Critical role of miR-9 in myelopoiesis and EVI1-induced leukemogenesis.

MicroRNA-9 (miR-9) is emerging as a critical regulator of organ development and neurogenesis. It is also deregulated in several types of solid tumors; however, its role in hematopoiesis and leukemogenesis is not yet known. Here we show that miR-9 is detected in hematopoietic stem cells and hematopoietic progenitor cells, and that its expression increases during hematopoietic differentiation. Ectopic expression of miR-9 strongly accelerates terminal myelopoiesis and promotes apoptosis in vitro and in vivo. Conversely, in hematopoietic progenitor cells, the inhibition of miR-9 with a miRNA sponge blocks myelopoiesis. Ecotropic viral integration site 1 (EVI1), required for normal embryogenesis, is considered an oncogene because its inappropriate up-regulation induces malignant transformation in solid and hematopoietic cancers. Here we show that EVI1 binds to the promoter of miR-9-3, leading to DNA hypermethylation of the promoter and repression of miR-9. Moreover, miR-9 expression reverses a myeloid differentiation block that is induced by EVI1. Our findings indicate that EVI1, when inappropriately expressed, delays or blocks myeloid differentiation at least in part by DNA hypermethylation and down-regulation of miR-9. It was reported that Forkhead box class O genes (FoxOs) inhibit myeloid differentiation and prevent differentiation of leukemia-initiating cells. Here we identify both FoxO1 and FoxO3 as direct targets of miR-9 in hematopoietic cells and find that up-regulation of FoxO3 inhibits miR-9-induced myelopoiesis. These results reveal a unique role of miR-9 in myelopoiesis and in the pathogenesis of EVI1-induced myeloid neoplasms and provide insights into the epigenetic regulation of miR9 in tumorigenesis.

Activation of Wnt/ß-catenin protein signaling induces mitochondria-mediated apoptosis in hematopoietic progenitor cells.

The canonical Wnt/ß-catenin signaling is activated during development, tumorigenesis, and in adult homeostasis, yet its role in maintenance of hematopoietic stem/progenitor cells is not firmly established. Here, we demonstrate that conditional expression of an active form of ß-catenin in vivo induces a marked increase in the frequency of apoptosis in hematopoietic stem/progenitor cells (HSCs/HPCs). Activation of Wnt/ß-catenin signaling in HPCs in vitro elevates the activity of caspases 3 and 9 and leads to a loss of mitochondrial membrane potential (ΔΨ(m)), indicating that it induces the intrinsic mitochondrial apoptotic pathway. In vivo, expression of activated ß-catenin in HPCs is associated with down-regulation of Bcl2 and expression of Casp3. Bone marrow transplantation assays reveal that enhanced cell survival by a Bcl2 transgene re-establishes the reconstitution capacity of HSCs/HPCs that express activated ß-catenin. In addition, a Bcl2 transgene prevents exhaustion of these HSCs/HPCs in vivo. Our data suggest that activation of the Wnt/ß-catenin pathway contributes to the defective function of HPCs in part by deregulating their survival.

RUNX1 regulates corepressor interactions of PU.1.

The transcription factor (TF) RUNX1 cooperates with lineage-specifying TFs (eg, PU.1/SPI1) to activate myeloid differentiation genes, such as macrophage and granulocyte macrophage colony-stimulating factor receptors (MCSFR and GMCSFR). Disruption of cooperative gene activation could contribute to aberrant repression of differentiation genes and leukemogenesis initiated by mutations and translocations of RUNX1. To investigate the mechanisms underlying cooperative gene activation, the effects of Runx1 deficiency were examined in an in vitro model of Pu.1-driven macrophage differentiation and in primary cells. Runx1 deficiency decreased Pu.1-mediated activation of Mcsfr and Gmcsfr, accompanied by decreased histone acetylation at the Mcsfr and Gmcsfr promoters, and increased endogenous corepressor (Eto2, Sin3A, and Hdac2) coimmunoprecipitation with Pu.1. In cotransfection experiments, corepressors were excluded from a multiprotein complex containing full-length RUNX1 and PU.1. However, corepressors interacted with PU.1 if wild-type RUNX1 was replaced with truncated variants associated with leukemia. Histone deacetylase (HDAC) enzyme activity is a major component of corepressor function. HDAC inhibition using suberoylanilide hydroxamic acid or MS-275 significantly increased MCSFR and GMCSFR expression in leukemia cell lines that express PU.1 and mutated or translocated RUNX1. RUNX1 deficiency is associated with persistent corepressor interaction with PU.1. Thus, inhibiting HDAC can partly compensate for the functional consequences of RUNX1 deficiency.

