bindung von AT- zu GC-reichen Arealen der DNA bedingt. Zudem erwies sich ein C3-Linker für die Verknüpfung des Naphthalimids mit dem rinnenbindenden Strukturelement als am besten geeignet, während bei den Acridon-Derivaten die Verbindungen mit einem N-terminalen Buttersäure-Linker die höchste DNA-Affinität aufwiesen. Dies ist sehr wahrscheinlich auf die im Vergleich zum Naphthalimid-Molekül geringere y-Achsen-Ausdehnung (bzgl. eines x/y-Koordinatensystems) des Acridons zurückzuführen. Die ermittelten Struktur-Wirkungsbeziehungen können dazu herangezogen werden, das rationale Design neuer DNA-Liganden mit potentiell stärkerer DNA-Bindung zu optimieren.

Ruolo dei recettori tirosinchinasici in oncologia animale

Disregolazioni dei recettori tirosinchinasici (RTK) sono di frequente riscontro nei tumori dell’uomo e in molti casi sono indicatori biologici che permettono di definire in maniera più accurata la prognosi dei pazienti. Possono rappresentare inoltre marker predittivi per la risposta a terapie antitumorali con farmaci a bersaglio molecolare. Numerosi inibitori tirosinchinasici (TKI) sono attualmente in corso di studio o già disponibili per l’utilizzo in oncologia umana, e molti di questi hanno dimostrato una significativa efficacia utilizzati singolarmente o in combinazione a terapie convenzionali. Studi recenti indicano che un quadro analogo di disregolazione dei recettori tirosinchinasici è presente anche nelle neoplasie dei piccoli animali, e ne suggeriscono in molti casi un’implicazione prognostica. Gli inibitori tirosinchinasi sono da poco entrati nell’arena dell’oncologia veterinaria, ma i primi risultati lasciano supporre che siano destinati ad essere integrati definitivamente nei protocolli terapeutici standard. La tesi consiste in una parte introduttiva in cui sono trattate le principali funzioni biologiche dei recettori tirosinchinasici, la loro struttura e il loro ruolo nell’oncogenesi e nella progressione tumorale in medicina umana e veterinaria. Si affrontano inoltre le principali metodiche di laboratorio per l’analisi molecolare in oncologia e i meccanismi d’azione dei farmaci inibitori tirosinchinasici, con un cenno ai prodotti maggiormente utilizzati e alle loro indicazioni. Segue la presentazione e la discussione dei risultati di quattro studi relativi alla valutazione delle disregolazioni del recettore tirosinchinasico Kit (espressione aberrante e mutazioni genomiche) nel mastocitoma cutaneo del gatto e del recettore del fattore di crescita epidermico (EGFR) nel carcinoma squamocellulare cutaneo del gatto e nei tumori polmonari primitivi del cane, con particolare attenzione al loro ruolo prognostico.

Transgenic mice as a tool for depression research: examples from the endocannabinoid and the corticotropin-releasing hormone system

Major depression belongs to the most serious and widespread psychiatric disorders in today’s society. There is a great need for the delineation of the underlying molecular mechanisms as well as for the identification of novel targets for its treatment. In this thesis, transgenic mice of the endocannabinoid and the corticotropin-releasing hormone (CRH) system were investigated to determine the putative role of these systems for depression-like phenotypes in mice. In the first part of the thesis, we found that the endocannabinoid system was prominently involved in a brain region-specific and temporally controlled manner in acute as well as in chronic stress processing. Genetic deletion in combination with pharmacological intervention revealed the importance of a fully functional endocannabinoid system for efficient neuroendocrine and behavioral stress coping. Accordingly, cannabinoid type 1 (CB1) receptor-deficient mice displayed several depression-like symptoms and molecular alterations, including “behavioral despair”, stress hormone hypersecretion and decreased glucocorticoid receptor and brain-derived neurotrophic factor expression in the hippocampus. However, the endocannabinoid system was dispensable for the efficacy of currently used antidepressant drugs. To facilitate future endocannabinoid research, a transgenic mouse was generated, which overexpressed the CB1 receptor protein fused to a fluorescent protein. In the second part of the thesis, conditional brain region-specific CRH overexpressing mice were evaluated as a model for pathological chronic CRH hyperactivation. Mutant mice showed aberrant neuroendocrine and behavioral stress coping and hyperarousal due to CRH-induced activation of the noradrenergic system in the brain. Mutant mice appeared to share similarities with naturally occurring endogenous CRH activation in wild-type mice and were sensitive to acute pharmacological blockade of CRH receptor type 1 (CRH-R1). Thus, CRH overexpressing mice serve as an ideal in vivo tool to evaluate the efficacy of novel CRH-R1 antagonists. Together, these findings highlight the potential of transgenic mice for the understanding of certain endo-phenotypes (isolated symptoms) of depression and their molecular correlates.

