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Leukemia Research 37 (2013) 1557– 1564

Contents lists available at ScienceDirect

Leukemia Research

j o ur nal ho me page: www.elsev ier .com/ locate / leukres

he E3 ubiquitin ligase TRAF2 can contribute to TNF-� resistance inLT3-ITD-positive AML cells

lf Schnetzkea, Mike Fischera, Bärbel Spies-Weissharta, Elisabeth Zirma,ndreas Hochhausa, Jörg P. Müllerb, Sebastian Scholl a,∗

Abteilung Hämatologie/Onkologie, Klinik für Innere Medizin II, Universitätsklinikum Jena, Jena, GermanyInstitut für Molekulare Zellbiologie, Centre for Molecular Biomedicine, Universitätsklinikum Jena, Jena, Germany

r t i c l e i n f o

rticle history:eceived 28 April 2013ccepted 5 August 2013vailable online 12 August 2013

a b s t r a c t

TNF-� has pleiotropic effects on cell survival and apoptosis. The E3 ubiquitin ligase TRAF2 plays a crucialrole for TNF-� mediated signaling since NF-�B activation by TNF-� is at least partially mediated by TRAF2.The objective of this study was to investigate whether TNF-� can induce apoptosis in FLT3-ITD-positiveAML cells and to elucidate the influence of TRAF2. Stable lentiviral mediated down-regulation of TRAF2

eywords:MLNF-�RAF2

resulted in a decrease of phosphorylation of the anti-apoptotic protein AKT and its downstream targetGSK-3�. Induction of apoptosis and impaired proliferation after TNF-� exposure were observed. Co-treatment of FLT3-ITD-positive cells with the specific FLT3 inhibitor AC220 was able to overcome TNF-�resistance. Taken together, we conclude that TRAF2 plays an important role in signal transduction andsurvival of AML cells.

LT3-ITD

C220

. Introduction

Acute myeloid leukemia (AML) represents a heterogenous dis-ase with respect to a broad spectrum of possible molecularberrations that can contribute to the pathogenesis of this sub-ype of leukemia [1,2]. Internal tandem duplications of the FMS-likeyrosine kinase receptors (FLT3-ITDs) can be observed in about 25%f patients with AML [3,4]. Presence of an FLT3-ITD is associatedith an unfavorable prognosis [5–7]. Furthermore, FLT3-ITD results

n cytokine independent activation of downstream signaling path-ays and especially the constitutive phosphorylation of STAT5,KT and the activation of the MAPK pathway, playing a piv-tal role in FLT3-ITD-mediated transformation [8,9]. In addition,t has been demonstrated that different putative pathways arenvolved in NF-�B activation in leukemic stem cells of AML [10,11].rosjean-Raillard et al. showed that FLT3-ITD mediates IKK� phos-horylation and subsequent NF-�B activation in AML cells [12].

Two decades ago, the existence of receptors activated by theumor necrosis factor alpha (TNF-�) has been demonstrated for

everal hematological diseases including AML cells [13,14]. Impor-antly, different effects of TNF-� can be observed in AML blasts. Onhe one hand, TNF-� mediated down-regulation of the granulocyte

∗ Corresponding author at: Abteilung Hämatologie/Onkologie, Klinik für Innereedizin II, Universitätsklinikum Jena, Erlanger Allee 101, 07740 Jena, Germany.

el.: +49 3641 9 324573; fax: +49 3641 9 324267.E-mail address: sebastian.scholl@med.uni-jena.de (S. Scholl).

145-2126/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.leukres.2013.08.004

© 2013 Elsevier Ltd. All rights reserved.

colony-stimulating factor (G-CSF) receptor in AML cells in a proteinkinase C-dependent manner [15]. On the other hand, synergisticeffects between TNF-� and the granulocyte-macrophage colony-stimulating factor (G-CSF) receptor concerning the proliferation ofprimary AML cells have been reported [16].

The cytokine TNF-� is able to activate a broad spectrum of intra-cellular signaling cascades that can either induce apoptosis (e.g.activation of caspase-8) or protect various cell types from pro-grammed cell death (e.g. NF-�B pathway activation) [17]. NF-�Bactivation is responsible for the expression of anti-apoptotic, butalso some proapoptotic genes are targeted by NF-�B including Fas-ligand or TRAIL, TNF-� and p53 [18]. Since NF-�B is at the crossroadsof life and death TNF-� mediated induction of apoptosis is depend-ent on the level of NF-�B activity. Therefore, most tumor cells areresistant to TNF-�-mediated cell death [19,20].

In the TNF-� induced TNF-receptor-1 (TNFR1) signaling path-way several molecules are responsible for NF-�B activation. Fur-thermore, these enzymes are indispensable for downstream activa-tion of important signaling pathways [21]. TNF receptor-associatedfactor 2 (TRAF2) is an E3 ubiquitin ligase which is critically involvedin the NF-�B activation by TNF-� [22]. In detail, TNF-� inducedactivation of TNFR1 causes the stimulation of TRAF2 which resultsin the initiation of the classical NF-�B pathway by recruitmentof the I�B kinase (IKK) complex. I�B proteins are phosphorylated

by the activated IKK complex and NF-�B dimers are released inthe cytoplasm which can now translocate into the nucleus [23].The binding on DNA regulates the expression of genes that areamongst others important for cell proliferation and survival [24].

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ince TRAF2 is significantly up-regulated in AML blasts, we hypoth-sized that TRAF2 might contribute to TNF-�-mediated resistancen AML cells [25]. Thus TRAF2 was downregulated in FLT3-ITD-ositive hematopoietic cells and TNF-� mediated effects on signalransduction, apoptosis and cell proliferation were characterized.

. Materials and methods

.1. Cell cultivation

The human AML-derived, FLT3-ITD-expressing cell lines, MV4-11 and MOLM-3, the FLT3-WT expressing human monoblastic cell line THP-1 as well ashe HEK293T packaging cell line were purchased from the German Collectionf Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). MV4-11,OLM-13, and THP-1 cells were grown in RPMI 1640 with 2% l-glutamine (Gibco

RL, Wiesbaden, Germany), 10% heat-inactivated fetal-bovine serum (FBS, GibcoRL), 1% penicillin/streptomycin in a 5% CO2 air environment and 37 ◦C fully humid-

fied incubator. The day before stimulation cells were set up at 5 × 105/ml in freshulture media. HEK293T cells were maintained in DMEM/F12 (Gibco, Karlsruhe,ermany) with 10% FBS.

.2. Reagents and antibodies

Anti-phospho-AKT (S473), anti-phospho-GSK-3� (S9), anti-phospho p70 S6T389), anti-phospho p38 (T180/Y182), anti-phospho-ERK1/2 (T202/Y204), anti-hospho NF-�B (S536), anti p-STAT5 (Y694) and anti-TRAF2 and were purchasedrom Cell Signaling (Beverly, MA, USA). Anti-GAPDH as well as horse-radish per-xidase (HRP)-conjugated secondary antibodies used for immunoblotting werebtained from Santa Cruz Biotechnology (Heidelberg, Germany). Oligonucleotidesere purchased from TIB Molbiol (Berlin, Germany). TNF-� was purchased from

mmunotools (Friesoythe, Germany), AC220 (quizartinib) from Selleckchem (Hous-on, TX, USA).

.3. Stable shRNA-mediated down regulation of TRAF2

Mission shRNA bacterial glycerol stocks encoding shRNAs targeting TRAF2TRCN0000004571) or non-targeting control shRNA were purchased fromigma–Aldrich (MISSION® shRNA lentivirus mediated transduction system,aufkirchen, Germany). Most efficient down-regulation of TRAF2 was obtainedy using pLKO.1-derivative plasmid encoding the shRNA TRAF2 5′-CCGGCCC-TGCAGATTCCACGCCATCTCGAGATGGCGTGGAATCTGCAAGGGTTTTT-3′ . For pro-uction of lentiviral particles, HEK293T cells were transfected with pLKO.1erivative plasmids in combination with pRev, pEnv-VSV-G, and pMDLg. Pseudo-yped particles were used to infect MV4-11 and MOLM-13 cells three times in theresence of 8 �g/ml polybrene. The transduced cell pools were selected with 2 �g/mluromycin 48 h post-transduction for 10 days and subjected to analysis.

.4. Immunoblotting

For short-term incubation cells were washed with serum-free medium andtarved for 2 h in serum-free medium prior to stimulation for the indicated times.ong-term exposure was under serum condition as indicated. Subsequently, cellysates were prepared and subjected to western blot analyses as described previ-usly [26]. Briefly, for whole-cell extracts, cells were rinsed twice with ice-cold PBSnd lysed in lysis buffer (150 mM NaCl, 20 mM Tris–HCl (pH 8.0), 1% nonidet P-40,0% glycerol, 1 mM EDTA). Shortly before lysis, protease (aprotinin, pepstatin, leu-eptin, and PMSF) and phosphatase inhibitors (sodium orthovanadate and sodiumuoride) were added. Protein concentration in supernatants was determined usinghe Bradford reagent (Sigma–Aldrich). Protein samples (40 �g) were separated onis-Tris 10% XT gels with XT-MOPS buffer and transferred to PVDF membrane (all

rom Biorad, München, Germany) before blocking for 1 h in 4% BSA. Blots wererobed with the indicated antibodies overnight at 4 ◦C. After the incubation withhe secondary antibody, the HRP-activity was quantified using enhanced chemi-uminescence reagent (Millipore, Darmstadt, Germany). Digital images were takensing the Fujifilm LAS-3000 (Fuji, Düsseldorf, Germany).

.5. RNA preparation and PCR analyses

Total RNA was routinely isolated from 5 to 106 cells using RNAeasyit (Qiagen, Hilden, Germany) according to standard protocol. First-strandDNA synthesis was performed with 1 �g of total RNA that were reverselyranscribed using 200 U of Superscript II reverse transcriptase (Invitrogen, Karl-ruhe, Germany). For qRT-PCR (quantitative real time reverse transcriptionCR), the following primer sets were used: human I�B�: forward primer 5′-

CTCTATCCATGGCTACCTG-3′ and reverse primer 5′-ACTTCAACAGGAGTGACACC-3′;uman TRAF2: forward primer 5′-CAGTTCGGCCTTCCCAGATAA-3′ and reverserimer 5′-TCGTGGCAGCTCTCGTATTCTT-3′; human RPL13A: forward primer 5′-GCTCATGAGGCTACGGAAA-3′ and reverse primer 5′-CTTGCTCCCAGCTTCCTATG-3′ .RT-qPCR based on SYBRGreen was performed using the LightCycler system 1.5

arch 37 (2013) 1557– 1564

(Roche Diagnostics, Mannheim, Germany). Data were analyzed using REST-software(relative expression software tool) (Qiagen).

2.6. Apoptosis assays

To monitor apoptosis, cells were stained with Annexin V-allophycocyanin-(APC)and propidium iodide (PI) and subsequently analyzed by flow cytometry using aFACSCalibur using Cell QuestTM software (Becton Dickinson, Heidelberg, Germany)according to the manufacturer’s protocol (BD Pharmingen, Heidelberg, Germany).

2.7. Determination of cell proliferation

Metabolic activity was evaluated using tetrazolium compound based CellTiter96® AQueous One Solution Cell Proliferation (MTS) assay (Promega, Madison, WI,USA) according to the manufacturer’s instructions. In brief, cells were seededin triplicate at 2 × 104 cells per well and exposed to increasing concentrationsof either cytarabine or daunorubicin. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxphenyl-2-(4-sulfophenyl)-2H-tetrazolium)) uptake was assayedafter 48 h. IC50 values were calculated by SigmaPlot (Systat Software, San Jose, CA,USA).

2.8. Cell cycle analysis

Cells were fixed at −20 ◦C for 30 min with 70% (v/v) ethanol. Cells were thenanalyzed using PI staining. Briefly, cells were fixed in 2% paraformaldehyde for30 min, washed twice with cold PBS and incubated for 30 min with PI/RNase solu-tion (0.1 mg/ml) (Becton Dickinson). Samples were analyzed on a FACSCalibur usingCell QuestTM software (Becton Dickinson, Heidelberg, Germany.)

2.9. Statistics

The Student’s t-test was used for statistical analyses and p values <0.05 wereconsidered statistically significant. Unless otherwise indicated, all experiments wereperformed in triplicates.

3. Results

3.1. Impact of TRAF2 down-regulation on TNF- ̨ induced NF-�Bsignaling

To investigate the impact of TRAF2 on TNF-� mediated signaling,TRAF2 was down-regulated by stable lentivirus-mediated shRNAexpression in MV4-11 and MOLM-13 cells. In MOLM-13 cells,down-regulation of TRAF2 resulted in a reduction of protein lev-els by 81% (±5%) whereas in MV4-11 cells the knockdown wasless efficient since reduction was only 53% (±3%) as evaluatedby semi-quantitative analysis (Fig. 1A). Congruently, a decreasedTRAF2 gene expression analyzed by quantitative real-time PCR wasdetected. The reduction of TRAF2 level in MOLM-13 cells was 65%(±4%) and 22% (±4%) in MV4-11 cells, respectively (Fig. 1B).

In order to confirm TNF-�-mediated signaling, gene expressionlevels of the NF-�B target gene I�B� was measured in response toTNF-� stimulation by qRT-PCR (Fig. 1C). Exposure of MOLM-13 cellsto TNF-� resulted in a 13.5-fold (±0.32) increase of I�B� mRNAexpression in the control and a 9.2-fold (±0.88) increase in theTRAF2 knockdown cell line. Thus, NF-�B activity after TNF-� stim-ulation was significantly impaired by down-regulation of TRAF2 inMOLM-13 cells compared to the control cell line (p < 0.05).

3.2. TRAF2 does not affect TNF- ̨ mediated survival and cellsignaling in MV4-11 cells

MV4-11 cells were that are known to be resistant to TNF-�induced apoptosis even at higher doses were stimulated for 48 hwith TNF-�. Knockdown of TRAF2 did not result in induction ofapoptosis in our cell system (Fig. 2A). To investigate FLT3-ITDdownstream signaling and a potential TRAF2-dependent impact,activation of signaling proteins in response to TNF-� was per-

formed using phospho-specific antibodies. Exposure to TNF-� wasaccomplished after 2 h of serum starvation under serum-free con-ditions. TNF-� stimulation caused an activation of pp38 correlatingwith maximum phosphorylation of AKT and GSK-3� after 5 min of

U. Schnetzke et al. / Leukemia Research 37 (2013) 1557– 1564 1559

Fig. 1. Characterization of TRAF2 knockdown in MV4-11 and MOLM-13 cells and induction of NF-�B pathway by TNF-�. (A) TRAF2 protein levels of MOLM-13 and MV4-11 cells expressing a TRAF2 shRNA (shTRAF2) or a nontargeting control shRNA (shCtrl) were assessed by immunoblotting. One representative western blot out of threei y quane l) for

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ndependent experiments is shown. (B) mRNA knockdown of TRAF2 was analyzed bxpression of I�B� in MOLM-13 cells. Cells were incubated with TNF-� (30 ng/metermined by qRT-PCR in relation to RPL13A as housekeeping gene. Results shown

timulation (Fig. 2B). The phosphorylation status was not differentn MV4-11 TRAF2 knockdown cells compared to the control celline. Stimulation with TNF-� did also result in AKT/mTOR activations seen by increased phosphorylation of p70S6 kinase. In addition,NF-� mediated activation of the MAPK1/2 (p42/44) pathway wasndependent of TRAF2 level. GAPDH was used as loading control forqual protein loading.

.3. Cytoprotective effect of TRAF2 in MOLM-13 cells

In order to investigate a potential functional role of TRAF2 after

NF-� stimulation, induction of apoptosis and cell proliferationn MOLM-13 cells was characterized. Compared to control cells

OLM-13 shTRAF2 cells showed a significant reduction in cell via-ility after 48 h treatment at TNF-� concentration of 5 ng/ml and

titative real-time PCR. RPL13A was used as housekeeping gene. (C) TNF-�-induced5 h. Subsequently, cells were harvested and the relative mRNA level of I�B� washe mean ± SD of three independent experiments. *p < 0.05.

higher (Fig. 3A). At 25 ng/ml TNF-� 83% (±2.5%) of the controland 54% (±6.1%) of the TRAF2 knockdown cell line were countedas viable cells. Concordantly, proliferation rate 48 h after TNF-� stimulation was significantly higher in MOLM-13 control cells(0.97 ± 0.04) compared to TRAF2 knockdown cells (0.58 ± 0.02)(Fig. 3B).

Next, we analyzed the effect of TRAF2 suppression on cell cycledistribution in MOLM-13 cells after TNF-� stimulation. A significanthigher proportion of proliferating cells (S + G2/M) was detected athigher doses of TNF-� (125 ng/ml) in the control cell line comparedto TRAF2 knockdown cells (32 ± 2.1% vs. 25 ± 1.4%) as demonstrated

in Fig. 3C. At lower doses of TNF-� (5 and 25 ng/ml), there was atendency of being more cells in a proliferative phase (S + G2/M) ofthe cell cycle in the control cell line as compared to TRAF2-deficientcells without being statistically significant.

1560 U. Schnetzke et al. / Leukemia Research 37 (2013) 1557– 1564

Fig. 2. Impact of TRAF2 knockdown on TNF-� induced apoptosis and FLT3-ITD downstream signaling in MV4-11 cells. (A) Cell viability in MV4-11 cells was assessed at 48 hafter treatment with TNF-� at the indicated concentrations by annexin V/PI binding using flow cytometry analysis. Graph demonstrates % of viable cells (low annexin V/PIb h TNFp ts.

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inding). (B) MV4-11 control and shTRAF2 cells were serum starved and treated witrotein loading. Results shown are the mean ± SD of three independent experimen

.4. Differential phosphorylation status of AKT and GSK-3ˇ inRAF2 deficient MOLM-13 cells

To analyze the role of TRAF2 for TNF-�-dependent signaling inn FLT3-ITD dependent cell-model, activation of AKT and GSK-3�as monitored using phospho-specific antibodies. Serum starvedOLM-13 cells were stimulated with 30 ng/ml TNF-� and samplesere taken 5, 120 min and 24 h post-stimulation.

A strong increase of p38 phosphorylation was observed 5 minfter TNF-� stimulation. Activation did not change in response toRAF2 depletion (Fig. 4). Since shRNA-mediated down-regulationf TRAF2 resulted in increased AKT and GSK-3� phosphorylationfter 5 min treatment, PI3K/AKT signaling appeared to be TRAF2ependent. In addition, increased S536 phosphorylation in NF-�B65 in response to TRAF2 suppression was observed. Differential

ffects on phosphorylation were time dependent. Whereas no dif-erences in phosphorylation status of AKT, GSK-3� and NF-�B p65ould be detected after 24 h incubation of TNF-� in presence of 10%CS in the control cell line, an obvious decrease occurred in the

-� (30 ng/ml) for the indicated time periods. GAPDH was used as a control for equal

TRAF2 knockdown cell line. GAPDH was used as loading control forequal protein loading.

3.5. Impact of FLT3-ITD inhibition on TRAF2 dependentTNF-˛-induced apoptosis

Since proliferative capacity of MOLM-13 and MV4-11 cellsdepends on the constitutive activity of FLT3-ITD, we wanted toexplore a potential role for FLT3-ITD in response to TNF-� inducedcell death. MOLM-13 and MV4-11 cells were exposed to the specificFLT3-ITD inhibitor AC220 only or in combination with TNF-�. Toelucidate a potential role for TRAF2 we exposed MOLM-13 shTRAF2cells that have been validated for an effective TRAF2 knockdownand the MOLM-13 control cell line to both TNF-� and AC220 atindicated concentrations (Fig. 5A). Exposure to AC220 resulted in

significant loss of viability irrespective of TRAF2. Cell viability after48 h was 51 ± 1.8% in shCtrl cells and 56% ± 1.3% in TRAF2 knock-down cells. When cells were exposed to both TNF-� and AC220apoptosis was significantly increased compared to either substance

U. Schnetzke et al. / Leukemia Research 37 (2013) 1557– 1564 1561

Fig. 3. Impact of TRAF2 on TNF-� induced apoptosis, proliferation and cell cylcein MOLM-13 cells. Dose-dependent effect of TNF-� on induction of apoptosis (A),proliferation (B) and cell cycle (C) was analyzed in MOLM-13 cells expressing TRAF2shRNA or a non-targeting control shRNA. Cells were incubated with TNF-� at indi-cated concentrations for 48 h. (A) Cell viability was measured using annexin V/PIstaining and subsequent flow cytometry. (B) Cell proliferation was assessed by MTSincorporation. (C) Cells were fixed and subsequently stained with propidium iodide.Cell cycle analysis was analyzed by flow cytometry. Results shown are the mean ± SDof three independent experiments. *p < 0.05.

Fig. 4. Differential AKT and GSK-3� signaling in TRAF2-deficient MOLM-13 cells.MOLM-13 cells expressing TRAF2 shRNA or a nontargeting control shRNA wereeither serum starved for 2 h and stimulated under serum free conditions or FCS(10%) treated as indicated and exposed to TNF-� (30 ng/ml) for the indicated time

periods. Cell lysates were subjected to Western blot analysis. Site-specific phos-phorylation of the indicated signaling proteins was analyzed immunologically. Asloading control, levels of GAPDH were detected.

alone but again irrespective of TRAF2. In shCtrl cells viability was7 ± 1.2%, in TRAF2 knockdown cells 6 ± 0.4%.

In MV4-11 cells TNF-� alone at 30 ng/ml had no significant effecton cell survival within 48 h (Fig. 5B). After single treatment withAC220 viability in MV4-11 cells remained at 90 ± 1.0% at 2 nM and82 ± 2.0% at 10 nM while co-treatment with 30 ng/ml TNF-� led toa significant reduction in viability (31 ± 0.9% at 2 nM AC220 and8 ± 0.8% at 10 nM AC220, respectively) (Fig. 5B). As a control cellline expressing wild type FLT3, THP-1 cells were used to excludeoff-target effects of the FLT3-ITD inhibitor. Viability of THP-1 cellsremained unchanged after 48 h stimulation with TNF-� or AC220.

Since TRAF2 expression had no influence on the combinatoryeffect of TNF-� and AC220 treatment we analyzed protein signalingat the FLT3-ITD homozygous MV4-11 cell line only. Incubation withTNF-� decreased the phosphorylation level of the transcription fac-tor STAT5 in MV4-11 cells (Fig. 5C). Combinatory treatment withAC220 caused further inhibition of STAT5 phosphorylation in adose dependent manner. Due to expression of the FLT3 wild typereceptor, no phosphorylation of STAT5 could be detected in THP-1cells. Enhanced p38 phosphorylation demonstrated effective TNF-� stimulation irrespective of the mutational status of FLT3. GSK-3�plays an important role in promoting cell survival. AC220 effec-tively abrogates phosphorylation of GSK-3� in FLT3-ITD positivecells and co-stimulation with TNF-� did not result in differentialactivity of GSK-3�.

4. Discussion

Outcome of AML patients requires further improvement. Chanceof survival of younger patients treated with intensive chemother-apy and in part with allogeneic stem cell transplantation is notsatisfying [27]. Therefore, identification of novel molecular targetsto impair transforming capacity of leukemic cells is important todevelop alternative therapeutic strategies to treat AML.

The activation of the NF-�B pathway has been demonstratedin various tumors including AML cells. In addition, NF-�B activa-tion can also be demonstrated in leukemic stem cells (LSCs) of AMLthat are of special relevance regarding the cure of this malignancy

1562 U. Schnetzke et al. / Leukemia Research 37 (2013) 1557– 1564

Fig. 5. Disruption of constitutive FLT3-ITD signaling leads to susceptibility of TNF-�-induced apoptosis. FLT3-ITD expressing MOLM-13, MV4-11 and wild type FLT3 expressingTHP-1 cells were exposed to the FLT3-ITD inhibitor AC220 and/or in combination with TNF-� at indicated concentrations. (A and B) Induction of apoptosis in MOLM-13s exin VG nd suR

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hCtrl, MOLM-13 shTRAF2, MV4-11 and THP-1 cells was measured after 48 h by annSK-3� were assayed by Western blotting. After 24 h cells were harvested, lysed aesults shown are the mean ± SD of three independent experiments.

11]. In AML cells, several signaling pathways contribute to NF-B activation (e.g. FLT3-ITD) and also the stimulation of receptorselonging to the innate immune system (e.g. TLR-4) leads to the up-egulation of NF-�B response genes in AML blasts [10,28]. Recentlye could demonstrate a functional role of the E3 ubiquitin ligase

RAF6 in FLT3-ITD-positive AML cells. In detail, stable knockdownf TRAF6 resulted in a pronounced activation of AKT after stimula-

ion of TLR-4 with lipopolysaccharide (LPS). Furthermore, we couldemonstrate a reduced susceptibility of FLT3-ITD-positive MV4-11ells to the treatment with cytostatic drugs after TRAF6 knockdown28].

/PI staining and subsequent flow cytometry. (C) Phosphorylation of STAT5, p38 andbjected to immunoblotting. GAPDH was used as control for equal protein loading.

Activation of the NF-�B pathway does also play a crucial roleafter stimulation of tumor cells with TNF-� and represents a keymechanisms of TNF-� mediated resistance due to anti-apoptoticeffects of NF-�B [29].

The E3 ubiquitin ligase TRAF2 is critically involved in TNF-� signaling and highly important for NF-�B activation afterstimulation of TNF receptors [30]. Jackson-Bernitsas et al. have

demonstrated in a HNSCC (head and neck: squamous cell carci-noma) cell model with transcriptionally active NF-�B activationthat dominant-negative (DN)-TRAF2 is able to inhibit the NF-�Bactivation [31]. Furthermore, the expression of TRAF2 is higher

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n AML blasts compared to normal hematopoietic progenitor cells25]. So far, there are no reports on the functional role of the E3biquitin ligase TRAF2 in AML cells. In order to study the role ofRAF2 in leukemic cells, we elucidated TNF-� signaling in responseo suppression of TRAF2 in AML cell models being characterizedy FLT3-ITD-mediated constitutional activation of distinct down-tream pathways.

Quantitative measurement of I�B� was identified as a powerfulethod for assessing the NF-�B status [32]. In order to analyze

NF-� induced NF-�B response and the impact of TRAF2 I�B� waseasured. A more than ten-fold induction of I�B� was detected

n the control cell line of MOLM-13 cells while the knockdown ofRAF2 significantly impaired the induction of I�B�. We thereforeypothesize a functional role of TRAF2 for TNF-� resistance in theLT3-ITD-driven MOLM-13 cell model.

To study the role of TNF-� we characterized signal transductionnd cell viability in AML cells. In consideration that TNF-� can acti-ate AKT in acute lymphoblastic leukemia cells, we hypothesizedhat the E3 ubiquitin ligase TRAF2 can mediate the activation ofKT which is already constitutively activated in the homozygousLT3-ITD-positive AML cells [33].

In MV4-11 cells, TNF-� did not affect cell viability and stablenockdown of TRAF2 did not influence sensitivity toward TNF-� inhese AML cells. The activity of TNF-� was demonstrated by a signif-cant increase of p38 phosphorylation after stimulation with TNF-�n the MV4-11 control cell line as well as in TRAF2 knockdown cells.n addition, a strong increase of AKT phosphorylation was observedn TNF-�-stimulated MV4-11 control cells which correlated withn increased phosphorylation of the AKT downstream target GSK-�. TRAF2 expression had only marginal effects on AKT activation

n MV4-11 cells. Thus, we conclude that additional signaling path-ays than constitutional activation of the anti-apoptotic proteinKT are responsible for TNF-� resistance in this FLT3-ITD-positiveML cell line.

In contrast to the observations in MV4-11 cells, we couldemonstrate a significant impact of TRAF2 in the heterozygousLT3-ITD positive MOLM-13 cell line. In detail, stable knockdownf TRAF2 was associated with a significant higher susceptibilityf MOLM-13 cells to TNF-� while only slight effects in MOLM-13ontrol cells could be observed. This is in line with earlier studiesemonstrating a potential antiapoptotic role for the protein TRAF234,35].

This thesis was supported by signaling studies of TNF-� treatedOLM-13 cells under standard cell culture conditions. In detail,

mpaired activation of the AKT/GSK-3� pathway in the presence ofNF-� could be observed in TRAF2 depleted MOLM-13 only. Thiss of particular interest since the NF-�B pathway and especially

TRAF2-containing signaling complex can be involved in severalross-talks with other downstream pathways, e.g. AKT or MAPK36].

Surprisingly, under serum-free conditions, stimulation withNF-� in MOLM-13 cells induced a rapid increase of phosphory-ated NF-�B phosphorylation irrespective of the expression levelf TRAF2. In TRAF2 knockdown cells, presence of FCS resulted in aigher basal phosphorylation of NF-�B while stimulation with TNF-

resulted in a decrease of activated NF-�B. In contrast, TNF-� hado effect on NF-�B phosphorylation in MOLM-13 control cells. TheKT dependent modulation of NF-�B phosphorylation and accom-anying increase of transcriptional activity of p65 might act as aediator for cell survival in presence of TNF-�.The differential response of MOLM-13 cells which are sensitive

o TNF-� induced cell death in a TRAF2 knockdown model and

V4-11 cells that are resistant remains speculative. Possible

xplanations might be the genetic background of the cell model orell type specific activated signaling pathways that allow MV4-11ells to resist TNF-� induced apoptosis in a TRAF2 knockdown

arch 37 (2013) 1557– 1564 1563

model whereas apoptosis is initiated in MOLM-13 cells. Alsothe knockdown efficacy of TRAF2 was less in MV4-11 cells thanin MOLM-13 cells which may contribute to the preservation ofresistance of MV4-11 cells to TNF-�.

Given the high incidence of activating FLT3 mutations and theobservation that FLT3-ITD promotes NF-�B activation of AML cells,we further addressed the question whether TNF-� resistance mightbe affected by inhibition of constitutively active FLT3-ITD. Thus,we compared the human AML cell line THP-1 expressing wildtype FLT3 with MOLM-13 and MV4-11 cells harboring FLT3-ITDhomozygous or heterozygous respectively. Inhibition of FLT3 wascarried out using the tyrosine kinase inhibitor AC220 (quizartinib)that has been characterized as one of the most specific and sensi-tive inhibitors of FLT3 [37]. Combinatory treatment of AC220 andTNF-� revealed a strong induction of apoptosis in both FLT3-ITDcell models irrespective of TRAF2 knockdown. Thus, a disruptionof the FLT3-ITD-induced activation of NF-�B and the inhibition ofother anti-apoptotic pathways by AC220 could overcome the strongresistance of TNF-�. Since this treatment did not alter cell viabilityof FLT3 wild type THP-1 cells, it can be concluded that FLT3-ITD-mediated transformation of AML cells appears to be one criticalmechanism of TNF-� resistance.

In consideration of very low TNF-� levels during sepsis, systemiceffects of TNF-� might be associated with severe and potentiallylimiting side effects [38]. Another therapeutic approach to stim-ulate AML blasts with TNF-� in a more specific manner could berepresented by leukemia-recognizing antibodies that are conju-gated with the cytokine allowing to be released near the AML cells.On the other hand, the development of inhibitors directed againstTRAFs is of special interest to overcome TNF-� resistance in AML.

Taken together, we present experimental data for a functionalrole of TRAF2 in FLT3-ITD-positive AML cells. To our knowledge,this reflects the first report demonstrating that TRAF2 is at leastin part responsible for TNF-� resistance in AML blasts. The chanceto overcome TNF-� resistance by concomitant inhibition of FLT3-ITD might represent a promising therapeutic approach that needsfurther pre-clinical evaluation (e.g. using primary AML cells).

Conflict of interest statement

A.H. receives research support from Novartis, BMS, Ariad andPfizer.

Acknowledgements

This work was supported by a grant from the DeutscheJosé Carreras Leukämie-Stiftung (Project 11/03) and by the IZKF(Interdisziplinäres Zentrum für Klinische Forschung), Universität-sklinikum Jena, Germany. U.S. and M.F. contributed equally to thiswork.

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