Methylation and silencing of miRNA-124 by EVI1 and self-renewal exhaustion of hematopoietic stem cells in murine myelodysplastic syndrome.

By expressing EVI1 in murine bone marrow (BM), we previously described a myelodysplastic syndrome (MDS) model characterized by pancytopenia, dysmegakaryopoiesis, dyserythropoiesis, and BM failure. The mice invariably died 11-14 months after BM transplantation (BMT). Here, we show that a double point mutant EVI1-(1+6Mut), unable to bind Gata1, abrogates the onset of MDS in the mouse and re-establishes normal megakaryopoiesis, erythropoiesis, BM function, and peripheral blood profiles. These normal features were maintained in the reconstituted mice until the study was ended at 21 months after BMT. We also report that EVI1 deregulates several genes that control cell division and cell self-renewal. In striking contrast, these genes are normalized in the presence of the EVI1 mutant. Moreover, EVI1, but not the EVI1 mutant, seemingly deregulates these cellular processes by altering miRNA expression. In particular, the silencing of miRNA-124 by DNA methylation is associated with EVI1 expression, but not that of the EVI1 mutant, and appears to play a key role in the up-regulation of cell division in murine BM cells and in the hematopoietic cell line 32Dcl3. The results presented here demonstrate that EVI1 induces MDS in the mouse through two major pathways, both of which require the interaction of EVI1 with other factors: one, results from EVI1-Gata1 interaction, which deregulates erythropoiesis and leads to fatal anemia, whereas the other occurs by interaction of EVI1 with unidentified factors causing perturbation of the cell cycle and self-renewal, as a consequence of silencing miRNA-124 by EVI1 and, ultimately, ensues in BM failure.

Preferential nuclear accumulation of JAK2V617F in CD34+ but not in granulocytic, megakaryocytic, or erythroid cells of patients with Philadelphia-negative myeloproliferative neoplasia.

Recently, Dawson et al identified a previously unrecognized nuclear role of JAK2 in the phosphorylation of histone H3 in hematopoietic cell lines. We searched nuclear JAK2 in total bone marrow (BM) cells and in 4 sorted BM cell populations (CD34(+), CD15(+), CD41(+), and CD71(+)) of 10 myeloproliferative neoplasia (MPN) patients with JAK2V617F mutation and 5 patients with wild-type JAK2 MPN. Confocal immunofluorescent images and Western blot analyses of nuclear and cytoplasmic fractions found nuclear JAK2 in CD34(+) cells of 10 of 10 JAK2-mutated patients but not in patients with wild-type JAK2. JAK2 was predominantly in the cytoplasmic fraction of differentiated granulocytic, megakaryocytic, or erythroid cells obtained from all patients. JAK2V617F up-regulates LMO2 in K562 and in JAK2V617F-positive CD34(+) cells. The selective JAK2 inhibitor AG490 normalizes the LMO2 levels in V617F-positive K562 and restores the cyto-plasmic localization of JAK2.

Point mutations in two EVI1 Zn fingers abolish EVI1-GATA1 interaction and allow erythroid differentiation of murine bone marrow cells.

