The t[(11;19)(p22;q23)] translocation, which gives rise to the MLL-ENL fusion protein, is commonly found in infant acute leukemias of both the myeloid and lymphoid lineage. To investigate the molecular mechanism of immortalization by MLL-ENL we established a Tet-regulatable system of MLL-ENL expression in primary hematopoietic progenitor cells. Immortalized myeloid cell lines were generated, which are dependent on continued MLL-ENL expression for their survival and proliferation. These cells either terminally differentiate or die when MLL-ENL expression is turned off with doxycycline. The expression profile of all 39 murine Hox genes was analyzed in these cells by real-time quantitative PCR. This analysis showed that loss of MLL-ENL was accompanied by a reduction in the expression of multiple Hoxa genes. By comparing these changes with Hox gene expression in cells induced to differentiate with granulocyte colony-stimulating factor, we show for the first time that reduced Hox gene expression is specific to loss of MLL-ENL and is not a consequence of differentiation. Our data also suggest that the Hox cofactor Meis-2 can substitute for Meis-1 function. Thus, MLL-ENL is required to initiate and maintain immortalization of myeloid progenitors and may contribute to leukemogenesis by aberrantly sustaining the expression of a “Hox code” consisting of Hoxa4 to Hoxa11.

Translocations involving the mixed lineage leukemia (MLL) gene on chromosome band 11q23 are associated with leukemias of both the myeloid and lymphoid lineage (1, 2). MLL translocations are most prevalent in infant leukemia where they comprise 80% of cases of acute lymphoblastic leukemia and 60% of cases of acute myeloid leukemia (AML; ref. 3). Infant leukemias bearing MLL translocations tend to have a particularly poor prognosis (3).

MLL is the human homologue of the Drosophila trithorax (TRX) gene (4). Gene targeting studies in mice have revealed that MLL is essential for definitive hematopoiesis and that it is required to maintain, but not to initiate, the expression of multiple Hox genes during embryogenesis (59). Some Hox genes are oncogenic when overexpressed in hematopoietic progenitor cells (10, 11). Taken together, this suggests that aberrant regulation of Hox genes by MLL fusion proteins is the basis for leukemias involving MLL translocations (12). Several recent publications do indeed suggest that Hox genes may play an important role in leukemia induced by MLL fusion proteins (1316).

MLL translocations result in the generation of an in-frame chimeric fusion in which MLL is joined to one of over 40 different partner genes, of which the most common are ENL, AF9, and AF4 (17). The t[(11;19)(p22;q23)] translocation results in the fusion of the MLL gene to the eleven-nineteen-leukemia (ENL) gene. Different murine models have been generated which recapitulate MLL-ENL–mediated leukemia. Immortalized myeloid and B-cell lines, both capable of inducing leukemia in vivo, have been generated by retroviral transduction of hematopoietic progenitor cells with MLL-ENL (18, 19). An interchromosomal recombination model has also been developed in which the de novo translocation of the MLL and ENL loci occurred specifically in hematopoietic cells (20). These mice developed AML with high penetrance and short latency, suggesting that the MLL-ENL translocation is the only event required for the development of leukemia (20).

To gain more insight into the molecular mechanism of 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 with the Tet-Off conditional expression system to regulate the expression of the full-length MLL-ENL fusion protein. We determined whether continued MLL-ENL expression was required to maintain as well as initiate myeloid immortalization, and analyzed the expression profile of all 39 murine Hox genes in MLL-ENL immortalized cell lines.

