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Abstract

Gliomas are the most frequent adult central nervous system (CNS) tumors. The 2016 published WHO CNS classification update implemented the mutational status of the enzyme isocitrate dehydrogenase (IDH) as important diagnostic marker in glioma. Thus it became defining of IDH-mutant astrocytoma (IDH-mut A) WHO grade 2-4 and oligodendroglioma WHO grade 2-3. The genetic and epigenetic landscape of glioma with IDH-mutant vs. IDH wild-type varies significantly and is used for tumor classification. These glioma samples carry heterozygous point mutations in IDH1 or IDH2. The most frequently observed variant is IDH1 R132H mutation occurring in >90% of astrocytoma. In this case the arginine residue in codon 132 is substituted by a histidine causing a gain-of-function. While wild-type IDH enzymes naturally catalyze the production of alpha-ketoglutarate (α-KG) from isocitrate, mutant IDH1 convert α-KG into D-2-hydroxyglutartate (2-HG). 2-HG acts as a competitive inhibitor of α-KG dependent enzymes and in doing so influences several tumor-promoting processes and by this acts as an oncometabolite. Most important, it inhibits DNA demethylases leading to a global hypermethylation phenotype. IDH mutations are considered as an early event as well as tumor driver in the development of glioma. Besides this IDH-mut A harbor loss-of-function mutations in TP53 and ATRX in high frequencies. Much is known about the genetics of IDH-mut A. However, to gain deeper insights into their development and progression, a representative and biological relevant model is needed, which is still lacking. Especially how biological mechanisms behind common alterations synergize, is not fully understood yet. All current models have weak points regarding various aspects. But the opportunity to identify new targets and test treatment options also integrating the tumor microenvironment will allow research to progress and patients to benefit. To close this gap, I aimed to establish an innovative comprehensive mouse model harboring not only the typical mutations but crucial IDH-mut A features. To model IDH-mut A I used neural stem cells (NSCs) derived from genetically engineered mice. The mice harbor an inducible heterozygous Idh1R132H knock-in alteration as well as loxP based homozygous p53ko and Atrxko alleles in all possible combinations. Using this model, the effect of Idh1R132H and its associated alterations can be investigated in a well-defined in vitro setting. My findings showed that Idh1R132Hp53koAtrxko leads to a more tumorigenic behavior, evidenced by increased viability and decreased apoptosis. Furthermore, it points out that induction of Idh1R132H is sufficient to develop the typical hypermethylation phenotype over time. By comparing methylation data of murine and human samples the advanced analysis revealed important pathways related to differentiation and tumorigenicity being altered. Furthermore the proteome data reveal adaptions seen most clearly in the cells’ metabolism. The following in vivo part was divided into a pilot experiment and the main experiment. The pilot experiment mainly aimed at identifying a for my purpose suitable immunodeficient strain, what turned out to be important. Within the main experiment I injected the murine NSCs intracranial into the brains of immunocompetent animals. After 10 months a higher lesion developing rate in Idh1R132Hp53koAtrxko was detected by MRI. Although my current work focused on generation, validation and characterization of the model it offers first valuable insights into the complex interplay between genetic, epigenetic and proteomic changes in IDH-mut A. Moreover the established model itself provides the basic framework for a future profound understanding of these tumors with additional treatment testing option.