The role of EVI1 in myeloid malignancies

YBCMD-01792; No. of pages: 10; 4C: Blood Cells, Molecules and Diseases xxx (2014) xxx–xxx

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Review

The role of EVI1 in myeloid malignancies Carolyn Glass, Michael Wilson, Ruby Gonzalez, Yi Zhang, Archibald S. Perkins ⁎ Department of Pathology and Laboratory Medicine, University of Rochester School of Medicine, Rochester, NY 14642, USA

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Article history: Submitted 20 December 2013 Available online xxxx (Communicated by M. Lichtman, M.D., 26 December 2013) Keywords: Leukemogenesis Oncogenes EVI1 Myeloid leukemia Apoptosis Differentiation

a b s t r a c t The EVI1 oncogene at human chr 3q26 is rearranged and/or overexpressed in a subset of acute myeloid leukemias and myelodysplasias. The EVI1 protein is a 135 kDa transcriptional regulator with DNA-binding zinc finger domains. Here we provide a critical review of the current state of research into the molecular mechanisms by which this gene plays a role in myeloid malignancies. The major pertinent cellular effects are blocking myeloid differentiation and preventing cellular apoptosis, and several potential mechanisms for these phenomena have been identified. Evidence supports a role for EVI1 in inducing cellular quiescence, and this may contribute to the resistance to chemotherapy seen in patients with neoplasms that overexpress EVI1. Another isoform, MDS1– EVI1 (or PRDM3), encoded by the same locus as EVI1, harbors an N-terminal histone methyltransferase(HMT) domain; experimental findings indicate that this protein and its HMT activity are critical for the progression of a subset of AMLs, and this provides a potential target for therapeutic intervention. © 2014 Published by Elsevier Inc.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Locus structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromosomal abnormalities associated with EVI1 in myeloid malignancies . . Mouse models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cellular effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Differentiation . . . . . . . . . . . . . . . . . . . . . . . Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . Role of MECOM in hematopoietic stem cell quiescence . . . . . Role of MECOM in leukemia cell quiescence . . . . . . . . . . Signaling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . cJun/AP1 . . . . . . . . . . . . . . . . . . . . . . . . . . TGF-β . . . . . . . . . . . . . . . . . . . . . . . . . . . Downstream target genes . . . . . . . . . . . . . . . . . . Epigenetic modification . . . . . . . . . . . . . . . . . . . . . . . . . Epigenetic modifications at the promoter of the Evi1 gene . . . . Recruitment of chromatin-modifying enzymes by the EVI1 protein MDS1–EVI1/PRDM3 as a chromatin modifying enzyme . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction The EVI1 gene was identified in 1988 as a site of proviral insertion in murine myeloid leukemias [1], and after 25 years of research and scores ⁎ Corresponding author at: Box 626, 601 Elmwood Avenue, Rochester, NY 14642, USA. E-mail address: [email protected] (A.S. Perkins).

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of publications, major questions still remain regarding its exact role in leukemogenesis. The goal of this review is to provide a critical assessment of what is known regarding its role in leukemogenesis, and what gaps of knowledge still exist. The ecotropic virus integration site 1 (EVI1) protein is an oncogenic transcription factor associated with human myeloid malignancies and several epithelial cancers [2–4]. Aberrant EVI1 expression occurs in 8–

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Please cite this article as: C. Glass, et al., The role of EVI1 in myeloid malignancies, Blood Cells Mol. Diseases (2014), http://dx.doi.org/10.1016/ j.bcmd.2014.01.002

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10% of human adult acute myeloid leukemia (AML) and, strikingly, up to 27% of pediatric mixed lineage leukemia (MLL) rearranged leukemias [5]. When inappropriately expressed, EVI1 is an independent risk factor for a very poor prognosis in hematopoietic cancer (reviewed by [6]; Fig. 1 [7]). Locus structure The confusion over the role of EVI1 in leukemia is the result in part to the complexity of the EVI1 genetic locus. It was realized six years after its discovery, that the locus harbors two distinct transcription start sites (TSS) located almost half a megabase apart [8]. The downstream TSS is termed EVI1, while the upstream one is referred to as MDS1 (Fig. 2). As a result of alternative splicing of mRNA transcripts emanating from the EVI1 TSS, three different EVI1 protein isoforms have been identified, having sizes of 135 kDa (also reported as 145 kDa), 123 kDa (also known as Δ105), and 103 kDa (also known as Δ324; Fig. 1). All of these contain zinc finger motifs: for the 135 kDa and 123 kDa isoforms, there are seven in the N-terminal domain (termed ZF1) and three in the C-terminal domain (ZF2); the 103 kDa isoform lacks fingers 6 and 7 of ZF1, which are critical for DNA binding [9]. Whereas all three of these isoforms are present in most murine leukemias, the 135 kDa isoform is the one most commonly found in human leukemias, and, is the most commonly studied. The MDS1 TSS appears to generate two protein isoforms: one ca. 30 kDa isoform of unknown function, and a 160 kDa form that is extended by 188 aa at the N-terminus relative to the EVI1 isoforms. Within this 188 aa region is a 110 aa “PR” domain with homology to the SET domain with histone methyltransferase activity [10]. The specific roles of the different isoforms are not well understood; the study of mutant mouse strains can give some indication of the necessity of the different isoforms for survival. Lack of the p160, p135 and p123 isoforms is lethal at 11 dpc [11], whereas lack of p160 alone is viable [12]; lack of p135, p123, and p103, but retention of p160, is perinatal lethal [13]. Despite these mouse models, studies have not been reported that provide information as to the roles of the specific isoforms. Chromosomal abnormalities associated with EVI1 in myeloid malignancies A number of inversions and translocations have been described in human AML and myelodysplastic syndrome (MDS) marrow cells involving chromosome 3q26, where the human gene is located. The most common EVI1 chromosomal rearrangements include inv3;3, t(3;3), t(3;12) and t(3;21). The inv(3;3) and t(3;3) result in EVI1 overexpression and involve a region 50 kb downstream of the ribophorin I gene (RPN1) at band 21 on chromosome 3. RPN1 encodes a transmembrane protein critical for ribosome binding on rough endoplasmic reticulum, and in these chromosomal rearrangements, transcription of EVI1 may be regulated by RPN1 enhancer elements [14]. The t(3;12)(q26;p13) results in a fusion protein involving ETV6 (also termed TEL), an ETS family transcription factor required for normal hematopoiesis. This rearrangement has been associated with acute transformation of MDS, blast crisis of chronic myeloid leukemia (CML) and a very poor prognosis [15]. The t(3;21)(q26;q22) translocation results in the RUNX1/ MDS1/EVI1 (AME) transcription factor fusion protein (RUNX1 also termed AML1), and has been identified during the blast phase of CML, in therapy-related cases of MDS and AML. Some studies support that AME blocks myeloid differentiation in cooperation with BCR–ABL, while other reports show AME suppresses CEBPA, the myeloid transcription factor critical for normal granulopoiesis [16].