Mice. All mice were maintained in the animal facilities of the National 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 constructed by subcloning the flag-tagged 5′ MLL cDNA fragment (amino acids 1-1251; kindly provided by A. Biondi; ref. 21) into a modified version of pMSCV-neo (BD Clontech, Palo Alto, CA) upstream of the phosphoglycerate kinase (PGK) promoter and neor. The 3′ MLL-ENL cDNA (amino acids 1,252-1,444 of MLL and amino acids 5-559 of ENL; kindly provided by D.C. Tkachuk; ref. 22) was then ligated in-frame downstream of the 5′ MLL cDNA fragment. For some experiments, the flag tag was replaced with a Myc tag. The tetracycline transactivator (tTA) cDNA (BD Clontech) was subcloned into pMSCV-internal ribosome entry site (IRES)-enhanced green fluorescent protein (EGFP) upstream of the IRES-EGFP cassette to generate the MSCV-tTA vector (23). The MSCV-tetracycline response element (TRE) vector was generated by ligating the TRE (BD Clontech) downstream of the PGK-Neor in pMSCV-neo. The myc-tagged MLL-ENL cDNA fragment was then ligated into MSCV-TRE downstream of the TRE to generate the MSCV-TRE-M/E vector.

Isolation and infection of hematopoietic progenitor cells. Retroviral supernatants were produced as described previously (23). Hematopoietic progenitor cells were purified from single-cell suspensions of bone marrow, extracted from C57BL/10 or C57BL/6 mice 5 days after i.v. injection of 150 mg/kg 5-fluorouracil (Faulding Pharmaceuticals, Leamington Spa, United Kingdom). Magnetic activated cell sorting was used to purify c-Kit+ hematopoietic progenitor cells using monoclonal antibody (mAb) specific to c-Kit (2B8; BD Biosciences, PharMingen, San Diego, CA), and lineage depleted hematopoietic progenitor cells, using mAbs from a lineage panel kit (BD PharMingen). Hematopoietic progenitor cells were cultured with 100 ng/mL stem cell factor (SCF), 10 ng/mL interleukin-3 (IL-3), and 10 ng/mL IL-6 (Peprotech EC, London, United Kingdom) for 24 hours before infection. Hematopoietic progenitor cells were infected with retrovirus supplemented with the same growth factors on 2 consecutive days by spinoculation (centrifugation at 700 × g, 25°C, 45 minutes) in the presence of 5 μg/mL polybrene (Sigma-Aldrich, Poole, United Kingdom). Coinfections were done using unconcentrated MSCV-TRE-M/E and concentrated MSCV-tTA supernatants.

Colony-forming assays and generation of myeloid cell lines. Transduced hematopoietic progenitor cells were cultured in 1.1 mL duplicate methylcellulose cultures in 35 mm plates 24 hours after infection. Cells were plated in Methocult M3434 (Stem Cell Technologies, Vancouver, 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 10 days, colonies were scored and cells harvested and pooled. Subsequent rounds were done by replating 1 × 104 cells under identical conditions in the absence of G418. In some cases, 2 μg/mL doxycycline (BD Clontech) was added. Colonies were stained with 1 mg/mL p-iodonitrotetrazolium (Sigma-Aldrich) in PBS. Myeloid cell lines, generated from pooled tertiary round colonies, were cultured in RPMI 1640 (Life Technologies, Invitrogen) with 10% FCS, l-glutamine, and 50 μmol/L 2-mercaptoethanol (basic medium) in the presence of SCF, IL-3, and IL-6. One constitutive (C-ME1) and three conditional (TRE-ME2, TRE-ME3, and TRE-ME6) lines were generated. Differentiation of the cell lines was done in basic medium supplemented with either 10 ng/mL granulocyte colony-stimulating factor (G-CSF) or 10 ng/mL macrophage colony-stimulating factor (M-CSF). Cytospins were made using a Shandon cytospin 3 (Thermo Electron Corporation, Waltham, MA) and fixed and stained with May-Grunwald-Giemsa (MGG).

Flow cytometry. Cells were stained with phycoerythrin-conjugated mAbs specific for c-Kit (2B8), Mac-1 (M1/70), and Gr-1 (RB6-8CS), and isotype control antibodies (all from BD Biosciences, PharMingen) and F4/80 (Caltag, Burlingame, CA). Cells were resuspended in PBS, 0.5% bovine serum albumin, and 0.05% sodium azide, and preincubated with unlabeled anti-Fcγ III/II receptor mAb (2.4G2; BD Biosciences, PharMingen) before staining with primary antibody. Flow cytometry was done on a Beckman Coulter Epics XL analyzer (Beckman Coulter, High Wycombe, United Kingdom) and data analyzed using EXPO3 software.

