Histone 3 expression in H3K27M DIPG patient-derived cell lines
Diffuse intrinsic pontine glioma (DIPG) or diffuse midline glioma (DMG) is an aggressive, untreatable, and universally fatal brain cancer of the pons. DIPG is found primarily in children (median 6-8 years old). Due to the difficulty of standard resection and relative resistance to traditional chemotherapy and radiation, DIPG is characterized by its poor prognosis, with 10% survival at 2 years and only 2% survival at 5 years post-diagnosis. 60-70% of DIPG contain a substitution mutation of lysine for methionine at position 27 of histone 3 (H3K27M), leading to decreased trimethylation through the inhibition of the PRC2 pathway and increased acetylation of H3K27. These post-translational modifications can affect gene expression, with methylation leading to chromatin condensation and less gene expression, and acetylation leading to chromatin de-condensation and increased gene expression. Histone deacetylase inhibitors (HDACi) have shown clinical efficacy in prior studies. In this study, we analyzed two patient-derived xenograft models with the goal of determining the molar ratio of mutant H3K27M vs. wildtype histone 3 proteins through the Jess automated western blot system. We identified an antibody that specifically reacts to H3K27M and not wildtype H3 in total histone isolates. Further optimization of sample protein and antibody concentrations for the Jess system will determine the molar ratio of mutant vs. wildtype H3 expression in future studies of patient-derived cell lines. This project reinforced the specificity of the H3K27M primary antibody and aided in tool development by demonstrating that the Jess system can be used to detect histone 3 proteins. By comparing the efficacy of HDACi in each cell line to mutant vs. wildtype H3 ratios, a correlation between molar ratio, tumor phenotype, and HDACi effectiveness will be established.
