High Ecotropic Viral Integration Site 1 Expression Research Articles

The adverse impact of ecotropic viral integration site-1 (EVI1) overexpression on the prognosis of acute myeloid leukemia with KMT2A gene rearrangement in different risk stratification subtypes.

AML patients with KMT2A-MLLT3 and other 11q23 abnormalities belong to the intermediate and high-risk level groups, respectively. Whether the poor prognostic value of Ecotropic Viral Integration site-1 (EVI1) overexpression suits either the subtypes of KMT2A-MLLT3 or Non-KMT2A-MLLT3 AML patients (intermediate and high risk group) needs to be further investigated. We retrospectively analyzed the clinical characteristics of 166 KMT2A-r and KMT2A-PTD AML patients. For the Non-KMT2A-MLLT3 group, patients in the EVI1-high subgroup had shorter OS and DFS and higher CIR than those in the EVI1-low subgroup (p=.027, p=.018, and p=.020, respectively). Additionally, both KMT2A-MLLT3 and Non-KMT2A-MLLT3 patients who received chemotherapy alone had poorer prognosis than patients who also received allogeneic hematopoietic stem cell transplant (allo-HSCT) regardless of their EVI1 expression level (all p < .001). For transplanted patients with KMT2A-MLLT3 or Non-KMT2A-MLLT3 rearrangement, the EVI1-high subgroup had worse prognosis than the EVI1-low subgroup (all p < .05). The 2-year CIR of the KMT2A-MLLT3 and Non-KMT2A-MLLT3 groups with high EVI1 expression was high (52% and 49.6%, respectively). However, for patients with low EVI1 expression, the 2-year CIR of transplanted patients with KMT2A-MLLT3 and Non-KMT2A-MLLT3 was relatively low. Our study showed that for the Non-KMT2A-MLLT3 group, the EVI1-high group had shorter OS and DFS than the EVI1-low group. High EVI1 expression showed an adverse effect in AML with KMT2A rearrangement in different risk stratification subtypes. For the EVI1-high patients with non-KMT2A-MLLT3 rearrangement, other novel regimens towards relapse should be taken into consideration.

EVI1 expression in early-stage breast cancer patients treated with neoadjuvant chemotherapy

BackgroundOverexpression of the EVI1 (ecotropic viral integration site 1) oncogene has recently been implicated as a prognostic factor in breast cancer (BC), particularly in triple-negative BC (TNBC). In this study we aimed to investigate frequency and clinical relevance of EVI1 expression in newly diagnosed BC treated with neoadjuvant chemotherapy.MethodsEVI1 expression was determined by immunohistochemistry using H-score as a cumulative measurement of protein expression in pretherapeutic biopsies of BC patients treated with anthracycline/taxane based neoadjuvant chemotherapy within the GeparTrio trial. EVI1 was analyzed as a continuous variable and dichotomized into low or high based on median expression. Endpoints were pathological complete response (pCR), disease-free survival (DFS) and overall survival (OS).ResultsOf the 993 tumors analyzed, 882 had available subtype information: 50.8% were HR + /HER2-, 15% HR + /HER2 + , 9.8% HR-/HER2 + , and 24.5% TNBC. Median EVI1 H-score was 112.16 (range 0.5–291.4). High EVI1 expression was significantly associated with smaller tumor size (p = 0.002) but not with BC subtype. Elevated EVI1 levels were not significantly associated with therapy response and survival in the entire cohort or within BC subtypes. However, TNBC patients with high EVI1 showed a trend towards increased pCR rates compared to low group (37.7% vs 27.5%, p = 0.114; odds ratio 1.60 (95%CI 0.90–2.85, p = 0.110) and numerically better DFS (HR = 0.77 [95%CI 0.48–1.23], log-rank p = 0.271) and OS (HR = 0.76 [95% 0.44–1.31], log-rank p = 0.314) without reaching statistical significance.ConclusionEVI1 was not associated with response to neoadjuvant therapy or patient survival in the overall cohort. Further analyses are needed to verify our findings especially in the pathological work-up of early-stage HER2-negative BC patients.Trial registrationNCT00544765.

EVI1-Positive AML Cells Show Distinct Features and High Dependency on ETS Transcription Factor ERG