Western blot analysis. Cells were lysed using NP40 buffer [150 mmol/L NaCl, 0.5% NP40, 50 mmol/L Tris (pH 8.0)] containing 1× complete protease inhibitor tablets (Roche Applied Science, Indianapolis, IN). Protein samples were resolved on a 6% SDS polyacrylamide gel and transferred to a polyvinylidenefluoride membrane (Immobilon-P, Millipore, Billerica, MA). Proteins were detected with anti-myc (clone 9B11; Cell Signaling Technology, Inc., Beverly, MA) or anti–α-tubulin (clone YL1/2; Serotec, Oxford, United Kingdom), the appropriate secondary horseradish peroxidase–conjugated antibodies, and a chemiluminescent reagent (ECL, Amersham Biosciences, Arlington Heights, IL).

Real-time quantitative PCR. Quantitative PCR reactions, using TaqMan probe based chemistry (Applera, Foster City, CA), were carried out and murine target amplicons were analyzed in a similar manner to the human set, as previously described (24). All oligonucleotides were designed against GenBank published sequences (Supplementary Table) using Primer Express software (Applera). Nucleotide sequences for oligonucleotide primers and probes are available on request. Total RNAs isolated from all major adult and fetal tissues, including whole embryos, were pooled and used for the validation of the murine Hox TaqMan primers and probes. The quantitative PCR analysis of Hox cofactor (Fig. 6) and FLT3 (Supplementary Fig. S4) gene expression was done using predesigned TaqMan primers and probes (Applera) and an ABI prism 7000 sequence detection system (Applera).

Standard curve generation. Amplicons generated using the forward and reverse primer pairs of the original Hox targets were cloned into pCR2.1-TOPO TA (Invitrogen) or pGEM-T Easy (Promega, Southampton, United Kingdom) using standard protocols. Plasmid DNA was prepared using the Qiagen miniprep kit (Qiagen Ltd., Crawley, United Kingdom) according to the instructions of the manufacturer. Quantitation of plasmid DNA was determined spectrophotometrically. An A260 value of 0.1 or higher was deemed satisfactory and, in terms of plasmid purity, an A260/A280 of 1.7 to 2.0 was accepted. The plasmid DNA concentration was converted to copy numbers of plasmid using the following formula: V (μL) = {1 × 108 × 309 × [plasmid size + insert size (bp)]} / [plasmid concentration (μg/μL) × 1 × 10−6 × 6.02 × 1023]. Plasmids were diluted to 109 copies/μL and linearized with NotI (New England Biolabs, Beverly, MA). Quantitative PCR analyses were done in triplicate using serially diluted plasmids (in the 101-107 copies/μL range) as templates, a primer and probe mixture, and Universal Mastermix (Applied Biosystems). All standard curves, correlation coefficients, gradient, and intercept values were generated using the ABI 7700 sequence detection system and associated software (version 1.7) according to the instructions of the manufacturer.

Continued MLL-ENL expression is required for immortalization. The retroviral constructs used in this study are depicted in Fig. 1A. Cells cotransduced with MSCV-TRE-M/E and MSCV-tTA will express the MLL-ENL fusion gene as a consequence of tTA binding the TRE. Doxycycline inhibits tTA binding of the TRE and results in loss of MLL-ENL expression. Expression of the full-length MLL-ENL protein (>220 kDa) was confirmed by Western blot analysis of 3T3 cells cotransduced with MSCV-TRE-M/E and MSCV-tTA (Fig. 1B). Expression of MLL-ENL was not detected in cotransduced cells 24 hours after the addition of doxycycline. Hematopoietic progenitor cells transduced with MSCV-M/E or cotransduced with MSCV-TRE-M/E and MSCV-tTA, but not control vectors, formed large compact colonies in methylcellulose cultures, composed of myeloblasts (Fig. 1C-E), which replated indefinitely (data not shown). However, in the presence of doxycycline, the conditionally immortalized cells only formed a few small clusters, mostly composed of terminally differentiated macrophages and neutrophils (Fig. 1C-E). Addition of doxycycline to MSCV-M/E cultures did not significantly affect the number and size of colonies formed or their myeloblast composition (Fig. 1C-E). These data suggest that continued expression of MLL-ENL is required for colony formation and for maintenance of the myeloblast phenotype of the immortalized cells.

