Suhas S. Kharat

Degradation of 5hmC-marked stalled replication forks by APE1 causes genomic instability

Loss of DNA demethylase TET2 protects stalled replication forks from degradation and confers chemoresistance.

Characterization of BRCA2 R3052Q variant in mice supports its functional impact as a low-risk variant

AbstractPathogenic variants in BRCA2 are known to significantly increase the lifetime risk of developing breast and ovarian cancers. Sequencing-based genetic testing has resulted in the identification of thousands of BRCA2 variants that are considered to be variants of uncertain significance (VUS) because the disease risk associated with them is unknown. One such variant is p.Arg3052Gln, which has conflicting interpretations of pathogenicity in the ClinVar variant database. Arginine at position 3052 in BRCA2 plays an important role in stabilizing its C-terminal DNA binding domain. We have generated a knock-in mouse model expressing this variant to examine its role on growth and survival in vivo. Homozygous as well as hemizygous mutant mice are viable, fertile and exhibit no overt phenotype. While we did not observe any hematopoietic defects in adults, we did observe a marked reduction in the in vitro proliferative ability of fetal liver cells that were also hypersensitive to PARP inhibitor, olaparib. In vitro studies performed on embryonic and adult fibroblasts derived from the mutant mice showed significant reduction in radiation induced RAD51 foci formation as well as increased genomic instability after mitomycin C treatment. We observed mis-localization of a fraction of R3052Q BRCA2 protein to the cytoplasm which may explain the observed in vitro phenotypes. Our findings suggest that BRCA2 R3052Q should be considered as a hypomorphic variant.

5hmC enhances PARP trapping and restores PARP inhibitor sensitivity in chemoresistant BRCA1/2-deficient cells

Mutations in BRCA1 and BRCA2 genes are the leading cause of hereditary breast and ovarian cancer. BRCA1/2-mutant cells are defective in repairing damaged DNA by homologous recombination and are characterized by hypersensitivity to PARP inhibitors. PARP inhibitors can trap PARP proteins on the chromatin, a mechanism that can contribute to the death of BRCA1/2-deficient cells. The FDA has approved multiple PARP inhibitors for the treatment of metastatic breast and ovarian cancers, yet despite the success of PARP inhibitors in treating BRCA1/2-mutant cancers, drug resistance is a major challenge. Here, we report that 5hmC enhances PARP1 trapping on the chromatin in olaparib-treated cells. Elevated PARP trapping generates replication gaps, leading to the restoration of PARP inhibitor sensitivity in chemoresistant BRCA1/2-deficient cells. Our findings suggest that combining 5hmC with olaparib can restore the sensitivity of chemoresistant BRCA1/2-deficient cells.