Zebularine partially reverses GST methylation in prostate cancer cells and restores sensitivity to the DNA minor groove binder brostallicin.

Brostallicin is a DNA minor groove binder that shows enhanced antitumor activity in cells with high glutathione S-transferase (GST)/glutathione content. Prostate cancer cells present, almost invariably, methylation of the GSTP1 gene promoter and, as a consequence, low levels of GST-pi expression and activity. In these cells, brostallicin shows very little activity. We tested whether pretreatment of heavily GST-methylated prostate cancer cells with demethylating agents could enhance the activity of brostallicin. Human prostate cancer cells LNCaP and DU145 were used for these studies both in vitro and in vivo. The demethylating agent zebularine was used in combination with brostallicin. Methylation specific PCR and pyrosequencing were used to determine the level of GST methylation. Pretreatment with demethylating agents enhanced the in vitro activity of brostallicin in LNCaP cells. Zebularine, in particular, induced an enhancement of activity in vivo comparable to that obtained by transfecting the human GSTP1 gene in LNCaP cells in vitro. Molecular analysis performed on tumor xenografts in mice pretreated with zebularine failed to detect re-expression of GST-pi and demethylation of GSTP1. However, we found demethylation in the GSTM1 gene, with consequent re-expression of GST-mu at the mRNA level. These results indicate that zebularine, both in vitro and in vivo, enhances the activity of brostallicin and that this enhancement correlates with re-expression of GST-pi and GST-mu. These findings highlight the potential therapeutic value of combining demethylating agents and brostallicin in tumors with GST methylation that poorly respond to brostallicin.

The Polo-Like Kinase 1 (PLK1) inhibitor NMS-P937 is effective in a new model of disseminated primary CD56+ acute monoblastic leukaemia.

CD56 is expressed in 15-20% of acute myeloid leukaemias (AML) and is associated with extramedullary diffusion, multidrug resistance and poor prognosis. We describe the establishment and characterisation of a novel disseminated model of AML (AML-NS8), generated by injection into mice of leukaemic blasts freshly isolated from a patient with an aggressive CD56(+) monoblastic AML (M5a). The model reproduced typical manifestations of this leukaemia, including presence of extramedullary masses and central nervous system involvement, and the original phenotype, karyotype and genotype of leukaemic cells were retained in vivo. Recently Polo-Like Kinase 1 (PLK1) has emerged as a new candidate drug target in AML. We therefore tested our PLK1 inhibitor NMS-P937 in this model either in the engraftment or in the established disease settings. Both schedules showed good efficacy compared to standard therapies, with a significant increase in median survival time (MST) expecially in the established disease setting (MST = 28, 36, 62 days for vehicle, cytarabine and NMS-P937, respectively). Importantly, we could also demonstrate that NMS-P937 induced specific biomarker modulation in extramedullary tissues. This new in vivo model of CD56(+) AML that recapitulates the human tumour lends support for the therapeutic use of PLK1 inhibitors in AML.

Therapeutic efficacy of the pan-cdk inhibitor PHA-793887 in vitro and in vivo in engraftment and high-burden leukemia models.

OBJECTIVE: The aim of the work was to determine and characterize, in vitro and in vivo, the therapeutic activity of PHA-793887, a new potent pan-cdk inhibitor, in the context of hematopoietic neoplasms. MATERIALS AND METHODS: Thirteen leukemic cell lines bearing different cytogenetic abnormalities and normal hematopoietic cells were used in cytotoxicity and colony assays. The drug activity at the molecular level was analyzed by Western blotting. PHA-793887 was also tested in vivo in several leukemia xenograft models. RESULTS: PHA-793887 was cytotoxic for leukemic cell lines in vitro, with IC(50) ranging from 0.3 to 7 microM (mean: 2.9 microM), regardless of any specific chromosomal aberration. At these doses, the drug was not cytotoxic for normal unstimulated peripheral blood mononuclear cells or CD34(+) hematopoietic stem cells. Interestingly, in colony assays PHA-793887 showed very high activity against leukemia cell lines, with an IC(50) <0.1 microM (mean: 0.08 microM), indicating that it has efficient and prolonged antiproliferative activity. PHA-793887 induced cell-cycle arrest, inhibited Rb and nucleophosmin phosphorylation, and modulated cyclin E and cdc6 expression at low doses (0.2-1 microM) and induced apoptosis at the highest dose (5 microM). It was also effective in vivo in both subcutaneous xenograft and primary leukemic disseminated models that better mimic naturally occurring human disease. Interestingly, in one disseminated model derived from a relapsed Philadelphia-positive acute lymphoid leukemia patient, PHA-793887 showed strong therapeutic activity also when treatment was started after establishment of high disease burden. CONCLUSIONS: We conclude that PHA-793887 has promising therapeutic activity against acute leukemias in vitro and in vivo.

