Hoxa9 and Meis1 are key targets for MLL-ENL-mediated cellular immortalization

Abstract

MLL fusion proteins are oncogenic transcription factors that are associated with aggressive lymphoid and myeloid leukemias. We constructed an inducible MLL fusion, MLL-ENL-ERtm, that rendered the transcriptional and transforming properties of MLL-ENL strictly dependent on the presence of 4-hydroxy-tamoxifen. MLL-ENL-ERtm-immortalized hematopoietic cells required 4-hydroxy-tamoxifen for continuous growth and differentiated terminally upon tamoxifen withdrawal. Microarray analysis performed on these conditionally transformed cells revealed Hoxa9 and Hoxa7 as well as the Hox coregulators Meis1 and Pbx3 among the targets upregulated by MLL-ENL-ERtm. Overexpression of the Hox repressor Bmi-1 inhibited the growth-transforming activity of MLL-ENL. Moreover, the enforced expression of Hoxa9 in combination with Meis1 was sufficient to substitute for MLL-ENL-ERtm function and to maintain a state of continuous proliferation and differentiation arrest. These results suggest that MLL fusion proteins impose a reversible block on myeloid differentiation through aberrant activation of a limited set of homeobox genes and Hox coregulators that are consistently expressed in MLL-associated leukemias.

FIG. 1.

Construction of an inducible derivative of MLL-ENL. A) Schematic representation of the MLL-ENL-ERtm construct. Numbers denote the respective amino acid positions of the individual constituents. The amino acid sequence of the linker is given in the one-letter code. (B) Western blot analysis of MLL-ENL-ERtm expression. Phoenix-E retroviral packaging cells were transfected with the retroviral vectors pMSCV-MLL-ENL, coding for native MLL-ENL, and pMSCV-MLL-ENL-ERtm. Nuclear lysates were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted with an anti-MLL monoclonal antibody recognizing an N-terminal epitope. The star denotes the N-terminal cleavage product of endogenous MLL.

FIG. 2.

Characterization of MLL-ENL-ERtm properties. (A) Transcriptional transactivator capability of MLL-ENL versus that of MLL-ENL-ERtm. The respective retroviral plasmids were coelectroporated with a luciferase reporter driven by the murine Hoxa7 promoter into the pre-B-cell line REH. The transfected cells were cultivated for 24 h in the presence or absence of 100 nM 4-OHT and lysed, and luciferase levels were determined normalized to protein content. Transactivation is given as the fold increase compared to the background levels obtained when an empty retroviral vector was coelectroporated. Shown are means and standard deviations of triplicate experiments. (B) Reversibility of 4-OHT-induced transactivation by MLL-ENL-ERtm. Mouse M1 cells stably expressing MLL-ENL-ERtm were cultivated for 3 days, and then 100 nM 4-OHT was added before the cells were depleted of inductor again at day 7 and kept for 3 more days in unsupplemented medium. At the indicated time points cell samples were drawn and electroporated with the Hoxa7-luciferase reporter, and luciferase levels were determined as relative luciferase units per microgram of protein. Shown are the means and standard deviations of triplicate experiments. (C) Outline and results of a bone marrow replating assay to assess the transforming properties of MLL-ENL-ERtm. Primary bone marrow cells enriched in hematopoietic precursor cells by 5-fluorouracil (5-FU) treatment of the donors were transduced with retroviruses capable of expression of MLL-ENL, MLL-ENL-ERtm, or neomycin only. The infected cells were plated under drug selection in cytokine-supplemented methocel medium. Ten thousand cells from colonies arising after the first selection round were replated in cytokine-supplemented medium, and this procedure was repeated once more. Shown are stained third-round colonies transduced with the indicated retroviruses and cultivated with the given amounts of 4-OHT. This is one representative example of five independent transduction experiments.

FIG. 3.

