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

基本信息

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
项目类别：
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

项目摘要

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.

通过化学筛选靶向 KRAS 突变肺癌的 DNA 修复在下面的 7 x 7 英寸空间中，简明地总结您提出的研究，概述背景、目标/假设、具体目标、研究设计以及与癌症问题的相关性。当您将摘要准备为单独的文件时以电子方式提交您的申请。请参阅应用说明。如果申请获得资助，本摘要将成为公开信息。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%是肺腺癌携带KRAS突变，缺乏靶向治疗。化疗仍然存在 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造成大量 DNA 损伤，尤其是双链断裂 (DSB)。然而，细胞也进化出保护机制（DNA损伤反应和修复）来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治疗 KRAS 突变肺癌的有效化疗增敏剂。初步数据：天然产物表现出多种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 种具有各种结构的化合物）类型）并确定强心苷为有效的 DNA 损伤反应抑制剂。我们展示强心苷特异性抑制 5' 至 3' DSB 末端切除，这是 DNA 损伤反应的激活和忠实的 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虽然对正常肺成纤维细胞的影响要小得多，表明这些细胞具有癌症特异性作用化合物。这种疗法的增敏作用在小鼠异种移植肺癌中得到了证实。目的：该项目的目标是通过以下方法确定分子靶点和详细机制：哪种强心苷可使 KRAS 突变肺癌的化疗增敏。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。 DSB 末端切除由许多蛋白质控制，包括 53BP1、BRCA1、UHRF1、我们推测强心苷通过调节表达来抑制DSB末端切除这些关键 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) 相比，我们确定 UHRF1 为最佳候选者，因为 UHRF1 起着关键作用促进 DSB 5' 至 3' 末端切除，抑制 UHRF1 抑制 DSB 末端切除。这里我们将确定（1）UHRF1如何介导DNA损伤反应和DSB 在存在强心苷的情况下进行修复，以及（2）强心苷的分子细节糖苷调节 UHRF1 的表达水平，从而调节细胞对 DSB 诱导的敏感性抗癌药物。在目标 2 中，我们将确定强心苷直接作用的细胞靶点上通过化学蛋白质组分析（即分子捕获后进行质谱分析）。我们鉴定出 Cdc20，有丝分裂 E3 泛素连接酶后期促进的激活因子复合体/环体 (APC/C)，作为候选者，因为据报道 UHRF1 可能被降解取决于 Cdc20。 (1)我们将纯化各种Cdc20重组蛋白以进行(a)表面等离子共振 (SPR) 以确定结合动力学，以及 (b) 等温滴定量热法确定强心苷与 Cdc20 的结合化学计量、亲和力和焓。 (2) 我们将使用细胞热位移测定来确定培养细胞中 AT2 与 Cdc20 的相互作用。 (3)我们将进一步确定强心苷与Cdc20相互作用对 UHRF1 降解。在目标 3 中，我们将进行结构-活性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 DNA损伤抑制活性同时降低心脏毒性。这些化合物将是为进一步的临床应用提供线索。在目标 4 中，我们将确定以下药物的治疗潜力：强心苷增强化疗效果确认， KRAS 突变原位肺癌症患者来源的异种移植物和转基因小鼠肺癌模型。这些研究将为测试放疗或化疗联合治疗提供基础强心苷在临床肺癌治疗中的应用。癌症相关性：肺癌是一种毁灭性的疾病。治疗敏化剂的鉴定增强 KRAS 突变肺癌的化疗效果是非常可取的，这将提高美国肺癌患者的生存率，尤其是那些携带 KRAS 突变的患者。