A phase I dose-escalation study of danusertib (PHA-739358) administered as a 24-hour infusion with and without granulocyte colony-stimulating factor in a 14-day cycle in patients with advanced solid tumors.

PURPOSE: This study was conducted to assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of the i.v. pan-aurora kinase inhibitor PHA-739358, danusertib, in patients with advanced solid tumors. EXPERIMENTAL DESIGN: In part 1, patients received escalating doses of danusertib (24-hour infusion every 14 days) without filgrastim (granulocyte colony-stimulating factor, G-CSF). Febrile neutropenia was the dose-limiting toxicity without G-CSF. Further dose escalation was done in part 2 with G-CSF. Blood samples were collected for danusertib pharmacokinetics and pharmacodynamics. Skin biopsies were collected to assess histone H3 phosphorylation (pH3). RESULTS: Fifty-six patients were treated, 40 in part 1 and 16 in part 2. Febrile neutropenia was the dose-limiting toxicity in part 1 without G-CSF. Most other adverse events were grade 1 to 2, occurring at doses >or=360 mg/m(2) with similar incidence in parts 1 and 2. The maximum tolerated dose without G-CSF is 500 mg/m(2). The recommended phase 2 dose in part 2 with G-CSF is 750 mg/m(2). Danusertib showed dose-proportional pharmacokinetics in parts 1 and 2 with a median half-life of 18 to 26 hours. pH3 modulation in skin biopsies was observed at >or=500 mg/m(2). One patient with refractory small cell lung cancer (1,000 mg/m(2) with G-CSF) had an objective response lasting 23 weeks. One patient with refractory ovarian cancer had 27% tumor regression and 30% CA125 decline. CONCLUSIONS: Danusertib was well tolerated with target inhibition in skin at >or=500 mg/m(2). Preliminary evidence of antitumor activity, including a partial response and several occurrences of prolonged stable disease, was seen across a variety of advanced refractory cancers. Phase II studies are ongoing.

PHA-739358, a potent inhibitor of Aurora kinases with a selective target inhibition profile relevant to cancer.

PHA-739358 is a small-molecule 3-aminopyrazole derivative with strong activity against Aurora kinases and cross-reactivities with some receptor tyrosine kinases relevant for cancer. PHA-739358 inhibits all Aurora kinase family members and shows a dominant Aurora B kinase inhibition-related cellular phenotype and mechanism of action in cells in vitro and in vivo. p53 status-dependent endoreduplication is observed upon treatment of cells with PHA-739358, and phosphorylation of histone H3 in Ser(10) is inhibited. The compound has significant antitumor activity in different xenografts and spontaneous and transgenic animal tumor models and shows a favorable pharmacokinetic and safety profile. In vivo target modulation is observed as assessed by the inhibition of the phosphorylation of histone H3, which has been validated preclinically as a candidate biomarker for the clinical phase. Pharmacokinetics/pharmacodynamics modeling was used to define drug potency and to support the prediction of active clinical doses and schedules. We conclude that PHA-739358, which is currently tested in clinical trials, has great therapeutic potential in anticancer therapy in a wide range of cancers.

Magnetic resonance imaging and histopathological characterization of prostate tumors in TRAMP mice as model for pre-clinical trials.

