ORIGINAL ARTICLE

CITED2-mediated human hematopoietic stem cellmaintenance is critical for acute myeloid leukemiaPM Korthuis1, G Berger1, B Bakker1, M Rozenveld-Geugien1, J Jaques1, G de Haan2, JJ Schuringa1, E Vellenga1 and H Schepers1

As the transcriptional coactivator CITED2 (CBP/p300-interacting-transactivator-with-an ED-rich-tail 2) can be overexpressed in acutemyeloid leukemia (AML) cells, we analyzed the consequences of high CITED2 expression in normal and AML cells. CITED2overexpression in normal CD34+ cells resulted in enhanced hematopoietic stem and progenitor cell (HSPC) output in vitro, as well asin better hematopoietic stem cell (HSC) engraftability in NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. This was because of anenhanced quiescence and maintenance of CD34+CD38− HSCs, due in part to an increased expression of the cyclin-dependentkinase inhibitor CDKN1A. We demonstrated that PU.1 is a critical regulator of CITED2, as PU.1 repressed CITED2 expression in a DNAmethyltransferase 3A/B (DNMT3A/B)-dependent manner in normal CD34+ cells. CD34+ cells from a subset of AML patientsdisplayed higher expression levels of CITED2 as compared with normal CD34+ HSPCs, and knockdown of CITED2 in AML CD34+ cellsled to a loss of long-term expansion, both in vitro and in vivo. The higher CITED2 expression resulted from reduced PU.1 activity and/or dysfunction of mutated DNMT3A/B. Collectively, our data demonstrate that increased CITED2 expression results in better HSCmaintenance. In concert with low PU.1 levels, this could result in a perturbed myeloid differentiation program that contributes toleukemia maintenance.

Leukemia advance online publication, 30 September 2014; doi:10.1038/leu.2014.259

INTRODUCTIONThe transcriptional coactivator CITED2 (CBP/p300-interacting-transactivator-with-an ED-rich-tail 2) was originally discovered tobind the CH1 region of CBP/p300.1 More recently, we showed thatCITED2 is essential for adult hematopoietic stem cell (HSC)maintenance.2 CITED2 deletion in adult HSCs led to rapid animallethality, resulting from hematopoietic stem and progenitor cell(HSPC)-specific apoptosis. In contrast, lineage-specific deletion ofCITED2 did not result in such a marked phenotype, suggestingHSC-specific actions of CITED2.2 These actions might be mediatedthrough control of the INK4a/ARF locus by the PRC1 genes BMI1and MEL18.3 In addition, TFAP2A and MYC have been shown tocontrol cellular proliferation of, respectively, breast and lungcancer cells using CITED2 as a cofactor.4,5 The fact that CITED2expression is essential for HSC maintenance and functions as acofactor in oncogene-induced transformation suggests thatCITED2 could also have a role in leukemia initiation or leukemicstem cell maintenance. Gene expression data from Anderssonet al.6 suggest that acute myeloid leukemic (AML) cells can havean enhanced CITED2 expression, while in chronic myeloidleukemia, CITED2 expression is even further increased upontransition into blast crisis.7,8 How CITED2 expression may beincreased during leukemogenesis is unknown, but importanttranscription factors, such as HIF1α,9 FOXO3A10 and STAT5,5 havebeen shown to regulate its expression. Closer examination of theCITED2 promoter revealed that, besides HIF1α, FOXO3A andSTAT5, ETS transcription factor-binding sites are also present.The presence of ETS-binding sites is of interest, as a member of

the ETS family of transcription factors, PU.1, is frequently

inactivated in AML. Although point mutations of PU.1 are rarelyobserved,11,12 many leukemic oncogenes are known to reduce orinactivate PU.1 expression.11,13–17 Such decreased expression ofPU.1 has been shown to induce AML in mice.18–20 Furthermore,PU.1 haploinsufficiency cooperates with SOX4 to induce AML inmice.21 Based on these findings, we questioned whether PU.1modulates CITED2 expression and function.Here we report that a subset of CD34+ AML cells express

increased levels of CITED2. As in normal CD34+ cells, this likelyaffects quiescence as well as apoptosis of these cells. Wefurthermore demonstrate that reduced expression or activity ofPU.1 contributes to this high expression of CITED2.

MATERIALS AND METHODSCulture conditionsLong-term cultures on stroma, colony-forming cell (CFC), long-termculture-initiating cell (LTC-IC) and single-cell assays were performed asdescribed previously and in the Supplementary Methods.22,23

Flow cytometry analysisAll fluorescence-activated cell sorter (FACS) analyses were performed on anLSRII (Becton Dickinson (BD), Alphen a/d Rijn, The Netherlands) orMACSQuant (Miltenyi Biotec, Leiden, The Netherlands) and data wereanalyzed using FlowJo (Treestar, Ashland, OR, USA). Cells were sorted on aMoFLo XDP or Astrios (DakoCytomation, Carpinteria, CA, USA). Antibodieswere obtained from BD Bioscience (Breda, The Netherlands), BioLegend(Uithoorn, The Netherlands), eBioscience (Vienna, Austria), Milteny (Leiden),Nuclilab (Huissen, The Netherlands) or DAKO (Enschede, The Netherlands)and are described in the Supplementary Methods.

