Original Article

Leukemia (2006) 20, 1582–1592. doi:10.1038/sj.leu.2404298; published online 29 June 2006

MN1-TEL, the product of the t(12;22) in human myeloid leukemia, immortalizes murine myeloid cells and causes myeloid malignancy in mice

C Carella1,3, J Bonten1,3, J Rehg2 and G C Grosveld1

  1. 1Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, Memphis, TN, USA
  2. 2Department of Pathology, St Jude Children's Research Hospital, Memphis, TN, USA

Correspondence: Dr GC Grosveld, Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA. E-mail: gerard.grosveld@stjude.org

3These authors contributed equally to this work

Received 26 January 2006; Revised 21 April 2006; Accepted 18 May 2006; Published online 29 June 2006.

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Abstract

MN1-TEL is the product of the recurrent t(12;22)(p12;q11) associated with human myeloid malignancies. MN1-TEL functions as an activated transcription factor, exhibiting weak transforming activity in NIH3T3 fibroblasts that depends on the presence of a functional TEL DNA-binding domain, the N-terminal transactivating sequences of MN1 and C-terminal sequences of MN1. We determined the transforming activity of MN1-TEL in mouse bone marrow (BM) by using retroviral transfer. MN1-TEL-transduced BM showed increased self-renewal capacity of primitive progenitors in vitro, and prolonged in vitro culture of MN1-TEL-expressing BM produced immortalized myeloid, interleukin (IL)-3/stem cell factor-dependent cell lines with a primitive morphology. Transplantation of such cell lines into lethally irradiated mice rescued them from irradiation-induced death and resulted in the contribution of MN1-TEL-expressing cells to all hematopoietic lineages, underscoring the primitive nature of these cells and their capacity to differentiate in vivo. Three months after transplantation, all mice succumbed to promonocytic leukemia. Transplantation of freshly MN1-TEL-transduced BM into lethally irradiated mice also caused acute myeloid leukemia within 3 months of transplantation. We infer that MN1-TEL is a hematopoietic oncogene that stimulates the growth of hematopoietic cells, but depends on secondary mutations to cause leukemia in mice.

Keywords:

chromosome translocation, myeloid leukemia, oncogene, ETS transcription factor, retroviral transfer

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Introduction

Recurrent chromosome translocations in human leukemia frequently produce tumor-specific fusion proteins involving TEL (ETV6), a member of the E26 transforming sequence (ETS) family of transcription factors.1, 2 TEL's N terminus contains a pointed (PNT) domain, which interacts with other proteins and defines a subclass of ETS proteins.3, 4 TEL's PNT domain mediates homo-oligomerization,5 but also associates with the PNT domains of FLI16 and TEL27 and recruits the transcriptional corepressor N-Cor.8 The domain between the PNT and DNA-binding domains recruits the corepressors Sin3a and SMRT.9, 10 These interactions are responsible for TEL's transcriptional repressor function. TEL's C-terminal ETS domain binds to DNA at sites (e.g., 5'-GGAA-3') typical of those bound by ETS proteins.11, 12

Translocations affecting TEL, mostly results in fusion of the PNT domain with various partners,1, 13 including the phosphotyrosine kinases (PTKs) platelet-derived growth factor-beta receptor, ABL,14 janus kinase 215 and ARG16 in hematopoietic diseases, and NTRK3 in some solid tumors.17, 18 Oligomerization via the PNT domain constitutively activates the PTK activity of the fused partner protein,15, 19, 20 thereby tumorigenically transforming hematopoietic cells.19, 21, 22, 23, 24 In other PNT-domain fusions, such as TEL-acute myeloid leukemia1 (AML1), recruitment of mSin3A via the PNT domain creates a dominant-negative transcription factor.10, 25, 26, 27 The PNT domain may similarly affect other transcription factor fusion partners, including TEL-MDS1 (TEL-EVI1)28 and TEL-CDX2.29

