Identification of chemotherapeutic sensitizers

点击翻译按钮获取中文摘要

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. 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.