ORIGINAL ARTICLE Transcriptional Dysregulation Mediated by RUNX1-RUNX1T1 in Normal Human Progenitor Cells and in Acute Myeloid L

Transcriptional dysregulation mediated by RUNX1-RUNX1T1 in normal human progenitor cells and in acute myeloid leukaemia

A Tonks1, L Pearn1, M Musson2, A Gilkes1, KI Mills1, AK Burnett1 and RL Darley1

1Department of Haematology, School of Medicine, Cardiff University, Cardiff, UK and 2Department of Pathology, School of Medicine, Cardiff University, Cardiff, UK

The t(8;21)(q22;q22) occurs frequently in acute myelogenous inv(16) patients are predominantly associated with the M4 leukaemia and gives rise to the transcription factor fusion subtype.5 Both abnormalities are indicative of good prognosis.6 protein, RUNX1-RUNX1T1 (also known as AML1-ETO). To identify the genes dysregulated by the aberrant transcriptional In our previous work we showed that RUNX1-RUNX1T1 activity of RUNX1-RUNX1T1, we used microarrays to determine selectively inhibits the granulocytic and erythroid differentiation 7,8 the effect of this mutation on gene expression in human capacity of normal human progenitors. It is implicit that progenitor cells and during subsequent development. Gene RUNX1-RUNX1T1 disturbs the transcriptional processes that signatures of these developmental subsets were very dissimilar regulate haematopoietic development, and indeed it may inhibit indicating that effects of RUNX1-RUNX1T1 are highly context transcription of RUNX1 target genes by recruiting complexes dependent. We focused on gene changes associated with 9,10 the granulocytic lineage and identified a clinically relevant consisting of mSin3 and nuclear hormone co-repressors. On subset of these by comparison with 235 leukaemia patient the other hand, RUNX1-RUNX1T1 has also been reported to 11,12 transcriptional signatures. We confirmed the overexpression of promote gene transcription, for example, AP-1. Previous a number of significant genes (Sox4, IL-17BR, CD200 and studies have identified target genes dysregulated by RUNX1- c-catenin). Further, we show that overexpression of CD200 and RUNX1T1 in human leukaemia cell lines such as U93713,14 or c-catenin is also associated with the inv(16) abnormality which in L-G mouse cells.15 However, the consequences of RUNX1- like RUNX1-RUNX1T1 disrupts core binding factor activity. We investigated the functional significance of CD200 and c-catenin RUNX1T1 expression are likely to be critically dependent on the overexpression in normal human progenitor cells. The effect of transcriptional environment in which the fusion protein is IL17 on growth was also assessed. Individually, none of these expressed. changes were sufficient to recapitulate the effects of RUNX1- In this report, we determined the effect of RUNX1-RUNX1T1 RUNX1T1 on normal development. These data provide the most on gene expression in human haematopoietic progenitors as comprehensive and pertinent assessment of the effect of well as during their subsequent development into granulocytic, RUNX1-RUNX1T1 on gene expression and demonstrate the highly context-dependent effects of this fusion gene. monocytic and erythroid cells. To focus on genes relevant to Leukemia (2007) 21, 2495–2505; doi:10.1038/sj.leu.2404961; leukaemogenesis, we also performed microarray analysis of 235 published online 27 September 2007 AML patients to identify dysregulated genes of clinical Keywords: plakoglobin; AML1-ETO; gene profiling; significance. Using this strategy we identified molecular haematopoiesis; differentiation differences in human cells that may be responsible for the observed dysregulated development arising from RUNX1- RUNX1T1 as a single abnormality. We identified dysregulation of a number of genes with putative involvement in leukaemogenesis. We additionally performed analyses of a number of these, including g-catenin (also known as plakoglobin or Jup), Introduction which plays a role both in cell adhesion and in Wnt signalling.16 We show that g-catenin is specifically overexpressed in both One of the most common molecular abnormalities in acute RUNX1-RUNX1T1 cells and in human t(8;21) disease and myelogenous leukaemia (AML), observed at a frequency of inv(16) leukaemias and that unlike RUNX1-RUNX1T1, over- 1 approximately 12%, is the t(8;21)(q22;q22) translocation. This expression of g-catenin in human cells does not inhibit involves the fusion of RUNX1 (also known as AML1)on differentiation. chromosome 21 with the RUNX1T1 gene (also known as ETO) on chromosome 8, leading to the formation of the RUNX1- 2 RUNX1T1 fusion protein. Fusions arising from both t(8;21) and Materials and methods inv(16) disrupt the core binding factor function and the 3 transcriptional events regulating haematopoiesis. The impor- Cell culture and gene transduction tance of these abnormalities in leukaemogenesis is underscored þ Human CD34 cells (495% pure) were isolated, cultured and by the fact that each is closely associated with a particular 8 transduced as previously described. PINCO-g-catenin was subset of AML; 90% of patients with an t(8;21) are associated 17 kindly provided by Xiaomin Zheng (Frankfurt, Germany). with an inhibition in granulocytic differentiation equating with 4 cDNA for CD200 was kindly provided by I.M.A.G.E consortium an AML subtype, French–American–British (FAB)-M2 while 18 cDNA clone I.D. 5299899 and subsequently subcloned into the PINCO expression plasmid. Transduced cells were used for Correspondence: Dr A Tonks, Department of Haematology, Cardiff Affymetrix microarray analysis and for functional/phenotypic University, 7th Floor, Heath Park, Cardiff, S Wales CF14 4XN, UK. E-mail: [email protected] analysis at day 3 (n ¼ 4) and later time points (n ¼ 4). Colony Received 1 May 2007; revised 23 August 2007; accepted 23 August assays were initiated on day 3 by limiting dilution in 96U plates 2007; published online 27 September 2007 (0.3 cells per well) in liquid medium containing IL-3, SCF, RUNX1-RUNX1T1 dysregulated genes A Tonks et al 2496 G/GM-CSF at 5 ng mlÀ1 and IL-17B/E (1, 10 or 100 ng mlÀ1 P’ value indicating whether a transcript was reliably detected (R&D systems, Abingdon, UK). Cultures were incubated at 371 C (present), marginally detected (marginal) or not detected (absent) with 5% CO2. Individual colonies (450 cells) were harvested were exported into Genespring v7.3 (Agilent Technologies, after 7 days following plating. To calculate the growth of Wokingham, UK). Target intensity values from each array were myeloid cells at each time point indicated, a portion of each normalised per array (to median) and per gene (to 50th culture was analysed by flow cytometry (see below) to percentile). The resulting expression levels on each chip were determine the proportion of CD13 þ CD36lo cells present in normalised around 1, which enabled the comparison of different the culture. experiments. Data were filtered to remove all genes that RUNX1-RUNX1T1 and control matched transduced cells changed by o1.5-fold in RUNX1-RUNX1T1-expressing cells were also cultured until day 6 and were fractionated into the when compared to controls and an additional three-arm main progenitor groups for microarray analysis. A depletion approach was applied to identify differentially regulated genes. strategy using the Miltenyi Biotec MiniMACS system was used Statistically significant changing genes were identified using for this purpose. Briefly, myeloblasts (CD15 þ ) were enriched ANOVA (parametric test based on the cross gene error model) from mixed lineage transduced cells by serial depletion of (‘ACGEM’) (Po0.05) or a ‘paired t-test’ in which RUNX1- monoblasts (CD14hi) and erythroblasts (CD36 þ ) using immu- RUNX1T1-transduced human progenitor cells were normalised nomagnetic beads directed against those antigens. Fractionation to respective controls and filtered on confidence in Genespring efficiency and lineage identities were confirmed using cell (Po0.05). Where possible the Benjamini–Hochberg multiple surface phenotyping and RNA was extracted as described in testing correction was applied to both these strategies, otherwise Supplementary Methods. the level of significance was reduced to Po0.01. A third nonstatistical ‘pairing’ approach was applied to changes in gene expression (present or marginal and 41.5-fold differences) in Cell surface phenotype and differentiation analysis individually paired human progenitor samples. Each of these At the time points indicated, liquid cultures were analysed by three analytical approaches was combined in a Venn’s diagram four-colour immunophenotypic analysis. Cells were stained to identify biological and statistically significant changing genes with CD13-APC (Universal Biologicals Cambridge, UK) in by at least two of the three strategies (Figure 2a). combination with CD15-biotin (Sigma, Poole, Dorset) or CD36- biotin (Ancell, Bayport, MN, USA) and one of the following PE- labelled antibodies (Dako, Cambridgeshire, UK): CD11b or Protein expression analysis of some gene identified by CD34; biotinylated CD15 or CD36 were subsequently labelled microarray analysis with Streptavidin-PerCP-Cy5.5 (BD). All reactions were con- To confirm RUNX1-RUNX1T1 protein expression and trolled with the appropriate isotype-matched irrelevant anti- microarray overexpression of Sox4, IL-17BR and g-catenin in body. Incubations were performed at 41 C for 30 min in the RUNX1-RUNX1T1-transduced cells, we performed western blot presence of 0.5% human gamma globulin (Sigma). Reagent analysis. Detection of each protein was determined using a concentrations were as recommended by the manufacturer. monoclonal or polyclonal antibody in conjunction with an anti- Flow cytometric data (acquired using a FACSCalibur cytometer, mouse Amersham ECL Advance Western Blotting Detection Kit BD Biosciences, Oxford, UK) were analysed using FCS express (GE Healthcare, Bucks, UK) according to the manufacturer’s v3.0 (DeNovo Software, Ontario, Canada). The threshold for instruction. Antibodies used include ETO (1:1000, Oncogene, GFP positivity was determined from the autofluorescence of Darmstadt, Germany), g-catenin (1:1000, BD Biosciences), Sox4 GFP negative cells in mock-transduced cultures; for antibody (1:50 Abcam, Cambridge, UK) and IL-17BR (1:250 R&D labelled cells, this was determined from control stained cells (in Systems). In the case of CD200, a PE conjugated antibody (BD each case, set to 95% of controls). Significance of difference was Biosciences) was used and protein expression was determined tested using the Student’s t-test or ANOVA. Minitab software by flow cytometry using similar protocols outlined above (see version 12.0 (Minitab Inc., Coventry, UK) was used for all ‘Cell surface phenotype and differentiation analysis’). analyses.