EVI1 is an aggressive nuclear oncoprotein deregulated by recurring chromosomal abnormalities in myelodysplastic syndrome (MDS). The expression of the corresponding gene is a very poor prognostic marker for MDS patients and is associated with severe defects of the erythroid lineage. We have recently shown that the constitutive expression of EVI1 in murine bone marrow results in a fatal disease with features characteristic of MDS, including anemia, dyserythropoiesis, and dysmegakaryopoiesis. These lineages are regulated by the DNA-binding transcription factor GATA1. EVI1 has two zinc finger domains containing seven motifs at the N terminus and three motifs at the C terminus. Supported by results of assays utilizing synthetic DNA promoters, it was earlier proposed that erythroid-lineage repression by EVI1 is based on the ability of this protein to compete with GATA1 for DNA-binding sites, resulting in repression of gene activation by GATA1. Here, however, we show that EVI1 is unable to bind to classic GATA1 sites. To understand the mechanism utilized by EVI1 to repress erythropoiesis, we used a combination of biochemical assays, mutation analyses, and in vitro bone marrow differentiation. The results indicate that EVI1 interacts directly with the GATA1 protein rather than the DNA sequence. We further show that this protein-protein interaction blocks efficient recognition or binding to DNA by GATA1. Point mutations that disrupt the geometry of two zinc fingers of EVI1 abolish the protein-protein interaction, leading to normal erythroid differentiation of normal murine bone marrow in vitro.

Repression of RUNX1 activity by EVI1: a new role of EVI1 in leukemogenesis.

Recurring chromosomal translocations observed in human leukemia often result in the expression of fusion proteins that are DNA-binding transcription factors. These altered proteins acquire new dimerization properties that result in the assembly of inappropriate multimeric transcription complexes that deregulate hematopoietic programs and induce leukemogenesis. Recently, we reported that the fusion protein AML1/MDS1/EVI1 (AME), a product of a t(3;21)(q26;q22) associated with chronic myelogenous leukemia and acute myelogenous leukemia, displays a complex pattern of self-interaction. Here, we show that the 8th zinc finger motif of MDS1/EVI1 is an oligomerization domain involved not only in interaction of AME with itself but also in interactions with the parental proteins, RUNX1 and MDS1/EVI1, from which AME is generated. Because the 8th zinc finger motif is also present in the oncoprotein EVI1, we have evaluated the effects of the interaction between RUNX1 and EVI1 in vitro and in vivo. We found that in vitro, this interaction alters the ability of RUNX1 to bind to DNA and to regulate a reporter gene, whereas in vivo, the expression of the isolated 8th zinc finger motif of EVI1 is sufficient to block the granulocyte colony-stimulating factor-induced differentiation of 32Dcl3 cells, leading to cell death. As EVI1 is not detected in normal bone marrow cells, these data suggest that its inappropriate expression could contribute to hematopoietic transformation in part by a new mechanism that involves EVI1 association with key hematopoietic regulators, leading to their functional impairment.

EVI1 recruits the histone methyltransferase SUV39H1 for transcription repression.

EVI1 is an oncoprotein inappropriately expressed in acute myeloid leukemia and myelodysplastic syndrome cells. In vitro studies indicate that diverse biological properties can be attributed to this protein. Its role in leukemogenesis is still unclear but it is thought that overall EVI1 can act mostly as a transcription repressor through its interaction with a subset of histone deacetylases. Studies with histone deacetylase inhibitors have however indicated that EVI1-mediated repression can be only partially rescued by deacetylase inhibitor drugs, suggesting that additional chromosomal modifications might occur to induce gene repression by EVI1. To investigate whether histone methylation contributes to the repressive potential of EVI1, we examined a potential association between EVI1, the histone methyltransferase (HMT) SUV39H1, and methyltransferase activity in vitro. We find that EVI1 directly interacts with SUV39H1 and that the proteins form an active complex with methyltransferase activity in vitro. Our data indicate that SUV39H1 enhances the transcription repressive potential of EVI1 in vivo. We suggest that EVI1 affects promoters' activity in two different pathways, by association with histone deacetylases and by recruiting chromatin-modifying enzymes to impose a heterochromatin-like structure establishing a lasting transcription repression.

EVI1 induces myelodysplastic syndrome in mice.