Inappropriate expression of Ecotropic viral integration site 1 (EVI1) has been associated with dismal clinical outcomes in acute myeloid leukemia (AML), while EVI1 is indispensable for normal hematopoiesis. We have previously reported that EVI1 expression is restricted to hematopoietic stem cell fraction and EVI1-expressing cells show robust long-term reconstitution capacity using Evi1-IRES-GFP knock-in (EVI1-GFP) mice, which enable us to track Evi1 expression on a single cell basis. In this study, we tried to elucidate the functional implication of EVI1 expression in AML using these mice.We generated murine EVI1-GFP AML model by retrovirally transducing MLL-AF9 or -ENL fusion gene into Lineage- Sca-1+ c-kit+ (LSK) cells from EVI1-GFP mice followed by transplantation into lethally irradiated syngeneic mice. Clonogenic and leukemogenic potentials of AML cells, especially leukemic cells with a granulocyte-macrophage progenitor phenotype (L-GMPs) from these mice, were compared according to GFP expression. Remarkably, GFP-positive L-GMPs tended to show lower colony-forming activity in semi-solid media and lower leukemia-initiating potential than GFP-negative L-GMPs. GFP-positive L-GMPs, however, induced a more aggressive form of AML, characterized by shorter survival in the secondary transplantation model. When EVI1-GFP AML mice underwent cytotoxic chemotherapy with cytarabine, the GFP-positive fraction was enriched during myelosuppression, indicating the survival advantage of EVI1-positive cells.To investigate the downstream target of EVI1, we employed murine EVI1-AML model, where murine hematopoietic cells exogenously expressing 3×FLAG-tagged EVI1 were transplanted into syngeneic mice. Using EVI1-AML cells, we performed chromatin-immunoprecipitation coupled to next-generation sequencing (ChIP-seq) by anti-FLAG tag antibody. To identify leukemia-specific targets of EVI1, the result was compared with the result of ChIP-seq obtained from 32D-cl3 murine hematopoietic progenitor cells with 3×FLAG tag inserted into 3'-end of the coding region of the EVI1 gene. Gene ontology analysis revealed that genes involved in immune processes are explicitly enriched in the leukemia samples. Among the list of EVI1-bound genes, we tried to refine functional downstream targets of EVI1, which are upregulated in murine EVI1-AML cells and of which expressions are positively correlated with EVI1. By combining the ChIP-seq data with murine transcriptome data that compare hematopoietic progenitor cells expressing empty-vector and EVI1+ AML cells, and public gene expression datasets of human AML (Valk et al. NEJM 2004), we picked out 18 genes as candidate EVI1 downstream genes.Functional screening using EVI1-AML cells and shRNAs against these genes revealed that silencing of ETS transcription factor ERG (ETS-related gene) markedly suppressed proliferation and colony-forming activity of EVI1-AML cells, as well as rendered them vulnerable to cytotoxic agents. Normal c-kit+ hematopoietic progenitor cells were less affected by shRNAs against ERG. By comparing MLL-ENL immortalized murine hematopoietic cells with high and low EVI1 expression, EVI1-high MLL-ENL cells showed higher ERG dependency than EVI1-low MLL-ENL cells. Pharmacological inhibition of ERG also led to marked inhibition of EVI1-AML cells and EVI1-high MLL-ENL cells. Finally, knockdown of ERG remarkably delayed AML development in bone marrow transplantation model of EVI1-AML and EVI1-expressing MLL-ENL AML.Our data suggest that EVI1-positive AML cells are characterized by an aggressive nature and resistance to cytotoxic agents, as well as low leukemia stem cell capacity. ERG would be a novel downstream target of EVI1, on which survival of EVI1-expressing AML cells depends. DisclosuresMasamoto: Kyowa Hakko Kirin Co., Ltd.: Speakers Bureau; Chugai Pharmaceutical Company: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau; Janssen Pharmaceutical K.K.: Speakers Bureau; Eisai Co., Ltd.: Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Speakers Bureau; MSD K.K.: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Speakers Bureau; Takeda Pharmaceutical Company Limited.: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Speakers Bureau; AbbVie GK: Speakers Bureau; SymBio Pharmaceuticals: Speakers Bureau. Kurokawa: Daiichi Sankyo Company.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; MSD K.K.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau.

High EVI1 Expression Predicts Adverse Outcomes in Children With De Novo Acute Myeloid Leukemia

BackgroundA high ecotropic viral integration site 1 (EVI1) expression (EVI1 high) is an independent prognostic factor in adult acute myeloid leukemia (AML). However, little is known of the prognostic value of EVI1 high in pediatric AML. This study aimed to examine the biological and prognostic significance of EVI1 high in uniformly treated pediatric patients with AML from a large cohort of seven centers in China.MethodsA diagnostic assay was developed to determine the relative EVI1 expression using a single real-time quantitative polymerase chain reaction in 421 newly diagnosed pediatric AML patients younger than 14 years from seven centers in southern China. All patients were treated with a uniform protocol, but only 383 patients were evaluated for their treatment response. The survival data were included in the subsequent analysis (n = 35 for EVI1 high, n = 348 for EVI1 low).ResultsEVI1high was found in 9.0% of all 421 pediatric patients with de novo AML. EVI1 high was predominantly found in acute megakaryoblastic leukemia (FAB M7), MLL rearrangements, and unfavorable cytogenetic aberrance, whereas it was mutually exclusive with t (8; 21), inv (16)/t (16; 16), CEBPA, NPM1, or C-KIT mutations. In the univariate Cox regression analysis, EVI1 high had a significantly adverse 5-year event-free survival (EFS) and overall survival (OS) [hazard ratio (HR) = 1.821 and 2.401, p = 0.036 and 0.005, respectively]. In the multivariate Cox regression analysis, EVI1 high was an independent prognostic factor for the OS (HR = 2.447, p = 0.015) but not EFS (HR = 1.556, p = 0.174). Furthermore, EVI1 high was an independent adverse predictor of the OS and EFS of patients with MLL rearrangements (univariate analysis: HR = 9.921 and 7.253, both p < 0.001; multivariate analysis: HR = 7.186 and 7.315, p = 0.005 and 0.001, respectively). Hematopoietic stem cell transplantation (HSCT) in first complete remission (CR1) provided EVI1 high patients with a tendential survival benefit when compared with chemotherapy as a consolidation (5-year EFS: 68.4% vs. 50.8%, p = 0.26; 5-year OS: 65.9% vs. 54.8%, p = 0.45).ConclusionIt could be concluded that EVI1 high can be detected in approximately 10% of pediatric AML cases. It is predominantly present in unfavorable cytogenetic subtypes and predicts adverse outcomes. Whether pediatric patients with EVI1 high AML can benefit from HSCT in CR1 needs to be researched further.

