Pirin Downregulation Is a Feature of AML and Leads to Impairment of Terminal Myeloid Differentiation

Pirin downregulation is a feature of AML and leads to impairment of terminal myeloid differentiation

S Licciulli1, V Cambiaghi1, G Scafetta1, AM Gruszka1 and M Alcalay1,2

1Department of Experimental Oncology, Istituto Europeo di Oncologia, Milan, Italy and 2Dipartimento di Medicina, Chirurgia ed Odontoiatria, Universita` degli Studi di Milano, Milan, Italy

Terminal differentiation of blood cells requires the concerted factors subverts normal differentiation, and can ultimately lead action of a series of transcription factors that are expressed at to leukemogenesis.2 specific stages of maturation and function in a cell-type and dosage-dependent manner. Leukemogenic oncoproteins block Many studies have addressed the analysis of global gene differentiation by subverting the normal transcriptional status of expression in AML, but a clear identification of the specific hematopoietic precursor cells. Pirin (PIR) is a putative trans- effectors of the differentiation block that characterizes AML is criptional regulator whose expression is silenced in cells bearing still lacking. We previously analyzed the gene expression profile the acute myeloid leukemia-1 eight–twenty–one (AML1/ETO) and of U937 cells expressing the AML-associated fusion proteins, promyelocytic leukemia/retinoic acid receptor (PML/RAR) leuke- promyelocytic leukemia/retinoic acid receptor (PML/RAR), mogenic fusion proteins. A role for PIR in myeloid differentiation has not to date been reported. In this study we show that acute myeloid leukemia-1/eight–twenty–one (AML1/ETO) and PIR expression is significantly repressed in a large proportion of promyelocytic leukemia zinc-finger/RAR, with the aim of acute myeloid leukemias (AMLs), regardless of subtype or identifying transcriptional targets involved in leukemogenesis underlying karyotypic abnormalities. We show that PIR expres- and found upregulation of genes involved in stem cell sion increases during in vitro myeloid differentiation of primary maintenance and repression of differentiation-associated hematopoietic precursor cells, and that ablation of PIR in the genes.3 However, many of the genes strongly deregulated by U937 myelomonocytic cell line or in murine primary hematopoietic precursor cells results in impairment of terminal myeloid the fusion proteins were poorly characterized and had no differentiation. Gene expression profiling of U937 cells after obvious functional link with hematopoiesis or leukemogenesis. knockdown of PIR revealed increased expression of genes Pirin (PIR) is a highly conserved nuclear protein, originally associated with the early phases of hematopoiesis, in particular, isolated as an interactor of the nuclear factor I/CCAAT box homeobox A (HOXA) genes. Our results suggest that PIR is transcription factor.4 Although its function has not been fully required for terminal myeloid maturation, and its downregulation elucidated, a role for PIR in transcriptional regulation has been may contribute to the differentiation arrest associated with AML. Leukemia (2010) 24, 429–437; doi:10.1038/leu.2009.247; proposed on the basis of its interaction with nuclear factor published online 10 December 2009 I/CCAAT box transcription factor as well as with the proto- Keywords: pirin; acute myeloid leukemia; myeloid differentiation; oncoprotein B-cell chronic lymphocytic leukemia/lymphoma transcription 3,5 a member of the IkB family that modulates the activities of nuclear factor (NF)-kB/Rel transcription factors.6 PIR is a member of the functionally diverse cupin family of proteins,7 Introduction and has been shown to possess quercetinase enzymatic activity.8 Other studies have linked PIR with different functions, Hematopoietic differentiation is a multistage process in which such as apoptosis9,10 and response to stress,11 and suggest a pluripotent self-renewing hematopoietic stem cells give rise to cell-type and species-specific activity. all blood cell lineages and involves successive stages of PIR expression is silenced by both PML/RAR and AML1/ETO commitment and maturation. As cells progress through the in U937 cells as well as in blasts from patients bearing the differentiation cascade, they acquire specific functions and lose corresponding t(15;17) and t(8;21) translocations.3 On the basis self-renewal capacity through selective activation and silencing of its putative function as a transcriptional regulator, we decided of a set of genes. These gene expression programs are triggered to further analyze PIR expression in AML, and to functionally by both extrinsic and intrinsic signals, and are controlled by the assess the effect of PIR silencing in hematopoietic cells. In this orchestrated action of a series of transcription factors whose study we show that PIR downregulation is very frequent in AML, temporal and cell-type-specific expression is critical for their with no correlation with specific disease subtype or underlying unique functions.1 genetic abnormality. Furthermore, PIR knockdown by short Acute myeloid leukemias (AMLs) are characterized by the hairpin RNA (shRNA) results in incomplete maturation of both expansion of myeloid precursors blocked at a specific stage of established (U937) and primary hematopoietic cells, suggesting differentiation. The central role for transcription factor abnorm- that its silencing may be instrumental in the differentiation block alities in determining AML is supported by the high frequency of that characterizes AML. genetic alterations involving the corresponding genes, including chromosomal translocations and point mutations. Deregulated expression or structural abnormalities of these transcription Materials and methods