1Department of Experimental Hematology, Cancer Research Center Groningen, University Medical Center Groningen, University of Groningen, Groningen,The Netherlands and 2Department of Stem Cell Biology, European Research Institute for the Biology of Aging (ERIBA), University Medical Center Groningen, Universityof Groningen, Groningen, The Netherlands. Correspondence: Dr H Schepers, Department of Experimental Hematology, Cancer Research Center Groningen, University MedicalCenter Groningen, University of Groningen, Hanzeplein 1, Groningen 9713, The Netherlands.E-mail: h.schepers@umcg.nlReceived 6 May 2014; revised 1 August 2014; accepted 22 August 2014; accepted article preview online 3 September 2014

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In vivo transplantations into NSG miceEight- to ten-week-old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ)mice were purchased from Charles River Laboratory (L'Arbresle Cedex,France) and bred in house. Mouse experiments were performed inaccordance with national and institutional guidelines and all experimentswere approved by the Institutional Animal Care and Use Committee of theUniversity of Groningen (IACUC-RuG). Experiments are described in theSupplementary Methods.

Quantitative PCR and gene expression profilingRNA isolation is described in the Supplementary Methods. Target geneexpression was investigated by means of quantitative PCR (Q-PCR). Typicalexamples are shown and presented as mean with standard error, sequencescan be found in Supplementary Table 2. A detailed description of the geneexpression analysis workflow is described in the Supplementary Methods.

Reporter assaysFor cloning and transfection of constructs see SupplementaryMethods.24,25 Cells were harvested and lysed using Promega LuciferaseCell Culture Lysis Reagent (Promega, Leiden, The Netherlands) accordingthe manufacturer's recommendations. Luciferase signals were measuredusing a Synergy H4 Hybrid Reader (BioTek, Bad Friedrichshall, Germany).Alternatively, the mean fluorescent intensity of green fluorescent protein(GFP) was measured by FACS.

Chromatin immunoprecipitationsA total of 2.5 × 106 CD34+ cells or HL-60 cells were used for chromatinimmunoprecipitation studies as described before.26

RESULTSCITED2 enhances human HSPC cultures and engraftmentGene expression profiling suggested that CITED2 expression mightbe enhanced in AML cells. To study whether high CITED2expression affects HSPC activity, cord blood (CB) CD34+ cellswere lentivirally transduced with CITED2 (Supplementary Figures1A and B) and hematopoietic development was studied. Improvedlong-term expansion of hematopoietic cells on MS5 cocultureswas observed upon overexpression of CITED2 (Figure 1a; Po0.05),with a marked increase in the size (Figure 1b) and number (datanot shown) of week 5 cobblestone area-forming cells. Toinvestigate the effect of ectopic CITED2 expression on progenitorcells, transduced CD34+ cells were plated in methylcellulose eitherdirectly or after each subsequent week of culture on MS5 stromalcells. CITED2 overexpression increased the number of CFCs~ 4-fold directly after transduction (Figure 1c). CITED2 increasedCFC numbers from both the CD34+CD38+ and CD34+CD38+

compartments, but subsequent replating capacity was confined tothe CD34+CD38− compartment (data not shown). This increasedprogenitor persisted for at least 5 weeks of culture (Figure 1d).These CITED2-expressing colonies were ~ 5x larger than controlcolonies (Figure 1e, note magnification), in line with the observedincrease in suspension cell numbers. These colonies depicted aclear increase in colony-forming unit-granulocyte upon over-expression of CITED2, which was paralleled by a decrease in burst-

forming unit-erythrocytes (Supplementary Figure 1C). Similarly, inMS5 cocultures an increase in the percentage of CD15+

granulocytic cells was observed (Supplementary Figure 1D, upperpanel), in contrast to a decrease in CD14+ monocytes/macro-phages and GPA+ erythroid cells (Supplementary Figure 1D, upperand lower panels).To assess whether CITED2 overexpression also affected in vivo

engraftment of human cells, 1–2x105 transduced CD34+ cells weretransplanted into NSG mice (n= 6/7) and at the indicatedtimepoints contribution of human CD45+ cells to the peripheralblood was measured. Figures 1f and g demonstrate that CD34+

cells overexpressing CITED2 contributed significantly better tohuman engraftment than control cells in four out of seventransplanted mice (Po0.05). The higher engraftment in four micesuggested that CITED2 either affected migration/homing towardthe BM or better maintained primitive HSCs. CXCR4 expression onboth mRNA and protein level was not affected by CITED2(Supplementary Figures 1E and F) and also the migration towardSDF-1 was not affected (Supplementary Figure 1G). However,within the bone marrow of the high engrafting mice, veryprimitive lin−CD34+CD38−CD90+CD45RA− cells could be detectedat 28 weeks after transplantation, with a similar distribution ofHSCs, MPPs and LMPPs as previously published27 (SupplementaryFigure 1H). These CITED2-maintained HSCs contributed normallyto lineage differentiation, as CD33 myeloid, CD19 B cells andCD3 T cells were observed 24–27 weeks after transplantation(Figure 1h and Supplementary Figure 1I).

