Identification of chemotherapeutic sensitizers

化疗增敏剂的鉴定

基本信息

批准号：
8948391
负责人：
Kyungjae Myung
金额：
$ 45.62万
依托单位：
NATIONAL HUMAN GENOME RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8948391
关键词：
Biochemical Bioinformatics Biological Assay Biological Markers CHEK2 gene Categories Cell Death Cell Line Cells Chemicals Chimeric Proteins Collaborations Collection DNA Damage DNA Repair Pathway DNA damage checkpoint DNA repair protein Exhibits Future Gene Targeting Genes Hereditary Nonpolyposis Colorectal Neoplasms Human Cell Line In Vitro Inhibitory Concentration 50 Laboratories Lead Luciferases Malignant Neoplasms Mismatch Repair Molecular Molecular Bank Mus Pathway interactions Pharmaceutical Preparations Phosphotransferases Production Proliferating Proteins Proteolysis Radiation Radiation therapy Signal Transduction Small Interfering RNA Specificity Testing Translational Research Tumor Burden United States National Institutes of Health Xenograft procedure base cancer cell cell killing chemotherapeutic agent chemotherapy compound 30 endonuclease fighting genome-wide high throughput screening in vivo killings knockout gene mouse model novel response screening small molecule small molecule libraries tumor

项目摘要

Chemotherapeutic and radiation treatments cause a variety of genotoxic insults that lead to cell death in rapidly proliferating cancer cells. To survive genotoxic insults, cancer cells depend on multiple DNA repair pathways. Depending on the types of genotoxic insult, cells use a specific DNA repair pathway. When a DNA repair pathway is compromised, cancer cells become more sensitive to certain genotoxic insults. The identification of chemotherapeutic agents acting on compromised DNA repair pathways in cancer cells would result in more efficient treatment of cancer cells. Such agents are potential sensitizers for radiation therapy. We found that ATAD5 protein is stabilized in response to almost all genotoxic insults. Thus, we hypothesized that ATAD5 would be a good biomarker to detect genotoxic insults. We generated a cell line expressing the ATAD5-luciferase fusion protein and showed that the fusion protein is also stabilized in response to genotoxic insults. We used this novel cell-based quantitative high-throughput ATAD5-luciferase assay and successfully screened over 300,000 compounds in the NIH chemical library in collaboration with the National Center for Advancing Translational Sciences (NCATS) and found 300 potential chemotherapeutic compounds. To identify DNA repair pathways targeted by the genotoxic compounds, we used 8 isogenic human cell lines with targeted gene knockouts in specific DNA repair pathways. Approximately 300 compounds were tested in survival assays on these cells and group into sub-categories based on their IC50 to kill these cells. We found a small molecule that killed a mismatch repair deficient cancer cells and two small molecules that killed parp1 deficient cancer cells more efficiently. We investigated whether the compound killing mismatch repair deficient tumor (Lynch syndrome tumor) can reduce tumor burden in vivo using xenograft mice as well as gene targeted mice models. The compound showed potential selective killing effect in both mice models. We also used this compound to dissect molecular functions of mismatch repair pathway. We found mismatch repair defendant DNA damage checkpoint activation and characterized detail molecular mechanisms in vitro. Mismatch repair specifically inhibits double strand break formation by endonuclease, XPF by activating CHK2-depedent DNA damage checkpoint. We also found that similar molecular mechanisms were used for selective killing effect in mismatch repair deficient tumors by the compound. In collaboration with NCATS, we also used the same ATAD5-luciferase cell line to identify compounds and siRNAs that inhibit the ATAD5 stabilization in response to genotoxic insults and have identified >80 compounds and >30 siRNAs. Genes identified from these siRNA screens will unveil the unknown mechanisms that inhibit proteolysis of DNA repair proteins in response to genotoxic insults. Two compounds from initial hits could be potential radiation and chemotherapeutic sensitizers in tumors. We found one compound inhibits general DNA damage response by destabilizing DNA damage response kinase(s). We have continued to study to identify targets of these compounds among genes identified from siRNA screening using bioinformatic analysis, epistatic analysis, as well as biochemical interactions.

化学治疗和辐射处理引起多种遗传毒性损伤，导致迅速增殖的癌细胞中细胞死亡。为了生存遗传毒性损伤，癌细胞依赖于多个DNA修复途径。根据遗传毒性损伤的类型，细胞使用特定的DNA修复途径。当DNA修复途径受到损害时，癌细胞对某些遗传毒性损伤变得更加敏感。鉴定作用于癌细胞中DNA修复途径的化学治疗剂将导致对癌细胞的更有效治疗。这样的药物是对放射治疗的潜在敏感性。我们发现ATAD5蛋白几乎响应所有遗传毒性损伤而稳定。因此，我们假设ATAD5将是检测遗传毒性侮辱的好生物标志物。我们产生了表达ATAD5-荧光素酶融合蛋白的细胞系，并表明融合蛋白还响应于遗传毒性损伤而稳定。我们使用了这种新型基于细胞的定量高通量ATAD5-荧光素酶测定法，并在NIH化学库中成功筛选了300,000多种化合物，并与国家前进的转化科学中心（NCAT）合作，发现了300种潜在的化学治疗化合物。为了鉴定遗传毒性化合物靶向的DNA修复途径，我们在特定的DNA修复途径中使用了8个具有靶向基因敲除的等源性人类细胞系。大约300种化合物在这些细胞上的生存测定中进行了测试，并根据其IC50杀死这些细胞。我们发现了一个小分子，该分子杀死了不匹配的维修缺乏癌细胞和两个小分子，可更有效地杀死PARP1缺乏癌细胞。我们研究了复合杀死不匹配的维修缺乏肿瘤（林奇综合征肿瘤）是否可以使用异种移植小鼠以及基因靶向小鼠模型来减轻体内肿瘤负担。该化合物在两种小鼠模型中均显示出潜在的选择性杀伤作用。我们还使用这种化合物来剖析不匹配修复途径的分子功能。我们发现不匹配维修被告DNA损伤检查点激活和体外的细节分子机制表征。不匹配维修特异性地通过激活CHK2-DEPEDEDEND DNA损伤检查点来特异性抑制双链断裂的形成，XPF。我们还发现，使用该化合物的不匹配修复缺乏肿瘤的选择性杀死作用相似。通过与NCAT合作，我们还使用了相同的ATAD5-荧光素酶细胞系来识别抑制ATAD5稳定化的化合物和siRNA，以响应遗传毒性损伤，并鉴定出> 80种化合物和> 30 siRNA。从这些siRNA筛选中鉴定出的基因将揭示出对遗传毒性损伤抑制DNA修复蛋白蛋白质水解的未知机制。初始命中的两种化合物可能是肿瘤中潜在的辐射和化学治疗敏化剂。我们发现一种化合物通过破坏DNA损伤反应激酶（S）的稳定而抑制了一般的DNA损伤反应。我们继续研究，以使用生物信息学分析，上皮分析以及生化相互作用从siRNA筛选中鉴定出这些化合物的靶标。