Ecotropic Viral Integration Site 1 Expression Research Articles

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.

Advances of ecotropic viral integration site 1 gene in acute lymphoblastic leukemia

As an proto-oncogene, ecotropic viral integration site 1 (EVI1) gene is over-expressed in acute myeloid leukemia (AML) because of chromosomal translocation or other genetic abnormalities, finally cause poor prognosis. In recent years, an increasing research of the expression of EVI1 in acute lymphoblastic leukemia (ALL), especially in pediatric ALL. The overexpression of EVI1 gene also suggest a poor prognosis, which is closely relate to the age. But the relationship between the expression of EVI1 gene and mixed lineage leukemia (MLL) and BCR-ABL gene rearrangement needs to be further studied. Researching the expression of EVI1 gene in ALL and exploring targeted therapeutic agents are important research directions in the future. Key words: Proto-oncogene; Leukemia, lymphoid; Gene expression; Ecotropic viral integration site 1 gene

Overexpression of ecotropic viral integration site-1 is a prognostic factor of lung squamous cell cancer.

AimTo explore the expression and clinical significance of ecotropic viral integration site-1 (EVI1) of lung squamous cell cancer (SCC).MethodsThe expression of EVI1 in SCC was detected by immunohistochemistry and the validation cohort was divided into EVI1 high-expression group and low-expression group according to the cutoff of immunohistochemical score. The correlations between EVI1 expression and the clinicopathological factors were analyzed by χ2 test. The relation between EVI1 expression and overall survival rate was evaluated by univariate analysis with Kaplan–Meier method. The independent prognostic factor was identified by multivariate analysis with Cox regression model.ResultsIn this study, the EVI1 high-expression percentage was 32.32% (53/164). EVI1 high expression was significantly associated with a poorer overall 5-year survival rate of SCC (P=0.021). Moreover, EVI1 high expression was identified as an independent prognostic factor of SCC, predicting the unfavorable prognosis (P=0.013).ConclusionHigh expression of EVI1 was significantly associated with a poorer prognosis and it was identified as an independent prognostic factor of SCC.

Role of microRNA in EVI1 Expressing Acute Myeloid Leukemia (AML)

Acute Myeloid Leukemia (AML) is a blood disorder which can be identified by the accumulation of somatic gene mutations. AML is characterized by formation of immature myeloid blasts in bone marrow and blood. Micro-RNAs (miRNAs) are 18–22 nucleotides, non-coding RNAs that play an important role in normal hematopoiesis. Aberrant expression of miRNA has been observed in leukemia and other diseases. One of the genes associated with AML is EVI1 (Ecotropic Viral Integration Site-1). Over expression of EVI1 is an indication of leukemic disease prognosis and EVI1 associated AML has been deemed as poor prognosis. Ectopic expression of EVI1 in AML regulates the expression of miRNAs, which eventually alters the functional properties of leukemic cells. In this review, we will discuss the role and importance of miRNA in cancers especially in leukemia. We also discuss the role of oncogene EVI1 in leukemia and its expression affecting the miRNA status of leukemic cells. In this review, we suggest that miRNA can be used as tools for cancer therapy and diagnosis. MiRNAs have been used in classifying cancers which has eased the efforts of medical practitioners to develop a strategy for cancer treatment. However, further insights in miRNA's cellular function would provide us with more options in treating cancer.

Arsenic Trioxide Suppresses the Expression of EVI-1 Gene and Regulates Related Hematopoietic Transcription Factors in THP-1 Cells

