Abstract Pediatric high-grade gliomas (pHGGs) are the most lethal pediatric cancers, with survival rarely exceeding 2 years. Histone H3.3 (H3-3A) is the most commonly mutated gene in pHGG; nearly 50% exhibit gain-of-function mutations at either lysine 27 (K27M) or glycine 34 (G34R/V). H3.3 mutations often co-occur with loss of ATRX, a chromatin remodeler. We previously demonstrated that ATRX epigenetically dysregulates transcription of cell cycle regulators. p16, a critical G1/S checkpoint regulator, is epigenetically downregulated in preclinical H3K27M models and has significant implications for tumor proliferation and radiosensitization. However, the role of concurrent histone mutation (H3K27M or H3G34R) and ATRX loss on p16 has not been studied. We generated isogenic mouse and human cell lines to individually characterize H3.3K27M, H3.3G34R, and wild-type H3.3 (H3WT), both with and without ATRX. Murine cell lines created from in utero electroporation-generated tumors determined histone status and ATRX loss was generated with a CRISPR knockout plasmid in vitro. Western blot and qPCR analysis of human and murine models demonstrate an H3K27M-mediated decrease in p16, with comparatively high levels of p16 in H3WT. Surprisingly, ATRX loss further decreases p16 expression in H3K27M mouse and human glioma cells. Additionally, H3K27M-mutant mouse cells demonstrate loss of chromatin accessibility (ATAC-seq) that is further decreased by ATRX deficiency, which matches with H3K4me3 loss at the p16 promoter in both murine and human glioma models. Data also demonstrates that concurrent ATRX loss and H3K27M mutation is associated with increased mitotic index after radiation and increased susceptibility to CDK4/6 inhibition. Ongoing work includes assessing the impact of ATRX/H3 mutations on (1) epigenetic profiles (CUT&RUN-seq) and (2) in vivo CDK4/6 inhibitor studies. In summary, H3K27M and ATRX deficiency leads to H3K4me3 loss at the p16 promoter, downregulating p16 expression. This results in G1/S checkpoint disruption, increased proliferation rates, and increased response to CDK4/6 inhibition.
Messinger et al. (Fri,) studied this question.