Continuous MLL-ENL Expression Is Necessary to Establish a ''Hox Code'' and Maintain Immortalization of Hematopoietic Progenitor Cells

Research Article

Continuous MLL-ENL Expression Is Necessary to Establish a ‘‘Hox Code’’ and Maintain Immortalization of Hematopoietic Progenitor Cells

Sarah J. Horton,1 David G. Grier,3 Glenda J. McGonigle,3 Alexander Thompson,3 Michelle Morrow,1 Inusha De Silva,1 Dale A. Moulding,1 Dimitris Kioussis,2 Terence R.J. Lappin,3 Hugh J.M. Brady,1 and Owen Williams1

1Molecular Haematology and Cancer Biology Unit, Institute of Child Health, University College London; 2Division of Molecular Immunology, National Institute for Medical Research, London, United Kingdom; and 3Department of Child Health, Queen’s University, Belfast, United Kingdom

Abstract essential for definitive hematopoiesis and that it is required to The t[(11;19)(p22;q23)] translocation, which gives rise to the maintain, but not to initiate, the expression of multiple Hox genes MLL-ENL fusion protein, is commonly found in infant acute during embryogenesis (5–9). Some Hox genes are oncogenic when leukemias of both the myeloid and lymphoid lineage. To overexpressed in hematopoietic progenitor cells (10, 11). Taken investigate the molecular mechanism of immortalization by together, this suggests that aberrant regulation of Hox genes by MLL-ENL we established a Tet-regulatable system of MLL- MLL fusion proteins is the basis for leukemias involving MLL ENL expression in primary hematopoietic progenitor cells. translocations (12). Several recent publications do indeed suggest Immortalized myeloid cell lines were generated, which that Hox genes may play an important role in leukemia induced by are dependent on continued MLL-ENL expression for their MLL fusion proteins (13–16). survival and proliferation. These cells either terminally MLL translocations result in the generation of an in-frame differentiate or die when MLL-ENL expression is turned chimeric fusion in which MLL is joined to one of over 40 different off with doxycycline. The expression profile of all 39 murine partner genes, of which the most common are ENL, AF9, and AF4 Hox genes was analyzed in these cells by real-time (17). The t[(11;19)(p22;q23)] translocation results in the fusion of the MLL gene to the eleven-nineteen-leukemia (ENL)gene. quantitative PCR. This analysis showed that loss of MLL- ENL was accompanied by a reduction in the expression of Different murine models have been generated which recapitulate multiple Hoxa genes. By comparing these changes with Hox MLL-ENL–mediated leukemia. Immortalized myeloid and B-cell gene expression in cells induced to differentiate with lines, both capable of inducing leukemia in vivo, have been granulocyte colony-stimulating factor, we show for the first generated by retroviral transduction of hematopoietic progenitor time that reduced Hox gene expression is specific to loss of cells with MLL-ENL (18, 19). An interchromosomal recombination MLL-ENL and is not a consequence of differentiation. Our model has also been developed in which the de novo translocation data also suggest that the Hox cofactor Meis-2 can substitute of the MLL and ENL loci occurred specifically in hematopoietic for Meis-1 function. Thus, MLL-ENL is required to initiate cells (20). These mice developed AML with high penetrance and and maintain immortalization of myeloid progenitors and short latency, suggesting that the MLL-ENL translocation is the only event required for the development of leukemia (20). may contribute to leukemogenesis by aberrantly sustaining the expression of a ‘‘Hox code’’ consisting of Hoxa4 to To gain more insight into the molecular mechanism of Hoxa11. (Cancer Res 2005; 65(20): 9245-52) immortalization by the MLL-ENL fusion protein, we established a conditional system for MLL-ENL expression in murine hematopoietic progenitor cells. We used retroviral delivery in combination Introduction with the Tet-Off conditional expression system to regulate the Translocations involving the mixed lineage leukemia (MLL) gene expression of the full-length MLL-ENL fusion protein. We on chromosome band 11q23 are associated with leukemias of both determined whether continued MLL-ENL expression was required the myeloid and lymphoid lineage (1, 2). MLL translocations are to maintain as well as initiate myeloid immortalization, and most prevalent in infant leukemia where they comprise 80% of analyzed the expression profile of all 39 murine Hox genes in MLL- cases of acute lymphoblastic leukemia and 60% of cases of acute ENL immortalized cell lines. myeloid leukemia (AML; ref. 3). Infant leukemias bearing MLL translocations tend to have a particularly poor prognosis (3). Materials and Methods MLL is the human homologue of the Drosophila trithorax (TRX) Mice. All mice were maintained in the animal facilities of the National gene (4). Gene targeting studies in mice have revealed that MLL is Institute for Medical Research and experiments done according to National Institute for Medical Research institutional guidelines and United Kingdom Home Office regulations. Retroviral constructs. The pMSCV-MLL-ENL (MSCV-M/E) vector was Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). constructed by subcloning the flag-tagged 5VMLL cDNA fragment (amino Requests for reprints: Owen Williams, Molecular Haematology and Cancer acids 1-1251; kindly provided by A. Biondi; ref. 21) into a modified version of Biology Unit, Institute of Child Health, University College London, 30 Guilford Street, pMSCV-neo (BD Clontech, Palo Alto, CA) upstream of the phosphoglycerate London WC1N 1EH, United Kingdom. Phone: 44-20-7813-8192; Fax: 44-20-7813-8100; kinase (PGK) promoter and neor. The 3VMLL-ENL cDNA (amino acids 1,252- E-mail: [email protected]. I2005 American Association for Cancer Research. 1,444 of MLL and amino acids 5-559 of ENL; kindly provided by D.C. doi:10.1158/0008-5472.CAN-05-1691 Tkachuk; ref. 22) was then ligated in-frame downstream of the 5VMLL cDNA www.aacrjournals.org 9245 Cancer Res 2005; 65: (20). October 15, 2005