Correspondence: Professor M Alcalay, Department of Experimental Acute myeloid leukemia (AML) samples Oncology, Istituto Europeo di Oncologia, IFOM-IEO Campus, Via The AML-M3 cases were kindly provided by Professor Francesco Adamello 16, 20139, Milan, Italy. E-mail: [email protected] Lo Coco (University of Rome, Tor Vergata, Rome, Italy); all Received 20 June 2009; revised 6 October 2009; accepted 29 cases of subtypes other than M3 are de novo AML (age 15–60 October 2009; published online 10 December 2009 years) from the Gruppo Italiano Malattie Ematologiche Maligne Pirin downregulation in AML S Licciulli et al 430 dell’Adulto (GIMEMA) LAM 99P and GIMEMA/European PIR short hairpin RNA (shRNA) Organization for Research on Treatment of Cancer AML12 Short hairpin RNA constructs were generated using the pSICO- trials; all cases showed 470% of bone marrow infiltration R-PGK-puro lentiviral vector that constitutively expresses the by leukemic cells and have been characterized for subcellular shRNA oligonucleotides and puromycin selection gene under nucleophosmin localization, karyotype, reverse transcription– control of PGK promoter. The following pairs of shRNA-forming PCR for major fusion transcripts, mixed lineage leukemia status sequences were annealed and cloned into the vector backbone: and FMS-like tyrosine kinase 3 mutations. CD34 þ cells purified Human PIR 7-for: 50-TGAAGCCACTTTGTCTTAATTTCAAG from healthy donors were obtained from Professor Antonio AGAATTAAGACAAAGTGGCTTCTTTTTTC-30 Tabilio (University of Perugia, Italy) and Professor Carmelo Human PIR 7-rev: 50-TCGAGAAAAAAGAAGCCACTTTGT Carlo-Stella (Istituto Nazionale dei Tumori, Milan, Italy). CTTAATTCTCTTGAAATTAAGACAAAGTGGCTTCA-30 Human PIR 4-for: 50-TGGGAATAAAGTCCAAGGTTTTCA AGAGAAACCTTGGACTTTATTCCCTTTTTTC-30 Quantitative real-time PCR (qPCR) Human PIR 4-rev: 50-TCGAGAAAAAAGGGAATAAAGTCC Total RNAs were extracted using RNeasy Mini Kit (QIAGEN, AAGGTTTCTCTTGAAAACCTTGGACTTTATTCCCA-30 Valencia, CA, USA) and complementary DNA synthesis was Mouse PIR 1-for: 50-TGGAGAAGCCTTGGGAATAATTCA performed using the Superscript II reverse transcriptase (Invitrogen, AGAGATTATTCCCAAGGCTTCTCCTTTTTTC-30 Carlsbad, CA, USA) following the manufacturer’s instructions. Mouse PIR 1-rev: 50-TCGAGAAAAAAGGAGAAGCCTTG TaqMan Gene Expression Assay Hs01125825_m1 for human PIR GGAATAATCTCTTGAATTATTCCCAAGGCTTCTCCA-30 was included in a low-density array (Applied Biosystems, Foster Mouse PIR 5-for: 50-TAAATAGAATTCCACATTAATTCAAG City, CA, USA) for high-throughput screening of AML blasts. AGATTAATGTGGAATTCTATTTTTTTTTC-30 Analysis of homeobox (HOX) gene expression in U937-pSICO Mouse PIR 5-rev: 50-TCGAGAAAAAAAAATAGAATTCCAC and U937-shPIR cell lines was performed using the following ATTAATCTCTTGAATTAATGTGGAATTCTATTTA-30 TaqMan Gene Expression Assays: Hs00601076_m1 for HOXA3; Lentiviruses were packaged by transfection of pSICO-R Hs00430330_m1 for HOXA5; Hs00600844_m1 for HOXA7; together with the helper vectors in 293T cells;12 the supernatant Hs00600844_m1 for HOXA9; and Hs00426284_m1 for HOXA13. was concentrated, filtered and added to the target cells in the presence of polybrene. U937 cells underwent spin infection at 1800 r.p.m. for 1 h at 32 1C thrice, followed by puromycin Cell culture and differentiation selection (0.75 mg/ml). For lineage minus (LinÀ) cells, the U937 and NB4 cells were grown in RPMI-1640 medium viral supernatant was concentrated by ultra-centrifugation at supplemented with 10% fetal bovine serum (FBS) and 2 mM 26 000 r.p.m. for 90 min at 4 1C before infection. LinÀ cells glutamine. U937 clones expressing AML1/ETO and PML/RAR underwent two cycles of infection in complete medium in have been described elsewhere.3 For differentiation experi- 24-well retronectin-coated (Takara-Shuzo, Shiga, Japan), non- ments, U937 cells were treated with vitamin D3 (250 ng/ml) and tissue culture-treated plates by incubation for 4 h at 37 1C, transforming growth factor-b (1 ng/ml), and NB4 cells were followed by puromycin selection (3 mg/ml). treated with retinoic acid (RA) 10À6 M. Cells were harvested at the indicated time points and stained with the anti-CD14 antibody (Covance, Princeton, NJ, USA, for U937) or anti- CD11b antibody (BD Pharmingen, San Diego, CA, USA, for Isolation of lineage minus (LinÀ) cells from mouse bone NB4). Samples were acquired with FacsCalibur (FACSCanto II, marrow Becton Dickinson, Franklin Lakes, NJ, USA). 293T cells, Lineage minus cells were purified from the bone marrow of 8- to obtained from American Type Culture Collection center, were 12-week-old 129SvEv mice. After Histopaque 1083 (Sigma- grown in Dulbecco’s modied Eagle’s medium supplemented Aldrich) density separation, bone marrow mononucleated cells with 10% FBS and 2 mM glutamine. were enriched for stem and progenitor cells by depletion of differentiated cells using commercially available reagents (Stem Cell Technologies, Vancouver, BC, Canada). Cells were cultured Generation of rabbit polyclonal anti-PIR antibody in RPMI-1640 medium containing 10% FBS, interleukin 3 (IL-3, PIR full-length coding sequence was inserted downstream to 20 ng/ml), IL-6 (20 ng/ml) and stem cell factor (SCF, 100 ng/ml the glutathione S-transferase sequence in pGEX-4T1 vector cytokines from PeproTech, Rocky Hill, NJ, USA). (Amersham Pharmacia Biotech, Uppsala, Sweden) and after rabbit immunization with the glutathione S-transferase–PIR fusion protein, total antiserum was antigen-purified and eluted at a concentration of 440 mg/ml. Lineage minus (LinÀ) differentiation assays in methylcellulose shPIR and control LinÀ cells were plated (5000 cells per plate) Western blot in methylcellulose medium (Methocult SF M3236, Stem Cell Cells were lysed in JS buffer (50 mM Hepes, 150 mM sodium Technologies) containing FBS and the following cytokines: IL-3 chloride, 1% glycerol, 1% Triton X-100, 1.5 mM magnesium (2 ng/ml), IL-6 (2 ng/ml), SCF (50 ng/ml), granulocyte colony- chloride and 5 mM EDTA), 30–50 mg of extracts were loaded stimulating factor (G-CSF; 50 ng/ml) and granulocyte-macrophage onto polyacrylamide gels for electrophoresis and proteins were CSF (GM-CSF; 10 ng/ml; all from PeproTech). At 7–10 days transferred to nitrocellulose filter (Whatman, Kent, UK). Western after plating, colonies were scored, and cells were harvested for blots were revealed using the enhanced chemiluminescence western blotting and for morphological examination. Fluores- method (Amersham Biosciences, Fairfield, CT, USA). Antibodies cence-activated cell sorting (FACS) analysis of Mac1, Gr1, Sca-1 used in this study were: anti-PIR (concentration 1 in 5000), anti- and c-Kit expression was performed using murine antibodies actin AC-40 and anti-vinculin hVIN1 (both from Sigma-Aldrich, (Pharmingen BD, San Jose, CA, USA). Cells were analyzed by a St Louis, MO, USA, 1 in 10 000). Becton Dickinson FacsCalibur, using CellQuest software.