GFI1 Is a Downstream Target of EVI1 in Normal Hematopoiesis

Acute myeloid leukemia (AML) with high expression of a transcriptional factor, Ecotropic viral integration site 1 (EVI1), is associated with extremely poor prognosis. EVI1 is, however, also essential for maintaining normal hematopoietic stem cells (HSCs), rendering it potentially difficult to target this molecule. To overcome this therapeutic difficulty, it is important to comprehensively elucidate differentially regulated downstream targets between normal and leukemia cells. In this study, we searched downstream targets of EVI1 in normal hematopoiesis by combining a chromatin immunoprecipitation sequence (ChIP-seq) and RNA-sequence (RNA-seq) analysis using a mouse hematopoietic cell line 32D-cl3 with high EVI1 expression. We deleted Evi1 using CRISPR/Cas9 in 32D-cl3 cells. Evi1 knock-out (KO) 32D-cl3 cells showed comparable cell growth with parental cells in the presence of IL-3, which enables them to proliferate permanently without differentiation. When they are allowed to differentiate by adding G-CSF, the number of KO cells decreased sharply at day 5-6, compared with parental 32D-cl3 cells. Along with the decreased cell number, KO cells also demonstrated higher positive rate of Gr-1 at day 7, a typical marker of differentiation into granulocytes, indicating accelerated differentiation of KO cells. These results indicated that EVI1 is required to maintain undifferentiated status of 32D-cl3 cells in a differentiation-permissive conditions, which can model normal hematopoiesis. We knocked in 3×FLAG tag at the 3' end of the Evi1 gene to perform ChIP-seq using anti-FLAG antibody. By using these knock-in cells, ChIP-seq was performed on day 0 and day 3 of G-CSF treatment, when they had started to differentiate with still maintained EVI1 expression. The peaks observed in undifferentiated day 0 sample were considered to contain a group of genes involved in undifferentiated hematopoietic cells in cooperation with EVI1. Genes associated HDAC class I, RAC1 signaling were enriched in these genes. To investigate the functional implications of the result of ChIP-seq, RNA-seq data using two clones of KO cells and parental cells were combined. We found that 152 genes were significantly up-regulated, and 155 genes were down-regulated in the KO cells, with false discovery rate less than 0.05. Twenty-four genes were identified by extracting common genes between ChIP-seq and RNA-seq; namely, genes which had day 0-specific peaks in ChIP-seq, and whose expression were decreased in the KO cells. In order to further examine the physiological implications of 24 genes in vivo, we referred to the results of RNA-seq using murine bone marrow transplantation model, where murine hematopoietic progenitor cells retrovirally transduced with Evi1 were transplanted into irradiated syngeneic mice, finally leading to AML after a long latency. Samples obtained early after post transplantation and those after AML onset were compared to those of normal hematopoietic progenitor cells. Among the above 24 genes, the expression of 5 genes was increased early after transplantation and decreased after the onset of AML, that is, these genes were up-regulated by EVI1 but don't seem to be involved in AML maintenance. We functionally validated the role of these genes in 32D-cl3 cells. Of the above, CRISPR/Cas9-mediated knock-out of Gfi1(Growth Factor Independent 1 Transcriptional Repressor) and Mfsd2b (Major facilitator superfamily domain containing 2B) in 32D-cl3 cells led to high Gr-1 positivity at day 7 like Evi1-KO cells, suggesting that these genes are involved in the functions of EVI1 in the normal hematopoiesis. The mRNA expression of these genes was compared in LSK (Lineage- Sca1+ c-kit+) cells from the bone marrow of Evi1 conditional knockout (cKO) mice and control mice. The expression of Gfi1 and Mfsd2b was decreased in LSK cells from Evi1 cKO mice. Furthermore, retroviral expression of Gfi1 in LSK cells restored the reduced colony-forming ability of Evi1 cKO cells. These results collectively suggest that GFI1 is regulated by EVI1 and is involved in the function of EVI1 regulating the stemness of hematopoietic stem and progenitor cells in normal hematopoiesis. These findings provide us with the novel insights on EVI1-mediated HSC maintenance as well as on the therapeutic strategy that specifically targets leukemia-specific EVI1 effectors while preserving normal hematopoiesis. Disclosures Kurokawa: Shire Plc: Speakers Bureau; Jansen Pharmaceutical: Speakers Bureau; Ono: Research Funding, Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Eisai: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Teijin: Research Funding; Takeda: Research Funding, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Otsuka: Research Funding, Speakers Bureau; Pfizer: Research Funding; Sanwa-Kagaku: Consultancy; MSD: Consultancy, Research Funding, Speakers Bureau; Chugai: Consultancy, Research Funding, Speakers Bureau; Bioverativ Japan: Consultancy; Celgene: Consultancy, Speakers Bureau; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nippon Shinyaku: Research Funding, Speakers Bureau.

EVI1 as a Marker for Lymph Node Metastasis in HNSCC.

Background: HNSCC is the sixth most common cancer in humans and has still a very poor prognosis. The treatment methods so far are very often associated with mutilation and impairment in the quality of life. Except for p16 expression, there are no reliable prognostic markers in HNSCC so far. Ecotropic Viral Integration Site 1 (EVI1) is a well-described prognostic marker in leukemia and different types of solid cancers. In these, a high EVI1 expression is associated with a poor prognosis. In HNSCC, it is not known so far if EVI1 has any prognostic relevance. Materials and Methods: We used our representative tissue cohort of 389 primary HNSCCs, of which 57.2% had one or more lymph node metastases. Here EVI1 expression was analyzed via immunohistochemistry and correlated with the clinical characteristics of these patients. Results: Although in HNSCC EVI1 expression does not predict poor survival, a high EVI1 expression in the primary tumor correlates with a lymph node metastatic disease. Conclusion: Consequently, EVI1 may serve as a biomarker to predict an occult lymph node metastasis in a clinical nodal negative (cN0) HNSCC.