IntroductionAcute myeloid leukemia (AML) is a malignant clonal hematopoietic stem cell disease. Ecotropic viral integration site-1(EVI-1) has been recognized as one of the most dominant oncogenes associated with murine and human myeloid leukemia. EVI-1 harbors several hallmark functions that are normally associated with leukemogenesis. Overexpression EVI-1 upregulates the transcription of the transcription factor gene GATA-2, which plays a critical role in the maintenance of hematopoietic stem cells.EVI-1 also binds to the transcription factors GATA-1,PU.1,RUNX-1,SCL and LMO2 thereby inhibiting their activity and blocking the differentiation of hematopoietic progenitors. Arsenic trioxide (ATO), which was used as a traditional Chinese medicine, has shown excellent therapeutic efficiency for acutepromyelocyticleukemia. A previous study has demonstrated that ATO targets EVI-1 protein to induce apoptosis . However, the molecular mechanisms underlying this regulation of AML cells by ATO have not been fully elucidated. We aim to expand our understanding of the cellular regulation and effect of ATO on EVI-1 and several related transcription factors (such as GATA1, GATA2, RUNX-1, LMO2, PU-1) in human acute monocytic leukemia THP-1 cells.MethodsThe endogenous expression of EVI-1 is higher in THP-1 cells, which was confirmed through gene analysis sequencing and comparison with Genbank.THP-1 cells were treated with various concentrations of ATO( 0,1,3,5¦ÌM) for 24h, 48h and 72h. Expressions of EVI-1,and related transcription factor genes(GATA-2 ,GATA-1, RUNX-1,MPO,LMO,PU.1,SCL2) were determined by qRT-PCR.ResultsCompared to controls, THP-1 cells with high expression of EVI-1 gene were selected to confirm that overexpression of EVI-1 can promote the expression of GATA-2 , and reduce the level of GATA-1, RUNX-1, MPO, LMO, PU.1, SCL2 simultaneously in vitro(Fig.1).To determine whether ATO is able to directly regulate EVI-1 and related transcription factor genes expression in THP-1 cells, we examined their mRNA expression by qRT-PCR after treatment with different concentrations of ATO. We found that ATO has the ability to down regulate EVI-1 gene in a dose-dependent and time-dependent manner( Fig.2a).Moreover, GATA-2 gene expression decreased when THP-1 cells were treated with ATO(Fig.2b). We examined the related transcription factors expression and found that ATO suppressed the expression levels of PU.1, RUNX-1, SCL and LMO2 (Fig.2c, 2d, 2e) in THP-1 cells. Interestingly, exposure to increasing concentrations of ATO for increasing lengths of time was associated with gradual promotion in the expression of GATA-1 mRNA( Fig 2f).SummaryWe verified that the endogenous expression of EVI-1 is higher in THP-1 cells, which confirmed THP-1 cells can be a vitro model to investigate EVI-1 gene functions. Meanwhile, related transcription factor gene GATA-2, which is regarded as EVI-1 regulatory element, is upregulated in THP-1 cells. Moreover, GATA-1, which is essential for erythroid differentiation, and other related transcription factor genes (PU.1, RUNX-1, SCL and LMO2) decreased in THP-1 cells. ATO dose-and time-dependently decreased expressions of EVI-1 and GATA-2 gene and regulated those related transcription factors in EVI-1-overexpressing cells compared with control cells.Disclosure: No relevant conflicts of interest to declare. DisclosuresLang:National Natural Science Fundation of China (no.81470312),: Research Funding; Foundation of Shanghai Committee of Science and Technology (no. 14411950704): Research Funding. Chen:National Natural Science Fundation of China (no.81470312): Research Funding; Foundation of Shanghai Committee of Science and Technology (no. 14411950704): Research Funding. Zhu:Foundation of Shanghai Committee of Science and Technology (no. 14411950704): Research Funding; National Natural Science Fundation of China (no.81470312): Research Funding. Zhong:National Natural Science Fundation of China (no.81470312): Research Funding; Foundation of Shanghai Committee of Science and Technology (no. 14411950704): Research Funding.

Activation of EVI1 transcription by the LEF1/β-catenin complex with p53-alteration in myeloid blast crisis of chronic myeloid leukemia

