Interfering with NBS1-ATM interaction to inhibit the DNA damage response and sensitize tumor cells to radiation. Free

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Regulators of the DNA damage response include proteins mutated in several radiosensitive diseases. For example, NBS1, mutated in Nijmegan Break Syndrome, and ATM, mutation of which causes Ataxia-Telangiectasia, are two critical elements of an optimal DNA damage response. The C-terminal domain of NBS1 binds to ATM and is required for ATM activation in response to DNA damage. We hypothesized that small peptides containing the consensus sequence found in the C-terminal of NBS1 could inhibit NBS1-ATM interaction and induce a radiosensitive phenotype. These NBS1 inhibitory peptides (NIPs) could be fused to a polyarginine (R₉) internalization sequence for peptide delivery, and tagged with biotin for detection in vitro. We generated three peptides, one containing the R₉ sequence alone, one with the wild type C-terminal NBS1 sequence (wtNIP), and one with a scrambled sequence (scNIP). Immunofluorescent microscopy was incorporated to determine peptide internalization and half-life, where a significant uptake of the peptides was evident with no observable cytotoxicity. To determine the effects of the NIPs on the ATM mediated DNA damage response, we investigated γ-H2AX and NBS1 foci formation in irradiated cells treated with R₉, wtNIP or scNIP. γ-H2AX foci formation occurs at the site of double strand breaks within minutes of DNA damage and was significantly impaired in cells treated with wtNIP, compared to cells treated with R₉ or scNIP. NBS1 foci formation, an event that also occurs at the sites of DNA damage as part of the MRN complex, was also significantly diminished. These observations demonstrated a strong inhibitory effect by the wtNIP in the ATM-mediated DNA damage response. To establish radiosensitization, the colony-forming assay was incorporated, where a significant decrease in clonogenic survival was observed in irradiated wtNIP treated cells, compared to R₉ and scNIP treated cells. The sensitizing enhancement ratio for wtNIP treated cells was 1.7 to 3.1 over a dose range of 2-6Gy.In addition, since the extreme C-terminal of NBS1 is evolutionarily conserved, sharing sequence homology with the C-terminal of Ku80 and ATRIP, we investigated whether these peptides could radiosensitize cells with differing genetic backgrounds. Specifically, ATM and DNA-PK null cells were utilized, both demonstrating a significant decrease in clonogenic survival following treatment with wtNIP and irradiation, illustrating the ability of these peptides to radiosensitize cells lacking either functional ATM or DNA-PK. Collectively, these data provide strong evidence for the use of a conserved NBS1 C-terminal small peptide as a radiosensitizer. We have established a proof of principle in vitro to justify further investigation of these peptides in animal studies.