背景:胶质母细胞瘤是最常见和侵袭性的原发性脑肿瘤,预后极差,强调迫切需要开发新的治疗方案。识别癌细胞的表观遗传脆弱性可以为各种类型的癌症提供极好的治疗干预点。
方法:在本研究中,我们使用定制的sgRNA文库EpiDoKOL,通过基于CRISPR/Cas9的遗传消融筛选研究了胶质母细胞瘤细胞存活的表观遗传调节因子,靶向染色质修饰剂的关键功能结构域。
结果:在多个细胞系中进行的筛选显示ASH2L,组蛋白赖氨酸甲基转移酶复合物亚基,作为胶质母细胞瘤细胞活力的主要调节剂。ASH2L耗竭导致细胞周期停滞和细胞凋亡。RNA测序和greenCUT&RUN一起确定了一组细胞周期调控基因,例如TRA2B,BARD1,KIF20B,ARID4A和SMARCC1在ASH2L耗尽后下调。质谱分析显示ASH2L在胶质母细胞瘤细胞系中的相互作用伙伴为SET1/MLL家族成员,包括SETD1A,SETD1B,MLL1和MLL2。我们进一步表明,胶质母细胞瘤细胞对SET1/MLL家族成员的表达具有不同的依赖性。在原位体内模型中,ASH2L耗尽的胶质母细胞瘤细胞的生长明显慢于对照组。TCGA分析显示,与低级别神经胶质瘤相比,胶质母细胞瘤中的ASH2L高表达,免疫组织化学分析显示,证明其临床相关性。因此,高吞吐量,强大的和负担得起的屏幕与集中的图书馆,比如EpiDoKOL,对快速发现癌细胞存活的新型表观遗传调节因子有着巨大的希望,例如ASH2L。
结论:一起,我们建议靶向ASH2L可以作为胶质母细胞瘤的新治疗机会.视频摘要。
Glioblastoma is the most common and aggressive primary brain tumor with extremely poor prognosis, highlighting an urgent need for developing novel treatment options. Identifying epigenetic vulnerabilities of cancer cells can provide excellent therapeutic intervention points for various types of cancers.
In this study, we investigated epigenetic regulators of glioblastoma cell survival through CRISPR/Cas9 based genetic ablation screens using a customized sgRNA library EpiDoKOL, which targets critical functional domains of chromatin modifiers.
Screens conducted in multiple cell lines revealed ASH2L, a histone lysine methyltransferase complex subunit, as a major regulator of glioblastoma cell viability. ASH2L depletion led to cell cycle arrest and apoptosis. RNA sequencing and greenCUT&RUN together identified a set of cell cycle regulatory genes, such as TRA2B, BARD1, KIF20B, ARID4A and SMARCC1 that were downregulated upon ASH2L depletion. Mass spectrometry analysis revealed the interaction partners of ASH2L in glioblastoma cell lines as SET1/MLL family members including SETD1A, SETD1B, MLL1 and MLL2. We further showed that glioblastoma cells had a differential dependency on expression of SET1/MLL family members for survival. The growth of ASH2L-depleted glioblastoma cells was markedly slower than controls in orthotopic in vivo models. TCGA analysis showed high ASH2L expression in glioblastoma compared to low grade gliomas and immunohistochemical analysis revealed significant ASH2L expression in glioblastoma tissues, attesting to its clinical relevance. Therefore, high throughput, robust and affordable screens with focused libraries, such as EpiDoKOL, holds great promise to enable rapid discovery of novel epigenetic regulators of cancer cell survival, such as ASH2L.
Together, we suggest that targeting ASH2L could serve as a new therapeutic opportunity for glioblastoma. Video Abstract.