The presence of a BCR-ABL1 fusion gene is necessary for the pathogenesis of chronic myeloid leukemia (CML) through t(9;22)(q34;q11) translocation. Imatinib, an ABL tyrosine kinase inhibitor, is dramatically effective in CML patients; however, 30% of CML patients will need further treatment due to progression of CML to blast crisis (BC). Aberrant high expression of ecotropic viral integration site 1 (EVI1) is frequently observed in CML during myeloid-BC as a potent driver with a CML stem cell signature; however, the precise molecular mechanism of EVI1 transcriptional regulation during CML progression is poorly defined. Here, we demonstrate the transcriptional activity of EVI1 is dependent on activation of lymphoid enhancer-binding factor 1 (LEF1)/β-catenin complex by BCR-ABL with loss of p53 function during CML-BC. The activation of β-catenin is partly dependent on BCR-ABL expression through enhanced GSK3β phosphorylation, and EVI1 expression is directly enhanced by the LEF1/β-catenin complex bound to the EVI1 promoter region. Moreover, the loss of p53 expression is inversely correlated with high expression of EVI1 in CML leukemia cells with an aggressive phase of CML, and a portion of the activation mechanism of EVI1 expression is dependent on β-catenin activation through GSK3β phosphorylation by loss of p53. Therefore, we found that the EVI1 activation in CML-BC is dependent on LEF1/β-catenin activation by BCR-ABL expression with loss of p53 function, representing a novel selective therapeutic approach targeting myeloid blast crisis progression.

Ecotropic viral integration site 1, a novel oncogene in prostate cancer.

Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer in men in the western world. Mutations in tumor suppressor genes and in oncogenes are important for PCa progression, whereas the role of stem cell proteins in prostate carcinogenesis is insufficiently examined. This study investigates the role of the transcriptional regulator Ecotropic Viral Integration site 1 (EVI1), known as an essential modulator of hematopoietic and leukemic stem cell biology, in prostate carcinogenesis. We show that in healthy prostatic tissue, EVI1 expression is confined to the prostate stem cell compartment located at the basal layer, as identified by the stem cell marker CD44. Instead, in a PCa progression cohort comprising 219 samples from patients with primary PCa, lymph node and distant metastases, EVI1 protein was heterogeneously distributed within samples and high expression is associated with tumor progression (P<0.001), suggesting EVI1 induction as a driver event. Functionally, short hairpin RNA-mediated knockdown of EVI1 inhibited proliferation, cell cycle progression, migratory capacity and anchorage-independent growth of human PCa cells, while enhancing their apoptosis sensitivity. Interestingly, modulation of EVI1 expression also strongly regulated stem cell properties (including expression of the stem cell marker SOX2) and in vivo tumor initiation capacity. Further emphasizing a functional correlation between EVI1 induction and tumor progression, upregulation of EVI1 expression was noted in experimentally derived docetaxel-resistant PCa cells. Importantly, knockdown of EVI1 in these cells restored sensitivity to docetaxel, in part by downregulating anti-apoptotic BCL2. Together, these data indicate EVI1 as a novel molecular regulator of PCa progression and therapy resistance that may control prostate carcinogenesis at the stem cell level.

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.

Absolute Quantification of EVI1 Overexpression in Acute Myeloid Leukemia By RQ-PCR Analysis : A Study of the ALFA Group

