NUP98-HOXA9 Induces Long-Term Proliferation and Blocks Differentiation of Primary Human CD34+ Hematopoietic Cells Akiko Takeda, Charles Goolsby, and Nabeel R

Research Article

NUP98-HOXA9 Induces Long-term Proliferation and Blocks Differentiation of Primary Human CD34+ Hematopoietic Cells Akiko Takeda, Charles Goolsby, and Nabeel R. Yaseen

Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois

Abstract develops into AML. The nucleoporin NUP98 gene is the target of NUP98-HOXA9, the chimeric protein resulting from the numerous chromosomal rearrangements, predominantly in AML t(7;11)(p15;p15) chromosomal translocation, is a prototype and MDS, resulting in fusion to different partner genes, many of of several NUP98 fusions that occur in myelodysplastic which belong to the homeobox family (2). NUP98-HOXA9, a syndromes and acute myeloid leukemia. We examined its prototype of the resulting fusion proteins, consists of an NH2- effect on differentiation, proliferation, and gene expression in terminal fragment of NUP98 fused to a COOH-terminal fragment of primary human CD34+ hematopoietic cells. Colony-forming HOXA9. The NH2 terminus of NUP98 includes the FG repeat cell (CFC) assays in semisolid medium combined with domain that forms a docking site for nuclear transport carrier morphologic examination and flow cytometric immunophe- proteins while the COOH terminus of HOXA9 contains the DNA- notyping revealed that NUP98-HOXA9 increased the numbers binding homeodomain (2). of erythroid precursors and impaired both myeloid and Investigation of the role of NUP98-HOXA9 in pathogenesis of erythroid differentiation. In continuous liquid culture, cells leukemia has been carried out, to date, mostly using mouse transduced with NUP98-HOXA9 exhibited a biphasic growth models and cell lines. Mice transplanted with bone marrow cells curve with initial growth inhibition followed by enhanced that express retrovirally transduced NUP98-HOXA9 develop a long-term proliferation, suggesting an increase in the numb- preleukemic myeloproliferative disorder followed eventually by ers of primitive self-renewing cells. This was confirmed by a AML (3). Coexpression of another homeobox transcription factor, dramatic increase in the numbers of long-term culture- Meis1, or the oncogene BCR-ABL accelerates the development of initiating cells, the most primitive hematopoietic cells AML (2). In a transgenic mouse model, another NUP98-homeobox detectable in vitro. To understand the molecular mechanisms fusion, NUP98-HOXD13, caused severe MDS followed by AML (4). underlying the effects of NUP98-HOXA9 on hematopoietic cell These studies indicate that NUP98-HOXA9 creates a preleukemic proliferation and differentiation, oligonucleotide microarray state and that additional genetic events are required for the analysis was done at several time points over 16 days, starting development of AML. In vitro, NUP98-HOXA9 promotes the at 6 hours posttransduction. The early growth suppression growth of murine hematopoietic progenitors and blocks their was preceded by up-regulation of IFNB1 and accompanied by differentiation (5). marked up-regulation of IFN-induced genes, peaking at 3 days The first clues to the function of NUP98-HOXA9 at the posttransduction. In contrast, oncogenes such as homeobox molecular level came from studies with murine cell lines in which transcription factors, FLT3, KIT, and WT1 peaked at 8 days or the FG repeat region of NUP98 was shown to function as a beyond, coinciding with increased proliferation. In addition, transactivation domain when fused to a GAL4 DNA-binding several putative tumor suppressors and genes associated with domain (6). In human cells, the global effect of NUP98-HOXA9 on hematopoietic differentiation were repressed at later time gene transcription was studied using a myeloid cell line, K562 (7). points. These findings provide a comprehensive picture of These studies showed that NUP98-HOXA9 acts as an aberrant the changes in proliferation, differentiation, and global gene transcription factor. However, its effects on proliferation, expression that underlie the leukemic transformation of differentiation, and gene expression in primary human hemato- human hematopoietic cells by NUP98-HOXA9. (Cancer Res poietic cells and the mechanisms by which it transforms these 2006; 66(13): 6628-37) cells remain unclear. Here we identify the effects of NUP98-HOXA9 on primary human Introduction hematopoietic progenitor/stem cells and the changes in gene Acute myeloid leukemia (AML) and myelodysplastic syndromes expression that underlie those effects. We provide evidence that NUP98-HOXA9 has a biphasic effect on the proliferation of human (MDS) are hematologic malignancies involving myeloid precursors + (1). AML is characterized by a block in myeloid differentiation and CD34 hematopoietic cells. An initial suppression of proliferation is h unchecked proliferation of primitive blasts. MDS is characterized associated with increased expression of IFN 1andmarked by ineffective hematopoiesis, dysplasia, and cytopenia, and often induction of many IFN-inducible genes. This is followed by long- term proliferation, impaired differentiation, and dramatically increased numbers of primitive long-term culture-initiating cells (LTC-IC). This biphasic effect seems to recapitulate the transition of MDS to AML. Microarray analysis over a period of 16 days Note: Supplementary data for this article are available at Cancer Research Online pointed to likely mechanisms for the biological effects of NUP98- (http://cancerres.aacrjournals.org/). Requests for reprints: Nabeel R. Yaseen, Department of Pathology, Northwestern HOXA9 by showing extensive changes in the expression of University, Ward 6-011, 303 East Chicago Avenue, Chicago, IL 60611. Phone: 312-503- oncogenes, transcription factors, growth factors, cell cycle 2093; Fax: 312-503-8240; E-mail: [email protected]. I2006 American Association for Cancer Research. regulators, putative tumor suppressors, and factors involved in doi:10.1158/0008-5472.CAN-06-0458 hematopoietic differentiation.

