Identification of genetic interactors of Brca2

Brca2 遗传相互作用子的鉴定

• 批准号: 10926179
• 负责人: SHYAM SHARAN
• 金额: $ 119.01万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10926179
• 关键词: Adaptor Signaling Protein; Affect; Aliquot; Alleles; Apoptosis; BRCA deficient; BRCA1 gene; BRCA2 gene; Biological; Breast; CDC25A gene; CRISPR/Cas technology; Cell Cycle; Cell Cycle Arrest; Cell Cycle Checkpoint; Cell Death; Cell Survival; Cell division; Cells; Cisplatin; Complex; DNA; DNA Damage; DNA Repair; DNA replication fork; Data Set; Databases; Dideoxy Chain Termination DNA Sequencing; Dimethyl Sulfoxide; Drops; Drug resistance; Electroporation; Embryo; Engineering; Epithelium; Essential Genes; Gene Activation; Genes; Genetic; Genetic Screening; Genome; Genome Stability; Genotype; Germ-Line Mutation; Goals; Growth; Guide RNA; HSF1; Harvest; Human; Laboratories; Lentivirus; Libraries; Lower Organism; Malignant Neoplasms; Malignant neoplasm of ovary; Mediating; Mediator of activation protein; Mus; Mutation; Neoplasms; Ovarian; PARP inhibition; Pathway interactions; Patients; Phenotype; Physiological; Plasmids; Poly(ADP-ribose) Polymerase Inhibitor; Process; Proliferating; Promoter Regions; Protein Engineering; Proteins; RNA; Recombinants; Resistance; Rest; Role; Sampling; Signal Pathway; Somatic Cell; Structure; System; TP53 gene; Testing; The Cancer Genome Atlas; Tissues; Transcription Initiation Site; Transcriptional Activation; Trypsin; Validation; aptamer; cBioPortal; cell type; cellular transduction; coping; embryonic stem cell; expression vector; genome sequencing; genomic locus; malignant breast neoplasm; mutant; nuclease; overexpression; p65; protein complex; recruit; repaired; response; stem; transcriptome sequencing; tumor; tumorigenesis; whole genome

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.

我们实验室的一个主要目标是识别可能有助于细胞活力和肿瘤发生的 BRCA2 遗传相互作用因子。这一目标基于以下事实：胚胎干细胞中 BRCA2 等必需基因的缺失会诱导细胞周期停滞和细胞凋亡，而体细胞中这些基因的缺失会导致肿瘤发生。据信，TRP53 等基因的突变使这些细胞能够克服因丢失此类必需基因而引起的生长停滞。我们正在使用 mESC 进行遗传筛选，以确定有助于 BRCA2 缺陷细胞存活的基因。由于 ES 细胞的基因组即使经过多次传代也相当稳定，因此它们代表了此类筛选的强大、简单但生理相关的系统。最近，我们启动了基于 CRISPR/Cas9 的全基因组筛选，以鉴定支持 Brca2ko/ko mESC 活力并可能有助于肿瘤发生的基因。多年来，我们已经鉴定了许多遗传相互作用因子，例如 GIPC3、PARP1、TET2 和 BRE。我们已经证明 GIPC3 通过与 APPL1/2（调节多种信号传导途径的接头蛋白）相互作用来促进细胞活力。同样，BRCC45/BRE 通过招募 USP7（一种去泛素化酶）来稳定细胞周期调节剂 CDC25A，从而有助于细胞活力。最近，我们证明 TET2 缺失会导致对 PARP 抑制剂（例如奥拉帕尼）产生耐药性。我们的功能研究表明，TET2 的丢失会导致对 PARPi 的抵抗，因为它会影响停顿叉的稳定性。我们目前正在研究调节 TET2 活性的可能性，该活性可用于恢复患者耐药肿瘤的 PARPi 敏感性。 PARP 抑制剂对 HR 基因（包括 BRCA2）缺陷细胞的综合致死作用已得到充分证实。令人惊讶的是，我们发现，如果在 BRCA2 功能丧失之前对细胞进行处理，PARP 抑制也可以导致 BRCA2 缺陷细胞的活力。我们假设 PARPi 处理可以保护复制叉免受 MRE11 介导的降解，并有助于细胞活力。我们通过用味醂瞬时抑制 MRE11 来生成可行的 Brca2 null mESC 的能力支持了这一点。我们当前的目标之一是通过 RNA-seq 和全基因组测序方法进一步了解味醂介导的细胞活力的机制。我们已经确定了许多上调的生物途径。我们正在研究这些途径，以确定那些直接参与支持 BRCA2 缺陷的生存能力的途径。我们现在已经在 mESC 中进行了全面而严格的遗传筛选，以识别其他遗传相互作用因子。我们使用小鼠 CRISPR/Cas9 协同激活介体 (SAM) 混合文库，该文库利用工程蛋白复合物来转录激活内源基因。基于 CRISPR/Cas9 gRNA 的基因激活系统由三个不同的组件组成： 1. 核酸酶失活的 Cas9-VP64 融合体； 2.在四环和茎环2处掺入两个MS2 RNA适体的gRNA； 3.表达激活辅助蛋白的MS2-P65-HSF1质粒。 CRISPR-Cas9 复合物采用结构引导方法设计，可在内源基因组位点实现高效转录激活。我们设计了 PL2F7 mESC 以表达 Cas9-VP64 融合体和 MS2-P65-HSF1，以使稳定细胞适合激活剂库筛选。从 Feng Zhang 博士处获得的小鼠 gRNA 文库代表了表达 69,225 种不同 gRNA 的质粒库，这些 gRNA 靶向 23,439 个小鼠基因的启动子区域（转录起始位点上游 200bp）。使用NGS方法进一步确认用gRNA文库慢病毒转导的ES细胞的gRNA多样性（85%代表性）。随后用 Cre 表达载体电穿孔这些细胞以删除 Brca2 的条件等位基因，并在 HAT 培养基中选择重组克隆。我们从代表 5 个独立电穿孔的 10 个 10cm 板中挑选了大约 960 个活菌落。我们对菌落进行基因分型以鉴定 Brca2ko/ko 克隆，并鉴定出总共 332 个克隆被确认为 Brca2ko/ko。然后，我们通过桑格测序确定了 gRNA 的序列，并在 189 个克隆中鉴定了目标基因（这些将被称为 Southern 确认基因）。作为独立的二次筛选，我们使用基于 NGS 的方法来鉴定可以支持 Brca2ko/ko 细胞活力的基因。我们用代表 SAM gRNA 文库的慢病毒转导细胞。删除条件等位基因并选择重组 Brca2ko/ko 克隆后，我们对平板上的所有菌落进行胰蛋白酶消化。我们保存了一份（作为 HAT 样品），并用含有 DMSO、奥拉帕尼或顺铂的培养基处理其余部分 5 天。选择结束后，我们从 DMSO、奥拉帕尼和顺铂平板上收获细胞。我们从一组细胞中提取 DNA，通过 PCR 扩增 gRNA，并使用 Illumina Mi-seq 系统对其进行测序。基于我们的假设，即 Brca2ko/ko 细胞对奥拉帕尼和顺铂敏感，我们分析了 gRNA 序列以鉴定存在于 DMSO 处理板中但已从奥拉帕尼和顺铂选择板中剔除的基因。这些测序结果的分析正在进行中。有趣的是，当我们直接测试代表“Southern 确认基因”的 189 个 gRNA 时，我们发现其中 59 个和 61 个 gRNA 在奥拉帕尼和顺铂选择中分别被剔除，而 30 个 gRNA 在这两种选择中均被剔除。一旦我们通过不同的方法鉴定出 Brca2ko/ko mESC 中上调的基因，我们将选择 10-12 个候选基因进行进一步验证，并使用以下标准进行机制研究： 1）在人类 BRCA2 突变癌症中发现上调的基因基于 TCGA 数据集（使用 cBioportal 数据库）中的可用信息，2) 在 BRCA2 缺陷条件下过度表达对患者生存有显着影响的基因，3) 鉴定具有 BRCA2 缺陷的基因针对单个基因的多个 gRNA，4) 在 DNA 修复中具有已知作用但与 BRCA2 的遗传或功能相互作用未知的基因。我们将首先确认潜在候选者是否有能力挽救 Brca2 null mESC 的致死性，以排除脱靶效应。随后，我们将检查所选基因对 HR、复制叉稳定性、耐药性以及整体基因组稳定性的影响，以从机制上了解 Brca2ko/ko mESC 的拯救和存活。