Establishment of MLL-ENL-ERtm-transduced cell lines. (A) 4-OHT-dependent growth of MLL-ENL-ERtm-transduced cells. Cells from two independent transduction experiments were explanted from third-round methocel cultures and incubated in cytokine-supplemented media in the presence or absence of 100 nM 4-OHT. Cell numbers were determined by direct counting. Diamonds, experiment 1; triangles, experiment 2; filled symbols, 100 nM 4-OHT; open symbols, without inductor. (B) Southern blots for detection of integrated MLL-ENL-ERtm copies. Genomic DNA was isolated from the cell lines used for panel A, digested with either NheI or BamHI, blotted, and hybridized against an MLL-specific probe. The localization of the restriction site and the hybridization probe (black bar) is indicated by the graphics. Normal mouse genomic DNA was included as a control (lane c). (C) RT-PCR analysis of MLL-ENL-ERtm expression. RNA was isolated from lines 1 and 2 as for panels A and B, reverse transcribed, and subjected to PCR amplification with primers spanning the MLL-ENL breakpoint. RT-minus controls are labeled −. (D) Immunophenotype of MLL-ENL-ERtm cells. After 12 days of cultivation either in the presence or the absence of 4-OHT (as indicated) the cells used for panel A were stained with fluorophore-coupled antibodies against the surface markers c-kit, Gr-1, and Mac-1 and analyzed by FACS. The open line corresponds to the background staining with an isotype-matched control antibody.

FIG. 4.

Reversion of cellular immortalization after inactivation of MLL-ENL-ERtm. (A) Terminal differentiation of MLL-ENL-ERtm cells after inductor withdrawal. The cell lines generated from MLL-ENL-ERtm-transduced cells were cultivated in cytokine-supplemented media in the absence of 4-OHT. Surface marker composition was monitored by FACS staining for 10 days. Only the results for line 1 are shown, as line 2 gave virtually indistinguishable results. (B) Morphological analysis of MLL-ENL-ERtm cells grown in the presence of 4-OHT and after 10 days without inductor. May-Grünwald-Giemsa stain of cytospin slides is shown. Original magnification, ×63.

FIG. 5.

Quantitative real-time PCR analysis. Expression of Hoxa7, Hoxa9, Meis1, Pbx3, and Pbx1 as indicated was quantitated by real-time PCR in RNA samples isolated from cells grown in the presence of 4-OHT (dark columns) or 72 h after 4-OHT withdrawal (light columns). Means and standard deviations of triplicate experiments are shown.

FIG. 6.

Coexpression of the Bmi-1 repressor gene inhibits MLL-ENL. (A, left) Colony count in the third round of a methocel assay with cotransduction of MLL-ENL and Bmi-1. Hematopoietic progenitor cells were coinfected with retroviruses encoding MLL-ENL and Bmi-1 and subjected to a methocel replating assay. Third-round colonies obtained from 10,000 cells were counted. Shown are the means and standard deviations of three cotransduction experiments. (A, right) Colonies obtained in the first and the third round of plating during the cotransduction test. Results of one of three conducted experiments are shown. (B) Southern blot performed with DNA isolated from cells either transduced with the indicated constructs or control cells (lane c) and hybridized with a Bmi-1 specific probe. (C) Expression of MLL-ENL and Bmi-1 specific RNA in cotransduced cells as detected by RT-PCR.

FIG. 7.

Hoxa9 and Meis1 are crucial downstream targets of MLL-ENL-ERtm. (A) Description of the experimental outline. puro, puromycin; hygro, hygromycin. (B) FACS analysis. MLL-ENL-ERtm cells were overtransduced as indicated with retroviral constructs coding for Hoxa7, Hoxa9, Meis1,or a combination thereof. After 5 days in selective medium supplemented with 4-OHT the surface marker distribution and the percentage of GFP-positive cells were determined by FACS (left panel). The black outline in all graphs represents a control staining with an isotype-specific antibody. After a further 10 days in selective medium but now without 4-OHT the differentiation status of the respective cells was assessed again by FACS (middle panel) and compared to a subpopulation of the same cells treated identically except that 4-OHT was present during culture (right panel). The insets in the graphs represent the distribution of GFP-positive Hoxa9-positive and -negative cells where appropriate. A two-dimensional analysis of GFP/Hoxa9 expression versus Gr-1 staining is depicted for two samples (highlighted in blue).