Myeloid leukemia with high EVI1 expression is sensitive to 5-aza-2'-deoxycytidine by targeting miR-9.

High expression of ecotropic viral integration site 1 (EVI1) has been associated with a poor prognosis in leukemia patients, but the underlying mechanism remains unclear. Aberrant expression of microRNAs plays critical roles in leukemia development. MiR-9 is a putative potential target of EVI1. We have investigated the regulating mechanism of miR-9 by EVI1 in leukemia cells. We first examined the relationship between miR-9 and EVI1 expression levels in nine leukemia cell lines by RT-PCR. Then we forced high expression of EVI1 in UoCM1 and K562 cells to confirm the downregulation of miR-9 by EVI1. Methylation of the miR-9 promoter region was detected by DNA bisulfite sequencing. We treated the EVI1-overexpressing cells with the hypomethylating agent 5-aza-2'-deoxycytidine (5-AZA) to reverse EVI1-induced hypermethylation of miR-9. EVI1 and miR-9 expression was negative related. Forced expression of EVI1 downregulated miR-9 by inducing hypermethylation of the miR-9 promoter. 5-AZA reversed high EVI1-induced hypermethylation of the miR-9 promoter and restored the expression of miR-9. 5-AZA induced extensive apoptosis and inhibited proliferation through cell cycle arrest in EVI1-overexpressing leukemia cells. Our results suggest that EVI1 may be involved in leukemia cell proliferation and apoptosis via the regulation of miR-9 promoter methylation. 5-AZA may represent a promising therapeutic option for EVI1-high leukemia patients.

CGRP-CRLR/RAMP1 signal is important for stress-induced hematopoiesis

Ecotropic viral integration site-1 (EVI1) has a critical role in normal and malignant hematopoiesis. Since we previously identified high expression of calcitonin receptor like receptor (CRLR) in acute myeloid leukemia (AML) with high EVI1 expression, we here characterized the function of CRLR in hematopoiesis. Since higher expression of CRLR and receptor activity modifying protein 1 (RAMP1) was identified in immature hematopoietic bone marrow (BM) cells, we focused on calcitonin gene-related peptide (CGRP), a specific ligand for the CRLR/RAMP1 complex. To elucidate the role of CGRP in hematopoiesis, Ramp1-deficient (Ramp1−/−) mice were used. The steady-state hematopoiesis was almost maintained in Ramp1−/− mice; however, the BM repopulation capacity of Ramp1−/− mice was significantly decreased, and the transplanted Ramp1−/− BM mononuclear cells had low proliferation capacity with enhanced reactive oxygen species (ROS) production and cell apoptosis. Thus, CGRP is important for maintaining hematopoiesis during temporal exposures with proliferative stress. Moreover, continuous CGRP exposure to mice for two weeks induced a reduction in the number of BM immature hematopoietic cells along with differentiated myeloid cells. Since CGRP is known to be increased under inflammatory conditions to regulate immune responses, hematopoietic exhaustion by continuous CGRP secretion under chronic inflammatory conditions is probably one of the important mechanisms of anti-inflammatory responses.

Activated Pentose Phosphate Pathway Mediated By Fbp-1 Upregulation Supports Progression of Acute Myeloid Leukemia with High EVI-1 Expression

EVI-1 (ecotropic viral integration site 1) is a transcription factor essential for the development as well as progression of acute myeloid leukemia (AML). Since high expression of EVI-1, seen in approximately 10% of patients with AML (EVI-1high AML), is associated with chemoresistance and poor clinical outcome, development of a novel therapeutic strategy is definitely required. Recent studies suggest that high EVI-1 expression alters metabolic profiles of leukemia cells including glycolysis, oxidative phosphorylation and amino acid and nucleotide synthesis. However, it has yet to be elucidated how these metabolic properties are induced and affect the clinical features of EVI-1high AML cells.In this study, we investigated key molecules that are induced by aberrant EVI-1 expression and modulate the metabolomics of leukemia cells. We conducted whole transcriptome analysis by RNA-sequencing in murine leukemia cells induced by retroviral transduction of bone marrow cells with EVI-1 followed by transplantation into irradiated mice. We identified 45 genes with more than 10-fold expression in EVI-1-transduced bone marrow leukemia cells compared to those with the mock vector at both early (4 weeks) and late (6 months) time points after transplantation. Intriguingly, the fructose bisphosphatase 1 gene (Fbp1), which encodes a key enzyme of gluconeogenesis was highly upregulated in EVI-1-induced leukemic bone marrow cells. We confirmed that FBP1 expression is quickly upregulated at both mRNA and protein levels by ectopic expression of EVI-1 in mouse bone marrow cells as well as human leukemia cell lines. Moreover, we observed a significant enrichment of EVI-1 in the promoter region of Fbp1 by chromatin immunoprecipitation followed by qPCR analysis in murine hematopoietic cells, suggesting that Fbp1 expression is directly regulated by EVI-1. Although high FBP-1 expression suppresses glycolytic metabolism by catalyzing the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and negatively affects cellular proliferation in various types of solid tumor cells, its role in leukemia cells has not been investigated. In contrast to the results previously reported in other types of cancers, pharmacologic inhibition or short hairpin RNA (shRNA)-mediated knockdown of FBP-1 significantly decreased colony-forming cell (CFC) capacity in EVI-1-transduced murine LSK (Linneg, c-Kitpos, Sca-1pos) cells but not in normal LSK cells. Moreover, treatment with an FBP-1 inhibitor of the mice secondarily transplanted with EVI-1-overexpressing AML cells significantly delayed progression of leukemia without compromising normal hematopoiesis in vivo. These results suggest that aberrant expression of FBP-1 is important for progression of EVI-1high AML.Since high FBP-1 enzymatic activity negatively affects the glycolytic pathway, it in turn enhances the pentose phosphate pathway (PPP), one of the main bypassing pathways from the glycolytic process. PPP generates pentoses and then ribose-5-phosphates, resulting in enhanced nucleotide synthesis essential for rapidly dividing cells. In addition to FBP-1, we found that multiple PPP-related enzymes such as glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase and ribose-5-phosphate isomerase were transcriptionally upregulated in EVI-1-transduced leukemia cells compared with the normal bone marrow cells. Importantly, knockdown of these PPP enzymes by shRNA transduction significantly decreased the CFC capacity of EVI-1 transduced murine LSK cells, further confirming that PPP has an important role in driving proliferation of EVI-1high leukemia cells. Collectively, these results indicate that the activated PPP through transcriptional upregulation of FBP-1 as well as its catalyzing enzymes is crucial for progression of EVI-1-driven leukemia cells. Since inhibition of FBP-1 did not compromise normal hematopoiesis, targeting the enzyme can be a promising therapeutic approach for the EVI-1high AML. DisclosuresKurokawa:Kyowa Hakko Kirin: Honoraria, Research Funding; MSD: Honoraria, Research Funding; Nippon Sinyaku: Honoraria, Research Funding; Astellas Pharma: Research Funding; Takeda Pharmaceutical: Research Funding; Eizai: Research Funding; Pfizer: Research Funding; Chugai Pharmaceutical: Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Otsuka Pharmaceutical: Research Funding; Teijin Pharma: Research Funding.