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Materials and Methods StemCell Technologies) that is genetically engineered to produce human G-CSF and IL-3 was used as a feeder layer after irradiation with 8,000 cGy to Retrovirus for transduction. NUP98-HOXA9 cDNA with NH2-terminal support the long-term culture of hematopoietic cells. The culture medium À hemagglutinin (HA) tag was subcloned into the MSCV-IRES-green contained 12.5% horse serum, 12.5% FBS, 10 4 mol/L 2-mercaptoethanol, fluorescent protein (GFP) retroviral expression vector as previously 2 mmol/L L-glutamine, 0.16 mmol/L I-inositol, and 16 Amol/L folic acid described (7). PG13 (CRL-10686 from American Type Culture Collection, in MEMa (Myelocult H5100 from StemCell Technologies) with 1 Amol/L Manassas, VA), a packaging cell line that produces GaLV-pseudotyped hydrocortisone. The frequency of LTC-ICs was determined by seeding retrovirus, was transfected with MSCV-IRES-GFP using Lipofectamine and varying numbers of cells in methylcellulose cultures using Methocult GF+ as PLUS reagent (Invitrogen, Carlsbad, CA) and cultured 2 days in DMEM described above and counting colonies after 17 days (9). LTC-IC numbers containing 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine, and 100 per 10,000 cells, the cell number originally plated at the start of the 5-week units/mL penicillin/streptomycin. GFP-positive cells were isolated by four LTC-IC assay culture, were estimated by assuming an average CFC output of rounds of sorting using Elite ESP (Beckman Coulter, Fullerton, CA). Virus- eight per LTC-IC (9). j containing culture supernatants were collected after 1 to 2 days at 32 C. Nucleofection. HA-NUP98-HOXA9 cDNA (7) was subcloned into the PG13 cells producing MSCV-IRES-GFP/NUP98-HOXA9 retrovirus were pTracer-CMV/Bsd vector that expresses Cycle 3-GFP-blasticidin fusion gene kindly provided by Dr. Malcolm Moore (Sloan-Kettering Cancer Institute, (Invitrogen) using EcoRI and XbaI sites. Human CD34+ cells preactivated as New York, NY). described above were resuspended at 2 Â 106/100 AL in Nucleofector + Culture and retroviral transduction of CD34 primary cells. Frozen solution (Human CD34 cell Nucleofector kit from Amaxa Biosystems, + human CD34 cells purified from mobilized peripheral blood of two patients Geithersburg, MD), mixed with 5 Ag DNA (HA-NUP98-HOXA9 in pTracer- were obtained after Institutional Review Board approval from the Bone CMV/Bsd or pTracer-CMV/Bsd control), and subjected to nucleofection Marrow Transplant Laboratory at Northwestern Memorial Hospital. They according to the protocol of the manufacturer. The cells were then cultured were meant for autologous transplantation of the now-deceased patients as at 1 Â 105/mL in complete cytokine medium for 3.5 hours and GFP+ cells a treatment for multiple myeloma. Before cell harvest, the peripheral blood were sorted using a MoFlo. Sorted cells were cultured at 2 Â 105/mL in of the two patients did not contain plasma cells and their bone marrows complete cytokine medium for 2.5 hours and collected for antihemag- showed adequate multilineage hematopoiesis. The cells were cultured at glutinin immunoblotting and total RNA isolation. Â 5 1 10 /mL for 2 days to preactivate in Iscove’s modified Dulbecco’s medium RNA isolation and oligonucleotide array expression analysis. Total (IMDM) containing 20% FBS, 100 ng/mL Fms-related tyrosine kinase 3 RNA was isolated from GFP-sorted and cultured cells using the RNeasy mini (FLT-3) ligand, 20 ng/mL granulocyte/macrophage colony-stimulating kit (Qiagen, Valencia, CA). Samples were analyzed using U133+2.0 factor (GM-CSF), 100 ng/mL stem cell factor, 100 ng/mL thrombopoietin, expression array (Affymetrix, Santa Clara, CA) by the Genomic Core 50 ng/mL interleukin (IL)-3, 100 ng/mL IL-6 (all cytokines were from Laboratory of Sloan-Kettering Cancer Center as previously described (7). Peprotech, Rocky Hill, NJ), 2 mmol/L L-glutamine, and 100 units/mL Quantitative real-time PCR. Template cDNA was synthesized using penicillin/streptomycin (complete cytokine medium). The cells were SuperScript III First-Strand Synthesis System for reverse transcription-PCR Â 5 resuspended at 0.8 10 /mL in fresh complete cytokine medium, placed (RT-PCR) (Invitrogen) from a portion of the total RNA preparation used for in RetroNectin (Takara, Otsu, Shiga, Japan)-coated virus-preloaded plates Affymetrix array analysis. Real-time PCR mixtures (25 AL per reaction) (8), and cultured for 2 days. GFP-positive cells were isolated by sorting using contained cDNA, 0.2 Amol/L each of forward and reverse primers, and 12.5 a MoFlo (Dako, Glostrup, Denmark) and a portion was analyzed for AL iQ SYBRGreen Supermix (Bio-Rad,Hercules, CA). The reactions were expression of NUP98-HOXA9 by immunoblotting with antihemagglutinin done in triplicate in 96-well plates using the GeneAmp 5700 Sequence antibody 12CA5 (Roche Applied Science, Indianapolis, IN). Another portion Detection System (Applied Biosystems, Foster City, CA) and analyzed with was cultured at 2 Â 105/mL in complete cytokine medium for 18 hours and the software from the manufacturer. The amount of transcript was collected for total RNA isolation (3 days posttransduction samples). For determined based on a standard curve specific for each gene and long-term growth, sorted cells were cultured continuously in complete normalized to the amount of glyceraldehyde phosphate dehydrogenase cytokine medium with periodic cell counting and feeding. At 8, 10, and transcript in the same sample. 16 days after transduction, cells were collected for total RNA isolation. Flow cytometry. Antibodies (BD, Franklin Lakes, NJ) against CD11b (phycoerythrin-conjugated clone D12), CD14 (allophycocyanin-conjugated Results clone M5E2), CD34 (phycoerythrin-conjugated clone 581), CD45 (allophy- Retrovirally transduced NUP98-HOXA9 is expressed in cocyanin-conjugated clone HI30 or allophycocyanin-Cy7–conjugated clone human CD34+ cells. To study the role of NUP98-HOXA9 in human 2D1), CD71 (phycoerythrin-conjugated clone M-A712), and CD235a leukemogenesis, human CD34+ hematopoietic progenitors from (allophycocyanin-conjugated clone GA-R2) were used for flow cytometric mobilized peripheral blood were retrovirally transduced to express analysis. The cells were acquired with FACSCalibur (BD) or CyAn (Dako) the fusion protein. Flow cytometry analysis of a typical cell and analyzed using the CellQuest (BD), Summit (Dako), or FCS Express f (De Novo Software, Thornhill, Ontario, Canada) software. preparation just after thawing showed that 96% of the cells were + + f CFC assays. Virus-transduced sorted cells were resuspended in IMDM CD34 CD45dim progenitors. The preparation contained 1% À + + containing 2% FBS and mixed with Methocult GF+ (H4435, StemCell CD34 CD45bright lymphoid, 1% CD11b myelo/monocytic, and + Technologies, Vancouver, British Columbia, Canada), which consists of 9% CD235a erythroid lineage cells. À 1% methylcellulose, 30% FBS, 1% bovine serum albumin, 10 4 mol/L Hemagglutinin-tagged NUP98-HOXA9 was subcloned into the 2-mercaptoethanol, 2 mmol/L L-glutamine, 50 ng/mL stem cell factor, MSCV-IRES-GFP retroviral vector (Fig. 1A) as described (7) and 20 ng/mL GM-CSF, 20 ng/mL IL-3, 20 ng/mL IL-6, 20 ng/mL granulocyte GaLV-pseudotyped virus was produced in PG13 cells. CD34+ cells colony-stimulating factor (G-CSF), and 3 units/mL erythropoietin in IMDM. were transduced with the NUP98-HOXA9 virus or control virus One thousand cells suspended in 1.1 mL were plated in each 35-mm dish Â prepared with empty vector. GFP-positive cells were purified by and cultured for 14 days. Colonies were counted at 40 magnification and fluorescence-activated cell sorting (FACS). A typical FACS profile is classified into three categories: pure erythroid, myelomonocytic, and mixed. shown in Fig. 1B. Antihemagglutinin immunoblotting of the sorted Cells were then suspended in IMDM containing 2% FBS and were either transferred to slides using a Cytospin centrifuge and visualized by Giemsa GFP-positive cells confirmed the specific expression of NUP98- staining or stained with antibodies for flow cytometry. HOXA9 protein in cells transduced with NUP98-HOXA9 virus LTC-IC assays. Ten thousand virus-transduced sorted cells were divided (Fig. 1C). into two 35-mm dishes and cultured for 5 weeks with weekly one-half NUP98-HOXA9 impairs erythroid and myeloid differentia- medium changes. The murine fibroblast cell line M2-10B4 (a gift from tion and induces proliferation of immature cells. The effect of www.aacrjournals.org 6629 Cancer Res 2006; 66: (13). July 1, 2006