Leukemia Pirin downregulation in AML S Licciulli et al 431

Figure 1 Pirin (PIR) is downregulated in acute myeloid leukemia (AML). (a) PIR mRNA expression was analyzed by quantitative PCR (qPCR) in 42 AML samples using as normal control the average expression in CD34 þ cells from 11 healthy donors (CD34 þ avg). AML samples were divided by cytogenetic alterations: translocation 15;17 (t(15;17)), translocation 8;21 (t(8;21)), inversion of chromosome 16 (inv(16)), rearrangementsin mixed lineage leukemia (MLL) gene (MLL-r), AML with mutated nucleophosmin (NPM; NPMc) and normal karyotype. (b) PIR expression in U937 clones expressing promyelocytic leukemia/retinoic acid receptor (PML/RAR; PR9) and AML1/ETO (A1/E), compared with wild-type cells, analyzed using qPCR (left) and western blot (right). (c) PIR protein expression in AML mouse models. Proteins were extracted from bone marrow mononucleated cells of wild-type mice and AML mice carrying PML/RAR or AML1/ETO. Actin was used as a loading control.

CD34 þ cell differentiation washing, staining and scanning were performed according to CD34 þ cells were obtained from healthy donors (stimulated Affymetrix protocols. Two copies of the HG-U133 Plus 2.0 array with G-CSF) and isolated from leukaphereses using the EasySep were hybridized with each target. Data derived from U937-shPIR 7 magnetic positive selection procedure (StemCell, Vancouver, cells (sample) were compared with those of U937-pSICO cells BC, Canada). CD34 þ cell differentiation was carried out both (baseline) by ‘comparative analysis’ with GeneChip Operating in liquid and in semisolid medium. Methylcellulose medium software, and further analyzed using GenePicker software13 as (GF þ H4535), formulated to support growth of granulocyte- previously described,3,14 using a fold change cutoff of 42 and a P- macrophage progenitors was purchased from Stem Cells value of 0.05. Functional classiﬁcation of lists of regulated probe Technologies. A total of 2000 cells per 35 mm dish were seeded sets was performed using Ingenuity’s Ingenuity pathways analysis in triplicate. Colonies were harvested and cells were lysed for (IPA) version 7.5-2202 software (Ingenuity Systems, www.ingenui- qPCR and western blotting after 9 days. For differentiation ty.com, Redwood City, CA, USA). Genes from the data set that met in liquid culture, cells were maintained for 12 days at the fold change cutoff of two and were associated with biological 250–300 000/ml in Iscove’s modiﬁed Dulbecco’s medium functions and/or diseases in the Ingenuity knowledge base were supplemented with FBS and IL-6, SCF, Flt3 ligand, thrombo- considered for the analysis. Fischer’s exact test was used to poietin, G-CSF and GM-CSF, each at 50 ng/ml. At day 9, 10% calculate a P-value determining the probability that each biological human serum was added to the medium. function and/or disease assigned to that data set is due to chance alone. Raw microarray data can be accessed from the Gene Expression Omnibus repository (http://www.ncbi.nlm.nih.gov/geo/) RNA extraction, Affymetrix GeneChip hybridization with data set accession number GSE16798. and analysis Total RNA was extracted using the RNeasy Mini Kit (QIAGEN). For each of the U937 cell lines (pSICO and shPIR 7), three independent Results RNA extractions were performed, and an equal quantity of each of the three RNA preparations was then mixed to generate an RNA Pirin (PIR) expression is downregulated in acute myeloid pool for each sample. Biotin-labeled cRNA targets were obtained leukemia (AML) from 5 mg of each RNA pool using Affymetrix custom kit On the basis of our previous results, we decided to analyze PIR (Affymetrix, Santa Clara, CA, USA). GeneChip hybridization, expression in a larger cohort of AML samples to uncover a

