Validation and Functional Analysis of Novel Mutated Genes in Pediatric Gliomas

Introduction: Gliomas are the most common and aggressive type of primary malignant brain tumors both in adults and in pediatric patients. Data from various studies have indicated a genetic diversity between pediatric and adult gliomas that may imply for the need of dedicated therapeutic approaches for pediatric gliomas. Using whole-genome sequencing, RNASeq and public data bases, we have identified several genetic aberrations that are unique to pediatric high grade gliomas (pedHGG). These genetic aberrations include novel point mutations, gene fusions and duplications in oncogenes such as NTRK2 and FGFR1. In some cases these alterations also define distinct molecular subgroups of glioma as judged by DNA methylation profiling. In this project, we aim to examine the functional role and relevance of those mutations in tumor development in order to identify new therapeutic approaches for this malignant tumor.

Materials and Methods: In order to assess the role of these mutations in pedHGG tumorigenicity we cloned our candidate genetic aberrations into Cre/loxP-inducible lentiviral vectors alone or in combination with a second hit in the form of shRNAs targeting P53 or P16INK4/P19ARF. In order to generate a mouse model of human pedHGG, we injected the established lentiviral vectors directly into the brain of Cre transgenic post-natal 1 (PN1) pups. In parallel, we also used an alternative methodology based on CRISPR/Cas9 system and in uterus electroporation at E-14.5. We characterized the obtained tumors by immunohistochemistry (IHC) and WB analysis. We extracted RNA from the generated tumors for further molecular characterization (RNAseq) and generated tumor derived primary cell cultures for pre-clinical drug screens using specific pathway inhibitors.

Results and Discussion: We were able to obtain tumors using both methodologies with different genetic aberrations that also resulted in higher mortality rate in mice. We characterized the obtained tumors by IHC and confocal microscopy analysis and started to check the expression of known downstream target genes and different pathway phosphorylation points in order to further elucidate the mechanism of action of the candidate genetic aberrations studied.

Conclusion: We were able to generate novel murine pHGG models that will not only enable us to further understand the basic biology of the tumorigenic mechanisms, but will also provide an excellent opportunity to rapidly translate our pre-clinical results in a manner that we hope will directly benefit patients in the clinic.