Patients and control sample materials Results A subset of bone marrow samples (n ¼ 235) from patients who had enrolled in the MRC-UK AML clinical trial protocol and Generation of human progenitor cells expressing were sent to or collected locally and fully processed at UHW RUNX1-RUNX1T1 for microarray analysis Cardiff were used in this study. Sample collection, processing, Human CD34 þ haematopoietic cells (which constitute a mixed clinical and biological characteristics of normal healthy donors progenitor population) were retrovirally transduced with a (n ¼ 9), patients with a t(8;21)(n ¼ 11) and other FAB-M2 vector co-expressing RUNX1-RUNX1T1 and GFP. Using this patients (that is, without the t(8;21)) (n ¼ 28) are described in approach we generated control (GFP alone) and RUNX1- Supplementary Table 1. Following storage in tissue banks, RNA RUNX1T1 matched CD34 þ cultures (four independent repli- of sufficient quantity and quality were extracted (see Supple- cate sets) that were used for microarray analysis immediately mentary Methods). following retroviral transduction (day 3 of culture). Due to the high frequency of retroviral transduction (B70%), enrichment of transduced cells was unnecessary (Supplementary Figure 1). The Affymetrix microarray analysis expression of the fusion gene in this context has previously been The Affymetrix data are available as Supplementary material on established.8,19 In addition, control and RUNX1-RUNX1T1 http://www.ebi.ac.uk/arrayexpress/query/entry (accession num- matched populations constituting individual lineages (erythro- ber e-mexp-583). MAS software (target intensity) was used to cytic, granulocytic and monocytic) were isolated from day 6 translate the scanned images into expression analysis files. cultures (Figure 1). This enabled us to also examine lineage- Unique signal values for each gene together with a ‘Detection specific effects of RUNX1-RUNX1T1.

