7 Abstract Neuroblastoma is the most common extracranial solid tumor of childhood. Despite advances in cancer diagnosis and therapy, the treatment of some forms of neuroblastoma is still complicated. One of the major complications of the chemotherapy is a developed drug resistance. This master thesis deals with the effect of cytostatics on protein and gene expression of selected proteins, which may contribute to chemoresistance of the human neuroblastoma cell line UKF-NB-4. The sensitive line UKF-NB-4 and the resistant line UKF-NB-4CDDP , UKF-NB-4DOXO and UKF-NB-4ELLI were exposed to cisplatin, doxorubicin, ellipticine for 24, 48 and 72 hours. The Western blot analysis showed that cytostatic agents cisplatin, doxorubicin or ellipticine added to the sensitive neuroblastoma cell line UKF-NB-4 in amounts which are added to resistant neuroblastoma cell lines in order to maintain resistance induced expression of p53 and reduced expression of retinoblastoma protein pRb after 72 hours of cultivation. Differences in the expression of RAS protein, cytochrome P450 1A1, 3A4 and cytochrome b5 has not been shown. Changes in the expression of the studied proteins in resistant lines UKF-NB-4CDDP , UKF-NB-4DOXO and UKF-NB-4ELLI cultured with and without cytostatic agents were not detected by the Western blot analysis....

<div>Vertebrate neurogenesis is a multistep process that coordinates complex signaling pathways and chromatin-based regulatory machinery to generate highly specialized cells (Hsieh and Zhao 2016; Urban and Guillemot 2014; Alunni and Bally-Cuif 2016; Yao and Jin 2014; Schmidt, Strahle, and Scholpp 2013). Epigenetic factors play a fundamental role in underwriting neurogenesis in part by contributing to control of gene expression in differentiating neurons. A mechanistic understanding of the epigenetic machinery underlying neurogenesis in vertebrates is necessary both to fully understand biogenesis of neural tissue in this subphylum as well as to develop effective therapeutic strategies to treat diseased or damaged neural tissue. </div><div>An example of an epigenetic factor that is important for both neuronal differentiation and disease states is CHD5, a vertebrate-specific member of the CHD family of ATP-dependent chromatin remodeling proteins. Chromodomain / Helicase / DNA-binding (CHD) proteins play a variety of roles in vertebrate development, and misregulation or loss of CHD proteins has been linked to numerous diseases (Mayes et al. 2014; Marfella and Imbalzano 2007; Bartholomew 2014). CHD5 is expressed primarily in neural tissue, where it is thought to contribute to neurogenesis, and has been strongly linked to tumor suppression (Thompson et al. 2003; Vestin and Mills 2013). Loss of CHD5 plays a significant role in development of neuroblastoma, a devastating tumor that is a leading cause of cancer-related death in children (Jiang, Stanke, and Lahti 2011; Maris and Matthay 1999). Consistent with the disease phenotype associated with loss of CHD5, reduced expression of CHD5 impairs differentiation of neuronal cells (Egan et al. 2013b). However, ablation of CHD5 in mice surprisingly resulted in no detectable defects in brain development (Li et al. 2014; Zhuang et al. 2014). A subsequent report revealed that mice conditionally ablated for CHD5 in neural tissue exhibit symptoms consistent with an autism spectrum disorder (Pisansky et al. 2017). Much remains to be learned about the role of CHD5 in these processes to clarify these observations.</div><div>Further, Chd5 is unique among the family of Chd remodelers in that it provides a biochemical basis for crosstalk between the critical epigenetic marks H3K27me3 and DNA methylation. Chd5 and the closely related remodelers Chd3 and Chd4 are all components of the Mi-2/NuRD histone deacetylase complex that plays a critical role in mediating transcriptional repression in response to DNA methylation in mammals (Allen, Wade, and Kutateladze 2013). Only CHD5 is preferentially expressed in neural tissue, however, and only Chd5 remodelers have biochemical evidence of direct interaction with H3K27me3, which plays a critical role in enabling proper expression of transcriptional programs during neurogenesis (Egan et al. 2013b). Chd5 is thus unique among CHD remodelers in that it is biochemically linked to both DNA methylation and H3K27me3 in addition to being preferentially expressed in neural tissue.