Transcriptional Signatures of Homologous Recombination Deficiency for Targeted Ch

描述(申请人提供)：DNA修复途径维持基因组的完整性，因此，许多癌细胞在DNA修复的某些方面存在缺陷也就不足为奇了。幸运的是，这些癌症特异性DNA修复缺陷为肿瘤选择性治疗提供了新的方法，例如PARP1抑制剂对BRCA缺陷或同源重组(HR)缺陷肿瘤的合成致死性。因此，一个重要的目标和挑战是确定哪些肿瘤在DNA修复的各种特定途径中存在缺陷。据了解，DNA修复能力的变化将影响细胞功能，无论是在基线状态下，还是在应对遗传毒性应激时。此外，有人提出，DNA修复能力的这些变化可能会重新编程细胞的转录格局。最近的证据表明，这种重新编程发生在多个水平上，包括特定RNA的合成和稳定性的变化，这表明DNA修复缺陷可能提供独特的转录信号。我们期望与HR缺陷相关的RNA合成和稳定性特征将是独特的，并可能有助于在不知道任何潜在基因缺陷的情况下识别HR缺陷。我们已经开发了一种全基因组的方法来识别这样的签名，称为“Bru-Seq”，其中新生RNA的溴核苷酸脉冲标记与RNA-Seq相结合的方式揭示了对基因表达的大量影响，具有标准微阵列或RNA-Seq方法所不允许的清晰度和效率。为了促进我们对正常细胞中突然的(躯体)DNA修复缺陷的影响的理解，我们建议综合评估在DNA修复基因在每条DNA修复途径中表达缺失后，模型非致瘤上皮细胞系MCF-10A中所有编码和调控RNA的动态状态。我们假设DNA修复缺陷将影响mRNA/microRNA的表达和稳定性，DNA修复途径特异的特征可用于预测对某些化疗药物的反应。具体地说，我们将使用Bru-Seq技术来(1)测量在六个主要DNA修复途径中每一个缺乏关键基因的等基因MCF-10A细胞系中所有编码和调控RNA物种的合成、稳定性和结构含量，以及(2)评估遗传毒性应激诱导的相同细胞在电离辐射和化疗药物替莫唑胺、顺铂和PARP抑制剂作用下转录谱的变化。这些研究将是在肿瘤样本中识别与特定DNA修复途径缺陷相关的签名的关键的第一步，这些签名可能被用作预测对靶向化疗药物的反应的生物标记物。

DESCRIPTION (provided by applicant): DNA repair pathways maintain the integrity of the genome and it is therefore not surprising that many cancer cells are defective in some aspect of DNA repair. Fortunately, these cancer-specific DNA repair defects offer novel approaches for tumor-selective therapy such as the synthetic lethality of PARP1 inhibitors with BRCA-deficient or homologous recombination (HR)-deficient tumors. An important goal and challenge is thus to identify which tumors are defective in the various specific pathways of DNA repair. It is understood that changes in DNA repair capacity will impact cellular functions both at baseline and in response to genotoxic stress. Further, it has been suggested that these changes in DNA repair capacity may reprogram the cellular transcriptional landscape. Recent evidence suggests that this reprogramming occurs at multiple levels, including changes in the synthesis and stability of specific RNAs, suggesting that DNA repair deficiency may provide unique transcriptional signatures. It is our expectation that the RNA synthesis and stability signature associated with a defect in HR will be unique and may be useful in identifying HR defects without knowledge of any underlying gene defect. We have developed a genome-wide approach for identifying such a signature, termed "BrU-Seq", in which BromoUridine pulse-labeling of nascent RNAs is combined with RNA-Seq in a manner that reveals a remarkably large number of influences on gene expression with a clarity and efficiency not allowed by standard microarray or RNA-Seq approaches. To advance our understanding of the impact of a sudden (somatic) DNA repair defect in a normal cell, we propose to comprehensively evaluate the dynamic status of all coding and regulatory RNAs in the model non-tumorigenic epithelial cell line, MCF-10A, following loss of expression of DNA repair genes across each DNA repair pathway. We hypothesize that DNA repair defects will impact mRNA/microRNA expression and stability with DNA repair pathway-specific signatures that can be used to predict response to certain chemotherapeutic agents. Specifically, we will use BrU-Seq technology to (1) measure the synthesis, stability and structural content of all coding and regulatory RNA species in isogenic MCF-10A cell lines deficient in a key gene in each of the six major DNA repair pathways and (2) evaluate genotoxic stress-induced alterations in the transcriptional profile of the same cells in response to ionizing radiation and the chemotherapeutics temozolimide, cisplatin and PARP inhibitors. These studies will be a critical first step towards identifying signatures associated with specific DNA repair pathway defects in tumor samples that may be used as biomarkers to predict response to targeted chemotherapeutic agents.