William T. Richardson

Introduction. Glioblastoma Multiforme (GBM) is the most common and lethal primary brain tumor.¹ It has a dismal prognosis due to persistent tumor recurrence, with a mean survival of 14 months.¹ There are notable differences in prevalence between the adult and pediatric populations, with an occurrence rate of 3/100,000 and 0.05-0.33/100,000, respectively.^1,2 Recent genetic analyses have shown that different mutations underly GBM in the two populations, with mutations causing Histone 3 (H3) dysregulation isolated to pediatric GBM (pGBM) and the incidence of H3K27M mutations decreasing with age.^3–5 H3K27M mutations were also found to be limited to diffuse midline pontine gliomas, which are particularly invasive and devastating tumors that occur primarily in young children.⁶ These findings indicate the potential and necessity for individualized therapies for pGBM treatment. Methods. CRISPR-Cas9 gene editing was used to introduce K27M and G34R H3.3 mutations into human astrocytes and wild-type pediatric glioma cells.⁷ In parallel, H3.3K27M mutations in glioma cells were reverted to wild-type.⁷ After control and gene-edited cells were assayed for differences in cell growth and proliferation, xenograft assays were performed via stereotactic introduction of transfected cells into mice pons.⁷ The effects of histone deacetylation on tumor metabolism were analyzed via administration of HDAC inhibitors to GBM cells and measurement of ¹³C flux and MYC/PGC1a expression.⁸ Fluorescence anisotropy assay was carried out to measure the equilibrium constants of Polycomb repressive complex 2 (PRC2) binding to either H3K27 or H3K27M.⁹  A methyltransferase assay and crystallography were performed to understand the automethylation activity of PRC2, a necessary step for conformational changes that allow for H3K27 trimethylation.^9,10 Results. Tumor cells harvested from the K27M and G34 H3 mutation-based xenograft models showed a marked derepression of genes in the NOTCH and MYC pathways⁷. DAPT administration to GBM cells was followed by an increased sensitivity to irradiation.⁷ HDAC administration to GBM cells correlated with PGC1⍺ transcription and subsequent switch from glycolytic flux to oxidative phosphorylation.⁸ The methionine residue in H3K27M binds the catalytic domain of PRC2 with greater affinity than lysine in wild-type H3, causing inhibition of automethylation activity, thereby reducing histone methylation.⁹ Mutations to residues K510, K514, or K515 of PRC2 also inhibited automethylation.¹⁰ Conclusions. H3 dysregulation is a key driver of the aggressive biology of pGBM. Further research on the mechanistic relationships between age and H3 mutations is necessary to understand the pathophysiology of pGBM and discover potential treatment options.

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