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Cancer Research fragment. For some experiments, the flag tag was replaced with a Myc tag. Real-time quantitative PCR. Quantitative PCR reactions, using TaqMan The tetracycline transactivator (tTA) cDNA (BD Clontech) was subcloned probe based chemistry (Applera, Foster City, CA), were carried out and into pMSCV-internal ribosome entry site (IRES)-enhanced green fluorescent murine target amplicons were analyzed in a similar manner to the human protein (EGFP) upstream of the IRES-EGFP cassette to generate the MSCV- set, as previously described (24). All oligonucleotides were designed against tTA vector (23). The MSCV-tetracycline response element (TRE) vector was GenBank published sequences (Supplementary Table) using Primer Express generated by ligating the TRE (BD Clontech) downstream of the PGK-Neor software (Applera). Nucleotide sequences for oligonucleotide primers and in pMSCV-neo. The myc-tagged MLL-ENL cDNA fragment was then ligated probes are available on request. Total RNAs isolated from all major adult into MSCV-TRE downstream of the TRE to generate the MSCV-TRE-M/E and fetal tissues, including whole embryos, were pooled and used for the vector. validation of the murine Hox TaqMan primers and probes. The quantitative Isolation and infection of hematopoietic progenitor cells. Retroviral PCR analysis of Hox cofactor (Fig. 6) and FLT3 (Supplementary Fig. S4) gene supernatants were produced as described previously (23). Hematopoietic expression was done using predesigned TaqMan primers and probes progenitor cells were purified from single-cell suspensions of bone marrow, (Applera) and an ABI prism 7000 sequence detection system (Applera). extracted from C57BL/10 or C57BL/6 mice 5 days after i.v. injection of Standard curve generation. Amplicons generated using the forward 150 mg/kg 5-fluorouracil (Faulding Pharmaceuticals, Leamington Spa, and reverse primer pairs of the original Hox targets were cloned into United Kingdom). Magnetic activated cell sorting was used to purify c-Kit+ pCR2.1-TOPO TA (Invitrogen) or pGEM-T Easy (Promega, Southampton, hematopoietic progenitor cells using monoclonal antibody (mAb) specific to United Kingdom) using standard protocols. Plasmid DNA was prepared c-Kit (2B8; BD Biosciences, PharMingen, San Diego, CA), and lineage using the Qiagen miniprep kit (Qiagen Ltd., Crawley, United Kingdom) depleted hematopoietic progenitor cells, using mAbs from a lineage panel kit according to the instructions of the manufacturer. Quantitation of plasmid (BD PharMingen). Hematopoietic progenitor cells were cultured with 100 DNA was determined spectrophotometrically. An A260 value of 0.1 or higher ng/mL stem cell factor (SCF), 10 ng/mL interleukin-3 (IL-3), and 10 ng/mL was deemed satisfactory and, in terms of plasmid purity, an A260/A280 of IL-6 (Peprotech EC, London, United Kingdom) for 24 hours before infection. 1.7 to 2.0 was accepted. The plasmid DNA concentration was converted to Hematopoietic progenitor cells were infected with retrovirus supplemented copy numbers of plasmid using the following formula: V (AL) = {1 108 with the same growth factors on 2 consecutive days by spinoculation 309 [plasmid size + insert size (bp)]} / [plasmid concentration (Ag/AL) (centrifugation at 700 g,25jC, 45 minutes) in the presence of 5 Ag/mL 1 106 6.02 1023]. Plasmids were diluted to 109 copies/AL and polybrene (Sigma-Aldrich, Poole, United Kingdom). Coinfections were done linearized with NotI (New England Biolabs, Beverly, MA). Quantitative PCR using unconcentrated MSCV-TRE-M/E and concentrated MSCV-tTA super- analyses were done in triplicate using serially diluted plasmids (in the natants. 101-107 copies/AL range) as templates, a primer and probe mixture, and Colony-forming assays and generation of myeloid cell lines. Universal Mastermix (Applied Biosystems). All standard curves, correlation Transduced hematopoietic progenitor cells were cultured in 1.1 mL coefficients, gradient, and intercept values were generated using the ABI duplicate methylcellulose cultures in 35 mm plates 24 hours after infection. 7700 sequence detection system and associated software (version 1.7) Cells were plated in Methocult M3434 (Stem Cell Technologies, Vancouver, according to the instructions of the manufacturer. Canada) containing SCF, IL-3, and IL-6 and supplemented with 10 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) and 1 mg/mL G418 (Life Technologies, Invitrogen, Paisley, United Kingdom). After 6 to Results 10 days, colonies were scored and cells harvested and pooled. Subsequent Continued MLL-ENL expression is required for immortali- 4 rounds were done by replating 1 10 cells under identical conditions in zation. The retroviral constructs used in this study are depicted in the absence of G418. In some cases, 2 Ag/mL doxycycline (BD Clontech) was Fig. 1A. Cells cotransduced with MSCV-TRE-M/E and MSCV-tTA added. Colonies were stained with 1 mg/mL p-iodonitrotetrazolium (Sigma- will express the MLL-ENL fusion gene as a consequence of tTA Aldrich) in PBS. Myeloid cell lines, generated from pooled tertiary round binding the TRE. Doxycycline inhibits tTA binding of the TRE and colonies, were cultured in RPMI 1640 (Life Technologies, Invitrogen) with results in loss of MLL-ENL expression. Expression of the full-length 10% FCS, L-glutamine, and 50 Amol/L 2-mercaptoethanol (basic medium) in the presence of SCF, IL-3, and IL-6. One constitutive (C-ME1) and three MLL-ENL protein (>220 kDa) was confirmed by Western blot conditional (TRE-ME2, TRE-ME3, and TRE-ME6) lines were generated. analysis of 3T3 cells cotransduced with MSCV-TRE-M/E and Differentiation of the cell lines was done in basic medium supplemented MSCV-tTA (Fig. 1B). Expression of MLL-ENL was not detected with either 10 ng/mL granulocyte colony-stimulating factor (G-CSF) or in cotransduced cells 24 hours after the addition of doxycycline. 10 ng/mL macrophage colony-stimulating factor (M-CSF). Cytospins were Hematopoietic progenitor cells transduced with MSCV-M/E or made using a Shandon cytospin 3 (Thermo Electron Corporation, Waltham, cotransduced with MSCV-TRE-M/E and MSCV-tTA, but not control MA) and fixed and stained with May-Grunwald-Giemsa (MGG). vectors, formed large compact colonies in methylcellulose cultures, Flow cytometry. Cells were stained with phycoerythrin-conjugated mAbs composed of myeloblasts (Fig. 1C-E), which replated indefinitely specific for c-Kit (2B8), Mac-1 (M1/70), and Gr-1 (RB6-8CS), and isotype (data not shown). However, in the presence of doxycycline, the control antibodies (all from BD Biosciences, PharMingen) and F4/80 (Caltag, Burlingame, CA). Cells were resuspended in PBS, 0.5% bovine serum albumin, conditionally immortalized cells only formed a few small clusters, and 0.05% sodium azide, and preincubated with unlabeled anti-Fcg III/II mostly composed of terminally differentiated macrophages and receptor mAb (2.4G2; BD Biosciences, PharMingen) before staining with neutrophils (Fig. 1C-E). Addition of doxycycline to MSCV-M/E primary antibody. Flow cytometry was done on a Beckman Coulter Epics XL cultures did not significantly affect the number and size of colonies analyzer (Beckman Coulter, High Wycombe, United Kingdom) and data formed or their myeloblast composition (Fig. 1C-E). These data analyzed using EXPO3 software. suggest that continued expression of MLL-ENL is required for Western blot analysis. Cells were lysed using NP40 buffer [150 mmol/L colony formation and for maintenance of the myeloblast NaCl, 0.5% NP40, 50 mmol/L Tris (pH 8.0)] containing 1 complete phenotype of the immortalized cells. protease inhibitor tablets (Roche Applied Science, Indianapolis, IN). Protein Characterization of MLL-ENL immortalized cell lines. Three samples were resolved on a 6% SDS polyacrylamide gel and transferred to a conditional cell lines (TRE-ME2, TRE-ME3, and TRE-ME6) were polyvinylidenefluoride membrane (Immobilon-P, Millipore, Billerica, MA). Proteins were detected with anti-myc (clone 9B11; Cell Signaling generated from hematopoietic progenitor cells cotransduced with Technology, Inc., Beverly, MA) or anti–a-tubulin (clone YL1/2; Serotec, MSCV-TRE-M/E and MSCV-tTA in three independent experiments, Oxford, United Kingdom), the appropriate secondary horseradish peroxi- and one constitutive cell line (C-ME1) was generated from dase–conjugated antibodies, and a chemiluminescent reagent (ECL, hematopoietic progenitor cells transduced with MSCV-M/E. All of Amersham Biosciences, Arlington Heights, IL). the cell lines proliferated rapidly in suspension culture, with C-ME1