TEL fusion proteins that retain the ETS DNA-binding moiety are rare.11, 30, 31, 32 The first identified, MN1-TEL1, is the product of the t(12;22)(q12;q11) associated with human myeloid malignancy. MN1-TEL activates transcription of reporter genes via TEL-binding sites.11 MN1 was discovered through its involvement in a t(4;22)(p16;q11) in meningioma.33 MN1, a nuclear protein, is a transcriptional coactivator11 recruited by the retinoic acid coactivators (RAC)3 and p300 in retinoic acid receptor alpha (RARalpha) retinoid X receptor-(RXR)-mediated transcription.34 In NIH3T3 fibroblasts, MN1-TEL exhibits transforming activity that depends on DNA binding via TEL and on N-terminal transactivating sequences and C-terminal sequences in MN1. We showed that mice expressing MN1-TEL under the control of Aml1 regulatory sequences develop T-cell lymphoma after a long latency.35 Nonetheless, these mice can also develop AML provided they obtain the appropriate secondary mutation. AML results when MN1-TEL is combined with overexpression of HOXA9, a combination also found in patients with the t(12;22).35, 36 Here we assessed the in vitro and in vivo transforming activity of MN1-TEL in mouse BM, using a retroviral transduction/BM transplantation approach and identified MN1-TEL sequences necessary for transformation of myeloid cells.

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Materials and methods

Plasmids and retrovirus production

MN1-TEL cDNA was cloned into the unique EcoR1 cloning site of murine stem-cell virus-internal ribosome entry site-green fluorescent protein (MSCV-IRES-GFP) plasmid, and high-titer virus (5 times 105 – 1.5 times 106 cfu/ml) was obtained as described previously.37 As a control, we used empty MSCV-IRES-GFP vector.

BM extraction, isolation of Lin BM cells and retroviral transduction, and BM transplantation

BM was harvested from the femurs and tibiae of male, 8- to 12-week old, FVB-J, C57BL/6J, or C57BL/6/129svJ mice treated with 5-fluorouracil (5FU) (Sigma, St Louis, MO, USA). Isolation of Lin- cells, viral transduction and BM transplantation into female, 8- to 12-week-old C57BL/6J or C57BL/6/129svJ recipients was performed as described previously.37

Hematopoietic colony-forming assays and LTC-IC assays

These assays were performed as described previously.38

Cell cycle analysis by FACS

Cell cycle analysis of MMT3 and vector control cells was performed as described previously.37

In vitro differentiation of MMT3 cells

MMT3 cells in liquid culture were induced to differentiate by adding dimethyl sulfoxide (DMSO) (1 and 2%), vitamin D3 (1 times 10-8 mol/l), trichostatin A (10 mug/ml), retinoic acid (1 mM) or one of the following growth factors: granulocyte colony-stimulating factor (G-CSF), 5 ng/ml; granulocyte–macrophage colony-stimulating factor (GM-CSF), 30 ng/ml; macrophage colony-stimulating factor (M-CSF), 5 ng/ml; IL-3, 50 ng/ml; erythropoietin (EPO), 2 U/ml; and IL-6, 50 ng/ml (Preprotech, Rocky Hill, NC, USA). Cell-surface markers (Sca1, cKit, Mac1, Gr1, Thy1, B220, CD3, CD4, CD8), identified by fluorescence-activated cell sorting (FACS) at 0, 3 and 7 days of culture, were compared with those on MMT3 cells cultured with IL-3, IL-6 and stem cell factor (SCF) (50 ng/ml).

Secondary BM transplantation

Mice (C57BL/6/129svJ) were given a single sublethal dose (8 Gy) of radiation and the next day were injected in the tail vein with 5–8 times 105 primary leukemic BM or spleen cells. Mice were inspected daily for signs of hematopoietic disease.

Analysis of diseased mice and tissue preparation

All animal procedures were conducted in accordance with the US Public Health Service Policy on Humane Care and Use of Laboratory Animals. Retro-orbital cavity blood was collected monthly and analyzed by FACS to determine the percentage of GFP-expressing white blood cells (WBCs), erythrocytes and platelets. Blood counts were obtained with a Hemavet 3700 instrument (Drew Scientific, Cumbria, UK), and Giemsa-stained blood smears were examined for abnormal cells. Moribund animals were killed by CO2 asphyxiation after methoxyflurane inhalation and analyzed as described previously.37

FACS of Hoechst 33342-stained side population cells

Exponentially growing MMT3 cells were harvested in phosphate-buferred saline (1 times 106 cells/ml) containing 10 mug/ml Hoechst 33342 (Sigma) and incubated for 90 min at 37°C. To positively identify the SP cells, the ATP binding cassette transporter inhibitor reserpine (10 muM; Sigma) was added to a small aliquot of cells. Cells were counterstained with propidium iodide to mark dead cells and sorted in a BD Biosciences FACS Vantage SE/DiVa instrument (BD Biosciences, San Jose, CA, USA) using laser excitation at 488 and 357 nm. Hoechst-33342 fluorescence was collected at 424plusminus22 nm (blue) and >640 nm (red). A bivariate display of red versus blue fluorescence was used to identify SP cells, which were absent from the aliquot incubated with reserpine. Viable GFP+ cells displaying diminished Hoechst fluorescence were collected as the SP cells; all other cells were collected as the non-SP cells.39