of either lineage were overrepresented among cells of the NUP98- HOXA9 plates. Consistent with the morphologic data, flow cytometry analysis À showed an increased proportion of CD235a+ CD45 erythroid cells and a concomitant decrease of CD11b+ CD45+ myeloid cells in the NUP98-HOXA9 plates compared with control plates (Fig. 2C). The increase in CD235a+ erythroid cells in the NUP98-HOXA9 plates was mostly due to an increase in immature CD71bright+ CD235a+ erythroid precursors, indicating a block in erythroid differentiation. Similarly, 52% of the CD45+ myeloid population of the NUP98- À HOXA9 plates was CD11b , indicating a predominantly immature phenotype, whereas only 7.7% of the myeloid population of the À control plates was CD11b (Fig. 2C). In summary, these results show that NUP98 inhibits myeloid and erythroid differentiation while increasing the numbers of immature cells. NUP98-HOXA9 has a biphasic effect on CD34+ cell proliferation. To determine the effect of NUP98-HOXA9 on the proliferation of human hematopoietic cells, sorted NUP98- HOXA9-transduced human CD34+ cells and control cells were continually grown in liquid culture in the presence of cytokines with periodic cell counting. Typical growth patterns observed in one of three independent experiments are shown in Fig. 3. The growth of NUP98-HOXA9 cells was slower than that of control cells in the early stages of the culture (Fig. 3A). However, by day Figure 1. Retroviral transduction of CD34+ primary cells. A, MSCV-IRES-GFP 9, this trend was reversed in all three experiments and NUP98- retroviral construct for the expression of hemagglutinin-tagged NUP98-HOXA9. HD, homeodomain; MSCV, murine stem cell virus; IRES, internal ribosomal HOXA9 cells grew faster than control cells. By day 12, the entry site; LTR, long terminal repeat. B, a typical FACS profile of cells cumulative number of NUP98-HOXA9 cells exceeded that of transduced with either control or NUP98-HOXA9 retrovirus. The GFP-positive control cells. NUP98-HOXA9 cells continued to proliferate for an cells shown within the gate were collected. C, antihemagglutinin immunoblotting F of sorted GFP-positive cells to confirm the expression of HA-NUP98-HOXA9 average of 54.3 1.5 days; after which, their numbers slowly protein. declined. Control cells stopped growing significantly earlier, after an average of 27.3 F 1.2 days, and their numbers were 2 orders of magnitude lower at their peak (Fig. 3B). These data suggest NUP98-HOXA9 on cell differentiation was assessed by plating that after an initial period of growth suppression, NUP98-HOXA9 sorted cells in semisolid methylcellulose-based medium for CFC induces the proliferation of primitive cells that are capable of assays. After 2 weeks of culture, plates of cells transduced with long-term growth. NUP98-HOXA9 looked strikingly different from plates of GFP- NUP98-HOXA9 promotes proliferation of LTC-ICs. The vast positive control cells even without magnification: the NUP98- majority of CD34+ cells are progenitors with no long-term self- HOXA9 plates contained much more prominent red-colored renewal capabilities. On the other hand, a small minority of more colonies than the control plates (Fig. 2A). Although total colony primitive progenitors/stem cells, known as LTC-ICs, are capable of numbers were comparable between the NUP98-HOXA9 and the self-renewal and remain clonogenic after prolonged in vitro culture. control plates, the NUP98-HOXA9 plates contained approximately These are the most primitive hematopoietic cells that can be twice as many cells. Under low magnification, many of the red assayed in vitro (10). To determine the effect of NUP98-HOXA9 on colonies in NUP98-HOXA9-transduced plates were large in size and the number of LTC-ICs, virus-transduced and sorted cells were several of those showed irregular contours with a mixed red/ cultured on a feeder layer of irradiated M2-10B4 cells (9) for colorless morphology (Fig. 2A). In contrast, control cells showed 5 weeks with weekly one-half medium changes. Cells recovered small, tight, uniformly red erythroid colonies. Giemsa staining of after 5 weeks were plated for CFC assays and colonies were cells obtained from representative red colonies showed that counted 17 days later. Cells transduced with NUP98-HOXA9 colonies from control cells consisted almost entirely of mature showed a drastic increase in the number of colonies in four hemoglobinized erythroid precursors whereas colonies from separate experiments using cells from two unrelated individuals. NUP98-HOXA9-expressing cells contained a prominent population The data are summarized in Table 2 expressed as the average of large immature blasts admixed with maturing erythroid cells numbers of LTC-ICs per 10,000 original input cells. (Fig. 2A). The blasts had open chromatin, prominent nucleoli, and Extensive changes in gene expression underlie the biological moderate to abundant amounts of vacuolated cytoplasm. effects of NUP98-HOXA9. The results described above show that To examine the entire cell population, cells were recovered from NUP98-HOXA9 inhibits hematopoietic differentiation, has a the CFC plates by suspending in medium and subjected to either biphasic effect on proliferation, and induces a dramatic increase Giemsa staining or flow cytometry analysis. Morphologic exami- in the numbers of primitive self-renewing cells. To identify the nation of the Giemsa-stained slides revealed that cells in the changes in gene expression that underlie these effects, a time- NUP98-HOXA9 plates included a prominent population of blasts course microarray study was conducted. Cells were retrovirally and consisted of higher percentages of erythroid lineage cells and transduced with NUP98-HOXA9 or control virus and sorted for lower percentages of myeloid lineage cells compared with cells in GFP expression as described above. RNA was isolated 3, 8, 10, and the control plates (Fig. 2B; Table 1). Furthermore, immature cells 16 days after transduction and subjected to oligonucleotide array

