靶向mTORC2活性增强卵巢癌细胞对PARP抑制剂的敏感性研究

Although high response rates are achieved, most tumors rapidly become resistant to poly ADP ribose polymerase inhibitors (PARPi), including BRCA1/2 deficient ovarian cancers. Thus how to prevent or reverse PARPi resistance represents critically important questions. In previous study, we demonstrate that Kras-mutation ovarian cancer cells are resistant to PARPi. Furthermore, adaptive PARPi resistance is associated with increased RAS/MAPK pathway activity. Kras mutations could obviously induce mTORC2 activity. In contrast, knockout of Rictor/mTORC2 prevents tumor formation in Kras mutant tumors. And Rictor deletion also induces reactive oxygen species(ROS) and DNA damage in ovarian cancer cells. Here, we are trying to investigate whether targeting mTORC2 could prevent or reverse PAPRi resistance. Except to identify the function of mTORC2 on homologous recombination repair (HRR), we will use different kinds of methods to evaluate the effects when targeting mTORC2 in PARPi resistant tumors. This study will provide a new landscape of promising drug targets in PARPi resistance tumors.

尽管PARP抑制剂在临床治疗中获得巨大成功，但绝大多数卵巢癌很快对其产生抵抗。因此如何预防或逆转PARP抑制剂耐药非常关键。申请者前期研究发现PARP抑制剂对携带Kras突变的卵巢癌细胞普遍存在抵抗，且获得性PARP抑制剂抵抗的细胞RAS/MAPK通路异常活化。外源性导入Kras突变，显著激活mTORC2活性。反之，敲除Rictor/mTORC2可以阻止Kras突变导致的肿瘤发生；同时可显著升高细胞内的氧化应激水平，导致DNA损伤。基于此，本项目将围绕mTORC2活性和PARP抑制剂敏感性，一方面深入探讨mTORC2与同源重组修复之间的关系，一方面系统评价靶向mTORC2对逆转PARP抑制剂抵抗的可能性。完成本项目将为解决临床PARP抑制剂抵抗耐药提供可参考的方案。

本项目在前期研究的基础上，发现敲除或反义干扰mTORC2重要组份Rictor后，细胞内的氧化应激水平升高，细胞内DNA损伤增加。代谢组学分析显示敲除Rictor后，细胞内过高的氧化应激，在导致谷胱甘肽代谢受抑制的同时，会激活磷酸戊糖途径，产生更多的NADPH，用于抵抗过高的氧化应激。当运用PI3K/AKT/mTOR通路不同的抑制剂（包括mTORC1抑制剂Rapamycin，AKT抑制剂MK2006，mTORC1和mTORC2抑制剂AZD8055，PI3K抑制剂GDC0941，非选择性AKT抑制剂CX4945）处理细胞，RPPA高通量多蛋白靶点分析发现下游关键蛋白变化。在动物水平上，我们运用PDX模型，分别给予单独的PARPi、AKTi和mTORi，及两者的联合抑制剂，发现联合PARPi和AKTi或mTORi较单药相比，均能显著抑制肿瘤生长和通路抑制活性。最后，我们对606例来自TCGA人类卵巢癌样本数据系统分析了PI3K/AKT/mTOR 通路，发现约有16%卵巢癌患者中Rictor表达发生改变；我们对133例浆液性卵巢癌和15例子宫内膜样卵巢癌中的Rictor蛋白进行了免疫组织化学染色，显示高表达Rictor的患者显示较差的生存率。通过本研究，初步证实联合PARP抑制剂和AKT/mTOR激酶活性对于逆转PARP抑制剂抵抗具有重要临床应用价值。