</div><div>With regards to mechanism, much remains to be learned regarding how Chd5 remodelers contribute to gene expression and tumor suppression. However, the data to date do not show extensive transcript phenotypes and it is not clear how the biochemical action of CHD5 contributes to the neurological phenotypes ascribed to altered expression of CHD5. Therefore, it is critical to determine a suitable context to study the role of CHD5 in these processes. Identification of CHD5-dependent genes in neurons is likely to generate insight into how loss of CHD5 contributes to tumorigenesis, in particular with regards to development of neuroblastoma. Regulatory pathways that drive neurogenesis have been found to be extensively conserved between humans and zebrafish. Therefore, we have turned to the power of the zebrafish model system to characterize how loss of Chd5 alters brain development during embryogenesis.</div><div>Importantly zebrafish development, and neurogenesis in particular, occurs largely over the first 5-days of development. Zebrafish are born outside of the mother, which can produce large clutches of several hundred embryos per week, providing us with an accessible context to study the role of chd5, the zebrafish homolog of human CHD5. The central nervous system of the zebrafish develops rapidly, and shares many of the organization features of the mammalian brain (Kalueff, Stewart, and Gerlai 2014). In particular, neuroblastoma arises from a population of cells known as sympathetic ganglion cells that are derived from the neural crest (Pei et al. 2013). These cells are conserved in vertebrates, and several models to study how these cells transform into neuroblastoma exist in zebrafish (Zhu et al. 2017; Morrison et al. 2016; Zhu and Thomas Look 2016). However, our understanding of the mechanisms controlling ganglion cell differentiation is incomplete and requires further investigation to understand how epigenetic and transcriptional mechanisms contribute to development of these cells and how failure of these processes leads to cancer. The neural crest undergoes a series of differentiation steps to form mature sympathetic neurons that are guided by bone morphogenic protein signaling, and transcription changes (Ernsberger and Rohrer 2018). These cells express key enzymes for synthesizing dopamine and norephinephrine to control the sympathetic system throughout the central nervous system (Ernsberger and Rohrer 2018).</div><div>To address these questions about Chd5, we have used CRISPR/Cas9 to generate chd5-/- zebrafish that are protein nulls as determined by western blot. These chd5-/- fish are phenotypically indistinguishable from wild-type fish under standard growth conditions as was previously observed for mice lacking CHD5 (Zhuang et al. 2014; Li et al. 2014). By using zebrafish, we are able to perform transcriptome analyses to identify Chd5 target genes at stages much earlier than has previously been performed in mice because we can harvest large amounts of the tissue of interest from the readily accessible embryos. We have therefore undertaken RNA-seq analysis of isolated brains from wild-type and chd5-/- fish to identify chd5-dependent genes in predominantly differentiating (2-day old) and substantially differentiated (5-day old) neural tissue. These data provide a substantively different perspective from previous studies that examine the role of CHD5 in gene expression of differentiated SY-SH5Y cells (Egan et al. 2013a) or in the forebrain of 8-week-old mice (Pisansky et al. 2017). (Jiang, Stanke, and Lahti 2011). One role we identified from this data, is the promotion of development of sympathetic ganglion cells (detailed below), illuminating for the first time a role for chd5 in promoting differentiation of cells directly involved in neuroblatoma.</div><div>We observe not only extensive changes in gene expression, but also identify a novel role for Chd5 in enabling proper splicing during this critical window of neurogenesis in the zebrafish brain. We are further exploring the role of CHD5 in these processes by creating comparable cell culture-based models of loss of CHD5 to determine the conservation of molecular phenotypes observed in zebrafish. Furthermore, this model enables us to leverage the extensive biochemical tools available in cell culture to examine alterations to the chromatin that are difficult to interpret from studies of complex tissues such as the brain. </div><div>Herein I will describe the research progress we have made to understand the role of Chd5 in gene expression and splicing in zebrafish, as well as ongoing work to engineer mouse embryonic stem cells as an additional model to study the transcriptional consequences of loss of CHD5. Critically, the addition of the cell culture model will greatly enable biochemical characterization of the changes that are leading in particular to the changes in gene expression and splicing and will provide us with a context to test for a direct role of CHD5 in these processes. In addition, this thesis will detail the results from ongoing projects using the zebrafish model system, including: development of models in zebrafish to study the tumor suppressive role of Chd5, phenotypes observed using a targeted chemical-genetic screen, and advancement in developing new tools in zebrafish to engineer specific genomic modifications that will greatly expand the power of this vertebrate model.</div><div><br></div>

