The MYB proto-oncogene suppresses monocytic differentiation of acute myeloid leukemia cells via transcriptional activation of its target gene GFI1

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Abstract

The MYB gene is a master regulator of hematopoiesis and contributes to leukemogenesis in several species including humans. Although it is clear that MYB can promote proliferation, suppress apoptosis and block differentiation, the identities of the MYB target genes that mediate these effects have only been partially elucidated. Several studies, including our own, have collectively identified substantial numbers of MYB target genes, including candidates for each of these activities; however, functional validation, particularly in the case of differentiation suppression, has lagged well behind. Here we show that GFI1, which encodes an important regulator of hematopoietic stem cell (HSC) function and granulocytic differentiation, is a direct target of MYB in myeloid leukemia cells. Chromatin immunoprecipitation and reporter studies identified a functional MYB-binding site in the promoter region of GFI, whereas ectopic expression and small hairpin RNA-mediated knockdown of MYB resulted in concomitant increases and decreases, respectively, in GFI1 expression. We also demonstrate that GFI1, like MYB, can block the induced monocytic differentiation of a human acute myeloid leukemia cell line, and most importantly, that GFI1 is essential for MYB’s ability to block monocytic differentiation. Thus, we have identified a target of MYB that is a likely mediator of its myeloid differentiation-blocking activity, and which may also be involved in MYB’s activities in regulating normal HSC function and myeloid differentiation.

Introduction

The MYB gene was identified as a transforming oncogene in retroviruses causing chicken leukemias,1, 2 and is now known to actively contribute to leukemogenesis in several species.3, 4 Subsequently, the Myb locus has been identified as a recurrent site of retroviral insertional mutagenesis in a number of murine hematopoietic malignancies.5, 6, 7 Importantly, MYB has been found to be directly activated in human leukemias by genetic lesions,8, 9, 10, 11, 12, 13, 14 such as translocation, duplication and structural alteration, although these genetic lesions are not very common. Furthermore, MYB mRNA is expressed at high levels in most human myeloid leukemias and acute lymphocytic leukemias,15 suggesting that MYB may have a more general role in leukemogenesis, such as acting as an essential cofactor for other oncogenes in the induction and maintenance of these leukemias (reviewed in Pattabiraman et al.3).

MYB’s contribution to leukemogenesis includes promoting proliferation and suppressing apoptosis.16 As a transcription factor, MYB exerts these functions by binding to and regulating a plethora of target genes, many of which we17 and others (reviewed in Ramsay et al.4) have identified. For example, MYB promotes proliferation by activating its target genes Myc18 and CCNB1,19 and suppresses apoptosis via activation or repression of its targets BCL218, 20, 21 or DRAK2,20 respectively. MYB’s ability to block differentiation is also a critical component of its transforming/leukemogenic potential. It is well established that various forms of MYB can repress differentiation of leukemic cell lines representing several hematopoietic lineages.22, 23, 24, 25 It is also clear that MYB can suppress the differentiation of primary hematopoietic cells from mouse and chicken.26, 27 However, the molecular mechanisms underlying MYB’s ability to suppress differentiation are largely unknown, although our recent genome-wide analysis of MYB target genes highlighted its ability to repress several key positive regulators of myeloid differentiation.17

The transcriptional repressor growth factor independent 1 (GFI1)28 has a variety of roles at multiple stages of hematopoiesis.29 It maintains the hematopoietic stem cell (HSC) pool30, 31 by restraining the proliferation of HSCs. Within the myeloid compartment, GFI1 is essential for granulocytic differentiation, in that Gfi1 null mice are severely neutropenic32, 33 and GFI1 mutations in humans cause severe congenital neutropenia.34 Enforced expression of Gfi1 in a mouse myeloid progenitor cell line, PUER, blocks monocytic differentiation but does not interfere with granulocytic differentiation.35

Previously, we identified Gfi1 as one of 400 direct target genes activated by MYB in ERMYB, a Myb-transformed murine myeloid progenitor cell line.17 In the current report, we extend that study by demonstrating that Gfi1 promoter region is directly regulated by MYB, that GFI1 is also regulated by MYB in a human myeloid leukemia cell line, and importantly, that transactivation of GFI1 is essential for and largely accounts for MYB’s ability to block monocytic differentiation in this myeloid leukemia model.

