Targeting DNA repair in KRAS mutated lung cancer by chemical screening

通过化学筛选靶向 KRAS 突变肺癌的 DNA 修复

• 批准号: 10207541
• 负责人: YOU-WEI ZHANG
• 金额: $ 48.55万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10207541
• 关键词: AGTR2 gene; Affinity; Amino Acids; Antineoplastic Agents; Arrhythmia; BRCA1 gene; Binding; Binding Proteins; Biological; Biological Assay; Calorimetry; Cancer Etiology; Cancer Model; Cancer Patient; Cardiac Glycosides; Cardiac Myocytes; Cardiotoxicity; Cell Survival; Cells; Cessation of life; Chemicals; Cisplatin; Clinic; Clinical; Confidential Information; Cultured Cells; DNA Damage; DNA Double Strand Break; DNA Repair; Data; Development; Disease; Double Effect; Double Strand Break Repair; Drug toxicity; Excision; Fibroblasts; Foundations; Funding; Future; Genes; Goals; Growth; Histologic; Human; In Vitro; Instruction; KRAS2 gene; Kinetics; Knowledge; Lead; Libraries; Longevity; Lung; Lung Adenocarcinoma; Malignant Neoplasms; Malignant neoplasm of lung; Mass Spectrum Analysis; Mediating; Mitotic; Modeling; Molecular; Molecular Target; Mus; Mutate; Mutation; Na(+)-K(+)-Exchanging ATPase; Natural Products; Neoplasm Metastasis; Non-Small-Cell Lung Carcinoma; Patients; Penetrance; Pharmaceutical Preparations; Phenotype; Physiological; Play; Poison; Pre-Clinical Model; Process; Proteins; Proteomics; Radiation therapy; Recombinant Proteins; Regulation; Reporting; Research; Research Design; Role; Signal Transduction; Solubility; Specificity; Stable Isotope Labeling; Structure; Structure-Activity Relationship; Surface Plasmon Resonance; Testing; Therapeutic; Titrations; Toxic effect; Tumor Tissue; analog; anaphase-promoting complex; anti-cancer; anticancer activity; base; biomarker-driven; cancer cell; cancer therapy; cancer type; cell killing; cell type; cellular targeting; chemosensitizing agent; chemotherapy; clinical application; clinical development; drug discovery; drug resource; effective therapy; enthalpy; genome sequencing; heart function; improved; inhibitor/antagonist; insight; lung cancer cell; lung cancer screening; lung xenograft; mouse model; mutant; novel; p53-binding protein 1; patient derived xenograft model; pre-clinical; protein degradation; repaired; response; screening; small molecule; stoichiometry; targeted treatment; tumor; tumor growth; ubiquitin ligase; ubiquitin-protein ligase; whole genome

Targeting DNA repair in KRAS mutated lung cancer by chemical screening In the 7 x 7-inch space below, summarize concisely your proposed research, outlining background, objective/hypothesis, specific aims, study design, and relevance to the cancer problem. You will prepare the abstract as a separate file when you electronically submit your application. Refer to Application Instructions. If the application is funded, this Abstract will become public information. Therefore, do not include proprietary/confidential information. Background: Lung cancer is the leading cause of cancer death in the US. Around 30% lung adenocarcinoma carries KRAS mutation, which lacks targeted therapies. Chemotherapy remains the mainstay treatment for KRAS mutated lung cancers. Many chemotherapy kills cancer cells by causing massive DNA damage, particularly double strand breaks (DSBs). However, cells also evolved protective mechanisms (DNA damage response and repair) to evade the cell killing effect of chemotherapy. Hence, small molecules that inhibit DNA damage response and DSB repair can be repurposed into effective chemo-sensitizers for KRAS mutated lung cancers. Preliminary Data: Natural products display a wide variety of structural complexity and diversity, representing a rich resource for drug discovery. To identify chemo-sensitizers for KRAS mutated lung cancer, we screened a natural product library (~1000 compounds with various structural types) and identified cardiac glycosides as potent DNA damage response inhibitors. We demonstrate that cardiac glycosides specifically inhibit the 5' to 3' DSB end resection, a process that is required for the activation of DNA damage response and faithful DSB repair. Cardiac glycosides strongly enhanced the growth inhibition effect of DSB-inducing drugs on KRAS mutant lung cancer cells while having much less effect on normal lung fibroblasts, indicating a cancer specific effect of these compounds. This therapy sensitizing effect was confirmed in xenografted lung cancers in mice. Objective: The goal of this project is to determine the molecular targets and detailed mechanisms by which cardiac glycosides sensitize chemotherapy in KRAS mutated lung cancers. Study Design: In Aim 1, we will determine how cardiac glycosides inhibit the 5' to 3' DSB end resection. DSB end resection is controlled by many proteins including 53BP1, BRCA1, UHRF1, etc. We hypothesize that cardiac glycosides inhibit DSB end resection by regulating expression levels of these critical DSB genes. Through whole genome sequencing and stable isotope labeling with amino acids (SILAC), we identified UHRF1 as the top candidate as UHRF1 plays a critical role in promoting the 5' to 3' end resection of DSBs and inhibition of UHRF1 suppresses DSB end resection. Here we will determine (1) how UHRF1 mediates DNA damage response and DSB repair in the presence of cardiac glycosides, and (2) the molecular details by which cardiac glycosides regulate the expression level of UHRF1, and therefore cell sensitivity to DSB-inducing anticancer drugs. In Aim 2, we will identify cellular targets by which cardiac glycosides directly act on through chemical proteomic analysis (i.e., molecular capturing followed by mass spectrometry). We identified Cdc20, the activating factor for the mitotic E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C), as a candidate because UHRF1 was reported to be degraded likely dependent on Cdc20. (1) We will purify various Cdc20 recombinant proteins to perform (a) surface plasmon resonance (SPR) to determine the binding kinetics, and (b) isothermal titration calorimetry to determine the binding stoichiometry, affinity and enthalpy of cardiac glycosides with Cdc20. (2) We will use cell thermal shift assay to determine the interaction of AT2 with Cdc20 in cultured cells. (3) We will further determine the impact of the interaction of cardiac glycosides with Cdc20 on UHRF1 degradation. In Aim 3, we will perform structure-activity relationship studies to characterize and validate cardiac glycoside derivatives that have highest possible solubility and DNA damage inhibition activity while reducing the cardiac toxicity. These compounds will be the leads for further clinical applications. In Aim 4, we will determine the therapeutic potential of cardiac glycosides in enhancing the effect of chemotherapy using identify, KRAS mutated orthotopic lung cancer patient-derived xenografts and genetically modified mouse lung cancer models. These studies will provide the foundation for testing the combination of radiotherapy or chemotherapy with cardiac glycosides in lung cancer treatment in the clinic. Cancer Relevance: Lung cancer is a devastating disease. Identification of therapy sensitizers to enhance the effect of chemotherapy in KRAS mutated lung cancer is highly desirable, which will improve the survival of lung cancer patients, especially those with KRAS mutations, in the US.