Figure 1.

MLL-ENL is required to maintain as well as initiate immortalization of hematopoietic progenitor cells. A, diagram of the retroviral constructs used in this study. LTR, long terminal repeat; ψ+, viral packaging signal; tTA-I-E, tTA-IRES-EGFP; P, phosphoglycerate kinase promoter; N, neomycin resistance gene; TRE, tetracycline response element; M/E, MLL-ENL; Ampr, ampicillin resistance gene. B, Western blot analysis of protein extracts from NIH3T3 cells transduced with the indicated retroviral constructs. Cells were maintained with or without doxycycline (dox) for 24 hours and then lysed. Myc-tagged MLL-ENL 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 methylcellulose cultures of transduced hematopoietic progenitor cells, after culture in the presence or absence of doxycycline for 7 days. Hematopoietic progenitor cells transduced with the indicated retroviral constructs were cultured in methylcellulose. Colonies were counted every 7 to 10 days, and cells were harvested and replated into subsequent rounds. D, typical morphology of the colonies (original magnification, ×40); E, typical morphology of the cells (original magnification, ×400) from the fifth round. Cells were visualized by cytospin preparation followed by MGG staining.

Figure 1.

MLL-ENL is required to maintain as well as initiate immortalization of hematopoietic progenitor cells. A, diagram of the retroviral constructs used in this study. LTR, long terminal repeat; ψ+, viral packaging signal; tTA-I-E, tTA-IRES-EGFP; P, phosphoglycerate kinase promoter; N, neomycin resistance gene; TRE, tetracycline response element; M/E, MLL-ENL; Ampr, ampicillin resistance gene. B, Western blot analysis of protein extracts from NIH3T3 cells transduced with the indicated retroviral constructs. Cells were maintained with or without doxycycline (dox) for 24 hours and then lysed. Myc-tagged MLL-ENL 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 methylcellulose cultures of transduced hematopoietic progenitor cells, after culture in the presence or absence of doxycycline for 7 days. Hematopoietic progenitor cells transduced with the indicated retroviral constructs were cultured in methylcellulose. Colonies were counted every 7 to 10 days, and cells were harvested and replated into subsequent rounds. D, typical morphology of the colonies (original magnification, ×40); E, typical morphology of the cells (original magnification, ×400) from the fifth round. Cells were visualized by cytospin preparation followed by MGG staining.

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Characterization of MLL-ENL immortalized cell lines. Three conditional cell lines (TRE-ME2, TRE-ME3, and TRE-ME6) were generated from hematopoietic progenitor cells cotransduced with MSCV-TRE-M/E and MSCV-tTA in three independent experiments, and one constitutive cell line (C-ME1) was generated from hematopoietic progenitor cells transduced with MSCV-M/E. All of the cell lines proliferated rapidly in suspension culture, with C-ME1 showing the highest rate of proliferation (Fig. 2A). Intact MLL-ENL provirus was detected by Southern blotting (Supplementary Fig. S1A) and expression of the MLL-ENL transcript was confirmed by reverse transcription-PCR using primers that span the MLL-ENL breakpoint (Supplementary Fig. S1B). As previously reported for 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).

Figure 2.

Generation of MLL-ENL immortalized cell lines. A, log of the fold accumulation in cell number of TRE-ME2 (○), TRE-ME3 (⋄), TRE-ME6 (□) and C-ME1 (▵). 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.

Figure 2.

