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The DNA damage response (DDR) is a DNA damage surveillance and repair mechanism that can limit the effectiveness of radiotherapy and DNA-damaging chemotherapy, commonly used treatment modalities in cancer. Two related kinases, ataxia telangiectasia mutated (ATM) and ATM and Rad3-related kinase (ATR), work together as apical proteins in the DDR to maintain genome stability and cell survival in the face of potentially lethal forms of DNA damage. However, compromised ATM signaling is a common characteristic of tumor cells, which places greater reliance on ATR to mediate the DDR. In such circumstances, ATR inhibition has been shown to enhance the toxicity of DNA damaging chemotherapy to many cancer cells in multiple preclinical studies, while healthy tissue with functional ATM can tolerate ATR inhibition. ATR therefore represents a very attractive anticancer target. Herein we describe the discovery of VX-970/M6620, the first ATR inhibitor to enter clinical studies, which is based on a 2-aminopyrazine core first reported by Charrier ( J. Med. Chem. 2011 , 54 , 2320 - 2330 , DOI: 10.1021/jm101488z ).

The accurate measurement of total body and subcutaneous fat is essential if therapeutic interventions, aimed at preventing or reversing lipodystrophy syndrome, are to be adequately assessed. The aim of this study was to investigate the variability of dual-energy X-ray absorptiometry (DEXA) scans analysis performed at local sites compared to central analysis in a multicenter clinical trial.The PIILR study was a multicenter randomized clinical trial in which 80 HIV-infected patients with physician-documented lipodystrophy had serial measurements of body composition performed with Lunar DEXA scans. Scans were analyzed at local sites and then were reanalyzed centrally.DEXA scans from 73 patients who completed 24 weeks study were compared. Greater variation in the locally analyzed results than in the centrally reanalyzed data was noted, with arm, leg, and combined limb fat being most divergent between the local and centralized assessments (ratio of local to central standard deviation was 1.28, 1.31, and 1.35, respectively). The magnitude of this variance was enough to alter statistically relevant differences between study populations.Quality assurance is an important issue in the use of DEXA scans to determine body fat composition in multicenter research studies. A central quality assurance site should be incorporated to reduce variability in results.

The activator protein-1 (AP-1) transcriptional complex is made up of members of the Fos (c-Fos, FosB, Fra1, Fra2) and Jun (c-Jun, JunB, JunD) families and is stimulated by insulin in several cell types. The mechanism by which insulin activates this complex is not well understood but it is dependent on the activation of the Erk1 and Erk2 isoforms of mitogen-activated protein kinases. In the current study we show that the AP-1 complex isolated from insulin-stimulated cells contained c-Fos, Fra1, c-Jun and JunB. The activation of the AP-1 complex by insulin was accompanied by (i) a transient increase in c-fos expression, and the transactivation of the ternary complex factors Elk1 and Sap1a, in an Erk1/Erk2-dependent fashion; (ii) a substantial increase in the expression of Fra1 protein and mRNA, which was preceded by a transient decrease in its electrophoretic mobility upon SDS/PAGE, indicative of phosphorylation; and (iii) a sustained increase in c-jun expression without increasing c-Jun phosphorylation on serines 63 and 73 or activation of the stress-activated kinase JNK/SAPK. In conclusion, insulin appears to stimulate the activity of the AP-1 complex primarily through a change in the abundance of the components of this complex, although there may be an additional role for Fra1 phosphorylation.

Drug design is an iterative process requiring cycles of compound synthesis and testing, with each successive synthesis phase yielding molecules predicted to have improved characteristics over the previous set of compounds. In the field of cancer drug discovery, a key early-stage element of the drug design and testing process usually involves the screening of compounds in cell-based in vitro assays. One of the most frequent parameters assessed in cancer drug discovery is the impact of a given molecule on the proliferation of a cancer cell. The methyl-[3H]-thymidine incorporation assay is a widely used, gold standard, method for measuring inhibition of cell proliferation and has been used successfully to screen and optimize potential new cancer drugs. The assay is based on measuring incorporation of methyl-[3H]-thymidine (the radiolabeled form of the DNA precursor thymidine) into the DNA of dividing cancer cells. The screen is used to generate concentration effect relationships for test compounds and for the derivation of IC50 values. IC50 value is defined as the concentration of a test compound required to achieve half maximal inhibition of methyl-[3H]-thymidine incorporation, a parameter that is indicative of antiproliferative potency. IC50 values derived from cell-based assays help drive the medicinal chemistry efforts toward improved drug design, and it is, therefore, critical that the screen provides consistent, robust data over the lifetime of the project - a requirement that necessitates good-quality cell culture practices. The methyl-[3H]-thymidine incorporation assay has been adapted to high-throughput format to facilitate screening of large numbers of compounds. The detailed description of this method, exemplified using the COLO-205 colorectal cancer cell line in a 96-well format, should give the reader a thorough account of how to conduct proliferation assays, as well as some notes and tips on how to ensure success and avoid potential pitfalls.