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Effect of NUP98-HOXA9 on Human CD34+ Cells expression analysis. Only those genes that exhibited z2-fold IFN-inducible genes in the 3-day samples (Table 3). Most of these difference in their expression between the NUP98-HOXA9 cells genes belong to the IFNa/h pathways while some belong to the and the controls were judged to be significantly affected by NUP98- IFNg pathway. IFNs are known to suppress hematopoietic cell HOXA9. Among the numerous genes significantly affected by growth (11). Thus, activation of the IFN signaling cascade may NUP98-HOXA9, representative genes that are most likely to explain explain the early decrease in proliferation seen in NUP98-HOXA9- its effects on cell proliferation, differentiation, and transformation transduced cells. Similarly, cyclin-dependent kinase inhibitor 1C are listed in Table 3. The supplemental list of all affected genes is (CDKN1C; also known as p57, Kip2) is known to play a role in cell available online (Supplementary Tables S1-S5). cycle arrest in hematopoietic cells (12) and its early induction The most striking feature of the gene profiling results was the (Table 3) may play a role in the early proliferation block in response dramatic up-regulation by NUP98-HOXA9 of a large number of to NUP98-HOXA9. The expression of both IFN-inducible genes and

Figure 2. NUP98-HOXA9 impairs both erythroid and myeloid differentiation. A, left, CFC assay plates of cells transduced with either control or NUP98-HOXA9 retrovirus. Middle, low-power photomicrographs of representative red colonies found in the control or the NUP98-HOXA9 plate. Right, Giemsa-stained cytospins of representative red colonies. B, Giemsa-stained cytospins of cells recovered from the whole CFC plates. Morphology scores are shown in Table 1. C, flow cytometric analysis of total cells recovered from the CFC plates. Cells were stained with CD45, CD235a, and CD71 antibodies (left) or with CD45 and CD11b antibodies (right). Bottom left, CD235a+ cells were plotted on a histogram to show CD71 expression. Bottom right, CD45+ cells were plotted on a histogram to show CD11b expression.

