ORIGINAL ARTICLE

Notch and NF-kB signaling pathways regulate miR-223/FBXW7axis in T-cell acute lymphoblastic leukemiaV Kumar1,9, R Palermo2,9, C Talora1, AF Campese1, S Checquolo3, D Bellavia1, L Tottone1, G Testa1, E Miele2, S Indraccolo4, A Amadori4,5,E Ferretti6, A Gulino1,2,7, A Vacca6 and I Screpanti1,2,8

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, thetranscriptional regulators of miRNAs, as well as the relationships between Notch signaling and miRNA deregulation, are poorlyunderstood. To identify miRNAs regulated by Notch pathway, we performed microarray-based miRNA profiling of severalNotch-expressing T-ALL models. Among seven miRNAs, consistently regulated by overexpressing or silencing Notch3, we focusedour attention on miR-223, whose putative promoter analysis revealed a conserved RBPjk binding site, which was nested to an NF-kBconsensus. Luciferase and chromatin immunoprecipitation assays on the promoter region of miR-223 show that both Notch andNF-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-223could be involved in GSI sensitivity and its inhibition may be exploited in target therapy protocols.

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INTRODUCTIONNotch proteins are a family of ligand-activated single-passtransmembrane heterodimeric receptors, involved in cell prolif-eration, differentiation and cell fate in different tissues.1–3

Deregulated Notch signaling during T-cell development resultsin malignant transformation, leading to the development of T-cellacute lymphoblastic leukemia (T-ALL), which representsapproximately 15% and 25% of ALLs seen in children andadults, respectively.4–6 Misregulation of Notch signaling representsa prominent oncogenic pathway in T-ALL, as enforced Notchsignaling, sustained by constitutive activation of Notch1 orNotch3, is a strong inducer of T-ALL in mouse models.7,8

Moreover, more than 50% of human T-ALL patient samplesshow activating Notch1 mutations,9,10 whereas overexpression ofNotch3, irrespective of gross abnormalities in the Notch3 locus, isa common finding in human T-ALL.11 Furthermore, increasingevidence reveals a key role of the cross-talk between Notch andNF-kB pathways in T-ALL development,12,13 suggesting NF-kBsignaling as one of the major mediators of Notch-inducedoncogenic transformation.14,15 In the past years, miRNAs arebecoming increasingly appreciated for their ability to regulate awide range of physiological and pathological processes includinghuman leukemias.16–19 However, although there has beenconsiderable progress in the study of the miRNA biology and oftheir role in several human diseases including tumors,20–22 themechanisms and signaling pathways involved in the regulation ofmiRNA expression are still not fully understood. In the past few

years, a growing number of works revealed Notch signalingpathway-related molecules as posttranscriptional targets ofmiRNAs.23–28 Moreover, within the 17–92 cluster of miRNAs, whichis highly expressed in hematopoietic cancer, miR-19 has beenshown to enhance Notch1-induced T-ALL in vivo.29 However,whether Notch may directly regulate miRNA network in leukemiashas not been investigated as yet. To this end, we evaluated high-throughput profiling of miRNAs, which are regulated by the Notchsignaling pathway and that cooperate to its oncogenic activity inT-ALL context. Here, we show that Notch signaling and NF-kBare able to increase miR-223 gene expression, which in turndownregulates the expression of the oncosuppressor FBXW7,known to regulate negatively Notch signaling, thus suggestingthat the Notch/miR-223/FBXW7 axis may reinforce Notch signalingeffect in T-ALL. Finally, we show that miR-223 inhibition preventsT-ALL resistance to g-secretase inhibitor (GSI) treatment, suggestingthat miR-223 could be involved in the mechanism of GSI sensitivityand its inhibition may be exploited in target therapy protocols.

MATERIALS AND METHODSMiceThe generation and typing of transgenic intracellular domain (IC) of Notch3(TgN3IC) mice have been described previously.8 The studies involvinganimals have been conducted following the Italian National Guidelines forAnimal Care established in Decree number 116 of 27 January 1992, inaccord with the directive CEE 86/609.

1Laboratory of Molecular Pathology, Department of Molecular Medicine, Sapienza University, Rome, Italy; 2Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia,Rome, Italy; 3Department of Medico-Surgical Sciences and Biotechnology, Sapienza University, Latina, Italy; 4Istituto Oncologico Veneto-IRCCS-Padova, Padua, Italy; 5Departmentof Surgery, Oncology and Gastroenterology, University of Padua, Padua, Italy; 6Department of Experimental Medicine, Sapienza University, Rome, Italy; 7Neuromed Institute,Pozzilli, Italy and 8Institute 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.it9These 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

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Cell sortingThymocyte suspensions from wild-type (wt) and TgN3IC mice were stainedwith anti-CD4-FITC and anti-CD8-PE antibodies as described before.30

CD4þCD8þ double-positive (DP) were isolated (purity level X95%) withthe FACSAria cell sorter (BD Biosciences, San Jose, CA, USA).

Cell lines and drug treatmentsJurkat, Jurkat IKKg� /� , DND41, Molt3 and TALL-1 cells were cultured inRPMI-1640 (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum.HEK 293T and M31 cells were maintained in Dulbecco’s modified Eagle’smedium (Gibco) containing 10% fetal bovine serum. Cells were treatedwith: 10 mM of GSI IX (DAPT) (565770; Calbiochem, Darmstadt, Germany), or50 ng/ml of 12-O-tetradecanoyl phorbol-13-acetate (P-8139; Sigma-Aldrich,St Louis, MO, USA) or 10 nM BAY-11-7082 (196871; Calbiochem).