Myelodysplasia is a hematological disease in which genomic abnormalities accumulate in a hematopoietic stem cell leading to severe pancytopenia, multilineage differentiation impairment, and bone marrow (BM) apoptosis. Mortality in the disease results from pancytopenia or transformation to acute myeloid leukemia. There are frequent cytogenetic abnormalities, including deletions of chromosomes 5, 7, or both. Recurring chromosomal translocations in myelodysplasia are rare, but the most frequent are the t(3;3)(q21;q26) and the inv(3)(q21q26), which lead to the inappropriate activation of the EVI1 gene located at 3q26. To better understand the role of EVI1 in this disease, we have generated a murine model of EVI1-positive myelodysplasia by BM infection and transplantation. We find that EVI1 induces a fatal disease of several stages that is characterized by severe pancytopenia. The disease does not progress to acute myeloid leukemia. Comparison of in vitro and in vivo results suggests that EVI1 acts at two levels. The immediate effects of EVI1 are hyperproliferation of BM cells and downregulation of EpoR and c-Mpl, which are important for terminal erythroid differentiation and platelet formation. These defects are not fatal, and the mice survive for about 10 months with compensated hematopoiesis. Over this time, compensation fails, and the mice succumb to fatal peripheral cytopenia.

The distal zinc finger domain of AML1/MDS1/EVI1 is an oligomerization domain involved in induction of hematopoietic differentiation defects in primary cells in vitro.

AML1/MDS1/EVI1 (AME) is a chimeric transcription factor produced by the (3;21)(q26;q22) translocation. This chromosomal translocation is associated with de novo and therapy-related acute myeloid leukemia and with the blast crisis of chronic myelogenous leukemia. AME is obtained by in-frame fusion of the AML1 and MDS1/EVI1 (ME) genes. The mechanisms by which AME induces a neoplastic transformation in bone marrow cells are unknown. AME interacts with the corepressors CtBP and HDAC1, and it was shown that AME is a repressor in contrast to the parent transcription factors AML1 and ME, which are transcription activators. Studies with murine bone marrow progenitors indicated that the introduction of a point mutation that destroys the CtBP-binding consensus impairs but does not abolish the disruption of cell differentiation and replication associated with AME expression, suggesting that additional events are required. Several chimeric proteins, such as AML1/ETO, BCR/ABL, and PML/RARa, are characterized by the presence of a self-interaction domain critical for transformation. We report that AME is also able to oligomerize and displays a complex pattern of self-interaction that involves at least three oligomerization regions, one of which is the distal zinc finger domain. Although the deletion of this short domain does not preclude the self-interaction of AME, it significantly reduces the differentiation defects caused in vitro by AME in primary murine bone marrow progenitors. The addition of a point mutation that inhibits CtBP binding completely abrogates the effects of AME on differentiation, suggesting that AME induces hematopoietic differentiation defects through at least two separate but cooperating pathways.

Combinatorial action of RUNX1 and PU.1 in the regulation of hematopoiesis.

The hematopoietic stem cell (HSC) has the potential to differentiate into mature cells with distinct phenotypes and functions. As suggested in recent reports, this plasticity can expand to include nonhematopoietic lineages, and, indeed, the HSC may repopulate liver and muscle tissues, as well. Considering the flexibility in HSC differentiation, these processes are regulated by a relatively small number of factors, some of which are expressed in all lineages, whereas others are activated only in a specific cell type. Combined evidence from many studies suggests that alternative subsets of these factors work in a combinatorial manner to regulate specific promoters for the induction of a specific lineage. RUNX1 and PU.1 have a fundamental role in HSC differentiation in that multifactor complexes are assembled around these proteins leading to tissue-specific and synergistic gene activation. Here we describe the relationship of RUNX1 with PU.1 as a facet of the combinatorial relationships that determine hematopoietic lineage commitment.

EVI1 and hematopoietic disorders: history and perspectives.