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cells immortalized by MLL-ENL (18), MLL-ENL protein could not be detected in any of the immortalized cells by Western blot analysis. However, quantitative PCR analysis showed that MLL-ENL expression was significantly reduced by doxycycline treatment in all conditional cell lines, but not affected significantly in the constitutive cells (Fig. 2B). Interestingly, C-ME1 expressed more MLL-ENL than the conditional lines, probably due to the different MLL-ENL expression constructs used in each case. This may explain why C-ME1 had the highest rate of proliferation. The cytokine requirements of the conditional cell lines were very similar to those of the constitutive line (Supplementary Fig. S1C). All the lines were growth factor dependent and cells died within 48 hours of growth factor withdrawal (data not shown). The conditional cell lines, TRE-ME2 and TRE-ME3, both induced AML in vivo when injected into unirradiated nonobese diabetic/ severe-combined immunodeficient mice, as did the constitutive cell line C-ME1 (data not shown). The latency of leukemias induced by TRE-ME2 (132 F 34 days, n = 9) and TRE-ME3 (118 F 19 days, n = 5) was longer than for C-ME1 (80 F 9 days, n = 5), consistent with their lower proliferation rate in vitro (Fig. 2A). However, TRE- ME6 did not induce leukemia over a 7-month observation period (data not shown). This was surprising because this line proliferated in vitro almost as well as TRE-ME2 and better than TRE-ME3. MLL-ENL immortalized cell lines terminally differentiate when MLL-ENL expression is turned off. Addition of doxycycline caused an inhibition in the proliferation of all three conditional Figure 1. MLL-ENL is required to maintain as well as initiate immortalization cell lines and cells ceased to proliferate by day 10 of doxycycline of hematopoietic progenitor cells. A, diagram of the retroviral constructs used in this study. LTR, long terminal repeat; w +, viral packaging signal; tTA-I-E, treatment (Fig. 3A). In contrast, the constitutive C-ME1 line tTA-IRES-EGFP; P, phosphoglycerate kinase promoter; N, neomycin resistance proliferated continuously in the presence of doxycycline, albeit at a r gene; TRE, tetracycline response element; M/E, MLL-ENL; Amp , ampicillin marginally slower rate than without doxycycline (Fig. 3A). Both resistance gene. B, Western blot analysis of protein extracts from NIH3T3 cells transduced with the indicated retroviral constructs. Cells were maintained with TRE-ME2 and TRE-ME6 terminally differentiated when MLL-ENL or without doxycycline (dox) for 24 hours and then lysed. Myc-tagged MLL-ENL expression was turned off by doxycycline, shown by characteristic was detected using a high-affinity anti-myc antibody. Arrow, band corresponding to full-length MLL-ENL. C, p-iodonitrotetrazolium stains of the fifth round changes in cell-surface marker expression and morphology (Fig. 3B methylcellulose cultures of transduced hematopoietic progenitor cells, after and C). However, whereas TRE-ME2 differentiated into neutrophils culture in the presence or absence of doxycycline for 7 days. Hematopoietic only, TRE-ME6 produced both neutrophils and macrophages. In progenitor cells transduced with the indicated retroviral constructs were cultured in methylcellulose. Colonies were counted every 7 to 10 days, and cells were contrast, TRE-ME3 cells seem able to initiate, but not able to harvested and replated into subsequent rounds. D, typical morphology of complete, the terminal differentiation on loss of MLL-ENL because the colonies (original magnification, 40); E, typical morphology of the cells they expressed increased Gr-1 in response to doxycycline but died (original magnification, 400) from the fifth round. Cells were visualized by cytospin preparation followed by MGG staining. without any changes in morphology. As expected, the constitutive cell line did not differentiate in response to doxycycline. We then examined whether our cell lines were also able to respond to showing the highest rate of proliferation (Fig. 2A). Intact MLL-ENL G-CSF, as previously published for MLL-ENL immortalized cell provirus was detected by Southern blotting (Supplementary lines (18). Analysis of cellular morphology (Fig. 4A) and cell-surface Fig. S1A) and expression of the MLL-ENL transcript was confirmed antigen expression (data not shown) showed that all lines, apart by reverse transcription-PCR using primers that span the MLL-ENL from TRE-ME3, terminally differentiated into neutrophils in breakpoint (Supplementary Fig. S1B). As previously reported for response to G-CSF treatment. Importantly, the rate of G-CSF

Figure 2. Generation of MLL-ENL immortalized cell lines. A, log of the fold accumulation in cell number of TRE-ME2 (o), TRE-ME3 (d), TRE-ME6 (5) and C-ME1 (4). B, relative level of MLL-ENL mRNA expression in TRE-ME2, TRE-ME3, TRE-ME6, and C-ME1 cells, treated with (black columns) and without (gray columns) doxycycline. Quantitative PCR analysis was done using cDNA from cells 24 hours after treatment with doxycycline. Columns, mean of triplicate measurements; bars, SD.