Introduction.The Ecotropic viral integration site 1 (EVI1) gene is located on chromosome band 3q26.2, and is composed of 16 exons. Upon diagnosis, EVI1 overexpression has been implicated in 10% of patients with acute myeloid leukemia (AML) and confers a poor prognosis. It occurs with or without chromosomal rearrangements involving 3q26.2 and is often associated with adverse cytogenetic abnormalities, but can also be found in normal karyotypes. At this point, expression of the EVI1 mRNA relative to the housekeeping gene Abelson (ABL) is determined by real time quantitative PCR (RQ-PCR) with ΔCt method. Although this technique gives good results upon diagnosis, it is not powerful enough to monitor patients in minimal residual disease (MRD). We therefore decided to develop a technique of absolute quantification of EVI1 expression in RQ-PCR with plasmid synthesis in order to determine overexpression thresholds in bone marrow (BM) and peripheral blood (PB), to follow up on changes in AML disease, and to look for differences of overexpression according to cytogenetics abnormalities.Methods. This study focuses on 71 patients with AML overexpressing EVI1 included in the ALFA-0701 and ALFA-0702 trials for whom frozen material was available. BM and PB samples were collected at AML diagnosis (n=71) and during follow-up (n=152) as defined in the ALFA-0701 and ALFA-0702 trials. Threshold values in PB and BM were established by analyzing 31 normal PB and normal 19 BM samples. To achieve quantification, we cloned a sequence of EVI1 localized on exons 14 and 15, framing forward primer, reverse primer and TaqMan probe used by Groschel et al in 2010. The sequence was integrated into plasmids pCR2.1 TOPO (Invitrogen) which were used to determine the standard curve. RQ-PCR was performed on ABI7900 (Applied Biosystems). In parallel, the expression of the housekeeping gene ABL was evaluated by RQ-PCR for each sample. The results were expressed as a percentage of EVI1 copies by ABL copies. For every patient, Karyotype and follow-up data were confronted with the percentage of EVI1 expression.Results. To establish our overexpression threshold, we analyzed EVl1 expression in the population of negative witnesses. On PB, the median value of EVl1 expression was 0,11% [0,02 – 0,44], and the maximal value was 0,49%. On BM, the median value was 0,36% [0,01 – 0,96], and the maximal value was 0,96%. Thus, the overexpression threshold was fixed at 0,5% for PB and at 1% for BM. Upon diagnosis, we confirmed EVl1 overexpression for 62 samples out of 71. Mismatches were observed for 9 samples of PB which were ranked in weak positive in relative quantification. EVl1 median expression were 23,26% [4,29 – 516,1] on BM and 3,61% [0,63 – 34,3] on PB. We analyzed EVI1 expression upon diagnosis and MRD1 for 20 patients. In BM, expression varied from 21,61% [4,18 – 592,70] to 3,56% [0,48 – 85,63]. In PB, expression varied from 4% [0,61 – 58,71] to 0,22% [0,01 – 11,51]. Although reduction was significant (p < 0,001), it remained insufficient for MRD follow-up on this parameter.Otherwise, upon diagnosis, we found a statistically significant difference between patients with intermediate cytogenetic (n=21) and patients with adverse cytogenetic (n=41) (p < 0,01) with an EVl1 median expression of 2.75% [0,54 – 24,07] against 10,88% [1,63 – 218,7] (fig 1). Strongest EVI1 overexpression were found in the MLL rearrangement sub-group (n=11) (p < 0,01) : 49,18% [1,7 – 614,1] vs 6,93% [0,69 – 109,9].We did not reveal a strong link between rates and variations of EVI1 expression on the survival of patients, but a trend towards a less pejorative survival seems to appear when EVI1 overexpression is associated with a normal karyotype versus a complex karyotype (fig 2). [Display omitted] Conclusions. We developed a model of absolute quantification of EVI1 expression based on plasmids containing a target sequence located in exons 14 and 15. Nevertheless, the basal expression of EVI1 is high and variations are weak between diagnosis and follow-up. However, we observed higher expression in patients with adverse cytogenetics abnormalities. But, it seems that cytogenetic abnormalities associated with EVI1 overexpression confer a poor prognosis, rather than overexpression in itself. DisclosuresNo relevant conflicts of interest to declare.

FUNCTION OF ECOTROPIC VIRAL INTEGRATION SITE 1 (EVI1) GENE IN MOUSE NEURAL PRECURSOR CELLS AND HUMAN GLIOMA INITIATING CELLS

BACKGROUND: Ecotropic viral integration site 1 (EVI1) gene encodes a transcription factor, containing ten zinc finger motifs found within two domains of the protein. Abnormal expression of EVI1 is found in 5-10% of patients with acute myeloid leukemia (AML), and is associated with unfavorable outcome. EVI1 maintains the self-renewal capacity of hematopoietic stem cells (HSCs), of cell quiescence and stem cell-like phenotypes in leukemia cells. EVI1 knocked out mice die approximately 10.5 days post-coitus (d.p.c.; E10.5) with defect or hypoplasty in heart, cranial ganglia, peripheral nervous system. It is possible that EVI1 also regulates the fate of neural stem cells (NPCs) and glioma initiating cells (GICs) as well as HSCs. METHODS: We investigated the biological effect of knockdown and overexpression of EVI1 against NPCs and GICs. RESULTS: We found that NPCs from nestin-cre induced EVI1 (-/-) mouse differentiated to neuron and glial cells earlier than wild type NPCs. Notch signals were altered in EVI1 (-/-) NPCs. Knockdown experiment with shEVI1 in GICs showed the monolayerd GICs hard to stick to laminin-coated dish by alteration of integrin signals. Because laminin is a major component of niche of NPCs and GICs, EVI1 has an important role to maintain the stem cell capacity by committing to niche. Moreover overexpression of EVI1 in U87 MG and GICs decreased cell proliferation. CONCLUSIONS: Taken together, EVI1 regulates the cell fate and proliferation of NPCs and GICs. SECONDARY CATEGORY: n/a.