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Results

MN1-TEL increases the self-renewal capacity of primitive hematopoietic progenitors

Lin- BM from 5FU-treated FVB mice was transduced with MSCV carrying an IRES sequence linked to the GFP gene, without (control) or with MN1-TEL cDNA. FACS 2 days later revealed GFP expression in 30–40% of MN1-TEL-transduced and 50–60% of control BM cells (Figure 1a). After 2 weeks in methylcellulose (MC), MN1-TEL-expressing cells showed a proliferation advantage over vector-transduced cells, either owing to a shorter cell cycle traverse, reduced apoptosis or both. Serial replating increased this advantage: the percentage of GFP+ MN1-TEL-expressing cells steadily increased to 100%, whereas that of GFP+ control cells remained constant (around 60%; Figure 1a). Although the colony-forming activity of control cells was exhausted at MC3 or MC4, MN1-TEL-transduced cells produced colonies for two additional cycles (Figure 1b). Colonies produced by MN1-TEL-expressing cells were GFP+, larger and more densely packed than those of control cells, especially at later rounds of MC assays (Figure 1c). After 4, 5 or 6 weeks of long-term-culture-initiating cell culture (LTC-IC),40 the effect of MN1-TEL on colony formation was more pronounced: MN1-TEL cultures in initial MC assay produced, on average, 35 times as many colonies as did control cultures (Figure 1d). The Sca1+/cKit+/Lin- progenitor phenotype of MN1-TEL cells (determined by cell-surface marker analysis; not shown) suggested expansion of a primitive hematopoietic progenitor. However, MN1-TEL did not immortalize cells: no colonies were produced beyond MC 6 following LTC-IC. The same results were obtained with C57BL/6 BM.

Figure 1.
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MN1-TEL promotes growth of mouse myeloid progenitors. (a) Mouse BM was transduced with MSCV-MN1-TEL-IRES-GFP or MSCV-IRES-GFP and serially plated into MC1–6. The percentage of GFP-expressing cells at each round of plating is shown. (b) The numbers of MN1-TEL or vector-transduced colonies at each round of MC culture are shown. (c) Left two panels compare the size of an MC3 colony of vector cells (left) and MN1-TEL+ cells (right). Right two panels show immunofluorescence micrographs of a non-transduced GFP- colony (left) and a GFP+/MN1-TEL+ colony (right) in the same MC2 culture. (d) Colony-forming capacity of BM cells transduced with retrovirus encoding MN1-TEL, MN1-TEL mutants or GFP alone, in serial MC assays (LMC1–6) after 4, 5 or 6 weeks of LTC-IC (LMC1–6). A visual representation of the MN1-TEL mutants is shown at the right of the bar graph. Green represents MN1 sequences and interrupting white boxes represent deleted sequences. Gray represents TEL sequences with the PNT domain in orange and the ETS DNA-binding domain in yellow. The small red box indicates the mutation in the ETS domain of the DBDM mutant. The Western blot underneath shows expression of the different MN1-TEL mutants in transduced GFP+ BM cells (indicated by asterisks at the right of the bands) detected with a C-terminal peptide TEL antibody. The glyceraldehyde-3-phosphate dehydrogenase loading control is shown underneath. (e) Mouse BM transduced with MSCV-MN1-TEL-IRES-GFP was cultured in LTC-IC for 5 weeks, followed by liquid culture with IL-3, SCF and IL-6 (4 weeks). Growth curves were determined in the presence of IL-3/SCF/IL-6 (MN1-TEL+GF) or without added growth factors (MN1-TEL-GF).

Full figure and legend (258K)

When after 5 weeks of LTC-IC, cells were put in liquid culture in the presence of any of the following cytokines: IL-6, IL-3, SCF, GM-CSF, G-CSF, M-CSF, EPO or IL-3+SCF+IL6, they proliferated in the presence of IL-3+SCF +IL6 (Figure 1d), IL-3 and somewhat with SCF (not shown). Removal of cytokines completely inhibited proliferation (Figure 1e).