Gamma-secretase modulators and inhibitors in Alzheimer's disease: influence of genetic background on efficacy and mechanism of action

According to the amyloid hypothesis, Alzheimer’s disease (AD) is caused by aberrant production or clearance of the amyloid-β (Aβ) peptides, and in particular of the longer more aggregation-prone Aβ42. The Aβ peptides are generated through successive proteolytic cleavage of the amyloid precursor protein (APP) by the β-site APP cleaving enzyme (BACE) and γ-secretase. γ-secretase produces Aβ peptides with variable C-termini ranging from Aβ34 to Aβ48, presumably by sequential trimming of longer into shorter peptides. γ-secretase is a multiprotein complex consisting of at least four different proteins and the presenilin proteins (PS1 or PS2) contain the catalytic center of the complex. In 2001 several non-steroidal anti-inflammatory drugs were identified as the founding members of a new class of γ-secretase modulators (GSMs) that can selectively reduce production of Aβ42. Concomitantly, these GSMs increase Aβ38 production indicating closely coordinated generation of Aβ42 and Aβ38 and a potential precursor-product relationship between these peptides. GSMs seem to exert their activity by direct modulation of γ-secretase. Support for this hypothesis is drawn from the finding that some PS mutations associated with early-onset familial AD (FAD) can modulate the cellular response to GSMs and to γ-secretase inhibitors (GSIs), which inhibit production of all Aβ peptides and are known to directly interact with PS. A particularly interesting FAD PS mutation is PS1-ΔExon9, a complex deletion mutant that blocks endoproteolysis of PS1 and renders cells completely non-responsive to GSMs. Studies presented in this thesis show that the diminished response of PS1-ΔExon9 to GSMs is mainly caused by its lack of endoproteolytic cleavage. Furthermore, we were able to demonstrate that a reduced response to GSMs and GSIs is not limited to PS1-ΔExon9 but is a common effect of aggressive FAD-associated PS1 mutations. Surprisingly, we also found that while the Aβ42 response to GSMs is almost completely abolished by these PS1 mutations, the accompanying Aβ38 increase was indistinguishable to wild-type PS1. Finally, the reduced response to GSIs was confirmed in a mouse model with transgenic expression of an aggressive FAD-associated PS1 mutation as a highly potent GSI failed to reduce Aβ42 levels in brain of these mice. Taken together, our findings provide clear evidence for independent generation of Aβ42 and Aβ38 peptides, and argue that the sequential cleavage model might be an oversimplification of the molecular mechanism of γ-secretase. Most importantly, our results highlight the significance of genetic background in drug discovery efforts aimed at γ-secretase, and indicate that the use of cellular models with transgenic expression of FAD-associated PS mutations might confound studies of the potency and efficacy of GSMs and GSIs. Therefore, such models should be strictly avoided in the ongoing preclinical development of these promising and potentially disease-modifying therapeutics for AD.

adaptive capabilities of the pi3k/akt/mtor pathway in acute myeloid leukemia revealed by the use of selective inhibitors

