1. Drug and Genetic Knockout screen of Oncohistone-driven pediatric high-grade glioma

We investigate how oncohistones contribute to the tumorigenic process of pediatric glioblastoma.

(A) Oncohistone have either been introduced to mouse pedHGG tumors or occur endogenously in human pedHGG tumor biopsies. Co-mutated genes likely participating in tumorigenesis are indicated. (B) Panel on the left illustrates the RCAS mouse model of introduced oncohistones in an PDGFA overexpressing environment for tumor production and derived primary mouse tumorspheres. The right panel illustrates the human pedHGG samples that are propagated as PDOX in mice and then established as primary tumorsphere cell lines. (C) Cell lines lines from mouse and human will be used for the drug screen and the CRISPR/Cas9 genetic knockout screen.

2. Identification of DNMT-interacting oncometabolites.

In this project, we are isolating metabolites that bind the DNA methyltransferases. We use co-immunprecipitation to enrich for the metabolites and then perform untargeted liquid chromatography/mass-spectrometry to determine their identity.

a) Stromal cells are derived from select tumor and established in tissue-culture conditions. b) Recombinant epitope tagged DNA methyltransferase are purified with immunoprecipitation methods. Whole cell extracts are isolated from tumor cells and incubated with recombinant DNMT protein underphysiological conditions. c) With antibody pull-down methodology, an oncometabolite/DNMT complex will be purified and subject to mass-spectrometry that will generate a spectrogram of a interacting low-mass-ion (LMI) compounds, which will be matched to the human metabolome database (HMDB) for identification and quantification.

3. Metabolic pathways directed towards the nucleus.

There are a number of metabolites known to directly contribute to changes of chromatin and ultimately gene expression. Here is a summary of the metabolic pathways that participate, where the most common ones have a thicker arrow pointing towards the nucleus. Some metabolites are directly shuttled into the nucleus to participate in chromatin formation.

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