Here we report a comprehensive biological characterization of a potent and selective small-molecule inhibitor of the DNA damage response (DDR) kinase ATR. We show a profound synthetic lethal interaction between ATR and the ATM-p53 tumor suppressor pathway in cells treated with DNA-damaging agents and establish ATR inhibition as a way to transform the outcome for patients with cancer treated with ionizing radiation or genotoxic drugs.

After lactation, the mouse mammary gland undergoes apoptosis and tissue remodelling as the gland reverts to its prepregnant state. This complex change was investigated using 2-DE. An integrated database was produced from lactation and involution proteomes. Forty-four molecular cluster indexes (MCIs) that showed altered expression from lactation to involution were selected for MS analysis. Of these, 32 gave protein annotations, 18 of which were unequivocal proteins. Selected proteins were then studied across all of development, including pregnancy, using data integrated from another proteome database. Two proteins, the RNA polymerase B transcription factor 3 (BTF3) and the minichromosome maintenance protein 3 (MCM3), although initially selected on the basis of the lactation/involution criteria, had expression profiles that indicated an additional role in mammary development and were further analysed. BTF3, a transcription factor previously not described in the mammary gland, was up-regulated strongly in pregnancy, indicating an involvement in alveolar growth. MCM3's expression was greatest in pregnancy and late involution, decreasing through lactation. Immunohistochemistry localised MCM3 to the mammary epithelium, where a greater proportion of cells stained than for the proliferation marker Ki67. MCM3 expression during lactation may identify cells that are licensed to repopulate the gland during cell loss in lactation and following involution.

Ductal morphogenesis in the mouse mammary gland occurs mainly postnatally and is driven by specialized structures at the ends of the developing ducts, the terminal end buds (TEBs), which later regress once ductal growth is complete. To identify proteins that are specifically associated with migration of TEBs we developed a novel method of isolating TEBs, which eliminated the mammary stroma. The protein expression profile of the TEBs was then compared with that of isolates taken from the 4th inguinal mammary gland of adult virgin mice using two-dimensional (2-D) gel electrophoresis and mass spectrometry (MS) analysis (matrix-assisted laser desorption/ionization and quadrupole time of flight). Following construction of an integrated protein expression database, 44 protein features which showed differential expression levels between the two sets were chosen for MS analysis. Of these, 24 gave protein annotations whereas the other 20 produced unidentified peptides. Fourteen unequivocal proteins were identified from these 24, whereas the remaining 10 matched more than one protein within a single 2-D gel feature. Several of the identified proteins were associated with the cytoskeleton and have previously been reported in axonal growth cones, suggesting that they may influence cell shape and motility within the advancing TEBs, in a similar fashion to migrating axons.

Adipose tissue plays a crucial endocrine role in controlling whole body glucose homeostasis and insulin sensitivity. Given the substantial rise in obesity and obesity-related diseases such as diabetes, it is important to understand the molecular basis of adipocyte differentiation and its control. Many studies have successfully exploited gene array technology to monitor changes in the profile of expressed genes during adipocyte differentiation, although this method only measures changes at the level of individual mRNA species. Using two-dimensional polyacrylamide gel electrophoresis, high-throughput image analysis, and candidate picking coupled with sequencing mass spectrometry, we have followed the changes in protein expression profile that occur during the differentiation of 3T3-L1 fibroblasts into adipocytes in response to dexamethasone, isobutyl methyl xanthine and insulin, or to the PPARgamma agonist, ciglitazone. Using this technique we have found alterations in the profile of over 2000 protein species during adipogenesis. Our studies reveal previously unknown alterations during adipogenesis in the expression or mobility (on sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of coactosin, which promotes actin filament destabilization, several signalling molecules, including RhoGDI-1, RhoGDI-2 and EHD1, and NEDD5 a protein involved in cytokinesis.

The precision error of the bone densitometer is used to interpret significant change in bone mineral density (BMD) in serial studies. The precision error can be expressed as standard deviation (SD) or coefficient of variation (CV). The aims of this study are to determine the precision error over a range of BMD values and to demonstrate the application of the precision error in clinical practice. A bone phantom was used consisting of a perspex block with eight compartments containing varying amounts of hydroxyapatite powder to simulate a range of bone densities. The block was scanned 21 times and manual regions placed over each compartment to measure the BMD in each compartment. There were no significant differences in the variances or SD for all eight compartments, that is, over the range of BMD normally encountered in clinical practice. However, the calculated CV show a progressive fall in values as the BMD rises. Therefore, the SD should be used to calculate significant BMD change. In a practise with quality control procedures in place to detect calibration drift and with appropriately trained personnel, a change of approximately 0.05 g/cm2 is generally regarded as being a significant change at a 95% confidence level.