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all lines, the pattern of expression was skewed towards the 5V members of the Hoxa gene cluster, Hoxa9 being the most highly expressed gene in each line (Fig. 5). Interestingly, the Hox cofactor Meis-1 was only expressed in C-ME1 and was completely absent in all of the conditional cell lines (Fig. 5). Hoxb3, Hoxb4, and Hoxb13 were the only Hoxb cluster genes expressed in the MLL-ENL immortalized cell lines. These genes were expressed at much lower levels than the 5VHoxa genes (Fig. 5 and Supplementary Fig. S3). Genes of the Hoxc and Hoxd clusters were not expressed at significant levels in any of the cell lines (Supplementary Fig. S3). The Hox gene expression profile of each cell line was then analyzed after treatment with doxycycline. TRE-ME2, TRE-ME3, and C-ME1 were analyzed 48 hours, and TRE-ME6 24 hours, after addition of doxycycline because TRE-ME6 terminally differentiated more rapidly on loss of MLL-ENL expression (Fig. 3C). Doxycycline caused a significant decrease in the expression (P < 0.05) of Hoxa4, Hoxa5, Hoxa6, Hoxa7, Hoxa9, Hoxa10, and Hoxa11 in all of the conditional cell lines (Fig. 5). In contrast, Hoxa gene expression was either unchanged or slightly increased following doxycycline treatment of C-ME1. This suggests that expression of Hoxa4-a11 genes may be maintained by MLL-ENL. In contrast, because doxycycline caused similar changes in the expression of Hoxb3, Hoxb4, and Hoxb13 genes in C-ME1 as in the conditional cell lines

Figure 3. MLL-ENL immortalized cell lines terminally differentiate on loss of MLL-ENL expression. A, log of the fold accumulation in cell number following maintenance of TRE-ME2 (circles), TRE-ME3 (diamonds), TRE-ME6 (squares), and C-ME1 (triangles) with or without doxycycline. Open symbols, without doxycycline; filled symbols, 2 Ag/mL doxycycline. B, flow cytometric analysis of cell surface antigen expression following culture of the cell lines with doxycycline (thick black lines) or without doxycycline (gray lines) for 4 days. C, morphology of the cells following culture with or without doxycycline for 4 and 8 days (original magnification, 400). The morphology of TRE-ME3 after 8 days with doxycycline is not shown because there were no viable cells left in the culture. induced differentiation was equivalent to that induced by doxycycline for TRE-ME2 and TRE-ME6, as judged by the comparable up-regulation in Gr-1 expression (Fig. 4B). In addition, M-CSF induced macrophage differentiation of only the TRE-ME6 line and of clones derived from TRE-ME6 (Fig. 4A). MLL-ENL cells express a pattern of Hoxa genes. To establish which Hox genes are expressed in MLL-ENL immortalized cell lines, we analyzed the expression profile of all 39 murine Hox genes and the Meis and Pbx Hox cofactor genes by quantitative PCR. The Hox genes were cloned and standard curves generated for 38 of the 39 genes (Supplementary Table and Fig. S2) and for the Meis-1 Hox cofactor gene. These curves were then used to convert the Figure 4. MLL-ENL cell lines differentiate in response to G-CSF. A, quantitative PCR data into Hox gene copy number. This enabled morphology of the cell lines and a representative clone of TRE-ME6 following culture with the indicated cytokines (original magnification, 400). B, Gr-1 us to compare the expression level of each Hox gene with that of expression of C-ME1, TRE-ME2, and TRE-ME6 following culture with either other Hox genes in the same cell line and with that of Hox genes in doxycycline (gray line) or G-CSF (thick black line). These cell lines were cultured with or without doxycycline or with G-CSF to compare their Hoxa gene the other cell lines. expressions by quantitative PCR (see Fig. 5). C-ME1 and TRE-ME2 were The MLL-ENL immortalized cell lines expressed a broadly analyzed after 48 hours and TRE-ME6 was analyzed after 24 hours. The similar pattern of Hoxa genes (Fig. 5). TRE-ME2 and TRE-ME6 mean fluorescence intensities of Gr-1 expression for control cells (cultured in SCF, IL-3, and IL-6), cells treated with doxycycline (plus SCF, IL-3, and IL-6), expressed similar levels of Hoxa genes to C-ME1, whereas all Hoxa and cells treated with G-CSF were (in arbitrary units) 68, 41, and 158 for C-ME1; genes were expressed at significantly lower levels by TRE-ME3. In 219, 305, and 298 for TRE-ME2; and 103, 222, and 225 for TRE-ME6.

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Figure 5. MLL-ENL maintains the expression of a Hoxa code. The number of copies of each Hoxa gene per 25 ng of total RNA following culture of the cell lines in SCF, IL-6, and IL-3 without doxycycline (gray columns), with 2 Ag/mL doxycycline (black columns), or with 10 ng/mL G-CSF (white columns). Cells were harvested at a time point at which the amount of differentiation induced by doxycycline or G-CSF was comparable. The level of differentiation was assessed by flow cytometric analysis of Gr-1 expression (see Fig. 4B). C-ME1 and TRE-ME2 were analyzed after 48 hours and TRE-ME6 was analyzed after 24 hours. TRE-ME3 was analyzed following culture with or without doxycycline for 48 hours but the Hoxa gene expression profile was not determined after culture in G-CSF because the cells died in response to this cytokine. Columns, mean of triplicate readings; bars, SD.

