c-Myb is an essential downstream target for homeobox-mediated transformation of hematopoietic cells

Abstract

Abdominal-type HoxA genes in combination with Meis1 are well-documented on-cogenes in various leukemias but it is unclear how they exert their transforming function. Here we used a system of conditional transformation by an inducible mixed lineage leukemia-eleven-nineteen leukemia (MLL-ENL) oncoprotein to overexpress Hoxa9 and Meis1 in primary hematopoietic cells. Arrays identified c-Myb and a c-Myb target (Gstm1) among the genes with the strongest response to Hoxa9/Meis1. c-Myb overexpression was verified by Northern blot and quantitative reverse transcription-polymerase chain reaction (RT-PCR). Also MLL-ENL activated c-Myb through up-regulation of Hoxa9 and Meis1. Consequently, short-term suppression of c-Myb by small inhibitory RNA (siRNA) efficiently inhibited transformation by MLL-ENL but did not impair transformation by transcription factor E2A-hepatic leukemia factor (E2A-HLF). The anti c-Myb siRNA effect was abrogated by coexpression of a c-Myb derivative with a mutated siRNA target site. The introduction of a dominant-negative c-Myb mutant had a similar but weaker effect on MLL-ENL-mediated transformation. Hematopoietic precursors from mice homozygous for a hypo-morphic c-Myb allele were more severely affected and could be transformed neither by MLL-ENL nor by E2A-HLF. Ectopic expression of c-Myb induced a differentiation block but c-Myb alone was not transforming in a replating assay similar to Hoxa9/Meis1. These results suggest that c-Myb is essential but not sufficient for Hoxa9/Meis1 mediated transformation.

Figure 1.

Experimental setup for identification of Hoxa9/Meis1 target genes. (A) Immunoblot of nuclear extracts from packaging cells used to produce pMSCV-HA-Hoxa9 and pMSCV-flag-Meis1 viruses. Control indicates cells transfected with empty vector. (B) Schematic representation of experimental outline. MLL-ENL-ERtm is a fusion of MLL-ENL with the ligand binding domain of a mutated estrogen receptor. MLL-ENL-ERtm is only active in the presence of 4-OHT.

Figure 2.

Verification of array results. (A) Northern blot and qRT-PCR. RNA from Hoxa9/Meis1-transduced MLL-ENL-ERtm cells was isolated 72 hours after inactivation of MLL-ENL and compared with control RNA from cells transduced with empty vectors. Hybridization was done with c-Myb- and β-Actin-specific probes. Specific bands are labeled. qRT-PCR was performed with an intron-spanning primer pair, and expression levels were determined with the ΔΔC_t method normalizing to actin. Means and standard deviations of triplicates are given. c-Myb RNA concentration in control cells was arbitrarily set to 1. (B) qRT-PCR testing for changes in Meis1, Hoxa9, and c-Myb expression in MLL-ENL-ERtm cells after inactivation of MLL-ENL. RNA was isolated from cells in the presence of 4-OHT (0-hour value, ▪), and 96 hours after 4-OHT withdrawal (□). The experiment was conducted as before except that 0-hour expression values were set arbitrarily to 1.

Figure 3.

Effect of c-Myb inhibition on MLL-ENL mediated transformation. (A) A retroviral construct in a “self-inactivating” (SIN) viral backbone (pSIRENretroQ) expressing a small hairpin RNA (shRNA) specifically directing against c-Myb was tested for efficacy by cotransfection with a flag-c-Myb expression clone. A similar virus with a luciferase-directed shRNA sequence was used as control. shRNAs are processed in the cell to siRNA. Knockdown was verified by Western blotting with anti-flag antibodies (top panel) and by transduction of primary hematopoietic cells with Myb shRNA viruses followed by qRT-PCR of c-Myb RNA (bar graph gives mean and standard deviation of 3 samples). Primary hematopoietic precursors were simultaneously transduced with a pMSCV-MLL-ENL virus and either the luciferase or the c-Myb shRNA construct. The cells were tested for enhanced replicative capacity as a marker for transformation in a methocel serial-replating assay under appropriate antibiotic selection. First-round and third-round colonies are shown in a representative example of 3 independent experiments. A numeric evaluation of relative colony numbers (n = 3) is shown in the bottom panel. (B) In a parallel experiment, c-Myb activity was reduced by coexpression of a dominant-negative c-Myb derivative (DNmyb). For this purpose the DNA binding domain of c-Myb (amino acids 1-200) was fused to a KRAB repressor domain. Correct expression of the flag-tagged construct was verified by an anti-flag immunoblot. Representative colony formation of cells expressing MLL-ENL and DNmyb or as control MLL-ENL and flag-c-Myb is shown as in panel A. Colonies were phtographed with a Canon Coolpix 990 camera (Canon, Tokyo, Japan) in nanomode after staining with iodonitro tetrazolim violet. Image processing was done with standard Windows (Microsoft, Redmond, WA) processing software.

Figure 4.

Specificity controls for siRNA mediated c-Myb knockdown. (A) A flag-tagged c-Myb mutant (Myb^*) with a silent mutation in the siRNA target sequence was constructed by site-directed mutagenesis. Myb^* was inserted into pMSCV-MLL-ENL under control of the plasmid-borne phosphoglycerate kinase promoter and tested for expression by anti-flag immunoblot with wild-type c-Myb as control. When tested under identical conditions, Myb^* was significantly more resistant than wild-type Myb to the knockdown effect of anti-Myb shRNA (compare with Figure 3A). The constructs coding for MLL-ENL or MLL-ENL/Myb^* were cotransduced with shRNA expression constructs as indicated into hematopoietic precursors and tested for colony-forming ability after serial replating. A representative example of third-round colonies is shown. (B) The effect of shRNA-mediated c-Myb suppression on E2A-HLF-mediated transformation was tested in an experiment analogous to that in panel A.

Figure 5.

Effect of a c-Myb-deficient genetic background on hematopoietic transformation. Mice heterozygous for a hypomorphic allele of c-Myb (Myb^kd) were intercrossed and the genotype of the offspring was determined by PCR. Myb^kd is a floxed-neo cassette inserted into intron 6 of the c-Myb gene. This modification leads to a reduction of c-Myb protein expression to approximately 5% to 10% of the wild-type level. Hematopoietic precursors from 5-FU-treated bone marrow harvested from 8- to 10-week-old mice of the indicated phenotype were transduced with MLL-ENL- or E2A-HLF-expressing retroviruses. Cells were subject to standard serial replating in methocel. A typical example of third-round colonies is shown. In total the experiment was performed 3 times.

Figure 6.

Transformation properties of c-Myb. (A) Serial replating assay with hematopoietic precursors transduced with empty virus or viruses coding for MLL-ENL, Hoxa9, Meis1, and c-Myb singly or in combination as indicated. Typical results of third-round colonies are shown. (B) MLL-ENL-ERtm cells were transduced with c-Myb or empty control virus. Surface markers c-kit and Gr-1 as well as proliferation rates of the resulting cells were analyzed by FACS (left panel) and cell counts (right panel) before and after inactivation of MLL-ENL by withdrawal of 4-OHT.