Because of its aberrant activation, the PI3K/AKT/mTOR signaling pathway represents a pharmacological target in blast cells from patients with acute myelogenous leukemia (AML). Using Reverse Phase Protein Microarrays (RPMA), we have analyzed 20 phosphorylated epitopes of the PI3K/Akt/mTor signal pathway of peripheral blood and bone marrow specimens of 84 patients with newly diagnosed AML. Fresh blast cells were grown for 2 h, 4 h or 20 h untreated or treated with a panel of phase I or phase II Akt allosteric inhibitors, either alone or in combination with the mTOR kinase inhibitor Torin1 or the broad RTK inhibitor Sunitinib. By unsupervised hierarchical clustering a strong phosphorylation/activity of most of the sampled members of the PI3K/Akt/mTOR pathway was observed in 70% of samples from AML patients. Remarkably, however, we observed that inhibition of Akt phosphorylation, as well as of its substrates, was transient, and recovered or even increased far above basal level after 20 h in 60% samples. We demonstrated that inhibition of Akt induces FOXO-dependent insulin receptor expression and IRS-1 activation, attenuating the effect of drug treatment by reactivation of PI3K/Akt. Consistent with this model we found that combined inhibition of Akt and RTKs is much more effective than either alone, revealing the adaptive capabilities of signaling networks in blast cells and highliting the limations of these drugs if used as monotherapy.

Spezifische Inhibition des Wnt-Signalwegs und die Analyse der Konsequenzen auf zellbiologischer Ebene

Der kanonische Wnt Signalweg ist durch Regulation einer Vielzahl von Zielgenen in unterschiedliche Prozesse wie Entwicklung, Wachstum und Differenzierung involviert. Fehlregulation des Signalwegs kann zur Tumorentstehung führen. Die exakte Rolle des Wnt Signalwegs und seiner Zielgene in der karzinogenen Kaskade ist noch nicht genau bekannt. In dieser Arbeit sollte die Beteiligung der Wnt Zielgene c-MYC, CCND1 (kodiert Cyclin D1) und VEGF an der Karzinogenese untersucht werden. Um die Funktionen der Wnt Zielgene und ihre zellulären Effekte unabhängig voneinander untersuchen zu können, wurden die Mengen der entsprechenden Transkriptionsprodukte durch siRNA (short interfering RNA) gezielt verringert. Die Konsequenzen der Inaktivierung wurden in Kolon- und Zervixkarzinomzelllinien untersucht, wobei die zellulären Parameter Proliferation, Apoptose, Metabolismus sowie Migration und Adhäsion untersucht wurden. Dabei konnte beobachtet werden, dass der Wnt Signalweg mit seinen Zielgenen Cyclin D1 und c-MYC die Proliferation mit dem Energiemetabolismus von Tumorzellen verknüpft. Darüber hinaus konnte gezeigt werden, dass Cyclin D1 an der Regulation der zelluläre Migration und Adhäsion beteiligt ist, während VEGF die Apoptose abhängig vom zellulären Kontext inhibiert. Diese Ergebnisse liefern erste Hinweise auf die funktionelle Rolle der verschiedenen Zielgene im Prozess der Karzinogenese in Tumoren mit aktiviertem Wnt Signalweg. Damit ist diese Arbeit ein möglicher Ausgangspunkt für Studien mit dem Ziel der gezielten therapeutischen Beeinflussung des Wnt Signalwegs auf Ebene der Zielgene.