RT-PCRRNA was extracted with TRIzol (Invitrogen, Carlsbad, CA, USA) as describedpreviously.31 Reverse transcription (RT) of total mRNA was performed withrandom primers and Superscript II (Invitrogen) according to the instructionmanual. Real-time PCR on coding genes was performed with TaqmanPrimers (Applied Biosystems, Foster City, CA, USA). The expression levels ofmiR-223 were determined by TaqMan real-time quantitative RT-PCR(qRT-PCR) using TaqMan MicroRNA Assay Kits (002295; Ambion, Austin,TX, USA), according to the manufacturer’s protocols. Data were analyzedby the DDCt method and b-actin or U6 small nuclear RNA (001973;Ambion) was used to normalize the expression levels of mRNA or miRNAs.The codec of the coding gene primers used (Applied Biosystems) arereported in Supplementary Table 1.

MiRNA profiling by Megaplex qRT-PCRMiRNAs were profiled using stem-loop RT-PCR-based 384-well Taqmanlow-density array (TLDA) cards (Applied Biosystems). Five hundrednanograms of total RNA was reverse transcribed, using multiplex-specificlooped miRNA primers for pools A and B (both human and mouse). Theproduct of the megaplex reaction was mixed with TaqMan Universal PCRMaster Mix, No AmpErase UNG (CAT. 4364341; Applied Biosystems) (2� ).Hundred microliters of the reaction mix were dispensed into each port ofTLDA and subjected to the microfluidic RT-PCR technology by 7900HTSystem (Applied Biosystems).

Data from TLDA runs of thymocytes and M31, Jurkat and Molt3 wereanalyzed by RQ Manager Software (Applied Biosystems). miRNAs havingthe Ct value 435 was deleted manually. Normalization of the Ct values ineach sample was carried out with U6 small nuclear RNA control: (deltaCt¼Ct(miR)�Ct (U6 small nuclear RNA)). The fold change for each miRNAprobe was calculated: fold change¼ 2^� ((meanDeltaCt (experimental)�meanDeltaCt (control)). The heat map was generated using SpotfireSoftware (TIBCO Software, Boston, MA, USA).

Statistical analysis of TLDA dataA comparative statistical analysis of RQ Manager data from wt andNotch3 transgenic mouse-derived thymocyte samples was made byRealTime StatMiner v3 software (Integromics SL, Granada, Spain), usingthe Mann–Whitney (Wilcoxon’s) test.

Transfections and plasmidsHEK 293T and M31 cells were transfected by Lipofectamine 2000 Kit(Invitrogen) according to the manufacturer’s instructions. Expressionvectors used were: Flag-N3IC,30 HA-N3IC,8 N1IC,32 RBPjk and MAM,33 p6534

and C/EBPa.35 Transient transfection of Jurkat cells, with siNotch3(sc-37135; SCBT, Santa Cruz B., Santa Cruz, CA, USA) and its control(AM4611; Ambion), was carried out with Amaxa-Nucleofection (Lonza,Walkersville, MD, USA). Jurkat and Molt3 cells were transfectedwith 100 nM of Control (CN-001000-01-05; Thermo Scientific) or Mimic(C-300580-07-0005; Thermo Scientific) of hsa-miR-223 or Control(IN-001005-01-05; Thermo Scientific) or anti-miR (IH-300580-0005; ThermoScientific) of hsa-miR-223, using NeonTransfection System (Invitrogen).

Western blot and antibodiesWhole-cell lysates were prepared in RIPA buffer. Immunoblottings wereperformed with antibodies against: Notch3 (2889; Cell Signalling, Beverly,MA, USA), c-Myc (SC-40; SCBT), Notch1Val1744 (no. 2421; Cell Signalling),

P-p65 (no. 3033; Cell Signalling), IkBa (no. 9242; Cell Signalling), cyclin E(sc-247; SCBT), FBXW7 (sc-33196; SCBT), C/EBPa (no. 2295; Cell Signalling),b-actin (sc-47778; SCBT), Notch1 (sc-6014-R; SCBT) and a-tubulin (sc-803;SCBT). Primary antibodies used for ChIP assays: NF-kB(p65) (17-10060;Millipore, Billerica, MA, USA), anti-RBPjk (ab25949; Abcam, Cambridge, MA,USA), Notch1 (no. 4147; Cell Signalling) and Notch3 (no. 2889; CellSignalling). To inhibit Notch1 activity, we added 10 mg/ml of blockinganti-human Notch1 (352104; BioLegend, San Diego, CA, USA) to the culturemedium for 48 h. Mouse IgG1 (401404; BioLegend) was used as an isotypecontrol.

MiR-223 promoter construct and luciferase assayThe fragment containing the putative miR-223 promoter region wasamplified by PCR from human genomic DNA using the primers: forward,50-CAAAGTCAACTACTTTCTTCTCCCTT-30 and reverse, 50-CCAGATGGAATTGGGCTTT-30 .

The PCR products were subcloned in Topo TA cloning vector(Invitrogen), and cleaved by NdeI and EcoRV and inserted upstream ofthe luciferase cDNA of pGL4-basic vector (Promega, Madison, WI, USA) togenerate the miR-223 promoter luciferase reporter. Luciferase and Renillaactivities were assayed with a Dual Luciferase Assay System (Promega).

