Abstract

Background

Residual disease of glioblastoma (GBM) causes recurrence. However, targeting residual cells has failed, due to their inaccessibility and our lack of understanding of their survival mechanisms to radiation therapy. Here we deciphered a residual cell–specific survival mechanism essential for GBM relapse.

Methods

Therapy resistant residual (RR) cells were captured from primary patient samples and cell line models mimicking clinical scenario of radiation resistance. Molecular signaling of resistance in RR cells was identified using RNA sequencing, genetic and pharmacological perturbations, overexpression systems, and molecular and biochemical assays. Findings were validated in patient samples and an orthotopic mouse model.

Results

RR cells form more aggressive tumors than the parental cells in an orthotopic mouse model. Upon radiation-induced damage, RR cells preferentially activated a nonhomologous end joining (NHEJ) repair pathway, upregulating Ku80 and Artemis while downregulating meiotic recombination 11 (Mre11) at protein but not RNA levels. Mechanistically, RR cells upregulate the Su(var)3-9/enhancer-of-zeste/trithorax (SET) domain and mariner transposase fusion gene (SETMAR), mediating high levels of H3K36me2 and global euchromatization. High H3K36me2 leads to efficiently recruiting NHEJ proteins. Conditional knockdown of SETMAR in RR cells induced irreversible senescence partly mediated by reduced H3K36me2. RR cells expressing mutant H3K36A could not retain Ku80 at double-strand breaks, thus compromising NHEJ repair, leading to apoptosis and abrogation of tumorigenicity in vitro and in vivo. Pharmacological inhibition of the NHEJ pathway phenocopied H3K36 mutation effect, confirming dependency of RR cells on the NHEJ pathway for their survival.

Conclusions

We demonstrate that the SETMAR-NHEJ regulatory axis is essential for the survival of clinically relevant radiation RR cells, abrogation of which prevents recurrence in GBM.

中文翻译：

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残留疾病细胞中SETMAR-H3K36me2-NHEJ修复轴的抑制作用可防止胶质母细胞瘤复发。

摘要

背景

胶质母细胞瘤（GBM）的残留疾病导致复发。但是，靶向残留细胞失败了，因为它们难以接近并且我们对放射治疗的生存机制缺乏了解。在这里，我们破译了GBM复发必不可少的残余细胞特异性生存机制。

方法

从主要患者样本和模仿放射抵抗临床情况的细胞系模型中捕获了具有治疗抵抗力的残留（RR）细胞。使用RNA测序，遗传和药理学扰动，过表达系统以及分子和生化分析鉴定了RR细胞中抗性的分子信号传导。在患者样品和原位小鼠模型中验证了发现。

结果

在原位小鼠模型中，RR细胞比亲代细胞形成更具侵略性的肿瘤。受到辐射诱导的损伤后，RR细胞优先激活非同源末端连接（NHEJ）修复途径，上调Ku80和Artemis，同时在蛋白水平而非RNA水平下调减数分裂重组11（Mre11）。从机制上讲，RR细胞上调Su（var）3-9 /增强的Zeste /三胸（SET）域和水手转座酶融合基因（SETMAR），介导高水平的H3K36me2和整体重染色。高H3K36me2导致有效募集NHEJ蛋白。在RR细胞中条件性抑制SETMAR诱导了不可逆的衰老，部分由还原的H3K36me2介导。表达突变型H3K36A的RR细胞无法在双链断裂处保留Ku80，从而损害了NHEJ的修复，在体外和体内导致细胞凋亡和致瘤性消除。NHEJ途径的药理抑制表现出H3K36突变效应，证实RR细胞对NHEJ途径具有生存依赖性。

结论

我们证明，SETMAR-NHEJ调节轴对于临床相关的放射RR细胞的存活至关重要，而后者的废除可防止GBM复发。