High EVI1 Expression Predicts Poor Outcomes in Adult Acute Myeloid Leukemia Patients with Intermediate Cytogenetic Risk Receiving Chemotherapy.

BackgroundAcute myeloid leukemia with intermediate cytogenetic risk (ICR-AML) needs to be stratified. The abnormal gene expression might be prognostic, and its cutoff value for patient grouping is pivotal.Material/MethodsEcotropic viral integration site 1 (EVI1) transcripts were assessed in 191 adult ICR-AML patients at diagnosis who received chemotherapy only. MLL-PTD, WT1 transcript levels, FLT3-ITD, and NPM1 mutations were simultaneously evaluated, and 27 normal bone marrow samples were tested to define normal threshold.ResultsThe normal upper limit of EVI1 transcript levels was 8.0%. Receiver operating characteristic curve analysis showed that 1.0% (a 0.9-log reduction from the normal limit) was the EVI1 optimal cutoff value for significantly differentiating relapse (P=0.049). A total of 23 patients (12%) had EVI1 levels ≥1.0%. EVI1 ≥1.0% had no effect on CR achievement, whereas it was significantly associated with lower 2-year relapse-free survival (RFS), disease-free survival (DFS), and overall survival (OS) rates in the entire cohort (P=0.0003, 0.0017, and 0.0009, respectively), patients with normal karyotypes (P=0.0032, 0.0047, and 0.0007, respectively), and FLT3-ITD (−) patients (all P<0.0001). Multivariate analysis showed that EVI1 ≥1.0% was an independent adverse prognostic factor for RFS, DFS, and OS in the entire cohort. In addition, patients with EVI1 transcript levels between 1.0% and 8.0% had 2-year RFS rates similar to those with EVI1 ≥8.0%, and they both had significantly lower RFS rates than those with EVI1 <1.0% (P=0.0005 and 0.027).ConclusionsHigh EVI1 expression predicts poor outcome in ICR-AML patients receiving chemotherapy. The optimal cutoff value for patient stratification is different from the normal limit.

Abstract 2034: Amplification and overexpression of EVI1 suppresses growth inhibition mediated by TGF-β in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. EVI1 (ecotropic viral integration site 1) is one of the most aggressive oncogenes associated with myeloid leukemia. The EVI1 gene codes for a zinc finger transcriptional factor that plays an important role in normal development and in oncogenesis. In humans, rearrangements of chromosome 3q26 often activate EVI1 expression in acute myeloid leukemia, chronic myeloid leukemia, and myelodysplastic syndrome. Importantly, high EVI1 expression is an independent negative prognostic indicator of survival in acute myeloid leukemia. The oncoprotein EVI1 has been reported to influence a number of signaling pathways. Thus, EVI1 activates the PI3K/AKT and RAS/ERK signaling pathways. Moreover, EVI1 has been reported to suppress TGF-β signaling by inhibiting Smad3. TGF-β acts as a tumor suppressor by arresting the growth of cells in the early stages of cancer. Whereas the majority of investigations have focused on the contribution of EVI1 to the pathogenesis and clinical characteristics of hematopoietic malignancies, little is known about its relevance for HCC. To identify genes potentially involved in HCC, we investigated DNA copy number aberrations in human HCC cell lines using high-resolution single nucleotide polymorphism (SNP) arrays (Affymetrix). We found that a novel amplification at the chromosomal region 3q26 occurs in a HCC cell line and that MECOM (MDS1 and EVI1 complex locus), which lies within the 3q26 region, was amplified. Amplification of DNA in certain regions of chromosomes plays a crucial role in the development and progression of human malignancies, specifically when proto-oncogenic target genes within those amplicons are overexpressed. Quantitative PCR analysis of the three transcripts transcribed from MECOM indicated that only EVI1, but not the fusion transcript MDS1-EVI1 or MDS1, was overexpressed in HCC cell lines and was significantly up-regulated in 22 (61%) of 36 primary HCC tumors when compared with their non-tumorous counterparts. A copy number gain of EVI1 was observed in 24 (36%) of 66 primary HCC tumors. Knockdown of EVI1 in HCC cells resulted in increased induction of the cyclin-dependent kinase inhibitor p15INK4B by TGF-β and decreased expression of c-Myc, cyclin D1, and phosphorylated Rb in TGF-β-treated cells. Consequently, knockdown of EVI1 led to reduced cell proliferation of HCC. Collectively, our results suggest that EVI1 is a probable target gene that acts as a driving force for the amplification at 3q26 in HCC and that the oncoprotein EVI1 suppresses TGF-β-mediated growth inhibition of HCC cells. Citation Format: Kohichiroh Yasui, Yasuyuki Gen, Osamu Dohi, Akira Tomie, Tomoko Kitaichi, Yoshito Itoh. Amplification and overexpression of EVI1 suppresses growth inhibition mediated by TGF-β in hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2034. doi:10.1158/1538-7445.AM2015-2034