MN1-TEL sequences necessary for increased proliferation in LTC-IC assays

MN1-TEL's ability to transform NIH3T3 fibroblasts depends on TEL-mediated DNA binding and functions provided by N-terminal and C-terminal MN1 sequences. We assessed these sequences' involvement in growth promotion of BM by examining the effects of MN1-TEL mutants described previously:11 MN1-TEL-DBDM (does not bind TEL-binding sites); MN1-TELDelta229–1223 (retains N-terminal part of MN1 sequences interacting with p300 and Rac3);34 the complementary mutant MN1-TEL12–228 (retains C-terminal part f MN1 sequences interacting with p300 and Rac3); MN1-TELDelta692–1123 (retains all sequences interacting with p300 and Rac3); and MN1-TELDelta18–1123 (missing almost all MN1 sequences). We transduced Lin- BM cells with MSCV-IRES-GFP virus-expressing mutant MN1-TEL, wild-type (WT) MN1-TEL or neither. Western blotting of sorted GFP+ cells with a C-terminal TEL antibody11 showed the expression of all MN1-TEL proteins, although the MN1-TEL, MN1-TEL-DBDM and MN1-TELDelta692–1123 were expressed at a lower level than MN1-TEL12–228 and MN1-TELDelta229–1223 and at much lower level than MN1-TELDelta18–1123 (Figure 1d). Given that vector-transduced and non-transduced BM produce few MC colonies after LTC-IC, this analysis allowed us to identify MN1-TEL mutants that retained the capacity to stimulate BM self-renewal activity.

Combining data from two LTC-IC experiments after 4, 5 and 6 weeks of LTC-IC, we determined average numbers of colonies in serial MC assays (Figure 1d). All cells producing colonies in serial MC assays were GFP+. Only MN1-TEL, MN1-TELDBDM and, to a lesser extent, MN1-TELDelta692–1123 cells showed increased self-renewal activity. Cells expressing Delta229–1223, Delta12–228 and Delta18–1123 MN1-TEL produced few colonies (Figure 1d). We concluded that N-terminal MN1 sequences but, surprisingly, not DNA binding by the ETS domain are important for MN1-TEL's growth-promoting activity.

BM cells expressing MN1-TEL can be immortalized in culture

Confirming our previous finding that BM from MN1-TEL knock-in mice can be immortalized,35 BM cells transduced with MN1-TEL retrovirus became immortalized upon 3–5 weeks of liquid culture with IL-3, IL-6 and SCF using cells recovered from the MC 1 (2 weeks) after 4 weeks of LTC-IC. May–Grunwald–Giemsa staining revealed small, blast-like cells and large cells with basophilic granules all expressing GFP and MN1-TEL (Figure 2a, c and e). Immunophenotyping by FACS showed they were cKit+, Sca+, partly Thy1+ and Mac1low (Figure 2c). We obtained numerous morphologically and immunophenotypically similar MN1-TEL cell lines from independently transduced BM isolates and examined one line, MMT3, for its cell cycle characteristics and cytokine dependence. FACS cell cycle analysis after 2 months of liquid culture of MMT3 cells and vector-transduced cells showed that 44.5% of the former and only 3% of the latter were in the S-phase (Figure 2b), indicating that MN1-TEL cells cycled much faster than vector control cells. We next cultured MMT3 cells with IL-3; SCF; IL-6; M-CSF; G-CSF; GM-CSF; EPO; IL-3, IL-6 or SCF (Figure 2d). Cells proliferated well with IL-3, to some extent with SCF, vigorously with IL-3+SCF+IL-6, and slowly with EPO. Non-proliferating cells were remarkably resistant to apoptosis induced by cytokine withdrawal and died slowly (half the population in 5 days). We compared the level of antiapoptotic BclXL and Bcl2 proteins expressed in MMT3 and control cells cultured for 2 months (Figure 2e). MMT3 cells expressed a moderately increased amount of BclXL and a substantially increased amount of Bcl2.