Control of protein degradation pathways by BAG proteins and changes during aging

Many age-related neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and polyglutamine disorders, including Huntington’s disease, are associated with the aberrant formation of protein aggregates. These protein aggregates and/or their precursors are believed to be causally linked to the pathogenesis of such protein conformation disorders, also referred to as proteinopathies. The accumulation of protein aggregates, frequently under conditions of an age-related increase in oxidative stress, implies the failure of protein quality control and the resulting proteome instability as an upstream event of proteinopathies. As aging is a main risk factor of many proteinopathies, potential alterations of protein quality control pathways that accompany the biological aging process could be a crucial factor for the onset of these disorders.rnrnThe focus of this dissertation lies on age-related alterations of protein quality control mechanisms that are regulated by the co-chaperones of the BAG (Bcl-2-associated athanogene) family. BAG proteins are thought to promote nucleotide exchange on Hsc/Hsp70 and to couple the release of chaperone-bound substrates to distinct down-stream cellular processes. The present study demonstrates that BAG1 and BAG3 are reciprocally regulated during aging leading to an increased BAG3 to BAG1 ratio in cellular models of replicative senescence as well as in neurons of the aging rodent brain. Furthermore, BAG1 and BAG3 were identified as key regulators of protein degradation pathways. BAG1 was found to be essential for effective degradation of polyubiquitinated proteins by the ubiquitin/proteasome system, possibly by promoting Hsc/Hsp70 substrate transfer to the 26S proteasome. In contrast, BAG3 was identified to stimulate the turnover of polyubiquitinated proteins by macroautophagy, a catabolic process mediated by lysosomal hydrolases. BAG3-regulated protein degradation was found to depend on the function of the ubiquitin-receptor protein SQSTM1 which is known to sequester polyubiquitinated proteins for macroautophagic degradation. It could be further demonstrated that SQSTM1 expression is tightly coupled to BAG3 expression and that BAG3 can physically interact with SQSTM1. Moreover, immunofluorescence-based microscopic analyses revealed that BAG3 co-localizes with SQSTM1 in protein sequestration structures suggesting a direct role of BAG3 in substrate delivery to SQSTM1 for macroautophagic degradation. Consistent with these findings, the age-related switch from BAG1 to BAG3 was found to determine that aged cells use the macroautophagic system more intensely for the turnover of polyubiquitinated proteins, in particular of insoluble, aggregated quality control substrates. Finally, in vivo expression analysis of macroautophagy markers in young and old mice as well as analysis of the lysosomal enzymatic activity strongly indicated that the macroautophagy pathway is also recruited in the nervous system during the organismal aging process.rnrnTogether these findings suggest that protein turnover by macroautophagy is gaining importance during the aging process as insoluble quality control substrates are increasingly produced that cannot be degraded by the proteasomal system. For this reason, a switch from the proteasome regulator BAG1 to the macroautophagy stimulator BAG3 occurs during cell aging. Hence, it can be concluded that the BAG3-mediated recruitment of the macroauto-phagy pathway is an important adaptation of the protein quality control system to maintain protein homeostasis in the presence of an enhanced pro-oxidant and aggregation-prone milieu characteristic of aging. Future studies will explore whether an impairment of this adaptation process may contribute to age-related proteinopathies.

HIF1α regulates Mitochondrial biogenesis and Cellular senescence induced by Gamma Radiation

Cellular response to γ-rays is mediated by ATM-p53 axis. When p53 is phosphorylated, it can transactivate several genes to induce permanent cell cycle arrest (senescence) or apoptosis. Epithelial and mesenchymal cells are more resistant to radiation-induced apoptosis and respond mainly by activating senescence. Hence, tumor cells in a senescent state might remain as “dormant” malignant in fact through disruption of p53 function, cells may overcome growth arrest. Oncocytic features were acquired in the recurring neoplasia after radiation therapy in patient with colonrectal cancer. Oncocytic tumors are characterized by aberrant biogenesis and are mainly non-aggressive neoplasms. Their low proliferation degree can be explained by chronic destabilization of HIF1α, which presides to adaptation to hypoxia and also plays a pivotal role in hypoxia-related radio-resistance. The aim of the present thesis was to verify whether mitochondrial biogenesis can be induced following radiation treatment, in relation of HIF1α status and whether is predictive of a senescence response. In this study was demonstrate that mitochondrial biogenesis parameters like mitochondrial DNA copy number could be used for the prediction of hypoxic status of tissue after radiation treatment. γ-rays induce an increase of mitochondrial mass and function, in response to a genotoxic stress that pushes cells into senescence. Mitochondrial biogenesis is only indirectly regulated by p53, whose activation triggers a MDM2-mediated HIF1α degradation, leading to the release of PGC-1β inhibition by HIF1α. On the other hand, this protein blunts the mitochondrial response to γ-rays as well as the induction of p21-mediated cell senescence, indicating prevalence of the hypoxic over the genotoxic response. Finally in vivo, post-radiotherapy mtDNA copy number increase well correlates with lack of HIF1α increase in the tissue, concluding this may be a useful molecular tool to infer the trigger of a hypoxic response during radiotherapy, which may lead to failure of activation of senescence.