(Supplementary Fig. S3), this effect is probably not connected with C-ME1 (Fig. 6). All of the cell lines expressed Meis-3 although C-ME1 loss of MLL-ENL expression. Thus, Hoxb genes are unlikely to be expressed lower levels than the conditional lines. Pbx-1 was only targets of MLL-ENL. expressed by TRE-ME2, and all of the cell lines expressed similar Although these data suggest that MLL-ENL may regulate the levels of Pbx-2 and Pbx-3. To determine whether these Hox cofactors expression of most 5VHoxa cluster genes, there is an alternative were regulated by MLL-ENL, their expression was analyzed following explanation for these results. Hox gene expression normally treatment of the cell lines with doxycycline or G-CSF. Doxycycline decreases as myeloid progenitors terminally differentiate (25, 26). had no significant effect on Meis-2 expression. Although Meis-3 We chose early time points to minimize differentiation-associated expression did decrease in the conditional cell lines in response to changes in Hox gene expression. However, we cannot exclude that doxycycline, G-CSF caused similar, if not greater, decreases. These the decreases we observe in Hox gene expression in our experiment changes are therefore likely to be due to differentiation of the cells. are due to the differentiation of immortalized cells on loss of MLL- Because doxycycline caused an increase in the expression of Pbx-2 in ENL expression rather than as a direct result of losing MLL-ENL C-ME1 as well as in the conditional cell lines, this is probably due to expression. Because TRE-ME2 and TRE-ME6 differentiated into an effect of doxycycline per se rather than to a decrease in MLL-ENL neutrophils in response to G-CSF, we decided to use this observation expression. Doxycycline caused a decrease in Pbx-3 expression in all in addressing whether the Hoxa4-a11 genes are targets of MLL-ENL. of the conditional cell lines. However, Pbx-3 expression increased in The Hoxa4-a11 genes are targets of MLL-ENL. We compared C-ME1 in response to doxycycline and was unchanged in all of the the Hoxa gene expression profile of cells stimulated to differentiate cell lines in response to G-CSF. This suggests that Pbx-3 expression is with G-CSF, despite continued expression of MLL-ENL, and maintained by MLL-ENL. compared it with that of cells treated with doxycycline. Cells were Although Meis-1 has been recently shown to induce FLT3 treated with G-CSF for the same length of time as they had been expression in Hoxa9 immortalized myeloid cells (27), FLT3 was not treated with doxycycline in the previous analyses (Fig. 4B). expressed at significant levels by either the conditional or Strikingly, doxycycline caused a much greater reduction in Hoxa constitutive MLL-ENL cell lines (Supplementary Fig. S4). gene expression than G-CSF in both conditional cell lines (Fig. 5). G-CSF treatment actually caused an increase in the expression of Hoxa4-a11 genes in TRE-ME2, as it did in C-ME1 (Fig. 5). In TRE- Discussion ME6, the only change in Hoxa gene expression caused by G-CSF We have generated immortalized murine hematopoietic progen- was a decrease in the expression of Hoxa7, Hoxa10, and Hoxa11.Of itor cells with conditional expression of MLL-ENL to establish these, only the Hoxa11 decrease was of similar magnitude to that whether continued MLL-ENL expression is required to maintain caused by doxycycline (Fig. 5). Taken together, these data suggest immortalization. Using the Tet-Off conditional expression system, that the decrease observed in the expression of Hoxa4-a11 genes we showed that cells immortalized by MLL-ENL failed to self- on loss of MLL-ENL is not a secondary result of differentiation, renew in methylcellulose on loss of MLL-ENL expression. but rather that the expression of these Hoxa genes is directly Furthermore, we generated immortalized cell lines that either maintained by MLL-ENL. terminally differentiated or died on switching off MLL-ENL We then analyzed the pattern of Hox cofactor genes expressed by expression by the addition of doxycycline. This result is consistent the cell lines using commercially available predesigned primers and with previous studies in which hematopoietic progenitor cells were probes. This analysis revealed that Meis-2 was expressed by all of the immortalized with an estrogen-regulated inducible MLL-ENL conditional cell lines whereas it was expressed at very low levels by fusion protein (13, 15). www.aacrjournals.org 9249 Cancer Res 2005; 65: (20). October 15, 2005