Results and Discussion

We previously identified Gfi1 as a likely direct MYB target gene by chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) and expression profiling17 in ERMYB, a mouse myeloid progenitor cell line.36 In this cell line, we confirmed that MYB protein both binds to the proximal promoter region of Gfi1 and regulates its expression.17 Consistently, we found that Gfi1 is upregulated in mouse primary bone marrow cells transformed by MYB.17 Furthermore, the Gfi1 level is reduced in the granulocyte–macrophage progenitor population isolated from Myb-hypomorphic booreana mice.17 To further confirm direct regulation, we examined a detailed time course of Gfi1 expression in ERMYB cells (Supplementary Figure S1). Gfi1 expression started to fall as early as 1 h after MYB inactivation by estrogen withdrawal, and markedly increased 1 h after estrogen readdition. These rapid kinetics make it very unlikely that regulation is indirect.

We next examined whether MYB binds to the human GFI1 promoter in human myeloid cells. The MYB-binding region (MBR) identified in our previous ChIP-Seq study is located 1.8 kb upstream of the transcriptional start site (TSS) of mouse Gfi1 variant 1.17 Because of the different TSS usage between the mouse and human genes, the identified mouse MBR corresponds to a conserved region 0.8 kb (GFI1+0.8 kb region) downstream of the TSS of human GFI1 variant 1 (Figure 1a). Using ChIP followed by quantitative PCR (ChIP-qPCR), we showed that MYB-protein binding is enriched in this region in U937 human acute myeloid leukemia (AML) cells (Figure 1b). We then searched this MYB-binding region for MYB-binding site (MBS) using the canonical binding motif as confirmed in our previous study.17 We found that this region indeed contains a potential MBS conserved between multiple vertebrate species (Figure 1c), including human, mouse and frog.

Figure 1
figure1

MYB binds to and regulates expression of GFI1 via a conserved MBS in the proximal promoter. (a) Schematic diagram showing the gene structures of mouse and human GFI1. The location of a MYB-binding region identified in our previous ChIP-Seq study,17 the potential MBS and the ChIP-qPCR amplicon in (b) are depicted. The mouse Gfi1 promoter region (−4.1 kb) that was tested in (d) is also illustrated. (b) MYB occupies a region 0.8 kb downstream of the TSS of human GFI1 variant 1. ChIP-qPCR was performed with anti-MYB sera on U937 cells. ChIP was performed as described17 using Myb 1-1 antibody (05-175, Millipore, Kilsyth, VIC, Australia) and Thelma anti-Myb serum (mixed 1:1).54 The TFF1 TSS region served as a negative control, whereas the MPO+1.0 kb region was a positive control. (c) The potential MBS is conserved between multiple vertebrate species. The core-binding sequence CNGTT is bold and boxed. (d) MYB binds to a conserved MBS to regulate Gfi1 expression. The indicated mouse Gfi1 promoter fragments were cloned into the pXPG firefly luciferase plasmid,55 following which K562 cells were transfected with the reporter constructs using Lipofectamine 2000 (Invitrogen, Melbourne, VIC, Australia). A TK-renilla construct (a gift from Dr Melissa Brown) was included as an internal control to normalize for transfection efficiency. Cells were harvested 48 h post transfection and cell lysates were assayed for firefly and renilla luciferase activity using the Dual Luciferase assay kit (Promega, Sydney, NSW, Australia). Luciferase reporter activities are expressed relative to that of the−4.1 kb construct. (e, f) Expression of MYB and GFI1, respectively, in MYB overexpressing and control U937 cells with 4 days’ treatment with 10 ng/ml PMA or vehicle control. U937/MYB cells were generated by first cloning the MYB-coding sequence into the lentiviral vector pLV41156; U937/Vector cells carry the empty vector. Lentivirus was produced by co-transfection of 293T cells with pLV411 vectors and packaging plasmids using Lipofectamine 2000 essentially as described.57 The supernatant was collected 48 h post transfection and used to transduce U937 cells. MYB-overexpressing cells were isolated using a MoFlo XDP cell sorter (Beckman Coulter, Lane Cove, NSW, Australia) based on the expression of vector-encoded green fluorescent protein. Gene expression was measured by quantitative RT-PCR (qRT-PCR) starting with 1 μg of total RNA prepared using Trizol (Invitrogen), which was reverse transcribed using Superscript III reverse transcriptase (Invitrogen). qRT-PCR was performed using iQ SYBR Green Supermix (Bio-Rad, Gladesville, NSW, Australia) on an ABI Prism 7900 HT instrument (Applied Biosystems, Mulgrave, VIC, Australia), and relative expression was calculated using ΔΔCt method with 18S rRNA as the internal control. (g, h) Expression of MYB and GFI1, respectively, in U937 cells carrying Tet-inducible MYB or scrambled control shRNAs as indicated, analyzed as in (e) and (f) above following 3 days’ treatment with 1 μg/ml Doxycycline (Dox) or vehicle. These cell lines have been described previously.20 Where relevant, data are presented as mean±s.e. ns, P>0.05, *P<0.05, **P<0.01, ***P<0.001, Student’s t-test. All primer sequences are in Supplementary Table 1.