Enhanced CITED2 expression increases quiescence of human HSCsAs CITED2 significantly improved the long-term in vitro and in vivooutput, it suggested that CITED2 has a profound impact on moreimmature human HSCs. LTC-IC assays with the total CD34+

compartment confirmed that CITED2 overexpression increases theLTC-IC frequency (Supplementary Figure 2A). This coincided with athreefold increase in the percentage of immature CD34+CD38−

HSCs after 3 days of culture (Figures 2a and b). To gain furtherinsight into how CITED2 increases the number of human HSCs,we analyzed whether these cells have a changed apoptotic or cellcycle profile. Figure 2c demonstrates that CITED2 decreases thepercentage of CD34+CD38− cells that are AnnexinV+. As murineHSCs cycle faster upon CITED2 deletion,28 we assumed thatoverexpression of CITED2 might also affect human HSCs quiescenceor proliferation. To confirm this, we performed Hoechst 33342/PyroninY stainings to measure G0, G1 and G2/S/M phases of the cellcycle. As shown in Figure 2d, an increased fraction of CITED2-transduced CD34+CD38− HSCs remained in the G0 phase of the cellcycle. Q-PCR analysis demonstrated that increased CITED2 expres-sion led to an increased expression of the cyclin-dependent kinaseinhibitors CDKN1B, CDKN1C and especially CDKN1A in the CD34+

CD38− compartment (Figure 2e), which is consistent with theenhanced quiescence of these cells. To asses directly theinvolvement of CDKN1A in the CITED2-mediated quiescence ofHSCs, and to verify this observation in an independent assay, CD34+

cells were doubly transduced with a lentivirus expressing CITED2

Figure 1. CITED2 enhances human HSPC cultures and engraftment. (a) Expansion on MS5 stromal coculture of CB CD34+ cells lentivirallytransduced with CITED2 or control lentivirus (n= 4). Each week cultures were demipopulated, fresh media were added and cells were counted.The average of four independent experiments is shown. Error bars denote standard deviation (*Po0.05). (b) Image of typical cobblestoneforming areas (at x10 magnification) after 5 weeks of culture on MS5 stromal cells. (c) CFC numbers from CD34+ cells directly aftertransduction with lentiviral CITED2 (n= 8, error bars denote standard deviation *Po0.05). (d) CFC numbers after the indicated weeks on MS5stromal cells. Each week suspension cells were harvested and interrogated for CFC activity (n= 3, error bars denote standard deviation).Relative CFC numbers are depicted for comparison, with average CFC numbers per 10 000 cells plated given underneath the figure (*Po0.05).(e) Typical images of control and CITED2-overexpressing colonies. Note that the control colonies have been taken at a x20 magnification,whereas the CITED2-transduced colonies have been taken at a x4 magnification. (f) Typical FACS plots showing human engraftment in NSGmice 24 weeks posttransplant with control or CITED2-transduced CD34+ cells. (g) Human engraftment in NSG mice transplanted with control-(n= 6) or CITED2- (n= 7) transduced CD34+ cells (*Po0.05). (h) Typical FACS plots showing multilineage CD3 (T-cell), CD19 (B-cell) and CD33(myeloid cell) engraftment at 24 weeks posttransplant in a CITED2-engrafted mouse.

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and a lentivirus-expressing a short hairpin against CDKN1A(Supplementary Figures 2B and C). Subsequently, CD34+CD38−

HSCs were single cell sorted and their proliferation was micro-scopically evaluated. For 4 days, each well was inspected for thepresence of one cell (quiescence) or more than one cell(proliferation). CITED2 expression indeed induced more cells to

remain quiescent (Figure 2f). Furthermore, this experiment demon-strated that the CITED2-induced quiescence is partially reversible onknockdown of CDKN1A. This indicates that CITED2 can modulatethe quiescence of HSCs by regulating the expression of CDKN1Aand thereby maintain HSCs. Although this CITED2-induced HSCquiescence is consistent with the enhanced long-term engraftment

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in vivo, it seems contradictory to the observed expansion in ourin vitro assays. However, performing the single-cell quiescence/proliferation assay also on CITED2-transduced CD34+CD38+ pro-genitor cells demonstrated that CD34+CD38+ progenitors areactually induced to proliferate (Supplementary Figure 2D) upontransduction with CITED2, demonstrating differential effects ofCITED2 on the HSC and progenitor compartments.

CITED2 is a direct repressive target of PU.1Next, we investigated the mechanisms underlying the enhancedCITED2 expression. Besides the well studied HIF1α-,24 FOXO3A-9

and STAT5-10 binding sites, the human CITED2 promoter containes

five potential PU.1-binding sites (Figure 3a), suggesting that PU.1has a role in regulating CITED2 expression. ENCODE29 ChIP-seqdata for PU.1 in hematopoietic cell lines confirmed this hypothesis(Supplementary Figure 3A). In addition, we first assessed PU.1binding to the CITED2 promoter under relevant conditions, byperforming chromatin immunoprecipitations (ChIPs) for endogen-ous PU.1 in CB-derived CD34+ cells. This demonstrated thatendogenous PU.1 indeed binds to the CITED2 promoter(Figure 3b). PU.1 binding to the CITED2 promoter was indepen-dently verfied in PU.1-overexpressing 293T lysates with strepta-vidin pulldown assays. Biotinylated oligos containing thePU.1-binding sites from the CITED2 promoter bound PU.1 asefficiently as a bona fide PU.1-binding site from the JUNB

