DNA repair deficient cells for analysis

描述（由申请方提供）：I期的成功完成导致开发了一组人细胞系，每种细胞系缺乏11种DNA糖基化酶之一。靶mRNA的消耗高达95%，靶蛋白水平和酶活性相应地消耗。为了扩大背景多样性，还使用相同的shRNA慢病毒在不同的肿瘤背景（包括神经胶质瘤和乳腺癌细胞系）中开发平行的细胞系组，证明了在不同的肿瘤细胞背景中类似的靶mRNA消耗。DNA糖基化酶的基因表达敲低揭示了DNA修复缺陷对人类转录组的影响。作为一组DNA修复缺陷细胞系的深远潜力的一个例子，我们表明，DNA糖基化酶缺陷调制的转录组和表观基因组，涉及一些DNA糖基化酶的甲基化维护和基因组表达多样性。此外，通过结合DNA糖基化酶和BRCA 1敲低，我们已经开始研究在BRCA 1敲低肿瘤细胞系中PARP抑制剂的有效性对DNA糖基化酶的需求。该项目的第二阶段将利用在第一阶段中优化的成功的工作流程范例，用于所有已知DNA修复基因缺陷的同基因人类细胞系的开发、功能表征、细胞库和转录组分析。这些包括参与碱基切除修复、损伤的直接修复、错配切除修复、核苷酸切除修复、同源末端置换、非同源末端连接、核苷酸库的调节、DNA聚合酶、编辑和加工核酸酶、Rad 6途径、染色质结构、疾病中有缺陷的DNA修复基因和保守的DNA损伤应答基因的基因。目的1中描述的研究涉及制备表达shRNA的慢病毒，转导和产生所有已知DNA修复基因（>150）的三种不同的人肿瘤细胞敲低组，然后是敲低细胞系的mRNA表达表征（qRT-PCR）和优化的放大和逐步表征以准备细胞系分布（细胞建库）。在目标2中，将通过蛋白质表达谱分析和遗传毒素挑战验证细胞系的预期DNA修复功能缺陷。最后（目的3），将进行全基因组转录谱分析，以定量内源性DNA修复能力变化介导的转录重编程，适当时，在特定遗传毒性胁迫后。随着DNA修复能力自发地和响应于遗传毒性应激而影响基本细胞功能，改变转录和表观遗传景观并决定细胞对应激的反应的预期，跨多种背景的一组完整的等基因DNA修复缺陷细胞系的开发将是基因和药物发现的有价值的平台，抑制剂特异性的验证和响应生物标志物的鉴定以及基因/药物合成致死性组合的新靶点。这组细胞系的现成可用性将允许学术和药学科学家研究肿瘤基因组不稳定性的分子病因学，并在肿瘤学研究中利用它。我们设想对与全球转录组相关的细胞系和信息的强劲市场需求。 公共卫生相关性：在这个II期提案中，我们计划利用在I期中优化的成功的工作流程范例，用于所有已知DNA修复基因缺陷的同基因人类细胞系的细胞系开发和转录组分析。这些高度表征和注释的同基因细胞系将形成用于基因和药物发现、抑制剂特异性验证以及响应生物标志物和基因/药物合成致死性组合的新靶点的鉴定的平台的基础。

DESCRIPTION (provided by applicant): Successful completion of Phase I led to the development a panel of human cell lines, each deficient in one of the eleven DNA glycosylase enzymes. Depletion of target mRNA was as high as 95%, with corresponding depletion of target protein levels and enzymatic activity. To expand background diversity, the same shRNA lentiviruses were also used to develop parallel cell line panels in diferent tumor backgrounds, including glioma and breast cancer cell lines, demonstrating similar target mRNA depletion across different tumor cell backgrounds. Gene expression knockdown of the DNA glycosylases exemplify the impact of DNA repair defects on the human transcriptome. As an example of the far reaching potential for a panel of DNA repair deficient cell lines, we show that DNA glycosylase deficiency modulated both the transcriptome and epigenome, implicating some DNA glycoylases in methylation maintenance and genome expression diversity. Further, by combining both DNA glycosylase and BRCA1 knockdown, we have begun to investigate the requirement for DNA glycosylases in the effectiveness of PARP inhibitors in a BRCA1 knockdown tumor line. Phase II of the project wil utilize the successful work-flow paradigm optimized in Phase I for the development, functional characterization, cell banking and transcriptome analysis of isogenic human cel lines deficient in all known DNA repair genes. These include genes involved in Base Excision Repair, Direct Reversal of Damage, Mismatch Excision Repair, Nucleotide Excision Repair, Homologous Recombination, Non- homologous End-Joining, the modulation of nucleotide pools, DNA polymerases, editing and processing nucleases, the Rad6 pathway, Chromatin Structure, DNA Repair genes defective in diseases and conserved DNA Damage Response genes. The studies described in Aim 1 involve the preparation of the shRNA expressing lentiviruses, transduction and generation of three different human tumor cell knockdown panels for all known DNA repair genes (>150), followed by the mRNA expression characterization (qRT-PCR) of the knockdown cell lines and optimized scale-up and step-wise characterization to prepare for cell line distribution (Cell Banking). In aim 2, the cell lines will be validated for the expected DNA repair functional deficiency by protein expression profiling and genotoxin challenge. Finally (Aim 3), whole-genome transcriptional profiles will be conducted to quantitate transcriptional reprogramming mediated by changes in endogenous DNA repair capacity and where appropriate, following specific genotoxic stress. With the expectation that DNA repair capacity impacts basic cellular functions both spontaneously and in response to genotoxic stress, alters the transcriptional and epigenetic landscape and dictates the cellular response to stress, the development of a complete panel of isogenic DNA repair deficient cell lines across multiple backgrounds will be a valuable platform for gene and drug discovery, validation of inhibitor specificity and the identification of response biomarkers and novel targets for gene/drug synthetic-lethality combinations. The ready availability of this panel of cell lines will permit both academic and pharmaceutical scientists to study the molecular etiology of tumor genomic instability and to exploit it in oncology research. We envision robust market demand for the cell lines and information that relates to the global transcriptome. PUBLIC HEALTH RELEVANCE: In this Phase II proposal we plan to utilize the successful work-flow paradigm optimized in Phase I for the cell-line development and transcriptome analysis of isogenic human cells lines deficient in all known DNA repair genes. These highly characterized and annotated isogenic cell lines will form the basis for a platform for gene and drug discovery, validation of inhibitor specificity and the identification of response biomarkers and novel targets for gene/drug synthetic-lethality combinations.