Leukemia RUNX1-RUNX1T1 dysregulated genes A Tonks et al 2497

Figure 1 Purification of individual lineages expressing RUNX1-RUNX1T1 used in Affymetrix microarray analysis. Representative density plots of RUNX1-RUNX1T1 transduced human progenitor cells enriched for specific lineages on day 6 of culture using a MiniMACS depletion strategy. Quadrants delimit background fluorescence of control-stained/mock-transduced cells. Similar data were generated from control cultures expressing GFP alone (not shown).

Identiﬁcation of genes dysregulated by RUNX1- strategy (pairing), which took into account changes in individual RUNX1T1 in normal human haematopoietic cells pairs of cord blood (see Materials and methods). We analysed Following total RNA isolation, cDNA synthesis and in vitro dysregulated probe sets that passed at least two of these three transcription, biotinylated cRNA from each sample was criteria (Figure 2a and Supplementary Table 2). The most striking hybridised to Affymetrix human 133A oligonucleotide arrays, feature of these lists (#1–#4) was their dissimilarity with only six which allowed the simultaneous analysis of approximately probe sets common to all four lists (Figure 2b). These genes 14 500 genes (represented by 22 283 probe sets) in both included CYP1A1, TM4SF1, CRHBP, HSP27 and Sox4 (repre- individual lineages and also on temporal changes (over days sented by two probe sets) (Table 1). These data suggest that the 3–6). Supplementary Figure 2 shows an unsupervised hierarch- effects of RUNX1-RUNX1T1 are highly context dependent. ical cluster tree, which demonstrates the reproducibility of Consistent with a previous study using mouse L-G cells,20 the individual samples between transduced cultures of different majority of gene changes were increases in expression (list #1: lineage subsets. To identify statistically signiﬁcant changes in 50% up; list #2: 76% up; list #3: 77% up; list #4: 89% up); expression, we performed a t-test, ACGEM and a nonstatistical therefore, though RUNX1-RUNX1T1 may directly repress the

Leukemia RUNX1-RUNX1T1 dysregulated genes A Tonks et al 2498

Figure 2 Identification of probe sets dysregulated by RUNX1-RUNX1T1. (a) Dysregulated probe sets were identified from at least two of the three MAS analyses, as outlined in Methods. The probe sets identified in day 3 were filtered for common probe sets identified in day 6 lineage data sets. For gene identities see Supplementary Table 2. (b) Mixed progenitor CD34 þ day 3 transduced cells and day 6 subsets (granulocytes, monocytes and erythrocytes) were compared to each other by Venn’s diagram to identify common dysregulated probe sets. Parentheses indicate the number of dysregulated probe sets in the gene list as identified in (a) list numbers. Areas of overlap are not proportional to the number of dysregulated probe sets.

expression of some genes,21 these data may not support the view changes (22 genes) were sustained in the granulocyte-enriched that the overall consequence of RUNX1-RUNX1T1 expression is population (Table 1 and Figure 2a, list #5). Each of these transcriptional repression. A caveat to this is that the presence of coincident changes was co-directional (that is, they were low levels of untransduced cells within the AML1-ETO similarly dysregulated on day 6 compared with the day 3 list). transduced population will to some extent reduce the sensitivity Features of this list included strong up regulation of TM4SF1 of detection of negatively regulated genes. Further, in day 3 (L6), which is commonly overexpressed in solid tumours22 and progenitors, we did observe repression (approximately twofold) the inducible cytochrome P450 isozyme, CYP1A1, which is of a number of developmentally significant transcription factors known to be developmentally regulated in haematopoiesis.23 (CEBPa, GATA-1, PU.1 and RARa) as well as repression of a number of genes associated with erythroid development (NF-E2, EKLF and GATA2). However, none of these changes were Correlative analysis of gene changes in AML patients sustained in any of the individual lineages, suggesting that these We next established which of the above gene changes were changes were correlative with the developmental disruption common to leukaemia patients. Using our database of 235 AML provoked by RUNX1-RUNX1T18,19 rather than being causative. patients we identified which of these changes were dysregulated We reasoned that we could enrich for RUNX1-RUNX1T1 in t(8;21) patients and in other AML subtypes including t(8;21)- target genes likely to be causal in mediating developmental negative M2 patients (other M2). A high proportion of the probe dysregulation by looking for gene changes, which were sets dysregulated in human progenitor/granulocyte cells were sustained in the granulocyte lineage (which is most closely also dysregulated in t(8;21) patients (12/26; 46%) representing associated with t(8;21) leukaemias). Using this approach, 26 9/22 genes (Table 1). These genes were thereby defined by this