Figure 2.
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MN1-TEL-expressing BM cells are immortalized during liquid culture. (a) FVB BM was transduced with MSCV-MN1-TEL-IRES-GFP, seeded into LTC-IC, harvested 4 weeks later and plated in MC. Two weeks later, MC colonies were seeded into liquid cultures containing IL-3/SCF/IL-6 and 5 weeks later cell lines containing morphologically variable cells emerged (left panel). Immunolabeling of these cells with the 2F2 monoclonal MN1 antibody revealed nuclear localization of MN1-TEL (lower right panel). Nuclear staining with 4',6'-diamidino-2-phenylindole (DAPI) is shown (upper right panel). (b) FACS cell cycle analysis of MMT3 cells and vector-transduced cells, both in culture for 2 months, showed that MMT3 cells cycled faster. (c) Cell-surface marker analysis of the MN1-TEL cell line MMT3. (d) MMT3 cells are growth-factor dependent, as shown by proliferation assays with different cytokines. (e) MMT3 cells and WT FVB BM cells grown in liquid culture with IL-3/SCF/IL-6 for 8 weeks were lysed and the proteins immunoblotted with anti-MN1, anti-actin, anti-BclX and anti-Bcl2 antibodies.

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Transplantation of MMT3 cells into lethally irradiated mice

Attempts to induce differentiation of MMT3 cells in vitro by adding vitamin D3, retinoic acid, DMSO, trichostatin A or various cytokines (IL-6, G-CSF, GM-CSF, M-CSF, IL-7 and IL-2) failed and the cells remained immunophenotypically and morphologically unchanged (not shown). We next tested whether leukemia developed in lethally irradiated mice given MMT3 cell transplants. Surprisingly, transplantation rescued all (n=10) recipients from radiation-induced BM failure; two mock-transplanted irradiated control mice died at 12 and 14 days after irradiation, respectively. One month after transplantation, the WBC counts of recipients were low (0.26–1.04 times 103/mul) but reached normal values (2.3–7.5 times 103/mul) after 2 months; 50–70% of cells were GFP+ at each time point (Figure 3a and b). Gating and immunophenotyping by FACS of the peripheral blood of one transplant recipient killed at 2 months determined the proportions of erythrocytes (3%), B cells (53%), T cells (7%), granulocytes (68%), macrophages (83%) and platelets (35%) expressing GFP (Figure 3c). This analysis also identified GFP+bright and GFP+dim populations (Figure 3c, right). The former mainly consisting of cKit+ cells that differentiated partially into immunoglobulin (Ig)M+ B cells and more poorly into Mac1+ monocytes/macrophages and Gr1+ granulocytes. The GFP+dim population (cKit/Sca1) differentiated better into Mac1+, Gr1+ and B220+ WBCs. The BM of this mouse (15% Sca1+, 61% cKit+, 4% Cd34+, 9% Thy1+, 9% B220+, 2% IgM+, 22% Mac1+ and16% Gr1+) showed a GFP+bright population consisting of cKit+ and partly Sca1+/Thy1+ cells and the GFP+dim population consisting of Mac1+/Gr1+ cells IgM-. (Figure 3d). Some cells in each population were B220+ but IgM-. Immunolabeling of cytospin preparations of this BM revealed 60% of cells expressing MN1-TEL (Figure 3e). FACS of eye blood from the nine remaining MMT3-transplant recipients also showed the two GFP+ populations (Figure 3c, right). To avoid killing more transplant recipients, we deduced the percentage of GFP+ cells in their peripheral blood from the FACS scatter plots. On average, erythrocytes were 3% GFP+ and platelets, 50%. The aberrant size distribution of the MMT3-derived cells precluded the determination of GFP+ contributions to the different WBC lineages.

Figure 3.
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Hematopoiesis in lethally irradiated mice is temporarily restored by transplantation with MMT3 cells. (a) Average percentage of GFP+ WBCs in the peripheral blood of 10 MMT3-transplanted mice 1, 2 and 3 months after transplantation. (b) Average WBC counts of MMT3-transplant recipients 1, 2 and 3 months after transplantation. Error bars indicate variation among mice. (c) Cell-surface marker analysis of peripheral blood from a mouse 2 months after transplantation with MMT3 cells (left eight panels). At the right is the GFP fluorescence in the peripheral blood of MMT3-transplant recipients, showing separate peaks representing GFP+dim and GFP+bright lymphocyte and monocyte populations and GFP+dim granulocytes. (d) Cell-surface marker analysis of BM from the same mouse 2 months after transplantation with MMT3 cells, also showing GFP+dim and GFP+bright populations. (e) MN1-TEL immunofluorescence in BM from a mouse 2 months after transplantation with MMT3 cells, using an MN1 antibody. Left, DAPI staining; middle, MN1-TEL detected with an MN1 antibody; right, vector-transduced BM stained with an MN1 antibody.

