NU 7026

NU 7026 is a selective inhibitor of  DNA-PK with IC50 value of 0.23 μM [1].
DNA-dependent protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase and involves in a variety of cellular processes, like, DNA double-strand break (DSB) repair, V(D)J recombination apparatus, chromatin structure and telomere maintenance. It has been shown that increased DNA-PK expression resulted in tumor cells resistance to radio- or chemo- therapy [2].
NU 7026 is a potent DNA-PK inhibitor and often is used combined with PARP-1 inhibitor AG14361 to sensitize tumor cells to radio- or chemo-therapy. When tested with primary PARP-1-/- and cells PARP-1+/+ cells, NU 7026 treatment (<50 μM) sensitized cells to IR-induced cytotoxicity and reduced clonogenic survival by inhibiting DNA-PK [1]. In N87 gastric cancer cells, administration NU 7026 combined with radiation increased DNA double-srand break, cell apoptosis and reduced cell survival through inhibiting DNA-PK [2]. When tested with CH1 human ovarian cancer cells, administration of NU 7026 combined with radiation reduced cell survival rate and clonogenic ability [3].
In female BALB/c mice model with CH1 cells subcutaneous xenograft, administration of NU 7026 orally or intraperitoneally sensitized mice to the radiotherapy with the dose of 100 mg/kg [3].
It has also been reported that NU 7026 is a potent inhibitor of PI-3K, ATM and ATR with IC50 value of 13 μM, >100μM and >100μM, respectively [1].
References:
[1].    Veuger, S.J., et al., Radiosensitization and DNA repair inhibition by the combined use of novel inhibitors of DNA-dependent protein kinase and poly(ADP-ribose) polymerase-1. Cancer Res, 2003. 63(18): p. 6008-15.
[2].    Niazi, M.T., et al., Effects of dna-dependent protein kinase inhibition by NU7026 on dna repair and cell survival in irradiated gastric cancer cell line N87. Curr Oncol, 2014. 21(2): p. 91-6.
[3].    Nutley, B.P., et al., Preclinical pharmacokinetics and metabolism of a novel prototype DNA-PK inhibitor NU7026. Br J Cancer, 2005. 93(9): p. 1011-8.

DNA-Dependent Protein Kinase As Molecular Target for Radiosensitization of Neuroblastoma Cells.[Pubmed:26716839]

PLoS One. 2015 Dec 30;10(12):e0145744.

Tumor cells might resist therapy with ionizing radiation (IR) by non-homologous end-joining (NHEJ) of IR-induced double-strand breaks. One of the key players in NHEJ is DNA-dependent protein kinase (DNA-PK). The catalytic subunit of DNA-PK, i.e. DNA-PKcs, can be inhibited with the small-molecule inhibitor NU7026. In the current study, the in vitro potential of NU7026 to radiosensitize neuroblastoma cells was investigated. DNA-PKcs is encoded by the PRKDC (protein kinase, DNA-activated, catalytic polypeptide) gene. We showed that PRKDC levels were enhanced in neuroblastoma patients and correlated with a more advanced tumor stage and poor prognosis, making DNA-PKcs an interesting target for radiosensitization of neuroblastoma tumors. Optimal dose finding for combination treatment with NU7026 and IR was performed using NGP cells. One hour pre-treatment with 10 muM NU7026 synergistically sensitized NGP cells to 0.63 Gy IR. Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on. Combined treatment of NGP cells with 10 muM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone. Inhibition of IR-induced DNA-PK activation by NU7026 confirmed the capability of NGP cells to, at least partially, resist IR by NHEJ. NU7026 also synergistically radiosensitized other neuroblastoma cell lines, while no synergistic effect was observed for low DNA-PKcs-expressing non-cancerous fibroblasts. Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells. Taken together, the current study shows that DNA-PKcs is a promising target for neuroblastoma radiosensitization.

Combining carbon ion irradiation and non-homologous end-joining repair inhibitor NU7026 efficiently kills cancer cells.[Pubmed:26553138]

Radiat Oncol. 2015 Nov 9;10:225.

BACKGROUND: Our previous data demonstrated that targeting non-homologous end-joining repair (NHEJR) yields a higher radiosensitivity than targeting homologous recombination repair (HRR) to heavy ions using DNA repair gene knockouts (KO) in mouse embryonic fibroblast (MEF). In this study, we determined if combining the use of an NHEJR inhibitor with carbon (C) ion irradiation was more efficient in killing human cancer cells compared with only targeting a HRR inhibitor. METHODS: The TP53-null human non-small cell lung cancer cell line H1299 was used for testing the radiosensitizing effect of NHEJR-related DNA-dependent protein kinase (DNA-PK) inhibitor NU7026, HRR-related Rad51 inhibitor B02, or both to C ion irradiation using colony forming assays. The mechanism underlying the inhibitor radiosensitization was determined by flow cytometry after H2AX phosphorylation staining. HRR-related Rad54-KO, NHEJR-related Lig4-KO, and wild-type TP53-KO MEF were also included to confirm the suppressing effect specificity of these inhibitors. RESULTS: NU7026 showed significant sensitizing effect to C ion irradiation in a concentration-dependent manner. In contrast, B02 showed a slight sensitizing effect to C ion irradiation. The addition of NU7026 significantly increased H2AX phosphorylation after C ion and x-ray irradiations in H1299 cells, but not B02. NU7026 had no effect on radiosensitivity to Lig4-KO MEF and B02 had no effect on radiosensitivity to Rad54-KO MEF in both irradiations. CONCLUSION: These results suggest that inhibitors targeting the NHEJR pathway could significantly enhance radiosensitivity of human cancer cells to C ion irradiation, rather than targeting the HRR pathway.