Adulte ou pédiatrique, les tumeurs solides ont besoin d’oxygène et de nutriments pour se développer et métastaser. Leur apport est assuré par la néo-vascularisation tumorale issue d’un processus multifactoriel appelé l’angiogénèse. Son équilibre est maintenu par une balance entre facteurs pro- et anti-angiogéniques. Elle fait partie des principales cibles pour traiter les cancers et l’inhibition de la voie VEGF en est un facteur clé. Cependant, la réponse aux agents anti-angiogéniques a montré, malgré des résultats encourageants, un effet transitoire associé à l’apparition d’une résistance adaptative de la tumeur.Nous avons étudié l’inhibition de l’angiogénèse et les mécanismes potentiels d’échappement dans les tumeurs pédiatriques solides, et principalement dans le neuroblastome. Le neuroblastome est une tumeur originaire de la crête neurale et atteint généralement l’enfant jeune. Nous avons exploré l’effet anti-tumoral de l’inhibition sélective des récepteurs 1, 2, 3 du VEGF à l’aide de l’inhibiteur à tyrosine kinase axitinib dans divers modèles précliniques de neuroblastome. L’axitinib a montré une activité anti-tumorale modérée associée à une inhibition de la vascularisation. Néanmoins, après un traitement prolongé in vitro, les cellules tumorales IGR-N91-Luc deviennent résistantes à l’axitinib. Elles prolifèrent normalement mais secrètent de «l’ hepatocyte growth factor» (HGF) et activent la voie MAPK. In vivo, le traitement prolongé par axitinib entraîne le développement d’un phénotype plus agressif de la tumeur avec l’augmentation du nombre d’animaux présentant des métastases, associée à une activation de la voie SRC. Ceci nous a conduit à explorer l’effet d’une inhibition ciblant principalement VEGFR2 et MET (récepteur à l’HGF) avec le cabozantinib. Ainsi nous avons contrôlé le développement tumoral en inhibant la néo-vascularisation et l’activation de SRC, et induit la mort cellulaire dans le modèle orthotopique IGR-N91-Luc et inhibé le développement métastatique dans le modèle systémique IMR-32-Luc. Par ailleurs, nous avons étendu notre exploration à d’autres facteurs jouant un rôle dans l’angiogénèse comme FGFR ou PDGFR car ils représentent, comme MET, de puissants oncogènes. Pour cibler simultanément VEGFR et PDGFR, nous avons utilisé l’inhibiteur multi-kinase regorafenib. In vivo, à des doses bien tolérées qui permettent un traitement prolongé, le regorafenib a montré une activité anti-tumorale significative. Cet effet a été associé principalement à une forte inhibition de la vascularisation mais également à l’induction de la mort cellulaire. En élargissant notre étude à d’autres modèles de tumeurs pédiatriques, nous avons observé que son activité est indépendante du type histologique. Compte tenu du caractère oncogénique de PDGFR, nous avons évalué cet inhibiteur dans des modèles présentant une amplification du gène PDGFRA, qui entraine une surexpression et une activation forte du récepteur. Combiné avec des thérapies standards capables d’induire des dommages à l’ADN telles que l’irradiation ou l’irinotecan, l’effet du regorafenib a été potentialisé, notamment dans les modèles amplifiés pour le gène PDGFRA se traduisant par des régressions tumorales. Ces évaluations précliniques soutiennent le développement d’une nouvelle stratégie thérapeutique pour les enfants atteints de tumeurs solides. / Solid tumors either adult or pediatric need oxygen and nutrients to grow and metastasize. Their input is provided by tumor neovascularization after a multifactorial process called angiogenesis. Balance is maintained by equilibrium between pro and anti-angiogenic factors. It is one of the main targets for treating cancers and the inhibition of the VEGF pathway is a key factor. However, despite encouraging results, the response to anti-angiogenic agents showed a transient effect associated with the development of an adaptive tumor resistance. We studied the inhibition of angiogenesis and potential escape mechanisms in solid pediatric tumors, mainly in neuroblastoma. Neuroblastoma is a solid tumor derived from the neural crest and it usually affects childhood. We investigated the anti-tumor effect of selective inhibition of VEGF receptors 1, 2, 3 using the tyrosine kinase inhibitor axitinib in various preclinical neuroblastoma l models. Axitinib showed a moderate anti-tumor activity associated with the inhibition of vascularization. However, after prolonged treatment in vitro, tumor cells IGR-N91-Luc become resistant to axitinib. They proliferate normally but secrete the "hepatocyte growth factor" (HGF) and activate the MAPK pathway. In vivo, prolonged treatment by axitinib results in the development of a more aggressive tumor phenotype with an increase in the number of animals exhibiting metastases associated with an activation of SRC signaling. This led us to explore the effect of inhibiting concomitant VEGFR2 and MET (HGF receptor), main cabozantinib targets. So we stabilized tumor growth by inhibiting the neovascularization and activation of SRC, induced cell death in the orthotopic model IGR-N91-Luc and inhibited metastatic development in the IMR-32-Luc systemic model. In addition, we extended our exploration of other factors that play a role in angiogenesis like FGFR or PDGFR because they represent, like MET, powerful oncogenes. To simultaneously target VEGFR and PDGFR, we used the multi-kinase inhibitor regorafenib. In vivo, at well-tolerated doses that allow prolonged treatment, regorafenib showed significant anti-tumor activity. This effect was mainly associated with a strong inhibition of vascularization, but also (with) induction of cell death. By expanding our study to other models of pediatric tumors, we observed that its activity was independent of histologic type. Given the oncogenic character of PDGFR, we evaluated the inhibitor in models which present a PDGFRA gene amplification, which results in a strong activation of the receptor. Combined with standard therapies that can induce DNA damages such as irinotecan or radiation, the effect of regorafenib was potentiated, mainly in PDGFRA gene amplified models, where tumor regressions were obtained. These preclinical evaluations support the development of a new therapeutic strategy for children with solid tumors.

Das Neuroblastom (NB) ist ein solider, extrakranieller Tumor neuroektodermalen Ursprungs, der sich im Kleinkindalter manifestiert. Ein etabliertes Zielantigen für die passive Immuntherapie beim NB ist das Disialogangliosid GD2. Aufgrund der geringen oder fehlenden Expression von MHC Klasse I Molekülen sowie der Tatsache, dass die Lyse von NB-Zellen durch verschiedene Mechanismen der natürlichen Zytotoxizität von NK-Zellen vermittelt werden kann, stellt eine auf NK-Zellen basierende Therapie einen vielversprechenden Ansatz zur Behandlung dieser Erkrankung dar. Auf dieser Grundlage wurde eine NK-Zelllinie generiert, die einen GD2-spezifischen chimären Antigenrezeptor (CAR) exprimiert (NK-92-scFv(ch14.18)-zeta). Die Hauptbestandteile dieses CARs sind ein Einzelkettenantikörper, welcher die variablen Regionen des GD2-spezifischen Antikörpers ch14.18 enthält, und die CD3ζ-Kette als signaltransduzierende Komponente. Im Rahmen dieser Arbeit konnte gezeigt werden, dass NK-92-scFv(ch14.18)-zeta in der Lage sind, auch Chemotherapie-resistente GD2-positive NB-Zelllinien effektiv abzutöten und dass dabei die Interaktion des CARs mit GD2 den Hauptmechanismus darstellt. Die anti-tumorale Wirkung von NK-92-scFv(ch14.18)-zeta in vivo wurde in einem Chemotherapie-resistenten GD2-positiven Xenograft-Mausmodell gezeigt. Die wiederholte Applikation von NK-92-scFv(ch14.18)-zeta in Kombination mit IL-2 resultierte in einem signifikant verlangsamten Tumorwachstum und einem verbesserten Überleben. Die Ergebnisse dieser Arbeit belegen, dass GD2-spezifische NK-92 das Potential für eine zukünftige klinische Anwendung besitzen. Demnach stellt der Einsatz einer solchen GD2-spezifischen NK-Zelllinie, die unter GMP-Bedingungen expandiert werden kann und zu jeder Zeit in einer standardisierten Qualität verfügbar wäre, eine vielversprechende Alternative zur Behandlung von Hochrisikopatienten dar, deren Erkrankung nicht mehr auf die Standardtherapie anspricht. / Neuroblastoma (NB) is a solid extracranial childhood malignancy of neuroectodermal origin. The Disialoganglioside GD2 is an established antigen for passive immunotherapy of NB. Cellular therapy of NB with natural killer (NK) cells is especially appealing because MHC class I expression is absent or low in most NB, rendering this tumor sensitive to NK cell recognition. Additionally, natural cytotoxicity of NK cells, mediated by interaction of activating NK cell receptors and their respective ligands on tumor cells, has been shown to play a role in lysis of NB cells. It is therefore tempting to assume that a combination of passive immunotherapy with GD2-specific antibodies and adoptive transfer of NK effector cells would result in an improved NB therapy. To achieve this goal an NK cell line expressing a GD2-specific chimeric antigen receptor (CAR) was engineered: NK-92-scFv(ch14.18)-zeta. This CAR consists of a GD2-specific scFv-fragment, which was generated from ch14.18, and the CD3ζ-chain as intracellular signal-transducing domain. Within this thesis, GD2-specificity of NK-92-scFv(ch14.18)-zeta as well