Figure 2. Enhanced CITED2 expression increases quiescence of human HSCs. (a) CD34+ CB cells were lentivirally infected with control orCITED2 and cultured in hematopoietic progenitor growth medium (HPGM) with 100 ng/ml of stem cell factor (SCF), thrombopoietin (TPO) andFLT3. After 2–3 days, cultures were analyzed for expression of CD34 and CD38. A typical FACS analysis of HSC (CD34+CD38−) or progenitor cells(CD34+CD38+) after transduction is shown (n= 15). Cells were first gated through CD271 (tNGFR, transduced cells) and DAPI (viability).(b) Average percentages of CD34+CD38− HSCs of 15 CBs. Error bars denote standard deviation. (c) CB cells were transduced, cultured andstained for CD34, CD38, CD271 and AnnexinV. The average of five CBs is shown, with error bars denoting standard deviation. (d) Cell cycleFACS analysis of transduced CD34+CD38− cells. A representative example is shown. In the right scatter plot, the average and standarddeviation of three samples is shown. (e) Q-PCR analysis for CDKN1A, CDKN1B and CDKN1C after overexpression of CITED2 in CD34+CD38− HSCs.(f) CB CD34+ cells were lentivirally infected with control, CITED2 and a lentiviral short hairpin against CDKN1A. One hundred and twenty singleCD34+CD38− HSCs were subsequently sorted into terasaki plates in Iscove's modified Dulbecco's medium (IMDM) plus 20% fetal calf serum(FCS) and 10 ng/ml IL-3 and SCF. For 4 days of culture, each well was microscopically analyzed for cells that had divided or cells that had notdivided (n= 3). Individual experiments were normalized to compare. Error bars depict standard deviation.

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promoter (Supplementary Figure 3C). Subsequently, SPI1 (thegene encoding human PU.1) was downregulated by means of ashort hairpin Mir-based lentivirus (Figure 3c and SupplementaryFigure 3D). This resulted in an increase in CITED2 expression inboth HL-60 and CB CD34+ cells (Figures 3c and e). In contrast,overexpression of a 4-hydrotamoxifen (4-OHT)-inducible PU.1(PU.1-ERt2; Figure 3d and Supplementary Figure 3E) in HL-60 cellsled to ~ 2-fold decrease in CITED2 expression (Figure 3d).To study the molecular interaction between PU.1 and CITED2 in

more detail, the human kidney cell line 293T was used. This cell

line expresses high levels of CITED2 and no PU.1. 293T cells weretransduced with PU.1-ERt2 and activation of PU.1 resulted inexpression of its target genes NCF4 (Supplementary Figure 3B) andin a twofold reduction in CITED2 expression, validating this modelfor further studies. To prove that CITED2 is indeed a direct targetof PU.1, the CITED2 promoter was subcloned upstream of aluciferase and/or GFP construct. After mutating the strongestChIP-binding sites (sites 3 and 5), reporter activity was assessed(Supplementary Figure 3F). Supplementary Figure 3G shows thatactivation of PU.1 reduces activity of the wild-type CITED2 (pCT2-

Figure 3. CITED2 expression is repressed by PU.1. (a) Schematic representation of the human genomic CITED2 locus. Black bars indicate thetwo exons. The arrow in exon 2 indicates the start of the CITED2 CDS. Black dots indicate the positions of the five putative ETS-binding sites(GAGGAA), with arrows indicating ChIP primers. B= BamHI, E= EcoRI, H=HinDIII, Hi=HincII and N=NotI are indicated for reference. Thebottom numbers indicate numbers of nucleotides away from the start ATG. (b) ChIP for PU.1 on CB CD34+ cells. Two to five million CD34+ cellswere crosslinked for 15min and ChIP was performed with anti-PU.1 ab (Santa Cruz; sc-352) or control preimmune serum (IgG control).Recovered genomic DNA was subjected to Q-PCR with the indicated primers specific for the five putative ETS-binding sites. A region 10 kbupstream was taken along as the negative control. Data are presented as relative enrichment over IgG control, and are the average of fourindependent experiments. Error bars denote standard deviation. (c) HL-60 cells were transduced with a lentivirus carrying a shRNAmirtargeting human SPI1. Western blot analysis indicates proper knockdown (upper panel). mRNA was isolated and Q-PCR against CSF3R andCITED2 was performed (lower panel). (d) HL-60 cells were transduced with lentiviral PU.1-ERt2 construct. After 4-OHT (100 nM) treatment for4 days, nuclear lysates were isolated and analyzed by western blot (upper panel). Bottom panel: mRNA was isolated and Q-PCR was performedfor CSF3R mRNA (to indicate proper PU.1 activation) and CITED2 mRNA. (e) Primary CB-derived CD34+ cells were transduced with SPI1shRNAmir and subjected to mRNA isolation and Q-PCR analysis for SPI1 and CITED2.

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wt) reporter. This reporter thus responds in the same manner asthe endogenous CITED2 promoter, validating it for furtherexperiments. Mutation of binding site 3 showed a moderateeffect (Supplementary Figures 3H and I), but mutation of site 5(pCT2-Δ5 and pCT2-Δ3Δ5) rescued the repression of CITED2 byPU.1 to a large extent (Supplementary Figures 3H and I).Taken together, these data demonstrate that CITED2 is adirect target of PU.1 and that activation of PU.1 leads torepression of CITED2.

