CRISPR activation screen identifies GNAO1 as a driver of glioblastoma cell differentiation

Inducing tumor cell differentiation is a potential strategy for treating malignant cancers, including glioma. Here we perform a genome-scale CRISPR activation screen and identify that G Protein Subunit Alpha O1 (GNAO1) as a critical regulator of glioma cell differentiation. GNAO1 overexpression in combination with a small molecular inhibitor cocktail, including CHIR99021, SB431542, RepSox, and Y27632, markedly promotes neuronal differentiation of glioblastoma cells. GNAO1 is high expressed in normal neuronal tissues but low expressed in gliomas. High expression of GNAO1 is correlated with a better prognosis. GNAO1 overexpression decreases cell proliferation, cell migration, and orthotopic GBM tumor xenograft growth. GNAO1 overexpression activates neuronal differentiation pathways by down-regulation of transcript factor HES1, a key mediator of neuronal differentiation. GNAO1 recruits TRIM21 and increases TRIM21-mediated ubiquitination and degradation of CREB, leading to decreased p300-mediated H3K27ac levels of HES1 promoter and HES1 downregulation. In addition, GNAO1 mRNA stability is regulated by METTL3-regulated N6-methyladenosine (m6A). Overexpression of GNAO1 enhances glioblastoma response to Temozolomide (TMZ) and extends the survivals of animals bearing orthotopic GBM xenograft tumors. Our finding suggests that inducing neuronal differentiation of glioblastoma cells by engineering GNAO1 overexpression is a potential therapy strategy, which not only reduces tumor cell proliferation but also enhances tumor cell response to chemotherapy. Overall design: To understand why GNAO1 is important for glioma cell differentiation, we performed transcriptome analysis in GSC R83/WT and GSC R83/GNAO1 cells using RNA-seq.

诱导肿瘤细胞分化是治疗包括胶质瘤在内的恶性肿瘤的潜在策略。本研究开展全基因组CRISPR激活筛选（CRISPR activation screen），鉴定出G蛋白亚基αO1（G Protein Subunit Alpha O1, GNAO1）是调控胶质瘤细胞分化的关键因子。将GNAO1过表达与CHIR99021、SB431542、RepSox及Y27632组成的小分子抑制剂组合联合使用，可显著促进胶质母细胞瘤细胞向神经元分化。GNAO1在正常神经组织中高表达，而在胶质瘤中低表达；其高表达与更佳的预后相关。GNAO1过表达可抑制细胞增殖、迁移，并延缓原位胶质母细胞瘤（GBM）异种移植瘤的生长。GNAO1通过下调作为神经分化关键调控介质的转录因子HES1（HES1），激活神经元分化通路；GNAO1可招募TRIM21，增强TRIM21介导的CREB泛素化与降解，进而降低p300介导的HES1启动子区域H3K27乙酰化（H3K27ac）水平，最终导致HES1表达下调。
此外，METTL3调控的N6-甲基腺苷（N6-methyladenosine, m6A）修饰可影响GNAO1 mRNA的稳定性。GNAO1过表达可增强胶质母细胞瘤对替莫唑胺（Temozolomide, TMZ）的敏感性，并延长携带原位胶质母细胞瘤异种移植瘤的实验动物的生存期。
本研究结果表明，通过工程化手段诱导GNAO1过表达以促进胶质母细胞瘤细胞向神经元分化，是一种潜在的治疗策略——该策略不仅可抑制肿瘤细胞增殖，还可增强肿瘤细胞对化疗药物的响应。
实验整体设计：为阐明GNAO1调控胶质瘤细胞分化的分子机制，本研究利用RNA测序（RNA-seq）对胶质瘤干细胞（glioma stem cell, GSC）R83/WT及GSC R83/GNAO1细胞开展了转录组分析。