Generation of MLL-ENL immortalized cell lines. A, log of the fold accumulation in cell number of TRE-ME2 (○), TRE-ME3 (⋄), TRE-ME6 (□) and C-ME1 (▵). 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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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 ± 34 days, n = 9) and TRE-ME3 (118 ± 19 days, n = 5) was longer than for C-ME1 (80 ± 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 cell lines and cells ceased to proliferate by day 10 of doxycycline treatment (Fig. 3A). In contrast, the constitutive C-ME1 line proliferated continuously in the presence of doxycycline, albeit at a marginally slower rate than without doxycycline (Fig. 3A). Both TRE-ME2 and TRE-ME6 terminally differentiated when MLL-ENL expression was turned off by doxycycline, shown by characteristic changes in cell-surface marker expression and morphology (Fig. 3B and C). However, whereas TRE-ME2 differentiated into neutrophils only, TRE-ME6 produced both neutrophils and macrophages. In contrast, TRE-ME3 cells seem able to initiate, but not able to complete, the terminal differentiation on loss of MLL-ENL because they expressed increased Gr-1 in response to doxycycline but died 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 G-CSF, as previously published for MLL-ENL immortalized cell lines (18). Analysis of cellular morphology (Fig. 4A) and cell-surface antigen expression (data not shown) showed that all lines, apart from TRE-ME3, terminally differentiated into neutrophils in response to G-CSF treatment. Importantly, the rate of G-CSF 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).

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 μg/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.

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 μg/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.

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Figure 4.

MLL-ENL cell lines differentiate in response to G-CSF. A, morphology of the cell lines and a representative clone of TRE-ME6 following culture with the indicated cytokines (original magnification, ×400). B, Gr-1 expression of C-ME1, TRE-ME2, and TRE-ME6 following culture with either 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 expressions by quantitative PCR (see Fig. 5). C-ME1 and TRE-ME2 were analyzed after 48 hours and TRE-ME6 was analyzed after 24 hours. The 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), and cells treated with G-CSF were (in arbitrary units) 68, 41, and 158 for C-ME1; 219, 305, and 298 for TRE-ME2; and 103, 222, and 225 for TRE-ME6.

Figure 4.

MLL-ENL cell lines differentiate in response to G-CSF. A, morphology of the cell lines and a representative clone of TRE-ME6 following culture with the indicated cytokines (original magnification, ×400). B, Gr-1 expression of C-ME1, TRE-ME2, and TRE-ME6 following culture with either 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 expressions by quantitative PCR (see Fig. 5). C-ME1 and TRE-ME2 were analyzed after 48 hours and TRE-ME6 was analyzed after 24 hours. The 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), and cells treated with G-CSF were (in arbitrary units) 68, 41, and 158 for C-ME1; 219, 305, and 298 for TRE-ME2; and 103, 222, and 225 for TRE-ME6.

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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 quantitative PCR data into Hox gene copy number. This enabled us to compare the expression level of each Hox gene with that of other Hox genes in the same cell line and with that of Hox genes in the other cell lines.

The MLL-ENL immortalized cell lines expressed a broadly similar pattern of Hoxa genes (Fig. 5). TRE-ME2 and TRE-ME6 expressed similar levels of Hoxa genes to C-ME1, whereas all Hoxa genes were expressed at significantly lower levels by TRE-ME3. In all lines, the pattern of expression was skewed towards the 5′ 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 5′ Hoxa 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).

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 μg/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.

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 μg/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.

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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 (Supplementary Fig. S3), this effect is probably not connected with loss of MLL-ENL expression. Thus, Hoxb genes are unlikely to be targets of MLL-ENL.

Although these data suggest that MLL-ENL may regulate the expression of most 5′ Hoxa cluster genes, there is an alternative explanation for these results. Hox gene expression normally decreases as myeloid progenitors terminally differentiate (25, 26). We chose early time points to minimize differentiation-associated changes in Hox gene expression. However, we cannot exclude that the decreases we observe in Hox gene expression in our experiment are due to the differentiation of immortalized cells on loss of MLL-ENL expression rather than as a direct result of losing MLL-ENL expression. Because TRE-ME2 and TRE-ME6 differentiated into neutrophils in response to G-CSF, we decided to use this observation in addressing whether the Hoxa4-a11 genes are targets of MLL-ENL.

