TP53突变体与H3K27M协同增强DNA损伤后的生存并驱动DIPG的放疗抵抗

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.