Many of the agents commonly used for cancer therapy kill tumor cells by damaging cellular DNA, and thus DNA repair proteins and their interactions represent potential targets for enhancing such therapy. Double- strand breaks (DSBs) in DNA, which are the major cytotoxic lesions formed by ionizing radiation as well as by topoisomerase II inhibitors, are repaired primarily by nonhomologous end joining (NHEJ) and, to a lesser extent, by homologous recombination (HRR). Thus, the efficiency of these repair systems is a critical factor in the effectiveness of radio- and chemotherapy. XLF is the most recently discovered core protein in the NHEJ pathway. Although genetic studies show that XLF is essential for efficient NHEJ, its precise role in the pathway is poorly understood. In addition, because the interaction between XLF and another NHEJ protein, XRCC4, is critical for NHEJ, but is transient and has only moderate (low ?M) affinity, it represents an attractive target for chemo/radiosensitization by disruption of NHEJ. The primary objectives of this application are, therefore, to elucidate in more detail the role of XLF in NHEJ, and to develop XLF/XRCC4 interaction inhibitors and evaluate them as chemo/radiosensitizers. In order to better clarify XLF's role and test the hypothesis that XLF and XRCC4 polymerize into a filament that aligns DNA ends, photoactivatable DNA substrates will be added to cell extracts to map the interactions of XLF with DNA near a DSB as a function of time and distance from the break. Experiments with a fluorescent NHEJ reporter integrated into the genome will determine whether alignment-based gap filling at DNA ends is strictly dependent on XLF in intact cells, as has been already demonstrated in cell extracts. For the inhibitor development, the novel in vitro selection technique, mRNA display, will be used to isolate small (Mr 1000-2000), cell-penetrating, drug-like cyclic peptides that bind to XRCC4 at its interface with XLF. Using mRNA display, an extremely diverse library of 10 trillion semi- random peptides fused to the mRNAs that encode them will be prepared using in vitro translation. Those that bind to the XLF binding pocket on XRCC4 will be selectively captured and their mRNA amplified with RT-PCR. After several rounds of iterative translation, selection, and amplification, candidate peptides that bloc the XLF/XRCC4 interaction will be identified. These peptides will be screened for NHEJ inhibition using a cell extract-based assay wherein joining of partially complementary DSB ends is stringently dependent on XLF. The strongest inhibitory peptides will be chemically synthesized with addition of cell-penetrating tags, and tested for sensitization of triple-negative breast tumor cells to two DSB-inducing agents: ionizing radiation and etoposide. The most potent sensitizers will be used in further experiments to determine to what extent DSB repair is inhibited in several cell lines, chosen either for their known radioresistance, or because they are deficient in DSB repair by the alternate HRR pathway. Based on their unique mode of action, these peptides or subsequent derivatives thereof may be clinically useful in sensitizing tumor cells for radio- and chemotherapy.

Public Health Relevance

Radiotherapy and certain forms of chemotherapy kill tumor cells by forming breaks in cellular DNA. In this project, a novel iterative synthesis and selection technique known as mRNA display will be used to identify peptides (small, protein-like molecules) that block the binding of XLF to its partner XRCC4, and disrupt the essential function of these proteins in repairing cellular DNA breaks. By blocking repair, these peptides may be useful in improving the effectiveness of radiotherapy and chemotherapy.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Basic Mechanisms of Cancer Therapeutics Study Section (BMCT)
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Pelroy, Richard
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Virginia Commonwealth University
Schools of Medicine
United States
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Chalasani, Sri Lakshmi; Kawale, Ajinkya S; Akopiants, Konstantin et al. (2018) Persistent 3'-phosphate termini and increased cytotoxicity of radiomimetic DNA double-strand breaks in cells lacking polynucleotide kinase/phosphatase despite presence of an alternative 3'-phosphatase. DNA Repair (Amst) 68:12-24
Iqbal, Emil S; Dods, Kara K; Hartman, Matthew C T (2018) Ribosomal incorporation of backbone modified amino acids via an editing-deficient aminoacyl-tRNA synthetase. Org Biomol Chem 16:1073-1078
Hacker, David E; Hoinka, Jan; Iqbal, Emil S et al. (2017) Highly Constrained Bicyclic Scaffolds for the Discovery of Protease-Stable Peptides via mRNA Display. ACS Chem Biol 12:795-804
Menon, Vijay; Povirk, Lawrence F (2017) XLF/Cernunnos: An important but puzzling participant in the nonhomologous end joining DNA repair pathway. DNA Repair (Amst) 58:29-37
Ring, Christine M; Iqbal, Emil S; Hacker, David E et al. (2017) Genetic incorporation of 4-fluorohistidine into peptides enables selective affinity purification. Org Biomol Chem 15:4536-4539
Alotaibi, Moureq; Sharma, Khushboo; Saleh, Tareq et al. (2016) Radiosensitization by PARP Inhibition in DNA Repair Proficient and Deficient Tumor Cells: Proliferative Recovery in Senescent Cells. Radiat Res 185:229-45
Gewirtz, David A; Alotaibi, Moureq; Yakovlev, Vasily A et al. (2016) Tumor Cell Recovery from Senescence Induced by Radiation with PARP Inhibition. Radiat Res 186:327-332
Almohaini, Mohammed; Chalasani, Sri Lakshmi; Bafail, Duaa et al. (2016) Nonhomologous end joining of complex DNA double-strand breaks with proximal thymine glycol and interplay with base excision repair. DNA Repair (Amst) 41:16-26
Hacker, David E; Almohaini, Mohammed; Anbazhagan, Aruna et al. (2015) Peptide and peptide library cyclization via bromomethylbenzene derivatives. Methods Mol Biol 1248:105-17
White, E Railey; Sun, Luxin; Ma, Zhong et al. (2015) Peptide library approach to uncover phosphomimetic inhibitors of the BRCA1 C-terminal domain. ACS Chem Biol 10:1198-208

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