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q Table 1. NUP98-HOXA9 enhances blast formation and regulation of RARa leading to down-regulation of C/EBP , which impairs both erythroid and myeloid differentiation results in down-regulation of granule proteins and other markers of terminal myeloid differentiation. Control NUP98-HOXA9 P The microarray data also point to a block in megakaryocytopoiesis. For example, pro-platelet basic protein (PPBP; h-thromboglo- Blasts/promyelocytes 1.35 F 2.06 10.70 F 3.70 0.028 bulin), a component of platelet a granules (17), and FLI1, an Ets Intermediate erythroid 3.80 F 4.44 27.55 F 7.39 0.007 family transcription factor that drives megakaryocytopoiesis (18), Mature erythroid 9.05 F 2.74 30.75 F 12.03 0.036 were down-regulated whereas the megakaryocytopoiesis inhibitor Myelocytes/metamyelocytes 28.55 F 7.17 12.10 F 5.02 0.010 thrombospondin 1 (19) was up-regulated (Table 3). Neutrophils/band cells 36.45 F 12.21 10.25 F 3.04 0.023 Most hemoglobin genes were up-regulated, consistent with the F F Macrophages 20.80 3.59 8.65 1.09 0.011 increased numbers of erythroid precursors observed in CFC Total 100 100 cultures of cells transduced with NUP98-HOXA9 (Table 1). Some genes associated with erythroid differentiation, such as APOE, NOTE: Cells collected from CFC plates were transferred to slides and AQP1, and ETS1 (20–22), were down-regulated. As globin genes are visualized by Giemsa staining. Five hundred cells found in randomly expressed early during erythroid differentiation (23), these data are chosen areas of each slide were categorized and the numbers were consistent with the block in erythroid maturation observed in expressed as percentages of total cells. The data shown are averages F NUP98-HOXA9-transduced cells by morphology and flow cyto- SD of four independent experiments. The P values obtained from a metry (Fig. 2B and C). paired Student’s t test show a significant difference between control To verify the microarray results, RNA levels of selected genes and NUP98-HOXA9 samples for all categories. were determined by quantitative RT-PCR. The fold changes in gene expression (shown between brackets in Table 3) were similar to those obtained by microarray analysis. Early NUP98-HOXA9 target genes include IFNB1 and many CDKNC1 declined or returned to baseline later on as NUP98- transcription factors. NUP98-HOXA9actsasanaberrant HOXA9-transduced cells entered the proliferative phase. leukemogenic transcription factor that probably binds DNA While expression of the IFN-inducible genes peaked on day 3, through its HOXA9 homeodomain (6, 7), yet its direct target genes many genes associated with cell proliferation and oncogenesis peaked at day 8 or beyond (Table 3), approximately coinciding with the beginning of the proliferative phase of NUP98-HOXA9- expressing cells (Fig. 3). These include a number of homeobox transcription factors, growth factors, receptor tyrosine kinases, and other oncogenes. Among the induced genes were several that are known to be involved in myeloid leukemogenesis, including HOXA9,HOXA7,MEIS1,FLT3,KIT,WT1,andANGPT1 (see Discussion). In addition, several genes with putative tumor suppressor functions were down-regulated, especially at later time points. At the cell cycle level, the increased cell proliferation was accompanied by up-regulation of cyclin A1, a cell cycle regulator that is overexpressed in AML (13), and down-regulation of several cell cycle inhibitors (Table 3). Microarray results (Table 3) confirmed the inhibition of myelomonocytic differentiation by NUP98-HOXA9 and suggested possible mechanisms. Genes encoding secondary neutrophil granule proteins such as lactoferrin, transcobalamin 1, and ABP1, which are expressed later in myeloid differentiation (14), were down-regulated. On the other hand, some genes encoding primary granule proteins, such as neutrophil elastase and cathepsin G (14), were up-regulated, whereas others, such as BPI and cathepsin B (14), were down-regulated. These findings are consistent with a partial differentiation block at around the blast/promyelocyte stage (14). Indeed, most cases of AML associated with NUP98 gene rearrangements belong to subcategories M2, M4, and M5 in which myeloid differentiation is only partially blocked (2). Interestingly, CCAAT/enhancer binding protein q (C/EBPq), a transcription factor that is essential for terminal granulocytic differentiation and is important for the expression of secondary granule proteins (15), Figure 3. NUP98-HOXA9 has a biphasic effect on cell proliferation. Cells were transduced with either control or NUP98-HOXA9 retrovirus (day 0), sorted was down-regulated by NUP98-HOXA9 (Table 3). Further, the (day 2), and continually cultured in the presence of cytokines with periodic cell down-regulation of C/EBPq was preceded by down-regulation of counting and feeding. The experiment was repeated thrice and the results retinoic acid receptor a (RARa), a transcription factor known to of a representative experiment are shown. A, cumulative numbers of q NUP98-HOXA9 cells up to day 19 expressed as percentage of cumulative control promote the expression of C/EBP (16). Thus, the inhibition of cell numbers. B, growth patterns of control (o) or NUP98-HOXA9 (n)cells myeloid differentiation by NUP98-HOXA9 may involve down- displayed as cumulative cell numbers originating from one cell of the day 2 culture.

