Vitalyi Senyuk

The role of EVI1 in normal and leukemic cells

One of the genes associated with both murine and human myeloid leukemia is EVI1 (ecotropic viral integration 1 site). EVI1 was first identified as a common locus of retroviral integration in myeloid tumors found in AKXD mice. The exact mechanism by which EVI1 induces leukemogenesis is not clear. Studies of the function of EVI1 in the bone marrow and in cell lines have shown that the inappropriate expression of EVI1 prohibits terminal differentiation of the bone marrow progenitor cells in granulocytes and erythroid cells, but strongly favors hematopoietic differentiation along the megakaryocytic lineage. We summarize recent data showing that EVI1 is a complex transcription factor with multiple functions, and this complexity is further demonstrated by the ability of EVI1 to interact with coactivators and corepressors and to abrogate cellular response to cytokines.

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.

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.

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.

Activation of Wnt/β-Catenin Protein Signaling Induces Mitochondria-mediated Apoptosis in Hematopoietic Progenitor Cells

Background: Wnt/β-catenin signaling plays a strong role in maintaining homeostasis of hematopoietic progenitor cells (HPCs). Results: Activated β-catenin deregulates the survival of HPCs by induction of the mitochondrial apoptotic pathway. Conclusion: Wnt/β-catenin signaling is critical for HPC pool maintenance. Significance: This study clarifies the role of the Wnt/β-catenin signaling in HPCs by showing that it has negative impact on the function and survival of the cells. The canonical Wnt/β-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 β-catenin in vivo induces a marked increase in the frequency of apoptosis in hematopoietic stem/progenitor cells (HSCs/HPCs). Activation of Wnt/β-catenin signaling in HPCs in vitro elevates the activity of caspases 3 and 9 and leads to a loss of mitochondrial membrane potential (ΔΨm), indicating that it induces the intrinsic mitochondrial apoptotic pathway. In vivo, expression of activated β-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 β-catenin. In addition, a Bcl2 transgene prevents exhaustion of these HSCs/HPCs in vivo. Our data suggest that activation of the Wnt/β-catenin pathway contributes to the defective function of HPCs in part by deregulating their survival.

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.

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 Promotes Cell Proliferation by Interacting with BRG1 and Blocking the Repression of BRG1 on E2F1 Activity

EVI1 is a complex protein required for embryogenesis and inappropriately expressed in many types of human myeloid leukemia. Earlier we showed that the forced expression of EVI1 in murine hematopoietic precursor cells leads to their abnormal differentiation and increased proliferation. In this report, we show that EVI1 physically interacts with BRG1 and its functional homolog BRM in mammalian cells. We found that the C terminus of EVI1 interacts strongly with BRG1 and that the central and C-terminal regions of BRG1 are involved in EVI1-BRG1 interaction. Using reporter gene assays, we demonstrate that EVI1 activates the E2F1 promoter in NIH3T3 cells but not in BRG1-negative SW13 cells. Ectopic expression of BRG1 is able to repress the E2F1 promoter in vector-transfected SW13 cells but not in EVI1-transfected SW13 cells. Finally, we show that EVI1 up-regulates cell proliferation in BRG1-positive 32Dcl3 cells but not in BRG1-negative SW13 cells. Taken together, these data support the hypothesis that the interaction with BRG1 is important for up-regulation of cell-growth by EVI1.

The oncoprotein EVI1 and the DNA methyltransferase Dnmt3 co-operate in binding and de novo methylation of target DNA.

EVI1 has pleiotropic functions during murine embryogenesis and its targeted disruption leads to prenatal death by severely affecting the development of virtually all embryonic organs. However, its functions in adult tissues are still unclear. When inappropriately expressed, EVI1 becomes one of the most aggressive oncogenes associated with human hematopoietic and solid cancers. The mechanisms by which EVI1 transforms normal cells are unknown, but we showed recently that EVI1 indirectly upregulates self-renewal and cell-cycling genes by inappropriate methylation of CpG dinucleotides in the regulatory regions of microRNA-124-3 (miR-124-3), leading to the repression of this small gene that controls normal differentiation and cell cycling of somatic cells. We used the regulatory regions of miR-124-3 as a read-out system to investigate how EVI1 induces de novo methylation of DNA. Here we show that EVI1 physically interacts with DNA methyltransferases 3a and 3b (Dnmt3a/b), which are the only de novo DNA methyltransferases identified to date in mouse and man, and that it forms an enzymatically active protein complex that induces de novo DNA methylation in vitro. This protein complex targets and binds to a precise region of miR-124-3 that is necessary for repression of a reporter gene by EVI1. Based on our findings, we propose that in cooperation with Dnmt3a/b EVI1 regulates the methylation of DNA as a sequence-specific mediator of de novo DNA methylation and that inappropriate EVI1 expression contributes to carcinogenesis through improper DNA methylation.

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.