EVI1, a target gene for amplification at 3q26, antagonizes transforming growth factor-β-mediated growth inhibition in hepatocellular carcinoma.

EVI1 (ecotropic viral integration site 1) is one of the most aggressive oncogenes associated with myeloid leukemia. We investigated DNA copy number aberrations in human hepatocellular carcinoma (HCC) cell lines using a high-density oligonucleotide microarray. We found that a novel amplification at the chromosomal region 3q26 occurs in the HCC cell line JHH-1, and that MECOM (MDS1 and EVI1 complex locus), which lies within the 3q26 region, was amplified. Quantitative PCR analysis of the three transcripts transcribed from MECOM indicated that only EVI1, but not the fusion transcript MDS1–EVI1 or MDS1, was overexpressed in JHH-1 cells and was significantly upregulated in 22 (61%) of 36 primary HCC tumors when compared with their non-tumorous counterparts. A copy number gain of EVI1 was observed in 24 (36%) of 66 primary HCC tumors. High EVI1 expression was significantly associated with larger tumor size and higher level of des-γ-carboxy prothrombin, a tumor marker for HCC. Knockdown of EVI1 resulted in increased induction of the cyclin-dependent kinase inhibitor p15INK4B by transforming growth factor (TGF)-β and decreased expression of c-Myc, cyclin D1, and phosphorylated Rb in TGF-β-treated cells. Consequently, knockdown of EVI1 led to reduced DNA synthesis and cell viability. Collectively, our results suggest that EVI1 is a probable target gene that acts as a driving force for the amplification at 3q26 in HCC and that the oncoprotein EVI1 antagonizes TGF-β-mediated growth inhibition of HCC cells.

EVI1 is critical for the pathogenesis of a subset of MLL-AF9–rearranged AMLs

AbstractThe proto-oncogene EVI1 (ecotropic viral integration site-1), located on chromosome band 3q26, is aberrantly expressed in human acute myeloid leukemia (AML) with 3q26 rearrangements. In the current study, we showed, in a large AML cohort carrying 11q23 translocations, that ∼ 43% of all mixed lineage leukemia (MLL)–rearranged leukemias are EVI1pos. High EVI1 expression occurs in AMLs expressing the MLL-AF6, -AF9, -AF10, -ENL, or -ELL fusion genes. In addition, we present evidence that EVI1pos MLL-rearranged AMLs differ molecularly, morphologically, and immunophenotypically from EVI1neg MLL-rearranged leukemias. In mouse bone marrow cells transduced with MLL-AF9, we show that MLL-AF9 fusion protein maintains Evi1 expression on transformation of Evi1pos HSCs. MLL-AF9 does not activate Evi1 expression in MLL-AF9–transformed granulocyte macrophage progenitors (GMPs) that were initially Evi1neg. Moreover, shRNA-mediated knockdown of Evi1 in an Evi1pos MLL-AF9 mouse model inhibits leukemia growth both in vitro and in vivo, suggesting that Evi1 provides a growth-promoting signal. Using the Evi1pos MLL-AF9 mouse leukemia model, we demonstrate increased sensitivity to chemotherapeutic agents on reduction of Evi1 expression. We conclude that EVI1 is a critical player in tumor growth in a subset of MLL-rearranged AMLs.

The Increased Expression of Integrin α6 (ITGA6) Enhances Drug Resistance in EVI1high Leukemia

Ecotropic viral integration site-1 (EVI1) is one of the candidate oncogenes for human acute myeloid leukemia (AML) with chromosomal alterations at 3q26. High EVI1 expression (EVI1high) is a risk factor for AML with poor outcome. Using DNA microarray analysis, we previously identified that integrin α6 (ITGA6) was upregulated over 10-fold in EVI1high leukemia cells. In this study, we determined whether the increased expression of ITGA6 is associated with drug-resistance and increased cell adhesion, resulting in poor prognosis. To this end, we first confirmed the expression pattern of a series of integrin genes using semi-quantitative PCR and fluorescence-activated cell sorter (FACS) analysis and determined the cell adhesion ability in EVI1high leukemia cells. We found that the adhesion ability of EVI1high leukemia cells to laminin increased with the increased expression of ITGA6 and integrin β4 (ITGB4). The introduction of small-hairpin RNA against EVI1 (shEVI1) into EVI1high leukemia cells reduced the cell adhesion ability and downregulated the expression of ITGA6 and ITGB4. In addition, the overexpression of EVI1 in EVI1low leukemia cells enhanced their cell adhesion ability and increased the expression of ITGA6 and ITGB4. In a subsequent experiment, the introduction of shRNA against ITGA6 or ITGB4 into EVI1high AML cells downregulated their cell adhesion ability; however, the EVI1high AML cells transfected with shRNA against ITGA6 could not be maintained in culture. Moreover, treating EVI1high leukemia cells with neutralizing antibodies against ITGA6 or ITGB4 resulted in an enhanced responsiveness to anti-cancer drugs and a reduction of their cell adhesion ability. The expression of ITGA6 is significantly elevated in cells from relapsed and EVI1high AML cases; therefore, ITGA6 might represent an important therapeutic target for both refractory and EVI1high AML.