Fig. 1. High EVI1 expression associates with poor survival outcome in AML. Kaplan Meier analysis of (A) overall survival (OS), (B) event-free survival (EFS), and (C) disease-free survival (DFS) shows an inferior outcome for EVI1 + patients in comparison with patients without EVI1 overexpression in a total cohort of 534 AML patients. Taken from Ref. [84].

Please cite this article as: C. Glass, et al., The role of EVI1 in myeloid malignancies, Blood Cells Mol. Diseases (2014), http://dx.doi.org/10.1016/ j.bcmd.2014.01.002

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Fig. 2. Protein isoforms encoded by the MECOM locus. A. Structure of the MECOM locus, showing the two starts of transcription and splicing pattern. Also shown are the sites of proviral insertion in murine tumors. B. Diagrams of the 160, 135, 123, and 103 kDa isoforms, with the zinc fingers designated as hatched. Also shown is the binding site for CtBP corepressor, and the acidic domain. C. Western blot analyses of EVI1 isoforms with N-terminus-specific antiserum. Lanes 1–4, 293T cells transfected with empty vector (lane 1), 103 kDa isoform (lane 2), 123 kDa isoform (lane 3) or 135 kDa isoform (lane 4). Lanes 6 and 7, extracts from DA-1 and FDCP-mix, respectively.

The rearrangements at 3q26 involving EVI1 can be detected by fluorescence in-situ hybridization using a combination of three bacterial artificial chromosome (BAC) probes that span the gene, a strategy worked out by several groups [17–19] (Fig. 3). Mouse models Mechanistic understanding of a gene's function in cancer is often advanced significantly by the availability of mouse models that closely mimic the human disease. For leukemogenic oncogenes, a standard approach is to transduce marrow-derived murine hematopoietic progenitors with a retrovirus bearing the oncogene of interest, and transplanting the transduced cells into irradiated recipient mice, which are then scored for development of leukemia. For EVI1, this approach has not proven straightforward, with different investigators reporting different results. Ren and Cuenco reported that transduction of the 135 kDa isoform of EVI1 into murine marrow cells and transplantation into irradiated recipients resulted in no disease [20]. Subsequent studies by Perkins, Spence, Jenkins, and Copeland found similar results (unpublished). Buonamici et al. used an MSCV retroviral system carrying the 135 kDa isoform of human EVI1, and transduced murine lineagenegative marrow cells, and transplanted these into irradiated recipients, with engraftment percentages of 75%. They documented expression of the epitope-tagged EVI1 by Western analysis of marrow cells from two moribund transplant recipients. They found that at 8–12 months

after transplant, recipients developed pancytopenia, particularly anemia and thrombocytopenia; spleen weights remained normal. Their analysis noted dyserythropoiesis, as characterized by irregular nuclear contours and nuclear budding. In the blood of the EVI1-positive transplanted mice, they noted anisopoikilocytosis, though no red cell distribution width value was given. None of the mice progressed to leukemia. They stated that apoptosis was the cause of the pancytopenia, but this explanation was not fully investigated [21], being based only on immunostaining of spleen for activated caspase 3. Colony formation studies were also performed, suggesting that EVI1 causes decreased responsiveness to the cytokines erythropoietin and granulomonocyte colony-stimulating factor (GM-CSF), though this finding was the subject of limited characterization. Flow cytometric analysis for lineage markers of marrow cells from 3 moribund and 2 age-matched control transplanted mice showed that there were comparable percentages for all lineages except for Ter119-positive cells, which showed 2-fold higher levels in EVI1-positive transplants, but data were only shown for one mouse. Their analysis of pre-anemic mice purportedly also showed increased erythroid progenitors by flow cytometry for Ter119, suggesting that this effect was not due to anemia. RNA analysis suggested lower levels of EpoR and cMpl in EVI1-transduced marrow. Yoshimi et al. [22] were able to generate AML in mice by retroviral transduction of EVI1 into 5-fluoruracil-treated marrow cells and subsequent transplantation into sub-lethally irradiated recipients. AML was defined by large numbers of myeloblast cells in the marrow (average

Please cite this article as: C. Glass, et al., The role of EVI1 in myeloid malignancies, Blood Cells Mol. Diseases (2014), http://dx.doi.org/10.1016/ j.bcmd.2014.01.002