The Hoxa4-a11 genes are targets of MLL-ENL. We compared the Hoxa gene expression profile of cells stimulated to differentiate with G-CSF, despite continued expression of MLL-ENL, and compared it with that of cells treated with doxycycline. Cells were treated with G-CSF for the same length of time as they had been treated with doxycycline in the previous analyses (Fig. 4B). Strikingly, doxycycline caused a much greater reduction in Hoxa 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-ME6, the only change in Hoxa gene expression caused by G-CSF was a decrease in the expression of Hoxa7, Hoxa10, and Hoxa11. Of these, only the Hoxa11 decrease was of similar magnitude to that caused by doxycycline (Fig. 5). Taken together, these data suggest that the decrease observed in the expression of Hoxa4-a11 genes on loss of MLL-ENL is not a secondary result of differentiation, but rather that the expression of these Hoxa genes is directly maintained by MLL-ENL.

We then analyzed the pattern of Hox cofactor genes expressed by the cell lines using commercially available predesigned primers and probes. This analysis revealed that Meis-2 was expressed by all of the conditional cell lines whereas it was expressed at very low levels by C-ME1 (Fig. 6). All of the cell lines expressed Meis-3 although C-ME1 expressed lower levels than the conditional lines. Pbx-1 was only expressed by TRE-ME2, and all of the cell lines expressed similar levels of Pbx-2 and Pbx-3. To determine whether these Hox cofactors were regulated by MLL-ENL, their expression was analyzed following treatment of the cell lines with doxycycline or G-CSF. Doxycycline had no significant effect on Meis-2 expression. Although Meis-3 expression did decrease in the conditional cell lines in response to doxycycline, G-CSF caused similar, if not greater, decreases. These changes are therefore likely to be due to differentiation of the cells. Because doxycycline caused an increase in the expression of Pbx-2 in C-ME1 as well as in the conditional cell lines, this is probably due to an effect of doxycycline per se rather than to a decrease in MLL-ENL expression. Doxycycline caused a decrease in Pbx-3 expression in all of the conditional cell lines. However, Pbx-3 expression increased in C-ME1 in response to doxycycline and was unchanged in all of the cell lines in response to G-CSF. This suggests that Pbx-3 expression is maintained by MLL-ENL.

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 cell lines in SCF, IL-6, and IL-3 without doxycycline (gray columns), with doxycycline (black columns), or with 10 ng/mL G-CSF (white columns). Cells 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 in G-CSF because the cells died in response to this cytokine. Columns, mean of triplicate readings; bars, SD.

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 cell lines in SCF, IL-6, and IL-3 without doxycycline (gray columns), with doxycycline (black columns), or with 10 ng/mL G-CSF (white columns). Cells 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 in G-CSF because the cells died in response to this cytokine. Columns, mean of triplicate readings; bars, SD.

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Although Meis-1 has been recently shown to induce FLT3 expression in Hoxa9 immortalized myeloid cells (27), FLT3 was not expressed at significant levels by either the conditional or constitutive MLL-ENL cell lines (Supplementary Fig. S4).

We have generated immortalized murine hematopoietic progenitor cells with conditional expression of MLL-ENL to establish whether continued MLL-ENL expression is required to maintain immortalization. Using the Tet-Off conditional expression system, we showed that cells immortalized by MLL-ENL failed to self-renew in methylcellulose on loss of MLL-ENL expression. Furthermore, we generated immortalized cell lines that either terminally differentiated or died on switching off MLL-ENL expression by the addition of doxycycline. This result is consistent with previous studies in which hematopoietic progenitor cells were immortalized with an estrogen-regulated inducible MLL-ENL fusion protein (13, 15).