Proteins associated with cancer cell plasma membranes are rich in known drug and antibody targets as well as other proteins known to play key roles in the abnormal signal transduction processes required for carcinogenesis. We describe here a proteomics process that comprehensively annotates the protein content of breast tumor cell membranes and defines the clinical relevance of such proteins. Tumor-derived cell lines were used to ensure an enrichment for cancer cell-specific plasma membrane proteins because it is difficult to purify cancer cells and then obtain good membrane preparations from clinical material. Multiple cell lines with different molecular pathologies were used to represent the clinical heterogeneity of breast cancer. Peptide tandem mass spectra were searched against a comprehensive data base containing known and conceptual proteins derived from many public data bases including the draft human genome sequences. This plasma membrane-enriched proteome analysis created a data base of more than 500 breast cancer cell line proteins, 27% of which were of unknown function. The value of our approach is demonstrated by further detailed analyses of three previously uncharacterized proteins whose clinical relevance has been defined by their unique cancer expression profiles and the identification of protein-binding partners that elucidate potential functionality in cancer.

We have recently reported (1) that two naturally occurring mutants of the insulin receptor tyrosine kinase domain, Arg-1174 --Gln and Pro-1178 --Leu (Gln-1174 and Leu1178, respectively), both found in patients with inherited severe insulin resistance, markedly impaired receptor tyrosine autophosphorylation, with both mutant receptors being unable to mediate the stimulation of glycogen synthesis or mitogenesis by insulin when expressed in Chinese hamster ovary cells. However, these mutations did not fully prevent IRS-1 phosphorylation in response to insulin in these cells, suggesting that IRS-1 alone may not be sufficient to mediate insulin's metabolic and mitogenic effects. In the present study, we have demonstrated that these mutations also impair the ability of the insulin receptor to activate the transcription factor Elk-1 and promote GLUT4 translocation to the plasma membrane. Although at low concentrations of insulin, the mutant receptors were impaired in their ability to stimulate the tyrosine phosphorylation of IRS-1, at higher insulin concentrations we confirmed that the cells expressing the mutant receptors showed significantly increased tyrosine phosphorylation of IRS-1 compared with parental nontransfected cells. In addition, at comparable insulin concentrations, the association of the p85alpha subunit of phosphoinositide 3-kinase (PI3-kinase) with IRS-1 and the enzymatic activity of IRS-1-associated PI3-kinase were significantly enhanced in cells expressing the mutant receptors. In contrast, no significant stimulation of the tyrosine phosphorylation of Shc, GTP loading of Ras, or mitogen-activated protein kinase phosphorylation was seen in cell lines expressing these mutant receptors. Thus, no activation of any measurable mitogenic or metabolic response was detectable, despite significant insulin-induced phosphorylation of IRS-1 and its association with PI3-kinase in cells stably expressing the mutant insulin receptors. These findings suggest that PI3-kinase activation alone may be insufficient to mediate a wide range of the metabolic and mitogenic effects of insulin. Additionally, the data provide support for the notion that insulin activation of Ras is more closely linked with Shc, and not IRS-1, phosphorylation.

DNA damaging agents have been the cornerstone of solid cancer therapy for decades yet they provide only modest benefit for patients with many tumor types. This reflects, in part, the efficient repair of DNA damage via a complex signaling and repair network known as the DNA damage response (DDR). Key regulators of the DDR are the phosphoinositol 3-kinase-like serine/threonine protein kinase (PIKK) family members ATR, ATM and DNA-PK. The DDR acts to detect DNA lesions, enforce checkpoints to halt cell cycle progression, and stimulate repair. Recent data have shown that elements of the DDR are commonly defective in cancer cells. It is widely believed that these cells become dependent on the remaining DDR pathways for survival from DNA damage. Inhibitors have been reported for a number of DDR enzymes, including ATM, DNA-PK, CHK1 and PARP, however there are no reports of drug-like ATR inhibitors. Here we disclose the in vitro characterization of a potent and highly selective ATR inhibitor (VE-821). This compound selectively blocks ATR signaling in cells (IC50 = 0.7 µM), but has little impact on ATM or DNA-PK signaling (IC50 >10 µM). Treatment with 10 µM VE-821 for 144 h causes little cell death in normal cell lines (5-11 %) but markedly higher death in cancer cell lines (28-46 %). VE-821 also dramatically sensitizes many cancer cells to multiple classes of genotoxic agents including antimetabolites, topoisomerase inhibitors and crosslinking agents; with over 10-fold increases genotoxic potency observed in some cases. In a panel of 36 lung cancer cell lines, VE-821 sensitized the cytotoxic effect of cisplatin to a far greater magnitude and over a broader subset of these lines than potent inhibitors of ATM, Chk1, or PARP. In over half of these cell lines, the IC50 of cisplatin was reduced by greater than 5 fold upon the addition of VE-821. We show that a basis for the cancer-selective effects of VE-821 is a synthetic lethal interaction between loss of ATM signaling (a frequent event in cancer resulting from loss of function of proteins such as ATM or p53) and ATR inhibition when cells encounter DNA damage. In keeping with this, ATR inhibition does not sensitize normal cells (with functional ATM) to the cytotoxic effects of genotoxic therapy. In this case a compensatory DDR is activated that is associated with marked activation of ATM, which in turn leads to reversible checkpoint arrest and a strong survival response. These studies show for the first time that a selective ATR inhibitor can preferentially sensitize cancer cells to genotoxic drugs by exploiting a synthetic lethal interaction between ATM and ATR signaling. This underpins the broad potential of ATR inhibition as a highly promising new strategy to improve the efficacy of genotoxic therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5491. doi:10.1158/1538-7445.AM2011-5491