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Fig. 3. Fluorescent in situ hybridization (FISH) of chromosome 3q26 and 3q21 loci reveal hidden 3q26 aberrations. BAC clone localization from centromere (Cen) to telomere (Tel) (A). A metaphase from EVI1+ patient no. 28 revealed a cryptic inv(3)(q21q26) (inv3) and a normal chromosome 3 (nor3) using EVI1 (RP11-82C9) and MDS1 (RP11-141C22) (B) and RPN1 (RP11-456 K4) BAC clones (C). Micrographs after FISH were acquired by imaging with a fluorescence microscope. Taken from Ref. [84].

of 55.8%), marked splenomegaly, and expression of c-kit and Mac-1 on green fluorescent protein (GFP)-positive leukemic cells. All EVI1 recipient mice succumbed to disease 6–11 months following transplantation [22]. Yoshimi et al. used the pMYs retroviral vector system [23] in their studies, whereas the previous reported marrow transplant models utilized the MSCV vector systems. The pMYs vector is derived from two modifications to the pMSCV vector: 1) replacement of the U3 region of the 5′ LTR of FMEV and MPEV with that of MMLV, and 2) a 3′ LTR consisting of U3 of MPSV and U5 of MMLV. These changes were made to increase the viral titer [23]. However, it is unclear if these modifications are responsible for the difference in experimental results reported using the two vector systems. In summary, mouse retroviral transduction experiments have yielded different results, likely due to experimental details such as vector and mouse strain employed, and perhaps on the type of cell that was transduced. It is likely that the oncogenic effects of EVI1 are context-dependent, with only certain cells of the hematopoietic hierarchy being susceptible. It is also likely that there are threshold effects, with only certain high levels of expression yielding disease. Also of note is that several investigators have found that EVI1 induces cellular quiescence in hematopoietic cells [24,25], thus providing a fundamental experimental barrier to oncogenic transformation. This finding also suggests that EVI1 needs a cooperating event in order to induce leukemia. Indeed, Jin et al. demonstrated that in retrovirallytransduced marrow cell recipient mice, EVI1 cooperates with MEIS1 and HOXA9 to accelerate leukemogenesis [26]. This combination may be relevant to human leukemogenesis, since MLL-fusion proteins, such as MLL-AF9 activate both EVI1 and HOX genes, and both of these have been shown to be essential for MLL-AF9-induced leukemogenesis (discussed below). Louz et al. [27] reported the creation of a set of transgenic mice bearing the Evi1 cDNA for the 135 kDa isoform under the control of the Sca-1 promoter. Three lines were established, and although no mice developed leukemia, two lines showed decreased numbers of colonyforming units-erythroid (CFU-E) from plated marrow cells; there was also shortened latency to retrovirally-induced leukemogenesis in

transgenic mice relative to non-transgenic. One of the lines showed perturbed hematopoiesis, sterility, and early demise in male mice only, but it is possible that these features were due to transgene disruption of an essential gene on the X chromosome rather than EVI1 expression [27]. Cellular effects Differentiation One of the earliest reported activities of EVI1 is its ability to interfere with the differentiation of myeloid cells and in this way it likely contributes to the leukemia phenotype. In 32Dcl3 cells, treatment with granulocyte-colony-stimulating factor (G-CSF) led to differentiation into mature granulocytes; transduction of 32Dcl3 cells with retrovirus encoding the EVI1 135 kDa isoform resulted in cells that failed to respond to G-CSF and undergo cell death [28]. While these data were clear and reproducible, subsequently it was shown that 32Dcl3 cells had a proviral insertion at the endogenous Evi1 locus and overexpressed multiple EVI1 isoforms [29]. It is likely that proviral activation of Evi1 in 32Dcl3 cells contributed to immortalized growth of the cell line, perhaps by preventing spontaneous differentiation. Although the cell line responded to G-CSF, Evi1 expression in this cell line does not completely abrogate responsiveness to cytokine-induced differentiation. The fact that further overexpression causes a loss of responsiveness to G-CSF indicates that a threshold exists for the biological effects of Evi1 in myeloid cells. Consistent with this effect on the differentiation potential, primary hematopoietic cells from mouse marrow transduced with a retrovirus encoding the 135 kDa EVI1 isoform showed decreased ability to form burst-forming units-erythroid (BFU-E) in semisolid medium [30]. Thus, an inhibitory effect on the erythroid differentiation potential of hematopoietic cells is a potential mechanism for the leukemogenic and/or myelodysplastic effects of Evi1. Louz et al. found that in transgenic mice overexpressing Evi1 under a Sca-1 promoter that one of three mouse lines showed disruption of erythropoiesis [27]. Laricchia-

Please cite this article as: C. Glass, et al., The role of EVI1 in myeloid malignancies, Blood Cells Mol. Diseases (2014), http://dx.doi.org/10.1016/ j.bcmd.2014.01.002