In vivo consequences of AML1-ETO fusion protein expression for hematopoiesis

The t(8;21) (q22;q22) translocation fusing the ETO (also known as MTG8) gene on human chromosome 8 with the AML1 (also called Runx1 or CBFα) gene on chromosome 21 is one of the most common genetic aberrations found in acute myeloid leukemia (AML). This chromosomal translocation occurs in 12 % of de novo AML cases and in up to 40 % of the AML-M2 subtype of the French-American-British classification. To date, the in vivo function of aberrant AML1-ETO fusion protein expression has been investigated by several groups. However, in these studies, controversial results were reported and some key issues remain unknown. Importantly, the consequences of aberrant AML1-ETO expression for self-renewing hematopoietic stem cells (HSCs), multipotent hematopoietic progenitors (MPPs) and lineage-restricted precursors are not known. rn The aim of this thesis was to develop a novel experimental AML1-ETO in vivo model that (i) overcomes the current lack of insight into the pre-leukemic condition of t(8;21)-associated AML, (ii) clarifies the in vivo consequences of AML1-ETO for HSCs, MPPs, progenitors and more mature blood cells and (iii) generates an improved mouse model suitable for mirroring the human condition. For this purpose, a conditional tet on/off mouse model expressing the AML1-ETO fusion protein from the ROSA26 (R26) locus was generated. rn Aberrant AML1-ETO activation in compound ROSA26/tetOAML1-ETO (R26/AE) mice caused high rates of mortality, an overall disruption of hematopoietic organs and a profound alteration of hematopoiesis. However, since the generalized activity of the R26 locus did not recapitulate the leukemic condition found in human patients, it was important to restrict AML1-ETO expression to blood cell lineages. Therefore, bone marrow cells from non-induced R26/AE mice were adoptively transplanted into sublethal irradiated RAG2-/- recipient mice. First signs of phenotypical differences between AML1-ETO-expressing and control mice were observed after eight to nine months of transgene induction. AML1-ETO-expressing mice showed profound changes in hematopoietic organs accompanied by manifest extramedullary hematopoiesis. In addition, a block in early erythropoiesis, B- and T-cell maturation was observed and granulopoiesis was significantly enhanced. Most interestingly, conditional activation of AML1-ETO in chimeric mice did not increase HSCs, MPPs, common lymphoid precursors (CLPs), common myeloid progenitors (CMPs) and megakaryocyte-erythrocyte progenitors (MEPs) but promoted the selective amplification of granulocyte-macrophage progenitors (GMPs). rn The results of this thesis provide clear experimental evidence how aberrant AML1-ETO modulates the developmental properties of normal hematopoiesis and establishes for the first time that AML1-ETO does not increase HSCs, MPPs and common lineage-restricted progenitor pools but specifically amplifies GMPs. The here presented mouse model not only clarifies the role of aberrant AML1-ETO for shaping hematopoietic development but in addition has strong implications for future therapeutic strategies and will be an excellent pre-clinical tool for developing and testing new approaches to treat and eventually cure AML.rn

Validation of a new method to assess the long-term Hypothalamic-Pituitary-Adrenal Activity using hair glucocorticoids as biomarkers. - Studies on pigs and laboratory Sprague-Dawley Rats

The evaluation of chronic activity of the hypothalamic-pituitary-adrenal (HPA) axis is critical for determining the impact of chronic stressful situations. The potential use of hair glucocorticoids as a non-invasive, retrospective, biomarker of long term HPA activity is of great interest, and it is gaining acceptance in humans and animals. However, there are still no studies in literature examining hair cortisol concentration in pigs and corticosterone concentration in laboratory rodents. Therefore, we developed and validated, for the first time, a method for measuring hair glucocorticoids concentration in commercial sows and in Sprague-Dawley rats. Our preliminary data demonstrated: 1) a validated and specific washing protocol and extraction assay method with a good sensitivity in both species; 2) the effect of the reproductive phase, housing conditions and seasonality on hair cortisol concentration in sows; 3) similar hair corticosterone concentration in male and female rats; 4) elevated hair corticosterone concentration in response to chronic stress manipulations and chronic ACTH administration, demonstrating that hair provides a good direct index of HPA activity over long periods than other indirect parameters, such adrenal or thymus weight. From these results we believe that this new non-invasive tool needs to be applied to better characterize the overall impact in livestock animals and in laboratory rodents of chronic stressful situations that negatively affect animals welfare. Nevertheless, further studies are needed to improve this methodology and maybe to develop animal models for chronic stress of high interest and translational value in human medicine.

