DNA repair deficient cells for analysis

描述(申请人提供)：第一阶段的成功完成导致开发了一组人类细胞株，每一株都缺乏11种DNA糖基酶中的一种。目的基因的缺失高达95%，相应的目的蛋白水平和酶活性也相应降低。为了扩大背景多样性，相同的shRNA慢病毒也被用来在不同的肿瘤背景中开发平行的细胞系面板，包括胶质瘤和乳腺癌细胞系，在不同的肿瘤细胞背景中显示出相似的靶mRNA缺失。DNA糖基酶的基因表达下调例证了DNA修复缺陷对人类转录组的影响。作为一组DNA修复缺陷细胞系的一个例子，我们发现DNA糖基酶缺陷同时调节了转录组和表观基因组，暗示了一些DNA糖基酶在甲基化维持和基因组表达多样性方面的作用。此外，通过结合DNA糖基酶和BRCA1基因敲除，我们已经开始研究在BRCA1基因敲除肿瘤系中对PARP抑制剂的有效性所需的DNA糖基酶。该项目的第二阶段将利用在第一阶段优化的成功的工作流程范例，对缺乏所有已知DNA修复基因的等基因人类细胞系进行开发、功能鉴定、细胞库和转录组分析。这些基因包括碱基切除修复、损伤的直接逆转、错配切除修复、核苷酸切除修复、同源重组、非同源末端连接、核苷酸池的调节、DNA聚合酶、编辑和处理核酸酶、Rad6途径、染色质结构、疾病中存在缺陷的DNA修复基因和保守的DNA损伤反应基因。目标1中描述的研究包括制备表达慢病毒的shRNA，转导和产生针对所有已知DNA修复基因的三种不同的人类肿瘤细胞敲除面板(&gt；150)，随后是敲除细胞系的信使核糖核酸表达特征(qRT-PCR)，以及优化的放大和分步特征，为细胞系分布做准备(Cell Banking)。在目标2中，将通过蛋白质表达谱和基因毒素攻击来验证细胞系是否存在预期的DNA修复功能缺陷。最后(目标3)，将进行全基因组转录图谱，以量化内源性DNA修复能力的变化以及在适当情况下特定的遗传毒性应激之后的转录重编程。人们期望DNA修复能力自发地影响细胞的基本功能，并响应基因毒性应激，改变转录和表观遗传格局，并决定细胞对应激的反应，因此开发一组跨越多种背景的等基因DNA修复缺陷细胞系将为基因和药物的发现、抑制剂特异性的验证以及响应生物标记物和基因/药物合成-致死组合的新靶点的确定提供一个有价值的平台。这组细胞系的现成将允许学术和制药科学家研究肿瘤基因组不稳定的分子病因学，并将其用于肿瘤学研究。我们预计，市场对与全球转录组相关的细胞系和信息的需求强劲。 公共卫生相关性：在这个第二阶段的提案中，我们计划利用在第一阶段优化的成功的工作流程范例，对缺乏所有已知DNA修复基因的同基因人类细胞系进行细胞系开发和转录组分析。这些高度特征化和注解的等基因细胞系将形成一个平台，用于基因和药物的发现、抑制剂特异性的验证以及识别响应生物标记物和基因/药物合成-致死组合的新靶点。

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.