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Robbio et al., noting that EVI1 might downregulate the EpoR gene, hypothesized that EVI1 protein may interfere with this gene's expression via interaction with GATA1 protein. To this end, they performed a series of co-immunoprecipitation studies in overexpression systems that suggested an interaction. To determine the interaction domain on EVI1, they showed that a crippling of zinc fingers 1 and 6 (via mutations of critical cysteines and histidine: H39A/C44A/C190A/C193A) resulted in loss of this interaction. However, it is clear from other works that these mutations result in loss of DNA binding [9,31], which raises the possibility that the EVI1:GATA1 interaction is actually due to DNA bridging. This possibility of the “interaction” being due to bridging via DNA is consistent with the finding that the interaction domain on the GATA1 side is also its DNA binding domain. Many of the conclusions of the paper are based on the use of the H39A/C44A/C190A/C193A mutant, which cripples the zinc finger region, and likely prevents not only DNA binding but also binding to other proteins including SMADs [32,33], histone deacetylase [34], jun N-terminal kinase [35], and PU.1 [36]. In summary, it appears that EVI1 does have some effect on GATA1 function; how much of this is due to a protein:protein interaction between EVI1 and GATA1, and how much is due to competition of EVI1 for GATA1 at sites of DNA binding is not evident. Also what is not clear is how much this contributes to myeloid leukemogenesis or MDS in humans. Further work by this group [37] tested the ability of the H39A/C44A/ C190A/C193A mutant to induce myelodysplastic changes in transplanted recipient mice receiving bone marrow transduced with EVI1. While wt EVI1 induced dysplastic-like changes as described previously [21], not surprisingly, the H39A/C44A/C190A/C193A mutant, being defective in multiple types of macromolecular interactions, did not induce such changes. The study also looked into transcriptional and protein changes that accompany transduction of marrow cells with EVI1. These changes include upregulation of cyclin D3, increased phosphorylation of Rb protein, upregulation of Bmi, and downregulation of miRNA-124. It appears that miR-124 may have some functional effects, since adding it back to EVI1-overexpressing cells can reverse certain defects in differentiation, colony formation, and gene expression induced by EVI1 [37]. However, what these changes have to do with interaction with GATA1 is unclear; it is also not clear whether miR-124 constitutes a direct or indirect target of EVI1. Finally, whether these changes play a role in human myeloid malignancies has yet to be confirmed. EVI1 binds and deregulates a major terminal myeloid differentiation gene, Cebpε. CCAAT/enhancer binding protein-ε (C/EBP-ε) is a regulator of myeloid lineage differentiation and is critical for the terminal differentiation of granulocytes [38–40]. Seven significant EVI1 binding sites, two of which were within the promoter region, were identified for Cebpε, which was associated with a two-fold downregulation of Cebpe in two different Evi1-overexpressing leukemic cell lines. As a master regulator of terminal myeloid differentiation, C/EBP-ε binds and activates several downstream gene targets to produce mature granulocytes. We found that several C/EBP-ε downstream gene targets (Epx, Lcn2, Mmp8 and Prg2), were also significantly down-regulated in EVI1 leukemic cells. Koeffler et al. demonstrated that neutrophils of Cebpε knockout mice are blocked at the myelocyte and metamyelocyte stages. Clonogenic assays revealed a significant decrease in the number of myeloid colonies, and a significant increase in Lin−/Sca1+/c-Kit+ colonies [38]. Neutrophils with Cebpε knockout have bilobed nuclei, lack secondary granules and mRNA for secondary granule proteins, and exhibit aberrant chemotaxis [41]. EVI1 has also been shown to interact with PU.1, a master regulator of early hematopoiesis, and to disrupt PU.1's ability to activate a myeloid promoter; while EVI1 does not interfere with PU.1's binding to DNA, it appears to disrupt the interaction between PU.1 and c-jun, which is known to be necessary for PU.1 to act on certain promoters [36]. These findings suggest that EVI1 represses PU.1 by blocking interaction between PU.1 and c-Jun, and thereby blocks myeloid differentiation; however, the causality in this chain of events has yet to be proven.

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The relevance of these studies to myeloid malignancy and EVI1mediated transcriptional regulation remains unclear for two reasons: one ambiguity is that the mutations that disrupt interaction between EVI1 and PU.1 (C190A/C193A/C219A/C222A) also disrupt binding to DNA [9], GATA1 [42], and perhaps SMADs [32]; a second concern is whether the PU.1 interaction is involved in leukemogenesis: whereas the C190A/C193A/C219A/C222A mutant was assessed in the context of a colony formation assay, it was not assessed for myeloid transformation to AML or MDS. Apoptosis Failure of apoptosis is a possible mechanism of EVI1-induced malignant transformation, as demonstrated in several studies. In one of the earliest studies, Kurokawa et al. demonstrated that EVI1 physically interacts with and inhibits c-Jun N-terminal kinase (JNK) via the first zinc finger domain (ZF1-7) to confer resistance to stressinduced apoptosis [35]. JNK is a class of mitogen-activated protein (MAP) kinases associated with stress responses of cells. The effect of EVI1 on the activity of JNK1 was evaluated in a kinase assay where the phosphorylation of the JNK1 substrate, c-Jun (GST-cJun), was evaluated (SDS-PAGE). In this assay, Hemagglutinin (HA)-tagged JNK1 was expressed alone or with EVI1 in COS-7 cells, and cells exposed to UV prior to JNK1 immunoprecipitation. Phosphorylation of c-Jun (documented by SDS-PAGE and immunoblotting) showed that JNK1 lost the ability to phosphorylate c-Jun substrate in the presence of EVI1. This study also showed that EVI1 inhibits JNK independent of the type JNK activator. Exposing COS-7 cells to other JNK activators, such as, the protein synthesis inhibitor anisomycin, sorbitol, and TNFα, in the presence of both EVI1 and JNK, resulted in suppression of JNK activity (measured by its ability to phosphorylate GST-c-Jun). These effects were also seen in another experiment where Flag-tagged EVI1 was expressed in COS-7 cells, pulled down on protein G-Sepharose conjugated with anti-Flag antibody and eluted with Flag peptide. A titration of HA-JNK1 was done with the resulting precipitates. The activity of JNK was inhibited in a dose-dependent manner. Furthermore, EVI1 specifically targets JNK activity and not other MAP kinases activated by stress stimuli. While the inhibitory effect of EVI1 on JNK activity appears real, it remains to be demonstrated as to whether this contributes to leukemogenesis. DeWeer et al. demonstrated significant downregulation of the microRNA MIR449A in 38 EVI1 rearranged leukemia patient samples by miRNA expression profiling. MIR449A expression in EVI1-rearranged cell lines induced apoptosis and was associated with decreased cell viability [43]. Further studies showed that direct promoter occupation of the EVI1 is critical for MIR449A repression. These studies provided evidence that the EVI1–MIR449A–NOTCH1/BCL2 regulatory axis may serve as a potential regulator of apoptosis in EVI1-induced leukemia. Another study provides further evidence that EVI1 induces the expression of BCL-XL and inhibits apoptosis, specifically by binding via the first set of zinc fingers [44]. In this study, ChIP analysis was used to demonstrate that EVI1 directly binds to the BCL-XL promoter. However this was done in HT-29 cells, a colon carcinoma cell line which overexpresses EVI1, and not in leukemic cells. HT-29 cells treated with EVI1 siRNA showed a down-regulation of BCL-XL expression. EVI1positive chronic myeloid leukemia patient samples were also used and showed a significantly higher BCL-XL expression compared to EVI1 negative samples. Furthermore, this study demonstrated that acetylation (a post-translational modification) of the EVI1 protein prevents its binding to the promoter region of BCL-XL and induces apoptosis. The relevance of these findings to leukemogenesis is unknown. We also reported the first genome-wide study of EVI1 DNA binding sites in leukemic cells [45] and confirmed EVI binding to and deregulation of a select number of novel EVI target genes involved in apoptosis. We identified EVI1 binding and significant downregulation of several

