Purpose EMT has been associated with metastatic spread and EGFR inhibitor resistance. We developed and validated a robust 76-gene EMT signature using gene expression profiles from four platforms using NSCLC cell lines and patients treated in the BATTLE study. Methods We conducted an integrated gene expression, proteomic, and drug response analysis using cell lines and tumors from NSCLC patients. A 76-gene EMT signature was developed and validated using gene expression profiles from four microarray platforms of NSCLC cell lines and patients treated in the BATTLE (Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination) study, and potential therapeutic targets associated with EMT were identified. Results Compared with epithelial cells, mesenchymal cells demonstrated significantly greater resistance to EGFR and PI3K/Akt pathway inhibitors, independent of EGFR mutation status, but more sensitivity to certain chemotherapies. Mesenchymal cells also expressed increased levels of the receptor tyrosine kinase Axl and showed a trend towards greater sensitivity to the Axl inhibitor SGI-7079, while the combination of SGI-7079 with erlotinib reversed erlotinib resistance in mesenchymal lines expressing Axl and in a xenograft model of mesenchymal NSCLC. In NSCLC patients, the EMT signature predicted 8-week disease control in patients receiving erlotinib, but not other therapies. Conclusion We have developed a robust EMT signature that predicts resistance to EGFR and PI3K/Akt inhibitors, highlights different patterns of drug responsiveness for epithelial and mesenchymal cells, and identifies Axl as a potential therapeutic target for overcoming EGFR inhibitor resistance associated with the mesenchymal phenotype

Tissue factor (TF) is an essential cofactor for the activation of blood coagulation in vivo. We now report that quiescent human platelets express TF pre-mRNA and, in response to activation, splice this intronic-rich message into mature mRNA. Splicing of TF pre-mRNA is associated with increased TF protein expression, procoagulant activity, and accelerated formation of clots. Pre-mRNA splicing is controlled by Cdc2-like kinase (Clk)1, and interruption of Clk1 signaling prevents TF from accumulating in activated platelets. Elevated intravascular TF has been reported in a variety of prothrombotic diseases, but there is debate as to whether anucleate platelets—the key cellular effector of thrombosis—express TF. Our studies demonstrate that human platelets use Clk1-dependent splicing pathways to generate TF protein in response to cellular activation. We propose that platelet-derived TF contributes to the propagation and stabilization of a thrombus.

New activities of human platelets continue to emerge. One unexpected response is new synthesis of proteins from previously transcribed RNAs in response to activating signals. We previously reported that activated human platelets synthesize B-cell lymphoma-3 (Bcl-3) under translational control by mammalian target of rapamycin (mTOR). Characterization of the ontogeny and distribution of the mTOR signaling pathway in CD34 ؉ stem cell-derived megakaryocytes now demonstrates that they transfer this regulatory system to developing proplatelets. We also found that Bcl-3 is required for condensation of fibrin by activated platelets, demonstrating functional significance for mTOR-regulated synthesis of the protein. Inhibition of mTOR by rapamycin blocks clot retraction by human platelets. Platelets from wild-type mice synthesize Bcl-3 in response to activation, as do human platelets, and platelets from mice with targeted deletion of Bcl-3 have defective retraction of fibrin in platelet-fibrin clots mimicking treatment of human platelets with rapamy- IntroductionPlatelets are critical effector cells in hemostasis and inflammation in health and disease. [1][2][3] Previously unrecognized responses of human platelets continue to emerge. 1,2,4 One of the most unexpected is synthesis of new proteins from constitutively expressed but silenced messenger RNAs (mRNAs) by activated platelets. [4][5][6][7][8][9] Activation-dependent synthesis of proteins is contrary to conventional expectations because platelets are anucleate. Nevertheless, early 10,11 and more recent 4,5,8,9,[12][13][14] observations demonstrate that mature circulating human platelets have a significant complement of mRNAs generated by precursor megakaryocytes. Furthermore, platelets translate some of these constitutive transcripts in response to activating signals, 9 providing a previously unrecognized mechanism to alter the platelet proteome. 15 Activated human platelets also process pre-mRNAs to mature, translatable transcripts. 4 The diversity of regulatory mechanisms in the platelet repertoire suggests that posttranscriptional gene expression is an important feature of their biology [4][5][6][7][8][9] and is again contrary to previous dogma that they are functionally simple cells.B-cell lymphoma-3 (Bcl-3), a member of the Ik␤␣ family of factors, 16,17 is an index example of signal-dependent synthesis of a specific protein by human platelets. [5][6][7]9 Its translation from constitutively repressed mRNA, which is rapid and yields new protein within minutes, is triggered by cellular activation via thrombin receptors and is modulated by engagement of integrin ␣ IIb ␤ 3 . [5][6][7] Inhibition of synthesis of Bcl-3 by the therapeutic macrolide rapamycin provided evidence that its translation in activated platelets is controlled by mammalian target of rapamycin (mTOR). 5 This conserved phosphatidylinositol kinaserelated kinase is a key regulator of cell-cycle progression, growth, and nutrient balance. [18][19][20] It was, however, not known to control expression of...

Epigenetic modification of DNA leads to changes in gene expression. DNA methyltransferases (DNMTs) comprise a family of nuclear enzymes that catalyze the methylation of CpG dinucleotides, resulting in an epigenetic methylome distinguished between normal cells and those in disease states such as cancer. Disrupting gene expression patterns through promoter methylation has been implicated in many malignancies and supports DNMTs as attractive therapeutic targets. This review focuses on the rationale of targeting DNMTs in cancer, the historical approach to DNMT inhibition, and current marketed hypomethylating therapeutics azacytidine and decitabine. In addition, we address novel DNMT inhibitory agents emerging in development, including CP-4200 and SGI-110, analogs of azacytidine and decitabine, respectively; the oligonucleotides MG98 and miR29a; and a number of reversible inhibitors, some of which appear to be selective against particular DNMT isoforms. Finally, we discuss future opportunities and challenges for next-generation therapeutics.

