抗癌药物PARP抑制剂获得性耐药的进化研究

BRCA1- and BRCA2-deficient human breast cancers are defective for the major DNA double strand break repair pathway homologous recombination (HR). Exploitation of this HR vulnerability has led to the recent development of poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) as a new class of anti-tumor drugs for the treatment of BRCA1- and BRCA2-deficient breast cancers. PARPi also show utility for treating cancers not deficient for HR. Despite preliminary success in clinical trials, acquired resistance to PARPi may invariably occur and, in fact, is beginning to be observed, posing a serious clinical problem for this PARPi-based cancer therapy. According to the tumor evolution theory, acquisition of resistance to PARPi is a fundamentally evolutionary process. However, as the evolutionary aspect has long been overlooked in the field, it is no surprise that little is known about the evolution of acquired resistance to different PARPi. Due to varying properties of different PARPi, heterogeneity of targeted tumors and different mechanisms of action of combining drugs, we hypothesize: the selective pressure imposed by different PARPi may influence the evolution of acquired resistance to PARPi by selecting or inducing certain gene mutations, resulting in different resistance traits. Interestingly, our preliminry studies raise a possibility that HR enhancement may be a common outcome of primary resistance mutations selected or induced by treatment with PARPi. Based on the concept of fitness cost in the evolution theory, primary resistance mutations tend to impair cell proliferation of resistant clones, likely resuming tumor sensitivity to PARPi in clinics after acquiring resistance to PARPi. However, because of long-term continuous treatment with PARPi, resistant tumor cells can acquire additional compensatory mutations to compensate for the impairment of their proliferation, resulting in persistent resistance in patients. To further elucidate the molecular mechanisms of the evolution of acquired resistance to PARPi and address the related clinical problems, we will: 1) Analyze HR during development of acquired resistance to different PARPi; 2) Determine effect of combining drugs on HR during development of acquired resistance to PARPi ; 3) Analyze cell proliferation of PARPi-resistant clones; 4) Identify both primary resistance mutations and compensatory mutations that promote acquired resistance to PARPi. This project will not only advance the study of the evolution of acquired resistance to PARPi at the molecular level, but also have significant clinical relevance.

聚（ADP-核糖）聚合酶小分子抑制剂（PARPi）是一类新型有效的抗肿瘤靶向药物，但其获得性耐药是一个严重的临床问题，并且我们对PARPi 获得性耐药进化机制知之甚少。基于多种选择压力的存在，我们提出假说：不同来源的选择压力影响PARPi 获得性耐药的进化过程，导致不同耐药特征。初期研究显示：作为一条重要的DNA 双链断离修复途径，同源重组（HR）参与PARPi 获得性耐药进化。此外，根据进化适应成本理论，主要耐药突变会损害细胞增殖，导致临床耐药性和敏感性之间的反复。因此，耐药细胞往往需要补偿突变以弥补受损的细胞增殖能力，导致持续性耐药。为了深入探索PARPi 获得性耐药进化机制及相应临床问题，我们将：1）分析 HR 在对不同PARPi 获得耐药后的变化；2）分析辅助药物对HR 水平的影响；3）检测PARPi 获得性耐药克隆的细胞增殖能力；4）鉴定PARPi 获得性耐药主突变和补偿突变。

哺乳动物细胞中有两条主要的DNA双链断裂修复途径，即同源重组（HR）和非同源末端连接（NHEJ）。HR缺陷导致基因组不稳定性，引起包括BRCA1/BRCA2突变和ATM突变的恶性肿瘤。PARP抑制剂（PARPi）是一类针对HR缺陷肿瘤的新型有效的靶向药物，近几年已经FDA批准上市，但其获得性耐药仍然是一个严重的临床问题。为解析PARPi 获得性耐药进化机制，我们分析了BRCA1突变和ATM突变细胞中HR缺陷状态与PARPi 获得性耐药的关系，并利用HR和NHEJ报告系统分析方式，结合CRISPR基因编辑技术及各种遗传学和细胞生物学方法，解析其中潜在的分子机制，鉴定PARPi 获得性耐药进化中可能产生的耐药突变。..我们发现：1）BRCA1/2缺陷细胞和ATM缺失细胞仍保留正常的、甚至超激活的γH2AX-MDC1信号轴介导的HR亚途径；2）抑制γH2AX-MDC1信号轴介导的HR亚途径可以提高癌细胞对PARPi的敏感性，帮助克服耐药；3）针对这条亚途径，我们利用计算机辅助虚拟配体筛选技术，结合我们的HR报告系统验证平台，获得2 个具有很强的HR抑制活性的先导化合物；4）在调查γH2AX-MDC1信号轴介导的HR亚途径的过程中，还揭示了这个信号轴调节NHEJ的分子机制；5）在研究BRCA1/BRCA2缺失细胞的PARPi耐药进化过程时，发现RECQL解旋酶缺失导致PARPi的敏感性；6）由于该项目在利用CRISPR基因编辑技术时遭遇了CRISPR基因编辑技术的效率和精准度不足的问题，我们因此针对性地开发了一个可以提高效率和精准度的基于配对Cas9-sgRNA的CRISPR基因编辑技术。..该项目不仅揭示了一个HR缺陷肿瘤PARPi耐药的一个新机制，而且筛选出2个具有潜力成为克服PARPi耐药性临床药物的小分子先导化合物，为临床克服PARPi耐药性的研究及应用提供新途径和新策略。