Leukemia Table 1 Identities of the 22 gene changes common to RUNX1-RUNX1T1 day 3 progenitors and day 6 granulocytes

Affymetrix ID GenBank Fold Common GO biological process Coincident Coincident changes in FAB classiﬁed AML accession no. change names changes in other (day 6) lineages t(8;21) M0 M1 Other M3 M4 M5 Inv(16) M2

205749_at NM_000499 8.53m CYP1A1 Electron transport M, E 20938 (6/7)_ata M90657 5.74m TM4SF1 Biological_process unknown: integral to plasma M, E || || membrane 205984_at NM_001882 5.56m CRHBP Learning and/or memory; pregnancy; signal M, E | transduction 213506_at BE965369 4.02m F2RL1 Protease-activated receptor that couples to || | guanosine-nucleotide-binding proteins. 201015_s_at NM_021991 2.46m c-Catenin Cell adhesion ||||| | 219255_x_at NM_018725 2.34m IL-17BR Regulation of cell growth E | 212614_at BG285011 2.31m ARID5B Negative regulation of transcription, DNA-dependent regulation of transcription, DNA-dependent 212224_at NM_000689 2.31m RALDH Aldehyde metabolism 220532_s_at NM_014020 2.14m LR8 Histogenesis and organogenesis E | 201841_s_at NM_001540 2.00m HSP27 Regulation of translational initiation M, E || ||

208965_s_at BG256677 1.94m IFI16 Regulation of nucleobase, nucleoside, nucleotide || Tonks A genes dysregulated RUNX1-RUNX1T1 and nucleic acid metabolism a m ||||

20141(6/7/8)_at NM_003107 1.93 Sox4 Regulation of transcription, DNA-dependent M, E al et 209583_s_at AF063591 1.93m CD200 Immune response; cell-to-cell responses E || | 221942_s_at AI719730 1.77m GUCY1A3 Signal transduction, cell communication ||||| 208937_s_at D13889 1.72m Id-1H Development; regulation of transcription from Pol II E promoter 211734_s_at BC005912 2.56k FCER1A Immune response 20243(6/7)_s_ata NM_000104 2.55k CYP1B1 Electron transport; visual perception M ||| 207067_s_at NM_002112 2.53k HDC Responsible for histamine synthesis M 210139_s_at L03203 2.28k GAS3 Mechanosensory behavior; negative regulation of cell | proliferation; development; synaptic transmission 204174_at NM_001629 1.98k ALOX5AP Inflammatory response; leukotriene |||||||| biosynthesis 204912_at NM_001558 1.88k IL-10R Signal transduction, cell communication | 204392_at NM_003656 1.84k CAMK1 Protein amino acid phosphorylation; signal transduction Dysregulated genes are listed according to their expression level with respect to day 6 controls. Changes coincident in other lineages (Supplementary Tables 4 and 5) are denoted by: E (erythroid) and M (monocytic). Changes coincident in FAB classified patients (vs normals) are also indicated (|). Other M2: AML M2 patients without a t(8;21). Clinically significant RUNX1-RUNX1T1 target genes are in bold. Individual genes are represented by numbered probes sets on the Affymetrix HG-U133A oligonucleotide GeneChip. aDenotes genes with multiple probe sets where average fold regulation is displayed. Leukemia 2499 RUNX1-RUNX1T1 dysregulated genes A Tonks et al 2500

