PROJECT SUMMARY
Pediatric high-grade gliomas (pHGG) are the most common and malignant primary brain tumors in children.
Mutations in H3F3A, one of the genes that encodes histone variant H3.3, are definitional for pHGG and H3.3
mutation status correlates with clinical variables such as survival and tumor location. pHGG with a lysine 27 to
methionine substitution (H3.3K27M) occur in the brain stem and correlate with particularly poor survival. H3.3K27M
is insufficient to drive tumorigenesis in mouse models. However, it co-occurs in humans with PDGFRA activation
and TP53 and ATRX deletion, suggesting that H3.3K27M cooperates with these oncogenic mutations to induce
tumors. Further, the phenotypic differences between H3.3K27M pHGG and other pHGG suggest differing
epigenetic alterations and mechanisms of tumorigenesis. This proposal is focused on identifying the epigenetic
and transcriptomic changes induced in murine neural stem cells (NSC) by H3.3K27M and its co-occurring
mutations and how those mutations lead to glioma cell phenotypes. To model H3.3K27M pHGG as accurately as
possible, I will use an RCAS-TVA-based model in which NSC harvested from Nestin tv-a; Trp53fl/fl or Nestin tv-
a; Trp53fl/fl; Atrxfl/fl mice are infected with RCAS viruses encoding Cre recombinase, PDGFß, and H3.3K27M. The
resulting NSC will be used to characterize the global and local epigenetic and transcriptomic changes induced
by H3.3K27M and its co-occurring mutations in NSC using western blots, ChIP-seq, and RNA-seq. Further, I will
characterize the phenotypic changes caused by these alterations using immunocytochemistry, extreme limiting
dilution, proliferation assays, and orthotopic allografts. Finally, I will validate the epigenetic and transcriptomic
signatures of H3.3K27M mouse NSC in a panel of H3.3WT and H3.3K27M patient derived xenograft (PDX) cell lines
via ChIP-seq and RNA-seq, as well as with existing datasets from human tumor samples. These studies will
identify mechanisms of pathogenesis for H3.3K27M pHGG and potential epigenetic therapeutic targets that are
both tractable in mice and relevant to humans. By completing this research proposal, I will gain technical skills
applicable to cancer biology, neuroscience, epigenetics, and biochemistry while also developing my critical
thinking, scientific communication, and ethical research skills.