PU.1 represses CITED2 expression through DNMT3A and DNMT3BAs PU.1 has been shown to mediate target gene repression viaDNA methylation,30 we investigated whether methylation was alsoinvolved in repression of CITED2. Therefore, CB CD34+ cells weretransduced with PU.1-ERt2 and treated for 4 days with 100 nM4-OHT in the presence or absence of 0.5 μM decitabine, aDNA methyltransferase inhibitor. Decitabine completely abrogatedPU.1-mediated repression of CITED2 (Figure 4a), suggestingthat PU.1 recruits DNA methyltransferases (DNMTs) to repress

Figure 4. PU.1 represses CITED2 expression through DNMT3A and DNMT3B. (a) CB CD34+ cells were transduced with PU.1-ERt2 or controlvector and stimulated with 100 nM 4-OH tamoxifen up to 96 h, with or without 0.5 μM decitabine (DAC). Q-PCR analysis was performed toinvestigate CITED2 mRNA expression. (b) The 293T cells infected with PU.1-ERt2 or control vector were transduced with pGIPZ SFFV shRNAmirscrambled (SCR), DNMT3A or DNMT3B and stimulated with 100 nM 4-OH tamoxifen up to 96 h. Data are normalized to controls. Q-PCR analysiswas performed to investigate CITED2 mRNA expression (n= 3). (c) The 293T cells infected with PU.1-ERt2 or control vector were transducedwith a FLAG-tagged DNMT3A construct and stimulated with 100 nM 4-OH tamoxifen up to 96 h. ChIP for DNMT3A ChIP was performed withanti-FLAG (M2), an antibody that specifically recognizes methylated DNA (MeDIP) or control preimmune serum (IgG control). Recoveredgenomic DNA was subjected to Q-PCR with the indicated primers specific for the three PU.1-binding sites that showed the strongest PU.1binding. (d) The OCI-AML3 cell line, harboring a DNMT3AR882C mutation, was transduced with lentiviral PU.1-ERt2 and stimulated with 100 nM

4-OHT for 4 days. mRNA was isolated to investigate CITED2 expression (n= 3).

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CITED2 expression. As CITED2 expression is highest in immaturecells and gradually decreases during myelopoiesis,2 the de novomethylases DNMT3A or DNMT3B are potentially involved. To thisend, PU.1-ERt2-expressing 293Ts were transduced with lentivirusesencoding short hairpins targeting either DNMT3A or DNMT3B(Supplementary Figures 4A–C). The scrambled control-infectedcells showed repression of CITED2 expression upon activation ofPU.1 (Figure 4b, control groups). In contrast, suppressing DNMT3Aor DNMT3B by means of RNA interference rescued the repressionof the CITED2 promoter by PU.1 either partially (DNMT3A) oralmost completely (DNMT3B) (Figure 4b).Both DNMT3A and DNMT3B function synergistically in the same

complex,31 but DNMT3A is the most frequently mutated DNMT3gene in AML.32 As deletion of Dnmt3a in mice also resulted inexpansion of HSC numbers,33 we further focused on DNMT3A.Next, we cotransduced PU.1-ERt2-expressing 293Ts with lenti-viruses encoding a FLAG-tagged DNMT3A construct and treatedfor 4 days with 100 nM 4-OHT. A subsequent ChIP against FLAGdemonstrated that activation of PU.1 led indeed to recruitment ofDNMT3A to the CITED2 promoter at the sites that showed thestrongest PU.1 binding (Figure 4c, top panels). This PU.1-mediatedrecruitment of DNMT3A coincided with a small increase in DNAmethylation as measured through ChIP for methylated DNA(Figure 4c, bottom panels). Based on these data we hypothesizedthat in cells bearing mutations in DNMT3A, PU.1 should no longerbe able to repress CITED2 expression (or to a lesser extent). To testthis, the OCI-AML3 cell line with a DNMT3AR882C mutation wastransduced with PU.1-ERt2 and stimulated with 4-OHT for 4 days.Indeed, PU.1 did not repress CITED2 expression in this cell line(Figure 4d), although PU.1 was activated as measured throughCSF3R expression. Taken together, these data show that DNMT3Aand DNMT3B have nonredundant functions in the PU.1-mediatedrepression of CITED2.

CITED2 and SPI1 expression are inversely correlated in leukemicCD34+ cellsMyeloid transcription factors such as C/EBPα and PU.1 arefrequently inactivated in AML.12,23 Having established that PU.1represses CITED2 expression, we investigated whether CITED2expression was enhanced in AML. CITED2 mRNA expression wasanalyzed by Q-PCR in AML CD34+ cells (n = 28) and comparedwith CITED2 expression in CD34+ cells from normal bonemarrow (n = 9). Figure 5a shows a wide range of CITED2expression in AML CD34+ cells, as compared with normal bonemarrow CD34+. Thirteen out of 28 AML patients displayed higherthan normal expression. Similar results were obtained whencompared with CD34+ cells from CB (n = 4, data not shown) orgranulocyte-colony stimulating factor-mobilized peripheralblood stem cells (n = 5, data not shown). No apparent correlationbetween CITED2 mRNA expresssion and patient characteristicscould be observed in our data set (Table 1). To verify theseresults, we analyzed two independent data sets. First, thedifference in CITED2 expression between normal human bonemarrow subsets and CD34+ selected AML samples withdefined translocations (n = 142) was investigated using theHemaExplorer website (http://servers.binf.ku.dk/hemaexplorer/).34

