The long-term objective of Dr. Myung's research has been to identify and understand the molecular mechanisms of DNA repair pathways preserving genomic integrity and to develop therapeutic approaches that target these pathways. Genomes are constantly challenged by stresses that destabilize genomic integrity. Living organisms have developed DNA repair mechanisms to maintain genomic integrity. Given that failure of DNA repair results in genomic instability, the identification of novel genes/pathways involved in these processes is a priority. To identify new genes/pathways for DNA repair, Dr. Myung's group screened the entire yeast genome for mutations that increase the rate of genomic instability. RAD5 and ELG1 were subsequently selected since mutations of these genes had the highest impact upon genomic instability. Dr. Myung's group demonstrated that suppression of genomic instability depends on the polyubiquitylation of proliferating cell nuclear antigen (PCNA) by yeast Rad5p that promotes a homologous recombination pathway. Furthermore, Dr. Myung's group found that sumoylation of PCNA antagonizes Rad5p-dependent genomic stability. Dr. Myung's group extended their findings to higher organisms by discovering two mammalian RAD5 orthologs, SHPRH and HLTF. These studies were especially significant since the identification of such RAD5 homologs remained elusive in the DNA repair field for the two decades following the identification of the yeast RAD5 gene.
Dr. Myung's group found that yeast Elg1p suppresses genomic instability through its function in the DNA damage response. They identified the human ELG1 ortholog, ATAD5 and demonstrated that ATAD5 preserves genomic integrity similar to yeast Elg1p. In response to DNA damage, ATAD5 is stabilized and localizes at stalled DNA replication forks through currently unknown mechanisms that inhibit proteolysis of ATAD5. We demonstrated that ATAD5 interacts with a deubiquitylating enzyme for PCNA, USP1, and assists deubiquitylation of PCNA after translesion synthesis-dependent DNA repair. Dr. Myung's group observed high levels of tumorigenesis and genome instability in heterozygous null atad5+/m mice and discovered somatic mutations of human ATAD5 in endometrial tumors suggesting ATAD5 as a novel tumor suppressor gene.
Dr. Myung's current research on the RAD5 protein stems from the preliminary observation that the SHPRH protein is localized at the promoter of rDNA in the nucleolus and that SHPRH regulates rDNA expression through the interaction with histone H3. Dr. Myung's research on the ATAD5 protein is based on the observation of lethal phenotype of homozygous atad5-/- mice at embryonic day 8.5. Dr. Myung hypothesized that the function of ATAD5 in DNA replication, rather than PCNA deubiquitylation, is essential for mouse survival during embryogenesis. Dr. Myung's group recently found that ATAD5 functions to regulate the level of PCNA in chromatin by its role in unloading PCNA from chromatin. Dr. Myung's group is currently investigating the functional consequence of ATAD5 somatic mutations found in different tumors.
Chemotherapeutic 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. Dr. Myung's group found that ATAD5 protein is stabilized in response to almost all genotoxic insults. They 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 cell line, and taking advantage of the unique resources within the National Center for Advanced Translational Science (NCATS), Dr. Myung's group identified ~300 compounds that stabilized ATAD5-luciferase in a dose dependent-manner from 300,000 compounds in the NIH chemical library. To identify DNA repair pathways targeted by the genotoxic compounds, Dr. Myung's group used isogenic 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. Dr. Myung's group will further investigate whether these compounds can reduce tumor burden in vivo using xenograft mice as well as gene targeted mice models.
In collaboration with NCATS, Dr. Myung's group 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 chemotherapeutic sensitizers in tumors that depend on pathways of protein stabilization in response to chemotherapy-induced DNA damage.
Last Updated: March 27, 2014