because TRE-ME6 differentiated more rapidly in response to doxycycline or G-CSF than any of the other lines. Several members of the Hoxa cluster, of which expression is regulated by MLL, are overexpressed in leukemias associated with MLL translocations (29–31). Because wild-type MLL has also been shown to control the expression of genes from the Hoxb and Hoxc clusters (5–8, 32), we examined the expression profile of all 39 murine Hox genes and Hox cofactors in MLL-ENL immortalized cell lines. We show that despite the phenotypic and functional differences of the different MLL-ENL cell lines, they all express a similar pattern of Hoxa genes or a ‘‘Hox’’ code consisting of Hoxa4, Hoxa5, Hoxa6, Hoxa7, Hoxa9, Hoxa10, and Hoxa11. It is important to note that although C-ME1, TRE-ME2, and TRE-ME6 expressed similar levels of Hoxa genes, all the Hoxa genes were expressed at significantly lower levels by TRE-ME3. This is not due to the expression level of MLL-ENL in TRE-ME3 because this is similar to that in TRE-ME2 and TRE-ME6. The data may be explained by differences in the levels of Hoxa genes expressed by the myeloid progenitor immortalized by MLL-ENL. In this case, MLL-ENL would act by maintaining preexisting levels of Hoxa gene expression rather than by directly transactivating Hoxa genes. This suggests that it is the pattern, or relative transcription level, of Hoxa gene expression maintained by MLL-ENL, rather than their absolute expression levels, which is important for MLL-ENL activity. Our data show that expression of Hox genes by the MLL-ENL immortalized cells is largely limited to the Hoxa cluster, the only other Hox genes which are significantly expressed being Hoxb3, Hoxb4, and Hoxb13. Myeloid cell lines immortalized by MLL-ENL have previously been shown to express some of these Hoxa genes (13, 14). However, Hoxa Figure 6. MLL-ENL maintains the expression of Pbx-3. Relative level of expression of the Pbx and Meis Hox cofactor genes following culture of the gene expression is not restricted to MLL-ENL cell lines because some cell lines in SCF, IL-6, and IL-3 without doxycycline (gray columns), with Hoxa genes are also expressed by myeloid cell lines immortalized by doxycycline (black columns), or with 10 ng/mL G-CSF (white columns). Cells E2A-HLF (33). It is possible, therefore, that these cells express were analyzed at time points identical to that used in the previous Hoxa gene expression analysis (see Fig. 5). TRE-ME3 was not analyzed following culture particular Hoxa genes because of a differentiation block imposed by in G-CSF because the cells died in response to this cytokine. Columns, mean MLL-ENL, and that this depends on the Hox gene expression pattern of triplicate readings; bars, SD. of the original immortalized myeloid progenitors. Only one previous study has shown that loss of MLL-ENL expression in immortalized Two of three of our conditional cell lines terminally differenti- myeloid cells results in a reduction in the expression of Hox genes; in ated on loss of MLL-ENL expression. TRE-ME2 differentiated into this case, Hoxa7 and Hoxa9 (15). However, this work did not neutrophils and TRE-ME6 exhibited both neutrophil and macro- distinguish between a direct role for MLL-ENL in maintenance of phage differentiation in response to doxycycline. The third Hox gene expression and the possibility that the reduction in Hoxa7 conditional cell line, TRE-ME3, failed to complete differentiation and Hoxa9 expression was due to differentiation of the myeloid on loss of MLL-ENL expression. The differentiation of each cell line progenitors on loss of MLL-ENL expression. This is an important on treatment with G-CSF and M-CSF reflected their response to consideration because expression of Hoxa genes does decrease doxycycline. Interestingly, clones of TRE-ME6 retained the ability to during differentiation of hematopoietic progenitors and terminal differentiate into both neutrophils and macrophages, suggesting differentiation of myeloid cells (25, 26). Our data show that the that it is truly bipotent. These data suggest that MLL-ENL expression of multiple Hoxa genes, in addition to Hoxa7 and Hoxa9, immortalizes myeloid progenitors at or immediately downstream is directly maintained by MLL-ENL. The decrease in expression of the granulocyte-monocyte progenitor hematopoietic stage, in levels of these genes on loss of MLL-ENL was not a secondary result agreement with a recent study (28). However, they also indicate of differentiation because treatment of the cells with G-CSF did not that the pool of myeloid progenitors susceptible to immortalization result in an equivalent decrease. This conclusion is consistent with by MLL-ENL does have a degree of heterogeneity. This pool experiments using the MLL-FKBP fusion that, in its transforming, includes both bipotent progenitors, with neutrophil and macro- dimerized form, up-regulates expression of Hoxa7 and Hoxa9 in phage differentiation potential, and unipotent progenitors, only immortalized myeloblastic cell lines (34). The demonstration by capable of neutrophil differentiation. The heterogeneity of myeloid chromatin immunoprecipitation that MLL-FKBP (34) and MLL- progenitors susceptible to MLL-ENL–induced immortalization may AF10 (35) bind to regulatory regions of Hoxa7 and Hoxa9 in explain the differences in cell-surface marker expression and transformed cells further supports this interpretation. proliferation rate of the different cell lines generated in our study. Although several studies suggest that Hoxa gene expression is The failure of TRE-ME6 to induce leukemia in vivo is more difficult important for leukemogenesis mediated by MLL fusion proteins, it is to explain. One possibility is that the differentiation block imposed unclear whether this is mediated by individual Hoxa genes or the 5V by MLL-ENL is less severe in TRE-ME6 than in the other cell lines Hoxa cluster as a whole (13, 15, 16, 33). It is possible that in certain

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. MLL-ENL Induced Hox Gene Expression experimental models, loss of specific Hoxa genes may be activity may be conferred by Meis-2. Thus, although the constitutive compensated by expression of other members of the 5VHoxa cluster MLL-ENL cell line expressed Meis-1, the conditional MLL-ENL cell (16, 33). Recently, it has been suggested that, together with Hoxa9, lines expressed Meis-2 in its place. In addition, all of the lines Meis-1 is a critical downstream mediator of MLL-ENL activity and examined expressed Meis-3, Pbx-2, and Pbx-3. We found that Pbx-3 that enforced expression of Hoxa9 and Meis-1 can substitute for was the only Hox cofactor of which expression was maintained by MLL-ENL function (15). Whereas this study suggests an important MLL-ENL. This finding is consistent with that of a previous study in role for Hoxa9 and Meis-1 in mediating MLL-ENL activity, it should which Pbx-3 expression decreased on loss of MLL-ENL expression in be noted that myeloid cells immortalized by Hoxa9 plus Meis-1 myeloid immortalized cell lines (15). Interestingly, Pbx-3 is overex- exhibit striking differences from MLL-ENL cells. For example, Meis-1 pressed in patients with MLL translocations when compared with expression in the former inhibits G-CSF–induced differentiation other AML or acute lymphoblastic leukemia patients (40). It has (27, 36), which occurs readily in the MLL-ENL cells generated in this been reported that, in the presence of Pbx-3, either Meis-1 or Meis-2 and previous studies (18). Furthermore, Hoxa9 and Meis-1 immor- can interact with Hoxa9 on target enhancers and that over- talized cells express FLT3 (27), whereas none of the MLL-ENL cell expression of Hoxa9, Meis-1, or Meis-2 alone can block G-CSF– lines in the present study were found to express this gene. These induced differentiation of 32Dcl3 cells (41). Meis-2 is also expressed differences may be explained by differences in the levels of Meis-1 in some murine (42) and human leukemic cells (43). Thus, Meis-2 expressed in each case. It is also possible that Meis-1 is not a direct may substitute for Meis-1 activity in our conditional MLL-ENL cell target of MLL-ENL. Thus, although wild-type MLL has been shown lines. Although Meis-1 overexpression is frequently found in to regulate the expression of multiple Hox genes, it does not regulate leukemias associated with MLL translocations (29, 30, 44), it is not Meis-1 expression (32). Furthermore, a recent study suggests that overexpressed in all patient samples (45). It will be interesting to Meis-1 expression in the myeloid progeny of transduced progenitors examine if Meis-2 is expressed, instead of Meis-1, in these patients. may be a nonspecific effect of retroviral transduction per se (37). It has been suggested that MLL fusion proteins specify a An alternative possibility is that MLL-ENL activity is mediated by particular ‘‘Hox code’’ (16) and that this may be the basis for their multiple Hoxa genes, as opposed to Hoxa9 alone, with the Hox activity. In the present study, we show directly that MLL-ENL does proteins synergizing with Pbx and Meis Hox cofactors normally regulate the expression of 5VHoxa genes. The pattern of expression expressed in myeloid progenitors. The Pbx and Meis cofactors form of Hoxa genes is similar in all of the MLL-ENL immortalized cells heterotrimeric complexes with Hox proteins (38) and are thought and may represent the ‘‘Hox code’’ specified by MLL-ENL. It will be to increase the specificity with which Hox proteins bind to their important to discover how this ‘‘Hox code’’ translates into myeloid target DNA sequences (39). Therefore, rather than being a direct transformation and leukemogenesis. target of MLL-ENL, it may be that Meis-1 expression provides a selection advantage to myeloid cells expressing the MLL-ENL Acknowledgments fusion, resulting in an outgrowth of cells coexpressing Meis-1 and the MLL-ENL Hoxa targets. A recent study has shown that Meis-1 Received 5/17/2005; revised 7/11/2005; accepted 8/3/2005. Grant support: Leukaemia Research Fund (S.J. Horton, M. Morrow, I. De Silva, D.A. provides just such a clonal selection advantage to myeloid cells Moulding, H.J.M. Brady, O. Williams), the Northern Ireland Leukaemia Research Fund immortalized by Hoxa9 and Meis-1 (27). This study also found that (A. Thompson, G.J. McGonigle), The Research and Development Office of the Health & COOH-terminal Meis-1 motifs are required for this activity. Personal Social Services in Northern Ireland (D.G. Grier), the Elimination of Leukaemia Fund (T.R.J. Lappin), and the Children with Leukaemia (H.J.M. Brady, Interestingly, these motifs are conserved in Meis-1, Meis-2, and O. Williams), United Kingdom. Meis-3. It is also possible that similar selection pressure may result The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance in increased expression of Meis cofactors in MLL-ENL leukemic with 18 U.S.C. Section 1734 solely to indicate this fact. cells in vivo. This in turn may lead to increases in expression of We thank Trisha Norton for excellent animal husbandry, Jo Sinclair for help with Meis target genes, such as FLT3. flow cytometry, A. Biondi (Milan University, Italy) and D.C. Tkachuk (Princess Margaret Hospital, Toronto, Canada) for the MLL fusion cDNAs, and Alexandre Our data indicate that the requirement for Meis-1 expression in Ptocnik (National Institute for Medical Research) and Jasper de Boer (Institute of MLL-ENL–induced leukemia is not absolute and that Hox cofactor Child Health) for their help and discussions.

