BIOLOGICALLY-BASED THERAPEUTICS FOR THE TREATMENT OF DIFFUSE INTRINSIC PONTINE GLIOMAS

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Abstract

BACKGROUND: Diffuse intrinsic pontine gliomas (DIPGs) in children carry a dismal prognosis despite the use of aggressive multi-modality treatment. Recent results have identified a somatic mutation in the H3F3A gene, resulting in replacement of lysine 27 by methionine in its encoded histone H3.3 protein (H3.3-K27M), which occurs primarily in DIPGs. The K27M mutation results in both increased and decreased gene expression. This specific mutation is an attractive therapeutic target for the treatment of DIPGs. METHODS: Using cell lines and tissues from two pediatric DIPGs and one pediatric GBM tumor obtained from surgical biopsies, H3.3-K27M mutation status was determined by direct sequencing. Histone H3.3 lysine 27 (H3K27) methylation status was evaluated by western blotting with antibodies specific for mono-, di-, and trimethylated H3K27. A selective inhibitor of the H3K27 demethylase JMJD3, GSK-J4, was used to examine cell proliferation and survival. GSK-J4 was found to inhibit JMJD3-induced H3K27 demethylation, resulting in an increase of H3K27 methylation. RESULTS: The H3.3-K27M mutation was identified in the two DIPG cell lines, but not in the pediatric GBM cells, nor in other adult glioma cells. H3.3-K27M mutant DIPG cells showed rapid growth in vitro, with a doubling time of approximately 30 hrs. In contrast, a pediatric GBM cell line with wild-type H3.3 grew much more slowly, with a doubling time of 72 hrs. H3.3-K27M mutant DIPG cells showed elevated H3K27 methylation in comparison to H3.3 wild-type glioma cells. GSK-J4 induced a marked dose-dependent inhibition of growth in H3.3-K27M mutant DIPG cells while H3.3 wild-type glioma cells showed no response to GSK-J4. GSK-J4 appears to have a potent and specific effect on tumor cells with the K27M mutation. CONCLUSIONS: Altered histone H3.3 K27 methylation is associated with rapid tumor cell growth in vitro, and is associated withn increased tumor cell sensitivity to GSK-J4. SECONDARY CATEGORY: Tumor Biology.

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