ORIGINAL ARTICLE Notch and NF-kB signaling pathways regulate miR-223/FBXW7 axis in T-cell acute lymphoblastic leukemia V Kumar 1,9 , R Palermo 2,9 , C Talora 1 , AF Campese 1 , S Checquolo 3 , D Bellavia 1 , L Tottone 1 , G Testa 1 , E Miele 2 , S Indraccolo 4 , A Amadori 4,5 , E Ferretti 6 , A Gulino 1,2,7 , A Vacca 6 and I Screpanti 1,2,8 Notch signaling deregulation is linked to the onset of several tumors including T-cell acute lymphoblastic leukemia (T-ALL). Deregulated microRNA (miRNA) expression is also associated with several cancers, including leukemias. However, the transcriptional regulators of miRNAs, as well as the relationships between Notch signaling and miRNA deregulation, are poorly understood. To identify miRNAs regulated by Notch pathway, we performed microarray-based miRNA profiling of several Notch-expressing T-ALL models. Among seven miRNAs, consistently regulated by overexpressing or silencing Notch3, we focused our attention on miR-223, whose putative promoter analysis revealed a conserved RBPjk binding site, which was nested to an NF-kB consensus. Luciferase and chromatin immunoprecipitation assays on the promoter region of miR-223 show that both Notch and NF-kB are novel coregulatory signals of miR-223 expression, being able to activate cooperatively the transcriptional activity of miR- 223 promoter. Notably, the Notch-mediated activation of miR-223 represses the tumor suppressor FBXW7 in T-ALL cell lines. Moreover, we observed the inverse correlation of miR-223 and FBXW7 expression in a panel of T-ALL patient-derived xenografts. Finally, we show that miR-223 inhibition prevents T-ALL resistance to g-secretase inhibitor (GSI) treatment, suggesting that miR-223 could be involved in GSI sensitivity and its inhibition may be exploited in target therapy protocols. Leukemia(2014) 28, 2324–2335; doi:10.1038/leu.2014.133 INTRODUCTION Notch proteins are a family of ligand-activated single-pass transmembrane heterodimeric receptors, involved in cell prolif- eration, differentiation and cell fate in different tissues. 1–3 Deregulated Notch signaling during T-cell development results in malignant transformation, leading to the development of T-cell acute lymphoblastic leukemia (T-ALL), which represents approximately 15% and 25% of ALLs seen in children and adults, respectively. 4–6 Misregulation of Notch signaling represents a prominent oncogenic pathway in T-ALL, as enforced Notch signaling, sustained by constitutive activation of Notch1 or Notch3, is a strong inducer of T-ALL in mouse models. 7,8 Moreover, more than 50% of human T-ALL patient samples show activating Notch1 mutations, 9,10 whereas overexpression of Notch3, irrespective of gross abnormalities in the Notch3 locus, is a common finding in human T-ALL. 11 Furthermore, increasing evidence reveals a key role of the cross-talk between Notch and NF-kB pathways in T-ALL development, 12,13 suggesting NF-kB signaling as one of the major mediators of Notch-induced oncogenic transformation. 14,15 In the past years, miRNAs are becoming increasingly appreciated for their ability to regulate a wide range of physiological and pathological processes including human leukemias. 16–19 However, although there has been considerable progress in the study of the miRNA biology and of their role in several human diseases including tumors, 20–22 the mechanisms and signaling pathways involved in the regulation of miRNA expression are still not fully understood. In the past few years, a growing number of works revealed Notch signaling pathway-related molecules as posttranscriptional targets of miRNAs. 23–28 Moreover, within the 17–92 cluster of miRNAs, which is highly expressed in hematopoietic cancer, miR-19 has been shown to enhance Notch1-induced T-ALL in vivo. 29 However, whether Notch may directly regulate miRNA network in leukemias has not been investigated as yet. To this end, we evaluated high- throughput profiling of miRNAs, which are regulated by the Notch signaling pathway and that cooperate to its oncogenic activity in T-ALL context. Here, we show that Notch signaling and NF-kB are able to increase miR-223 gene expression, which in turn downregulates the expression of the oncosuppressor FBXW7, known to regulate negatively Notch signaling, thus suggesting that the Notch/miR-223/FBXW7 axis may reinforce Notch signaling effect in T-ALL. Finally, we show that miR-223 inhibition prevents T-ALL resistance to g-secretase inhibitor (GSI) treatment, suggesting that miR-223 could be involved in the mechanism of GSI sensitivity and its inhibition may be exploited in target therapy protocols. MATERIALS AND METHODS Mice The generation and typing of transgenic intracellular domain (IC) of Notch3 (TgN3 IC ) mice have been described previously. 8 The studies involving animals have been conducted following the Italian National Guidelines for Animal Care established in Decree number 116 of 27 January 1992, in accord with the directive CEE 86/609. 1 Laboratory of Molecular Pathology, Department of Molecular Medicine, Sapienza University, Rome, Italy; 2 Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Rome, Italy; 3 Department of Medico-Surgical Sciences and Biotechnology, Sapienza University, Latina, Italy; 4 Istituto Oncologico Veneto-IRCCS-Padova, Padua, Italy; 5 Department of Surgery, Oncology and Gastroenterology, University of Padua, Padua, Italy; 6 Department of Experimental Medicine, Sapienza University, Rome, Italy; 7 Neuromed Institute, Pozzilli, Italy and 8 Institute Pasteur-Foundation Cenci Bolognetti, Sapienza University, Rome, Italy. Correspondence: Professor I Screpanti, Laboratory of Molecular Pathology, Department of Molecular Medicine, Sapienza University, viale Regina Elena 291, 00161 Rome, Italy. E-mail: isabella.screpanti@uniroma1.it 9 These authors contributed equally to this work. Received 23 October 2013; revised 28 April 2014; accepted 3 April 2014; accepted article preview online 14 April 2014; advance online publication, 13 May 2014 Leukemia (2014) 28, 2324–2335 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu