5-lipoxygenase is a candidate target for therapeutic ...tumor and stem cell biology 5-lipoxygenase...

Tumor and Stem Cell Biology

5-Lipoxygenase Is a Candidate Target for TherapeuticManagement of Stem Cell–like Cells in Acute MyeloidLeukemia

Jessica Roos1, Claudia Oancea1, Maria Heinssmann1, Dilawar Khan1, Hannelore Held1, Astrid S. Kahnt2,Ricardo Capelo2, Estel la Buscat�o2, Ewgenij Proschak2, Elena Puccetti3, Dieter Steinhilber2, Ingrid Fleming4,Thorsten J. Maier2,5, and Martin Ruthardt1

AbstractNonsteroidal anti-inflammatory drugs such as sulindac inhibit Wnt signaling, which is critical to maintain

cancer stem cell–like cells (CSC), but they also suppress the activity of 5-lipoxygenase (5-LO) at clinically feasibleconcentrations. Recently, 5-LO was shown to be critical to maintain CSC in amodel of chronic myeloid leukemia.For these reasons, we hypothesized that 5-LO may offer a therapeutic target to improve the managementof acute myeloid leukemia (AML), an aggressive disease driven by CSCs. Pharmacologic and geneticapproaches were used to evaluate the effects of 5-LO blockade in a PML/RARa-positive model of AML.As CSC models, we used Sca-1þ/lin� murine hematopoietic stem and progenitor cells (HSPC), which wereretrovirally transduced with PML/RARa. We found that pharmacologic inhibition of 5-LO interfered stronglywith the aberrant stem cell capacity of PML/RARa-expressing HSPCs. Through small-molecule inhibitorstudies and genetic disruption of 5-LO, we also found that Wnt and CSC inhibition is mediated by theenzymatically inactive form of 5-LO, which hinders nuclear translocation of b-catenin. Overall, our findingsrevealed that 5-LO inhibitors also inhibit Wnt signaling, not due to the interruption of 5-LO–mediated lipidsignaling but rather due to the generation of a catalytically inactive form of 5-LO, which assumes a newfunction. Given the evidence that CSCs mediate AML relapse after remission, eradication of CSCs in thissetting by 5-LO inhibition may offer a new clinical approach for immediate evaluation in patients with AML.Cancer Res; 74(18); 5244–55. �2014 AACR.

IntroductionActually, one of the major clinical challenges in oncology is

to target the cancer stem cell–like cells (CSC). Current ther-apeutic strategies, regardless of conventional cytotoxic ornovel molecular approaches, often fail to eradicate cancercompletely because they are unable to efficiently target CSCs.

In this respect, a key signaling pathway for the maintenanceof CSCs in many cancers, including colorectal, breast, andpancreatic cancer, is the Wnt signaling pathway (1–4).

In acute and chronicmyeloid leukemias (AML and CML) therole of the Wnt signaling activation for the pathogenesis andthe maintenance of the CSCs is well understood (5–7). The factthat in leukemia inhibition of theWnt signaling directly targetsthe CSCs (leukemic stemcell, LSC)means that compounds andrelated signaling pathways that are able to inhibit Wnt sig-naling offer considerable potential within the setting of main-tenance therapy of leukemia. Deregulated activation of theWnt signaling leads to aberrant self-renewal of the LSCs inAML and CML and is fundamental to its maintenance (7–9).The activation of Wnt signaling in leukemia is mainly due toeither a direct deregulation of key effectors such as APC, Axin,b-catenin, g-catenin, or TCF/Lef by, i.e., mutations, or func-tional inhibition of their key regulators by leukemia-associatedfusion proteins (LAFP), including PML/RARa, AML-1/ETO,MLL/AF9, or BCR/ABL. These LAFPs are generated by chro-mosomal translocations such as t(15;17) (PML/RARa), t(8;21)(AML-1/ETO), t(9;11)(MLL/AF9), or t(9;22) (BCR/ABL; refs. 6,7, 10–12). The aberrant self-renewal of the LSCs depends on theaberrant activation ofWnt signaling by the LAFPs (6, 7, 10). Theoverexpression of g-catenin, one of the key players in Wntsignaling in HSPCs, leads to the induction of leukemia in vivo(12).

Nonsteroidal anti-inflammatory drugs (NSAID) developedas COX-1/2 inhibitors are efficient Wnt signaling inhibitors

1Department of Hematology, Goethe University, Frankfurt, Germany.2Department of Pharmaceutical Chemistry, Goethe University, Frankfurt,Germany. 3Institute for Molecular Biology and Tumor Research, PhilippsUniversity, Marburg, Germany. 4Centre of Molecular Medicine, GoetheUniversity, Frankfurt, Germany. 5Institute of Biomedicine, Pharmacology,Aarhus University, Aarhus C, Denmark.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

T.J. Maier and M. Ruthardt contributed equally to this article.

Corresponding Author: Martin Ruthardt, Labor f€ur Tumorstammzellbio-logie, Med. Klinik II/H€amatologie, Klinikum der Goethe Universit€at Frank-furt, Theodor Stern Kai 7, Frankfurt 60590, Germany. Phone: 4969-6301-5338; Fax: 4969-6301-6131; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-3012

�2014 American Association for Cancer Research.

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(13–15). Therefore, compounds such as sulindac, indometha-cin, or ibuprofen have been employed in therapeuticapproaches to stem cell therapy in models of AML and CML(6, 16). In fact, sulindac inhibits aberrant stem cell capacityinduced by PML/RARa or PLZF/RARa (X-RARa) in human aswell as in primary murine HSPCs. Sulindac targets bothb-catenin and g-catenin in X-RAR–expressing progenitor cells(16), whereas indomethacin is effective in the model of MLL/AF9-induced AML (6).However, for targeting Wnt signaling and stem cell activity

in AML or CML models, NSAID concentrations were requiredthat highly exceeded those sufficient for COX-1/2 inhibition(5, 6, 16). This calls into question whether NSAIDs target COX-1/2 to inhibit Wnt signaling in AML and CML or whetheranother player(s) is/are involved.Recently, it has been shown that NSAIDs, such as sulindac or

celecoxib, which are capable of inhibiting Wnt signaling, alsosuppress 5-LO activity. Interestingly, 5-LO–inhibitory concen-trations were similar to those for suppression of Wnt signalingand the aberrant self-renewal of LSCs (16, 17).Together with COX-1/2, 5-LO is a key enzyme in arachidonic

acid (AA) metabolism. 5-LO is crucial for the synthesis ofproinflammatory leukotrienes. Initial evidence for a role of5-LO in the regulation of LSC activity was provided by thefinding that 5-LO activity is indispensable for the induction ofa CML-like disease by BCR/ABL in vivo. Both genetic targetingof 5-LO in Alox�/� mice and pharmacologic targeting by aselective 5-LO inhibitor abolished the leukemogenic potentialof BCR/ABL. In thismodel, 5-LOhas been identified as a criticalregulator of LSCs in CML-like disease (18). In the present work,we investigated (i) whether effects of 5-LO targeting of LSCs inCML are extendible to the LSCs in AML, (ii) whether 5-LO isinvolved in the regulation of Wnt signaling, to better under-stand howNSAIDs inhibit bothWnt signaling and the aberrantself-renewal of LSCs in AML and CML; and (iii) whether andhow 5-LO represents a novel target for stem cell therapyapproach in AML.

Materials and MethodsPlasmidsThe retroviral vectors, PINCO- PML/RARa and PINCO-HA-

b-cateninS33A, as well as the expression vector, pCDNA3.1-5LO, have been described elsewhere (16). The TopFlash/Fop-Flash system and the Renilla luciferase pRL-TK construct(Promega) have also been described previously (19). ThepGL3-g-catenin construct and the pGL3basic and pRL-cyto-megalovirus (pRL-CMV) vectors were already described (12).

Cell lines and chemicals293T, NB4, and THP-1 cells were obtained from German

Resource Centre for Biological Material. 293T cells were cul-tured in DMEM (Gibco), supplemented with 10% fetal calfserum (FCS; Gibco). NB4, THP-1, and U937-PR9 cells weremaintained in RPMI medium supplemented with 10% FCS(Gibco). The identity of these cells was routinely controlled byimmunodetection (immunofluorescence or Western blotting)of PML/RARa (NB4 and U937-PR9) or the induction of 5-LOactivity (THP-1) and the response to all-trans retinoic acid–

induced granulocytic differentiation (NB4), respectively. CJ-13,610 was synthesized in-house (see SupplementaryMaterialsand Methods) and calcium ionophore A23187, arachidonicacid, zileuton, indomethacin, and diclofenac were purchasedfrom Sigma–Aldrich. The sEH inhibitor trans-4-[4-(3-adaman-tan-1-ylureido) cyclohexyloxy]-benzoic acid (t-AUCB) 1 waskindly provided by BruceHammock (University of California atDavis, Davis, CA).

Isolation of Sca1þ/lin� hematopoietic stem andprogenitor cells

Sca1þ/lin� HSPCs were isolated from either 8- to 12-week-old female C57BL/6N mice (Janvier) or male and femaleB6.129S2-Alox5tm1Fun/J (Jackson Laboratory) and B6.129X-Ephx2tm1Gonz/J mice (provided by Frank Gonzalez, NIH,Bethesda, MD) after killing by CO2 asphyxiation. The isolationof Sca1þ/lin� cells was performed as described previously (20).Two days after the isolation cells were retrovirally transfected.

Retroviral infectionEcotropic Phoenix packaging cells were transfected with

PINCO-PML/RARa or empty vector by calcium phosphateprecipitation according to widely established protocols. Ret-roviral supernatant was collected at days 2 and 3 after trans-fection. Infection of the target cells was performed as describedpreviously (12). The minimal accepted infection efficiency was70%, as assessed by the detection ofGFP-positive cells by FACS.Differences in the infection efficiency between samples did notexceed 10%.

Determination of 5-LO product formation in THP-1 andSca1þ/lin� cells

To induce 5-LO expression, differentiation of THP-1 ortransduced Sca1þ/lin� cells were either induced by the addi-tion of TGFb1 (1 ng/mL) and 1, 25-dihydroxyvitamin D3 (50nmol/L) or by addition of GM-/G-CSF (20 ng/mL, 60 ng/mL;Cell Concepts) for 4 days. Determination of 5-LO productformation was performed as previously described (21). ForSca1þ/lin� cells, leukotriene formation in the supernatant wasmeasured with a LTB4-ELISA, according to the manufacturer'sinstructions (Enzo Life Science). 5-LO product formations ofCJ-13,610-, zileuton-, and sulindac-treated THP-1 cells wereanalyzed by high-performance liquid chromatography (HPLC)as described previously (22). For indomethacin, leukotrienelevels in the supernatant were analyzed by liquid chromatog-raphy/ tandem mass spectrometry (LC/MS-MS) as describedpreviously (23). 5-LO product formation included 5-HETE forCJ-13,610 and sulindac and LTB4 for indomethacin.

ImmunohistochemistryThe spleens were embedded in paraffin blocks according to

a conventional tissue processing procedure. Immunohis-tochemistry was performed on 5 mm sections of each paraffinblock. The sections were deparaffinized with xylol (Sigma) andrehydrated through graded alcohol series (100%, 95%, 80%,50%, H2O). Antigen unmasking was achieved through heating(95�C) with 0.25 mmol/L EDTA buffer for 50 minutes. Theendogenous peroxidase activity was blocked using 3%

5-Lipoxygenase and Leukemic Stem Cells

www.aacrjournals.org Cancer Res; 74(18) September 15, 2014 5245




hydrogen peroxide (5minutes, room temperature), followed bypreincubation with antibody mix (TBS, 2% BSA, 2% normalgoat serum, 0.02% Tween 20) for 20 minutes at roomtemperature. The primary antibody (mouse monoclonalanti–b-catenin; BD Biosciences) and biotinylated isolectinB4 (dilution 1/25; Sigma) were diluted in the antibody mix.This antibody mixture was applied and samples were thenincubated for 2 hours at room temperature. After washing withTBS, the secondary antibody (DAKO Envision Dual-Link Sys-tem HRP; DAKO) was added and samples were further incu-bated for 30 minutes at room temperature. DAKO AEC-highsensitivity substrate chromogen was applied for 15 minutesand after intensewashingwithH2O, slideswere counterstainedwith hematoxylin (Sigma) for 15 seconds and washed underrunning H2O. Immunohistomount (Santa Cruz BiotechnologyInc.) was used for mounting.

Competitive repopulation assayThe competitive repopulation assay (CRA) was based on the

CD45.1-CD45.2 chimerism of C57BL/6N mice, as describedpreviously (24). CD45.2þ/Sca1þ/lin� were retrovirally trans-duced and cultivated in DMEM supplemented with 10% FCS(Gibco), mIL6,mSCF, andmIL3 upon exposure of either 0.001%DMSO, 0.3mmol/L, and 3mmol/L CJ-13,610 for 7 days. A total of1� 104 CD45.2þ/Sca1þ/lin� cells were injected into lethally (11Gy) irradiated CD45.1þ and CD45.2þ female recipients(B6DJLF1/JRj; Janvier) together with 5 � 105 normal Ly5.1þ

bone marrow (BM) cells. The proportion of CD45.2þ donorcell–derived hematopoietic cells was determined by FACSanalysis of cells from the peripheral blood or spleen. The cellswere stained with conjugated monoclonal antibodies specificfor CD45.1 and CD45.2 or unstained control (Miltenyi Biotec).

Transactivation assayspCDNA3 or pC3-5LO expression plasmids were either

cotransfected with pRT-LK, pGL3-OT, or pGL3-OF or pGL3-g-catenin, pGL3-basic, and pRT-CMV into U937-PR9 cells bynucleofection according to the manufacturer's instructions(Nucleofector Kit C, Lonza). Two hours later, transgene expres-sion was induced by the exposure of the cells to 100 mmol/LZnSO4 (Sigma) as described (25). Twenty-four hours after Zn2þ

treatment, luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega) accordingly andall assays were normalized to cotransfected Renilla activity.

ImmunofluorescenceCells were applied on positively charged coverslips (Menzel-

Gl€aser), washed with TBS (10 mmol/L Tris-HCl pH 8, 150mmol/L NaCl) and fixed in 4% paraformaldehyde (Applichem)for 30minutes followed by blocking and permeabilization with5% (w/v) nonfat dry milk (Roth) and 0.2% Triton X-100 (Roth)for 30 to 45 minutes. Cells were incubated with monoclonalanti–5-LO (BD Biosciences), polyclonal anti–b-catenin (cloneH102; Santa Cruz Biotechnology Inc.), and polyclonal anti-RARa (Santa Cruz Biotechnology Inc.) antibodies, respectively.After extensive washing in TBS, cells were stained with AlexaFluor 488-conjugated goat anti-mouse or Alexa Fluor 594-conjugated goat anti-rabbit Ig antibodies (Invitrogen). Nucleusstaining was obtained using TO-PRO 3 (Invitrogen). The cover-slips were mounted with Moviol (Sigma). Images wereacquired by a Leica TCS-SP5 confocal microscope (Leica)under identical conditions for pinhole opening, laser power,photomultiplier tension, and layer number. During data elab-oration by Fiji software (www.fiji.sc), identical parameterswere applied for all samples.

Statistical analysisStatistical significance was determined using one-way

ANOVA with Bonferroni posttest using GraphPad Prism 5.0(GraphPad).

Results5-LO is inhibited by NSAIDs and is expressed in normaland malignant hematopoietic stem cell compartments

Some NSAIDs, such as sulindac, indomethacin, or celecoxib,are able to inhibit Wnt signaling, but only at high concentra-tions, exceeding those for the suppression of COX-1/2 (Table 1;refs. 6, 7, 13–16, 26–29). It has recently been shown thatsulindac sulfide (activemetabolite of sulindac) is able to inhibit5-LO at clinically relevant concentrations (17). Therefore, weinvestigated whether other NSAIDs such as indomethacin areable to inhibit 5-LO. Monocytic THP-1 cells, known to express

Table 1. COX-1/2 IC50 values of NSAIDs used as Wnt signaling inhibitors

NSAIDSCOX-1 IC50

valuesa (mmol/L)COX-2 IC50

valuesa (mmol/L)

Concentrationsused for Wntsignaling

inhibition (mmol/L)b Literature

Sulindac sulfidec 1.02 10.43 50–200 (13, 15, 16, 26)Indomethacin 0.16 0.46 40–600 (5, 6, 14, 15, 26, 28)Diclofenac 0.14 0.05 >100 (15, 26)Ibuprofen 4.75 >30 1,000 (26, 28)Celecoxib 21.9 0.2 60–100 (27, 29)

aIn human whole-blood assay (endotoxin-induced PGE2 synthesis).bIn different cell systems (see literature).cActive metabolite of sulindac.

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Cancer Res; 74(18) September 15, 2014 Cancer Research5246




5-LO and to have inducible 5-LO activity (30), were preincu-bated with inhibitors. 5-LO product formation was induced byexposure to Ca2þ ionophore A23187 (21). CJ-13,610, a selective5-LO inhibitor, was used as a positive control. We found thatindomethacin was able to suppress 5-LO product formation ina concentration-dependent manner (Fig. 1A). Indomethacinsuppressed 5-LO activity at higher concentration as neededfor COX inhibition. (Fig. 1A). Noteworthy for both inhibitors,indomethacin and sulindac sulfide, the 5-LO–inhibitory con-centrations were similar to those required for suppressionof Wnt signaling and the aberrant self-renewal of LSCs(16, 17). Therefore, we investigated the subpopulation of cellsthat should be targeted in a "stem cell therapy" approach.We studied 5-LO product formation in a highly enrichedSca1þ/lin� population to disclosewhether 5-LO is expressed inHSPCs. Differentiation was induced by GM/G-CSF. 5-LO activ-ity was assessed by the formation of LTB4, by ELISA. Here, weshow for the first time that the HSPC compartment not onlyexpressed 5-LO, but also exhibited 5-LO enzyme activity, whichwas only slightly induced by the GM-CSF–induced differenti-ation process (Fig. 1B). These findings were confirmed byWestern blot analysis of Sca1þ/lin� HSPCs (data not shown).

Targeting 5-LO by selective inhibitors reduces the stemcell capacity of PML/RARa-positive HSPCsBecause NSAID concentrations that are able to inhibit

aberrant LSC capacity lie within the range needed to suppress5-LO, we speculated whether the effects of the NSAIDs on LSCsare mediated by inhibition of 5-LO. Therefore, we studied theeffects of CJ-13,610, a potent and selective inhibitor of 5-LO, onLSC capacity.The effects of 5-LO inhibition on aberrant LSC capacity were

addressed in serial replating efficiency assays in PML/RARa-

positive Sca1þ/lin� HSPCs. Serial replating revealed the com-bined effect of PML/RARa onproliferation, differentiation, andself-renewal of HSPCs (12, 31). Therefore, we retrovirallyexpressed PML/RARa in Sca1þ/lin�, murineHSPCs and platedthem in semisolid medium supplemented with mIL3, mIL6,and mSCF in the presence/absence of 0.3 and 3 mmol/L of CJ-13,610 (Fig. 2A). Empty vector–transduced cells were used ascontrols. Serial replatings were performed as graphicallydescribed in Fig. 2A.

CJ-13,610 abolished the aberrant serial replating capacityof PML/RARa-positive HSPCs at a concentration of 0.3mmol/L, starting from the second plating (Fig. 2B). An effectof the treatment was already seen at the first plating, asrevealed by the modifications in the morphology of thecolonies in the presence of 3 mmol/L CJ-13,610 (Fig. 2C).The rare colonies in the controls also disappeared upontreatment (Fig. 2A).

In addition to the 5-LO and COX pathway, there is also athird arachidonic acid pathway, CYP (cytochrome P450)/sEH(soluble epoxide hydrolase) pathway, known to be involved inWnt signaling, proliferation, and mobilization of HSC (32). Toinvestigate the role of CYP/sEH in the effects of stem cellsuppression and to confirm that only 5-LO inhibition isresponsible for the observed effects, we extended the treatmentof PML/RARa-positive Sca1þ/lin� cells, to sulindac sulfide,in COX-inhibitory concentrations, as well as to tAUCB,an sEH inhibitor (Supplementary Fig. S1; ref. 33). In fact,treatment with neither sulindac sulfide or tAUCB alone norin combination was able to suppress the replating efficiency ofPML/RARa. Only the presence of CJ-13,610 led to a completeinhibition of the aberrant replating efficiency of PML/RARa(Supplementary Fig. S1). Also, the genetic inhibition of sEH,using murine Sca1þ/lin� cells with a sEH�/� background,

Figure 1. Effects of NSAIDs on 5-LO product formation and expression of 5-LO in PML/RARa-expressing murine HSCs. A, effect of NSAIDs on 5-LO productformation in THP-1 leucocytes. Cells were preincubated with CJ-13,610, sulindac sulfide (pharmacologic active form of sulindac), or indomethacin for15 minutes. 5-LO product formation was triggered by addition of 5 mmol/L A23187 (Ca2þ -ionophore) and 10 mmol/L AA. Downstream 5-LO products [LTB4,5-HETE (5(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid)] were analyzed with reverse HPLC (CJ-13,610, sulindac sulfide) and LC/MS-MS(indomethacin). Data are given as means � SEM versus vehicle control (100%) of four (CJ-13,610, sulindac sulfide) and two (indomethacin) independentexperiments. B, Sca1þ/lin� HSCs were transduced with PML/RAR. Empty vector–transduced cells were used as controls. Cells were treated with� GM/G-CSF to induce granulocytic/monocytic differentiation. After 4 days in culture, cells were stimulated with 2.5 mmol/L A23187 and 20 mmol/L AA for5-LO activation. Leukotriene formation was measured with LTB4-ELISA. Data are shown from a single representative experiment of two independentexperiments performed, which yielded similar results.






showed no effect on the replating efficiency of PML/RARa-expressing HSPCs (Supplementary Fig. S2).

In summary, these data indicate that the pharmacologicinhibition of 5-LO is sufficient to interfere with the aberrantreplating capacity of PML/RARa-positive HSPCs.

The abolition of the stem cell capacity of PML/RARa-positive HSPCs by CJ-13,610 is accompanied by theinhibition of Wnt signaling

CJ-13,610 not only inhibited serial replating capacity ofPML/RARa-positive but also inhibited colony formation ofempty vector–transduced HSPCs. Thus, we investigatedwhether the inhibition of 5-LO generally suppresses stemcells or only LSCs. First, we studied the effects of CJ-13,610on short-term hematopoietic stem cells (ST-HSC) and earlyimmature progenitors in a colony-forming unit spleen day 12(CFU-S12) assay (34). Therefore, Sca1þ/lin� HSPCs wereretrovirally transduced with PML/RARa or with emptyvector and exposed in liquid culture to 0.3 or 3 mmol/LCJ-13,610. After 7 days, cells were counted, harvested, andinoculated into lethally irradiated mice (Fig. 3A). On day 12,the mice were sacrificed, spleens were fixed, and the spleencolonies were counted. As reported in Fig. 3B, the exposureto CJ-13,610 led to a concentration-dependent and signifi-

cant reduction of the number of spleen colonies expressingPML/RARa. The exposure to 3 mmol/L CJ-13,610 almostcompletely abolished these colonies (Fig. 3B and C). Inter-estingly, 3 mmol/L CJ-13,610 allowed the formation of a fewcolonies in the controls, indicating that CJ-13,610 did notexert stem cell toxicity (Fig. 3B).

To determine which stem cell compartment is targetedby CJ-13,610, we studied its effects on the frequency ofshort-term (ST) and long-term (LT) HSC in a CRA. In thisassay, the ST-HSC population was detected at 12 weeks (Fig.3F, left) and the LT-HSC population after 6 months (Fig. 3F,right). Therefore, Sca1þ/lin� HSPCs were retrovirally trans-duced with PML/RARa or with empty vector and exposed inliquid culture to 0.3 or 3 mmol/L CJ-13,610. After 7 days, cellswere counted, harvested, and inoculated into lethally irra-diated mice (Fig. 3E). As shown in Fig. 3F, exposure to CJ-13,610 led to a reduction of both ST- and LT-HSC, asrevealed by the percentage of CD45.2þ cells, with respectto untreated controls at 12 weeks and 6 months, respec-tively (Fig. 3F). Treatment of control cells led to a higherfrequency of ST- and LT-HSCs, confirming that targeting of5-LO is not stem cell toxic. Very similar results wereobtained using another selective 5-LO inhibitor, zileuton(Supplementary Fig. S3).

Figure 2. Effect of CJ-13,610 onreplating efficiency of PML/RARa-expressing murine HSCs.A, experimental strategy forstudying the influence ofCJ-13,610 on the biology ofPML/RARa-expressing murineHSCs. B, serial replating(I–V) of empty vector control andPML/RARa-positive cells treatedwith 0.3 mmol/L, 3 mmol/LCJ-13,610, or vehicle control(DMSO). C, colony morphology ofplatings I–V. Data are shown from asingle representative experiment ofthree independent experimentsperformed, which yielded similarresults.

Roos et al.





Interestingly, the effects of CJ-13,610 were not related to theinduction of apoptosis, excluding a cytotoxic effect of the 5-LOinhibitor (data not shown).The effects of NSAIDs on cancer stem cells are mainly

attributed to an inhibitory activity on b-catenin and the Wntsignaling. Therefore, we investigated whether suppression ofthe PML/RARa-induced aberrant stem cell capacity by CJ-13,610 was related to an inhibitory effect on b-catenin andWntsignaling. Thus, we performed immunohistochemical stainingof the spleen colonies induced by PML/RARa, using an anti-body raised against human b-catenin (Fig. 3D). To confirm thespecificity of the b-catenin staining, we used an unstainedcontrol, only incubated with the secondary antibody. PML/RARa activation ofWnt signalingwas reversed byCJ-13,610. Asshown in Fig. 3D, spleen colonies derived from PML/RARa-

positive HSPCs exposed to solvent exhibited a strongb-cateninsignal (red staining) in comparison with controls and coloniesderived from PML/RARa-positive HSPCs treated with CJ-13,610. To confirm these data, we performed real-time quan-titative PCR analysis of the expression of the direct Wnt targetgenes Axin 2 and Cyclin D1 in the PML/RARa-positive cell lineNB4 after treatment with 3 mmol/L CJ-13,610. As a control forWnt signaling inhibition, we used indomethacin. We couldobserve that treatment with CJ-13,610 led clearly to a reductionof mRNA expression levels of Axin 2 as well as cyclin D1(Supplementary Fig. S4).

Taken together, these data demonstrate a selective effect ofCJ-13,610 on the LSC compartment, accompanied by theinhibition of Wnt signaling without toxicity to the normalhematopoietic stem cell compartment.

Figure 3. Effect of CJ-13,610 on stem cell capacity in PML/RARa-positive HSPCs. A, experimental strategy for studying the influence of CJ-13,610 on short-term stem cell capacity of PML/RARa-expressingmurine HSPCs (CFU-S12). CFU-S12 colony numbers (B) and spleen (C)morphology from cells treatedwith0.3 mmol/L and 3 mmol/L CJ-13,610 (3 mice/group). Statistical analysis was performed by one-way ANOVA with the Bonferroni posttest (��, P < 0.001). Dataare shown from a single representative experiment of three independent experiments performed, which yielded similar results. D, immunohistochemicalstaining of b-catenin in CFU-S12 spleen slices. Empty vector control and PML/RARa-expressing spleens of a CFU-S12 experiment in which the Sca1þ/lin�

HSPCs were retrovirally transduced and treated with 3 mmol/L CJ-13,610. Slides were stained with either an anti–b-catenin and/or only secondary antibody(unstained). E, experimental strategy for studying the influence of CJ-13,610 on ST- and LT-stem cell capacity of PML/RARa-expressing murine HSCs(CRA). F, CRA. Sca1þ/lin� BM cells from CD45.2 mice were retrovirally infected with empty vector control or PML/RARa. Cells were treated with 3 mmol/LCJ-13,610. After 7 days in liquid culture, cells were cotransplanted with CD45.1 BM cells into lethally (11Gy) irradiated CD45.1þ2 recipient mice(4–5 mice/group). An analysis of donor chimerism was performed at 12 weeks and 6 months after transplantation. The plots show a representativedonor-derived chimerism in (left) peripheral blood after 12 weeks and (right) spleen after 6 months from an individual mouse.






Loss of 5-LO expression does not recapitulate theinhibitory effects of the inhibitors on the leukemicphenotype by PML/RARa

To further investigate the role of 5-LO in themaintenance ofnormal and leukemic stem cells, we investigated the effect oftargeted genetic inhibition of 5-LO on leukemogenic potentialand the aberrant stem cell capacity induced by PML/RARa inAlox5�/� mice (35). PML/RARa was retrovirally expressed inSca1þ/lin�, Alox5�/� and Alox5þ/þ HSPCs, respectively. First,we performed serial replating assays. As shown in Fig. 4A, theloss of 5-LO expression not only failed to inhibit the replatingefficiency of PML/RARa-expressing HSPCs, but also led toincreased proliferation of colony-forming cells, as revealed byan increased number of colonies in comparison with Alox5þ/þ

controls expressing PML/RARa (Fig. 4 A and B).Next, we tested the LT-HSC capacity of Alox5�/� and

Alox5þ/þ HSPCs expressing PML/RARa in a CRA. Alox5�/�

and Alox5þ/þ HSPCs did not show significant differencesin the frequency of the PML/RARa-positive LT stem cellpopulation (CD45.1þ), as revealed 9 months after transplan-tation into lethally irradiated recipient mice (CD45.1 and2 population; Fig. 4C).

Taken together, these data show that loss of 5-LO expressiondoes not interferewith the replating efficiency and LT stem cellcapacity of PML/RARa-positive HSPCs. The data strongly

suggest that stem cell inhibition may be mediated by theinhibited/inactive form of 5-LO, indicating a pathophysiologi-cally relevant difference between enzymatically active andinactive 5-LO.

Coexpression of catalytically inactive 5-LO inhibits thePML/RARa-induced activation of Wnt signaling inhuman leukemic U937 cells

Assuming that the catalytically inactive form of 5-LO isresponsible for stem cell inactivation, it should, like theNSAIDs, also inhibit Wnt signaling. Therefore, we investigatedactivation of Wnt signaling in a cellular model in the absenceand presence of 5-LO. We took advantage of the fact that PR9cells, promonocytic U937 cells expressing PML/RARa underthe control of the Zn2þ-inducible methallothionein1 (MT-1)promoter do not express endogenous 5-LO due to the meth-ylation of the 5-LO promoter (36). PML/RARa activates Wntsignaling in these cells through the activation of b-catenin andg-catenin (10, 12). The lack of 5-LO expression in both controland P/R9 cells was confirmed by Western blotting and by PCR(data not shown). Noteworthy, transfected 5-LO is unable tosynthesize leukotrienes in undifferentiated U937 cells (37).This may relate to cell type–specific features governing 5-LOactivity, such as the redox state, the phosphorylation status of5-LO, the activity of intracellular tyrosine kinases, structural

Figure 4. Effect of 5-LO�/� (Alox5�/�) on replating efficiency and long-term stem cell capacity of PML/RARa-expressing HSCs. A, Sca1þ/lin� BM cells (wild-type and Alox5�/�) were retrovirally infected with empty vector control and PML/RARa and plated in methylcellulose medium supplemented with mIL3,mIL6, and mSCF to assess primary colony formation. The colony numbers were counted on day 10. After determining the colony number, the cells wereharvested and serially replated every 10 days. B, colony morphology of platings I–VI. Data are shown from a single representative experiment of three thatyielded similar results. C, CRA. Sca1þ/lin� BM cells from wild-type and Alox5�/� CD45.2þ mice were retrovirally infected with empty vector control andPML/RARa. After 7 days in liquid culture, cells were cotransplanted with CD45.1þ BM cells into lethally (11Gy) irradiated CD45.1 and 2þ recipient mice (7–8mice/group). An analysis of donor chimerism was performed at 9 months after transplantation to determine the LT-stem cell capacity. The graph shows adonor-derived chimerism at 9 months in peripheral blood. Statistical significance was tested using the Student t test (��, P < 0.001). ns, nonsignificant.

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characteristics in the cytoskeleton, and the individual expres-sion of further enzymes involved in leukotriene biosynthesis(38). Notably, in myeloid cells, the activity of 5-LO was alreadyreported by us to depend on the differentiation status and theactivity of cellular peroxidases, whichmay also be true in 5-LO–transfected U937 cells (22). We expressed 5-LO in these cellsunder the control of a CMV promoter. Activation of Wntsignaling by PML/RARa was addressed by the transactivationof the "Topflash/Fopflash" reporter system, in which theactivation of the reporter is under the control of a TCF/LEFresponsive element (19). Herewe show that the expression of 5-LO prevented the activation of the Wnt target promoter byPML/RARa (Fig. 5A).To further understand the mechanism by which 5-LO sup-

presses Wnt signaling, we investigated the effect of 5-LO ong-catenin–dependent transactivation. b- and g-catenin areclosely related and are both key mediators of Wnt signaling.In contrast to b-catenin, which exhibits transforming potentialonly upon constitutive stabilization by mutations, the over-expression of g-catenin alone is able to transform cells (39).Weused a construct in which the luciferase reporter was con-trolled by the g-catenin promoter, which is activated byg-catenin itself (12). As reported in Fig. 5B, only a slightreduction of the transactivation of g-catenin promoter byPML/RARa was seen in the presence of 5-LO (Fig. 5B). Insummary, these results suggest that 5-LO interferes with thePML/RARa-induced activation of Wnt signaling by inhibitingb-catenin- and not g-catenin–dependent transactivation. Thisability is mediated by inactive 5-LO.

5-LO and b-catenin show a direct interactionTo test the possibility of a direct influence of 5-LO on Wnt

signaling, we investigated the interaction between 5-LO and

b-catenin. Therefore, we coexpressed 5-LO in 293T cells togeth-er with a HA-tagged constitutive active b-catenin mutant,S33A, which lacks the phosphorylation sites for proteasomaldegradation (Supplementary Fig. S5, right). Coimmunopreci-pitation using anti–5-LO or high affinity anti-HA antibodiescoupled to magnetic beads, followed by Western blotting,revealed a direct interaction between 5-LO and active b-cate-nin (Supplementary Fig. S5, left, IP 5-LO and IP HA). The slightbands of 5-LO in b-catenin in the respective anti–b-catenin oranti–5-LO precipitates weremost likely due to specific bindingof endogenous proteins. 293T cells expressed both endogenousb-catenin and 5-LO (data not shown). Nevertheless, uponoverexpression of both proteins the amount of precipitated5-LO or b-catenin, respectively, was clearly higher than thatseen in the controls showing a direct interaction between thetwo proteins.

In summary, our data show a direct interaction between 5-LO and b-catenin, the key regulator of the Wnt signalingpathway, which may account for the inhibitory effect ofinactive 5-LO on Wnt signaling.

5-LO colocalizes with b-catenin and hinders b-cateninfrom entering the nucleus

To confirm the biologic relevance of the interaction between5-LO and b-catenin revealed in vitro, we sought to disclose themechanism by which this interaction interferes with Wntsignaling. Therefore, we studied the influence of the expressionof 5-LO on the localization of b-catenin in PR9 cells by indirectimmunofluorescence. Thus, we expressed 5-LO in PR9 cellsand induced them to express PML/RARa by Zn2þ treatment.To investigate the colocalization of 5-LO and b-catenin, weperformed double anti–5-LO/anti–b-catenin staining. As con-trols, we used empty vector–transfected P/R9 cells.

Figure5. Effect of 5-LOonPML/RARa-mediatedWntpathwayactivation. Transactivation assay forWnt signaling–related transcription (A)b-catenin (TCF/LEF)or g-catenin–dependent transcription (B). The expression vectors pCDNA3.1 or pCDNA3.1-5-LO were cotransfected by nucleofection with either Topflash(OT) or Fopflash (OF) reporter constructs (A) or pGL3-g-catenin or pGL3 basic constructs (B) into 5-LO negative U937 cells expressing PML/RARa under thecontrol of a Zn2þ-inducible metallothionein1 (MT-1) promoter. The transgene was induced by exposure to 100 mmol/L ZnSO4. Luciferase activity wasmeasured 24 hours later and normalized withRenilla activity. The data are expressed as themeans of two (A) and three (B) independent experiments with SD.Statistical analysis was performed by the Student t test (�, P < 0.05).






The expression of PML/RARa upon Zn2þ induction wasconfirmed by anti-RARa staining, which revealed the typicalmicrospeckled pattern of PML/RARa (Supplementary Fig.S6; refs. 40, 41). The b-catenin expression in U937 cellsshowed a more scattered pattern than in other cell lines,most often fibroblasts, where the distribution of b-catenin ismore diffuse (42–44). As shown in Fig. 6A, upon Zn2þ

induction, the proportion of nuclear b-catenin (red fluoro-chrome) increased, confirming activation of Wnt signalingupon PML/RARa induction. In contrast, in the absence ofPML/RARa, anti–b-catenin staining was seen exclusively inthe cytoplasm. The expression of 5-LO in the PR9 cells led toperinuclear anti–5-LO staining (green fluorochrome),whereas empty vector–transfected cells did not reveal anyspecific anti–5-LO staining, further confirming that thesecells do not express 5-LO. The anti–5-LO staining wasnot influenced by the presence of PML/RARa. In contrast,in the presence of 5-LO, no nuclear anti–b-catenin stainingwas seen. The superimposition of anti 5-LO (green fluoro-chrome) and anti–b-catenin (red fluorochrome) stainingshowed partial colocalization of 5-LO and b-catenin (yellow)(Fig. 6A). Four confocal sections (Fig. 6B, a–d) of the samecell taken on the horizontal axis of the microscope (0.25–0.33mm interval) are shown in Fig. 6B. To view the colocalizationin greater detail, a further electronic 4-fold magnificationof the indicated area of each layer is shown (Fig. 6B, a'–d').A three-dimensional reconstruction of all the layers of thecell (data not shown) illustrated the same colocalizationseen in Fig. 6B. The apparently small amount of colocalizing

5-LO and b-catenin accords with the data obtained bycoimmunoprecipitation.

To investigate whether CJ-13,610 is capable of inducingcolocalization of 5-LO and b-catenin, human NB4 acutepromyelocytic leukemia cells exhibiting well-defined cyto-solic 5-LO expression were exposed to 3 mmol/L CJ-13,610.As can be seen treatment with CJ-13,610 led to partialcolocalization of 5-LO and b-catenin (Supplementary Fig.S7) strengthening the hypothesis of a 5-LO–mediated sup-pression of Wnt signaling induced by CJ-13,610. Takentogether, immunofluorescence studies could confirm thedirect interaction of 5-LO with b-catenin, triggered by CJ-13,610, suggesting also a novel mechanism for Wnt signalinginhibition by which b-catenin is prevented by 5-LO fromentering the nucleus.

DiscussionThe aim of our study was to determine whether the inhi-

bition of 5-LO may account for the effects of NSAIDs on theWnt signaling pathway and to assess their capacity to suppressCSCs in leukemia. We could show that the pharmacologicinhibition of 5-LO at clinically feasible concentrations ofselective inhibitors, CJ-13,610 or zileuton (45–47) suppressesthe aberrant stem cell capacity of PML/RARa-positive HSPCsvia inhibition of Wnt signaling. In addition, inhibitors of theother arachidonic acid metabolizing enzymes, COX and sEH,failed to suppress PML/RARa-positive HSPCs. Finally, a num-ber of subsequent experiments substantiated the CJ-13,610–independent suppressive action of ectopically expressed 5-LO

Figure 6. Colocalization between 5-LO and b-catenin in PR9 cells. Cells were transfected by nucleofectionwith either empty vector control or 5-LO. Two hoursafter nucleofection, transgene expressionwas inducedby 100mmol/L ZnSO4. A, twenty-four hours later, cells were imaged in phase contrast (gray, a), stainedwith TO-PRO-3 (blue, b), and stained with anti–5-LO and anti–b-catenin antibody, respectively. Alexa Fluor 594–conjugated anti-rabbit-Ig (red, c) andAlexa Fluor 488, anti-mouse-Ig (green, d) were used for immunostaining. Merged images (e) were obtained by electronic overlapping of images recordedin a–d. B, cells, treated and stained as in A, were scanned in 30 layers (0.25–0.33 mm interval) on z-axis. Electronic overlapped merged images of phasecontrast, TO-PRO-3 (nuclei), red fluorescence (b-catenin), and green fluorescence (5-LO) are shown in B. Left, four confocal sections (a–d) are shown.Colocalizations are indicated with arrows. A �4 magnification (a0–d0 ) is reported on the right side of B.

Roos et al.





on Wnt signaling and leukemic cells. Thus, suppression ofaberrant stem cell capacity should mainly result from thedrug's ability to specifically target 5-LO. However, pleiotropicoff-target effects of CJ-13,610 cannot ultimately be excluded.Nevertheless, even the existence of such possible off-targeteffects, contributing to the antileukemic effect of the drug,wouldnot detract from the documented keyfinding of a pivotalrole of 5-LO in AML.On the basis of our findings that the 5-LO directly interacts

with b-catenin, we conclude that the Wnt inhibitory effect ofNSAIDs in our leukemiamodel is mediated not by inhibition ofthe COX-1/2 and prostaglandin E2 synthesis as observed inmodels of colorectal cancer (48). This is supported by thefinding that the activation of Wnt signaling in LSC is notrelated to upregulation of COX, but is related to the presence ofaberrant chimeric transcription factors resulting from chro-mosomal translocations such as t(15;17)-PML/RARa or t(8;21)AML-1/ETO fusion proteins (10, 12). The inhibitory effect ontumor stem cells of the inhibition of 5-LO is not limited to ourleukemia model but has also been described in glioblastoma.Here, the selective 5-LO inhibitor, Nordy, was able to reducesphere formation and the frequency of stem cells in xenografttumors (49).Both the genetic disruption and pharmacologic inhibition

of 5-LO suppress the tumor-initiating cells of BCR/ABL-driven CML-like disease in mice (18). This is, at leastpartially, in contrast with our findings on the PML/RARa-driven AML model, in which the genetic targeting of 5-LOdid not replicate the effects of pharmacologic inhibition onthe stem cell capacity. This is in accordance with recentfindings showing full leukemogenic potential of AML-1/ETOex9–expressing HSPCs in an Alox5�/� backgroundalthough an impairment of the in vitro self-renewal of HSPCsexpressing either AML-1/ETOex9, PML/RARa or MLL/AF9was seen (50). CML is a myeloproliferative disease charac-terized by proliferation of hematopoietic progenitors, whichare still able to fully differentiate. PML/RARa induces anacute leukemia characterized by aberrant self-renewal of theHSCs and differentiation block. The stem cell compartmentof these two diseases differ being more mature for the CML-like disease and very primitive HSPCs for PML/RARa(31, 51). Furthermore, the induction and the maintenanceof AML-LSC depend on the activation of Wnt signaling,whereas for the induction of CML-like disease, Wnt signalingseems to be nonessential (5). Therefore, we hypothesize thatthe presence of the enzymatically inactive form of 5-LO isneeded to inhibit Wnt signaling in the PML/RARa-positiveHSPCs by blocking the transition of b-catenin to the nucleus.That 5-LO is able to alter nuclear trafficking of a protein, wasshown for p53, as 5-LO antagonized genotoxic stress-induced apoptosis by inhibiting binding of p53 to thepromyelocytic leukemia protein (PML) and its relocalizationinto PML-nuclear bodies (52).The exact molecular mechanism by which inhibitor-

bound and catalytically inactive 5-LO interacts with b-cate-nin and possibly other Wnt components remains unclear.Possibly, 5-LO inhibitors, such as CJ-13,610 and zileuton,induce certain conformational changes in the 5-LO enzyme,

thereby allowing suppressive interactions of the enzymewith other proteins of the Wnt signaling pathway. Similarly,AA and competitive 5-LO inhibitors mimicking AA wereobserved to trigger changes in subcellular localization of5-LO that may relate to the effects observed in this studywith CJ-13,610 (53). As a consequence, cytosolic and peri-nuclear b-catenin bound to 5-LO may be trapped andhindered from entering the nucleus, suggesting that theprotein cannot drive anymore the proleukemic transcriptionof Wnt target genes, as demonstrated in our study. Notably,in PR9 cells, ectopically transfected 5-LO is capable ofsuppressing Wnt signaling, whereas in other cell types,including the PML/RARa-positive HSPCs, the presence ofCJ-13,610 is required to convert 5-LO to an active Wntsuppressor. We conclude that the environment of PR9 cellsinto which the ectopically transfected 5-LO is imbeddedrenders 5-LO catalytically inactive by still unknown mechan-isms thereby allowing inhibition of Wnt signaling. Furtherbiophysical studies on protein structure are required addres-sing in detail the possible conformational changes in 5-LOafter binding to 5-LO inhibitors allowing interaction withb-catenin.

Our study demonstrated a crucial role for 5-LO in themaintenance of LSCs in AML, confirming recent data inmurine models of AML (50). The fact that, in a subset oftumors, the inhibition of 5-LO is able to suppress the tumorstem cell renders feasible a stem cell therapy as amaintenanceregimen after induction and consolidation therapy protocols.The use of 5-LO inhibitors, instead of COX inhibitors, wouldfurther avoid the adverse effects of COX inhibitors, such asgastrointestinal bleeding and ulcerations or cardiovascularside effects, including hypertension, myocardial infarction,and stroke. Taken together, our data establish pharmacologicinhibition of 5-LO as a novel approach of stem cell therapy inleukemia, by targeting Wnt signaling.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J. Roos, D. Steinhilber, I. Fleming, T.J. Maier,M. RuthardtDevelopment of methodology: C. Oancea, D. Khan, E. Puccetti, T.J. MaierAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Roos, C. Oancea, M. Heinssmann, D. Khan, H. Held,A.S. Kahnt, R. Capelo, M. RuthardtAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Roos, C. Oancea, A.S. KahntWriting, review, and/or revision of the manuscript: J. Roos, E. Proschak,I. Fleming, T.J. Maier, M. RuthardtAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): H. HeldStudy supervision: E. Proschak, D. Steinhilber, T.J. Maier, M. RuthardtPerformed experiments: D. KhanOther (synthesis of the compound): E. la Buscato

AcknowledgmentsThe authors thank Prof. Dr. Mike Parnham for critically proofreading the

article and Carlo Angioni for excellent technical assistance.

Grant SupportThis work was funded by the German Jos�e Carreras Leukemia Foundation

(DJCLS RF-11/15; M. Ruthardt and I. Fleming, by LOEWE (Landesinitiative zu






F€orderung €Okonomischer und Wissenschaftlicher Exzellenz) in the Center ofLipid Research Frankfurt (LIFF; T.J. Maier and M. Ruthardt), and by the GermanResearch Foundation (DFG-MA-5825/1-1). T.J. Maier is recipient of a Heisen-berg fellowship of the German Research Foundation (DFG-MA-5825-2-1).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received October 30, 2013; revised June 9, 2014; accepted June 21, 2014;published OnlineFirst July 31, 2014.

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2014;74:5244-5255. Published OnlineFirst July 31, 2014.Cancer Res Jessica Roos, Claudia Oancea, Maria Heinssmann, et al.

like Cells in Acute Myeloid Leukemia−of Stem Cell 5-Lipoxygenase Is a Candidate Target for Therapeutic Management

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