The modulating effect of ATM, ATR, DNA-PK inhibitors on the cytotoxicity and genotoxicity of benzo[a]pyrene in human hepatocellular cancer cell line HepG2.[Pubmed:26595742]

Environ Toxicol Pharmacol. 2015 Nov;40(3):988-96.

The effect of inhibitors of phosphatidylinositol-3-kinase-related kinases (PIKK): ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK) on response of HepG2 human liver cancer cells to benzo[a]pyrene (BaP) was investigated. PIKK inhibitors: KU55933 (5 muM), NU7026 (10 muM) or caffeine (1 and 2mM) when used as single agents or in combinations (KU55933/NU7026 and caffeine/NU7026) did not significantly influence the BaP (3 muM) cytotoxicity (MTT reduction test). BaP induced a weak proapoptotic effect which was moderately enhanced by both inhibitor combinations. HepG2 cells exposed to BaP showed a strong S-phase arrest which was considerably diminished by both inhibitor combinations. The DNA damage (comet assay) induced after continuous 24h exposure to BaP was significantly diminished by both inhibitor combinations. Weak induction of reactive oxygen species by BaP was observed, which was not modulated by the inhibitor combinations. Similarly, no modulation of the glutathione levels was observed.

DNA-PK activity is associated with caspase-dependent myogenic differentiation.[Pubmed:27513301]

FEBS J. 2016 Oct;283(19):3626-3636.

Differentiation of myoblasts into myotubes is essential for skeletal muscle development and regeneration. Caspase-3 and caspase-9 are required for efficient myoblast differentiation. The caspase-activated endonuclease activity, CAD, and the DNA-damage repair protein XRCC1 have also been shown to be required to complete differentiation. DNA-damage associated with differentiation is accompanied by phosphorylation of Histone 2AX, an event normally catalysed by kinases ATR, ATM or DNA-PK. However, the kinase responsible for phosphorylation during differentiation is not known. Here we show that inhibition of DNA-PK, but not of ATR or ATM, blocked histone phosphorylation during differentiation. We also show that DNA-PK inhibition and siRNA-mediated DNA-PK knockdown blocked cell fusion. These data implicate a new role for DNA-PK in myogenic differentiation.

micorRNA-101 silences DNA-PKcs and sensitizes pancreatic cancer cells to gemcitabine.[Pubmed:27988337]

Biochem Biophys Res Commun. 2017 Jan 29;483(1):725-731.

Gemcitabine sensitization is important for the treatment of pancreatic cancer. We have previously shown that DNA-dependent protein kinase catalytic subunit (DNA-PKcs) over-expression causes Akt activation and gemcitabine resistance in pancreatic cancer cells. Here, we aim to downregulate DNA-PKcs via introduction of micorRNA-101 ("miR-101"). We showed that forced-expression of miR-101 downregulated DNA-PKcs and potentiated gemcitabine-induced PANC-1 pancreatic cancer cell death and apoptosis. Contrarily, miR-101 depletion through expressing antagomiR-101 in PANC-1 cells resulted in DNA-PKcs upregulation and gemcitabine resistance. DNA-PKcs downregulation is the primary reason of gemcitabine-sensitization by miR-101. DNA-PKcs inhibition (by NU7026) or silence (by targeted siRNAs) disabled miR-101-mediaetd gemcitabine sensitization. Significantly, Akt Ser-473 phosphorylation in PANC-1 cells was also inhibited by miR-101, but was augmented with antagomiR-101 expression. Importantly, we showed that miR-101 level was downregulated in gemcitabine-resistant human pancreatic cancer tissues, which was correlated with DNA-PKcs upregulation. Together, these results suggest that miR-101 sensitizes PANC-1 cells to gemcitabine possibly via downregulating DNA-PKcs.

Chlorambucil cytotoxicity in malignant B lymphocytes is synergistically increased by 2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026)-mediated inhibition of DNA double-strand break repair via inhibition of DNA-dependent protein kinase.[Pubmed:17351105]

J Pharmacol Exp Ther. 2007 Jun;321(3):848-55.

Chlorambucil (CLB) treatment is used in chronic lymphocytic leukemia (CLL) but resistance to CLB develops in association with accelerated repair of CLB-induced DNA damage. Phosphorylated histone H2AX (gammaH2AX) is located at DNA double-strand break (DSB) sites; furthermore, it recruits and retains damage-responsive proteins. This damage can be repaired by nonhomologous DNA end-joining (NHEJ) and/or homologous recombinational repair (HR) pathways. A key component of NHEJ is the DNA-dependent protein kinase (DNA-PK) complex. Increased DNA-PK activity is associated with resistance to CLB in CLL. We used the specific DNA-PK inhibitor 2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026) to sensitize CLL cells to chlorambucil. Our results indicate that in a CLL cell line (I83) and in primary CLL-lymphocytes, chlorambucil plus NU7026 has synergistic cytotoxic activity at nontoxic doses of NU7026. CLB treatment results in G(2)/M phase arrest, and NU7026 increases this CLB-induced G(2)/M arrest. Moreover, a kinetic time course demonstrates that CLB-induced DNA-PK activity was inhibited by NU7026, providing direct evidence of the ability of NU7026 to inhibit DNA-PK function. DSBs, visualized as gammaH2AX, were enhanced 24 to 48 h after CLB and further increased by CLB plus NU7026, suggesting that the synergy of the combination is mediated by NU7026 inhibition of DNA-PK with subsequent inhibition of DSB repair.