Guan Xiaowen, Xu Xinke, Mo Xiaolan, Deng Houliang
School of Life Sciences, Zhengzhou University, Zhengzhou, Henan, China.
Department of Neurosurgery, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, Guangdong, China.
Front Oncol. 2025 Jun 30;15:1589396. doi: 10.3389/fonc.2025.1589396. eCollection 2025.
Pediatric high-grade glioma (pHGG) with the histone H3 Lys27Met substitution (H3K27M) is a devastating disease with a high mortality rate in children and adolescents (from birth to 19 years of age). No effective treatments have been developed for this tumor type. Thus, a better understanding of the underlying complex mechanisms and identify more potential drugs targeting H3K27M-mutant pHGG are urgently needed.
In the current study, we established pHGG cell models harboring H3K27M by transfecting two pHGG cell lines, SF188 and Res259, with the H3K27M mutant and H3 wild-type (WT) plasmids and then performed drugs screening. Then we employed an EJ5 reporter assay to measure nonhomologous end joining (NHEJ) activity. Western blotting was used to analyze DNA damage markers (γ-H2AX and PLK1), and cell cycle progression was assessed. Additionally, we utilized whole-exome sequencing and CRISPR/Cas9 genome editing to generate Res259 cell lines with stable deficiencies in ARID1A, P53, or PALB2, followed by viability assays to evaluate drug sensitivity.
Notably, BMN673 (talazoparib) was identified as a synthetic lethal hit in the H3K27M-mutant SF188 cell model. However, BMN673 did not affect the constructed H3K27M-mutant Res259 cells. Moreover, we showed that the H3K27M mutation induced an aberrant increase in NHEJ activity. Furthermore, BMN673 treatment increased the protein levels of γ-H2AX and PLK1, induced cell cycle arrest, and promoted PARP1 trapping in H3K27M-mutant SF188 cells. In addition, the results of a series of viability assays revealed that the H3K27M mutation combined with PALB2 deficiency sensitized H3K27M-mutant Res259 cells to BMN673. However, deficiencies in ARID1A or P53 did not produce similar effects.
Overall, our results may provide some reference value for further study of the effects of BMN673 and PALB2 deficiency in the treatment of H3K27M-mutant pHGG.
携带组蛋白H3赖氨酸27甲硫氨酸替代（H3K27M）的儿童高级别胶质瘤（pHGG）是一种在儿童和青少年（从出生到19岁）中死亡率很高的毁灭性疾病。针对这种肿瘤类型尚未开发出有效的治疗方法。因此，迫切需要更好地了解其潜在的复杂机制，并确定更多靶向H3K27M突变型pHGG的潜在药物。
在本研究中，我们通过用H3K27M突变体和H3野生型（WT）质粒转染两种pHGG细胞系SF188和Res259，建立了携带H3K27M的pHGG细胞模型，然后进行药物筛选。然后我们采用EJ5报告基因检测法来测量非同源末端连接（NHEJ）活性。蛋白质印迹法用于分析DNA损伤标记物（γ-H2AX和PLK1），并评估细胞周期进程。此外，我们利用全外显子测序和CRISPR/Cas9基因组编辑来生成在ARID1A、P53或PALB2中稳定缺陷的Res259细胞系，随后进行活力测定以评估药物敏感性。
值得注意的是，BMN673（他拉唑帕尼）在H3K27M突变的SF188细胞模型中被鉴定为合成致死性药物。然而，BMN673对构建的H3K27M突变的Res259细胞没有影响。此外，我们表明H3K27M突变导致NHEJ活性异常增加。此外，BMN673处理增加了H3K27M突变的SF188细胞中γ-H2AX和PLK1的蛋白水平，诱导细胞周期停滞，并促进PARP1捕获。此外，一系列活力测定的结果表明，H3K27M突变与PALB2缺陷相结合使H3K27M突变的Res259细胞对BMN673敏感。然而，ARID1A或P53的缺陷没有产生类似的效果。
总体而言，我们的结果可能为进一步研究BMN673和PALB2缺陷在治疗H3K27M突变型pHGG中的作用提供一些参考价值。