Subsequently 4 kbp of the promoter region of mouse Gfi1 containing the potential MBS was inserted into a luciferase reporter and tested for activity in transfected K562 cells, another myeloid leukemia cell line that expresses substantial levels of MYB. As shown in Figure 1d, the activity of the −1.4 kb promoter, which lacks the potential MBS, is significantly lower than that of the −4.1 and−2.1 kb constructs, both of which contain this site. Similarly, the activity of the −4.1 kb mut promoter, in which the potential MBS is mutated, is significantly lower than that of the −4.1 kb wild-type promoter.

To demonstrate that MYB also regulates the expression of endogenous human GFI1, a derivative of the U937 human AML cell line ectopically expressing MYB (subsequently referred to U937/MYB) was constructed by lentiviral transduction. The U937 cell line has the advantage that it undergoes phorbol 12-myristate 13-acetate (PMA)-inducible monocytic differentiation, allowing us to study the effects of MYB and GFI1 on this process (see below). Figure 1e shows that the U937/MYB cells modestly (approximately twofold) overexpressed MYB in the absence of PMA compared with controls (U937/Vector) and maintained about half that level following PMA treatment, presumably because the expression of endogenous MYB was switched off. In contrast, MYB expression was almost undetectable in U937/Vector cells (as expected) following PMA treatment. Importantly, the expression of GFI1 was increased in U937/MYB cells compared with U937/Vector cells and, whereas the expression decreased in both following PMA treatment, the level of GFI1 was substantially higher after treatment in U937/MYB cells (Figure 1f).

We also examined the effects of MYB knockdown using a Tet-on inducible small hairpin RNA (shRNA) system.37, 38 U937 cells were transduced with lentivirus encoding either a scrambled control shRNA (shScr) or an shRNA targeting MYB (shMYB). In the presence of Doxycycline, the expression of shRNA is induced, and MYB mRNA expression was reduced to about 40% of the original level in U937/shMYB cells (Figure 1g) with, as we have shown previously,20 a similar or greater reduction in MYB protein. As shown in Figure 1h, MYB knockdown resulted in a corresponding reduction in the level of GFI1 mRNA. These results have been replicated with two independent small interfering RNAs (siRNAs) against MYB (Supplementary Figures S2a and b). Taken together, the evidence presented above and previously17 demonstrates that MYB binds to a conserved site in the proximal promoter region of GFI1/Gfi1 and positively regulates its expression in myeloid cells.