化学筛选KRAS突变肺癌的靶向DNA修复 在下面7 x 7英寸的空间中，简要总结您提出的研究，概述背景、目标/假设， 具体目标、研究设计和与癌症问题的相关性。当您需要将摘要作为单独的文件准备时， 电子提交您的申请。请参阅应用说明。如果申请得到资助，本摘要将 成为公共信息。因此，不包括专有/机密信息。 背景：肺癌是美国癌症死亡的主要原因。大约30%的肺 腺癌携带KRAS突变，缺乏靶向治疗。化疗仍然是 KRAS突变肺癌的主要治疗方法。许多化疗通过以下方式杀死癌细胞： 导致大量的DNA损伤，特别是双链断裂（DSB）。然而，细胞也 进化出保护机制（DNA损伤反应和修复）以逃避细胞杀伤效应 化疗。因此，抑制DNA损伤反应和DSB修复的小分子可以 被重新利用为KRAS突变肺癌的有效化学增敏剂。 初步数据：天然产物显示出各种各样的结构复杂性和多样性， 代表了药物发现的丰富资源。鉴定KRAS突变的化学增敏剂 肺癌，我们筛选了一个天然产物库（约1000种具有不同结构的化合物）， 类型），并确定强心苷作为有效的DNA损伤反应抑制剂。我们证明 强心苷特异性抑制5'至3' DSB末端切除，这是一个需要的过程， 激活DNA损伤反应和忠实的DSB修复。强心苷 增强DSB诱导药物对KRAS突变肺癌细胞的生长抑制作用 而对正常肺成纤维细胞的影响小得多，表明这些细胞的癌症特异性作用。 化合物.这种治疗增敏作用在小鼠异种移植肺癌中得到证实。 目的：本课题的目标是通过以下方法来确定其分子靶点和详细的机制： 其强心苷使KRAS突变的肺癌中的化疗敏感。 研究设计：在目标1中，我们将确定强心苷如何抑制5'至3' DSB末端 切除术DSB末端切除受许多蛋白质控制，包括53 BP 1，BRCA 1，UHRF 1， 等我们假设强心苷通过调节表达抑制DSB末端切除 这些关键的DSB基因的水平。通过全基因组测序和稳定同位素标记 与氨基酸（SILAC），我们确定UHRF 1作为首选候选人，因为UHRF 1起着关键作用 在促进DSB的5'端至3'端切除和抑制UHRF 1抑制DSB末端 切除术在这里，我们将确定（1）UHRF 1如何介导DNA损伤反应和DSB 在强心苷的存在下修复，和（2）心脏的分子细节， 糖苷调节UHRF 1的表达水平，因此调节细胞对DSB诱导的细胞凋亡的敏感性。 抗癌药在目标2中，我们将确定强心苷直接作用的细胞靶点 on through 化学蛋白质组学分析（即，分子捕获，随后质谱法）。 我们 鉴定了Cdc 20，有丝分裂E3泛素连接酶后期促进的激活因子 复合体/细胞周期体（APC/C）作为候选者，因为据报道UHRF 1可能被降解 依赖于Cdc 20。(1)我们将纯化各种Cdc 20重组蛋白，以进行（a）表面修饰， 等离子体共振（SPR）测定结合动力学，和（B）等温滴定量热法 以确定强心苷与Cdc 20的结合化学计量、亲和力和焓。（二） 我们将使用细胞热位移分析来确定AT 2与Cdc 20在培养细胞中的相互作用。 (3)我们将进一步确定强心苷与Cdc 20的相互作用对 UHRF 1降解。在目标3中，我们将进行构效关系研究， 表征和验证具有最高可能溶解度的强心苷衍生物， 抑制DNA损伤活性，同时降低心脏毒性。这些化合物将是 用于进一步的临床应用。在目标4中，我们将确定 强心苷类药物增强化疗效果的研究 识别， KRAS突变原位肺 癌症患者来源的异种移植物和遗传修饰的小鼠肺癌模型。这些 这些研究将为检测放射治疗或化学治疗的组合提供基础， 强心苷在肺癌治疗中的临床应用。 肺癌是一种常见的恶性肿瘤。治疗致敏物的鉴定， 增强化疗在KRAS突变肺癌中的效果是非常可取的，这将 改善美国肺癌患者的生存率，尤其是那些KRAS突变的患者。