as efficacy towards GD2-expressing NB cell lines, including relapse cell lines that exhibit partial or multidrug resistance were demonstrated. Blocking the interaction between the CAR and GD2 resulted in almost complete abrogation of NK-92-scFv(ch14.18)-zeta-mediated lysis of GD2-positive NB cell lines in vitro, indicating that this interaction is the main mechanism of activation of NK-92-scFv(ch14.18)-zeta. Importantly, repeated application of NK-92-scFv(ch14.18)-zeta in combination with IL-2 significantly decreased tumor growth and prolonged survival of mice in an aggressively growing drug-resistant xenograft NB mouse model. These findings suggest that GD2-specific NK-92 has potential for a future clinical application as NB-specific effector cells that would be ready on demand in a standardized quality.

Das Neuroblastom, ein embryonaler Tumor des sympathischen Nervensystems, stellt durch seine außerordentliche Heterogenität klinisch eine große Herausforderung dar. Ziel dieser Arbeit war es, die Expression der GATA-Transkriptionsfaktoren GATA-2, -3, -4 und des Kofaktors friend-of-GATA (FOG)-2 im Neuroblastom und im sich entwickelnden sympathischen Nervensystem zu vergleichen. Davon ausgehend wurde die Rolle der Proteine im Neuroblastom näher untersucht. Es wurde gezeigt, dass alle vier Proteine in humanem Neuroblastom-Gewebe sowie in einer humanen Neuroblastom-Zelllinie (SH-SY5Y) exprimiert werden und nukleär lokalisiert sind. Lediglich Gata-4 wurde jedoch im sich entwickelnden sympathischen Nervensystem der Maus nicht exprimiert. Die Einzigartigkeit von GATA-4 bestätigte sich auch durch Microarray-Analysen von 251 Neuroblastom-Proben. Während GATA-2, -3 und FOG-2 signifikant mit Markern für eine günstige Prognose assoziiert wurden, korrelierte die GATA-4 Expression mit MYCN-Amplifikation. Interessanterweise führte die lentivirale Überexpression von GATA-4 zu einer Proliferationsinhibition humaner Neuroblastomzellen (SH-SY5Y und SH-EP) sowie zu der verstärkten Expression von DPYSL3 und Bcl-2. Zudem konnte durch das Differenzierungsagens Retinsäure die GATA-4 Expression induziert werden. So wurde in dieser Arbeit bestätigt, dass normale Entwicklungsprozesse in prognostisch günstigen Neuroblastomen intakt sind. Umgekehrt sind in Tumoren mit schlechterer Prognose diese Prozesse gestört. Die in vitro verlangsamte Proliferation sowie die Induktion von Bcl-2 nach Überexpression von GATA-4 könnten in vivo bei der schlechteren Therapierbarkeit der prognostisch ungünstigen Neuroblastome eine Rolle spielen. Es ist bekannt, dass die Behandlung mit Retinsäure u. a. durch Bcl-2 zu einer Chemoresistenz führen kann. Da die Expression von GATA-4 durch Retinsäure induziert werden und GATA-4 die Expression von Bcl-2 verstärken kann, könnte GATA-4 an der Chemoresistenz beteiligt sein. / Neuroblastoma, an embryonal tumor of the sympathetic nervous system, remains clinically challenging due to its extreme heterogeneity. The aim of this study was to compare the expression of GATA transcription factors GATA-2, -3, -4 and the cofactor friend-of-GATA (FOG)-2 in neuroblastoma and in the developing sympathetic nervous system. The functional role of these proteins in neuroblastoma was subsequently investigated based on the results of the GATA expression studies. The analysis showed that all four proteins are expressed in human neuroblastoma tissue as well as in a human neuroblastoma cell line (SH-SY5Y) and are localized in the cell nuclei. Only Gata-4, however, was not expressed in the developing murine sympathetic nervous system. Its uniqueness was also confirmed by microarray analyses of 251 neuroblastoma specimens. While GATA-2, -3 and FOG-2 were significantly associated with favorable prognostic markers, GATA-4 expression correlated with MYCN-amplification. Interestingly, lentiviral GATA-4 overexpression led to inhibited proliferation of human neuroblastoma cells (SH-SY5Y and SH-EP) as well as to increased expression of DPYSL3 and Bcl-2. In addition, GATA-4 expression could be induced by the differentiation agent retinoic acid. In conclusion, it was confirmed that normal developmental molecular pathways are intact in prognostically favorable neuroblastoma. In contrast, these developmental processes seem to be defective in tumors with unfavorable prognosis. The slowed proliferation, as observed in vitro, as well as the induction of Bcl-2 brought about by GATA-4 overexpression may contribute in vivo to the difficult treatability of prognostically unfavorable neuroblastoma. It is known that treatment of neuroblastoma with retinoic acid can lead to chemoresistance, mediated by Bcl-2 amongst others. Since retinoic acid can induce the expression of GATA-4 and GATA-4 itself can enhance the expression of Bcl-2, GATA-4 could be involved in chemoresistance.

