基于全基因组RNAi筛选鉴定HABP1及其对PARP抑制剂敏感机制研究

Poly ADP-ribose polymerase (PARP) plays an important role in various DNA damage repair pathways. BRCA deficient cells treated with PARP inhibitor (PARPi) display synthetic lethal effect. Theoretically, any gene mutation that causes homologous recombination (HR) repair defects can induce synthetic lethality after treated with PARPi. And PARPi can be applied to treat tumor patients harboring gene mutations that disrupt HR repair pathway by synthetic lethality with PARPi. However, up to date, the clinical application of PARPi is mainly confined to BRCA-deficient tumor patients, which are HR repair defects, severely restricting its scope of use. Therefore, it is urgent to identify new PARPi-sensitive gene mutations to expand the clinical application scope of PARPi. In this project, we screened PARPi-sensitive genes through a genome-wide RNAi library in model animal C. elegans. I focus on F59A2.3 from dozens of candidate genes identified from the screening. The knock-out cell line of HABP1, a human homologues gene of C. elegans F59A2.3, also shows a sensitivity to PARPi. In this project, we will explore the molecular and pathology mechanism of PARPi sensitivity caused by HABP1 deficiency from the following three levels: cells, animals and tumor tissues, respectively. And we will also study the expression level and mutation status of this gene in different tumor tissues. The revelation of these questions will produce important theoretical significance and application value for us to deepen the understanding of the regulatory network of HR repair and will also help us to expand the clinical application of PARPi in tumor patients.

多聚腺苷二磷酸-核糖聚合酶（PARP）在多种DNA损伤修复通路中发挥重要功能。用PARP抑制剂（PARPi）处理BRCA缺陷细胞会产生合成致死效应。理论上讲，任何导致HR缺陷的基因突变都会对PARPi敏感。然而，临床上对PARPi的应用还主要局限在BRCA缺陷的肿瘤病人中，严重制约了它的使用范围。因此亟需探索新的对PARPi敏感的基因。本项目使用秀丽线虫通过全基因组RNAi文库来筛选对PARPi敏感的基因。在几十个备选基因中，我选择其中一个基因F59A2.3重点研究。该基因在人中的同源基因HABP1的敲除细胞株也表现出对PARPi的敏感性。本项目将分别从细胞、动物、肿瘤组织三个层次对HABP1缺陷导致PARPi敏感性的机制展开研究并追踪不同肿瘤组织中该基因的表达、突变情况。这些问题的揭示将为我们加深对同源重组修复调控网络的理解以及拓展PARPi的适用人群，都具有重要理论意义和应用价值。

细胞内无时无刻不在面临外源和内源的基因毒性物质对基因组的损伤，这些损伤最严重的的是双端DNA断裂。双链NDA断裂主要依赖于能进行精确修复的同源重组（Homologous recombination，HR）修复通路和不精确的非同源末端连接（Non-Homologous recombination End Joining, NHEJ）修复通路。参与HR修复的蛋白比如BRCA1/2功能失活后，会导致细胞的基因组不稳定，最终导致肿瘤易感性。作为新的靶向药物，PARP抑制剂（PARPi）能够和BRCA1/2缺陷的肿瘤细胞合成致死。所以鉴定能够调控HR修复效率的新蛋白可以提高PRAP抑制剂以及其他治疗HR缺陷肿瘤化疗药物。我在前期的工作中，利用模式动物线虫为研究对象，通过全基因组RNAIi筛选，鉴定出HABP1缺陷后可以增强对PARPi的敏感性。随后我们也证明在细胞中HABP1缺失也具有同样的表型。在机制研究中，我们发现：HABP1可以结合MRN复合体中的MRE11以及RAD50，HABP1通过结合MRE11来稳定MRE11的表达，保护MRE11免受蛋白酶体途径的降解。HABP1低表达会使MRE11依赖的同源重组修复途受到抑制，从而表现出对PARPi的敏感性。进一步的研究显示，在裸鼠身上的HABP1敲除细胞的种植瘤模型显示出对常用化疗药物PARPi以及CPT-11的敏感性。在取自肿瘤病人的组织样本中，我们也发现了HABP1和MRE11的表达水平呈现正相关，且HABP1的低与病人较好的预后呈正相关。这一研究结果提示我们HABP1低表达的病人可以使用CPT-11、PARPi进行化疗，而且HABP1可以作为肿瘤治疗中的一个潜在靶点进行开发。