Misregulation of the tumour suppressor gene CYLD drives hyperactivation of T cells leading to intestinal pathology

The tumour suppressor gene cyld is mutated in familial cylindromatosis, an autosomal-dominant condition that predisposes to multiple skin tumours. The deubiquitinase CYLD acts as a negative regulator of NF-κB signaling. To analyse the function of CYLD in vivo we used the CYLDex7/8 mice, which are characterized by loss of the full-length transcript and overexpression of a short splice variant of CYLD (sCYLD). In CYLDex7/8 mice the overexpression of sCYLD results in splenomegaly and lymphadenopathy. Additionally, the B cell population in spleen and lymph nodes is increased at the expense of T cells. Analysis of CYLDex7/8 T cells showed a significant reduction of CD4 single positive (SP) and CD8 SP T cells in the thymus and in the periphery. By investigating the impact of sCYLD in TCR signaling in thymocytes, we could demonstrate that sCYLD partially inhibited the activation of Zap70 and thereby negatively regulated TCR signaling. In vitro as well as in vivo we could show that CD4+ T cells displayed a hyperactive phenotype, proliferated to a better extent than WT cells and expressed high amounts of inflammatory cytokines such as IL-6 and IL-17A. Western Blots of steady state thymocytes and peripheral CD4+ T cells were performed, showing that the noncanonical pathway was highly upregulated visualized by the expression levels of RelB and p100 leading to a hyperactive phenotype of CD4+ T cells. In order to investigate the contribution of sCYLD in positive and negative selection in the thymus in vivo, the HY-TCR transgene (HYtg) was crossed to CYLDex7/8 mice. The analysis of CYLDex7/8 HYtg males revealed an increase in CD4+CD8+ DP as well as in CD8+ SP thymocytes, suggesting a less pronounced negative selection in CYLD mutant mice compared to HYtg control mice. Interestingly, the impaired negative selection in the thymus was accompanied by a strong colitis phenotype at early ages (4 weeks). Since medullary TECs (mTECs) play an important role in the late stage of T cell development by negatively selecting autoreactive thymocytes, the levels of mTECs in the medullary compartment was investigated. Of note, low numbers of mTECs were observed, combined with decreased expression levels of the mTEC markers UEA-1, keratin-5, claudin-3 and claudin-4. The reduction of mTECs in the medullary compartment could explain the inflammatory phenotype of CD4+ T cells in CYLDex7/8 mice leading to the severe intestinal pathology observed in these mice. Taken together, these results show an important role of sCYLD in T cell development and function as well as in NF-кB signaling of T cells.

Caratterizzazione e ruolo di PKCε e PKCδ in modelli di differenziamento megacariocitario normale e patologico

La PKCε e la PKCδ, chinasi ubiquitariamente distribuite e ad azione pleiotropica, sono implicate del differenziamento, sopravvivenza e proliferazione cellulare. Esse sono coinvolte nel processo differenziativo delle cellule staminali ematopoietiche e in fenomeni patologici associati al compartimento sanguigno. In questa tesi sono presentati i risultati riguardanti lo studio in vitro del ruolo di PKCε e PKCδ nel contesto del differenziamento megacariocitario, in particolare si caratterizza l’espressione e la funzione di queste chinasi nel modello umano e nel modello murino di Megacariocitopoiesi, normale e patologica. Confrontando le cinetiche dei due modelli presi in analisi nello studio è stato possibile osservare come in entrambi PKCε e PKCδ dimostrino avere una chiara e specifica modulazione nel progredire del processo differenziativo. Questi dati, se confrontati, permettono di affermare che PKCε e PKCδ presentano un pattern di espressione opposto e, nel modello umano rispetto a quello murino, reciproco: nell’uomo i livelli di PKCε devono essere down-modulati, mentre nel topo, al contrario, i livelli della chinasi risultano up-modulati durante lo stesso processo. Analogamente, le CD34+ in differenziazione presentano una costante e maggiore espressione di PKCδ durante la maturazione MK, mentre nel modello murino tale proteina risulta down-modulata nella fase più tardiva di formazione della piastrina. Le chinasi mostrano in oltre di agire, nei due modelli, attraverso pathways distinti e cioè RhoA nel topo e Bcl-xL nell’uomo. È stato inoltre verificato che l’aberrante differenziamento MK osservato nella mielofibrosi primaria (PMF), è associato a difetti di espressione di PKCε e di Bcl-xL e che una forzata down-modulazione di PKCε porta ad un ripristino di un normale livello di espressione di Bcl-xL così come della popolazione di megacariociti formanti propiastrine. I dati ottenuti indicano quindi che PKCε e PKCδ svolgono un ruolo importante nel corretto differenziamento MK e che PKCε potrebbe essere un potenziale nuovo target terapeutico nelle PMF.

