Poly(ADP-ribose) polymerase (PARP1) facilitates DNA repair by binding to DNA breaks and attracting DNA repair proteins to the site of damage. Nevertheless, PARP1-/- mice are viable, fertile and do not develop early onset tumours. Here, we show that PARP inhibitors trigger gamma-H2AX and RAD51 foci formation. We propose that, in the absence of PARP1, spontaneous single-strand breaks collapse replication forks and trigger homologous recombination for repair. Furthermore, we show that BRCA2-deficient cells, as a result of their deficiency in homologous recombination, are acutely sensitive to PARP inhibitors, presumably because resultant collapsed replication forks are no longer repaired. Thus, PARP1 activity is essential in homologous recombination-deficient BRCA2 mutant cells. We exploit this requirement in order to kill BRCA2-deficient tumours by PARP inhibition alone. Treatment with PARP inhibitors is likely to be highly tumour specific, because only the tumours (which are BRCA2-/-) in BRCA2+/- patients are defective in homologous recombination. The use of an inhibitor of a DNA repair enzyme alone to selectively kill a tumour, in the absence of an exogenous DNA-damaging agent, represents a new concept in cancer treatment.
The serine/threonine protein kinase ATM signals to cell cycle and DNA repair components by phosphorylating downstream targets such as p53, CHK2, NBS1, and BRCA1. Mutation of ATM occurs in the human autosomal recessive disorder ataxia-telangiectasia, which is characterized by hypersensitivity to ionizing radiation and a failure of cells to arrest the cell cycle after the induction of DNA double-strand breaks. It has thus been proposed that ATM inhibition would cause cellular radio-and chemosensitization. Through screening a small molecule compound library developed for the phosphatidylinositol 3-kinase-like kinase family, we identified an ATP-competitive inhibitor, 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (KU-55933), that inhibits ATM with an IC 50 of 13 nmol/L and a Ki of 2.2 nmol/L. KU-55933 shows specificity with respect to inhibition of other phosphatidylinositol 3-kinase-like kinases. Cellular inhibition of ATM by KU-55933 was demonstrated by the ablation of ionizing radiation-dependent phosphorylation of a range of ATM targets, including p53, ␥H2AX, NBS1, and SMC1. KU-55933 did not show inhibition of UV light DNA damage induced cellular phosphorylation events. Exposure of cells to KU-55933 resulted in a significant sensitization to the cytotoxic effects of ionizing radiation and to the DNA double-strand break-inducing chemotherapeutic agents, etoposide, doxorubicin, and camptothecin. Inhibition of ATM by KU-55933 also caused a loss of ionizing radiation-induced cell cycle arrest. By contrast, KU-55933 did not potentiate the cytotoxic effects of ionizing radiation on ataxia-telangiectasia cells, nor did it affect their cell cycle profile after DNA damage. We conclude that KU-55933 is a novel, specific, and potent inhibitor of the ATM kinase.
Dysregulation of DNA damage repair and signalling to cell cycle checkpoints, known as the DNA damage response (DDR), is associated with a predisposition to cancer and affects responses to DNA-damaging anticancer therapy. Dysfunction of one DNA repair pathway may be compensated for by the function of another compensatory DDR pathway, which may be increased and contribute to resistance to DNA-damaging chemotherapy and radiotherapy. Therefore, DDR pathways make an ideal target for therapeutic intervention; first, to prevent or reverse therapy resistance; and second, using a synthetic lethal approach to specifically kill cancer cells that are dependent on a compensatory DNA repair pathway for survival in the context of cancer-associated oxidative and replicative stress. These hypotheses are currently being tested in the laboratory and are being translated into clinical studies.
AG14361 is, to our knowledge, the first high-potency PARP-1 inhibitor with the specificity and in vivo activity to enhance chemotherapy and radiation therapy of human cancer.
DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by ionizing radiation and topoisomerase II poisons, such as etoposide and doxorubicin. A major pathway for the repair of DSB is nonhomologous end joining, which requires DNA-dependent protein kinase (DNA-PK) activity. We investigated the therapeutic use of a potent, specific DNA-PK inhibitor (NU7441) in models of human cancer. We measured chemosensitization by NU7441 of topoisomerase II poisons and radiosensitization in cells deficient and proficient in DNA-PK CS (V3 and V3-YAC) and p53 wild type (LoVo) and p53 mutant (SW620) human colon cancer cell lines by clonogenic survival assay. Effects of NU7441 on DSB repair and cell cycle arrest were measured by ;H2AX foci and flow cytometry. Tissue distribution of NU7441 and potentiation of etoposide activity were determined in mice bearing SW620 tumors. NU7441 increased the cytotoxicity of ionizing radiation and etoposide in SW620, LoVo, and V3-YAC cells but not in V3 cells, confirming that potentiation was due to DNA-PK inhibition. NU7441 substantially retarded the repair of ionizing radiation-induced and etoposide-induced DSB. NU7441 appreciably increased G 2 -M accumulation induced by ionizing radiation, etoposide, and doxorubicin in both SW620 and LoVo cells. In mice bearing SW620 xenografts, NU7441 concentrations in the tumor necessary for chemopotentiation in vitro were maintained for at least 4 hours at nontoxic doses. NU7441 increased etoposide-induced tumor growth delay 2-fold without exacerbating etoposide toxicity to unacceptable levels. In conclusion, NU7441 shows sufficient proof of principle through in vitro and in vivo chemosensitization and radiosensitization to justify further development of DNA-PK inhibitors for clinical use.
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