Identification of chemotherapeutic sensitizers

化疗和放射治疗引起多种遗传毒性损伤，导致快速增殖的癌细胞的细胞死亡。为了在遗传毒性损伤中存活，癌细胞依赖于多种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修复途径受损时，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受损DNA修复途径的化疗剂将导致癌细胞的更有效治疗。这类药物是放射治疗的潜在增敏剂。我们发现，ATAD 5蛋白是稳定的，几乎所有的遗传毒性的侮辱。因此，我们假设ATAD 5将是检测遗传毒性损伤的良好生物标志物。我们产生了表达ATAD 5-荧光素酶融合蛋白的细胞系，并显示融合蛋白也在响应于遗传毒性损伤时稳定。 使用这种新的基于细胞的定量高通量ATAD 5-荧光素酶测定，我们与NIH化学基因组学中心（NCGC）合作，从市售化合物库以及国家毒理学计划（NTP）库中筛选了4，000多种化合物，该中心现在是国家推进转化科学中心（NCATS）和国家环境健康科学研究所（NIEHS）的一部分。我们确定了22种抗氧化剂，包括白藜芦醇，染料木素和黄芩素，目前正在使用或研究用于治疗心血管疾病，2型糖尿病，骨质疏松症，慢性肝炎和抗衰老。用这些化合物处理分裂的细胞诱导DNA损伤并导致细胞死亡。 尽管白藜芦醇、染料木素和黄芩素具有遗传毒性作用，但它们不会引起致突变，而致突变是传统抗癌药物的主要副作用。 此外，白藜芦醇和染料木黄酮杀死多药耐药癌细胞。 因此，我们建议，白藜芦醇，染料木素，黄芩素是有吸引力的候选人，改善化疗药物。 此外，我们与NCGC合作，从NIH化学文库中的300，000种化合物中鉴定出200种以剂量依赖性方式稳定ATAD 5-荧光素酶的化合物。为了鉴定遗传毒性化合物靶向的DNA修复途径，我们使用了8个等基因人类细胞系以及10个等基因鸡DT 40 B细胞系，这些细胞系在特定的DNA修复途径中具有靶向基因敲除。在对这些细胞的存活测定中测试了大约200种化合物，并基于它们杀死这些细胞的IC 50将其分组为亚类。我们目前正在确认这些化合物对在相同DNA修复途径中有缺陷的癌细胞的杀伤潜力。我们将使用异种移植小鼠以及基因靶向小鼠模型进一步研究这些化合物是否可以降低体内肿瘤负荷。每种化合物都将成为剖析不同DNA修复途径分子功能的良好工具。 与NCATS合作，我们还使用相同的ATAD 5-荧光素酶细胞系来鉴定响应于遗传毒性损伤而抑制ATAD 5稳定化的化合物和siRNA，并且已经鉴定了>80种化合物和>30种siRNA。从这些siRNA筛选中鉴定的基因将揭示抑制DNA修复蛋白对遗传毒性损伤的蛋白水解的未知机制。此外，从筛选中鉴定的化合物将是肿瘤中的放射和化学治疗增敏剂，所述肿瘤依赖于响应于放射/化学治疗诱导的DNA损伤的蛋白质稳定化途径。我们目前正在研究，以确定这些化合物的靶基因之间的siRNA筛选使用生物信息学分析，上位性分析，以及生化相互作用。

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. Using this novel cell-based quantitative high-throughput ATAD5-luciferase assay, we screened over 4,000 compounds from commercially available compound library as well as National Toxicology Program (NTP) library in collaboration with the NIH Chemical Genomics Center (NCGC) that is now part of National Center for Advancing Translational Science (NCATS) and National Institute of Environmental Health Sciences (NIEHS). We identified 22 antioxidants, including resveratrol, genistein, and baicalein, that are currently used or investigated for the treatment of cardiovascular disease, type 2 diabetes, osteopenia, osteoporosis, and chronic hepatitis, and for anti-aging. Treatment of dividing cells with these compounds induced DNA damage and resulted in cell death. Despite their genotoxic effects, resveratrol, genistein, and baicalein did not cause mutagenesis, which is a major side effect of conventional anti-cancer drugs. Furthermore, resveratrol and genistein killed multi-drug resistant cancer cells. We therefore propose that resveratrol, genistein, and baicalein are attractive candidates for improved chemotherapeutic agents. Furthermore, we identified 200 compounds that stabilized ATAD5-luciferase in a dose dependent-manner from 300,000 compounds in the NIH chemical library in collaboration with the NCGC. To identify DNA repair pathways targeted by the genotoxic compounds, we used 8 isogenic human cell lines as well as 10 isogenic chicken DT40 B cell lines with targeted gene knockouts in specific DNA repair pathways. Approximately 200 compounds were tested in survival assays on these cells and group into sub-categories based on their IC50 to kill these cells. We are currently confirming the killing potential of these compounds on cancer cells defective in the same DNA repair pathways. We will further investigate whether these compounds can reduce tumor burden in vivo using xenograft mice as well as gene targeted mice models. Each compound will become a good tool to dissect molecular functions of different DNA repair pathways. 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. In addition, compounds identified from the screening will be radiation and chemotherapeutic sensitizers in tumors that depend on pathways of protein stabilization in response to radiation/chemotherapy-induced DNA damage. We are currently studying to identify targets of these compounds among genes identified from siRNA screening using bioinformatic analysis, epistatic analysis, as well as biochemical interactions.