References transcriptional maintenance factor in morphogenesis. 13. Ayton PM, Cleary ML. Transformation of myeloid Proc Natl Acad Sci U S A 1998;95:10632–6. progenitors by MLL oncoproteins is dependent on 1. Ayton PM, Cleary ML. Molecular mechanisms of 8. Yagi H, Deguchi K, Aono A, Tani Y, Kishimoto T, Hoxa7 and Hoxa9. Genes Dev 2003;17:2298–307. leukemogenesis mediated by MLL fusion proteins. Komori T. Growth disturbance in fetal liver 14. So CW, Lin M, Ayton PM, Chen EH, Cleary ML. Oncogene 2001;20:5695–707. hematopoiesis of Mll-mutant mice. Blood 1998;92: Dimerization contributes to oncogenic activation of 2. Daser A, Rabbitts TH. Extending the repertoire of the 108–17. MLL chimeras in acute leukemias. Cancer Cell 2003;4: mixed-lineage leukemia gene MLL in leukemogenesis. 9. Ernst P, Fisher JK, Avery W, Wade S, Foy D, Korsmeyer 99–110. Genes Dev 2004;18:965–74. SJ. Definitive hematopoiesis requires the mixed-lineage 15. Zeisig BB, Milne T, Garcia-Cuellar MP, et al. Hoxa9 3. Pui CH, Kane JR, Crist WM. Biology and treatment of leukemia gene. Dev Cell 2004;6:437–43. and Meis1 are key targets for MLL-ENL-mediated infant leukemias. Leukemia 1995;9:762–9. 10. Thorsteinsdottir U, Sauvageau G, Hough MR, et al. cellular immortalization. Mol Cell Biol 2004;24:617–28. 4. Tkachuk DC, Kohler S, Cleary ML. Involvement of a Overexpression of HOXA10 in murine hematopoietic 16. Kumar AR, Hudson WA, Chen W, Nishiuchi R, Yao Q, homolog of Drosophila trithorax by 11q23 chromosomal cells perturbs both myeloid and lymphoid differentia- Kersey JH. Hoxa9 influences the phenotype but not the translocations in acute leukemias. Cell 1992;71:691–700. tion and leads to acute myeloid leukemia. Mol Cell Biol incidence of Mll-AF9 fusion gene leukemia. Blood 2004; 5. Yu BD, Hess JL, Horning SE, Brown GA, Korsmeyer SJ. 1997;17:495–505. 103:1823–8. Altered Hox expression and segmental identity in Mll- 11. Kroon E, Krosl J, Thorsteinsdottir U, Baban S, 17. Scandura JM, Boccuni P, Cammenga J, Nimer SD. mutant mice. Nature 1995;378:505–8. Buchberg AM, Sauvageau G. Hoxa9 transforms Transcription factor fusions in acute leukemia: varia- 6. Hess JL, Yu BD, Li B, Hanson R, Korsmeyer SJ. Defects primary bone marrow cells through specific collabo- tions on a theme. Oncogene 2002;21:3422–44. in yolk sac hematopoiesis in Mll-null embryos. Blood ration with Meis1a but not Pbx1b. EMBO J 1998;17: 18. Lavau C, Szilvassy SJ, Slany R, Cleary ML. Immortal- 1997;90:1799–806. 3714–25. ization and leukemic transformation of a myelomono- 7. Yu BD, Hanson RD, Hess JL, Horning SE, Korsmeyer SJ. 12. Hess JL. MLL: a histone methyltransferase disrupted cytic precursor by retrovirally transduced HRX-ENL. MLL, a mammalian trithorax-group gene, functions as a in leukemia. Trends Mol Med 2004;10:500–7. EMBO J 1997;16:4226–37. www.aacrjournals.org 9251 Cancer Res 2005; 65: (20). October 15, 2005