EVI-1 Mediates Apoptosis Resistance Via CD261 Induction and Enhances Leukemogenic Potential in Human Acute Lymphoblastic Leukemia

Abstract 1356The transcriptional regulator ecotropic viral integration site-1 (EVI-1) has mainly been studied for its role in myeloid malignancies, in which high EVI-1 levels are associated with particularly aggressive disease. The role of EVI-1 in lymphoid cells, however, is largely unknown. Here, we show that EVI-1 is indeed expressed and functional in lymphoid malignancies. EVI-1 expression was analyzed by real-time PCR in 5 lymphoid cell lines and primary cells from patients with pediatric and adult acute lymphoblastic leukemia (ALL; n=32), chronic lymphocytic leukemia (CLL; n=19) and acute myeloid leukemia (AML; n=20) as well as healthy bone marrow (BM; n=3) as control. High EVI-1 expression was detected in 5/32 pediatric ALL, 2/3 adult ALL, 2/19 CLL and 2/20 studied AML samples, but in none of the healthy BM controls. Interestingly, high EVI-1 expression appeared associated with poor clinical prognosis (p=0.017) as previously reported in myeloid malignancies. Next, EVI-1 shRNA and non-coding shRNA lentiviruses were used to generate EVI-1 knockdown and control cells from NALM-16 and REH cells. Stably transduced knockdown EVI-1 and control lines were obtained and EVI-1 knockdown was confirmed on gene and protein level by real-time PCR and western blot as well as intracellular FACS analyses. Knock-down of EVI-1 expression suppressed cell growth and metabolic activity and lead to increased spontaneous apoptosis. Moreover, exposure to staurosporine, Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL)- or Etoposide-induced apoptosis was significantly stronger in knockdown versus control cells, as shown by measurement of DNA-content, Annexin V and caspase-3/7 activity as well as western blot for activated caspase 3 and PARP. Molecularly, apoptotic response to TRAIL was mediated by induction of its receptor CD261 in EVI-1 knockdown cells, as shown by experiments performed with TRAIL and CD261 blocking antibodies. No significant proliferation differences were noted in cell cycle analyses and BrdU assays performed with EVI-1 knockdown and control treated NALM-16 and REH cells, which is in contrast to previous reports with mouse hematopoietic stem cells where EVI-1 regulates proliferation through Gata2.Furthermore, in vivo leukemogenic capacity was assessed by transplantation of EVI-1 knockdown and control NALM-16 cells in immune-permissive NOD/SCID/IL2Rγnull (NSG) mice. Inhibition of EVI-1 severely impaired leukemogenic properties of NALM-16 cells, resulting in suppression of leukemic engraftment rates and dramatic inhibition of organ infiltration by leukemic cells. Consistently, overall survival was prolonged in mice transplanted with EVI-1 knockdown versus control cells. Taken together, our data show that EVI-1 is not only expressed in myeloid but also lymphoid leukemias and might contribute to the leukemogenic potential and apoptosis resistance of ALL cells. Experiments underway in our laboratory are further investigating the molecular mechanisms by which EVI-1 regulates apoptosis sensitivity and putative differences between effects in cells of myeloid and lymphoid origin. Future studies in larger patient cohorts will be needed to explore the prognostic impact of EVI-1 expression in lymphoid neoplasia. Disclosures:No relevant conflicts of interest to declare.

Angiopoietin1 contributes to the maintenance of cell quiescence in EVI1high leukemia cells

Ecotropic viral integration site-1 (EVI1) is an oncogenic transcription factor in human acute myeloid leukemia (AML) associated with poor prognosis. Because the drug-resistance of leukemia cells is partly dependent on cell quiescence in the bone marrow niche, EVI1 may be involved in cell cycle regulation in leukemia cells. As a candidate regulator of the cell cycle in leukemia cells with high EVI1 expression (EVI1high), we analyzed angiopoietin1 (Ang1), which is a down-regulated gene in EVI1-deficient mice and is involved in the quiescence of hematopoietic stem cells. The results of real-time PCR analyses showed that Ang1 is highly expressed in leukemia cell lines and primary AML cells with EVI1high expression. Introduction of shRNA against EVI1 into EVI1high leukemia cells down-regulated Ang1 expression. Moreover, knockdown of Ang1 in EVI1high leukemia cells promoted cell cycle progression and down-regulated the CDK inhibitor p18 (INK4c). Treatment with a decoy Tie2/Fc protein also down-regulated the expression of p18. These results suggest that Ang1/Tie2 signaling may suppress cell cycle progression via maintenance of G0/G1 phase through up-regulation of p18 expression. This mechanism may help to maintain EVI1high leukemia cells in the bone marrow niche and promote resistance to anti-cancer drugs.

