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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Small Business Innovation Research Grants (SBIR) - Phase II (R44)
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Special Emphasis Panel (ZRG1-IMST-E (15))
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Hagan, Ann A
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Trevigen, Inc.
United States
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Li, Jianfeng; Svilar, David; McClellan, Steven et al. (2018) DNA Repair Molecular Beacon assay: a platform for real-time functional analysis of cellular DNA repair capacity. Oncotarget 9:31719-31743
Braganza, Andrea; Li, Jianfeng; Zeng, Xuemei et al. (2017) UBE3B Is a Calmodulin-regulated, Mitochondrion-associated E3 Ubiquitin Ligase. J Biol Chem 292:2470-2484
Godin, Stephen K; Zhang, Zhuying; Herken, Benjamin W et al. (2016) The Shu complex promotes error-free tolerance of alkylation-induced base excision repair products. Nucleic Acids Res 44:8199-215
Quiñones, Jason L; Thapar, Upasna; Yu, Kefei et al. (2015) Enzyme mechanism-based, oxidative DNA-protein cross-links formed with DNA polymerase ? in vivo. Proc Natl Acad Sci U S A 112:8602-7
Wang, Jingnan; Li, Jianfeng; Santana-Santos, Lucas et al. (2015) A novel strategy for targeted killing of tumor cells: Induction of multipolar acentrosomal mitotic spindles with a quinazolinone derivative mdivi-1. Mol Oncol 9:488-502
Brown, M F; Leibowitz, B J; Chen, D et al. (2015) Loss of caspase-3 sensitizes colon cancer cells to genotoxic stress via RIP1-dependent necrosis. Cell Death Dis 6:e1729
Chandran, Uma R; Luthra, Soumya; Santana-Santos, Lucas et al. (2015) Gene expression profiling distinguishes proneural glioma stem cells from mesenchymal glioma stem cells. Genom Data 5:333-336
Shen, John Paul; Srivas, Rohith; Gross, Andrew et al. (2015) Chemogenetic profiling identifies RAD17 as synthetically lethal with checkpoint kinase inhibition. Oncotarget 6:35755-69
Wendell, Stacy Gelhaus; Golin-Bisello, Franca; Wenzel, Sally et al. (2015) 15-Hydroxyprostaglandin dehydrogenase generation of electrophilic lipid signaling mediators from hydroxy ?-3 fatty acids. J Biol Chem 290:5868-80
Fang, Qingming; Inanc, Burcu; Schamus, Sandy et al. (2014) HSP90 regulates DNA repair via the interaction between XRCC1 and DNA polymerase ?. Nat Commun 5:5513

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