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.

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.

Le neuroblastome à haut risque est caractérisé par un faible taux de survie (~30%) et des récidives fréquentes, malgré les traitements multimodaux existants. Généralement, les études d’évaluation des médicaments utilisent la culture cellulaire en 2D, mais elle ne reflète pas la biologie tumorale du neuroblastome in situ, incluant les caractéristiques associées à l’hypoxie et la densité cellulaire. En comparaison aux patients, la culture cellulaire conventionnelle semble altérer le phénotype cellulaire du neuroblastome, le transcriptome et l’épigénome qui affecteront, à leur tour, les résultats des études pharmacologiques. Ainsi, un nouveau modèle de culture cellulaire en 3D a été développé avec plusieurs lignées cellulaires de neuroblastome afin d’atteindre une culture de sphéroïdes à long terme (un mois) avec un taux de viabilité convenable. L’hypothèse de recherche est que les changements épigénétiques et transcriptionnels seront induits par l’adaptation en 3D et vont s’amplifier dans le temps lors de la culture en 3D. Ces changements ont été mesurés en 3D dans le temps pour identifier le moment où l’épigénome des sphéroïdes ressemble le plus à celui des patient à haut risque. Le changement de l’expression des régulateurs épigénétiques survient 24 jours après la mise en sphéroïde, ce qui se traduit par une différence dans la sensibilité aux médicaments épigénétiques par rapport à la culture 2D. En conclusion, notre étude nous a permis de dériver des nouveaux modèles de neuroblastome qui sont plus représentatifs des patients d’un point de vue épigénétique et pharmacologique. / High-risk neuroblastoma is characterized by a low survival rate (~30%) and frequent recurrences, despite all the multimodal treatments available to date. Typically, drug evaluation studies use 2D cell culture which does not reflect well the tumor biology of neuroblastoma in situ, including features associated with hypoxia and cell density. Thus, compared to patients data, the conventional 2D cell culture seems to alter the neuroblastoma cell phenotype, transcriptome and epigenome which in turn will affect the results of pharmacological studies. A new 3D cell culture model was developed with several neuroblastoma cell lines to achieve long term spheroid culture (up to one month) with a suitable viability rate. We hypothesized that transcriptional and epigenetic changes will be induced by 3D adaptation and will amplify over time in the cells cultured in 3D. All these changes were measured by Western Blot in 2D, in short term 3D and in long term 3D to identify the timepoint when the epigenome of the spheroids most closely resembles that of the patient. We found that the occurrence of changes in epigenetic regulator expression occurs after 24 days of spheroid culture, resulting in a difference in a drug sensitivity compared to 2D culture. In summary, we developed new neuroblastoma models that are more representative of patient’s epigenome and pharmacological sensitivity.