The Transcription Factor Evi-1 Is Overexpressed, Promotes Proliferation, and Is Prognostically Unfavorable in Infratentorial Ependymomas

Ependymomas are glial tumors of presumably radial glial origin that share morphologic similarities with ependymal cells. The molecular genetics of ependymomas of supratentorial, infratentorial, and spinal location is heterogeneous. We aimed at identifying pathways operative in the development of infratentorial ependymomas. To do so, gene expression profiles of tumor cells laser microdissected from infratentorial ependymomas (n = 15) were compared with that of nonneoplastic ependymal cells laser microdissected from autopsy tissue (n = 7). Among 31 genes significantly overexpressed (>5-fold) in ependymomas, transcription factor EVI1 (ecotropic viral integration site 1) showed the highest overexpression (35-fold). Evi-1 protein expression could be confirmed in formalin-fixed, paraffin-embedded samples of 26 of 28 infratentorial ependymomas but only in 7 of 47 nonependymal glial tumors (P < 0.001). Furthermore, MDS1/EVI1 fusion transcripts were detectable in 17 of 28 infratentorial ependymomas and significantly correlated with MGMT (O6-methylguanine-DNA-methyltransferase) promoter hypermethylation (P < 0.05). In primary infratentorial ependymoma cells, transfection with EVI1-specific siRNAs resulted in significant growth inhibition [48 hours: 87% ± 2% and 74% ± 10% as compared with control (mean ± SD; P < 0.001)]. The prognostic role of EVI1 could further be validated in an independent cohort of 39 infratentorial and 26 supratentorial ependymomas on the basis of mRNA expression profiling. Although in supratentorial ependymomas EVI1 expression status had no prognostic impact, in infratentorial ependymomas, high EVI1 expression was associated with shorter overall survival and progression-free survival. To conclude, the transcription factor Evi-1 is overexpressed in infratentorial ependymomas, promotes proliferation of ependymal tumor cells, and is prognostically unfavorable.

CD52 as a molecular target for immunotherapy to treat acute myeloid leukemia with high EVI1 expression

Ecotropic viral integration site 1 (EVI1) is an oncogenic transcription factor in human acute myeloid leukemia (AML) with chromosomal alterations at 3q26. Because a high expression of EVI1 protein in AML cells predicts resistance to chemotherapy with a poor outcome, we have searched for molecular targets that will treat these patients with AML. In this study, we determined that CD52, which is mainly expressed on lymphocytes, is highly expressed in most cases of AML with a high EVI1 expression (EVI1(High)). CAMPATH-1H, a humanized monoclonal antibody against CD52, has been used to prevent graft-versus-host disease and treat CD52-positive lymphoproliferative disorders. Here, we investigated the antitumor effect of CAMPATH-1H on EVI1(High) AML cells. CAMPATH-1H significantly inhibited cell growth and induced apoptosis in CD52-positive EVI1(High) leukemia cells. Furthermore, CAMPATH-1H induced complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity against CD52-positive EVI1(High) leukemia cells. After an intravenous injection of CAMPATH-1H into NOD/Shi-scid/IL-2Rγ;null mice with subcutaneous engraftment of EVI1(High) leukemia cells, tumor growth rates were significantly reduced, and the mice survived longer than those in the phosphate-buffered saline-injected control group. Thus, CAMPATH-1H is a potential therapeutic antibody for the treatment of patients with EVI1(High) leukemia.

Evi-1 as a critical regulator of leukemic cells

Ecotropic viral integration site-1 (EVI-1) has been recognized as one of the dominant oncogenes associated with murine and human myeloid leukemia. Recent clinical studies demonstrated that high EVI-1 expression was an independent negative prognostic indicator of survival in leukemia patients. In addition, gene-targeting studies in mice reveal that Evi-1 is preferentially expressed in hematopoietic stem cells (HSCs) and plays an essential role in proliferation/maintenance of HSCs. Proteins associated with EVI-1, signaling pathways regulated by EVI-1, and downstream mediators of EVI-1 transcriptional regulation have been described and characterized. In this study, we summarize current knowledge regarding biochemical properties and biological functions of EVI-1, which provides a foundation for the development of novel therapeutic strategies.

Acetylation of Lysine 564 Adjacent to the C-terminal Binding Protein-binding Motif in EVI1 Is Crucial for Transcriptional Activation of GATA2

Ecotropic viral integration site 1 (EVI1) is an important transcription factor for leukemogenesis. EVI1 is a member of a group of transcription factors with C-terminal binding protein (CtBP)-binding motifs that act as transcriptional co-repressors; however, we recently found that EVI1 directly activates GATA2 transcription, which is an important gene for the maintenance of hematopoietic stem cells. We show here that EVI1-activated GATA2 transcripts derive from exon 1S of GATA2, which is specifically activated in neural and hematopoietic cells. EVI1 was acetylated by the histone acetyltransferase p300/CBP association factor (P/CAF) in myeloid leukemia cells and hematopoietic progenitor cells. Acetylation at Lys(564), which is adjacent to the CtBP-binding consensus sequence of EVI1, was found to be important for transcriptional activation of GATA2. Mutation of Lys(564) to alanine (K564A) markedly reduced the ability of EVI1 to bind DNA and activate transcription of GATA2. Furthermore, we confirmed that Lys(564) in EVI1 was specifically acetylated in leukemia and primary hematopoietic cells by using an antibody directed against an acetylated Lys(564) EVI1 peptide. Moreover, co-transfection of P/CAF with EVI1 overcame the suppressive effect of the CtBP co-repressor and resulted in GATA2 transcriptional activation; nonetheless, CtBP2 was still included in the protein complex with EVI1 and P/CAF on the EVI1-binding site in the GATA2 promoter region. Thus, acetylation of EVI1 at Lys(564) by P/CAF enhances the DNA binding capacity of EVI1 and thereby contributes to the activation of GATA2.