Figure 4 The effects of g-catenin or CD200 overexpression on the protein expression of RUNX1-RUNX1T1 target genes in human progenitor cells. (a) Western blot analysis of Sox-4, IL-17BR and Figure 3 Confirmation of RUNX1-RUNX1T1 target gene overexpres- g-catenin expression in day 6 human progenitor cells transduced with sion in human progenitor cells. (a) Western blot analysis of Sox4, IL- g-catenin or CD200. (b) CD200 protein expression was confirmed by 17BR, RUNX1-RUNX1T1 and g-catenin overexpression in day 6 flow cytometric analysis of progenitor cells transduced with g-catenin human progenitor cells transduced with RUNX1-RUNX1T1. GAPDH or CD200. MFI, mean fluorescence intensity. was used as a loading control. In the case of g-catenin, multiple banding results from differential phosphorylation. Densitometric fold increases compared to control (GFP) are shown when corrected for acts as a stimulatory haematopoietic cytokine by expanding loading. (b) Overexpression of CD200 was confirmed by flow 29 cytometric analysis using a PE conjugated antibody. MFI, mean myeloid progenitors and initiating granulocyte proliferation. fluorescence intensity. Since the overexpression of its receptor might in part explain the manifestation of RUNX1-RUNX1T1-associated disease in this lineage, we determined whether RUNX1-RUNX1T1-expressing criterion as a potentially clinically relevant subset. This selection human progenitor cells had an altered response to IL-17. Colony excluded probe sets with the largest fold differences in assays were performed in the presence of IL-17B and E alone or particular, TM4SF1; however, previous reports have also shown in combination (the ligands for the B-type IL-17 receptor). As we this gene not to be overexpressed in t(8;21) patients.24 In have previously shown RUNX1-RUNX1T1-suppressed initial comparison to other AML subsets, two of these (IL-17BR and IL- colony forming potential8 compared with control cells (Supple- 10R) were exclusive to t(8;21) patients, while only one gene mentary Table 3); however, neither IL-17B nor IL-17E nor a (ALOX5AP) was dysregulated in all subtypes. Of the remainder, combination of both cytokines significantly affected their colony a high proportion were also found to be similarly dysregulated in forming potential at any concentration tested (Supplementary the M0-M2 subtypes and inv(16) patients (Table 1). Table 3). Further, when RUNX1-RUNX1T1-expressing cells Four of these potentially clinically relevant gene changes were cultured in the presence of these cytokines (either (Sox4, IL-17BR, CD200 and g-catenin) have been shown from individually or in combination), there was no change in the previous work to be functionally significant in the haemato- growth profile of these cells (Supplementary Figure 3). These poietic context.14,25–28 Each was shown to be also over- data suggest that while RUNX1-RUNX1T1 may increase the expressed at the protein level, thereby validating the expression of IL17B receptor, this does not apparently correlate microarray data (Figure 3). Further, we overexpressed CD200 with an altered response to known ligands of this receptor. or g-catenin in human CD34 þ progenitor cells (Supplementary CD200 is a membrane-associated antigen, which is broadly Figure 1) to determine whether their overexpression influenced expressed on many different cell types and appears to have the expression of the other clinically relevant proteins (Sox-4, IL- immunosuppressive function.30 Since its expression is associated 17BR, CD200 or g-catenin). Figure 4 demonstrates that neither with poor prognosis in both multiple myeloma and AML,28,31 we CD200 nor g-catenin overexpression modulated expression of examined whether CD200 affected the development of haemato- these proteins, indicating that the overexpression of these poietic progenitor cells. We overexpressed CD200 in human proteins was not inter-dependent. Sox4 is a transcription factor, CD34 þ progenitor cells (Supplementary Figure 1) and followed which has not been previously shown to be overexpressed in the development of haematopoietic progenitor cells in bulk liquid AML but has been shown to have the capacity to inhibit culture and in colony assays. Overexpression of CD200 had no granulocytic differentiation in 32D cells.25 Overexpression of significant effect on the myeloid colony forming potential interleukin-17 receptor (IL-17BR) has been previously asso- (Supplementary Table 3). Further, we observed no changes in ciated with t(8;21) leukaemias.26 It has been reported that IL-17 proliferation or in the differentiation of cells overexpressing CD200

Leukemia RUNX1-RUNX1T1 dysregulated genes A Tonks et al 2501 (data not shown). Taken together, these data suggest that while differentiation of granulocytic cells, thereby extending their CD200 is dysregulated by RUNX1-RUNX1T1, it does not proliferative capacity.7,8 It has previously been shown that recapitulate any of the effects observed with the fusion gene on g-catenin overexpression promotes self-renewal of haemato- haematopoietic development. poietic progenitor cells,17 an observation we were able to g-Catenin (also known as JUP, plakoglobin) functions both as corroborate by limiting dilution/serial replating (data not a major structural protein in the adherens junction complex and shown). However, it is not known whether this protein can as a transcription factor when translocated to the nucleus.32 It affect the development of human haematopoietic cells. We, has previously been shown to be upregulated by both RUNX1- therefore, compared the effect of g-catenin with RUNX1- RUNX1T1 and PML-RARa in U937 cells.14 These authors also RUNX1T1 on the development of these cells in bulk liquid indicated that this protein was upregulated in AML patients culture. Using the same retroviral vector as employed for expressing these fusions (using a pooled group of 9 patients RUNX1-RUNX1T1 overexpression (Supplementary Figure 1), compared to 53 patients without these translocations). Using our we overexpressed g-catenin in human CD34 þ progenitor cells larger cohort of 235 AML patients, we were able to correlate (Figure 6a). Consistent with our previous observations, RUNX1- g-catenin with each AML subtype (Figure 5). We found t(8;21) RUNX1T1-expressing cells displayed an initial inhibition in patients significantly (Po0.01) overexpressed g-catenin by 3.0- growth (threefold reduction on day 6 compared with controls) fold when compared to FAB-M2 patients without this cytoge- followed by extended proliferation of differentiation-blocked netic abnormality. Expression of g-catenin in patients with PML- myeloid cells (Figure 6b). In contrast, cells overexpressing RARa (M3 subtype) was significantly lower that that observed in g-catenin proliferated in a similar manner to control cells. The t(8;21) patients and otherwise not significantly different from morphology of these cultures was examined on day 17 of closely related subtypes. One striking observation was that, culture (Figure 6c). As expected, RUNX1-RUNX1T1-expressing patients expressing an inv(16) mutation (generally associated cells displayed an immature myeloid phenotype as previously with FAB-M4) also significantly overexpressed g-catenin ex- described.8 In contrast, cultures overexpressing g-catenin had pression by 2.9-fold when compared to M4 individuals without normal morphology dominated by mature neutrophils. To an inv(16) mutation (Po0.0001). This data, therefore, show for confirm these data we examined the development of these the first time that overexpression of g-catenin is particularly cultures over 15 days following retroviral transduction by using associated with leukaemias with mutations affecting core multiparameter flow cytometry. Normal myeloid development binding factor activity such as t(8;21) and inv(16), both of is associated with loss of CD34 expression and upregulation of which are associated with good prognosis. CD14, CD15 and CD11b. We found that like RUNX1- RUNX1T1, g-catenin overexpression caused significant reten- tion of CD34 expression on myeloid cells (Figure 6d; left panel) supporting the previous observation that g-catenin overexpres- Unlike RUNX1-RUNX1T1, overexpression of g-catenin 17 does not inhibit granulocytic development sion was able to promote self-renewal. However, while A defining feature of RUNX1-RUNX1T1 is its ability to promote RUNX1-RUNX1T1-expressing cells delayed upregulation of the self-renewal of human cells and selectively inhibit the markers associated with granulocytic development, CD15 and CD11b (Figure 6d; middle panels), overexpression of g-catenin showed no delay in the upregulation of these antigens compared with control cells (and was indeed somewhat enhanced at some time points). Neither g-catenin, nor (as previously shown) RUNX1-RUNX1T1, suppressed the expression of the monocytic antigen CD14 (Figure 6d; right panel). Taken together, these data suggest that unlike RUNX1-RUNX1T1, overexpression of g-catenin does not significantly impair subsequent granulocytic development.