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genes that encode for ligand gated ATP-dependent P2 purinoreceptors, specifically P2rx3, Prx4, and P2rx7 in EVI1 leukemic cells. Failure of apoptosis due to P2 purinoreceptor dysfunction has been implicated in previous studies [46,47]. P2RX7 is an extracellular ATP receptor involved in rapid cell death via calcium influx and is of particular interest due to its well established role in regulating apoptosis in macrophages [48–50]. P2RX7 activation has also been associated with increased caspase-1 and caspase-3 activity [49]. Humphreys et al. demonstrated that P2RX7 stimulation with ATP rapidly elevates caspase-3 protease activity associated with DNA fragmentation, and is also strongly linked to upregulation of the c-Jun N-terminal kinase pathway [51]. We found that EVI1 binds to 3 sites within the P2rx7 gene promoter region with significant reduction of P2rx7 transcription in AML. We also investigated the physiological effects of Evi1 knockdown in murine AML cells, and found a significant increase in apoptosis via the intrinsic pathway (a change in mitochondrial potential via activation of caspases 9 and 3) and not the extrinsic pathway (no activation of caspase 8). Apoptosis was characterized by DNA fragmentation, histone

release, and a reduction in mitochondrial membrane potential. Furthermore, procaspases 3 and 9, but not caspase 8 or Bid, was cleaved following EVI1 knockdown [52]. Role of MECOM in hematopoietic stem cell quiescence We previously reported in our Mds1–Evi1 KO mice that hematopoietic stem cells (HSC) have a higher rate of proliferation than HSC from WT mice. HSC quiescence was dysregulated, at least in part, through diminishing Cdkn1c, encoding p57, in KO mice, indicating that MECOM is essential for the maintenance of stem cell quiescence and survival [53]. To date, the mechanism by which MDS1–EVI1, which contains all of EVI1 plus the PR domain at the N-terminus (see Fig. 2B), regulates Cdkn1c transcription has not been resolved, nor has the importance of this pathway to leukemogenesis been elucidated. Similar effects have been seen with EVI1 isoforms that lack the PR domain. To assess the effect of Evi1 overexpression in HSCs, we generated mice in which EVI1 could be induced in the HSC compartment. In this

Fig. 4. Significant EVI1 DNA binding peaks. Analysis using the UCSC Genome Browser showed the ChIP-Seq EVI1 binding sites demonstrated 10.9% alignment with the whole mouse genome (approximately 5 million reads). 16,745 significant peaks, defined as a difference in number of reads between the control rabbit IgG and EVI1 antiserum antibody yielding a p-value b0.001, were identified based on a Poisson distribution. a) Of the 16,745 generated significant peaks (p b 0.001), 45.5% were within introns, 35.0% within distal intergenic region, and 7.1% were within the proximal (within 1 kb) of the TSS. b) A 500 bp DNA sequence extracted around each significant peak was matched to de novo consensus sequence discovery programs. The AGGAAG ETS-like motif was identified and refined in 88% of the significant EVI1 ChIP-Seq binding sites. Taken from Ref. [45].