Das Neuroblastom ist der am weitesten verbreitete solide, extrakranielle Tumor im Kindesalter. Trotz intensiver Forschung sind die Überlebensraten von Patienten mit fortgeschrittenem Tumorwachstum nach wie vor schlecht. Die Idee, eine zelluläre, langanhaltende Immunantwort im Körper zu induzieren, vermittelt durch zytotoxische CD8+T-Zellen, die sich gegen den Tumor richten, scheint dabei besonders attraktiv. Als tumorassoziiertes Antigen (TAA) wurde zu diesem Zweck für diese Arbeit die murine Tyrosinhydroxylase (mTH), das Schrittmacherenzym der Katecholaminbiosynthese, gewählt, da sie in der Mehrzahl der Neuroblastome stark überexprimiert ist. Für die Impfexperimente wurden sog. DNA-Minigen-Vakzine, die für Peptide aus der mTH-Sequenz kodieren, konstruiert. Die Auswahl Minigen-Peptide erfolgte mit dem MHC-Klasse-I-Liganden-Vorhersageprogramm syfpeithi, welches drei vorhergesagte starke H2-Kk-Liganden lieferte (mTH3k). Außerdem wurden zwei weitere Vakzine hergestellt: als Negativkontrolle das Vakzin mTHlowest, dessen mTH-Peptide laut syfpeithi schlechte MHC-Klasse-I-Liganden darstellen und das Vakzin Ersatzepis, dessen Peptide auf der Oberfläche von murinen NXS2-Neuroblastomzellen aus MHC-Klasse-I-Komplexen isoliert werden konnten. Sowohl in prophylaktischen als auch therapeutischen Impfversuchen in Mäusen konnte das Tumorwachstum und die spontane Metastasierung in sekundäre Organe wie die Leber signifikant verhindert werden. Außerdem konnte gezeigt werden, daß der Antitumoreffekt auf der Induktion mTH-spezifischer, zytotoxischer CD8+T Zellen (CTLs) beruht. Zusätzlich und insbesondere interessant für eine eventuelle klinische Anwendung eines auf der TH basierenden DNA-Vakzins verursachte das mTH-Minigen-Vakzin zumindest in Mäusen keine Aktivierung selbst-reaktiver CD8+T-Zellen. Alles in allem lassen die in dieser Arbeit erhaltenen Ergebnisse den Schluß zu, daß sich die Tyrosinhydroxylase als TAA in Form eines DNA-Vakzins zur adjuvanten Therapie des Neuroblastoms eignet. / Therapeutic vaccination against tumor antigens without induction of autoimmunity remains a major challenge in cancer immunotherapy. Here, we demonstrate for the first time effective therapeutic vaccination followed by eradication of established spontaneous neuroblastoma metastases using a tyrosine hydroxylase (TH) DNA minigene vaccine. We identified three novel mouse TH (mTH3) derived peptides with high predicted binding affinity to MHC class I H2-Kk according to prediction program syfpeithi and computer modeling of epitopes into MHC class I binding groove. Subsequently, a DNA minigene vaccine based on pCMV-F3Ub encoding for mutated ubiquitin (G76 to A76) and mTH3 was generated. Prophylactic and therapeutic efficacy of this vaccine was established following oral delivery using attenuated Salmonella typhimurium SL7207. Only mice immunized with mTH3 were free of spontaneous liver metastases. This effect was clearly dependant on ubiquitin and high affinity of the mTH epitopes to MHC class I. Specifically, we demonstrated a crucial role for minigene expression as a stable ubiquitin-Ala76 fusion peptide for vaccine efficacy. Interestingly, the unstable wild type ubiquitin-Gly76 vaccine was completely ineffective. The immune response following mTH3 DNA minigene vaccination was mediated by CD8+ T-cells as indicated by infiltration of primary tumors and TH specific cytolytic activity in vitro. Importantly, no infiltration was detectable in TH expressing adrenal medulla, indicating the absence of auto immunity. In summary, we demonstrate effective therapeutic vaccination against neuroblastoma with a novel rationally designed tyrosine hydroxylase minigene vaccine without induction of autoimmunity providing an important base line for clinical application of this strategy.