Two of three of our conditional cell lines terminally differentiated on loss of MLL-ENL expression. TRE-ME2 differentiated into neutrophils and TRE-ME6 exhibited both neutrophil and macrophage differentiation in response to doxycycline. The third conditional cell line, TRE-ME3, failed to complete differentiation on loss of MLL-ENL expression. The differentiation of each cell line on treatment with G-CSF and M-CSF reflected their response to doxycycline. Interestingly, clones of TRE-ME6 retained the ability to differentiate into both neutrophils and macrophages, suggesting that it is truly bipotent. These data suggest that MLL-ENL immortalizes myeloid progenitors at or immediately downstream of the granulocyte-monocyte progenitor hematopoietic stage, in agreement with a recent study (28). However, they also indicate that the pool of myeloid progenitors susceptible to immortalization by MLL-ENL does have a degree of heterogeneity. This pool includes both bipotent progenitors, with neutrophil and macrophage differentiation potential, and unipotent progenitors, only capable of neutrophil differentiation. The heterogeneity of myeloid progenitors susceptible to MLL-ENL–induced immortalization may explain the differences in cell-surface marker expression and proliferation rate of the different cell lines generated in our study. The failure of TRE-ME6 to induce leukemia in vivo is more difficult to explain. One possibility is that the differentiation block imposed by MLL-ENL is less severe in TRE-ME6 than in the other cell lines 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 (2931). Because wild-type MLL has also been shown to control the expression of genes from the Hoxb and Hoxc clusters (58, 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 gene expression is not restricted to MLL-ENL cell lines because some Hoxa genes are also expressed by myeloid cell lines immortalized by E2A-HLF (33). It is possible, therefore, that these cells express particular Hoxa genes because of a differentiation block imposed by MLL-ENL, and that this depends on the Hox gene expression pattern of the original immortalized myeloid progenitors. Only one previous study has shown that loss of MLL-ENL expression in immortalized myeloid cells results in a reduction in the expression of Hox genes; in this case, Hoxa7 and Hoxa9 (15). However, this work did not distinguish between a direct role for MLL-ENL in maintenance of Hox gene expression and the possibility that the reduction in Hoxa7 and Hoxa9 expression was due to differentiation of the myeloid progenitors on loss of MLL-ENL expression. This is an important consideration because expression of Hoxa genes does decrease during differentiation of hematopoietic progenitors and terminal differentiation of myeloid cells (25, 26). Our data show that the expression of multiple Hoxa genes, in addition to Hoxa7 and Hoxa9, is directly maintained by MLL-ENL. The decrease in expression levels of these genes on loss of MLL-ENL was not a secondary result of differentiation because treatment of the cells with G-CSF did not result in an equivalent decrease. This conclusion is consistent with experiments using the MLL-FKBP fusion that, in its transforming, dimerized form, up-regulates expression of Hoxa7 and Hoxa9 in immortalized myeloblastic cell lines (34). The demonstration by chromatin immunoprecipitation that MLL-FKBP (34) and MLL-AF10 (35) bind to regulatory regions of Hoxa7 and Hoxa9 in transformed cells further supports this interpretation.

Although several studies suggest that Hoxa gene expression is important for leukemogenesis mediated by MLL fusion proteins, it is unclear whether this is mediated by individual Hoxa genes or the 5′ Hoxa cluster as a whole (13, 15, 16, 33). It is possible that in certain experimental models, loss of specific Hoxa genes may be compensated by expression of other members of the 5′ Hoxa cluster (16, 33). Recently, it has been suggested that, together with Hoxa9, Meis-1 is a critical downstream mediator of MLL-ENL activity and that enforced expression of Hoxa9 and Meis-1 can substitute for MLL-ENL function (15). Whereas this study suggests an important role for Hoxa9 and Meis-1 in mediating MLL-ENL activity, it should be noted that myeloid cells immortalized by Hoxa9 plus Meis-1 exhibit striking differences from MLL-ENL cells. For example, Meis-1 expression in the former inhibits G-CSF–induced differentiation (27, 36), which occurs readily in the MLL-ENL cells generated in this and previous studies (18). Furthermore, Hoxa9 and Meis-1 immortalized cells express FLT3 (27), whereas none of the MLL-ENL cell lines in the present study were found to express this gene. These differences may be explained by differences in the levels of Meis-1 expressed in each case. It is also possible that Meis-1 is not a direct target of MLL-ENL. Thus, although wild-type MLL has been shown to regulate the expression of multiple Hox genes, it does not regulate Meis-1 expression (32). Furthermore, a recent study suggests that Meis-1 expression in the myeloid progeny of transduced progenitors may be a nonspecific effect of retroviral transduction per se (37).

