Interruption of squalene epoxidase and DNA damage response in cancer therapy

项目摘要 非小细胞肺癌(NSCLC)是最常见的肺癌。目前对这种疾病的治疗方法 仍然不足，迫切需要新的治疗策略。角鲨烯环氧酶(SQLE) 通过将角鲨烯转化为氧化角鲨烯来控制胆固醇生物合成的酶经常被 在非小细胞肺癌中高表达。这种蛋白的高表达与预后不良有关。因此，目标是 这一应用是为了寻找治疗高表达Sqle的非小细胞肺癌的新方法。SQLE抑制剂是 目前临床上用于治疗真菌感染，部分是由于角鲨烯的蓄积。引人注目的是，我们最近 全基因组功能丧失筛查和初步数据表明，Sqle被敲除后受到抑制 增强了对靶向DNA损伤反应(DDR)激酶CHK1及其上游的抑制剂的敏感性 因数ATR。ATR-CHK1轴是复制应激反应的关键组成部分。ATR和CHK1的抑制作用 导致复制叉崩溃和DNA双链断裂(DSB)的产生，DSB是一种主要的DNA结构 可以激活自动柜员机的激活剂。鉴于ATM在DSB修复和细胞周期检查点中的关键作用，细胞 ATR/CHK1活性受抑制的患者严重依赖ATM生存。我们的初步数据显示，Sqle 基因敲除会导致Wip1的增加，Wip1是一种抑制ATM活动的磷酸酶。因为它已经 此前已有报道称角鲨烯的积累会导致Wip1蛋白表达增加，我们 假设SQLE抑制抑制ATM活性，从而使细胞对ATR和ATR敏感 Chk1抑制剂。因此，表达高SqLE的NSCLC细胞的子集可以被 SQLE和ATR或CHK1的联合抑制作用。提出了两个具体目标，即确定(1) Sqle抑制增强NSCLC细胞对ATR和CHK1抑制剂的敏感性的机制 (2)SQLE与ATR或CHK1联合抑制的协同抗肿瘤作用。在目标1中，我们将 确定SQLE抑制是否会抑制ATM活动，从而导致DDR受损，包括DSB 修复和细胞周期检查点，以一种依赖于Wip1和角鲨烯的方式。一种体外激酶法，DSB 修复记者说，将使用细胞遗传学分析和细胞生物学技术。要确定牵涉其中 在Wip1和角鲨烯中，将评估Sqle抑制导致的ATM活性缺陷和随后的DDR 在表达野生型和失活Wip1的细胞中，以及在有或没有角鲨烯合成的细胞中 抑制力。在目标2中，我们将评估SQLE和ATR或CHK1联合抑制的抗肿瘤效果 使用体外分析以及基于细胞系和患者来源的异种移植(PDX)模型。如果成功，我们的研究 通过寻找新的治疗方法，将对提高肺癌患者的存活率产生重大影响 从同时抑制胆固醇生物合成所需蛋白质的角度探讨 和DDR。

Project Summary Non-small cell lung cancer (NSCLC) is the most common lung cancer. Current treatments for this disease remain inadequate, and novel treatment strategies are urgently needed. Squalene epoxidase (SQLE), an enzyme controlling cholesterol biosynthesis by converting squalene to oxidosqualene, is frequently overexpressed in NSCLC. High expression of this protein is associated with poor prognosis. Thus, the goal of this application is to identify new approaches to treat high SQLE-expressing NSCLC. SQLE inhibitors are currently used in clinic for treating fungal infection partially by accumulation of squalene. Strikingly, our recent genome-wide loss-of-function screen and preliminary data suggest that SQLE inhibition by knockdown enhanced the sensitivity to inhibitors targeting the DNA damage response (DDR) kinase CHK1 and its upstream factor ATR. ATR-CHK1 axis are the key component of replication stress response. Inhibition of ATR and CHK1 leads to replication fork collapse and generation of DNA double strand breaks (DSBs), a major DNA structure that can activate ATM kinase. Given the critical role of ATM in DSB repair and cell cycle checkpoints, the cells with inhibited ATR/CHK1 activity rely heavily on ATM for survival. Our preliminary data suggest that SQLE knockdown leads to an increase in WIP1, which is a phosphatase that suppresses ATM activity. Since it has been reported previously that squalene accumulation lead to increase in WIP1 protein expression, we hypothesize that SQLE inhibition suppresses ATM activity, thereby rendering the cells sensitive to ATR and CHK1 inhibitors. Thus, a subset of NSCLC cells expressing high SQLE can be specifically targeted by the combined inhibition of SQLE and ATR or CHK1. Two Specific Aims are proposed, which are to determine (1) the mechanisms by which SQLE inhibition potentiates NSCLC cell sensitivity to ATR and CHK1 inhibitors and (2) the synergistic antitumor efficacy of combined inhibition of SQLE and ATR or CHK1. In Aim 1, we will determine whether SQLE inhibition suppresses ATM activity, thereby leading to impaired DDR, including DSB repair and cell cycle checkpoint, in a manner dependent on WIP1 and squalene. An in vitro kinase assay, DSB repair reporters, a cytogenetic assay and cell biological techniques will be used. To determine the involvement of WIP1 and squalene, SQLE inhibition-induced defects in ATM activity and subsequent DDR will be evaluated in cells expressing wild type and inactivated WIP1, and in cells with or without with squalene syntheses inhibition. In Aim 2, we will assess the antitumor efficacy of the combined inhibition of SQLE and ATR or CHK1 using in vitro assays and cell line-based and patient-derived xenograft (PDX) models. If successful, our studies will have a significant impact on improving the survival of lung cancer patients by identifying novel therapeutic approaches from the perspective of simultaneously inhibiting the proteins required for cholesterol biosynthesis and DDR.