Three-dimensional CRISPR screening reveals epigenetic interaction with anti-angiogenic therapy

Angiogenesis underlies development, physiology and pathogenesis of cancer, eye and cardiovascular diseases. Inhibiting aberrant angiogenesis using anti-angiogenic therapy (AAT) has been successful in the clinical treatment of cancer and eye diseases. However, resistance to AAT inevitably occurs and its molecular basis remains poorly understood. Here, we identify novel molecular modifiers of the blood endothelial cell (EC) response to a widely used AAT bevacizumab by performing a pooled genetic screen using three-dimensional microcarrier-based cell culture and CRISPR–Cas9. Functional inhibition of the epigenetic reader BET family of proteins BRD2/3/4 shows unexpected mitigating effects on EC survival and/or proliferation upon VEGFA blockade. Moreover, transcriptomic and pathway analyses reveal an interaction between epigenetic regulation and anti-angiogenesis, which may affect chromosomal structure and activity in ECs via the cell cycle regulator CDC25B phosphatase. Collectively, our findings provide insight into epigenetic regulation of the EC response to VEGFA blockade and may facilitate development of quality biomarkers and strategies to overcome resistance to AAT. One cell line, six conditions, three biological replicates per condition.

血管生成（Angiogenesis）是发育进程、生理稳态以及癌症、眼部疾病与心血管疾病发病机制的核心基础。采用抗血管生成治疗（Anti-angiogenic Therapy, AAT）抑制异常血管生成，已在癌症与眼部疾病的临床治疗中获得成功。然而，抗血管生成治疗耐药性不可避免地产生，其分子机制至今仍未被充分阐明。本研究通过基于三维微载体的细胞培养体系与CRISPR–Cas9技术开展池化遗传筛选，鉴定出可调控血管内皮细胞（Blood Endothelial Cell, EC）对临床常用抗血管生成治疗药物贝伐单抗（Bevacizumab）应答的新型分子修饰因子。对表观遗传阅读器（Epigenetic Reader）BET家族蛋白BRD2/3/4进行功能性抑制，可在VEGFA（血管内皮生长因子A, Vascular Endothelial Growth Factor A）阻断时，对血管内皮细胞的存活与/或增殖产生意料之外的缓解作用。此外，转录组学与通路分析揭示了表观遗传调控与抗血管生成之间的相互作用，该作用可通过细胞周期调控因子CDC25B磷酸酶（CDC25B Phosphatase）影响血管内皮细胞的染色体结构与活性。综上，本研究的发现为解析血管内皮细胞对VEGFA阻断应答的表观遗传调控机制提供了新见解，同时可为开发高质量生物标志物以及攻克抗血管生成治疗耐药性的相关策略提供理论支撑。本实验采用1种细胞系，设置6种实验条件，每个条件包含3次生物学重复。