Chromatin immunoprecipitationChromatin immunoprecipitation (ChIP) was performed as describedpreviously.13 The relative enrichment of the miR-223 promoter wasdetermined by subsequent SYBR green Q-PCR by using the followingprimers: miR-223-For (50-GGTATTCCAGGTTTCCCTCAA-30) and miR-223-Rev(50-TTCCAAGCTGCAGAGAGAGAG-30), and was normalized to GAPDH usingprimers: GAPDH-For (50-GCCACCCAGAAGACTGTGGAT-30) and GAPDH-Rev(50-TAGACGGCAGGTCAGGTCCAC-30). PCR reactions were run at 95 1C for10 min, followed by 40 cycles at 95 1C for 15 s and 60 1C for 30 s. Melting-curve analysis was performed at the end of each SYBR green Q-PCR run.Melting-curve temperature profile was generated through the cycle of95 1C for 1 min, 60 1C for 1 min and heating to 95 1C for 20 min.

Transduction of cells with lentiviral vectorsThe silencing of Notch3 in Molt3 cells was carried out with pLKO-puro-IPTG-1xLacO lentivirus particles expressing short hairpin RNA for humanNotch3 (TRCN0000020234; Sigma-Aldrich) according to the manufacturer’sinstruction. The infected cells were selected for 7 days with puromycin(2mg/ml) followed by the treatment with IPTG (isopropylthio-b-galacto-side) for induction of short hairpin RNA against Notch3. Validation ofNotch3 silencing was carried out by western blot or qRT-PCR.

Cell growth, cell viability and caspase activity assaysTo assess cell growth, trypan blue was used to count viable cells. Cellproliferation was monitored by 2-(4, 5-dimethyltriazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) (CGD-1; Sigma-Aldrich) according to theinstruction manual. Cleaved caspase-3 and -7 activity was determined byluminescence using the Caspase-Glo 3/7 assay from Promega.

RESULTSMicroarray profiling of Notch-regulated miRNAs in T-ALLTo characterize miRNAs that are regulated by Notch signaling inT-ALL, we carried out miRNA expression profiling of freshlyisolated CD4þCD8þ DP) thymocytes from 6-week-old wtand TgN3IC mice, a mouse model of T-ALL, overexpressingthe constitutively active N3IC, we established previously.8 Wefocused the profiling studies on sorted DP thymocytes, as we andothers previously demonstrated that accumulation of DP cells inthe thymus, spleen, lymph nodes and peripheral blood representsa pathognomonic feature of T-cell leukemias sustained byenforced expression of NotchIC in pre-T cells or in the bonemarrow of mice.7,8,33

In wt and TgN3IC mice, the thymocyte subset distribution issimilar with respect to CD4 and/or CD8 expression(Supplementary Figure 1A), and the overexpression of the N3IC

in the DP cells from the TgN3IC mice (Supplementary Figure 1B)correlates with the increased mRNA expression of known Notch

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target genes such as Hes136 and pTa37 when compared with wtlittermates (Supplementary Figure 1C). Total RNA was thenisolated and the expression of 768 miRNAs was analyzed byqRT-PCR using rodent TLDA. Relative miRNA expression wasnormalized against the endogenous control U6 using thecomparative DDCT method and calculated by using SDS RQmanager ver. 1.2 software (Applied Biosystems).

A comparative statistical analysis of wt and TgN3IC mouse-derived thymocytes, made by RealTime StatMiner v3 software(Integromics), revealed a significant upregulation, of up totwofold, of 38 miRNAs and a considerable downregulation of 2miRNAs (Supplementary Table 2). The unsupervised hierarchicalclustering analysis shows that the strongest significantly upregu-lated miRNAs, in TgN3IC when compared with the wt DPthymocytes, were miR-337-5p, miR-31 and miR-223, and the mostsignificantly downregulated miRNAs were miR-150 and miR-700(Figure 1). To confirm the pattern of regulated miRNAs in culturedcell contexts, we transiently transfected immature murine M31T cells,38 which we previously reported do not express Notch333

with an N3IC expression vector (Figure 2a) to simulate the Notchpathway activation. As shown in Figure 2b, the enforcedexpression of N3IC resulted in the increased expression of theNotch target genes Hes1 and c-Myc when compared with thecontrol vector-transfected cells. Total RNA from these samples was

analyzed for the expression of differentially regulated miRNAs byqRT-PCR using rodent TLDA. Amplification plots that did notamplify in all the samples, or/and had very high variation, or/andwhose expression was too low were excluded from the analysis bymanual inspection. Relative miRNA expression was normalizedagainst the endogenous control U6 using the comparative DDCTmethod and calculated by using SDS RQ manager ver. 1.2software. As shown in Supplementary Table 3, 92 miRNAs wereupregulated and 60 miRNAs were downregulated when comparedbetween N3IC- and pcDNA-transfected M31 T cells, as theirexpression levels varied up to twofold. The unsupervisedhierarchical clustering analysis showed that, in N3IC-transfectedM31 T cells when compared with the control vector-trans-fected cells, the strongest significantly upregulated miRNAs weremiR-223, miR-546 and miR-875-3p, and the most significantlydownregulated miRNAs were miR-679, miR-675-3p and miR-363(Figure 2c and Supplementary Table 3).