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Table 2. NUP98-HOXA9 promotes proliferation of provide robust assays for the further dissection of those LTC-ICs mechanisms and for preliminary testing of potential therapies. Initial Antiproliferative Effect of NUP98-HOXA9 LTC-IC Continuous culture of NUP98-HOXA9-transduced CD34+ cells in liquid medium revealed initial growth inhibition (Fig. 3). Because Control 1.6 F 2.8 the vast majority of CD34+ cells are lineage-committed progenitors NUP98-HOXA9 43.8 F 13.7 (24), the cells that failed to grow and/or died at the initial stage consist mostly, if not entirely, of these progenitors. IFNs are known NOTE: Cells transduced with either control or NUP98-HOXA9 virus for their growth-inhibitory effects on cells, including those of the were assayed for LTC-ICs. The numbers shown are average numbers of hematopoietic lineage (11). Therefore, the early induction of IFNh1 LTC-IC per 10,000 original input cells F SD. Four independent and IFN-inducible genes (Table 3) may explain the antiproliferative experiments were carried out using cells from two unrelated effect of NUP98-HOXA9 in the first days after transduction. individuals. The difference between the control and NUP98-HOXA9 Interestingly, the oncoprotein P210/BCR-ABL has also been samples was highly significant with P = 0.006 based on a paired Student’s t test. reported to up-regulate a group of IFN-responsive genes (25). In addition, IFN-inducible genes are up-regulated in preleukemic conditions such as aplastic anemia, paroxysmal nocturnal hemo- globinuria, and MDS (26). Thus, induction of IFN pathways may in primary human hematopoietic cells are not known. While the reflect a common response during leukemogenesis. The mecha- gene changes observed 3 days or more after NUP98-HOXA9 nism by which NUP98-HOXA9 up-regulates IFNh1 expression transduction yielded important information relevant to its remains to be determined, and it is not clear whether the IFN biological effects (Table 3), many of these genes are probably response is part of the transformation process or a reaction affected by NUP98-HOXA9 indirectly, rather than being direct against it. transcriptional targets of NUP98-HOXA9. In addition, the mechanism of the strong induction of IFN-inducible genes by NUP98- Leukemogenesis and Induction of Long-term HOXA9 at 3 days posttransduction was not clear. To address these Proliferation by NUP98-HOXA9 issues, a microarray analysis needed to be done within hours of The initial suppression of proliferation by NUP98-HOXA9 was NUP98-HOXA9 expression in cells, which is not possible with followed by several weeks of sustained proliferation (Fig. 3), retroviral transduction. We therefore used the Amaxa Nucleofector suggesting an expansion of primitive progenitors and/or stem cells. device that introduces DNA into primary cells efficiently and allows This notion was confirmed by the dramatic increase in the rapid expression of the protein of interest. CD34+ cells were numbers of LTC-ICs (Table 2). This biphasic effect of NUP98- transfected with a NUP98-HOXA9 construct that expresses a GFP HOXA9 on hematopoietic cells, with early growth inhibition and marker from a separate promoter; control cells were transfected late proliferation of primitive cells, is reminiscent of the with the empty vector. GFP-positive cells were purified by FACS progression of MDS to AML. Indeed, NUP98-HOXA9 and other after 3.5 hours, followed by further culture for 2.5 hours. Expression NUP98 fusions have been observed in cases of MDS (2), and mice of NUP98-HOXA9 protein was confirmed by immunoblotting (not transgenic for NUP98-HOXD13, another leukemogenic NUP98-HOX shown) and total RNA was isolated. Microarray analysis showed fusion, develop severe MDS followed by acute leukemia (4). that NUP98-HOXA9 up-regulated IFNh1 expression within 6 hours Interestingly, NUP98-HOXA9 induced proliferation of erythroid posttransfection (Table 3). In contrast, all but one of the IFN- precursors (Table 1; Fig. 2), which is often a feature of human MDS inducible genes that were up-regulated after 3 days were still at (27). In addition, erythroid proliferation has been reported when baseline at this time point. These results indicate that the early leukemogenic proteins such as BCR-ABL, constitutively active induction of IFNh1 expression led to the subsequent induction of STAT5A, and AML1-ETO are overexpressed in human CD34+ cells the genes involved in IFN-signaling pathways. Overexpression of (28–30). Similar to NUP98-HOXA9, AML1-ETO caused initial IFNh1 was confirmed by quantitative real-time PCR(Table 3). inhibition of cell proliferation followed by increased long-term Another interesting observation is that many transcription proliferation and expansion of primitive cells (30–32). However, the factors, particularly homeobox factors, were already induced by initial growth inhibition lasted f4 weeks with AML1-ETO as NUP98-HOXA9 at the 6-hour time point. This suggests that the opposed to only 9 days with NUP98-HOXA9 (Fig. 3). In addition, effects of NUP98-HOXA9 on CD34+ cells may be mediated, at least human CD34+ cells transduced with AML1-ETO continued to grow in part, by the induction of other transcription factors, especially for over 7 months in culture whereas cells transduced with NUP98- those of the homeobox family. HOXA9 stopped growing after f54 days. It remains to be determined whether NUP98-HOXA9 increases the number of LTC-ICs by stimulating the proliferation of multipotent progeni- Discussion tors/stem cells or whether it endows more mature progenitors with In the present study, we undertook a comprehensive investiga- self-renewal capacity. tion of the biological effects of NUP98-HOXA9 on primary human Microarray analysis revealed marked changes in gene expression CD34+ progenitor/stem cells and correlated those effects with that point to the mechanisms behind the long-term proliferation of changes in global gene expression. These studies followed the cells transformed by NUP98-HOXA9. The IFN response, which effects of NUP98-HOXA9 from the earliest time point within peaks at day 3, began to subside at day 8 as evidenced by dramatic 6 hours of the introduction of the oncogene up to several weeks decreases in the levels of IFN-responsive genes. This coincided with thereafter. The results provide insights into the mechanisms by the beginning of the proliferative phase of NUP98-HOXA9- which NUP98-HOXA9 transforms human hematopoietic cells and transduced cells at around day 9 (Fig. 3). In contrast to IFN www.aacrjournals.org 6633 Cancer Res 2006; 66: (13). July 1, 2006

Table 3. NUP98-HOXA9 induces changes in gene Table 3. NUP98-HOXA9 induces changes in gene expression that account for its effects on proliferation expression that account for its effects on proliferation and differentiation and differentiation (Cont’d)