To begin to dissect the molecular mechanisms by which MYB suppresses the differentiation of leukemic cells, we first established MYB’s ability to block PMA-induced monocytic differentiation in U937 cells. As expected, PMA treatment of U937/Vector cells resulted in strong upregulation of monocytic differentiation marker genes, such as CD11b (Figure 2a) and CD14 (Figure 2b), and in morphological features associated with mature monocytes (Figure 2c). In contrast, the upregulation of CD11b (Figure 2a) and CD14 (Figure 2b) upon PMA treatment was significantly dampened by enforced MYB expression (that is, in U937/MYB cells). Consistent with this, the morphology of PMA-treated U937/MYB cells indicated they were refractory to terminal differentiation although they did show signs of somewhat enhanced differentiation compared with untreated cells (Figure 2c). Conversely, knockdown of MYB in U937/shMYB cells was sufficient to trigger substantial upregulation of CD11b (Figure 2d) and CD14 (Figure 2e), and to cause morphological changes indicative of a degree of differentiation (Figure 2f). These results have been replicated with two siRNAs targeting MYB (Supplementary Figures S2c and d).

Figure 2
figure2

MYB expression opposes monocytic differentiation of U937 cells. (a and b) Enforced expression of MYB suppresses PMA-induced expression of monocytic differentiation markers. U937/MYB and U937/Vector cells were treated with either vehicle or PMA for 4 days, after which CD11b (a) and CD14 (b) expression was analyzed by qRT-PCR as described for Figure 1. (c) May-Grunwald Giemsa staining of cytocentrifuged vehicle- and PMA-treated U937/MYB and U937/Vector cells. (df) MYB knockdown primes U937 cells for monocytic differentiation. U937/shMYB and U937/shScr cells were treated with either vehicle or Dox for 3 days and then analyzed by qRT-PCR for CD11b and CD14 expression and by May-Grunwald Giemsa staining, as indicated. qRT-PCR primer sequences are shown in Supplementary Table 1. Data are presented as mean±s.e. ns, P>0.05, **P<0.01, ***P<0.001, Student’s t-test. Scale bar=20 μm.

Taken together, the data of Figures 1 and 2 show that MYB expression opposes monocytic differentiation of U937 cells and simultaneously regulates the expression of GFI1 in these cells, in agreement with GFI1 being a direct target of MYB. These observations further raise the possibility that GFI1 may in fact mediate, at least in part, MYB’s suppressive effects on monocytic differentiation.

To assess the functional relevance of GFI1 in the context of U937 differentiation, we next examined the effects of enforced GFI1 expression on PMA-induced monocytic differentiation of U937 cells. To this end, we generated stable lines overexpressing either GFI1 or P2A, a loss-of-function GFI1 mutant28 (Supplementary Figure S3a). Enforced expression of GFI1 strongly suppressed the PMA-induced increase in the proportion of cells displaying the monocytic differentiation markers CD11b or CD14 (Figures 3a and b), whereas the P2A mutant failed to do so. A similar pattern was observed when we examined CD11b and CD14 mRNA (Supplementary Figures S3b and c). Furthermore, unlike PMA-treated U937/Vector and U937/P2A cells that displayed morphology typical of mature monocytes, PMA-treated U937/GFI1 cells showed morphology indicative of only partial differentiation, including relatively larger nuclei and less cytoplasm (Figure 3c).

Figure 3
figure3

Enforced GFI1 expression suppresses PMA-induced monocytic differentiation of U937 cells. Wild-type and P2A mutant GFI1 were cloned into the MSCV-IRES-mCherry vector, and VSV-G pseudotyped retrovirus was produced by co-transfection of 293T cells with these vectors and V-Pack packaging plasmids (Stratagene/Agilent, Mulgrave, VIC, Australia). Transduction and isolation of U937 cells were carried out as described for Figure 1, except that isolation was on the basis of mCherry fluorescence. Cells were treated with either vehicle control or PMA for 4 days and analyzed. (a and b) Enforced GFI1 expression strikingly reduced the proportion of CD11b+ and CD14+ cells, as indicated, upon PMA induction. Cells were stained with anti-human CD11b PE and anti-human CD14 APC antibodies (eBioscience, San Diego, CA, USA) and analyzed by flow cytometry on a Gallios flow cytometer (Beckman Coulter). Data are presented as mean±s.e. ns, P>0.05, **P<0.01, ***P<0.001, Student’s t-test. (c) May-Grunwald Giemsa staining of cytocentrifuged vehicle- and PMA-treated U937/Vector, U937/GFI1 and U937/P2A cells. Scale bar=20 μm.

