Identification of genetic interactors of Brca2

Division Of Basic Sciences - Nci

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Abstract

A major aim of our laboratory is to identify genetic interactors of BRCA2 that may contribute to cell viability and tumorigenesis. This aim is based on the fact that loss of essential genes such as BRCA2 in embryonic stem cells induces cell cycle arrest and apoptosis yet in somatic cells loss of these genes result in tumorigenesis. It is believed that mutations in genes such as TRP53 enables these cells to overcome the growth arrest induced by the loss of such essential genes. We are using mESC to perform genetic screens to identify genes that can contribute to the survival of BRCA2-deficient cells. Because the genome of ES cells is quite stable even after several passages, they represent a powerful and simple yet physiologically relevant system for such screens. More recently, we have initiated a CRISPR/Cas9-based whole genome screen to identify genes that support viability of Brca2ko/ko mESC and may contribute to tumorigenesis. Over the years, we have identified a number of genetic interactors such as GIPC3, PARP1, TET2 and BRE. We have shown GIPC3 to contribute to cell viability via its interaction with APPL1/2, adaptor proteins that regulate multiple signaling pathways. Similarly, BRCC45/BRE contributes to cell viability by recruiting USP7, a deubiquitylase, to stabilize cell cycle regulator CDC25A. More recently, we demonstrated that TET2 loss results in resistance to PARP inhibitors, such as olaparib. Our functional studies revealed that TET2 loss contributed to resistance to PARPi because of its impact on stability of stalled forks. We are currently examining the possibility of modulating TET2 activity that can be used for restoring PARPi sensitivity of resistant tumors in patients. Synthetic lethality of cells defective in HR genes including BRCA2 by PARP inhibitors is well established. Surprisingly, we uncovered that PARP inhibition can also lead to the viability of BRCA2-deficient cells if cells are treated prior to the loss of BRCA2 function. We postulated that PARPi treatment protects replication forks from MRE11-mediated degradation and contributes to cell viability. This was supported by our ability to generate viable Brca2 null mESC by transiently inhibiting MRE11 with mirin. One of our current aims is to further understand the mechanism of mirin mediated cell viability by RNA-seq and whole genome sequencing approaches. We have identified a number of biological pathways that are upregulated. We are examining these pathways to identify those that are directly involved in supporting viability of BRCA2-deficient). We have now undertaken a comprehensive and rigorous genetic screen in mESC to identify additional genetic interactors. We are using the mouse CRISPR/Cas9 Synergistic Activation Mediator (SAM) pooled library, which utilizes an engineered protein complex for the transcriptional activation of endogenous genes. The CRISPR/Cas9 gRNA-based gene activation system consists of three different components: 1. nuclease inactive Cas9-VP64 fusion; 2. a gRNA incorporating two MS2 RNA aptamers at the tetraloop and stem-loop 2; 3. the MS2-P65-HSF1 plasmid which expresses the activation helper protein. The CRISPR-Cas9 complex has been engineered by a structure-guided approach to result in highly efficient transcriptional activation at endogenous genomic loci. We have engineered PL2F7 mESC to express a Cas9-VP64 fusion and the MS2-P65-HSF1 to make the stable cells suitable for the activator library screen. The mouse gRNA library obtained from Dr. Feng Zhang represents a pool of plasmids expressing 69,225 different gRNAs targeting the promoter region (200bp upstream of the transcription start site) of 23,439 mouse genes. The ES cells transduced with the gRNA library lentiviruses were further confirmed for the gRNA diversity(85% representation) using an NGS approach. Subsequently these cells were electroporated with Cre expression vector to delete the conditional allele of Brca2 and the recombinant clones were selected in HAT media. We picked around 960 viable colonies from ten 10cm plates representing 5 independent electroporation. We genotyped the colonies to identify Brca2ko/ko clones and identified a total of 332 clones confirmed to be Brca2ko/ko. We then determined the sequence of gRNAs by Sanger sequencing and identified the target gene in 189 clones (these will be referred to as Southern confirmed genes). As an independent secondary screen, we have used an NGS-based approach to identify the genes that can support viability of Brca2ko/ko cells . We transduced the cells with the lentiviruses representing the SAM gRNA library. After deletion of the conditional allele and selection of the recombinant Brca2ko/ko clones, we trypsinized all the colonies on the plates. We saved an aliquot (as HAT sample) and treated the rest with media containing DMSO, olaparib or cisplatin for 5 days. After the selection was over, we harvested the cells from DMSO, olaparib and cisplatin plates. We extracted the DNA from a pool of cells, amplified the gRNA by PCR and sequenced them using Illumina Mi-seq system. Based on our hypothesis that the Brca2ko/ko cells will be sensitive to olaparib and cisplatin, we analyzed the gRNA sequences to identify the genes, that were present in the DMSO treated plate but had dropped out of the olaparib and cisplatin selection plates. The analysis of these sequencing results is in progress. Interestingly, when we directly tested the 189 gRNAs representing the "Southern confirmed genes", we found that 59 and 61 of those had dropped out in olaparib and cisplatin selections, respectively, and 30 gRNAs dropped in both selections. Once we identify genes that are upregulated in Brca2ko/ko mESCs by different approaches, we will select 10-12 candidates for further validation and 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 will start by first confirming the ability of potential candidates to rescue the lethality of Brca2 null mESC to rule out off target effects. Later, we will examine the selected genes for 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.