<p>Neuroblastoma is a childhood cancer that originates from neuroblasts in the peripheral nervous system. Neuroblastoma show considerable heterogeneity with respect to location, responsiveness to treatment and prognosis. Since current therapy involves drugs with risk of serious side effects in the growing child, there is a clinical need for more effective and less toxic treatment strategies.</p><p>Angiogenesis, the formation of new blood vessels, is critical for tumor progression. Specific inhibition of tumor-induced angiogenesis should restrict growth of most solid tumors and thereby provide a new treatment strategy. The aim of this study was to investigate the effects of angiogenic inhibition in experimental neuroblastoma in mice.</p><p>We found that experimental neuroblastomas expressed the perhaps most potent angiogenic growth factor, VEGF-A, and that plasma VEGF-A levels correlated with tumor size. SU5416, a novel antagonist of VEGFR-1 and 2, reduced angiogenesis and tumor growth in our model. We also investigated the properties of SU11657, a new, orally available, synthetic small molecule multi-targeted tyrosine kinase inhibitor. SU11657, at a well-tolerated dose, was more potent than SU5416 in reducing tumor growth rate and angiogenesis, even in MYCN-amplified tumors. Chemotherapeutics can also inhibit angiogenesis, when administrated daily in a non-toxic dose. CHS 828, a new chemotherapeutic, given orally, alone induced complete neuroblastoma regression in 44 % of the animals. Furthermore, the bisphosphonate zoledronic acid, developed to reduce bone resorption, showed anti-tumor activity in our model. Zoledronic acid was more potent than the angiogenic inhibitor TNP-470. Thus bisphosphonates may have other beneficial properties in patients with cancer apart from preventing bone resorption.</p><p>In conclusion, SU5416, SU11657, CHS 828, and zoledronic acid represent new drugs with potent anti-tumor effects. Angiogenic inhibition as single therapy or in combination with chemotherapeutics may be beneficial in the treatment of rapidly growing and highly vascularized solid tumors of childhood such as neuroblastoma.</p>

The oncogene Anaplastic Lymphoma Kinase (ALK) is a Receptor Tyrosine Kinase (RTK) and was initially discovered as the fusion protein NPM (nucleophosmin)-ALK in a subset of Anaplastic Large Cell Lymphomas (ALCL). Since then more fusion proteins have been identified in a variety of cancers. Further, overexpression of ALK due to gene amplification has been observed in many malignancies, amongst others neuroblastoma, a pediatric cancer. Lately, activating point mutations in the kinase domain of ALK have been described in neuroblastoma patients and neuroblastoma cell lines. In contrast, the physiological function of ALK is still unclear, but ALK is suggested to play a role in the normal development and function of the nervous system. By employing cell culture based approaches, including a tetracycline-inducible PC12 cell system and the in vivo D. melanogaster model system, we aimed to analyze the downstream signalling of ALK and its role in neuroblastoma. First, we wished to analyze whether ALK is able to activate the small GTPase Rap1 contributing to differentiation/proliferation processes. Activated ALK recruits a complex of the GEF C3G and CrkL and activates C3G by tyrosine phosphorylation. This activated complex is able to activate Rap1 resulting either in neurite outgrowth in PC12 cells or proliferation of neuroblastoma cells suggesting a potential role in the oncogenesis of neuroblastoma driven by gain-of-function mutant ALK. Next, we could show that seven investigated ALK mutations with a high probability of being oncogenic (G1128A, I1171N, F1174L, F1174S, R1192P, F1245C and R1275Q), are true gain-of-function mutations, respond differently to ALK inhibitors and have different transforming ability. Especially the F1174S mutation correlates with aggressive disease development. However, the assumed active germ line mutation I1250T is in fact a kinase dead mutation and suggested to act as a dominant-negative receptor. Finally, ALK mutations are most frequently observed in MYCN amplified tumours correlating with a poor clinical outcome. Active ALK regulates mainly the initiation of MYCN transcription in human neuroblastoma cell lines. Further, ALK gain-of-function mutants and MYCN synergize in transforming NIH3T3 cells. Overall, somatic mutations appear to be more aggressive than germ line mutations, implying a different impact on neuroblastoma. Further, successful application of ALK inhibitors suggests a promising future for the development of patient-specific treatments for neuroblastoma patients.