Supplementary Figure 5A demonstrates that CITED2 expressionwas significantly higher in most AML samples as compared withnomal cellular bone marrow subsets. Second, also in CD34+ cellsfrom pediatric AML samples (n= 17, profiled by Andersson et al.6)CITED2 expression was found to be significantly higheras compared with CD34+ normal bone marrow cells(Supplementary Figure 5B). Taken together, these data indicatethat CD34+ cells from AML patients have higher CITED2 expressionthan normal CD34+ cells.Next, we examined whether a negative correlation exists

between PU.1 and CITED2 expression. We therefore plotted

SPI1 expression against CITED2 expression and we observeda significant inverse correlation between SPI1 and CITED2expression (Figure 5b and Supplementary Figure 5C). Toexclude differentiation-dependent effects, we phenotypeda panel of these AML samples for a lymphoid-primed multi-potent progenitor- or granulocyte–macrophage progenitor-likephenotype.35 CD45RA was strongly expressed in all AMLsanalyzed, in both CD38− and CD38+ compartments, suggestingthat all AMLs displayed a granulocyte–macrophage progenitor-like mature phenotype (Supplementary Figure 5D). As CITED2expression is low in normal granulocyte–macrophage progeni-tors (Supplementary Figure 5A and data not shown), thissupported our finding that CITED2 expression is aberrantly highin these AMLs. Subsequent bisulfate conversion and pyrose-quencing of 12 AML samples demonstrated a general hypo-methylation of the CITED2 promoter around the PU.1-bindingsites (Supplementary Figure 5E), consistent with the higherexpression of CITED2.Next, we investigated whether PU.1 also regulates CITED2

expression in patient AML cells. AML CD34+ cells were transducedwith PU.1-ERt2. AML numbers 1 and 29 responded with adecreased expression of CITED2 upon 4-OHT treatment(Figure 5c and Table 1). In contrast, 4 of the 29 AML patientsamples (AML numbers 7, 10, 11 and 21) contained aDNMT3AR882C/H mutation (Table 1), and did not show a decreasein CITED2 expression upon PU.1-ERt2 activation (Table 1), similar tothe results in the OCI-AML3 cell line (Figure 4d). This supports ourdata that DNMT3A is critical for PU.1 to repress CITED2, andtogether these data indicate that loss of PU.1 or DNMT3A is acontributing factor in elevating CITED2 levels.

CITED2 is required for maintenance of leukemic culturesGene set enrichment analysis on gene expression profiles ofCD34+CD38− HSCs after CITED2 upregulation indicated thatvarious leukemia-related signatures are enriched upon over-expression of CITED2 (Supplementary Table 1), suggesting thatCITED2 expression has a functional role in AML maintenance. Weperformed RNA interference against CITED2 in CD34+ AML cells(n= 14). AML CD34+ cells, transduced with lentiviral RNAinterference vectors targeting CITED2 or a scrambled hairpin(control) (Figures 5d and e), were cultured on MS5 stromal cells, asdescribed previously,36 and observed for several weeks. Twodifferent hairpins were used to knockdown CITED2, to minimizeoff target effects, both with similar efficiencies and cell biologicresults. (Supplementary Figures 6A and D, Supplementary Table 2).Control cells from AML numbers 6, 10 and 20 failed to expand onMS5 stromal cultures and were not analyzed further. Eighty-twopercent of the AMLs (9/11) showed a severely impaired expansionupon knockdown of CITED2 (Figure 5f and Table 1). This mostlikely is the result of combined effects on cell cycle as well as onapoptosis. We have also analyzed the differentiation of theseAMLs in vitro, but did not observe any differences betweenshCITED2- and control-transduced cells (n= 11, data not shown).From several AMLs we compared the level of knockdown to therelative decrease in leukemic expansion, but we could not detect asignificant correlation between the two (Supplementary Fig. 6E).Lastly, we addressed whether knockdown of CITED2 also inhibitsAML engraftment in vivo. We transduced CD34+ AML cells fromAML numbers 30–32 with lentiviral control or CITED2 RNAi andtransplanted 40, 25 or 18% GFP-sorted cells into NSG mice(N= 17). At 9 weeks post-transplant the peripheral blood of thesemice was analyzed for the presence of CD45+GFP+ cells.Supplementary Figures 6F and G demonstrate that the mice thatreceived CITED2-knockdown AML cells had a significant lowercontribution of GFP+ cells, as compared to the mice that receivedcontrol transduced AML cells. As the phenotype of the trans-planted AML cells was CD34+CD33+ (Supplementary Figure 6G),

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these cells represent cells of leukemic origin. Taken together,these data demonstrate that CITED2 is highly expressed in asubset of AML CD34+ cells, and that CITED2 has a critical functionin maintaining long-term leukemic growth both in vitro andin vivo.

DISCUSSION

We present data showing that enhanced expression of CITED2influences the function of normal human HSPCs in vitro and in vivoand that interfering with CITED2 expression has a profound

Figure 5. CITED2 expression is necessary for leukemic stem cell maintenance. (a) CD34+ cells from AML (n= 28) and CD34+ cells from normalbone marrow (NBM, n= 9) were isolated and investigated for CITED2 mRNA expression by Q-PCR. (b) Scatter plot analysis of CITED2expression within CD34+ cells from AMLs with varying degrees of SPI1 expression. The Spearman’s ranked correlation coefficient is indicated.(c) CD34+ cells from AML nos. 1 and 29 were transduced with lentiviral PU.1-ERt2 or control, 4-OHT treated for 3 days and mRNA was isolated.Subsequently, Q-PCR was performed for CITED2. (d) Representative example of AML CD34+ cells transduced with control hairpins (scrambledcontrol) or short hairpins against CITED2, indicating similar transduction efficiencies. (e) Sorting lentivirally transduced CD34+ cells from AMLpatients (n= 7) demonstrates an ~ 55% knockdown of CITED2 mRNA expression, as measured by Q-PCR. Mean of the AMLs is presented witherror bars denoting standard deviation. (f) Lentiviral knockdown of CITED2 in CD34+ AML cells demonstrate that CITED2 expression in thesecells is essential for the inititation of long-term leukemic cultures on MS5 stromal layers. Five representative growth curves are shown. (g) Onerepresentative AML from the non-responsive group with low CITED2 expression demonstrates that CITED2 is not essential for the initiation ofleukemic growth in this particular AML.

