Targeting DNA repair in KRAS mutated lung cancer by chemical screening

KRAS基因突变肺癌靶向DNA修复的化学筛选 在下面7x7英寸的空白处，简明扼要地总结你提出的研究，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。你将把摘要准备成单独的文件，当你 以电子方式提交您的申请。请参阅应用说明。如果申请得到资助，本摘要将 成为公开信息。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，缺乏有针对性的治疗。化疗仍在继续 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，特别是双链断裂(DSB)。然而，细胞也 进化的保护机制(DNA损伤反应和修复)以逃避细胞杀伤效应 化疗的结果。因此，抑制DNA损伤反应和DSB修复的小分子可以 被重新用于治疗KRAS突变肺癌的有效化学增敏剂。 初步数据：天然产物表现出广泛的结构复杂性和多样性， 为药物发现提供了丰富的资源。突变KRAS化疗敏感剂的鉴定 肺癌，我们筛选了一个天然产物文库(~1000个具有不同结构的化合物 类型)，并确定心脏糖苷是有效的DNA损伤反应抑制剂。我们展示了 心脏苷类特别抑制5‘到3’DSB末端切除，这一过程是 激活DNA损伤反应和忠实的DSB修复。强心苷类 增强DSB诱导药物对KRAS突变型肺癌细胞的生长抑制作用 而对正常肺成纤维细胞的影响要小得多，这表明这些物质具有癌症特异性 化合物。在小鼠肺癌移植瘤中证实了该疗法的增敏作用。 目的：本项目的目标是通过以下方式确定分子靶点和详细的机制 在KRAS基因突变的肺癌中，哪些心脏苷类药物使化疗增敏。 研究设计：在目标1中，我们将确定心苷如何抑制5‘到3’DSB末端 切除手术。DSB末端切除受多种蛋白质控制，包括53BP1、BRCA1、UHRF1、 等。我们假设心脏糖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 在氨基酸(SILAC)中，我们将uhrf1确定为首选候选，因为uhrf1起着关键作用。 在促进DSB的5‘端到3’端切除和抑制uhrf1抑制DSB端 切除手术。在这里，我们将确定(1)uhrf1如何介导dna损伤反应和dsb。 在存在心脏糖苷的情况下进行修复，以及(2)心脏 糖苷调节uhrf1的表达水平，因此细胞对dsb诱导的敏感性。 抗癌药物。在目标2中，我们将确定心脏糖苷直接作用的细胞靶点。 从上到下 化学蛋白质组学分析(即分子捕获和质谱分析)。 我们 有丝分裂E3泛素连接酶后期促进活性因子Cdc20的鉴定 复合体/环体(APC/C)，因为据报道uhrf1可能被降解 依赖于CdC20。(1)我们将提纯各种cdc20重组蛋白以进行(A)表面处理 等离子体共振(SPR)测定结合动力学，以及(B)等温滴定量热法 测定强心苷类化合物与CdC20的结合化学计量学、亲和力和结合热。(2) 我们将使用细胞热位移实验来确定AT2与培养细胞中的Cdc20的相互作用。 (3)我们将进一步确定强心苷与CdC20相互作用的影响。 Uhrf1降解。在目标3中，我们将进行结构-活性关系研究，以 鉴定和验证具有最高可能的溶解度和 DNA损伤抑制活性，同时降低心脏毒性。这些化合物将成为 为进一步的临床应用提供线索。在目标4中，我们将确定其治疗潜力 强心苷在提高化疗疗效中的作用 识别， KRAS突变的原位肺 癌症患者来源的异种移植和转基因小鼠肺癌模型。这些 研究将为测试放射治疗或化疗与 心苷类药物在肺癌治疗中的临床应用。 癌症相关性：肺癌是一种毁灭性的疾病。治疗敏感剂的鉴定 提高化疗对KRAS突变肺癌的疗效是非常可取的，这将是 提高美国肺癌患者的存活率，特别是那些带有KRAS突变的患者。

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.