As we showed that enforced expression of GFI1 has similar effects to enforced MYB expression in that both suppress PMA-induced monocytic differentiation of U937 cells, we next asked whether GFI1 contributes to or mediates this activity of MYB. To this end, we superinfected U937/MYB cells with lentiviruses-expressing shRNAs targeting GFI1 or a non-targeting control shRNA (shNT). Two different GFI1 shRNAs (shGFI1-B and -E) each knocked down GFI1 protein efficiently in the absence or presence of PMA (Figure 4a; compare the respective ‘PMA −’ and ‘PMA +’ lanes). We then asked whether knockdown of GFI1 in U937/MYB cells would abolish MYB’s ability to suppress PMA-induced differentiation. Indeed, GFI1 knockdown in MYB-overexpressing U937 cells restored the upregulation of CD11b and CD14 expression induced by PMA treatment (Figures 4b and c). Consistently, PMA treatment induced adherence (to tissue culture plastic), which is characteristic of monocytic differentiation, of the parental U937 cells but not of U937/MYB cells, whereas induced adherence was restored in the shGFI1 cells (Supplementary Figure S4). Furthermore, GFI1-knockdown U937/MYB cells displayed morphology similar to mature macrophages when treated with PMA, closely resembling the parental U937 cells, whereas the U937/MYB/shNT cells largely retained an undifferentiated morphology (Figure 4d).

Figure 4
figure4

MYB suppresses monocytic differentiation of U937 cells by activating GFI1 expression. U937/MYB cells were additionally transduced with shNT (control), shGFI1-B or shGFI1-E GIPZ lentiviruses (Open Biosystems/Thermo Scientific, Scoresby, VIC, Australia) generated with the Trans-Lentiviral Packaging System constructs form the same manufacturer. Transduction was as described above, following which cells were selected with puromycin (2 μg/ml; Sigma-Aldrich, Sydney, NSW, Australia), expanded and then treated with either vehicle control or PMA for 4 days. Parental U937 cells are also shown as a control. (a) Western blot showing efficient knockdown of GFI1 protein by shGFI1-B or shGFI1-E. Western blotting was performed essentially as previously described,20 using Myb 1-1 antibody (05-175, Millipore), GFI1 antibody (N-20, Santa Cruz, Carlsbad, CA, USA), or β-tubulin antibody (T0198, Sigma-Aldrich) in 5% skim milk/TBS-T and were subsequently developed by using Westpico chemiluminescent substrate (Thermo Scientific). (b and c) Knockdown of GFI1 largely restored PMA-induced upregulation of CD11b and CD14 as determined by flow cytomtery. (d) May-Grunwald Giemsa staining of cytocentrifuged vehicle- and PMA-treated U937/MYB cells carrying control shNT, shGFI1-B or shGFI1-E lentiviruses and parental U937 cells as indicated. Data are presented as mean±s.e. ***P<0.001, Student’s t test. Scale bar=20 μm.

In this report, we have demonstrated that in an AML cell line, MYB blocks PMA-induced differentiation largely by activating its target gene GFI1. GFI1 may subsequently suppress monocytic differentiation by binding to and antagonizing PU.1,35 as well as by directly repressing the genes encoding monocytic cytokine colony-stimulating factor 1 (CSF1) and its receptor (CSF1 and CSF1R, respectively;39 see also40 for review). It is very likely that MYB utilizes this mechanism to block differentiation in other systems. For instance, it has been shown that, in another AML cell line THP-1, siRNA-mediated MYB knockdown resulted in a significant decrease of GFI1, and GFI1 knockdown induced pro-differentiative changes largely shared by MYB knockdown.25 These findings corroborate much of the current study. Note that these two AML cell lines bear different oncogenic mutations, being CALM/AF10 in U937 cells41 and possibly MLL/MLL-T3 in THP-1 cells,42 suggesting that GFI1 may constitute a common effector used by MYB to block differentiation, at least in a subset of AML of the monocyte–macrophage lineage. Nevertheless, GFI1 is unlikely to account for MYB’s differentiation-blocking ability in all myeloid leukemia, as it may have roles other than differentiation-blocking in other lineages (see below). For example, MYB also suppresses granulocytic differentiation (and that of other hematopoietic lineages), implying that target genes other than GFI1 contribute to MYB’s differentiation-suppressing capacity; these may include the MYB-repressed-positive regulators of myeloid differentiation identified in our previous study,17 such as RUNX1 and PU.1.