An alternative possibility is that MLL-ENL activity is mediated by multiple Hoxa genes, as opposed to Hoxa9 alone, with the Hox proteins synergizing with Pbx and Meis Hox cofactors normally expressed in myeloid progenitors. The Pbx and Meis cofactors form heterotrimeric complexes with Hox proteins (38) and are thought to increase the specificity with which Hox proteins bind to their target DNA sequences (39). Therefore, rather than being a direct target of MLL-ENL, it may be that Meis-1 expression provides a selection advantage to myeloid cells expressing the MLL-ENL fusion, resulting in an outgrowth of cells coexpressing Meis-1 and the MLL-ENL Hoxa targets. A recent study has shown that Meis-1 provides just such a clonal selection advantage to myeloid cells immortalized by Hoxa9 and Meis-1 (27). This study also found that COOH-terminal Meis-1 motifs are required for this activity. Interestingly, these motifs are conserved in Meis-1, Meis-2, and Meis-3. It is also possible that similar selection pressure may result in increased expression of Meis cofactors in MLL-ENL leukemic cells in vivo. This in turn may lead to increases in expression of Meis target genes, such as FLT3.

Our data indicate that the requirement for Meis-1 expression in MLL-ENL–induced leukemia is not absolute and that Hox cofactor activity may be conferred by Meis-2. Thus, although the constitutive MLL-ENL cell line expressed Meis-1, the conditional MLL-ENL cell lines expressed Meis-2 in its place. In addition, all of the lines examined expressed Meis-3, Pbx-2, and Pbx-3. We found that Pbx-3 was the only Hox cofactor of which expression was maintained by MLL-ENL. This finding is consistent with that of a previous study in which Pbx-3 expression decreased on loss of MLL-ENL expression in myeloid immortalized cell lines (15). Interestingly, Pbx-3 is overexpressed in patients with MLL translocations when compared with other AML or acute lymphoblastic leukemia patients (40). It has been reported that, in the presence of Pbx-3, either Meis-1 or Meis-2 can interact with Hoxa9 on target enhancers and that overexpression of Hoxa9, Meis-1, or Meis-2 alone can block G-CSF–induced differentiation of 32Dcl3 cells (41). Meis-2 is also expressed in some murine (42) and human leukemic cells (43). Thus, Meis-2 may substitute for Meis-1 activity in our conditional MLL-ENL cell lines. Although Meis-1 overexpression is frequently found in leukemias associated with MLL translocations (29, 30, 44), it is not overexpressed in all patient samples (45). It will be interesting to examine if Meis-2 is expressed, instead of Meis-1, in these patients.

It has been suggested that MLL fusion proteins specify a particular “Hox code” (16) and that this may be the basis for their activity. In the present study, we show directly that MLL-ENL does regulate the expression of 5′ Hoxa genes. The pattern of expression of Hoxa genes is similar in all of the MLL-ENL immortalized cells and may represent the “Hox code” specified by MLL-ENL. It will be important to discover how this “Hox code” translates into myeloid transformation and leukemogenesis.

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Grant support: Leukaemia Research Fund (S.J. Horton, M. Morrow, I. De Silva, D.A. Moulding, H.J.M. Brady, O. Williams), the Northern Ireland Leukaemia Research Fund (A. Thompson, G.J. McGonigle), The Research and Development Office of the Health & 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, O. Williams), United Kingdom.

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 with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Trisha Norton for excellent animal husbandry, Jo Sinclair for help with flow cytometry, A. Biondi (Milan University, Italy) and D.C. Tkachuk (Princess Margaret Hospital, Toronto, Canada) for the MLL fusion cDNAs, and Alexandre Ptocnik (National Institute for Medical Research) and Jasper de Boer (Institute of Child Health) for their help and discussions.

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