To further evaluate the mutual ability of Notch3 to regulatespecific miRNAs, Notch3 expression was also ectopically silencedin Notch1 mutation-bearing Molt3- and Notch1-unmutated Jurkathuman T-ALL cell lines, both constitutively expressing N3IC, asshown in Figures 3a and b, and known to express constitutivelyactive N1IC. The strength of Notch3 silencing was evaluated bywestern blot of N3IC (Figures 3a and b) and by RT-PCR of Notch

Figure 1. MiRNA expression profiling in DP thymocytes from TgN3IC mouse model of T-ALL heat map showing distinct miRNAs expressionprofiles in TgN3IC mice relative to wt counterpart. Each column represents one of three RNA samples and each row represents one miRNA.Expression levels are depicted according to the color scale at the bottom with increased expression ranging from green to red.

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target genes (Figures 3c and d). Notably, the levels of N1IC areunchanged in either cell line, whereas the status of Notch1activation, as revealed by the immunoreactivity to theanti-Notch1Val1744 antibody, is not affected in Jurkat cells andappears slightly decreased in Molt3 cells after Notch3 silencing(Figures 3a and 3b). The total RNA from the above samples wassubjected to miRNA profiling by qRT-PCR using human TLDA andthen analyzed manually as described previously for the M31T cells. The result revealed 36 miRNAs upregulated and 189miRNAs downregulated in Molt3 T cells (Supplementary Table 4)and 34 miRNAs upregulated and 130 miRNAs downregulated inJurkat T cells (Supplementary Table 5). In Figures 3e and f, the heatmaps show expression profiles of differentially regulated miRNAs.

Interestingly, according to Venn diagram analysis, there were 11miRNAs, which were commonly upregulated in N3IC-overexpres-sing mouse models (Figure 4a). Moreover, the intersection of themiRNA profile of Molt3 and Jurkat human cell lines silenced forNotch3 revealed 95 miRNAs, which were commonly down-regulated in both samples (Figure 4b). Finally, as shown inFigure 4c, only seven miRNAs were significantly modulatedin response to either N3IC overexpression or Notch3 silencing, inboth mouse and human cells.

To investigate the direct transcriptional regulation of miRNAs byNotch3, we performed in silico analysis of the putative regulatoryregions of the seven selected miRNAs, by Genomatix Maltinspectorsoftware (Genomatix Software GmbH, Munich, Germany). Werevealed a putative RBPjk consensus site evolutionarily conservedbetween human and mouse only in miR-223 and in miR-139-5p(Figure 4d). Between them, miR-223 appeared particularlyinteresting to us, as it was previously shown to be preferentiallyexpressed in primary hematopoietic tissue,16 and a cross-comparison of miRNA expression profiles in human T-ALLidentified miR-223 among the highest expressed miRNAs inT-ALL.39 Taken together, the above results allowed us to focus ourstudy on the relationship between Notch signaling and miR-223 inT-ALL.

Notch and NF-kB regulate miR-223-expressionThe locus of miR-223 maps on the X chromosome andis transcribed independently of any recognized gene.40,41

Interestingly, our analysis revealed a novel evolutionarilyconserved putative RBPjk and NF-kB overlapping binding sitebetween 280 and 299 bp upstream the transcription start site of

Figure 2. N3IC ectopic overexpression regulates the expression of distinct miRNAs in M31 T-cell line. (a) Western blot confirms the expressionof transfected N3IC in M31 cells. (b) RT-PCR shows increased mRNA expression of Notch target genes in N3IC-transfected M31 cells comparedwith the empty vector (pcDNA). Results are expressed as the means average deviations of three separate experiments and bars indicate s.d.*Po0.05, ***Po0.001. (c) Heat map shows genomic miRNA expression profiles of N3IC-transfected vs empty vector-transfected M31 cells. Eachcolumn represents one RNA sample and each row represents one miRNA. Expression levels are depicted according to the color scale at thebottom with increased expression ranging from green to red.

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the pri-miR (Figure 5a), in addition to the previously describedC/EBPa and PU.1 binding sites.40,41 To investigate the role ofNotch signaling in the transcriptional regulation of the miR-223,

we generated a luciferase reporter vector containing 690 bp ofthe human pri-miR-223 50 proximal genomic region (Figure 5a).The bona fide promoter region was confirmed by the increased

Figure 3. Notch3 silencing modulates the expression of distinct set of miRNAs in human T-ALL cell lines. Western blots show the efficiency ofNotch3 silencing (a) by the isopropylthio-b-galactoside (IPTG) induction in Molt3 transduced with an IPTG-inducible lentivirus expressingshort hairpin RNA against Notch3 and (b) by the transient transfection of siRNA against Notch3 in Jurkat T cells. Notch1 and activated Notch1(Notch1Val1744) expression in response to Notch3 silencing (a and b). RT-PCRs show downmodulation of Notch3 and Notch target genes Hes1and cMyc in Notch3-silenced (c) Molt3 and (d) Jurkat cells. Results are shown as the means average deviations of three separate experimentsand bars indicate s.d. *Po0.05; **Po0.01. Heat maps show differentially regulated miRNAs of Notch3-silenced (e) Molt3 and (f ) Jurkat cellswith respect to the control samples. Each column represents one RNA sample and each row represents one miRNA. Expression levels aredepicted according to the color scale at the bottom with increased expression ranging from green to red.

