Beschreibung:
Abstract: Rhabdomyosarcomas are the most frequent soft tissue sarcomas in childhood and adolescence, but they can occur at any age with an overall survival of around 65%. Cured patients are often affected by long-term side effects of the multimodal treatment. Hence, it is necessary to improve existing therapies or develop new therapies. This thesis deepens the understanding of genomic drivers and metabolic interactions resulting in new targets for novel therapies. The sarcoma-relevant genes studied in this thesis were identified by Hettmer et al. in 2015, using a customized shRNA-based proliferation screen. Possible pharmacological intervention has been reported for the candidate genes ASNS, UBE2C, CENPE, HAS2 and CREB3L2. The effects of treatment with bortezomib, latrunculin A, sorafenib and UA62784 on candidate genes UBE2C, CENPE, HAS2 and CREB3L2 and sarcoma growth were studied in this thesis by gene expression analysis and proliferation assays. The chemicals bortezomib, latrunculin A and UA62784 inhibited the growth of human and mouse RMS cell lines, whereas sorafenib increased the proliferation of four out of six sarcoma cell lines. Additional experiments showed that treatment with latrunculin A resulted in disruption of the actin cytoskeleton. These results highlighted the need of further research aiming to improve the knowledge of sarcoma-relevant genes in RMS and their possible therapeutic value. In addition, in the underlying functional proliferation screen, asparagine synthetase was identified as the top hit. Based on this finding, we investigated the effects of asparagine synthetase expression and asparagine availability on the metabolism and growth of rhabdomyosarcoma. Therefore, a metabolomic approach using mass spectrometry in combination with cell and molecular biology methods was applied. Upon asparagine starvation the metabolism of rhabdomyosarcoma was strongly impaired in comparison to wild type cells, resulting in lower intracellular aspartate concentration, increased aspartate/glutamine ratio, reduced nicotinamide adenine dinucleotide (NAD+)/nicotinamide adenine dinucleotide hydroygen ratio (NADH), redirection of TCA cycle flux associated to lower concentrations of tricarboxylic acid cycle metabolites, and increased lactate/pyruvate ratio. All taken together, these changes indicated a redirection of the metabolism towards aspartate synthesis in the context of low asparagine availability. The reduced NAD+/NADH and the increased lactate/pyruvate ratios were connected to a shortage of NAD+ regeneration via the electron transport chain and could be rescued by the supplementation of pyruvate. Consequently, aspartate levels, NAD+/NADH ratios and lactate/pyruvate ratios were restored by pyruvate supplementation as well. Finally, the inhibition of the electron transport chain by complex 1 inhibitors blocked NAD+ regeneration and enhanced the effect of asparagine starvation. Hence, the combination of complex 1 inhibitors and asparaginase offers a new and effective treatment option for rhabdomyosarcoma