19. Zeisig BB, Garcia-Cuellar MP, Winkler TH, Slany RK. and a novel function of the Meis1 C terminus. Blood nisms of cooperativity with Hoxa9 in myeloid leukemia. The oncoprotein MLL-ENL disturbs hematopoietic 2005;106:254–64. Proc Natl Acad Sci U S A 2001;98:13120–5. lineage determination and transforms a biphenotypic 28. Cozzio A, Passegue E, Ayton PM, Karsunky H, Cleary 37. Ono R, Nakajima H, Ozaki K, et al. Dimerization of lymphoid/myeloid cell. Oncogene 2003;22:1629–37. ML, Weissman IL. Similar MLL-associated leukemias MLL fusion proteins and FLT3 activation synergize to 20. Forster A, Pannell R, Drynan LF, et al. Engineering arising from self-renewing stem cells and short-lived induce multiple-lineage leukemogenesis. J Clin Invest de novo reciprocal chromosomal translocations associ- myeloid progenitors. Genes Dev 2003;17:3029–35. 2005;115:919–29. ated with Mll to replicate primary events of human 29. Armstrong SA, Staunton JE, Silverman LB, et al. MLL 38. Shen WF, Rozenfeld S, Kwong A, Kom ves LG, cancer. Cancer Cell 2003;3:449–58. translocations specify a distinct gene expression profile Lawrence HJ, Largman C. HOXA9 forms triple com- 21. Caslini C, Alarcon AS, Hess JL, Tanaka R, Murti KG, that distinguishes a unique leukemia. Nat Genet plexes with PBX2 and MEIS1 in myeloid cells. Mol Cell Biondi A. The amino terminus targets the mixed lineage 2002;30:41–7. Biol 1999;19:3051–61. leukemia (MLL) protein to the nucleolus, nuclear matrix 30. Rozovskaia T, Ravid-Amir O, Tillib S, et al. Expression 39. Mann RS, Chan SK. Extra specificity from extra- and mitotic chromosomal scaffolds. Leukemia 2000; profiles of acute lymphoblastic and myeloblastic leuke- denticle: the partnership between HOX and PBX/EXD 14:1898–908. mias with ALL-1 rearrangements. Proc Natl Acad Sci homeodomain proteins. Trends Genet 1996;12:258–62. 22. Adler HT, Nallaseth FS, Walter G, Tkachuk DC. HRX U S A 2003;100:7853–8. 40. Andersson A, Eden P, Lindgren D, et al. Gene leukemic fusion proteins form a heterocomplex with the 31. Debernardi S, Lillington DM, Chaplin T, et al. expression profiling of leukemic cell lines reveals leukemia-associated protein SET and protein phospha- Genome-wide analysis of acute myeloid leukemia with conserved molecular signatures among subtypes tase 2A. J Biol Chem 1997;272:28407–14. normal karyotype reveals a unique pattern of homeobox with specific genetic aberrations. Leukemia 2005;19: 23. Morrow M, Horton S, Kioussis D, Brady HJ, Williams gene expression distinct from those with translocation- 1042–50. O. TEL-AML1 promotes development of specific hema- mediated fusion events. Genes Chromosomes Cancer 41. Fujino T, Yamazaki Y, Largaespada DA, et al. Inhi- topoietic lineages consistent with preleukemic activity. 2003;37:149–58. bition of myeloid differentiation by Hoxa9, Hoxb8, and Blood 2004;103:3890–6. 32. ErnstP,MabonM,DavidsonAJ,ZonLI, Meis homeobox genes. Exp Hematol 2001;29:856–63. 24. Thompson A, Quinn MF, Grimwade D, et al. Global Korsmeyer SJ. An Mll-dependent Hox program drives 42. Nakamura T, Jenkins NA, Copeland NG. Identifica- down-regulation of HOX gene expression in PML-RARa + hematopoietic progenitor expansion. Curr Biol 2004; tion of a new family of Pbx-related homeobox genes. acute promyelocytic leukemia identified by small-array 14:2063–9. Oncogene 1996;13:2235–42. real-time PCR. Blood 2003;101:1558–65. 33. So CW, Karsunky H, Wong P, Weissman IL, Cleary 43. Smith JE, Afonja O, Yee HT, Inghirami G, Takeshita K. 25. Sauvageau G, Lansdorp PM, Eaves CJ, et al. ML. Leukemic transformation of hematopoietic progen- Chromosomal mapping to 15q14 and expression Differential expression of homeobox genes in function- itors by MLL-GAS7 in the absence of Hoxa7 or Hoxa9. analysis of the human MEIS2 homeobox gene. Mamm ally distinct CD34+ subpopulations of human bone Blood 2004;103:3192–9. Genome 1997;8:951–2. marrow cells. Proc Natl Acad Sci U S A 1994;91:12223–7. 34. Martin ME, Milne TA, Bloyer S, et al. Dimerization of 44. Ferrando AA, Armstrong SA, Neuberg DS, et al. Gene 26. Pineault N, Helgason CD, Lawrence HJ, Humphries MLL fusion proteins immortalizes hematopoietic cells. expression signatures in MLL-rearranged T-lineage and RK. Differential expression of Hox, Meis1, and Pbx1 Cancer Cell 2003;4:197–207. B-precursor acute leukemias: dominance of HOX genes in primitive cells throughout murine hematopoi- 35. Okada Y, Feng Q, Lin Y, et al. hDOT1L links histone dysregulation. Blood 2003;102:262–8. etic ontogeny. Exp Hematol 2002;30:49–57. methylation to leukemogenesis. Cell 2005;121:167–78. 45. Drabkin HA, Parsy C, Ferguson K, et al. Quantita- 27. Wang GG, Pasillas MP, Kamps MP. Meis1 programs 36. Calvo KR, Knoepfler PS, Sykes DB, Pasillas MP, tive HOX expression in chromosomally defined sub- transcription of FLT3 and cancer stem cell character, Kamps MP. Meis1a suppresses differentiation by G-CSF sets of acute myelogenous leukemia. Leukemia 2002; using a mechanism that requires interaction with PBX and promotes proliferation by SCF: potential mecha- 16:186–95.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Continuous MLL-ENL Expression Is Necessary to Establish a ''Hox Code'' and Maintain Immortalization of Hematopoietic Progenitor Cells

Sarah J. Horton, David G. Grier, Glenda J. McGonigle, et al.

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