CITED2 is essential for leukemia maintenancePM Korthuis et al

8

Leukemia (2014) 1 – 11 © 2014 Macmillan Publishers Limited

Table1.

Patien

tch

aracteristics

AMLpa

tient

FAB

Risk

FLT3

NPM

DNMT3A

Cytogenetics

CITED2knockdow

nPU

.1overexpression

%Tran

sductio

n

Control

shCITED2

1M2

Good

WT

ND

WT

t(8.21

)Growth

impaired

CITED

2expressiondown

4535

2M1

Interm

ediate

WT

ND

WT

NK

3M5

Poor

WT

ND

WT

5q−;−

7;2p

−;1

7p4

M5

Unkn

own

ITD

WT

WT

ND

5M4

Interm

ediate

WT

ND

WT

NK

6M4

Interm

ediate

WT

WT

WT

Inv1

6Failedexpan

sion

2117

7M1

Interm

ediate

ND

WT

R88

2C

NK

Growth

impaired

NodownregulationofCITED

219

178

M1

Poor

WT

ND

WT

Inv.(3q),7−,1

0−

9M4

Good

WT

WT

ND

Inv1

6,c-kit+

10ND

Interm

ediate

WT

WT

R882H

NK

Failedexpan

sion

NodownregulationofCITED

250

5411

M1

Interm

ediate

ITD

Cyt

R88

2C

NK

Growth

impaired

NodownregulationofCITED

219

1712

M4e

oGood

WT

WT

ND

t(16

;16)(p13

;q22

),c-kit+

13M1

Poor

ITD

WT

WT

NK

14M5

Interm

ediate

WT

ND

WT

NK

15M0

Interm

ediate

ITD

WT

WT

NK

Growth

impaired

1213

16M5

Interm

ediate

ITD

ND

WT

NK

17M4

Interm

ediate

WT

ND

WT

NK

18M1

Unkn

own

ITD

ND

WT

NK

19M5

Interm

ediate

ITD

WT

WT

NK

Growth

impaired

2723

20M0

Poor

ND

WT

WT

5q−,T

risomy6

Failedexpan

sion

1416

21M2

Interm

ediate

ITD

Cyt

R882H

NK

NodownregulationofCITED

222

M2

Interm

ediate

ITD

WT

WT

NK

Noresponse

2219

23M5

Interm

ediate

ITD

Cyt

WT

NK

Growth

impaired

2824

24M5

Unkn

own

WT

WT

WT

NK

Growth

impaired

4848

25M2

Poor

WT

WT

WT

45,X

,−Y,t(8;21

),13q

−,14q

+[8]/46

,XY[2]

Growth

impaired

2221

26M2

Interm

ediate

ND

WT

WT

t(8;21

),t(q22

;q22

)27

M1

Poor

WT

WT

ND

45xx,3

p+,−7,

8p−,4

6xx

Noresponse

1010

28M1

Poor

ITD

WT

WT

NK

29M2

Poor

WT

Cyt

WT

−7,

−10

Growth

impaired

CITED

2expressiondown

2222

30M1

Poor

WT

ND

Inv.(3q),7−,10−

Growth

impairedin

vivo

35

31M2

Poor

ITD

WT

ND

47,X

Y,t(3;5)(q23

;q33

),+8[10

]Growth

impairedin

vivo

812

32ND

Poor

ITD

Cyt

ND

Complex

Growth

impairedin

vivo

1319

Abbreviations:AML,acute

myeloid

leukemia;C

ITED

2,CBP/p30

0-interacting-transactivator-with-anED

-rich-tail2

;DNMT,DNAmethyltran

sferase;

FAB,

Fren

ch–American

–British;ITD

,internal

tandem

duplication;

ND,n

otdetermined

;WT,wild

type.AMLno.

29gaveco

nsisten

tpoorqualityRNA,sonomRNAexpressionan

alysisco

uld

beperform

ed.Thebold

entriesareto

emphasizewhichmutationsarepresentin

those

particu

larAMLs

andwhether

downregulationofCITED

2ledto

growth

impairm

ent.