Leukemia Pirin downregulation in AML S Licciulli et al 432 possible correlation with one or more AML subtypes or alterations (Figure 1a and Supplementary Table 1). Down- cytogenetic alterations. To this aim, 42 AML samples represent- regulation or silencing of PIR expression is, therefore, a common ing different French–American–British classification subtypes molecular feature in human AML. and cytogenetic abnormalities (Supplementary Table 1) were Pirin expression was next analyzed in myeloid cells bearing screened for PIR mRNA expression and compared with the AML fusion proteins to assess whether these model systems average expression level in CD34 þ hematopoietic progenitor reflect our findings in primary AML blasts. We first analyzed cells derived from 11 healthy donors. qPCR analysis of these U937 cells expressing PML/RAR and AML1/ETO3 by qPCR and samples revealed a greater than twofold downregulation of western blot with anti-PIR antibody, and found that both mRNA PIR mRNA expression in 39/42 AML cases (in 36/42 cases, and protein expression decreased in the presence of the downregulation was greater than fivefold and in 21/42 cases, the fusion proteins (Figure 1b). Next, we analyzed PIR levels in expression was not detectable) and no correlation with French– primary leukemic blasts derived from the bone marrow of AML American–British classification subtype or specific genetic mouse models.15 PIR is expressed at low levels in normal

Figure 2 Pirin (PIR) expression increases during myeloid differentiation. PIR expression in human CD34 þ cells before and after in vitro myelomonocytic differentiation, analyzed using (a) quantitative PCR (qPCR) and (b) western blot. (c) Variation of CD34 (left) and CD11b (right) expression associated with differentiation was assessed using ﬂuorescence-activated cell sorting (FACS) analysis. (d) Cytospin preparations of human CD34 þ cells before and after in vitro myelomonocytic differentiation were stained with May–Grumwald–Giemsa (MGG) for morphological examination. Photographs were taken at an original magniﬁcation of Â 40. (e) FACS analysis of c-Kit, Mac1 and Gr1 expression to assess myeloid differentiation of lineage minus (LinÀ) cells before and after treatment with granulocyte and granulocyte-macrophage colony- stimulating factors (G-CSF and GM-CSF). (f) Analysis of PIR protein expression in LinÀ cells after 7 days of treatment with G-CSF and GM-CSF. (g) FACS analysis of CD11b expression in NB4 cells treated with retinoic acid (RA) for 48 h. (h) Analysis of PIR mRNA expression in NB4 cells after 24 and 48 h of RA treatment. Abbreviations: LC, liquid culture; MC, methylcellulose. Western blots were normalized with actin or vinculin as indicated.

Figure 4 Pirin (PIR) knockdown impairs differentiation of lineage minus (LinÀ) cells. (a) Western blot control of PIR knockdown in LinÀ cells before plating in methylcellulose. Vinculin was used as a loading control. (b) Representative plates of methylcellulose assay (one out of three) for each condition. Colonies were stained with nitroblue tetrazolium. (c) Average colony number from triplicate plates. (d) Cytospin preparations of cells forming the colonies were stained with May–Grumwald–Giemsa (MGG) for morphological examination. Photographs were taken at an original magnification of Â 100. Panels show one representative microscope field for each condition. Arrowheads indicate cells that were counted as immature. Scale bar ¼ 20 mm. (e) Relative presence of immature cells in the three conditions. (f, g) Fold increase in c-Kit and Sca-1 markers expression (respectively) in shPIR cells compared with pSICO-R control cells, as assessed using fluorescence-activated cell sorting (FACS) analysis. The experiment was performed twice giving similar results and data shown refer to one representative experiment.

Leukemia Pirin downregulation in AML S Licciulli et al 433 murine bone marrow, and is not detectable in the bone marrow Taken together, our results suggest that PIR transcriptional of mice with AML induced by either PML/RAR or AML1/ETO repression is a very frequent event in AML and leads to a (Figure 1c). decrease in PIR protein levels in both human and mouse models of the disease.

