Interruption of squalene epoxidase and DNA damage response in cancer therapy

癌症治疗中角鲨烯环氧酶和 DNA 损伤反应的中断

• 批准号: 10066331
• 负责人: Junran Zhang
• 金额: $ 18.23万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-06 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10066331
• 关键词: ATM Signaling Pathway; ATR gene; Antifungal Agents; Atlases; Binding; Biological; Biological Assay; CHEK1 gene; Cancer Patient; Cell Cycle Checkpoint; Cell Death; Cell Line; Cell Survival; Cells; Cholesterol; Clinic; Clinical Trials; Comet Assay; Cytogenetics; DNA Damage; DNA Double Strand Break; DNA Structure; DNA replication fork; Dangerousness; Data; Defect; Disease; Double Strand Break Repair; Drug Targeting; Drug usage; Enzymes; Flow Cytometry; G2/M Arrest; Generations; Genes; Goals; Growth; Human; Impairment; In Vitro; Interruption; Lead; Lung Adenocarcinoma; Lung Neoplasms; Malignant Neoplasms; Malignant neoplasm of lung; Mycoses; Non-Small-Cell Lung Carcinoma; Nonhomologous DNA End Joining; Oncology; Patients; Pharmacologic Substance; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Prognosis; Proteins; Reporter; Reporting; Role; Squalene; Techniques; Testing; United States Food and Drug Administration; antitumor agent; antitumor effect; ataxia telangiectasia mutated protein; base; biological adaptation to stress; cancer diagnosis; cancer therapy; cholesterol biosynthesis; epoxidase; genome-wide; homologous recombination; improved; in vitro Assay; in vitro activity; inhibitor/antagonist; knock-down; loss of function; lung cancer cell; mouse model; novel; novel strategies; novel therapeutic intervention; overexpression; patient derived xenograft model; protein expression; repaired; replication stress; response; small hairpin RNA; success; transcription factor; treatment strategy; tumor

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.

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 目前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NSCLC中过度表达。该蛋白的高表达与不良预后相关。因此， 本申请旨在鉴定治疗高SQLE表达的NSCLC的新方法。SQLE抑制剂是 目前在临床上部分通过角鲨烯的积累用于治疗真菌感染。引人注目的是，我们最近的 全基因组功能缺失筛选和初步数据表明，通过敲低抑制SQLE 增强了对靶向DNA损伤反应（DDR）激酶CHK 1及其上游的抑制剂的敏感性 ATR因子。ATR-CHK 1轴是复制应激反应的关键组成部分。ATR和CHK 1的抑制 导致复制叉崩溃和DNA双链断裂（DSB），一种主要的DNA结构 可以激活ATM激酶鉴于ATM在DSB修复和细胞周期检查点中的关键作用， ATR/CHK 1活性受到抑制的患者严重依赖ATM生存。我们的初步数据表明，SQLE 敲低导致WIP 1增加，WIP 1是一种抑制ATM活性的磷酸酶。因为它有 先前报道角鲨烯积累导致WIP 1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敏感， CHK 1抑制剂。因此，表达高SQLE的NSCLC细胞亚群可以被特异性靶向。 SQLE和ATR或CHK 1的联合抑制。提出了两个具体目标，即确定（1） SQLE抑制增强NSCLC细胞对ATR和CHK 1抑制剂敏感性的机制， (2)联合抑制SQLE和ATR或CHK 1的协同抗肿瘤功效。在目标1中，我们 确定SQLE抑制是否抑制ATM活动，从而导致DDR受损，包括DSB 修复和细胞周期检查点，以依赖于WIP 1和角鲨烯的方式。体外激酶试验，DSB 将使用修复报告基因、细胞遗传学测定和细胞生物学技术。以确定参与 WIP 1和角鲨烯，将评估SQLE抑制诱导的ATM活性缺陷和随后的DDR 在表达野生型和失活WIP 1的细胞中，以及在具有或不具有角鲨烯合成的细胞中， 抑制作用在目标2中，我们将评估SQLE和ATR或CHK 1的联合抑制的抗肿瘤功效。 使用体外测定和基于细胞系和患者来源的异种移植物（PDX）模型。如果成功，我们的研究 将通过发现新的治疗方法对提高肺癌患者的生存率产生重大影响， 从同时抑制胆固醇生物合成所需的蛋白质的角度来看， 和DDR。