Gene symbol Fold change Gene symbol Fold change

6h 3d* 8d 10d16d 6h 3d* 8d 10d 16d

b IFN pathway HOXA9 2.00 3.70 3.61 2.75 4.70 IFNB1 4.59 (4.46 F 0.42) c (7.10 F 0.73) HOXB2 2.00 IFI44L 531.00 238.86 52.00 4.29 HOXC6 2.64 IFI44 20.40 5.25 5.66 2.30 PBX3 2.14 2.86 IFI27 9.39 18.38 18.40 4.59 (2.76 F 0.36) IFIH1 4.01 2.64 2.46 MEIS1 2.00 2.46 2.00 2.46 IFI16 2.70 (2.12 F 0.37) IFI35 2.65 HOP 10.80 8.00 9.85 5.28 IFIT1 35.50 51.98 12.10 3.03 IRX3 3.42 24.25 21.10 9.85 IFIT2 10.20 3.48 6.46 2.64 DLX2 4.92 IFIT3 8.15 3.61 5.25 2.64 MSX1 2.14 IFIT5 5.47 2.64 HLX1 2.07 IFITM1 2.58 2.30 2.49 2.07 Growth factors MX1 39.40 13.00 9.19 4.00 REN (renin) 68.60 51.98 34.30 13.93 (83.20 F 3.89) (352.0 F 11.2) MX2 18.00 3.73 4.59 2.46 TSLP 4.63 2.30 36.80 4.59 G1P2 8.88 4.92 4.92 3.25 ANGPT1 2.64 5.66 4.00 G1P3 9.39 4.00 6.50 3.03 ANGPT2 2.76 14.90 OAS1 15.00 4.90 3.63 FGF18 14.20 2.46 2.46 OAS2 5.87 4.29 3.88 3.38 Oncogenes OAS3 5.13 4.00 4.00 2.00 FLT3 2.00 2.30 2.46 GBP1 3.55 2.67 2.58 (2.26 F 0.49) (1.97 F 0.42) BIRC4BP 5.06 2.83 3.83 2.14 KIT 2.14 2.83 EIF2AK2 2.65 2.14 WT1 2.30 IRF7 5.47 2.46 3.03 EVI1 2.38 2.27 2.00 (6.31 F 1.17) MEF2C 2.10 2.23 ISGF3G 3.74 SOX4 2.39 2.07 2.55 2.64 STAT1 2.65 2.07 2.04 2.00 MYCN 2.67 24.25 STAT2 2.15 MLLT3 (AF9) 2.07 INDO 4.16 3.73 13.90 6.96 ZNFN1A1 16.00 SP110 2.65 2.25 (Ikaros) DDX58 3.26 2.46 2.39 KLF5 2.00 2.67 USP18 5.88 10.56 6.50 4.92 PTGS2 (COX2) 2.71 2.30 2.30 2.14 TRIM22 2.49 Putative tumor RSAD2 8.07 4.09 11.60 3.62 suppressors À LY6E 3.38 7.46 6.96 3.73 CACNA1G 2.14 À À CD69 2.32 3.62 DOCK4 2.00 2.30 RARRES1 À3.03 À2.46 (3.32 F 0.59) (TIG1) CXCL10 3.26 2.00 Myelomonocytic IFRG28 2.83 differentiation Cell cycle ELA2A 21.40 5.10 3.25 CDKN1C 6.17 2.14 (elastase 2A) (p57, Kip2) (7.16 F 2.03) CTSG 2.01 3.25 CCNA1 3.03 (cathepsin G) (cyclin A1) BPI À6.06 À4.59 À2.30 CDC14B À5.66 CTSB À2.22 À2.15 CDC14A À2.00 (cathepsin B) KLF2 À2.52 À2.14 À2.83 DEFA4 À4.29 À4.59 À2.00 À À À LGALS12 2.64 2.00 2.14 (defensin, a4) Homeobox DEFA1/3 À4.00 À8.00 HOXA3 2.83 7.77 8.00 8.57 4.29 (defensin, a1/3) HOXA4 2.30 LTF (lactoferrin) À17.15 HOXA5 2.14 3.87 6.06 3.25 5.66 HOXA6 2.87 HOXA7 2.39 3.73 3.03 3.03 (Continued on the following page)