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luciferase activity obtained by transfecting HEK 293T cells with ahuman-C/EBPa expression vector (Figure 5b). As expected, giventhe presence of the nested RBPjk and NF-kB binding site, theectopic overexpression of either N3IC or N1IC or p65-NF-kBexpression vectors resulted in an increased activity of theluciferase reporter. Moreover, the co-transfection of N3IC or N1IC

with p65 displayed a synergic effect on the promoter activation(Figure 5b). Notably, the endogenous levels of miR-223 wereupregulated when N3IC or N1IC were transfected in M31 T cells(Figures 5c and d and Supplementary Figures 2A and B), whichneither expresses Notch1 nor Notch3 (Figure 5e). Moreover,Figure 5e shows that the transfection of either Notch1 or Notch3specifically induces only the increase of the respective protein,further demonstrating the positive effect of either activatedNotch1 and Notch3 on miR-223 expression.

To deepen the study of the relationship between Notch andNF-kB signaling pathways in the miR-223 transcriptional regulation,we directly analyzed the DNA binding of Notch3, Notch1, RBPjkand p65 to the miR-223 promoter region. For this purpose, weperformed an in vivo ChIP assay by using sonicated chromatinfrom wt Jurkat T cells and the IKKg-deficient Jurkat cells (JurkatIKKg� /� ), in which the stable deletion of IKKg prevents NF-kBactivation.42 The absence of NF-kB activation was confirmed bythe decreased phosphorylation of p65 and accumulation of IkBa inIKKg� /� when compared with wt Jurkat cells (Figure 5f).

Chromatin extracts were immunoprecipitated with either anti-Notch3, anti-Notch1, anti-RBPjk or anti-p65 antibodies andsubjected to RT-PCR amplification with specific primers amplifyingthe region containing the RBPjk/NF-kB nested binding site of

the miR-223 promoter. As shown in Figure 5g, in wt Jurkat cells allthe mentioned antibodies recovered the promoter fragmentcontaining the above-described binding site. Interestingly, nosignal was detected in anti-RBPjk, anti-Notch1 or anti-Notch3antibody-immunoprecipitated chromatin from Jurkat IKKg� /�

cells, although the levels of Notch1 and Notch3 proteins areincreased in Jurkat IKKg� /� when compared with Jurkat wt(Figure 5f), suggesting NF-kB activation as a key mediating eventfor the binding of Notch to this promoter region.

Consistently, Figure 5h and Supplementary Figure 2c clearlyshow decreased expression of miR-223 in Jurkat IKKg� /� cellswhen compared with the wt counterpart. Finally, the NF-kBpharmacological activation using 12-O-tetradecanoyl phorbol-13-acetate or inhibition using BAY-11-7082 resulted, respectively, inthe increased or decreased endogenous miR-223 expression inboth Jurkat and Molt3 cells (Figure 5i and SupplementaryFigure 2D).

Overall, our results confirm the close relationship betweenNF-kB signaling activity and miR-223 gene expression in T-ALLcontext and demonstrate that Notch and NF-kB are novelcoregulatory signals of miR-223.

Notch regulates T-ALL cell growth through miR-223-mediatedtargeting of FBXW7The recently identified ability of miR-223 to target the onco-suppressor FBXW7/hCdc4 (FBXW7)43 suggested that this axiscould mediate the effect of Notch signaling on T-ALL cellgrowth. Interestingly, we show that the enforced expression of

Figure 4. MiR-223 expression depends on Notch3 signaling in both human and mouse models of leukemia (a) The Venn diagram shows thenumber of miRNAs upregulated by Notch3 overexpression. The intersection gives a view of miRNAs that are induced in response to Notch3overexpression. (b) The Venn diagram shows the number of miRNAs downregulated upon Notch3 interference. The intersection gives a viewof miRNAs that are decreased in response to Notch3 silencing. (c) The Venn diagram shows the number of miRNAs modulated in response toeither Notch3 overexpression or silencing. The intersection gives a direct view of the miRNAs that are positively regulated by Notch3signaling. (d) Relative expression levels of the seven miRNAs regulated by Notch3 signaling in all the cell models analyzed and theirchromosomal localization both in human and in mouse genome. The last column indicates the presence (þ ) or the absence (� ) of acanonical RBPjk binding site in miRNAs putative promoter region.