Discussion

Certain fusion proteins, of which RUNX1-RUNX1T1 is an archetypal example, drive the leukaemogenic process to the extent that they give rise to discrete subsets of AML. Previously, we have devised a model based on normal human progenitors, which enabled us to analyse the consequences of RUNX1- RUNX1T1 expression as a single abnormality. These studies have shown that RUNX1-RUNX1T1 is able to inhibit the differentiation of myeloid cells and promote their self-renewal (consistent with its putative initiating role).8,19 Using Affymetrix microarray analysis, we have identified the genes dysregulated by RUNX1-RUNX1T1 in normal human progenitors and during subsequent dysregulated development of specific myeloid Figure 5 Overexpression of g-catenin in AML. Microarray data lineages. demonstrating the normalised expression of g-catenin in AML patients We found that RUNX1-RUNX1T1 induced a large number of ¼ (n 235) classified according to the FAB criteria. Horizontal bars gene changes in CD34 progenitor cells as well as in the indicate the average normalised expression of g-catenin within each AML subtype. Horizontal line represents an upper threshold of g- individual myeloid lineages. The number of probe sets catenin expression in normal individual donors (mean þ 2 s.d.; 0.82). dysregulated in each lineage varied considerably and there w M2 patients without a t(8;21). *M2 patients with a t(8;21). was little overlap between these data sets (Figure 2b). Together

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Figure 6 Effect of g-catenin overexpression on human myeloid development and differentiation. (a) Western blot analysis of g-catenin (upper) and GAPDH (below) in human progenitor cells transduced with g-catenin. Densitometric fold increases compared to control (GFP) are shown when corrected for loading. (b) Expansion of g-catenin transduced cells cultured in IL-3, SCF and G/GM-CSF. Data indicate mean71 s.d. (n43). Note the growth curves for g-catenin and control cells overlay exactly. (c) Representative cytocentrifuge preparations stained with Wright-Giemsa. (d) Immunophenotypic proﬁle of myelocytes (GFP þ , CD13 þ , CD36À) labelled with antibodies to one other determinate as indicated. Data indicate w mean71 s.d. (n ¼ 5). *Po0.05, Po0.001 indicate statistical signiﬁcance.

these data indicate that the effect of RUNX1-RUNX1T1 was four (CSF-1, EAR-2, NF-E2 and CEBPa) were dysregulated in highly context dependent. This, in turn, probably explains the t(8;21) patients. As has been previously reported,34 we observed poor concordance with RUNX1-RUNX1T1 targets identiﬁed in repression of CEBPa in day 3 progenitors (2.3-fold; see other systems (which mostly used cell lines13–15). Of the Supplementary Table 2); however, repression of CEBPa was RUNX1-RUNX1T1 target genes previously cited in a recent not sustained during subsequent development of any lineage review,33 only eight (ID2, CD11a, NF-E2, CSF-1, CEBPa, JUND, and consistent with this, there was also no signiﬁcant repression UBP43 and EAR-2) were dysregulated in any of our RUNX1- of this gene in t(8:21) patient blasts when compared with RUNX1T1 gene lists (Supplementary Table 2) and of these only normals (which are most closely developmentally matched with