BACKGROUND: The Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) develops progressive forms of prostate cancer. Due to the lack of a validated non-invasive methodology, pathology has been so far the most common parameter evaluated in efficacy studies. METHODS: We studied by magnetic resonance imaging (MRI) 210 mice that were repeatedly measured up to 33 weeks of age in order to stage prostate tumors and follow pathological progression in single animals. A pre-clinical trial with doxorubicin was also performed. RESULTS: Progressive forms of cancer (well and poorly differentiated (PD) adenocarcinomas) were easily recognized on MR images and MRI findings were validated against histopathological analysis. Age at tumor onset was different for the two tumoral forms. Doxorubicin treatment caused a strong reduction in tumor volume. CONCLUSIONS: Prostate cancer in TRAMP mice is multifocal and heterogeneous: a non-invasive methodology such as MRI facilitates the rational design of translational pre-clinical trials in this widely used animal model.

PHA-680632, a novel Aurora kinase inhibitor with potent antitumoral activity.

PURPOSE: Aurora kinases play critical roles during mitosis in chromosome segregation and cell division. The aim of this study was to determine the preclinical profile of a novel, highly selective Aurora kinase inhibitor, PHA-680632, as a candidate for anticancer therapy. EXPERIMENTAL DESIGN: The activity of PHA-680632 was assayed in a biochemical ATP competitive kinase assay. A wide panel of cell lines was evaluated for antiproliferative activity. Cell cycle analysis. Immunohistochemistry, Western blotting, and Array Scan were used to follow mechanism of action and biomarker modulation. Specific knockdown of the targets by small interfering RNA was followed to validate the observed phenotypes. Efficacy was determined in different xenograft models and in a transgenic animal model of breast cancer. RESULTS: PHA-680632 is active on a wide range of cancer cell lines and shows significant tumor growth inhibition in different animal tumor models at well-tolerated doses. The mechanism of action of PHA-680632 is in agreement with inhibition of Aurora kinases. Histone H3 phosphorylation in Ser10 is mediated by Aurora B kinase, and our kinetic studies on its inhibition by PHA-680632 in vitro and in vivo show that phosphorylation of histone H3 is a good biomarker to follow activity of PHA-680632. CONCLUSIONS: PHA-680632 is the first representative of a new class of Aurora inhibitors with a high potential for further development as an anticancer therapeutic. On treatment, different cell lines respond differentially, suggesting the absence of critical cell cycle checkpoints that could be the basis for a favorable therapeutic window.

Wild rats as monitors of environmental lead contamination in the urban area of Milan, Italy.

Twenty-one wild brown rats (Rattus norvegicus) were collected from four areas of Milan (Italy). Main organs were harvested from each rat, fixed in 10% neutral buffer formalin, and embedded in paraffin. Organ sections were stained with hematoxylin and eosin, kidney sections were also stained with the Ziehl-Neelsen method. Graphite furnace atomic absorption spectrophotometry was performed on all kidney samples. The presence of inclusion bodies and karyocytomegaly was scored. Six adult rats out of 21 (28.6%) showed kariocytomegaly and intranuclear inclusion bodies of the renal proximal tubules, characteristic of lead toxicity. The nuclear inclusion bodies were always Ziehl-Neelsen positive. The six rats which showed the characteristic histological alterations ascribed to lead, had lead concentrations statistically higher than the three control rats (4 microg/g wet wt. versus 0.09 microg/g wet wt., P<0.001) and than the captured wild rats. Intranuclear inclusion bodies were found in rats with kidney lead concentrations greater than 0.6 microg/g wet weight. There were significant differences between the four areas of capture. The incidence of intranuclear inclusion bodies and karyocytomegaly in proximal tubular cells was higher in rats with high lead levels detected by spectrophotometry. The present results indicate a renal lead concentration of 0.6 microg/g wet weight as a threshold to detect histologic kidney changes due to lead absorption. Results demonstrate that the examination of adult free-living rats may be considered an appropriate method to obtain a rapid, sensitive, easy, cost effective estimate of lead contamination in urban areas. Animal models allow us to evaluate potential human health risk associated with environmental contamination.