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model, EVI1 is induced through expressing the reverse tetracyclineinducible transactivator (rtTA), which is under the transcriptional control of a Rosa26 promoter, allowing for constitutive and ubiquitous expression. In this model, the addition of doxycycline leads to a 10,000-fold upregulation of Evi1 transcripts, but no induction of Mds1–Evi1 transcript. Our unpublished data indicated that this leads to cell cycle arrest [25]. This has been confirmed by a similar work from Baum's group in Germany, who used Rosa26rtTA-nls-Neo2 mouse, expressing the reverse tetracycline-inducible transactivator (rtTA) under the control of the ubiquitously active Rosa26 locus (Rosa26rtTA) for pharmacologically regulated induction of EVI1 with rtTA-responsive promoters (tetP). They reported that inducible expression of EVI1 leads to a pronounced arrest in G0/G1 through induction of the cell cycle inhibitory genes encoding p18, p27 and p57, and concomitant downregulation of all cyclins [24]. The role that this EVI1-induced quiescence phenotype plays in leukemogenesis is unclear, but may contribute to the establishment of a population of quiescent leukemia stem cells, and/or resistance to chemotherapeutic killing of dividing cells. Role of MECOM in leukemia cell quiescence John Dick's lab identified a gene expression signature from primary human leukemia cells that revealed that MECOM is one of a few genes in the core “stem cell” gene clusters shared by HSC and leukemia stem cells (LSC) [54]. Indeed, several lines of evidence point to a critical role for MECOM in regulating leukemia cell quiescence. Yamakawa et al. reported that EVI1 overexpressing U937 cells grew more slowly and accumulated in G0 to a greater extent than the corresponding control cell line [55]. Konrad et al. reported that induction of EVI1 in U937T cells strongly inhibited cell proliferation primarily by slowing the transit through the cell mitotic cycle. They set up a system to express EVI1 and MDS1–EVI1 in a tetracycline-regulated manner in the human monocytic leukemia cell line U937. Induction of either of these proteins caused cells to accumulate in the G0/G1 phase of the cell cycle; the rate of spontaneous apoptosis was also modestly increased [56]. Karakaya et al. reported that over expression of EVI1 in the U20S osteosarcoma cell line induced a partial cell cycle arrest in the G0/G1 phase; overexpression of EVI1 interfered with cytokinesis and led to the accumulation of cells with supernumerary centrosomes in the G0/ G1 phase [57,58]. Whereas this finding suggests a role for EVI1 in causing chromosomal instability, analysis of human AML cells with chromosome 3q26 rearrangements does not support this idea [58]. Furthermore, in HT-29 cells, which have high levels of endogenous MECOM, silencing of endogenous MECOM expression resulted in a marked increase in proliferation as compared to the wild type and control-siRNA transfected cells, indicating that MECOM mediates suppression of cellular proliferation in HT-29 cells, primarily by delaying cell cycle progression [56]. Drug-resistance of leukemia cells is partly dependent on cell quiescence in the marrow. In an inducible expression system in U937 human leukemia cells, Rommer et al. [59] and others [55,60] have reported that EVI1 partially protected human myeloid cells from the cytotoxic effects of chemotherapy. Several mechanisms are proposed including upregulation of CDKN1A/p21/WAF [59], and increased expression of Integrin α6 (ITGA6) [55] and angiopoietin1 [60]. Signaling pathways cJun/AP1 Hirai and coworkers found that EVI1 can upregulate reporter constructs bearing the AP-1-responsive promoter region. AP-1 activity (Fos/Jun heterodimer) is activated by a variety of growth signals, including phorbol esters (12-O-tetradecanoylphorbol 13-acetate (TPA)). Tanaka et al. [61] showed that EVI1 could markedly up-regulate

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reporter constructs with TPA-responsive elements (TRE). Similarly, this group also showed that RUNX1–MDS1–EVI1 (RME), the protein product of the leukemia-associated t(3;21), can also transactivate a TRE reporter. Through structure–activity-relationship (SAR) studies, they show that activation of AP-1 requires the second set of zinc fingers (zinc fingers 8–10; ZF2), and appears to occur via transcriptional upregulation of Fos, via an element within the promoter that does not appear to bind EVI1. The exact mechanism of Fos upregulation is not clear; nor is it clear whether AP-1 upregulation is required for leukemogenesis. Noting that transcriptional activation of Fos is lost upon deletion of ZF2, it is important to note that neither transformation of bone marrow progenitors by RME nor transformation of Rat1 fibroblasts requires DNA binding via ZF2 [62]. It is possible that a non-DNA binding function of ZF2 is required for both Fos activation and transformation. TGF-β Additional studies have shown that high-level expression of the 135 kDa isoform of EVI1 interferes with the growth-inhibitory effects of transforming growth factor β1 (TGF-β). Expression of this isoform represses TGFβ-mediated transactivation of a TGF-β reporter, and leads to diminished responsiveness to the growth inhibitory effects of TGF-β; importantly however, these studies were performed in nonhematopoietic cells [32,63]. This repressive effect may be mediated by the interaction of EVI1 via zinc fingers 1–7 with Smad3 [32], since deletions of broad and non-overlapping sections of this region result in loss of the ability to inhibit TGF-β activation of reporters and loss of ability to interact with SMAD3 [32]. However, it also appears that binding of EVI1 to CtBP via the EVI1 repression domain is also critical, since an EVI1 amino acid missense mutant that lacks CtBP binding can only partially inhibit TGF-β activation of a TGF-β reporter (Izutsu et al., 2001). One issue of concern is that several cell lines that express Evi1 due to retroviral insertion at the locus, such as NFS60 [1] and 32Dcl3 [29], are sensitive to the growth inhibitory effects of TGFβ [64], raising questions as to the importance of EVI1's interference with TGF-β signaling in leukemic cells. Alliston et al. [33] extended these findings, presenting data to suggest that EVI1 interacts with other SMADs, including SMAD2, and SMAD4. If true, this would implicate EVI1 as playing a regulatory role in signaling mediated by other TGF-β family members, including BMPs and activin, which signal through these other SMADs. Downstream target genes EVI1 directly binds target genes critical for normal hematopoiesis via two separate zinc finger DNA binding domains [9,65,66]. One of the mechanisms by which EVI overexpression can induce malignancy is through direct binding to these target genes. Early suggestions that EVI1 target genes serve a critical role in myeloid leukemia was provided by the significant overlap between the EVI1 and GATA core binding motifs [30,65,67]. However, while EVI1 binds to some classic GATA1 sites with high affinity, it binds most with low affinity [67]; the exact role of EVI1 in influencing the transcriptional regulation of GATA1 target genes has not been fully addressed. Pre-B-Cell leukemia homeobox 1 (Pbx1) has been identified as a key transcriptional target of EVI1. Pbx1 transcription increases with Evi1 overexpression in hematopoietic stem and progenitor cells, an effect diminished by the deletion of both EVI1-ZF1 and -ZF2. Furthermore, knockdown of Pbx1 inhibits the colony-forming ability of Evi1-transduced cells, but not in cells transduced with Aml1/Eto or E2a/Hlf [68]; these findings suggest a critical role in EVI1-induced leukemogenesis. There is some evidence implicating Gata2 as a key EVI1 transcriptional target as well. Evi1−/− mouse embryos display reduced hematopoietic proliferation, which correlates with lower Gata2 expression. EVI1 was shown to up-regulate Gata2 transcription, and addition of

