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...