The ecotropic viral integration site 1 (EVI1) gene was identified almost 20 years ago as the integration site of an ecotropic retrovirus leading to murine myeloid leukemia. Since its identification, EVI1 has slowly been recognized as one of the most aggressive oncogenes associated with human leukemia. Despite the effort of many investigators, still very little is known about this gene. The mechanism by which EVI1 operates in the transformation of hematopoietic cells is not known, but it is clear that EVI1 upregulates cell proliferation, impairs cell differentiation, and induces cell transformation. In this review, we summarize the biochemical properties of EVI1 and the effects of EVI1 in biological models.

SUV39H1 interacts with AML1 and abrogates AML1 transactivity. AML1 is methylated in vivo.

Acute myeloid leukemia 1 (AML1) belongs to a family of DNA-binding proteins highly conserved through evolution. AML1 regulates the expression of several hematopoietic genes and is essential for murine fetal liver hematopoiesis. We report here that the histone methyltransferase SUV39H1, a mammalian ortholog of the Drosophila melanogaster SU(VAR) 3-9, forms complex with AML1. SUV39H1 methylates lysine 9 of the histone protein H3 leading to the formation of the high-affinity binding site on chromatin for proteins of the heterochromatin protein 1 family (HP1). The interaction of AML1 with SUV39H1 requires the N-terminus of AML1 where the Runt domain is located. Binding of AML1 to SUV39H1 abrogates the transactivating and DNA-binding properties of AML1 and dissociates the net-like nuclear structure of AML1. It has been reported that AML1 is capable of interaction with histone acetyl transferases (CBP, p300, and MOZ) and with component of the histone deacetylase complex (Sin3), and that the interaction with these coregulators affects the strength of AML1 in promoter regulation. Our data suggest that other enzymes are also involved in gene regulation by AML1 activity by modulating the affinity of AML1 for DNA.

The leukemia-associated transcription repressor AML1/MDS1/EVI1 requires CtBP to induce abnormal growth and differentiation of murine hematopoietic cells.

The leukemia-associated fusion gene AML1/MDS1/EVI1 (AME) encodes a chimeric transcription factor that results from the (3;21)(q26;q22) translocation. This translocation is observed in patients with therapy-related myelodysplastic syndrome (MDS), with chronic myelogenous leukemia during the blast crisis (CML-BC), and with de novo or therapy-related acute myeloid leukemia (AML). AME is obtained by in-frame fusion of the AML1 and MDS1/EVI1 genes. We have previously shown that AME is a transcriptional repressor that induces leukemia in mice. In order to elucidate the role of AME in leukemic transformation, we investigated the interaction of AME with the transcription co-regulator CtBP1 and with members of the histone deacetylase (HDAC) family. In this report, we show that AME physically interacts in vivo with CtBP1 and HDAC1 and that these co-repressors require distinct regions of AME for interaction. By using reporter gene assays, we demonstrate that AME represses gene transcription by CtBP1-dependent and CtBP1-independent mechanisms. Finally, we show that the interaction between AME and CtBP1 is biologically important and is necessary for growth upregulation and abnormal differentiation of the murine hematopoietic precursor cell line 32Dc13 and of murine bone marrow progenitors.

Targeted therapies in myelodysplastic syndromes: ASH 2003 review.

The myelodysplastic syndromes (MDS) continue to pose conceptual and practical conundrums because of their heterogeneity and therapeutic challenges. They are not restricted to the presence of clonal cells that are prone to excessive proliferation and premature apoptosis. In MDS the bone marrow microenvironment also is abnormal and exhibits an excess of proinflammatory cytokines, especially tumor necrosis factor (TNF), neoangiogenesis, and poorly defined immune defects. Thalidomide, a drug with anti-TNF, antiangiogenic, and immunomodulatory activities, and other agents with anti-TNF effects, such as pentoxifylline, etanercept, and infliximab, have produced hematologic improvement in 20% to 40% of patients. These agents may provide effective therapy for a subset of lower-risk MDS patients, even if the drugs target the bone marrow microenvironment predominantly. However, in higher-risk MDS patients, especially those with more than 10% blasts, it is important to eliminate abnormal cell clones; drugs used for this purpose have included arsenic trioxide, topotecan, the farnesyl transferase inhibitor tipifarnib, and demethylating agents, such as 5-azacytidine and decitabine. To increase the therapeutic index, a combination strategy may be preferable for higher-risk MDS patients, in whom the seed (clone) and the soil (bone marrow microenvironment) must be targeted simultaneously. The challenge is to recognize the subset that is likely to respond to a given drug so that patients can be preselected for therapy.

