VCP (also known as p97 or Cdc48p in yeast) is an AAA(+) ATPase regulating endoplasmic reticulum-associated degradation. After high-throughput screening, we developed compounds that inhibit VCP via different mechanisms, including covalent modification of an active site cysteine and a new allosteric mechanism. Using photoaffinity labeling, structural analysis and mutagenesis, we mapped the binding site of allosteric inhibitors to a region spanning the D1 and D2 domains of adjacent protomers encompassing elements important for nucleotide-state sensing and ATP hydrolysis. These compounds induced an increased affinity for nucleotides. Interference with nucleotide turnover in individual subunits and distortion of interprotomer communication cooperated to impair VCP enzymatic activity. Chemical expansion of this allosteric class identified NMS-873, the most potent and specific VCP inhibitor described to date, which activated the unfolded protein response, interfered with autophagy and induced cancer cell death. The consistent pattern of cancer cell killing by covalent and allosteric inhibitors provided critical validation of VCP as a cancer target.

Fourier-transform infrared (FTIR) spectroscopy is a vibrational technique that gives information on the chemical composition of a sample, providing a "molecular fingerprint" of it. It is a powerful approach to study intact cells. The aim of the present study was to analyse and quantify apoptotic cells by using a FTIR approach based on attenuated total reflection (ATR). We incubated human HL60 leukaemic cells with camptothecin, a cytotoxic drug, and monitored apoptosis induction over a period of time. Several ATR-FTIR spectral changes occurred during the apoptotic process. In particular, we observed that the apoptotic index was inversely correlated with the spectral area in the region 1200-900 cm(-1), assigned to the absorption of nucleic acids. We therefore propose that ATR-FTIR spectral features may be used as a diagnostic marker of apoptotic cells.

Growth Factor Receptor. NTRK1 was originally isolated from a colorectal carcinoma (CRC) sample as component of a somatic rearrangement (TPM3-NTRK1) resulting in expression of the oncogenic chimeric protein TPM3-TRKA, but there has been no subsequent report regarding the relevance of this oncogene in CRC. The KM12 human CRC cell line expresses the chimeric TPM3-TRKA protein and is hypersensitive to TRKA kinase inhibition.We report the detailed characterization of the TPM3-NTRK1 genomic rearrangement in KM12 cells and through a cellular screening approach, the identification of NMS-P626, a novel highly potent and selective TRKA inhibitor. NMS-P626 suppressed TPM3-TRKA phosphorylation and downstream signaling in KM12 cells and showed remarkable antitumor activity in mice bearing KM12 tumors.Finally, using quantitative reverse transcriptase PCR and immunohistochemistry (IHC) we identified the TPM3-NTRK1 rearrangement in a CRC clinical sample, therefore suggesting that this chromosomal translocation is indeed a low frequency recurring event in CRC and that such patients might benefit from therapy with TRKA kinase inhibitors.

Quantification of BrdU incorporation into DNA is a widely used technique to assess the cell cycle status of cells. DNA denaturation is required for BrdU detection with the drawback that most protein epitopes are destroyed and classical antibody staining techniques for multiplex analysis are not possible. To address this issue we have developed a novel method that overcomes the DNA denaturation step but still allows detection of BrdU. Cells were pulsed for a short time by 5-ethynyl-2'-deoxyuridine, which is incorporated into DNA. The exposed nucleotide alkyne group of DNA was then derivatized in physiologic conditions by the copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) using BrdU azides. The resulting DNA-bound bromouracil moiety was subsequently detected by commercial anti-BrdU mAb without the need for a denaturation step. Continuous labeling with EdU showed a slightly increased anti-proliferative activity compared to BrdU. However, using a lower concentration of EdU for labeling can compensate for this. Alkynyl tags could be detected quickly by a highly specific reaction using BrdU azides. Fluorescence quenching by the DNA dye PI using both BrdU azides was negligible. Our labeling method is suitable for FCM and HCA and shows a higher signal to noise ratio than other methods. This method also allowed multiplex analysis by simultaneous detection of EdU-BrdU, caspase-3, and phospho-histone 3 mAbs, proving sensitivity and feasibility of this new technique. In addition, it has the potential for use in vivo, as exemplified for bone marrow studies. We have established a new method to determine the position of cells in the cell cycle. This is superior when compared to traditional BrdU detection since it allows multiplex analysis, is more sensitive and shows less quenching with PI. The method provides new opportunities to investigate changes in protein expression at different cell cycle stages using pulse labeling experiments.

