Identification of chemotherapeutic sensitizers

化疗和放射治疗会引起多种基因毒性损伤，导致快速增殖的癌细胞死亡。为了在基因毒性的侮辱下存活下来，癌细胞依赖于多条DNA修复途径。根据基因毒性损伤的类型，细胞使用特定的DNA修复途径。当DNA修复途径受损时，癌细胞对某些基因毒性侮辱变得更加敏感。识别作用于癌细胞中受损的DNA修复途径的化疗药物将导致对癌细胞的更有效的治疗。这类试剂是放射治疗的潜在增敏剂。我们发现，ATAD5蛋白对几乎所有的遗传毒性侮辱都有稳定的反应。因此，我们假设ATAD5将是检测遗传毒性侮辱的一个很好的生物标志物。我们建立了一个表达ATAD5-荧光素酶融合蛋白的细胞系，并表明融合蛋白对遗传毒性侮辱也是稳定的。 此前，我们使用了这种新颖的基于细胞的定量高通量ATAD5-荧光素酶分析方法，并与NIH化学基因组中心(NCGC)合作，成功地从商业可用化合物库以及国家毒理学计划(NTP)库中筛选出4,000多种化合物。NCGC现在是国家高级翻译科学中心(NCATS)和国家环境健康科学研究所(NIEHS)的一部分。在这个初步筛选中，我们报告了22种抗氧化剂，包括白藜芦醇、染料木素和黄芩素，它们诱导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，但它们不会引起突变，这是传统抗癌药物的主要副作用。因此，我们建议白藜芦醇、染料木素和黄芩素是有吸引力的改进化疗药物的候选药物。 此外，我们与NCGC合作，从NIH化学库中的300,000种化合物中鉴定了200种以剂量依赖的方式稳定ATAD5-荧光素酶的化合物。为了确定基因毒性化合物靶向的DNA修复途径，我们使用了8个在特定DNA修复途径中具有靶向基因敲除的等基因人类细胞系。大约200种化合物在这些细胞上进行了存活测试，并根据它们的IC50将其分组以杀死这些细胞。我们发现了一个小分子可以杀死错配修复缺陷的癌细胞，两个小分子可以更有效地杀死PARP1缺陷的癌细胞。我们正在通过异种移植小鼠和基因靶向小鼠模型来研究这些化合物是否可以降低体内的肿瘤负担。每一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。 在与NCATS的合作中，我们还使用相同的ATAD5-荧光素酶细胞系来鉴定抑制ATAD5稳定性的化合物和siRNAs，并鉴定了&gt；80化合物和&gt；30 siRNA。从这些siRNA筛选中发现的基因将揭示未知的机制，这些机制抑制DNA修复蛋白在应对遗传毒性侮辱时的蛋白分解。最初的HITS中有两种化合物可能是肿瘤的潜在放射和化疗增敏剂。我们发现有一种化合物通过破坏DNA损伤反应激酶(S)的稳定性来抑制一般的DNA损伤反应。我们目前正在研究利用生物信息学分析、上位性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。

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. Previously, we used this novel cell-based quantitative high-throughput ATAD5-luciferase assay and successfully 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). In this pilot screen, we reported 22 antioxidants, including resveratrol, genistein, and baicalein 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. 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 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 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 are investigating 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. 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 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.