Human endogenous retroviruses (HERVs) constitute about 8% of the human genome. There are around 4000 pol-containing retroviral integrations in the human genome, which makes it impractical to measure each of them separately. Therefore we developed a set of degenerate real time PCRs to detect major groups bearing sequence similarities to gammaretroviruses, one of the largest groups of human endogenous retrovirus, and betaretroviruses, some of which have integrated into the human genome most recently and which remain the most intact. It was found that, although both gammaretroviral and betaretroviral RNAs were broadly expressed in various healthy tissues including reproductive tissues and brain, a differential expression pattern was observed. My work further revealed that HERVE and HERVW, two gammaretroviral sequences, were ubiquitously and highly expressed in pathologic and normal female reproductive tissues with tissue specific patterns. Expression of HERVE was higher in endometriotic tissue than in normal endometrium. HERVE and HERVW RNAs were higher in normal ovarian tissue than in ovarian cancer. Besides these tissue- and neoplasia-related differences, there were wide differences in HERV expression among individuals. Next, a selective pattern of HERVW upregulation was demonstrated in SK-N-DZ, a neuroblastoma cell line, upon re-oxygenation after a period of hypoxia or with 5-azacytidine, a demethylating agent. Furthermore, broad and high expressions of gammaretrovirus-like transcripts in different brain areas analyzed were identified. The expression levels were variable among different donors. In conclusion a ubiquitous HERV expression was observed in tissues and cell lines, with various patterns. At this stage the data are not sufficient to conclude whether HERV has any physiological or pathological roles in humans. However, their differential expression patterns are compatible with functional roles of HERV in humans.

Neuroblastoma is a childhood cancer that originates from neuroblasts in the peripheral nervous system. Neuroblastoma show considerable heterogeneity with respect to location, responsiveness to treatment and prognosis. Since current therapy involves drugs with risk of serious side effects in the growing child, there is a clinical need for more effective and less toxic treatment strategies. Angiogenesis, the formation of new blood vessels, is critical for tumor progression. Specific inhibition of tumor-induced angiogenesis should restrict growth of most solid tumors and thereby provide a new treatment strategy. The aim of this study was to investigate the effects of angiogenic inhibition in experimental neuroblastoma in mice. We found that experimental neuroblastomas expressed the perhaps most potent angiogenic growth factor, VEGF-A, and that plasma VEGF-A levels correlated with tumor size. SU5416, a novel antagonist of VEGFR-1 and 2, reduced angiogenesis and tumor growth in our model. We also investigated the properties of SU11657, a new, orally available, synthetic small molecule multi-targeted tyrosine kinase inhibitor. SU11657, at a well-tolerated dose, was more potent than SU5416 in reducing tumor growth rate and angiogenesis, even in MYCN-amplified tumors. Chemotherapeutics can also inhibit angiogenesis, when administrated daily in a non-toxic dose. CHS 828, a new chemotherapeutic, given orally, alone induced complete neuroblastoma regression in 44 % of the animals. Furthermore, the bisphosphonate zoledronic acid, developed to reduce bone resorption, showed anti-tumor activity in our model. Zoledronic acid was more potent than the angiogenic inhibitor TNP-470. Thus bisphosphonates may have other beneficial properties in patients with cancer apart from preventing bone resorption. In conclusion, SU5416, SU11657, CHS 828, and zoledronic acid represent new drugs with potent anti-tumor effects. Angiogenic inhibition as single therapy or in combination with chemotherapeutics may be beneficial in the treatment of rapidly growing and highly vascularized solid tumors of childhood such as neuroblastoma.