In agreement with its involvement in repressing the monocytic differentiation in AML cell lines, GFI1 has been shown to be highly expressed in some cases of chronic myeloid leukemia (CML) and AML.43, 44, 45 Furthermore, Gfi1 was shown to be strongly upregulated during murine leukemogenesis in mouse models driven by two different human AML oncogenes.46 It is worth noting that GFI1 may have different roles in different subsets of leukemia. For instance, we found that GFI1 levels remain unchanged during retinoic acid-induced granulocytic differentiation of HL-60, a promyelocytic leukemia cell line (data not shown). This is consistent with GFI1’s essential role in granulocytic differentiation.33 It was also reported that ectopic GFI1 expression inhibited the proliferation and colony-forming ability of BCR/ABL-expressing CML cells but did not induce granulocytic differentiation.47 Even within AML, GFI1 may have different roles depending on the driving events, as indicated by the increased susceptibility of Gfi1−/− mice to develop AML in the presence of activated K-Ras.48 Finally, we note that Gfi1 was identified and is frequently found as a retroviral integration site in rodent T-lymphoid malignancies,49, 50 and very recently has been reported to be essential for the maintenance of human T cell-acute lymphocytic leukemia (T-ALL).51 Given that MYB activation can also induce lymphoid tumors, including T-ALL in humans,8, 9 it is tempting to speculate that upregulation of GFI1 might contribute to MYB’s ability to transform cells of this lineage also.

Although we have shown here and previously that in mammals MYB positively regulates GFI1, knockdown of zebrafish gfi1aa (also known as gfi1.1), the ortholog of mammalian GFI1, has been reported to decrease c-myb levels.52 Presumably such regulation is indirect as GFI proteins all function primarily as transcriptional repressors. This study does not contradict our findings as regulation of gfi1aa by c-myb was not studied in that report. Nevertheless, it would be interesting to investigate whether such regulation is conserved in mammals; if so, GFI1 and MYB may potentially form a positive feedback loop to enhance their own and each other’s expression at certain stages or cell types in mammalian hematopoiesis or leukemogenesis.

MYB is a master regulator of the entire hematopoietic system (reviewed in Ramsay et al4), whereas GFI1 has roles primarily in HSCs and the myelomonocytic lineage.29, 30, 31, 32, 33 The downregulation of MYB expression is a prerequisite for terminal differentiation in multiple lineages, including the monocytic lineage. In the current study, we show that GFI1 accounts for MYB’s ability to block monocytic differentiation in the context of an AML model. This mechanism may also hold true in normal monopoiesis. GFI1 has been proposed to have a dual role in myeloid development: it not only allows terminal maturation of granulocytes but also prevents differentiation of monocytes and macrophages.33 The requirement of GFI1 in granulocytic differentiation has been well established: (1) Gfi1−/− mice develop severe neutropenia33 and (2) reintroduction of Gfi1 in ex vivo cultured Gfi1−/− cells restores G-CSF-induced granulocytic differentiation.33 We suggest that in monopoiesis, downregulation of MYB may trigger the downregulation of GFI1, which in turn allows the terminal differentiation of monocytes and macrophages. In agreement with this hypothesis, only GFI1lo myeloid progenitors differentiate to monocytes, whereas GFI1hi myeloid progenitors differentiate to granulocytes.53

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Acknowledgements

This work was supported in part by a grant from the National Health and Medical Research Council of Australia (to TJG) and a University of Queensland Postdoctoral Fellowship (to PY).

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Correspondence to T J Gonda.

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Supplementary Information accompanies this paper on the Oncogene website

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Keywords

  • U937
  • MYB
  • GFI1
  • monocytic differentiation
  • target gene

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