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Development of myeloid leukemia after transplantation

Myeloid leukemia (Sca1+, Mac1+, cKitdim) developed in all mice 10–13 weeks after transplantation (Figure 4a and b). Peripheral WBC counts were high (100–180 times 103/mul) and GFP+ cells had invaded most organs, including liver, spleen, kidney, lung, heart and caused brain hemorrhages (Figure 4c). The invading blast cells had distorted or effaced the architecture of the spleen, liver and femoral BM in association with dysplastic megakaryocytes. Immunohistochemical staining of paraffin sections showed that blasts in the BM and the invaded organs were positive for cKit, CD34, with some also positive for GATA1 (not shown). The blasts were negative for CD3, CD45, TdT, Mac2, lysozyme, myeloperoxidase and Factor 8. These features would be consistent with a myeloid leukemia with megakaryocyte maturation.

Figure 4.
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Transplantation with MMT3 cells causes myeloid leukemia in mice. (a) Kaplan–Meier survival curve for nine lethally irradiated mice given transplants of MMT3 cells (MN1-TEL) and five lethally irradiated mice given transplants of MSCV-IRES-GFP-transduced BM (Vector). (b) Representative cell-surface marker analysis of BM from one of nine mice in which leukemia developed 3 months after transplantation with MMT3 cells. (c) Tissue sections (hematoxylin–eosin stain) from a mouse in which leukemia developed 3 months after transplantation with MMT3 cells. Left, hemorrhage in the brain (times 100 magnification), a common feature in these mice; middle, liver with invading tumor cells (times 200); right, spleen with leukemic cells (times 200).

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To determine which cells of the MMT3 cell line rescued the lethally irradiated mice, we incubated cells with Hoechst 33342 and sorted the unstained, GFP+ side population (SP) by FACS (Figure 5a). SP cells variably constituted 5–25% of all GFP+ cells. In normal BM, the SP is highly enriched in hematopoietic stem cells.39 The MMT3 SP consisted of mainly small cells with scant cytoplasm; non-SP cells were larger, with more abundant cytoplasm (Figure 5b). Transplantation of SP-MMT3 cells into lethally irradiated recipients (n=4) recapitulated the results obtained with unsorted MMT3 cells; mice were rescued from radiation-induced death but succumbed to myeloid leukemia (cKit+/Sca1+/Mac1+dim) 3.5–4 months after transplantation (Figure 5c, left). Non-SP MMT3 cell transplants also prolonged the lives of lethally irradiated recipients, but these mice (n=2) died of leukopenia 2 months after transplantation (Figure 5c, left panel). Clearly, the SP-MMT3 cells included primitive cells with repopulating and leukemogenic activity, whereas the non-SP cells included non-leukemic, short-term repopulating cells.

Figure 5.
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Lethally irradiated mice receiving transplants of MMT3 SP cells are rescued from radiation death but develop leukemia. (a) Left, a culture of fast-growing MMT3 cells was stained with Hoechst 33342 dye and sorted by two-color FACS (red and blue fluorescence) to separate stained (non-SP) and unstained (SP, boxed area in the left panel) cells, all expressing GFP (right). (b) Cytospin preparations of the MMT3 non-SP and SP fractions obtained in (a). (c, left) Kaplan–Meier survival curve for lethally irradiated mice given transplants of non-SP (n=2) and SP (n=4) MMT3 cells, rescuing them from hematopoietic failure. Non-SP recipients died of leukocytopenia 8 and 9 weeks later, whereas SP recipients died of myeloid leukemia between 15 and 18 weeks later. (Right) Leukemic cells were isolated from a moribund MMT3-transplant recipient and liquid cultured with IL-3/SCF/IL-6 (MMT3+) or without growth factors (MMT3-), and leukemic cells isolated from a recipient of MSCV-MN1-TEL-IRES-GFP BM (see Figure 6) were liquid cultured with IL-3/SCF/IL-6 (MN1-TEL+) or without growth factors (MN1-TEL-).

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Next, we addressed whether individual cells in the MMT3 cell line are multipotent or whether multiple sub-populations, each with limited differentiation capacity, together conferred multipotentiality. We sorted six single SP-MMT3 cells, and expanded each to a total of 3 times 106 cells. These were then transplanted into 6 lethally irradiated mice (5 times 105 cells/mouse), but all 36 recipients died 11–14 days later. This result might indicate that the repopulating cells represent a minor subset (<17% of cells) of the SP-MMT3 cells or that the cells were no longer multipotent owing to expansion in culture.