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Figure 5. MiR-223 expression is regulated by Notch3 and NF-kB (a) Graphical representation of miR-223 promoter region. Conserved regionsare shown as green peak as retrieved from USCS genome browser. RBPjk and NF-kB binding site in conserved region are shown, respectively,in dark and light gray and their intersection shows nested region. The rectangle shows the schematic representation of the miR-223 promoterreporter construct. The putative consensus site for C/EBPa from � 39 to 48 bp and the nested binding site RPBjk/NF-kB from � 280 to 299 bpfrom the start of transcription, fixed as þ 1bp, are indicated by boxes (miR-223 promoter). (b) Luciferase assay performed on HEK 293T cellsafter the co-transfection with a luciferase reporter construct containing the human miR-223 promoter and N3IC, N1IC or NF-kB-p65 (p65)driving vectors, alone or in combination. (C/EBPa is used as positive control). RT-PCR shows the relative miR-223 expression in M31 T cellstransfected with (c) N3IC or (d) with N1IC vs control vector-transfected cells. (e) Notch1 and Notch3 expression in M31 T cells transfected withN3IC or with N1IC expression vectors. (f ) Western blot analysis of whole-cell extracts from Jurkat wt and Jurkat IKKg� /� cells probed with anti-Notch1Val1744, anti-Notch3, anti-phospho-p65, anti-IKKg or anti-IkBa antibodies. The b-actin expression was used as loading control.(g) Crosslinked protein–DNA complexes from Jurkat wt and Jurkat IKKg� /� T cells were subjected to immunoprecipitation with antibodiesagainst Notch3, Notch1, RBPjk or p65. The immunoprecipitated DNA samples were amplified by RT-PCR using miR-223 promoter-specificprimers. Fold enrichment of miR-223 promoter region, containing the RBPjk and NF-kB overlapping binding site, are normalized toglyceraldehyde 3-phosphate dehydrogenase (GAPDH) (negative control). (h) RT-PCR shows decreased expression of endogenous miR-223 inJurkat IKKg� /� cells compared with wt counterpart. (i) Expression levels of endogenous miR-223 in Jurkat and Molt3 cells treated with either12-O-tetradecanoyl phorbol-13-acetate TPA, BA-Y11-7082 (BAY-11) or with the vehicle alone were examined by real-time RT-PCR as detailed inthe Material and methods. All the results shown in the figures are expressed as the mean average deviations of three separate experimentsand bars indicate s.d. *Po0.05; **Po0.01, ***Po0.001.

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the mimic-miR-223 in Jurkat and Molt3 cells resulted inremarkable decrease of the FBXW7 gene expression, andconversely, the inhibition of miR-223 by antago-miR-223increased the FBXW7 expression (Figure 6a and SupplementaryFigure 3A). This inverse correlation was also supported by thegene expression analysis carried out on DP thymocytes from wt vsTgN3IC mice (Figure 6b and Supplementary Figure 3B) and in a

panel of previously described T-ALL patient-derived xenografts,44

all expressing the wt FBXW7 sequence (Figure 6c). Mostinterestingly, Notch3 silencing in Molt3 and Jurkat cell lines,which both display activated Notch1 (Figures 3a and b), preventedthe upregulation and downregulation of miR-223 and FBXW7,respectively (Figure 6b and Supplementary Figure 3B). Moreover,Notch1 inhibition decreased miR-223 and increased FBXW7

Figure 6. MiR-223 fine tunes leukemia cell growth by repressing FBXW7. (a) Relative gene expression modulation of the endogenous FBXW7in response to ectopic activation (Mimic) or inhibition (Anti-miR) of miR-223 in Jurkat (left panel) and in Molt3 (right panel) cell lines. (b) Therelative percent expression of miR-223 and FBXW7 was compared by RT-PCR in DP thymocytes from TgN3IC vs wt mice, in Jurkat, Molt3 andTALL-1 cells, in Notch3 silenced vs controls and in (c) nine samples of T-ALL patient-derived xenografts. (d) Protein expression levels of FBXW7and its downstream target proteins Notch1, c-Myc and cyclin E in Jurkat (left panel) and in Molt3 (right panel) cells transiently transfected witheither miR-223 (Mimic) or antago-miR (Anti-miR) and relative controls. Decreased cell growth of Jurkat and Molt3 48 h after transfection withantago-miR against miR-223 (Anti-miR) compared with control cells as shown by the cell counts by using trypan blue staining (e) and by MTTproliferation assay (f ). Change in cleaved caspase-3 and -7 activities as determined by luminescence in the Caspase-Glo 3/7 assay in Jurkat andMolt3 cells transfected for 48 h with antago-miR against miR-223 (Anti-miR) compared with control cells (g). The MTT and the Caspase-Glo 3/7assays results are expressed as fold change±s.d. relative to controls. All the results shown in the figures are expressed as the means averagedeviations of three separate experiments and bars indicate s.d. *Po0.05; **Po0.01, ***Po0.001.

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expression in Jurkat cells (Supplementary Figure 4), but not inMolt3 cells (data not shown). In addition, decreased miR-223 andincreased FBXW7 were observed in Notch3-silenced TALL-1 cells,which neither bear Notch1-activating mutations nor displayNotch1 activation45 (Figure 6b and Supplementary Figure 3B).Taken together, these results allow us to suggest that both Notch3and Notch1 are able to regulate the miR-223/FBXW7 axis, indifferent T-ALL cell lines.

Intriguingly, while the above results consistently show theinverse correlation between miR-223 and FBXW7 expression in allmouse and in vitro human models analyzed, we were insteadunable to point out a direct correlation between miR-223expression levels and the upregulation of Notch and NF-kB targetgenes in primary human T-ALL samples (Supplementary Figure 5).

Notably, several studies have revealed multiple targets of theFBXW7 ubiquitin ligase activity,46 such as Notch1,47 c-Myc48 andcyclin E,49 which are often found to be deregulated in severalhuman cancers. Consistently, transient transfection with themimic-miR-223 in both Jurkat and Molt3 cells resulted in thedecreased levels of FBXW7 and in the increased protein levels ofN1IC, c-Myc and cyclin E. Conversely, the inhibition of the miRNAcaused the increased expression of FBXW7 and the subsequentdecrease of target proteins (Figure 6d).

Notably, the transient inhibition of miR-223 by antago-miRinduced a significant growth inhibition of both Jurkat and Molt3cells (Figures 6e and 6f). Such a growth inhibition may besubsequent to apoptosis activation, as suggested by the increasedactivation of caspase-3 and -7 (Figure 6g).