CITED2 is essential for leukemia maintenancePM Korthuis et al

9

© 2014 Macmillan Publishers Limited Leukemia (2014) 1 – 11

impact on primary human leukemic cells. We clearly demonstratethat CITED2 expression enhances in vitro long-term culture andin vivo engraftment into NSG mice. Our human data are thereforenot only consistent with observations in mice that CITED2 isrequired for proper murine HSCs function, but additionallydemonstrate that overexpression of CITED2 alone is sufficient tomaintain the primitive CD34+CD38− HSC pool by decreasingapoptosis and enhancing quiescence. This is in line with data fromCITED2Δ/Δ mice, where deletion of CITED2 resulted in activelycycling long-term HSCs with an increased apoptotic profile.2,28 Ourdata indicate that in human HSCs CITED2 induces the expressionof CDKN1A and CDKN1C. This is slightly different from theexperiments performed by Du et al.,28 as they have shown thatCITED2 controls the expression of the cyclin-dependent kinaseinhibitor Cdkn1c, but not of Cdkn1a.28 The mechanism behind thisis at present unclear. CITED2 probably does not bind to DNA itself,but most likely uses CBP/p300 in response to various externalsignals.1,5 We functionally validated these data demonstrating thatdownregulation of CDKN1A can rescue the CITED2-inducedquiescence in CD34+CD38− HSCs. Deletion of Cdkn1a or Cdkn1cin mice resulted in a loss of engrafting cells upon serialtransplantation.37,38 Thus by inducing CDKN1A/C-mediated quies-cence, CITED2 prevents exhaustion and hence increases overallhematopoiesis, as is demonstrated by our enhanced long-termculture and in vivo engraftment data.It appears that PU.1, one of the main transcription factors

involved in HSC maintenance and differentiation, affects CITED2expression. Lowering PU.1 activation in human CD34+ cells led toan increase in CITED2 expression, and conversely, activating PU.1led to a decrease in CITED2 expression. Previously, we observedthat HSCs from the PU.1-knockout mice also showed a fourfoldincrease in the expression of CITED2 (data not shown) andmaintained their long-term HSCs. This therefore suggested thatCITED2 contributes to the maintenance of these long-term HSCsand the subsequent development of AML.18,19,39

In AML, such a direct inverse correlation between CITED2 andSPI1 expression was observed in many, but not all cases(Figure 5b). This most likely stems from the fact that manyoncogenes affect the activation of PU.1 rather than itsexpression,11,13,17 as well as the interplay with various cytokinesand transcription factors that activate CITED2 expression.5,9,10

Nevertheless, the decreased expression of CITED2 upon PU.1reactivation in these AMLs is in line with our other data.In addition, our data demonstrated that both DNMT3A and

DNMT3B knockdown interfered with PU.1-mediated repression ofCITED2, similar to its repressive function on p16INK4A.30 Since inAML both DNMT3A and DNMT3B mutations have beenobserved32,40 and both factors are necessary for formingrepressive complexes,31 our data are consistent with suchobservations. Activation of PU.1 led to a ~ 2- to ~ 10-foldenrichment of DNMT3A binding around the PU.1 binding siteson the CITED2 promoter (Figure 4c), although because of technicalvariation in ChIP efficiency this was not significant. A similar trendwas observed toward an increase in methylation upon PU.1activation (Figure 4c), as could be expected from a de novomethyltransferase. Bisulfate conversion and pyrosequencing in 12of our AML samples demonstrated a general hypomethylationof the CITED2 promoter around the PU.1 binding sites(Supplementary Figure 5E), which was consistent with the overallenhanced CITED2 expression in AML. However, it must be notedthat the CITED2 promoter is a large CpG island, which are typicallyhypomethylated.41,42 Furthermore, we cannot exclude the possi-bility that PU.1 and DNMT3A affect the methylation of a currentlyunknown upstream enhancer of CITED2. Our observations that inAMLs with a DNMT3A mutation (discovered in ~ 20% of all AMLpatients32) PU.1 is unable to repress CITED2 expression isconsistent with such a possibility.

Whether CITED2 only contributes to AML maintenance down-stream of PU.1 or is also involved in leukemia initiation is notresolved. Our data functionally demonstrate that CITED2 isessential for long-term leukemic maintenance in vitro and in vivoand our GSEA results indicate that the CITED2-induced geneexpression programs at least partially overlap with publishedleukemia-related signatures (Supplementary Table 1).43–47

Although overexpression of CITED2 changed proliferation andapoptosis of HSCs and skewed myeloerythroid differentiation ofprogenitors, no leukemic transformation was observed. Thissuggests that additional alterations are necessary.Taken together, our findings indicate that CITED2 has a critical

role in regulating normal versus malignant hematopoiesis. Wepropose a model where CITED2 repression during myelopoiesis isamong others, regulated by PU.1, via DNMT3A/B. Together withdiminished activation of PU.1, this results in a perturbed myeloiddifferentiation program and is likely to contribute to leukemicdevelopment and/or maintenance.

CONFLICT OF INTERESTThe authors declare no conflict of interest.

ACKNOWLEDGEMENTSWe acknowledge RJ van der Lei, H Moes and G Mesander for help with flowcytometry, and Amgen Inc. and Kirin for providing cytokines. We greatly appreciatethe help of Dr A van Loon, Dr JJ Erwich and colleagues (Obstetrics departments fromthe Martini Hospital and UMCG) for collecting CB, Dr AB Mulder (Department ofLaboratory Medicine) and Professor E van den Berg (Department of Genetics) formutation analyzes and A Brouwers-Vos (Department of Experimental Hematology)for help with Illumina Bead Arrays. We also thank Dr T Plosch and M Zwiers from theDepartment of Obstetrics and Gynecology for their help with the pyrosequencing.This work was funded by an NWO VENI grant (91611105) awarded to HS.

AUTHOR CONTRIBUTIONSPMK, GB, BB, MR, JJ and HS performed experiments and analyzed data. GdH,JJS, EV and HS analyzed and discussed data. JJS, EV and HS designed theexperiments and wrote the manuscript.

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

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© 2014 Macmillan Publishers Limited Leukemia (2014) 1 – 11