Pirin (PIR) expression increases during myeloid differentiation of primary hematopoietic cells in vitro We next analyzed whether PIR expression is modulated during normal myeloid differentiation using human CD34 þ cells and murine LinÀ cells, which represent hematopoietic stem/ progenitor cells. CD34 þ cells were isolated from healthy donors using a positive selection procedure. To induce myelomonocytic differentiation, CD34 þ cells were either grown in liquid culture containing IL-6, SCF, Flt3 ligand, thrombopoietin, G-CSF and GM-CSF for 12 days, with addition of human serum at day 9, or plated in methylcellulose containing IL-6, SCF, GM-CSF, IL-3 and G-CSF for 9 days. PIR expression was assessed at both mRNA and protein levels, and resulted significantly upregulated upon myelomonocytic differentiation (Figures 2a and b). Maturation was assessed by FACS analysis of CD34 and CD11b expression (Figure 2c) and by Figure 3 Pirin (PIR) knockdown impairs differentiation of U937 cells. morphology (Figure 2d). (a) Western blot analysis of PIR knockdown in shPIR 4 and shPIR 7 Lineage minus cells were purified from the bone marrow of U937 cells compared with empty vector-infected cells. Actin was used wild-type 129SvEv mice by negative selection, and plated in as a loading control. (b) Fluorescence-activated cell sorting (FACS) methylcellulose in the presence of G-CSF and GM-CSF to drive analysis of CD14 expression on U937 cells after 24 and 48 h of myeloid differentiation. After 7 days, cells were harvested, vitamin D3 (VD3) and transforming growth factor-b (TGF-b); shPIR 4 and shPIR 7 cells were compared with control (empty vector) cells. differentiation was assessed by FACS analysis of c-Kit, Mac1 and Three independent experiments were performed and the statistical Gr1 expression (Figure 2e) and cells were lysed for protein significance of differences between shPIR and control cells were extraction. LinÀ cells grown in liquid culture in the absence of assessed using Student’s t-test (**Po0.05; *P ¼ 0.12). G-CSF and GM-CSF were used as control. Western blot analysis

Leukemia Pirin downregulation in AML S Licciulli et al 434 showed signiﬁcantly higher PIR levels in mature cells than in undifferentiated LinÀ cells (Figure 2f). We next analyzed whether PIR levels vary in a model of AML differentiation therapy. For this purpose, we used the RA-sensitive NB4 human cell line, derived from a patient with acute promyelocytic leukemia (APL). Expression of the CD11b surface antigen was measured after 48 h of RA treatment to control NB4 maturation (Figure 2g). Consistent with the results obtained in normal primary cells, PIR mRNA levels increased approximately threefold upon terminal differentiation (Figure 2h). The lower-fold increasecomparedwiththatobservedinprimarycellsafter myelomonocytic maturation is likely due to the fact that NB4 cells are promyelocytes, and therefore already partially differentiated. Taken together, these data show that PIR expression increases signiﬁcantly during myeloid maturation of both human and murine hematopoietic cells.

Pirin (PIR) knockdown partially impairs myeloid differentiation of U937 cells U937 cells differentiate to monocytes in response to treatment with vitamin D3 and transforming growth factor-b.16,17 U937 differentiation can be monitored by measuring the increase in expression of specific surface markers such as CD14. To directly measure the effects of PIR downregulation in myeloid matura- Figure 5 Expression levels of HOXA genes increase after pirin (PIR) tion, we performed PIR knockdown experiments in U937 cells. knockdown in U937 cells. (a) Top five functions identified by Two different PIR-shRNAs (shPIR 7 and shPIR 4) were cloned in Ingenuity’s Ingenuity pathways analysis (IPA) software for the list of the pSICO-R lentiviral vector that allows for puromycin upregulated genes in U937-shPIR 7 cells. (b) Expression levels of HOXA genes in U937-shPIR 7, U937-shPIR 4 and pSICO-R-infected selection of transduced cells. pSICO-R vector-transfected cells cells were analyzed using quantitative PCR (qPCR). Average induction were used as control. Western blot analysis showed that PIR in three independent measurements is reported. expression was blocked by both PIR-shRNAs with high efficiency (Figure 3a). CD14 expression was measured using FACS analysis in U937 shPIRmm 5 did not lead to a significant variation in colony control cells and in PIR-shRNA-transduced cells after treatment number (87%) (Figures 4b and c). In both conditions, however, the with vitamin D3 and transforming growth factor-b for 24 and 48 h. size of the colonies was smaller (Figure 4b). May–Grumwald– Control cells behaved similar to wild-type U937 cells (data not Giemsa staining showed that, as opposed to control colonies that shown), suggesting that lentiviral infection does not interfere with show a prevalence of mature myeloid cells (macrophages, the differentiation program. On the contrary, after 24 h of vitamin promyelocytes and neutrophils), shPIRmm 1 and shPIRmm 5 D3 and transforming growth factor-b treatment, we observed a 55 colonies contain variable proportions of undifferentiated, blast- and 42% decrease in CD14 expression in shPIR 7 and shPIR 4 like cells (18.5 and 13%, respectively), as indicated by cells, respectively; after 48 h, the reduction in CD14 expression morphology, low nucleus/cytoplasm ratio, basophilic staining was of 38% by both shPIR constructs (Figure 3b). These results and lack of cytoplasmic granules (Figures 4d and e). Representa- suggest that PIR silencing leads to an impairment in terminal tive cells are shown in Supplementary Figure 1. Furthermore, maturation of U937 cells and may contribute to the differentiation FACS analysis of surface markers revealed that the proportion of of block characteristic of AML blasts. cells expressing c-Kit and Sca-1 increased 3.9-fold and 1.7-fold, respectively, in shPIRmm 1 cells and 1.5-fold in shPIRmm 5, with respect to control cells (Figures 4f and g). Pirin (PIR) knockdown impairs the ability of LinÀ cells There are some differences in the specific phenotypes to respond to differentiation stimuli in vitro resulting from PIR ablation with the two shRNAs. These may The effect of PIR knockdown on myeloid differentiation was also be due to different off-target effects associated with each analyzed in primary murine hematopoietic cells. Two shRNAs for construct. The probability that two shRNAs yield similar off- the murine PIR homolog (shPIRmm 1 and shPIRmm 5) were target effects is, however, extremely low; therefore, these results cloned into the pSICO-R vector and used to infect LinÀ cells. After suggest that PIR ablation in LinÀ cells partially impairs the puromycin selection, a stable bulk population of cells was differentiation response to G-CSF and GM-CSF. obtained for each construct, and western blot analysis revealed a significant reduction of PIR expression in both cases (Figure 4a). We then performed a clonogenic assay by plating shPIR- Pirin (PIR) knockdown in U937 cells results in increased transduced LinÀ cells in methylcellulose in the presence of expression of HOXA genes G-CSF and GM-CSF. After 7 days, colonies were scored, cells We next analyzed the global gene expression profile of U937 from replica plates were pooled and differentiation was assessed cells that do not express PIR, with the aim of identifying genes visually using May–Grumwald–Giemsa staining and immuno- that may participate in the impairment of terminal differentia- phenotypically using FACS analysis of Sca-1 and c-Kit, which are tion. Total RNA was extracted from U937 cells transduced with characteristic markers of hematopoietic stem/progenitor cells. The shPIR 7 or with the empty pSICO-R vector and hybridized to number of colonies obtained after G-CSF and GM-CSF treatment Affymetrix GeneChip HG-U133 Plus 2.0 arrays as described in was significantly decreased only by shPIRmm 1 (54%), whereas Materials and methods. Results were analyzed as previously

