Transcriptional Signatures of Homologous Recombination Deficiency for Targeted Ch

University Of Pittsburgh At Pittsburgh, Pittsburgh PA

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Abstract

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.