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: 10436267
• 负责人: YOU-WEI ZHANG
• 金额: $ 47.58万
• 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/10436267
• 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-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; 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.

通过化学筛查靶向KRAS中的DNA修复肺癌 在下面的7 x 7英寸空间中，简单地总结了您提出的研究，概述背景，客观/假设， 具体目的，研究设计以及与癌症问题相关。您将在您时准备摘要作为单独的文件 以电子方式提交您的申请。请参阅申请说明。如果申请是资助的，则该摘要将 成为公共信息。因此，请勿包含专有/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 腺癌带有KRAS突变，缺乏靶向疗法。化学疗法仍然存在 KRAS突变肺癌的主要治疗方法。许多化学疗法通过 引起巨大的DNA损伤，尤其是双链断裂（DSB）。但是，细胞也 进化的保护机制（DNA损伤响应和修复）以逃避细胞杀戮效果 化学疗法。因此，抑制DNA损伤响应和DSB修复的小分子可以 将重新用于KRAS突变肺癌的有效化学敏感剂。 初步数据：天然产品显示出多种结构复杂性和多样性， 代表丰富的药物发现资源。鉴定突变的KRAS的化学敏感器 肺癌，我们筛选了一个天然产品库（〜1000种具有各种结构的化合物 类型）并确定心脏糖苷是有效的DNA损伤反应抑制剂。我们证明 这种心脏糖苷专门抑制5'至3'DSB终端切除术，这是 DNA损伤反应的激活和忠实的DSB修复。心脏糖苷强烈 增强了DSB诱导药物对KRAS突变肺癌细胞的生长抑制作用 虽然对正常肺成纤维细胞的影响要小得多，但表明这些癌症的特定作用 化合物。在小鼠的异种移植肺癌中证实了这种疗法敏化作用。 目的：该项目的目的是确定分子靶标和详细机制 哪种心脏糖苷使KRAS突变的肺癌中的化学疗法敏感。 研究设计：在AIM 1中，我们将确定心脏糖苷如何抑制5'至3'DSB端 切除。 DSB终端切除受许多蛋白质控制，包括53BP1，BRCA1，UHRF1， 我们假设心脏糖苷通过调节表达来抑制DSB终端切除 这些关键DSB基因的水平。通过整个基因组测序和稳定的同位素标记 使用氨基酸（SILAC），我们将UHRF1确定为顶级候选者，因为UHRF1起着至关重要的作用 在促进DSB的5'至3'末端切除和抑制UHRF1时，抑制了DSB端 切除。在这里，我们将确定（1）UHRF1如何介导DNA损伤响应和DSB 在存在心脏糖苷的情况下修复，以及（2）心脏的分子细节 糖苷调节UHRF1的表达水平，因此细胞对DSB诱导的敏感性 抗癌药。在AIM 2中，我们将确定心脏糖苷直接作用的细胞靶标 通过 化学蛋白质组学分析（即分子捕获，然后进行质谱法）。 我们 确定的Cdc20，促进有丝分裂E3泛素连接酶的激活因子促进 复合/循环体（APC/C）作为候选人，因为据报道UHRF1可能会降解 取决于CDC20。 （1）我们将净化各种CDC20重组蛋白以执行（a）表面 等离子体共振（SPR）确定结合动力学，（b）等温滴定量热法 为了确定与CDC20的心脏糖苷的亲和力和焓。 （2） 我们将使用细胞热移测定法确定AT2与Cdc20在培养细胞中的相互作用。 （3）我们将进一步确定心脏糖苷与Cdc20的相互作用的影响 UHRF1退化。在AIM 3中，我们将执行结构性关系研究 表征和验证具有最高溶解度的心脏糖苷衍生物 DNA损伤抑制活性，同时降低心脏毒性。这些化合物将是 引导进一步的临床应用。在AIM 4中，我们将确定 心脏糖苷在增强使用化学疗法的作用方面使用 确认， KRAS突变的原位肺 癌症患者衍生的异种移植物和基因改良的小鼠肺癌模型。这些 研究将为测试放疗或化疗与 诊所肺癌治疗中的心脏糖苷。 癌症相关性：肺癌是一种毁灭性疾病。鉴定治疗敏化剂 增强化学疗法对KRAS突变肺癌的作用是非常可取的，这将 改善美国肺癌患者，尤其是患有KRAS突变的患者的生存率。