Arsenic trioxide and thalidomide combination produces multi-lineage hematological responses in myelodysplastic syndromes patients, particularly in those with high pre-therapy EVI1 expression.

Twenty-eight myelodysplastic syndromes (MDS) patients were treated with arsenic trioxide (ATO) and thalidomide. Seven patients responded including one complete hematologic and cytogenetic response and one with regression in spleen size. Two trilineage responses were seen in patients with inv(3)(q21q26.2). Three of five patients who had high pre-therapy EVI1 levels showed unexpectedly good responses while two died early in the first cycle. In vitro studies using 32Dcl3 cells forced to express EVI1 confirmed increased sensitivity of these cells to ATO. Both low/high risk MDS may benefit significantly from therapy with ATO/thalidomide, and those with high pre-therapy EVI1 expression may be uniquely sensitive.

Association of 3q21q26 syndrome with different RPN1/EVI1 fusion transcripts.

BACKGROUND AND OBJECTIVES: Patients with acute myeloblastic leukemia (AML) with features of myelodysplastic syndrome and abnormalities of megakaryocytopoiesis often have cytogenetic aberrations of 3q21 and 3q26 bands involving the paracentric inversion [inv(3) (q21q26)] or a reciprocal translocation [t(3;3) (q21;q26)]. These abnormalities frequently cause inappropriate expression of the EVI1 gene located at 3q26. Other genes that have been implicated at the rearrangement breakpoint are GR6 and RPN1 (both on 3q21). The aim of this study was to investigate the expression of the EVI1 fusion genes in AML patients with 3q21q26 syndrome. DESIGN AND METHODS: We used reverse transcription polymerase chain reaction to evaluate the expression of EVI1 and GR6, and particularly of the fusion genes RPN1-EVI1 and GR6-EVI1 in 9 AML patients with either inv(3)(q21q26) (7 cases) or t(3;3)(q21;q26) (2 cases). RESULTS: EVI1 and GR6 were always expressed, as was RPN1-EVI1; GR6-EVI1 was absent. In 8/9 patients, the part of EVI1 retained in RPN1-DEVI1 contained blocks B and C of the PR domain commonly found in the MDS1-EVI1 gene. In the remaining patient [with inv(3) (q21q26)], only block C was retained: we named this variant fusion gene RPN1-DEVI1. This patient lacked the micromegakaryocytopoiesis frequently found in 3q21q26 syndrome. INTERPRETATION AND CONCLUSIONS: These findings support the hypothesis that EVI1 activation plays a dominant role in the pathogenesis of the 3q21q26 syndrome. EVI1 expression might occur either as a consequence of rearrangements leading to the formation of different fusion transcripts, such as RPN1-EVI1 and RPN1-DEVI1 or following disruption of the PR activation domain of the MDS1-EVI1 gene.

A new translocation that rearranges the AML1 gene in a patient with T-cell acute lymphoblastic leukemia.

The AML1 gene (also known as RUNX1 or CBFA2), located in chromosome band 21q22, encodes a transcription factor which heterodimerizes with the CBFbeta protein forming a complex called human core binding factor (CBF). The CBF complex appears to regulate a number of genes important for hematopoiesis. AML1 is one of the most common targets of chromosomal rearrangements in human leukemias and has been involved in 14 chromosomal translocations to date. Here we report a new chromosomal translocation, t(4;21)(q31;q22) that disrupts the AML1 gene in a 12-year-old boy with newly diagnosed T-cell acute lymphoblastic leukemia (ALL). This is the first reported chromosomal translocation where AML1 is rearranged in childhood T-cell ALL. By metaphase fluorescence in situ hybridization analysis, the AML1 breakpoint was mapped using recombinant phage clones, and shown to be either immediately upstream or downstream of exon 5.