MiR-223 confers resistance to GSIs in T-ALL cell linesThe results reported above suggest the involvement of Notch-mediated regulation of miR-223 in Jurkat and Molt3 cellproliferation through the inhibition of the oncosuppressor FBXW7,which in turn results in the increased stability of proproliferativeand oncogenic molecules. However, it was recently shown thatthe treatment with the Notch signaling inhibitor GSI-XXI increasesmiR-223 levels in several T-ALL cell lines, allowing the authors tosuggest that Notch signaling may inhibit miR-223 expression.50

To address this controversy, we treated several T-ALL cell lineswith the GSI IX (DAPT), known as a strong inhibitor of Notchsignaling. Surprisingly, while such a treatment increased theendogenous expression of miR-223 in both Molt3 and Jurkat,known as GSI-resistant T-ALL cell lines (Figure 7a andSupplementary Figure 7A), the GSI-sensitive DND41 cell linedisplayed decreased miR-223 expression upon DAPT treatment(Figure 7a). The mechanism of such a differential outcome hasbeen unveiled by the subsequent analysis of GSI treatment effects.As shown in Figure 7b and Supplementary Figures 6a, b and 7b, c,DAPT inhibited Notch signaling activation by blocking the NotchS3 cleavage, thus decreasing the expression of both Notch1 andNotch3 ICs in all the cell lines. However, at the same time itinduced the increased expression of the miR-223 transcriptionalactivator C/EBPa only in GSI-resistant cells (Figure 7b andSupplementary Figures 6A and 7B, C), leaving instead unchangedsuch an expression in GSI-sensitive cells (Figure 7b andSupplementary Figure 6B). Moreover, consistently with themiR-223 function, DAPT treatment resulted in the decrease ofFBXW7 protein levels in Molt3 and Jurkat, GSI-resistant T-ALL celllines (Figure 7b and Supplementary Figures 6A and 7B, C). On thecontrary, the same treatment did not affect FBXW7 expression inGSI-sensitive DND41 cells (Figure 7b and Supplementary Figure 6B).

This observation allowed us to hypothesize that resistance orsensitivity to GSI of different T-ALL cell lines could be linked to thedifferential regulation of miR-223 expression.

To confirm our hypothesis, we treated with DAPT or vehiclealone for 8 days Molt3 and DND41 cells, both bearingNotch1-activating mutations and wt FBXW7,45,51 transfected with

antago-miR-223 or mimic-miR-223, respectively, or scramblecontrols. Although DAPT treatment did not affect the growth ofscramble-transfected Molt3 cells (Figure 7c), a progressivedecrease in cell growth was observed by the fourth day oftreatment in Molt3 cells transfected with antago-miR (Figure 7d).This observation confirms that the GSI resistance in Molt3 cellsmay be mediated by miR-223.

The same results, even though with a delayed kinetic, were alsoobserved after DAPT treatment for 12 days of GSI-resistant Jurkatcells, bearing a wt Notch1 and a mutated FBXW7, transfected withscramble or antago-miR (Supplementary Figures 7D and E).

Finally, while DAPT treatment on scramble-transfected DND41cells resulted in decreased cell growth (Figure 7e), it did not affectthe growth of miR-223 mimic-transfected cells (Figure 7f), thussuggesting that miR-223 ectopic overexpression could revert GSIsensitivity of DND41 cells.

DISCUSSIONThe tumor-promoting role of Notch signaling in T-ALL was firstlyhypothesized by the observation of Notch1 translocations in T-cellleukemias.52 Subsequently, the enforced expression ofconstitutively active ICs of Notch1 and Notch3 has been shownto induce leukemia in mouse models.7,8 Finally, Notch1 gain-of-function mutations have been identified in more than 50% ofhuman T-ALL patients,9,53 whereas Notch3 overexpression has beenshown in most human T-ALL samples.11,44 Since then, increasingevidence has confirmed that dysregulated Notch signalingrepresents one of the major oncogenic signals in T-cellleukemogenesis,4–6,54 being also responsible for the activation ofa number of oncogenic pathways, such as NF-kB.4,12,55 However,neither all the molecular targets of Notch pathway nor their specificrole in T-ALL development have been fully elucidated.

In this work, by analyzing a mouse model of Notch3-inducedT-ALL8,11 and performing complementary gain-of-function andloss-of-function experiments in human and mouse T-cell lines, weidentified Notch signaling-regulated miRNAs. Out of sevenmiRNAs identified as putatively specific Notch3 targets, miR-223exhibited the most significant regulation accordingly to Notch3expression modulation. Furthermore, the presence of a conservedRBPjk and NF-kB overlapping binding site in its promoter, wepreviously have shown to be responsive to Notch3 in cooperationwith NF-kB,34 suggested this miRNA as a Notch- and NF-kB-regulated gene.

Consistent with our finding, a recent study identified miR-223among the most abundant miRNAs in T-ALL patients and in amouse model of Notch1-induced T-ALL.39 However, neither itsdirect link with Notch signaling deregulation nor the Notch-dependent molecular mechanisms that cause its deregulation inT-ALL have been clarified.