项目成果

Survival of BRCA2-Deficient Cells Is Promoted by GIPC3, a Novel Genetic Interactor of BRCA2.

GIPC3（一种 BRCA2 的新型遗传相互作用因子）可促进 BRCA2 缺陷细胞的存活。

• 发表时间: 2017-12
• 影响因子: 3.3
• 作者: Ding, Xia;Philip, Subha;Martin, Betty K;Pang, Yan;Burkett, Sandra;Swing, Deborah A;Pamala, Chinmayi;Ritt, Daniel A;Zhou, Ming;Morrison, Deborah K;Ji, Xinhua;Sharan, Shyam K
• 通讯作者: Sharan, Shyam K

The Indenoisoquinoline TOP1 Inhibitors Selectively Target Homologous Recombination-Deficient and Schlafen 11-Positive Cancer Cells and Synergize with Olaparib.

茚并异喹啉 TOP1 抑制剂选择性靶向同源重组缺陷和 Schlafen 11 阳性癌细胞，并与奥拉帕尼协同作用。

• 发表时间: 2019
• 作者: Marzi, Laetitia;Szabova, Ludmila;Gordon, Melanie;Weaver Ohler, Zoe;Sharan, Shyam K;Beshiri, Michael L;Etemadi, Moudjib;Murai, Junko;Kelly, Kathleen;Pommier, Yves
• 通讯作者: Pommier, Yves

RAD52 S346X variant reduces breast cancer risk in BRCA2 mutation carriers.

RAD52 S346X 变异可降低 BRCA2 突变携带者患乳腺癌的风险。

• 发表时间: 2020-06
• 影响因子: 6.6
• 作者: Biswas, Kajal;Sharan, Shyam K
• 通讯作者: Sharan, Shyam K

Exploring role of 5hmC as potential marker of chemoresistance.

探索 5hmC 作为化学耐药性潜在标志物的作用。

• 发表时间: 2020-10-22
• 影响因子: 2.1
• 作者: Kharat, Suhas S;Sharan, Shyam K
• 通讯作者: Sharan, Shyam K

BRCA2-DSS1 interaction is dispensable for RAD51 recruitment at replication-induced and meiotic DNA double strand breaks.

BRCA2-DSS1 相互作用对于复制诱导和减数分裂 DNA 双链断裂时 RAD51 的招募来说是可有可无的。

• 发表时间: 2022
• 影响因子: 16.6
• 作者: Mishra, Arun Prakash;Hartford, Suzanne A;Sahu, Sounak;Klarmann, Kimberly;Chittela, Rajani Kant;Biswas, Kajal;Jeon, Albert B;Martin, Betty K;Burkett, Sandra;Southon, Eileen;Reid, Susan;Albaugh, Mary E;Karim, Baktiar;Tessarollo, Lino;Keller, J
• 通讯作者: Keller, J