Investigation of tumor stem cell maintenance and cellular hierarchy in pediatric high-grade glioma

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Project Summary The project’s overall goal is to determine important biological characteristics and investigate therapeutic options for pediatric high-grade gliomas (PHGG), the most aggressive of childhood central nervous system tumors and most common cause of childhood cancer mortality. PHGG survival rates are less than 5% for the subtype diffuse midline glioma and 20% for hemispheric histone 3-wild type (H3-wt) PHGG. PHGG are highly invasive and often grow diffusely among normal cells, limiting surgery as a therapeutic option. Radiation therapy (RT) is transiently effective, but the tumors nearly always recur. Despite hundreds of clinical trials, no chemotherapy has shown a definitive survival benefit in PHGG. Effective PHGG therapies are critically needed. PHGG likely originates from stemlike tumor initiating cells (PICs). PHGG tumors comprise several distinct cell types of glial origin, in varying proportions. This tumor heterogeneity complicates understanding PHGG tumor biology and designing therapies. Aim 1 will investigate how each distinct cell type in PHGG contributes to overall tumorigenesis in a mouse model. Single-cell RNA-Seq (scRNA-Seq) analysis of orthotopic patient derived PHGG xenografts (PDX) will be used to define the cell types present and identify differentially regulated oncogenic pathways that drive their growth. Pathway expression will be knocked down by targeting key effector genes with shRNA using stable lentiviral transduction. The effect on tumor growth will be evaluated using survival, histology and single-cell RNA-Seq. Aim 2 will perform lineage tracing to determine whether a single PIC cell type produces all of the proliferating cell types that comprise PHGG. Lineage tracing will be performed in a mouse PDX model. Single- cell genomic DNA sequencing will be performed on PDX tumors. Mutational signatures consisting of single and multiple nucleotide variations as well as copy number variation will be used to define each cell type. Conserved patterns of mutation among cell types will be used to determine the hierarchical relationships among cell types. Once the lineage relationships are worked out, resistance to RT will be studied in the PDX model. RT is the most consistently effective therapy against PHGG but works only temporarily before cells regrow. RT resistance by cell type will be determined based upon differential survival of cell types versus control following RT. Drug screening of resistant cell types to identify radiation sensitizers will be performed. The candidate drugs will be combined with RT to investigate their effectiveness at increasing the duration of the RT effect.