Identification of genetic interactors of BRCA2

Division Of Basic Sciences - Nci

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Abstract

We have performed a genome wide screen and identified 189 gRNA that can rescue the lethality of Brca2ko/ko mESCs. We have examined the expression of corresponding genes and found several to be upregulated in Brca2ko/ko mESCs We have selected 10 candidates for further validation and to carry out mechanistic studies using the following criteria: 1) genes that are found to be upregulated in human BRCA2 mutant cancers based on available information from the TCGA dataset (using cBioportal database), 2) genes whose overexpression in BRCA2 deficient conditions has significant impact on survival of patients, 3) identification of genes with multiple gRNAs targeting a single gene, 4) genes with a known role in DNA repair but genetic or functional interaction with BRCA2 is unknown. We are now focusing on two genes, Ubl5 and Ndfip1. We have confirmed their ability to rescue the lethality of Brca2 null mESC to rule out off target effects. We have tested the selected genes for their impact on HR, replication fork stability, drug resistance as well as overall genomic stability to mechanistically understand the rescue and survival of Brca2ko/ko mESCs. As part of this project we had previously identified TET2 as genetic interactor of BRCA2. We uncovered a role for the TET proteins in restoring stability of stalled replication forks and resistance to PARPi (Kharat et al., Science Signaling). TET proteins catalyze the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) using alpha ketogluterate as a co-substrate. They further catalyze the conversion of 5hmC to 5formylcytosine (5fC) and 5carboxylcytosine (5caC) that is finally replaced by an unmodified cytosine. Therefore, TET proteins play an important role in DNA demethylation. Vitamin C acts as cofactor for TET proteins and increases global 5hmC levels in TET dependent manner. We observed a marked increase in 5hmC levels in BRCA2-deficient cells and the levels were further increased in the presence of vitamin C. Increase in 5hmC levels correlated with fork instability. Recently, we have shown that vitamin C treatment restores sensitivity of BRCA1/2-deficient cells that gain resistance to olaparib either due to restoration of HR or stability of stalled replication forks. We have demonstrated that vitamin C enhances olaparib dependent PARP1 trapping on chromatin by increasing 5hmC levels. Enhanced PARP1 trapping correlated with increase in replication gaps, that restored olaparib sensitivity in chemoresistant cells. Similar response is observed when cells are directly treated with 5hmC. Our findings show that Vitamin C/5hmC can restore sensitivity of olaparib resistant BRCA1/2-deficient cells.