Leukemic BM from MMT3-transplant recipients grew in culture without added cytokines, indicating that a secondary mutation(s) had occurred that rendered these cells IL3/SCF-independent (Figure 5c, right panel). The MMT3 leukemia was fully transplantable into secondary sublethally irradiated mice and caused disease (Sca1+, Mac1+, cKitdim Thy1dim) within 3–4 weeks after transplantation.

Mice receiving freshly transduced MN1-TEL BM also develop myeloid leukemia

We also tested whether C57BL/6/129svJ mice receiving transplants of C57BL/6 BM freshly transduced with MSCV-MN1-TEL-IRES-GFP (MN1-TEL-BM, 10 mice) or MSCV-IRES-GFP (vector-BM, 10 mice) developed leukemia. MN1-TEL-BM recipients died of myeloid leukemia 8–14 weeks after transplantation. No disease developed in vector-BM recipients, but two mice died of graft failure (Figure 6a). MN1-TEL-BM recipients had high peripheral WBC counts (1.5–2.0 times 105/mul), and most had brain hemorrhages. The peripheral blood contained blasts and poorly differentiated neutrophils; the BM consisted of monomorphic myeloid cells (Figure 6b). Leukemic cells invaded the spleen, liver, lungs, kidney, intestines and lymph nodes, showing mitotic figures in all organs. Leukemic BM of moribund animals was phenotypically variable: two samples contained GFP+dim and GFP+bright cell populations (Figure 6c, lower panels); the remainder contained a single GFP+dim population (Figure 6c, upper panels). In mice with two GFP+ populations, GFP+dim cells were mainly cKit+dim, whereas the GFP+bright population was mainly Mac1+/Gr1+/Thy1+. The leukemic cells in the BM of the other eight mice were cKit+/Mac1+/Thy1+ with 10% of the cells also Gr1+. Transplantation of the leukemic BM into sublethally irradiated secondary recipients generated fulminant myeloid leukemia 19–27 days after transplantation. Leukemic BM from these mice proliferated without added growth factors (Figure 5c), although not as vigorously as MMT3-derived leukemic cells.

Figure 6.
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Myeloid leukemia develops in mice given transplants of MN1-TEL-expressing BM. (a) Kaplan–Meier survival curve of 10 lethally irradiated C57BL/6/129svJ mice that received C57BL/6 BM transduced with MSCV-MN1-TEL-IRES-GFP (MN1-TEL) and 10 mice that received MSCV-IRES-GFP-transduced BM (Vector). (b) The peripheral blood of moribund MN1-TEL leukemic mice contained blast cells and partially differentiated neutrophils (upper left panel). Leukemic cells filled the BM (upper right panel) and invaded most organs such as spleen (lower right panel) and the liver (lower left panel). Note the presence of numerous mitotic figures in the BM and spleen (black arrowheads). (c) Cell-surface marker analysis of BM from mice with MN1-TEL leukemia. Upper row, a single GFP+dim population; lower row, GFP+dim and GFP+bright cell populations.

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Discussion

Previously, we established that MN1-TEL has weak transforming activity in NIH3T3 fibroblasts11 and causes hematopoietic disease in mice when expressed from the endogenous Aml1 locus.35, 36 Here we have shown that retrovirally expressed MN1-TEL has a more potent transforming activity than MN1-TEL expressed by Aml1. Retrovirally expressed MN1-TEL increased the self-renewal activity of myeloid progenitors, but serial MC cultures were always finite. Other oncogenic fusion transcription factors such as MLL-AF9 and AML1-ETO show similar activities.41, 42 Immortalized, cytokine-dependent cell lines arose only when MN1-TEL BM cells were expanded in liquid culture. Because the number of cells in liquid culture vastly exceeds that in MC assays, they are more likely to undergo secondary mutations causing immortalization. We have not yet attempted to identify these mutations.

Although cells of the MN1-TEL cell line MMT3 were impervious to differentiation stimuli in vitro, they displayed a remarkable capacity to differentiate along various lineages after transplantation into lethally irradiated mice. Although we analyzed the BM of only one mouse 2 months post-transplantation (i.e., 1 month before leukemia developed; Figure 3c), we obtained similar results from the peripheral blood of the nine remaining mice. However, results from recipients of MMT3-SP cells suggest that leukemic cells are not inherent to the cell line but are generated after transplantation. Given that SP cells (5 times 105 cells/mouse) were transplanted directly after FACS and, in this experiment, composed 25% of the MMT3 population, these mice received 2.5 times as many primitive cells as did mice receiving non-sorted MMT3 cells (8 times 105 cells/mouse). If the MMT3 line had contained leukemic cells, one would expect leukemia to have developed more rapidly in SP-MMT3-transplant recipients. However, there was no significant difference in disease onset (14–18 weeks versus 10–13 weeks; Figures 4a and 5c), consistent with the notion that MMT3 cells underwent additional leukemic events after transplantation. This suggestion was further supported by the observation that MMT3 cells recovered from the leukemic mice had become IL3-independent (Figure 5c), which could only have happened by additional mutation.