Background. MK-0457 (VX-680) is a small-molecule inhibitor of Aurora kinases A, B, and C, FLT3, and JAK-2 with nanomolar level broad spectrum pre-clinical anti-tumor activity. The T315I BCR-ABL mutation mediates high level resistance to imatinib, dasatinib and nilotinib. A Phase I study of MK-0457 is being conducted in patients with refractory hematologic malignancies. The study regime is a 5 day continuous IV regimen given every 2 to 3 weeks. Methods. Fifteen CML patients received MK-0457 dosed at 8, 12, 16, 20, 24, 28, and 32 mg/m2/hr (11/15 had detectable BCR-ABL T315I). Peripheral blood for biomarker studies was available from patients at the 12 mg/m2/hr dose and above. Specimens were drawn prior to initiation of the infusion and at the end of the infusion (Day 5) during Cycle 1. Patient specimens have been analyzed using flow cytometry assays to measure BCR-ABL inhibition (phospho-CRKL), FLT3 and JAK-2 inhibition (phospho-STAT5) and Aurora kinase inhibition (phospho-histone H3). Results. MK-0457 has in vitro activity against cells expressing wild-type or mutated BCR-ABL, including the T315I BCR-ABL mutation. In CML and ALL patients with the T315I BCR-ABL mutation, MK-0457 is capable of inhibiting BCR-ABL as measured by the phospho-CRKL assay. The observed decrease in BCR-ABL activity trends with plasma exposure to MK-0457. At dose levels less than 20 mg/m2/hr, plasma exposure of MK-0457 typically does not exceed 1 micromolar and inhibition of BCR-ABL has not been observed. At exposures that exceed 1 micromolar, MK-0457 inhibits BCR-ABL, with pCRKL abundance falling below the lower limit of detection in one patient dosed at 24 mg/m2/hr and decreasing compared to baseline in several other patients. There is also a trend correlating degree of BCR-ABL inhibition as measured by the pCRKL biomarker and clinical response to MK-0457. Conclusions. MK-0457 inhibits BCR-ABL activity in CML and ALL patients with the BCR-ABL T315I mutation. Objective responses have been observed in patients without demonstrable BCR-ABL inhibition, suggesting that inhibition of Aurora kinase may also contribute to the clinical efficacy of MK-0457 in CML and ALL patients with the BCR-ABL T315I mutation.

MK-0457 (VX-680) is a potent small molecule inhibitor of the Aurora-family of protein kinases that inhibits cell proliferation through aberrant mitosis and failed cytokinesis. Affected cells are subsequently deleted by apoptosis. Tumor regression and increased survival have been reported in multiple in-vivo models and in January 2005 MK-0457 entered Phase I clinical trials as the first in class Aurora inhibitor. Although MK-0457 demonstrates significant selectivity over the vast majority of kinases tested, it has been shown to potently inhibit Flt-3 and Abl, both of which are targets for anti-cancer therapies. Most notably MK-0457 potently inhibits a series of mutant Abl kinases including the multi-drug resistant T315I form that is commonly expressed in Gleevec refractory CML and ALL. We report here the first MK-0457 / Aurora co-complex crystal structure and show that it adopts a closed inactive conformation. By comparison with other reported kinase structures we provide a structural basis for the cross-reactivity with wild type and drug resistant mutants of the Abl kinase. We also show that MK-0457 blocks both Aurora and Abl cellular kinase activity and displays potent cytotoxic activity against a range of leukemic cell lines, including Gleevec resistant cells that are reliant on mutant Abl for growth and survival. Similar observations have recently been made in the clinic, where new data show that MK-0457 has activity in patients with BCR-ABL T315I mutant CML and Philadelphia positive ALL.