Purpose: Purpose of this study has been the assessment of nuclear factor-nB (NF-nB) as a survival factor in human mesothelial cells (HMC), transformed HMC and malignant mesothelioma (MMe) cells.We aimed at verifying whether the proteasome inhibitor Bortezomib could abrogate NF-nB activity in MMe cells, leading to tumor cell death and may be established as a novel treatment for this aggressive neoplasm. Experimental Design: In HMC and MMe cells, NF-nB nuclear translocation and DNA binding were studied by electrophoretic mobility shift assay, following treatment with tumor necrosis factor-a (TNF-a). The IKK inhibitor Bay11-7082 was also tested to evaluate its effects on HMC, transformed HMC, and MMe cell viability upon exposure to asbestos fibers. Following Bortezomib treatment, cytotoxicity of MMe cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, whereas apoptosis and cell-cycle blockade were investigated by high-content analysis. Bortezomib was also given to mice bearing i.p. xenografts of MMe cells, and its effects on tumor growth were evaluated. Results: Here, we show that NF-nB activity is a constitutive survival factor in transformed HMC, MMe cells, and acts as a survival factor in HMC exposed to asbestos fibers. Bortezomib inhibits NF-nB activity in MMe cells and induces cell cycle blockade and apoptosis in vitro as well as tumor growth inhibition in vivo. Conclusions: Inhibition of NF-nB constitutive activation in MMe cells by Bortezomib resulted in in vitro cytotoxicity along with apoptosis and in vivo tumor regression. Our results support the use of Bortezomib in the treatment of MMe and has led to a phase II clinical trial currently enrolling in Europe.

Chordomas are rare bone tumors with no approved therapy. These tumors express several activated tyrosine kinase receptors, which prompted attempts to treat patients with tyrosine kinase inhibitors. Although clinical benefit was observed in phase II clinical trials with imatinib and sorafenib, and sporadically also with EGFR inhibitors, therapies evaluated to date have shown modest activity. With the goal of identifying new drugs with immediate therapeutic potential for chordoma patients, we collected clinically approved drugs and other advanced inhibitors of MET, PDGFRβ, and EGFR tyrosine kinases, and assessed their antiproliferative activity against a panel of chordoma cell lines. Chordoma cell lines were not responsive to MET and PDGFRβ inhibitors. U-CH1 and UM-Chor1 were sensitive to all EGFR inhibitors, whereas the remaining cell lines were generally insensitive to these drugs. Afatinib was the only EGFR inhibitor with activity across the chordoma panel. We then investigated the molecular mechanisms behind the responses observed and found that the antiproliferative ICs correlate with the unique ability of afatinib to promote degradation of EGFR and brachyury, an embryonic transcription factor considered a key driver of chordoma. Afatinib displayed potent antitumor efficacy in U-CH1, SF8894, CF322, and CF365 chordoma tumor models In the panel analyzed, high EGFR phosphorylation and low AXL and STK33 expression correlated with higher sensitivity to afatinib and deserve further investigation as potential biomarkers of response. These data support the use of afatinib in clinical trials and provide the rationale for the upcoming European phase II study on afatinib in advanced chordoma..

Cell cycle analysis using flow cytometry (FC) to measure cellular DNA content is a common procedure in drug mechanism of action studies. Although this technique lends itself readily to cell lines that grow in suspension, adherent cell cultures must be resuspended in a cumbersome and potentially invasive procedure that normally involves trypsinization and mechanical agitation of monolayer cultures. High-content analysis (HCA), an automated microscopy-based technology, is well suited to analysis of monolayer cell cultures but provides intrinsically less accurate determination of cellular DNA content than does FC and thus is not the method of choice for cell cycle analysis. Using Cellomics's ArrayScan TM reader, the authors have developed a 4-color multiparametric HCA approach for cell cycle analysis of adherent cells based on detection of DNA content (4,6-diamidino-2-phenylindole [DAPI] fluorescence), together with the known cell cycle markers bromo-2-deoxyuridine (BrdU) incorporation, cyclin B1 expression, and histone H3 (Ser28) phosphorylation within a single cell population. Considering all 4 markers together, a reliable and accurate quantification of cell cycle phases was possible, as compared with flow cytometric analysis. Using this assay, specific cell cycle blocks induced by treatment with thymidine, paclitaxel, or nocodazole as test drugs were easily monitored in adherent cultures of U-2 OS osteosarcoma cells. (Journal of Biomolecular

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