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Table 3. NUP98-HOXA9 induces changes in gene Homeobox genes. These encode a group of transcription expression that account for its effects on proliferation factors well known for their involvement in normal hematopoiesis and differentiation (Cont’d) and leukemogenesis, including HOXA9, HOXA7, HOXA4, MEIS1, PBX3, and others (Table 3). Several homeobox transcription factors Gene symbol Fold change are expressed in primitive hematopoietic cells and down-regulated with differentiation (33). HOXA9 plays an important role in 6h 3d* 8d 10d16d hematopoietic stem cell proliferation (34) and its overexpression in mouse bone marrow causes AML, which is accelerated by TCN1 3.30 À2.00 À2.00 coexpression of MEIS1 (3). MEIS1 also accelerates the induction of (transcobalamin 1) AML by NUP98-HOXA9 in mice (3). Several homeobox genes, À À ABP1 4.92 2.64 including HOXA9, HOXA7, and MEIS1, are frequently overexpressed À À À MMP9 (gelatinase) 4.29 4.59 2.30 in human AML and are associated with worse prognosis (35–39). ALOX5 À3.21 À2.87 À2.70 Interestingly, a number of homeobox genes, particularly HOXA7 CEACAM6 (CD66c) À2.84 À6.28 À4.29 CD163 À2.07 À2.46 and HOXA9, are downstream targets of leukemogenic fusions of the CD24 À2.68 À2.77 À2.29 MLL gene at chromosome 11q23 and are thought to mediate the CD14 À2.00 leukemogenic effects of these fusions in AML (40). The early and RARA À3.03 sustained induction of homeobox transcription factors by NUP98- CEBPE À2.30 À2.46 À2.00 HOXA9 (Table 3) suggests that they may play a similar role in Megakaryocyte mediating leukemogenesis by NUP98 fusions, and their over- differentiation expression may contribute to the worse prognosis of patients with THBS1 2.91 2.14 3.71 these fusions. (thrombospondin 1) Other leukemogenic transcription factors. Several other PPBP (CXCL7, À3.62 À4.59 À2.46 transcription factors with a strong association with AML were h-thromboglobulin) F13A1 (coagulation À2.14 up-regulated by NUP98-HOXA9. These include EVI1, MEF2C, SOX4, factor XIII, A1) and WT1 (Table 3). EVI1 is overexpressed in some cases of AML in DAB2 À2.31 À2.43 which it is associated with a poor prognosis (41). Its overexpression FLI1 À7.17 in response to NUP98-HOXA9 may contribute to the poor Erythroid differentiation prognosis in patients with NUP98 gene rearrangements. Interest- HBD (hemoglobin, y) 3.73 3.48 10.56 ingly, EVI1 is thought to counteract the antiproliferative effects of HBB (hemoglobin, h) 2.22 2.35 4.73 IFNa (42) and may therefore contribute to the proliferation of HBG1/2 8.60 NUP98-HOXA9-expressing cells in spite of the activation of IFN (hemoglobin, gA/G) pathways. SOX4 can cooperate with either MEF2C or EVI1 to HBA1/2 3.03 induce AML in mice (43, 44). WT1 plays a key role in cell (hemoglobin, a1/2) RHAG (Rhesus 2.00 proliferation in many human neoplasms, particularly acute blood group– leukemia, and is used to monitor minimal residual disease AML associated and MDS (45). glycoprotein) Receptor tyrosine kinases. In addition to aberrant transcrip- APOE À2.07 À2.00 tional regulation, the pathogenesis of AML often involves AQP1 À2.00 abnormalities of signaling pathways. Two receptor tyrosine kinases KLF1 2.46 well known for their role in the pathogenesis of AML were up- À À ETS1 4.09 2.47 regulated by NUP98-HOXA9: FLT3 kinase and KIT (Table 3). À F ( 5.40 1.98) Activation of FLT3 by mutation and/or internal tandem duplica- tion is observed in about one third of AML cases and increased *For the 3 days posttransduction samples, the fold changes represent expression of wild-type FLT3 is seen in some AML patients in the average of two separate experiments using cells derived from two which it seems to be associated with a worse prognosis (39, 46). unrelated individuals. Similarly, KIT, the receptor for stem cell factor, is expressed in cNumbers in parentheses represent the results obtained by quantita- blasts from 80% of AML patients and is activated by mutation in tive real-time PCRdone in triplicate on cDNA derived from the same many cases of AML (46). preparations of total RNA used for the microarray analysis. Growth factors. Several secreted factors associated with cell b Oligonucleotide microarray probe sets and quantitative PCRprimers proliferation and leukemogenesis were up-regulated by NUP98- used to detect HOXA9 are not present in the sequence of the chimeric HOXA9, including renin, TSLP, angiopoietins, and fibroblast growth NUP98-HOXA9 construct; thus, the up-regulation of HOXA9 reflects factor 18 (FGF18) (Table 3). Among them, the induction of renin true induction of the endogenous HOXA9 gene. expression was particularly strong. Renin is a constituent of the renin-angiotensin system (RAS) and converts angiotensinogen to angiotensin I, which then is converted by angiotensin-converting enzyme to angiotensin II, a major regulator of blood pressure and pathway genes, the expression of most of the genes associated with electrolyte balance. In addition to its classic role, the RAS may play proliferation and oncogenesis peaked at later time points (8 days an important role in hematopoiesis (47). Indeed, angiotensin II has or more after transduction). As discussed below, several of these been shown to increase the proliferation of both primitive and genes are likely to play an important role in the pathogenesis of committed hematopoietic progenitors. Blasts from many AML AML in patients with NUP98-HOXA9 gene rearrangements. patients express renin whereas normal bone marrow cells do not. www.aacrjournals.org 6635 Cancer Res 2006; 66: (13). July 1, 2006

Thus, it is possible that the enhanced expression of renin is a part transcription factors such as PML-RARa and AML1-ETO in of the mechanism for uncontrolled cell growth in some leukemias. inducing leukemia in mice (49, 50). Therefore, early overexpression of FLT3 may similarly cooperate with NUP98-HOXA9 in the Early NUP98-HOXA9 Target Genes leukemic transformation of human cells. Examination of changes in gene expression 6 hours after + In summary, the present data show that NUP98-HOXA9 alters introduction of NUP98-HOXA9 into CD34 cells provided a list of gene transcription in human hematopoietic precursors, resulting in likely direct transcriptional targets of NUP98-HOXA9 (Table 3 and disruption of cell differentiation and a biphasic effect on cell Supplementary Table S1). In addition, it provided clues to the growth. The observed changes in gene expression identify likely earliest events in NUP98-HOXA9 oncogenesis. The leukemogenic direct target genes for NUP98-HOXA9 and provide clues to the genes induced by NUP98-HOXA9 within 6 hours are mostly mechanisms by which it transforms human hematopoietic cells. transcription factors, including members of the homeobox family The assays described, particularly the LTC-IC assay, can be used to and EVI1 (Table 3). This suggests that the initiating oncogenic further dissect these mechanisms and to test potential therapeutic event is an abnormal transcriptional program that leads to agents. secondary changes in gene expression, culminating in blocked differentiation and increased proliferation. For example, NUP98- HOXA9 induces renin at 3 days but not at 6 hours. Because the renin gene promoter contains HOX/PBX-binding sites and is Acknowledgments thought to be regulated by HOX/PBX proteins (48), its induction by Received 2/6/2006; revised 4/9/2006; accepted 4/28/2006. Grant support: NIH grant RO1 HL 82549 and Myeloid Malignancies Specialized NUP98-HOXA9 may be mediated by the early induction of Center of Research grant from the Leukemia and Lymphoma Society. homeobox transcription factors. The costs of publication of this article were defrayed in part by the payment of page Interestingly, the leukemogenic receptor tyrosine kinase FLT3 is charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. also up-regulated within 6 hours of NUP98-HOXA9 transduction. We thank Dr. Malcolm Moore for retrovirus-producing cells and Dr. Richard Burt Constitutively active FLT3 can cooperate with leukemogenic fusion and Marcelo Villa for help with obtaining CD34+ primary human hematopoietic cells.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. NUP98-HOXA9 Induces Long-term Proliferation and Blocks Differentiation of Primary Human CD34 + Hematopoietic Cells

Akiko Takeda, Charles Goolsby and Nabeel R. Yaseen

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