Leukemia Pirin downregulation in AML S Licciulli et al 435 Table 1 Common regulated genes between PIR silencing and expression of PML/RAR or AML1/ETO in U937 cells

Symbol Gene ID PIR-KD PR A1E Entrez gene name

PIR 8544 À23.2 À47.9 À22.4 Pirin (iron-binding nuclear protein) DCTN4 51164 À2.5 À2.2 Dynactin 4 (p62) ID1 3397 À2.0 À3.5 Inhibitor of DNA binding 1, dominant negative helix-loop-helix protein LITAF 9516 À2.4 À2.1 Lipopolysaccharide-induced TNF factor TSPAN14 81619 À2.2 À3.7 À2.1 Tetraspanin 14 ATP6V0D2 245972 3.7 3.4 ATPase, H+ transporting, lysosomal 38 kDa, V0 subunit d2 CARD16 114769 2.1 2.0 Caspase recruitment domain family, member 16 CCDC74A 90557 2.0 2.4 Coiled-coil domain containing 74A CD109 135228 3.7 11.8 CD109 molecule CD36 948 3.3 9.9 CD36 molecule (thrombospondin receptor) CD48 962 3.2 3.4 CD48 molecule CITED2 10370 2.0 2.2 Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2 CXCL11 6373 3.6 3.8 Chemokine (C-X-C motif) ligand 11 CYSLTR1 10800 2.2 2.0 Cysteinyl leukotriene receptor 1 FABP6 2172 4.6 2.6 Fatty acid binding protein 6, ileal FAR2 55711 3.3 2.0 Fatty acyl CoA reductase 2 GFPT1 2673 2.4 2.3 Glutamine-fructose-6-phosphate transaminase 1 HOXA5 3202 2.0 2.0 Homeobox A5 HOXA9 3205 2.0 2.1 Homeobox A9 IFI6 2537 2.1 2.3 Interferon, a-inducible protein 6 IL7R 3575 5.2 3.0 Interleukin 7 receptor KYNU 8942 3.5 19.2 Kynureninase (L-kynurenine Hydrolase) LY96 23643 2.6 3.0 Lymphocyte antigen 96 NEGR1 257194 2.8 2.4 Neuronal growth regulator 1 NLRP7 199713 2.1 7.3 NLR family, pyrin domain containing 7 OAS1 4938 4.0 2.9 20,50-oligoadenylate synthetase 1, 40/46 kDa PTPRC 5788 2.0 2.1 Protein tyrosine phosphatase, receptor type, C RASGRP2 10235 À2.0 À4.8 RAS guanyl releasing protein 2 (calcium and DAG regulated) SAMSN1 64092 3.0 2.4 2.1 SAM domain, SH3 domain and nuclear localization signals 1 SLC2A3 6515 2.1 2.3 2.0 Solute carrier family 2 (facilitated glucose transporter), member 3 SOX4 6659 2.0 2.3 2.6 SRY (sex-determining region Y)-box 4 TIAM1 7074 2.2 2.0 T-cell lymphoma invasion and metastasis 1 Abbreviations: AML1/ETO, acute myeloid leukemia-1/eight–twenty–one corepressor; PML/RAR, promyelocytic leukemia/retinoic acid receptor; PIR-KD, Pirin knock-down; PR, PML/RAR; A1E, AML1/ETO. Complete list of 32 genes (5 downregulated and 27 upregulated) that are common to the list of U937-shPIR 7 regulated genes and either U937- AML1/ETO or U937-PML/RAR regulated genes. Fold changes refer to up- or downregulation compared with respective control cells (that is, U937 cells infected with the empty cloning vectors).