Study of PI3K/Akt signaling pathway as potential molecular target for T-cell acute lymphoblastic leukemia (T-ALL) treatment: pan-inhibition of PI3K catalitic isoforms as better therapeutic approach

Class I phosphatidylinositol 3-kinases (PI3Ks) are heterodimeric lipid kinases consisting of a regulatory subunit and one of four catalytic subunits (p110α, p110β, p110γ or p110δ). p110γ/p110δ PI3Ks are highly enriched in leukocytes. In general, PI3Ks regulate a variety of cellular processes including cell proliferation, survival and metabolism, by generating the second messenger phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). Their activity is tightly regulated by the phosphatase and tensin homolog (PTEN) lipid phosphatase. PI3Ks are widely implicated in human cancers, and in particular are upregulated in T-cell acute lymphoblastic leukemia (T-ALL), mainly due to loss of PTEN function. These observations lend compelling weight to the application of PI3K inhibitors in the therapy of T-ALL. At present different compounds which target single or multiple PI3K isoforms have entered clinical trials. In the present research, it has been analyzed the therapeutic potential of the pan-PI3K inhibitor BKM120, an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T-lymphoblasts. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. BKM120 efficacy was confirmed in in vivo studies to a subcutaneous xenotransplant model of human T-ALL. Because it is still unclear which agents among isoform-specific or pan inhibitors can achieve the greater efficacy, further analyses have been conducted to investigate the effects of PI3K inhibition, in order to elucidate the mechanisms responsible for the proliferative impairment of T-ALL. Overall, these results indicated that BKM120 may be an efficient treatment for T-ALLs that have aberrant up-regulation of the PI3K signaling pathway and strongly support clinical application of pan-class I PI3K rather than single-isoform inhibitors in T-ALL treatment.

IGF system, CD99 and tumor-specific chromosomal translocations: evaluation of their cross-talk and definition of their role in Ewing sarcoma and prostate cancer

Aberrant expression of ETS transcription factors, including FLI1 and ERG, due to chromosomal translocations has been described as a driver event in initiation and progression of different tumors. In this study, the impact of prostate cancer (PCa) fusion gene TMPRSS2-ERG was evaluated on components of the insulin-like growth factor (IGF) system and the CD99 molecule, two well documented targets of EWS-FLI1, the hallmark of Ewing sarcoma (ES). The aim of this study was to identify common or distinctive ETS-related mechanisms which could be exploited at biological and clinical level. The results demonstrate that IGF-1R represents a common target of ETS rearrangements as ERG and FLI1 bind IGF-1R gene promoter and their modulation causes alteration in IGF-1R protein levels. At clinical level, this mechanism provides basis for a more rationale use of anti-IGF-1R inhibitors as PCa cells expressing the fusion gene better respond to anti-IGF-1R agents. EWS-FLI1/IGF-1R axis provides rationale for combination of anti-IGF-1R agents with trabectedin, an alkylator agent causing enhanced EWS-FLI1 occupancy on the IGF-1R promoter. TMPRSS2-ERG also influences prognosis relevance of IGF system as high IGF-1R correlates with a better biochemical progression free survival (BPFS) in PCa patients negative for the fusion gene while marginal or no association was found in the total cases or TMPRSS2-ERG-positive cases, respectively. This study indicates CD99 is differentially regulated between ETS-related tumors as CD99 is not a target of ERG. In PCa, CD99 did not show differential expression between TMPRSS2-ERG-positive and –negative cells. A direct correlation was anyway found between ERG and CD99 proteins both in vitro and in patients putatively suggesting that ERG target genes comprehend regulators of CD99. Despite a little trend suggesting a correlation between CD99 expression and a better BPFS, no clinical relevance for CD99 was found in the field of prognostic biomarkers.