We noticed that during a year in culture, the surface markers of the MMT3 cells changed from 100% cKit+/Sca1+ to 60% also Mac1+, a phenotype similar to that of the MMT3 leukemia cells in diseased mice. Leukemic cells in these mice might therefore have arisen from this sub-population of cells in the MMT3 line.

We addressed whether individual cells of the MMT3 line were multipotent or whether multiple sub-populations have limited differentiation capacity. The finding that none of the six single-cell-derived MMT3-SP cell batches rescued transplant recipients from radiation-induced death suggested either that only a small sub-population (<17%) of SP-MMT3 cells have repopulating activity, or that the cells' multipotency was lost during expansion to a population of 3 times 106 in culture. In either case, the outcome prevented us from determining whether individual MMT3-SP cells are multipotent.

Because MN1-TEL stimulates proliferation and slightly impairs differentiation of BM cells,36 a second genetic event is needed to immortalize them in vitro. Full leukemic transformation required at least one additional genetic event to confer cytokine independence. Therefore, full transformation requires two or more events in addition to MN1-TEL expression.

We mapped MN1-TEL sequences essential for stimulation of myeloid cell proliferation and found one substantial difference from the findings of a similar analysis in NIH3T3 fibroblasts:11 growth of myeloid progenitors in LTC-IC was independent of a functional TEL DNA-binding domain. We believe that MN1 is responsible for this finding, because it alone also promotes growth of myeloid cells of a slightly different phenotype in LTC-IC (J Bonten, C Carella and G Grosveld, unpublished observation) but not of fibroblasts.11 Therefore, the growth-stimulating effect of MN1-TEL-DBDM may be mediated entirely by its MN1 sequences. Further analysis is needed to resolve this issue.

We therefore concentrated on the contribution of the different MN1 (rather than TEL) sequences to the oncogenicity of the fusion protein. The growth-stimulating activity of MN1-TEL depended largely on the N-terminal 228 amino acids of MN1, a domain also important for interaction with p300 and Rac3 in HepB3 cells.34 Consistent with p300 and Rac3 interactions within the first 520 N-terminal amino acids of MN1,34 MN1-TELDelta692–1123 retained some transforming ability. However, whether loss of transforming activity of MN1-TELDelta12–228 is owing to the loss of interaction with these two coactivators remains to be determined. Experiments with additional MN1-TEL mutants and co-immunoprecipitation studies will resolve this issue.

MN1's contribution to oncogenicity also depends on sequences between amino acids 693 and 1123. Interestingly, this region contains an arginine-methyltransferase target sequence (amino acids 939–947).43 Arginine methylation is important for transcriptional activation of nuclear receptor transcription complexes,44 and given MN1's interaction with RARalpha-RXR dimers,34 this methylation sequence might contribute to MN1's function, a possibility we are investigating.

Finally, our finding that myeloid leukemia develops in mice given transplants of MN1-TEL-transduced BM (reported here) but not in mice carrying an MN1-TEL knockin construct35 may be explained by the much higher level of expression of MN1-TEL mediated by the retroviral vector than by the Aml1 knockin allele.

In summary, we have further established that MN1-TEL is a bona fide hematopoietic oncogene and that its function strongly depends on sequences in the N-terminal domain of MN1. We are determining which proteins interact with this domain and which downstream target genes mediate the growth-promoting effects of MN1-TEL in hematopoietic cells.

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Acknowledgements

We thank Blake McGourty for the supply of C57BL/6/129svJ-mixed background mice and technical assistance. We gratefully acknowledge Ann-Marie Hamilton Easton and Richard Ashmun for expert FACS analysis, and we thank Charlette Hill for editing the manuscript. We are grateful to Luc van Rompaey who initiated these experiments. This work was supported by NCI Grant CA72999, the cancer center (CORE) support grant CA217G and by the American Lebanese Syrian Associated Charities (ALSAC).