Please cite this article as: C. Glass, et al., The role of EVI1 in myeloid malignancies, Blood Cells Mol. Diseases (2014), http://dx.doi.org/10.1016/ j.bcmd.2014.01.002

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Evi1 or Gata2 into Evi1−/− hematopoietic cells restored proliferation in vitro [69]. Further studies revealed that Gata2 transcriptional activation and suppression of TGF-β signaling were both critical to EVI1mediated proliferation in hematopoietic stem/progenitor cells found in murine embryonic para-aortic splanchnopleural tissue [70]. Lastly, it was shown that acetylation of EVI1 protein at lysine 564 is critical for both DNA-binding ability and upregulation of Gata2, and that this residue is specifically acetylated in myeloid leukemias and primary hematopoietic cells [71]. However the relevance of Gata2 regulation by EVI1 in malignancy remains unknown, as the studies conducted did not examine a direct role in leukemogenesis. Chromatin immunoprecipitation (ChIP) studies have yielded insight into potential EVI1 target genes as well. A tetracycline-inducible EVI1VP16 fusion protein into NIH 3T3 cells was used to identify potential EVI1 transcriptional targets, and ChIP was performed in the myeloid leukemic cell lines 32Dcl3 and NFS 58 to confirm occupancy of endogenous EVI1; the results revealed Gad45g, Gata2, Zfpm2/Fog2, Ski1, Klf5, Den, and Map3k14 as target genes potentially relevant to EVI1-induced leukemogenesis [31]. Two genome-wide ChIP-Seq studies for EVI1 have been published. The first report identified global EVI1 target genes in a human ovarian cancer cell line [72]. This study demonstrated that over 25% of EVI1occupied genes were also bound by activator protein 1 (AP1), providing evidence for a synergistic cooperative interaction between EVI1 and AP1, specifically the FOS protein. AP1 controls important cellular processes such as apoptosis, cellular differentiation and proliferation and has been described as a “nuclear decision-maker” critical for determining life or death cell fate decisions. In a separate study, we reported the first genome wide EVI1 DNA binding sites in murine myeloid leukemic cell lines (Fig. 4) [45]. We confirmed EVI binding to and deregulation of a select number of previously reported EVI1 downstream gene targets (using ChIP-Seq and RNA-Seq, respectively), and also identified novel EVI target genes involved in EVI1-induced leukemogenesis (Fig. 5). The results revealed that 1) EVI1 binds to and suppresses Cebpε, a critical regulator of terminal myeloid differentiation; 2) EVI1 binds to and suppresses Serpinb2, and other genes involved in Jak-Stat signaling; and 3) EVI1 suppresses several genes involved in apoptotic mechanisms mediated by ATPdependent purinoreceptors. Importantly, an ETS-like motif was identified in 88% of target genes (Fig. 4).

Epigenetic modification Another potential mechanism of malignant transformation by MECOM proteins is through epigenetic modifications, specifically via chromatin modifying functions. There has been a growing body of

Fig. 5. Summary diagram of critical genes involved in EVI1 leukemia. Taken from Ref. [45].

evidence for epigenetic aberrations associated with EVI1 induced leukemia [44,73,74]. Epigenetic modifications, defined as heritable changes in gene expression without changes in the underlying DNA sequence, has been an increasing focus of research and drug development in blood and solid tumor malignancies [75–77]. Epigenetic changes largely occur via various chromatin-modifying processes such as DNA or histone methylation, histone acetylation, protein phosphorylation and sumoylation. There is also a growing interest in developing inhibitors of chromatin modifying enzymes for various cancers, as supported by numerous clinical trials for histone deacetylation and histone methylation inhibitors [78]. We review here the chromatin modifying mechanisms associated with EVI1. Epigenetic modifications at the promoter of the Evi1 gene Overexpression of EVI1 is associated with 3q26 chromosomal rearrangements and confers extremely poor prognosis in acute myeloid leukemia (AML). One study [73] of 476 AML patients reported that those with no basal expression of the EVI1 gene have a better prognosis than patients with EVI1 overexpression. Consistent with the fact that DNA methylation is generally associated with repression of gene transcription, EVI1 overexpression was associated with the absence of DNA methylation in the promoter region of EVI1. The EVI1 locus was also enriched with marks of active gene transcription, specifically histones H3 and H4 acetylation, and histone H3 lysine 4 (H3K4) trimethylation. Conversely, cells without EVI1 expression were associated with DNA hypermethylation and marks of gene repression such as trimethylation of histone H3 lysine 27 (H3K27) at the EVI1 promoter. Recruitment of chromatin-modifying enzymes by the EVI1 protein EVI1 may also recruit chromatin-modifying enzymes to regulate gene transcription. The Wieser lab [79] was the first to report that EVI1 (and the longer isoform MDS1–EVI1) directly interacts with a chromatin-modifying enzyme, specifically a histone deacetylase (HDAC), to repress gene transcription. The HDAC was found to associate with the EVI1 repression domain (define), and the activity of which was inhibited by an HDAC inhibitor. HDACs are a class of enzymes that remove acetyl groups from lysine amino acids on histones, which leads to tighter DNA winding around histones, inaccessibility by transcription factors and subsequent gene silencing. HDAC was identified to associate with the EVI1 zinc finger 1 DNA binding and the repression domains, whose activity was inhibited by the HDAC inhibitor Trichostain A. The role of HDAC1–EVI1 binding in the context of leukemogenesis was not assessed. EVI1 was also identified to directly bind two different histone acetyltransferase (HAT) enzymes: cAMP-responsive element-binding protein (CBP) and p300/CBP-associated factor (P/CAF) [34]. HATs are enzymes that acetylate lysine amino acids on histones, removing a positive charge to weaken the affinity between histones and negatively charged phosphate groups on DNA. Therefore, HATs are generally linked to open euchromatin and active transcription. It was determined that EVI1 physically interacts with P/CAF the EVI1 region between amino acids 283 and 514, whereas CBP binding to EVI1 occurs through the proximal zinc finger domain of EVI1. Although the direct relevance to leukemogenesis was not addressed through deletion analyses, it was demonstrated that the presence of CBP and P/CAF directs the ability of EVI1 to increase gene transcription in reporter gene assays. EVI1 was observed to directly recruit and interact with two histone methyltransferase (HMT) enzymes, G9A and SUV39H1 [80,81]. HMTs are generally associated with gene silencing and inactive heterochromatin. HMTs do not alter any charged bonds between histones and DNA but rather recruit “reader” molecules that recognize and bind methyl marks on histones via bromodomains. The reader molecules in turn induce gene silencing and formation of heterochromatin. G9A

