MODL-19. CREATION OF AN IN VITRO MODEL SYSTEM FOR THE STUDY OF H3.1K27M DMG

Abstract

Abstract Diffuse Midline Gliomas (DMG) are a devastating group of pediatric high-grade gliomas that occur in the brainstem with a median survival of less than 1 year. A greater understanding of the early tumorigenic events is essential for the development of effective therapeutics. DMG is characterized by founder mutations in histone H3, either H3.1K27M or H3.3K27M. These mutations cause global hypomethylation, resulting in aberrant gene expression. It is unknown how this mechanism contributes to tumorigenesis. Interestingly, H3.1K27M DMG show an increased incidence in females, whereas H3.3K27M DMG shows no sex difference. This illustrates that the tumorigenic potential of H3.1K27M may be different between the sexes. Few models of DMG incorporate the study of H3.1K27M, even though it represents a unique opportunity to obtain valuable information on the tumorigenesis of DMG through the study of the sex difference. Additionally, an important regulatory element, a stem loop structure, present in mammals is missing from all previous models studying H3.1K27M. Thus, we have created an in vitro model system for H3.1K27M DMG that incorporates the stem loop structure utilizing the RCAS mouse model system. This system utilizes RCAS vectors and a RCAS-ntva transgenic mouse line to deliver specific mutations to nestin expressing cells in the brainstem, including neural and glial progenitor cells, the potential cells of origin. Confirmed through immunofluorescence staining, we can isolate a pure population of NPCs and transduce them with RCAS vectors in vitro to produce stable expression of H3.1K27M. Verified via immunocytochemistry and flow cytometry, delivering H3.1K27M with the stem loop included limits its expression to S phase of the cell cycle, mimicking the human disease biology. These results demonstrate that we have created an in vitro H3.1K27M DMG model system for the study of sex differences and tumorigenesis in DMG.