Leukemia RUNX1-RUNX1T1 dysregulated genes A Tonks et al 2503 our day 6 granulocyte preparation). Previously, it has been that overexpressing Sox4 inhibits granulocytic differentiation.25 reported that t(8:21) patients have dramatically reduced levels of Whilst the dysregulation of Sox4 has not been confirmed in CEBPa compared with other types of AML35 and indeed in our human leukaemias, it has, however, been implicated in data sets t(8:21) patients did express lower levels than non- carcinoma of the colon39 suggesting that Sox4 may contribute t(8:21) M2 patients, though the repression was only twofold (as to cancer development. opposed to the sixfold previously reported using RT–PCR based Another upregulated gene with putative involvement in analysis). We compared part of our data set (Figure 2, gene list leukaemogenesis was CD200 (also known as Mox2). Ectopic #1 and Supplementary Table 2) with a previous study by Mulloy overexpression of CD200 did not, however, affect haemato- et al.,36 which examined the effect of RUNX1-RUNX1T1 on poietic progenitor development using the model described here, CD34 þ gene expression. Using the same statistical analysis in though this was not surprising given that CD200 has no intrinsic this present study, we were able to identify seven dysregulated signalling activity.40 Nevertheless, CD200 confers poor prog- probe sets representing the following five genes CYP1A1, nosis in noncore-binding factor-associated AMLsFpossibly by HRH1, FCN1, NPTX1 and CKB. The only genes common to provoking immunosuppression and inhibiting immunological our data were CYP1A1 and NPTX1. However, since Mulloy clearance of residual disease. From this perspective, it is et al. performed only three replicate analyses, few gene changes paradoxical that CD200 should be overexpressed in t(8;21) were significant. We, therefore, compared all gene changes and inv(16) leukaemias, which are generally associated with (41.5-fold) in probe sets common to both GeneChips and found good prognosis. It is possible that even within these subgroups that 43% of gene changes were present in both data sets. Apart the level of CD200 expression may influence survival; however, from the inclusion of nonstatistical gene changes, some of the we currently have insufficient patients within these groups to test discrepancies in the data sets may arise from the fact that the this possibility. CD34 þ culture/infection protocol performed by Mulloy et al. Of the nine genes (Table 1) that were determined to be was more protracted than that employed here and, as we have dysregulated by RUNX1-RUNX1T1, only g-catenin had pre- shown, differentiation strongly influence RUNX1T1-RUNX1T1- viously been associated with dysregulation in response to induced gene changes. In support of this, we found a greater RUNX1-RUNX1T1. Further, g-catenin was the only gene overlap in our developmentally sustained gene changes (Table 1) containing a RUNX1 consensus binding site (TG(T/C)GGT) in with a 75% concurrence rate. Four gene changes were not seen its promoter/enhancer region. g-Catenin is a close homologue of in the data set published by Mulloy et al.(CD200, ALOX5AP, b-catenin, which has been intensively studied because of its PMP22 and L6), whereas our data only demonstrated a important roles in development and epithelial cancer.41 Each of significant upregulation of TrkA in t(8;21) patients. Taking these these catenins appears to function both as a major structural data as a whole, our conclusion is that gene profiling for protein in the adherens junction complex and as a transcription RUNX1-RUNX1T1 target genes is only relevant to the context in factor when translocated to the nucleus.32 Our observations which it is performed and it is for this reason we have based our showed that g-catenin was overexpressed in normal human study on normal human progenitors cells, which is the most progenitor cells transduced with RUNX1-RUNX1T1 (B2.4- relevant model system available. fold), an observation confirmed at the protein level. This is This study is the first to employ the U133 GeneChips in this consistent with previous data based on U937 cells transduced context, which (containing over 22 000 probe sets) inevitably with RUNX1-RUNX1T1.14 We have extended these studies by meant that new RUNX1-RUNX1T1 target genes would be identifying overexpression of g-catenin as one of the funda- identified. Amongst these were a number of functionally mental gene changes associated with human progenitor significant genes which were also dysregulated in subsequent granulocytes. In addition, we examined its significance in 235 granulocytic development as well as in t(8;21) patients. Over- transcriptional signatures associated with each AML subtype. expression of both IL-17BR (encoding the receptor for IL-17B We have shown for the first time that the highest levels of and IL-17E37) and the Sox4 transcription factor have been g-catenin are associated not only with t(8;21) leukaemias but previously associated with myeloid or B- and T-cell leukaemias also with another core-binding factor-associated abnormality in BXH2 and AKXD recombinant inbred mice by virtue of the inv(16). Interestingly, g-catenin expression is also high in FAB fact they are preferential sites for proviral integration38 raising M0 patients. Since approximately 20% of FAB M0 patients have the concern that these may have been overexpressed in these Runx1 mutations, this may account for the high g-catenin data sets by the same mechanism. However, these genes were expressors in this subgroup. Unfortunately, the Affymetrix also overexpressed in t(8;21) patients and it is furthermore highly microarrays do not discriminate Runx mutations; consequently, unlikely that clonal populations overexpressing these genes we have no immediate way of determining this information. A could have emerged within the 48 h period the cells were further consideration is that these Runx mutations appear to exposed to virus. Neither of these genes has been previously have different functional consequences (compared with the 8;21 identified as a RUNX1-RUNX1T1 target genes, though over- translocation) in that they appear to act in a strongly dominant- expression of IL17BR has been previously associated with negative manner, which is hard to reconcile with enhanced t(8;21) leukaemias.26 It has been reported that IL-17 acts as a transcription of g-catenin. Both the t(8;21) and inv(16) groups stimulatory haematopoietic cytokine by initiating proliferation are associated with a favourable prognosis and it is interesting of neutrophils (reviewed Schwarzenberger and Kolls29). The that g-catenin overexpression is associated with tumour overexpression of its receptor might in part explain the suppression in epithelial cells.42,43 Furthermore, underexpres- manifestation of RUNX1-RUNX1T1-associated disease in this sion of g-catenin has been associated with poor prognosis.44 lineage. However, when human RUNX1-RUNX1T1-expressing There is also evidence that the very rare autosomal recessive CD34 þ progenitor cells were cultured with IL-17 (B or E isoform disorder in which functional g-catenin is absent may predispose or in combination), no significant effects on colony growth or to MDS and AML.45 On the other hand, there is evidence that bulk culture growth were observed, suggesting an increase in IL- g-catenin may also promote leukaemogenesis by promoting 17B receptor expression does not correlate with an increased stem cell self-renewal possibly via its role in Wnt signalling.17,46 response to known ligands. Sox4 is one of a family encoding Recent evidence shows that the inv(16) abnormality also HMG-box transcription factors. It has been shown in 32D cells promotes the self-renewal of human cells. It is, therefore, likely

