Ataxia telangiectasia mutated (ATM) kinase signals DNA double-strand breaks (DSB) to cell-cycle arrest via p53 and DNA repair. ATM-defective cells are sensitive to DSB-inducing agents, making ATM an attractive target for anticancer chemo-and radiosensitization. KU59403 is an ATM inhibitor with the potency, selectivity, and solubility for advanced preclinical evaluation. KU59403 was not cytotoxic to human cancer cell lines (SW620, LoVo, HCT116, and MDA-MB-231) per se but significantly increased the cytotoxicity of topoisomerase I and II poisons: camptothecin, etoposide, and doxorubicin. Chemo-and radiosensitization by ATM inhibition was not p53-dependent. Following administration to mice, KU59403 distributed to tissues and concentrations exceeding those required for in vitro activity were maintained for at least 4 hours in tumor xenografts. KU59403 significantly enhanced the antitumor activity of topoisomerase poisons in mice bearing human colon cancer xenografts (SW620 and HCT116) at doses that were nontoxic alone and well-tolerated in combination. Chemosensitization was both dose-and scheduledependent. KU59403 represents a major advance in ATM inhibitor development, being the first compound to show good tissue distribution and significant chemosensitization in in vivo models of human cancer, without major toxicity. KU59403 provides the first proof-of-principle preclinical data to support the future clinical development of ATM inhibitors. Mol Cancer Ther; 12(6); 959-67. Ó2013 AACR.

DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by topoisomerase II poisons. Nonhomologous end joining (NHEJ) is a major pathway for DSB repair and requires DNA-dependent protein kinase (DNA-PK) activity. DNA-PK catalytic subunit (DNA-PKcs) is structurally similar to PI-3K, which promotes cell survival and proliferation and is upregulated in many cancers. KU-0060648 is a dual inhibitor of DNA-PK and PI-3K in vitro. KU-0060648 was investigated in a panel of human breast and colon cancer cells. The compound inhibited cellular DNA-PK autophosphorylation with IC 50 values of 0.019 mmol/L (MCF7 cells) and 0.17 mmol/L (SW620 cells), and PI-3K-mediated AKT phosphorylation with IC 50 values of 0.039 mmol/L (MCF7 cells) and more than 10 mmol/L (SW620 cells). Five-day exposure to 1 mmol/L KU-0060648 inhibited cell proliferation by more than 95% in MCF7 cells but only by 55% in SW620 cells. In clonogenic survival assays, KU-0060648 increased the cytotoxicity of etoposide and doxorubicin across the panel of DNA-PKcs-proficient cells, but not in DNA-PKcs-deficient cells, thus confirming that enhanced cytotoxicity was due to DNA-PK inhibition. In mice bearing SW620 and MCF7 xenografts, concentrations of KU-0060648 that were sufficient for in vitro growth inhibition and chemosensitization were maintained within the tumor for at least 4 hours at nontoxic doses. KU-0060648 alone delayed the growth of MCF7 xenografts and increased etoposide-induced tumor growth delay in both in SW620 and MCF7 xenografts by up to 4.5-fold, without exacerbating etoposide toxicity to unacceptable levels. The proof-of-principle in vitro and in vivo chemosensitization with KU-0060648 justifies further evaluation of dual DNA-PK and PI-3K inhibitors.

Purpose: MetXia-P450 is a novel recombinant retroviral vector that encodes the human cytochrome P450 type 2B6 gene (CYP2B6), Escherichia coli lacZ , and neomycin resistance marker genes. Cytochrome P450 enzymes are primarily expressed in the liver and convert the prodrug cyclophosphamide to an active phosphoramide mustard and acrolein. Gene-based delivery of CYP2B6 to the tumor site leads to local prodrug activation and higher concentrations of the active metabolites at the target site.Experimental Design: MetXia-P450 was directly injected into metastatic cutaneous tumor nodules on days 1 and 2 and nodules biopsied on day 7. Oral cyclophosphamide (100 mg/m 2 ) was administered between days 8 and 22. Subsequent cycles of oral cyclophosphamide were repeated for 2 of 4 weeks. Gene transfer levels in biopsy samples were measured by histologic and quantitative PCR analyses. Safety assessments were made using PCR for vector dissemination to the blood after injection and using PCR and serologic analyses to detect replicating virus. Secondary end points included clinical response, toxicity, and evaluation of antitumor immune responses by measurement of carcinoembryonic antigen and 5T4 antibodies.Results: Twelve patients with breast cancer (n = 9) and melanoma (n = 3) received three dose levels of MetXia-P450 (f8 Â 10 5

Male Shotokan karate players (karateka) (N = 208) completed the Competitive State Anxiety Inventory-2 and the Profile of Mood States about 40 minutes before a competition. Single-factor multivariate analysis of variance of preperformance mood and anxiety scores indicated significant differences between winning and losing competitors. Winners scored higher on Vigor, Anger, and Self-confidence, and lower on Tension, Depression, Fatigue, Confusion, Cognitive Anxiety, and Somatic Anxiety. Discriminant function analysis showed that 91.96% of participants could be correctly classified as winners or losers on the basis of preperformance mood scores. This figure rose to 93.47% when scores on the anxiety subscales were also included in the discriminant function analysis. Anxiety scores alone produced 78.89% discrimination. Mood profiles for winning karateka were in line with the "mental health" profile of Morgan except for above-average scores on Anger. This result supports the view of McGowan and Miller that anger may facilitate performance in karate competition. The capacity of measures of psychological state to discriminate performance exceeds previous reports, suggesting that karate performance may be exceptionally mood-dependent. These results suggest that interventions which increase scores on Vigor and Anger and reduce scores on Tension, Depression, Fatigue, and Confusion may be particularly efficacious for Shotokan karate performance.

Human cytochrome P450 2B6 ( CYP2B6 ) metabolizes the prodrug cyclophosphamide ( CPA ) to produce phosphoramide mustard that cross -links DNA leading to cell death. We have constructed a novel retroviral vector encoding CYP2B6 ( designated``MetXia -P450'' ) and used it to transduce the human tumor cell lines HT29 and T47D. MetXia -P450 transduction sensitised these cells to the cytotoxic effects of the prodrug CPA. Results from in vitro experiments demonstrated adverse effects on the clonogenic survival of cyclophosphamide -treated cells transduced with MetXia -P450. Cytotoxic activity accompanied by bystander effect was particularly evident in 3 -D multicellular spheroid models suggesting that this in vitro system may be a more appropriate model for assessing the efficacy of gene directed -enzyme prodrug therapy ( GDEPT ). We have applied this approach in a clinically relevant gene therapy protocol on established subcutaneous tumor xenografts. These studies show for the first time the efficacy of a P450 -based GDEPT strategy mediated by a direct retroviral gene transfer in vivo. Cancer Gene Therapy ( 2001 ) 8, 473 ± 482