Abstract 7380: TP53 mutants cooperate with H3K27M to enhance survival after DNA damage and drive radioresistance in DIPG

Abstract Diffuse intrinsic pontine glioma (DIPG) is driven by the H3K27M oncohistone and frequently co-occurs with TP53 mutations, a genetic combination closely associated with radiation failure. Because H3K27M globally reprograms chromatin and alters promoter accessibility, we hypothesized that it creates a permissive epigenetic environment that enhances the stability and activity of specific TP53 mutants to promote radioresistance. To test this, we expressed TP53 wild-type (WT) or DNA-binding-domain hotspot mutants, including contact mutants (R273C, R273H, R248W) and a conformational mutant (R175H), in TP53-knockout HEK293 cells with either H3.3 or H3K27M. Sequence-specific DNA binding was first assessed using a transcription factor assay. Several mutants (R273H, R273C) retained DNA-binding activity, which increased substantially in the H3K27M context, whereas R175H remained low. Because these results suggested enhanced promoter engagement after DNA damage, we next performed ChIP-qPCR after 8 Gy irradiation. In the presence of H3K27M, multiple mutants (R273H, R273C, R248W) maintained occupancy at p53 target genes (MDM2, CDKN1A/p21), in contrast to reduced binding when H3.3 WT was expressed. Western blot analysis further revealed that several TP53 mutants, including R175H, were selectively stabilized in H3K27M-expressing cells, while WT p53 levels and transcriptional output were unchanged. We then examined transcriptional programs influenced by H3K27M in each TP53 background. Although several chaperone and chromatin-remodeling genes (HSPA14, BRD4, KDM7A) showed modest changes in R273C_H3K27M cells, the most prominent alterations were in stress-signaling pathways, including induction of the NKG2D stress-ligand RAET1E (logFC = +1.14, p = 0.0627). These gene-level changes indicate that H3K27M rewires cellular programs that cooperate with mutant p53 under genotoxic stress. Finally, we tested whether these chromatin-driven molecular effects translate into a functional phenotype. H3K27M provided only a modest proliferative advantage to WT TP53 cells, but markedly increased the survival and regrowth of TP53-mutant cells over 24-72 hours after irradiation. Taken together, these findings support a model in which H3K27M enhances mutant p53 DNA binding, stabilizes mutant p53 proteins, and reprograms stress-responsive pathways to amplify pro-survival responses after DNA damage. This cooperation between H3K27M and specific TP53 mutants provides a mechanistic explanation for radioresistance in DIPG and underscores the importance of considering both TP53 genotype and histone context when designing targeted radiosensitization strategies. Citation Format: Karol A. Arizaca Maquera, Viral Oza, Andrew Gaines, Colin Williams, Jessica Blackburn. TP53 mutants cooperate with H3K27M to enhance survival after DNA damage and drive radioresistance in DIPG [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7380.