Leukemia RUNX1-RUNX1T1 dysregulated genes A Tonks et al 2504 that g-catenin also contributes to enhanced self-renewing 5 Reilly JT. Pathogenesis of acute myeloid leukaemia and capacity in this context.3 inv(16)(p13;q22): a paradigm for understanding leukaemogenesis? Despite this obvious similarity with the effects of RUNX1- Br J Haematol 2005; 128: 18–34. RUNX1T1, there were also obvious contrasts: g-catenin over- 6 Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison G et al. The importance of diagnostic cytogenetics on expression did not result in initial growth inhibition or promote outcome in AML: analysis of 1612 patients entered into the MRC extended proliferative capacity. Consistent with this, g-catenin AML 10 trial. The Medical Research Council Adult and Children’s did not inhibit granulocyte differentiation. Taken together, these Leukaemia Working Parties. Blood 1998; 92: 2322–2333. results suggest that overexpression of g-catenin is able to 7 Mulloy JC, Cammenga J, MacKenzie KL, Berguido FJ, Moore MA, promote the self-renewal of progenitor cells to some extent, Nimer SD. The AML1-ETO fusion protein promotes the expansion but unlike RUNX1-RUNX1T1, overexpression of g-catenin does of human hematopoietic stem cells. Blood 2002; 99: 15–23. 8 Tonks A, Tonks AJ, Pearn L, Pearce L, Hoy T, Couzens S et al. not suppress the differentiation of granulocytic cells. It is also Expression of AML1-ETO in human myelomonocytic cells interesting to note that overexpression of g-catenin (or CD200) selectively inhibits granulocytic differentiation and promotes their did not modulate the protein expression of the RUNX1- self-renewal. Leukemia 2004; 18: 1238–1245. RUNX1T1 target genes Sox-4, IL17R or CD200 (g-catenin) 9 Lutterbach B, Westendorf JJ, Linggi B, Patten A, Moniwa M, indicating that their overexpression was not inter-dependent. Davie JR et al. ETO, a target of t(8;21) in acute leukemia, interacts One potential mechanism by which g-catenin may influence with the N-CoR and mSin3 corepressors. Mol Cell Biol 1998; 18: haematopoiesis is through its reported capacity to bind the Tcf 7176–7184. 10 Wang J, Hoshino T, Redner RL, Kajigaya S, Liu JM. ETO, fusion family of transcription factors, which regulate the transcription 47,48 partner in t(8;21) acute myeloid leukemia, represses transcription of genes such as c-myc and cyclin D1. Indeed recent studies by interaction with the human N-CoR/mSin3/HDAC1 complex. have linked increased b-catenin transcriptional activity with Proc Natl Acad Sci USA 1998; 95: 10860–10865. both AML and with chronic myelogenous leukaemia progres- 11 Frank RC, Sun X, Berguido FJ, Jakubowiak A, Nimer SD. The sion.49,50 However, g-catenin differs from b-catenin in that it is t(8;21) fusion protein, AML1/ETO, transforms NIH3T3 cells and reported to be a much less efficient transcriptional activator and activates AP-1. Oncogene 1999; 18: 1701–1710. 51 12 Rhoades KL, Hetherington CJ, Rowley JD, Hiebert SW, Nucifora G, may even decrease the affinity of some Tcfs for DNA. It has Tenen DG et al. Synergistic up-regulation of the myeloid-specific also been suggested that g-catenin may act as a competitor with promoter for the macrophage colony-stimulating factor receptor by b-catenin, implying that the relative levels of these catenins may AML1 and the t(8;21) fusion protein may contribute to leukemo- be important.52 In the model described here, however, we found genesis. Proc Natl Acad Sci USA 1996; 93: 11895–11900. no induction of b-catenin and no induction of the Tcf/LEF target 13 Alcalay M, Meani N, Gelmetti V, Fantozzi A, Fagioli M, Orleth A genes, c-myc and cyclin D1. Moreover, there was no associa- et al. Acute myeloid leukemia fusion proteins deregulate genes involved in stem cell maintenance and DNA repair. J Clin Invest tion with either RUNX1-RUNX1T1 or inv(16) AML patients and 2003; 112: 1751–1761. c-myc and cyclin D1 induction or indeed b-catenin expression. 14 Muller-Tidow C, Steffen B, Cauvet T, Tickenbrock L, Ji P, Currently, therefore we can provide no supporting evidence that Diederichs S et al. Translocation products in acute myeloid induction of either c-myc or cyclin D1 forms part of the leukemia activate the Wnt signaling pathway in hematopoietic mechanism of g-catenin action in this context. We are cells. Mol Cell Biol 2004; 24: 2890–2904. attempting to identify what the transcriptional targets of g- 15 Shimada H, Ichikawa H, Nakamura S, Katsu R, Iwasa M, catenin are in each of these contexts. Given the alternative role Kitabayashi I et al. Analysis of genes under the downstream control of the t(8;21) fusion protein AML1-MTG8: overexpression of g-catenin in cell adhesion, it is also possible that g-catenin of the TIS11b (ERF-1, cMG1) gene induces myeloid cell prolifera- overexpression may promote the capacity of RUNX1- tion in response to G-CSF. Blood 2000; 96: 655–663. 53,54 RUNX1T1-expressing cells to occupy the stem cell niche. 16 Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B et al. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science 1997; 275: 1787–1790. Acknowledgements 17 Zheng X, Beissert T, Kukoc-Zivojnov N, Puccetti E, Altschmied J, Strolz C et al. Gamma-catenin contributes to leukemogenesis We would like to thank Xiaomin Zheng and Martin Ruthardt induced by AML-associated translocation products by increasing (Johann Wolfgang Goethe-Universita¨t, Frankfurt, Germany) for the self-renewal of very primitive progenitor cells. 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