基于APEX技术解析中心粒旁物质蛋白质组及协同致死应用

协同致死效应为开发新的肿瘤靶向治疗方法开辟了新的思路，但是目前有效的协同致死靶点组合屈指可数。中心体是有吸引力的抵抗癌细胞疯狂增殖的靶点，中心体异常扩增是多种类型癌细胞的典型特征，其复制依赖蛋白激酶PLK4，然而许多癌细胞对PLK4抑制剂并不敏感。最近研究发现在缺失中心体时这些细胞的中心粒旁物质(PCM)可以自发聚集，从而能够促进纺锤体组装使细胞持续分裂。由此本申请项目提出了联合靶向PLK4与关键PCM蛋白协同致死癌细胞的新策略。由于PCM不能用传统的方法进行分离鉴定，人们对PCM的蛋白成分和组装机制仍缺乏了解，本项目首次利用APEX2邻近蛋白标记技术，在活细胞原位标记并解析PCM蛋白质组，之后利用CRISPR/Cas9敲除平台对PCM中的关键蛋白进行功能鉴定，从中筛选出PLK4抑制剂的协同致死靶点。本研究将拓展人们对PCM蛋白组成和功能的认知，为今后靶向中心体用药杀伤肿瘤提供理论基础。

Synthetic lethality drives the discovery of new cancer drugs, but there are few effective synthetic lethal interactions at present. Centrosome is an attractive target to resist the proliferation of cancer cells, and centrosome replication depends on protein kinase PLK4. However, many cancer cells are not sensitive to PLK4 inhibitor. Recent studies have found that the pericentriolar material (PCM) of these cells can spontaneously aggregate and promote spindle assembly to drive cell division in the absence of centrosome. Therefore, this study proposed a new strategy to kill cancer cells by targeting PLK4 and PCM protein. Because PCM can not be separated by traditional methods, the understanding of the protein composition and assembly mechanism of PCM is still limited. In this study, APEX2 adjacent protein labeling system was used for the first time to analyze the proteome of PCM in live cells in situ. Then, based on the CRISPR/Cas9 high-efficiency screening platform established previously, we screened the proteins that play a key role in PCM assembly and function, which could synthetically kill cancer cells with PLK4 inhibitor. This study will expand the knowledge of protein composition and function of PCM, and provide the theoretical basis for targeting centrosome drugs to kill tumor.