Xeroderma Pigmentosum Complementation Group C (XPC): Emerging Roles in Non-Dermatologic Malignancies.

Xeroderma pigmentosum complementation group C (XPC) is a DNA damage recognition protein essential for initiation of global-genomic nucleotide excision repair (GG-NER). Humans carrying germline mutations in the XPC gene exhibit strong susceptibility to skin cancer due to defective removal via GG-NER of genotoxic, solar UV-induced dipyrimidine photoproducts. However, XPC is increasingly recognized as important for protection against non-dermatologic cancers, not only through its role in GG-NER, but also by participating in other DNA repair pathways, in the DNA damage response and in transcriptional regulation. Additionally, XPC expression levels and polymorphisms likely impact development and may serve as predictive and therapeutic biomarkers in a number of these non-dermatologic cancers. Here we review the existing literature, focusing on the role of XPC in non-dermatologic cancer development, progression, and treatment response, and highlight possible future applications of XPC as a prognostic and therapeutic biomarker.

DNA Repair Capacity for Personalizing Risk and Treatment Response - Assay Development and Optimization in Human Peripheral Blood Mononuclear Cells (PBMCs).

DNA repair capacity (DRC) is the ability of a cell to repair DNA damage. Differential DRC plays an important role in human disease, including lung and other cancers. Measuring DRC could aid in translational disease research and in personalizing treatment. We developed and optimized a flow cytometry-based assay to measure individual DRC using GFP-expressing plasmids modified by ultraviolet (UV) light for nucleotide excision repair (NER) and restriction enzyme digestion to induce a blunt double-strand cut between promoter and GFP expression regions for nonhomologous end joining (NHEJ). Cryopreserved peripheral blood mononuclear cells (PBMCs) from healthy volunteers were used to measure DRC and optimize the assay. Pathway specificity of the NHEJ DRC assay was confirmed using Ku80-/- MEF cells, which showed a 6-fold reduction in NHEJ compared to Ku80+/+. Using a cell mixing assay, we show a linear correlation between NHEJ DRC and the expected concentration of Ku80. NHEJ DRC measurements in cryopreserved PBMCs are quantifiable with low interindividual and inter-assay variability, and a titratable decrease in NHEJ activity was observed in PBMCs treated with the DNA-PK inhibitor NU7441. Pathway specificity of the NER DRC assay was confirmed by a decrease in measured NER activity in human XPC deficient compared to XPC proficient fibroblasts, with a linear correlation measured between NER DRC and expected XPC concentration by cell mixing assay. NER DRC is quantifiable, reproducible, and titratable in PBMCs from healthy volunteers. We measured both NER and NHEJ DRC in PBMCs obtained from newly diagnosed, untreated lung cancer patients; measured DRC differed in these PBMCs compared to healthy volunteers. With further investigation, measurement of NER and NHEJ DNA repair capacity may be useful in personalizing disease risk and response to DNA damaging therapies and small molecular inhibitors of DNA repair pathways using readily available human PBMCs.

Discovery and development of novel DNA-PK inhibitors by targeting the unique Ku-DNA interaction.

DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) repair pathway and the DNA damage response (DDR). DNA-PK has therefore been pursued for the development of anti-cancer therapeutics in combination with ionizing radiation (IR). We report the discovery of a new class of DNA-PK inhibitors that act via a novel mechanism of action, inhibition of the Ku-DNA interaction. We have developed a series of highly potent and specific Ku-DNA binding inhibitors (Ku-DBi's) that block the Ku-DNA interaction and inhibit DNA-PK kinase activity. Ku-DBi's directly interact with the Ku and inhibit in vitro NHEJ, cellular NHEJ, and potentiate the cellular activity of radiomimetic agents and IR. Analysis of Ku-null cells demonstrates that Ku-DBi's cellular activity is a direct result of Ku inhibition, as Ku-null cells are insensitive to Ku-DBi's. The utility of Ku-DBi's was also revealed in a CRISPR gene-editing model where we demonstrate that the efficiency of gene insertion events was increased in cells pre-treated with Ku-DBi's, consistent with inhibition of NHEJ and activation of homologous recombination to facilitate gene insertion. These data demonstrate the discovery and application of new series of compounds that modulate DNA repair pathways via a unique mechanism of action.