Inactivation of the M2 form of pyruvate kinase (PKM2) in cancer cells is associated with increased tumorigenicity. To test the hypothesis that tumor growth may be inhibited through the PKM2 pathway, we generated a series of small-molecule PKM2 activators. The compounds exhibited low nanomolar activity in both biochemical and cell-based PKM2 activity assays. These compounds did not affect the growth of cancer cell lines under normal conditions in vitro, but strongly inhibited the proliferation of multiple lung cancer cell lines when serine was absent from the cell culture media. In addition, PKM2 activators inhibited the growth of an aggressive lung adenocarcinoma xenograft. These findings show that PKM2 activation by small molecules influences the growth of cancer cells in vitro and in vivo, and suggest that such compounds may augment cancer therapies.

The proto-oncogene proviral integration site for moloney murine leukemia virus (PIM) kinases (PIM-1, PIM-2, and PIM-3) are serine/threonine kinases that are involved in a number of signaling pathways important to cancer cells. PIM kinases act in downstream effector functions as inhibitors of apoptosis and as positive regulators of G1-S phase progression through the cell cycle. PIM kinases are upregulated in multiple cancer indications, including lymphoma, leukemia, multiple myeloma, and prostate, gastric, and head and neck cancers. Overexpression of one or more PIM family members in patient tumors frequently correlates with poor prognosis. The aim of this investigation was to evaluate PIM expression in low- and high-grade urothelial carcinoma and to assess the role PIM function in disease progression and their potential to serve as molecular targets for therapy. One hundred thirty-seven cases of urothelial carcinoma were included in this study of surgical biopsy and resection specimens. High levels of expression of all three PIM family members were observed in both noninvasive and invasive urothelial carcinomas. The second-generation PIM inhibitor, TP-3654, displays submicromolar activity in pharmacodynamic biomarker modulation, cell proliferation studies, and colony formation assays using the UM-UC-3 bladder cancer cell line. TP-3654 displays favorable human ether-à-go-go-related gene and cytochrome P450 inhibition profiles compared with the first-generation PIM inhibitor, SGI-1776, and exhibits oral bioavailability. In vivo xenograft studies using a bladder cancer cell line show that PIM kinase inhibition can reduce tumor growth, suggesting that PIM kinase inhibitors may be active in human urothelial carcinomas.

Stimulated inflammatory cells synthesize plateletactivating factor (PAF), but lysates of these cells show little enhancement in PAF synthase activity. We show that human neutrophils contain intracellular plasma PAF acetylhydrolase (PLA2G7), an enzyme normally secreted by monocytes. The esterase inhibitors methyl arachidonoylfluorophosphonate (MAFP), its linoleoyl homolog, and Pefabloc inhibit plasma PAF acetylhydrolase. All of these inhibitors induced PAF accumulation by quiescent neutrophils and monocytes that was equivalent to agonist stimulation. Agonist stimulation after esterase inhibition did not further increase PAF accumulation. PAF acetylhydrolase activity in intact neutrophils was reduced, but not abolished, by agonist stimulation. Erythrocytes, which do not participate in the acute inflammatory response, inexplicably express the type I PAF acetylhydrolase, whose only known substrate is PAF. Inhibition of this enzyme by MAFP caused PAF accumulation by erythrocytes, which was hemolytic in the absence of PAF acetylhydrolase activity. We propose that PAF is continuously synthesized by a nonselective acyltransferase activity(ies) found even in noninflammatory cells as a component of membrane remodeling, which is then selectively and continually degraded by intracellular PAF acetylhydrolase activity to modulate PAF production.-Chen, J., L. Yang, J. M. Foulks, A. S. Weyrich, G. K. Marathe, and T. M. McIntyre. Intracellular PAF catabolism by PAF acetylhydrolase counteracts continual PAF synthesis. J. Lipid Res.

Phospholipids containing sn-2 polyunsaturated fatty acyl residues are primary targets of oxidizing radicals, producing proapoptotic and membrane perturbing fragmented phospholipids. The only known phospholipases that specifically select these oxidized and/or short-chained phospholipids as substrates are mammalian group VII phospholipases A2s that were purified and cloned as PAF acetylhydrolases. Platelet-activating factor (PAF) is a short-chained phospholipid, and whether these enzymes actually are PAF hydrolases or evolved as oxidized phospholipid phospholipases is unknown. The fission yeast Schizosaccharomyces pombe, which does not form or use PAF as a signaling molecule, contains an open-reading frame potentially homologous to mammalian group VII phospholipase A2s. We cloned this SPBC106.11c locus and expressed it in distantly related Saccharomyces cerevisiae that lack homologous sequences. The S. pombe locus encoded a functional phospholipase A2, now renamed plg7+, that hydrolyzed PAF and a synthetic oxidized phospholipid. Expression of human type II PAF acetylhydrolase or S. pombe Plg7p enhanced the viability of S. cerevisiae subjected to oxidative stress. We conclude that a single-celled organism with an exceedingly spare genome still expresses an unusually discriminating phospholipase A2, and that selective hydrolysis of phospholipid oxidation products is an early, and critical, way to overcome oxidative membrane damage and oxidant-induced cell death.