Please cite this article as: C. Glass, et al., The role of EVI1 in myeloid malignancies, Blood Cells Mol. Diseases (2014), http://dx.doi.org/10.1016/ j.bcmd.2014.01.002

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adds a mono- or di-methyl group, while SUV39H1 adds a trimethyl to H3K9. The Delwel laboratory first demonstrated that the N-terminal portion of EVI1 is required to interact with the H3-specific HMT domain (also known as the SET domain) of the SUV39H1 enzyme, and that the presence of SUV39H1 results in gene repression in a dose dependent manner [80]. The Kurokawa laboratory also demonstrated EVI1 recruits and binds a polycomb group protein HMT called EZH2 via the EVI1 zinc finger 1 domain [22]. The EVI1/EZH2 protein complex was identified to be required for silencing of the Pten gene through methylation of H3K79. Furthermore, this study was the first to demonstrate that the chromatin modifying enzyme EZH2 is required for the serial replating capacity of EVI1-induced human AML bone marrow cells. Finally, EVI1 was discovered to physically interact with DNA methyltransferases 3a and 3b to form an enzymatically active complex for the induction of DNA methylation [82]. DNA methylation is generally associated with repression of gene transcription. Inappropriate EVI1 expression has been linked to improper DNA methylation and malignancy, via the EVI1/Dmnt protein complex. A parallel study [7] also showed EVI1 interaction with DNA methyl transferases 3A and 3B. In this study, the methylation signature at promoters of genes in EVI1 AML blast cells (hypermethylation) was significantly different from normal CD34(+) bone marrow cells and other non-EVI1 AMLs. MDS1–EVI1/PRDM3 as a chromatin modifying enzyme MDS1–EVI1 (PRDM3) was identified to possess HMT activity with the ability to modify chromatin by the addition of a mono-methyl group to H3K9 [10]. H3K9 monomethylation is required for H3K9 trimethylation, which generally leads to repression of gene transcription and heterochromatin formation. MDS1–EVI1 also functions as a bona-fide transcription factor, which binds sequence-specific DNA with high specificity via both ZF1 and ZF2 binding domains [67]. The unique combination of harboring both HMT enzymatic and transcription factor functions suggests that MDS1–EVI1 has the potential to not only decide what regions of the genome to bind, but also influence how and when to regulate gene transcription via histone modification. We demonstrated using genetic approaches that the HMT activity of MDS1–EVI1 is required for MLL-AF9-induced leukemogenesis [83]. Since HMTs have been inhibited with small molecules, it leaves open the possibility of treating MLL fusion protein leukemias with small molecule therapeutics that inhibit the HMT activity of MDS1–EVI1. Conclusion The MECOM locus and the EVI1 proteins play an important role in myeloid malignancy, in particular, poor prognosis AML. Roles have been well established for the protein causing a block to myeloid differentiation and apoptosis. In addition, a role for EVI1 in quiescence has also been found and this may explain the relative resistance of EVI1positive leukemias to chemotherapy. However, for each of these areas, the relative contribution of several molecular mechanisms is still not clear; to clarify this will require studies performed in the context of AML itself, rather than heterologous model systems. There is the potential of targeted therapies that block EVI1 function. Though some have been attempted [62], to date, we are at the beginning stages of this long-term goal. Conflict of interest The authors have no conflicts to declare. Acknowledgment Funding: This work was supported in part by the National Institutes of Health (NIH) (R01CA120313 to ASP), New York State Stem Cell Science (NYSTEM) (C026423 to YZ), Alex's Lemonade Stand Foundation

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(ALSF), Children's Leukemia Research Association (CLRA) and a National Heart Lung Blood Institute T32 post-doctoral fellowship grant under Dr. Thomas Pearson to the University of Rochester Clinical Translational Research Institute.

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Please cite this article as: C. Glass, et al., The role of EVI1 in myeloid malignancies, Blood Cells Mol. Diseases (2014), http://dx.doi.org/10.1016/ j.bcmd.2014.01.002