Here, for the first time, we demonstrate that Notch1, Notch3and p65 are directly recruited on the promoter region of miR-223and positively regulate its expression in T-ALL. Notably, the NF-kBinhibition in T-ALL cells results in decreased expression ofendogenous miR-223. Consistently, the abrogation of NF-kB path-way in Jurkat IKKg� /� , despite the preserved expression of highlevels of Notch1 and Notch3, leads to the downmodulation ofmiR-223 expression, as neither p65 nor RBPjk, or Notch1 or Notch3were recruited on its promoter region, demonstrating the existenceof a cooperative cross-talk between Notch and NF-kB in transcrip-tionally regulating the expression of miR-223. The unexpected lackof correlation observed between miR-223 levels and the upregula-tion of Notch and NF-kB target genes in T-ALL patient-derivedxenografts we analyzed may be caused by a higher complexity ofmiR-223 and different Notch and NF-kB target gene regulation inprimary tumor cells. In addition, we can hypothesize that a simplemodel Notch/NF-kB may be not able to drive both miR-223 and theselected target genes to the same extent.

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However, we show that the inhibition of miR-223expression/activity may be obtained with NF-kB inhibitorsor specific inhibition of Notch1 or Notch3. These data,together with previous43 and our present observations that

miR-223 silencing results in cell cycle arrest and apoptosisof T-ALL cells, further support the previously suggestedtarget therapy protocols combining Notch and NF-kBinhibitors.4,14

Figure 7. MiR-223 regulates GSI sensitivity of human T-ALL cell lines. (a) Relative expression of endogenous miR-223 in DAPT- vs dimethylsulfoxide (DMSO)-treated Molt3 and DND41 T-ALL cell lines. Results are expressed as the means average deviations of three separateexperiments and bars indicate s.d. **Po0.01. (b) Whole-cell extracts from DMSO- and DAPT-treated Molt3 and DND41 cells were subjected towestern blot by using antibodies against activated Notch1 (N1Val1744), Notch3, C/EBPa and FBXW7. b-Actin is used as a loading control. Growthcurves of Molt3 cells treated with DAPT or the vehicle alone and transfected with anti-miR-223-ctr (Scramble) (c) or with anti-miR-223 (d).Growth curves of DND41 cells treated with DAPT or the vehicle alone and transfected with mimic-miR223-ctr (Scramble) (e) or with mimic-miR-223 (f ). The relative cell number in percentage at each time point represents the mean value of triplicates normalized to the cell numberat day 8 of DMSO treatment. All the results shown in the figures are expressed as the mean average deviations of three separate experimentsand bars indicate s.d.

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While we were preparing this manuscript, two papers indicatedmiR-223 as a Tal-1 target gene in Tal-1-positive T-ALL cell lines.43,56

Interestingly, in one of them,43 it is shown that miR-223 is alsoexpressed in Tal-1-negative T-ALL cell lines, suggesting thatmiR-223 expression could be maintained by other pathways,known to be aberrantly activated in T-ALL development.Moreover, it is shown that Tal-1 regulates the FBXW7 tumorsuppressor through miR-223.

In keeping with this, our combinatorial computational analysisrevealed FBXW7 at the highest rank among the miR-223 targets.Here, we show that miR-223 regulates FBXW7 in both Jurkat andMolt3 T-ALL cell lines by directly inducing the decrease of itsmRNA and hence resulting in the downmodulation of FBXW7 atboth mRNA and protein levels. Moreover, we found an inversecorrelation between miR-223 and FBXW7 expression in a mousemodel of Notch3-dependent T-ALL, in human T-ALL cell linessilenced for Notch3 or Notch1 and, for the first time, in a panel ofprimary T-ALL patient-derived xenografts.

Finally, we show that the ectopic modulation of miR-223expression subverts the GSI sensitivity of GSI-resistant Molt3 andJurkat and GSI-sensitive DND41 T-ALL cell lines, possibly byregulating FBXW7. Moreover, our data suggest that the GSIresistance of Molt3 and Jurkat T-ALL cells is likely to reflect theGSI-induced increased C/EBPa expression, which leads to the lossof FBXW7 as an important consequence of miR-223 upregulation.

Notably, loss-of-function mutations of FBXW7 have alreadybeen suggested to be involved in GSI resistance of leukemiacells.45,57 However, such mutations are found only in about 15% ofT-ALL.58,59 Importantly, in the present work we analyzed primaryT-ALL patient-derived xenografts, all expressing the wt FBXW7sequence,44 in which we observed an inverse correlation betweenmiR-223 and FBXW7 expression. Additionally, we show thatspecific inhibition of miR-223 restores GSI sensitivity in GSI-resistant Molt3 cells carrying wt FBXW7. Therefore, upregulation ofFBXW7 through the specific inhibition of miR-223 could offer anattractive targeted therapy for GSI-resistant T-ALLs harboring wtFBXW7 and overexpressing miR-223.

CONFLICT OF INTERESTThe authors declare no conflict of interest.

ACKNOWLEDGEMENTSWe thank Dr Sonia Minuzzo for help in establishing T-ALL xenografts. This work wassupported by the Italian Association for Cancer Research (AIRC), the Italian Ministryof University and Research (MIUR), FIRB and PRIN Programs, the European Union(FP7-MC-ITN 215761-NotchIT).

AUTHOR CONTRIBUTIONSVK and RP designed and performed experiments, analyzed data and wrote thepaper; AFC, LT, EM and GT performed experiments; SC and DB analyzed data; SI,AA, CT, EF, AG, AV and IS designed experiments, analyzed data and wrote thepaper.

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Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)

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