described,3,18 and regulated genes (more than twofold) were Considering their known relevance in myeloid differentiation annotated and classified using the Ingenuity pathways analysis and leukemogenesis, we further analyzed by qPCR whether software. A total of 492 regulated genes were identified, 305 other HOXA genes were regulated after PIR silencing. A induced and 187 repressed, after knockdown of PIR protein comparison of HOXA mRNA levels in U937-shPIR 7 or U937- (Supplementary Table 2). The most striking enrichment that shPIR 4 with U937-pSICO-R confirmed the upregulation of emerged from functional classification was the significant HOXA5 and HOXA9 identified by microarray analysis, and upregulation of genes involved in immune response and further identified induction of HOXA3, HOXA7 and HOXA13 lymphocyte function (Figure 5a). However, no downregulation expression (Figure 5b). of genes involved in myeloid differentiation was observed (for Taken together, these results suggest that PIR does not complete functional classification, see Supplementary Table 3). modulate the expression of crucial regulators of myeloid The transcriptional response to PIR silencing may, however, differentiation, but may be involved in the repression of genes be affected in AML cells by the simultaneous expression that are expressed in early hematopoietic progenitor cells. of leukemogenic oncogenes. To identify which PIR targets may be of relevance in AML, we searched for genes that are concordantly regulated by both PIR ablation and expression of Discussion AML1/ETO or PML/RAR in U937 cells. Previously published data sets3,14,18 were reanalyzed using the same stringency Pirin is a transcriptional regulator previously identified as a parameters and annotated using Ingenuity pathways analysis. target of the AML-associated fusion proteins AML1/ETO and We thus identified 32 common target genes, 5 of which PML/RAR.3 In this report, we show that PIR expression levels are downregulated (including PIR), and 27 upregulated (Table 1). downregulated or silenced not only in AML with t(8;21) and Functional classification of this list (Supplementary Table 4) t(15;17), but in the vast majority of AML cases, independently of revealed seven genes involved in hematopoiesis: 6 upregulated cytogenetic alterations and subtype. To analyze the conse- (CXCL11, HOXA5, HOXA9, IL7R, PTPRC and SOX4) and one quences of PIR silencing in myeloid cells and its possible role in downregulated (ID1). Interestingly, several of the induced genes AML, we analyzed the effects of PIR ablation in U937 cells and are involved in the early phases of hematopoiesis and/or are in primary mouse hematopoietic cells. In both model systems, activated in AML. we observed an impairment of terminal myeloid differentiation,

Leukemia Pirin downregulation in AML S Licciulli et al 436 suggesting that PIR may be required for efficient myeloid Maligne dell’Adulto) Acute Leukemia Working Party for AML maturation. Consistent with this hypothesis, PIR expression samples. We are grateful to Manuela Capillo for assistance with increases during myeloid maturation of both human and murine mouse models, Giuseppe Ossolengo for the production of anti-PIR primary hematopoietic precursor cells, and in RA-induced antibody, to Elisa Venturini, Luca Rotta and Simone P Minardi for differentiation of an APL cell line. support in microarray experiments and analysis of results with IPA The mechanism through which PIR participates in the process (Ingenuity) software, to Loris Bernard, Laura Tizzoni and Valentina of differentiation is not immediately obvious, considering the Dall’Olio for support in qPCR experiments and to Ivan Muradore diversity of functions so far attributed to PIR. It has been defined and Simona Ronzoni for FACS analysis. This work was supported as a transcriptional regulator on the basis of its interaction with by grants from Associazione Italiana per la Ricerca sul Cancro B-cell chronic lymphocytic leukemia/lymphoma 35 and nuclear (AIRC) and Fondazione CARIPLO to MA. factor I/CCAAT box transcription factor.4 We therefore analyzed the transcriptional effects of PIR silencing in U937 cells and did not detect modulation of genes associated with myeloid References differentiation. Although this may be due to the cellular model under analysis, expression of AML1/ETO or PML/RAR in the 1 Rosenbauer F, Tenen DG. Transcription factors in myeloid same model system does indeed result in repression of essential development: balancing differentiation with transformation. Nat regulators of myeloid differentiation, such as genes encoding for Rev Immunol 2007; 7: 105–117. the PU.1 and CEBP family of transcriptional regulators. PIR 2 Look AT. Oncogenic transcription factors in the human acute silencing instead results in moderate induction of several leukemias. Science 1997; 278: 1059–1064. genes associated with early hematopoiesis, such as CITED2,19 3 Alcalay M, Meani N, Gelmetti V, Fantozzi A, Fagioli M, Orleth A 20 21 22,23 et al. Acute myeloid leukemia fusion proteins deregulate genes CD109, SOX4 and importantly, HOXA genes, sugges- involved in stem cell maintenance and DNA repair. J Clin Invest ting that the increase in PIR expression observed during myeloid 2003; 112: 1751–1761. differentiation may be required for repression of genes 4 Wendler WM, Kremmer E, Forster R, Winnacker EL. Identification associated with stem cell/progenitor functions. PIR may, there- of pirin, a novel highly conserved nuclear protein. J Biol Chem fore, be involved in rendering hematopoietic progenitors 1997; 272: 8482–8489. permissive to the expression of myeloid-specific genes. This 5 Dechend R, Hirano F, Lehmann K, Heissmeyer V, Ansieau S, Wulczyn FG et al. The Bcl-3 oncoprotein acts as a bridging factor view is further supported by the increased transcriptional between NF-kappaB/Rel and nuclear co-regulators. Oncogene levels of genes associated with lymphoid commitment and 1999; 18: 3316–3323. differentiation, such as IL7R, CXCL11 and CD69, observed upon 6 Baldwin Jr AS. The NF-kappa B and I kappa B proteins: new silencing of PIR. discoveries and insights. Annu Rev Immunol 1996; 14: 649–683. The mechanism through which downregulation of PIR 7 Pang H, Bartlam M, Zeng Q, Miyatake H, Hisano T, Miki K et al. expression is achieved in AML remains to be analyzed. PIR is Crystal structure of human pirin: an iron-binding nuclear protein 14 and transcription cofactor. J Biol Chem 2004; 279: 1491–1498. not a direct transcriptional target of AML1/ETO or PML/RAR 8 Adams M, Jia Z. Structural and biochemical analysis reveal (S Licciulli and M Alcalay, unpublished results) and other indirect pirins to possess quercetinase activity. J Biol Chem 2005; 280: mechanisms must therefore be responsible for its repression. 28675–28682. AML cells, unlike normal human CD34 þ progenitor cells, 9 Gelbman BD, Heguy A, O’Connor TP, Zabner J, Crystal RG. show activation of NF-kB,24 and inhibition of NF-kB activity in Upregulation of pirin expression by chronic cigarette smoking is AML blasts induces cell death.25 It has been recently shown that associated with bronchial epithelial cell apoptosis. Respir Res 2007; 8: 10. NF-kB physically interacts in the cytoplasm with mutated 10 Orzaez D, de Jong AJ, Woltering EJ. A tomato homologue of the nucleophosmin, which is found in approximately 30% of human protein PIRIN is induced during programmed cell death. AML,26 and that this interaction results in cytoplasmic seques- Plant Mol Biol 2001; 46: 459–468. tration of the active form of NF-kB.27 Concordantly, the absence 11 Hihara Y, Muramatsu M, Nakamura K, Sonoike K. A cyanobacterial of nucleophosmin mutations is one of the main characteristics of gene encoding an ortholog of Pirin is induced under stress patients with increased NF-kB activity. Although PIR down- conditions. FEBS Lett 2004; 574:101–105. 12 Rubinson DA, Dillon CP, Kwiatkowski AV, Sievers C, Yang L, regulation was a constant feature in AML in our study, it was less Kopinja J et al. A lentivirus-based system to functionally silence pronounced in samples derived from patients with mutated genes in primary mammalian cells, stem cells and transgenic mice nucleophosmin. One could therefore speculate that PIR expres- by RNA interference. Nat Genet 2003; 33: 401–406. sion may be partially controlled by NF-kB, which is an 13 Finocchiaro G, Parise P, Minardi SP, Alcalay M, Muller H. intriguing possibility in consideration of the putative role of GenePicker: replicate analysis of Affymetrix gene expression PIR itself in NF-kB regulation. microarrays. Bioinformatics 2004; 20: 3670–3672. 14 Gardini A, Cesaroni M, Luzi L, Okumura AJ, Biggs JR, Minardi SP Our results show that PIR downregulation is a common et al. AML1/ETO oncoprotein is directed to AML1 binding regions molecular feature of AML and may be functionally linked with and co-localizes with AML1 and HEB on its targets. PLoS Genet the pathogenesis of the disease. 2008; 4: e1000275. 15 Viale A, De Franco F, Orleth A, Cambiaghi V, Giuliani V, Bossi D et al. Cell-cycle restriction limits DNA damage and maintains self- renewal of leukaemia stem cells. Nature 2009; 457: 51–56. Conflict of interest 16 Rigby WF, Shen L, Ball ED, Guyre PM, Fanger MW. Differentiation of a human monocytic cell line by 1,25-dihydroxyvitamin D3 The authors declare no conflict of interest. (calcitriol): a morphologic, phenotypic, and functional analysis. Blood 1984; 64: 1110–1115. 17 Testa U, Masciulli R, Tritarelli E, Pustorino R, Mariani G, Martucci Acknowledgements R et al. Transforming growth factor-beta potentiates vitamin D3-induced terminal monocytic differentiation of human leukemic cell lines. J Immunol 1993; 150: 2418–2430. We are grateful to Pier Giuseppe Pelicci for helpful discussions. 18 Meani N, Minardi S, Licciulli S, Gelmetti V, Coco FL, Nervi C et al. We thank Francesco Lo Coco (Tor Vergata University, Rome, Italy) Molecular signature of retinoic acid treatment in acute promye- and the GIMEMA (Gruppo Italiano Malattie Ematologiche locytic leukemia. Oncogene 2005; 24: 3358–3368.

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

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