Aberrant expression of ecotropic viral integration site-1 in acute myeloid leukemia and acute lymphoblastic leukemia

Ecotropic viral integration site-1 (EVI1) proto-oncogene expression in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) requires further investigation. Here, EVI1 expression levels were measured in 216 Chinese patients with AML and 67 with ALL via quantitative real-time polymerase chain reaction. We found that EVI1 expressed at a high level (H-EVI1) was present in 11.1% of patients with AML versus 20.9% with ALL. Low levels of EVI1 expression occurred in 23.1% with AML versus 43.3% with ALL. This suggested that alteration of EVI1 expression was more profound in ALL than in AML. H-EVI1 was significantly enriched in 30–60-year-old patients. French–American–British (FAB) M3 subtype was significantly correlated with H-EVI1. Interestingly, we found that EVI1 expression was negatively associated with presence of the Philadelphia chromosome (Ph+) and MLL rearrangements in AML. However, Ph+, but not MLL rearrangements, was inversely correlated with EVI1 expression in B-ALL. These results for the first time suggest a mutually exclusive relationship between EVI1 expression and Ph+ karyotype.

Prognostic Significance Of EVI1 expression In Acute Myeloid Leukemia Patients With Intermediate and Adverse Cytogenetic Risk Undergoing Allogeneic Hematopoietic Cell Transplantation With Reduced-Intensity Conditioning

Allogeneic hematopoietic cell transplantation (HCT) represents a post-remission therapy offering potential cure for acute myeloid leukemia (AML) patients (pts). Reduced-Intensity Conditioning (RIC) is increasingly used in AML pts undergoing HCT ineligible for conventional conditioning.The ecotropic viral integration site 1 (EVI1) gene maps to chromosome 3q26 and encodes a transcription factor that has an important role during embryogenesis. EVI1 activation, e.g. through chromosome 3 translocations, is found in several human myeloid disorders. The presence of EVI1 expression has been described as a predictor of poor outcome in pts treated with standard cytarabine based chemotherapy. Whether the expression of EVI1 also associates with outcome in AML pts undergoing RIC-HCT, with a therapeutic approach mainly based on a graft-versus-leukemia effect, remains unknown.Here we tested the prognostic impact of EVI1 expression in RIC-HCT treated AML pts. We analyzed 57 AML pts (median age, 61 years [y]; range 27–74y) who received RIC (Fludarabin 30mg/m^2 at day-4 to -2 & 2Gy total body irradiation at day 0)-HCT at the University of Leipzig, with pretreatment bone marrow material available. Donors were human leucocyte antigen (HLA)-matched related (n=6, 10.5%) or HLA-matched (n=41; 72%) or mismatched (>= 1 antigen; n=10; 17.5%) unrelated. At HCT 82.4% (n=47) of the pts were in complete remission (CR). 28.6% (n=14) had acute graft-versus-host disease (GvHD; >= grade 2) and 80.5% (n=33) (31.7% (n=13) limited; 48.8% (n=20) extensive) chronic GvHD. Median follow-up was 7.0 y for pts alive. Medical research council (MRC) genetic classification was: intermediate (n=39; 73.5%) or adverse (n=14; 26.5%). The pts were also characterized for CEBPA and NPM1 mutations, as well as presence of an FLT3-ITD at diagnosis. EVI1 expression was measured with quantitative reverse transcription polymerase chain reaction and normalized to 18S.71.9% (n=41) of our pts were EVI1 expressers. The presence of EVI1 expression did not significantly associate with any clinical or biological characteristics. Still, by trend EVI1 expression associated with an adverse karyotype (P=.08) and NPM1 mutations (P=.16).The presence of EVI1 expression significantly associated with shorter overall survival (OS; P=.04) and event-free survival (EFS; P=.03; Figure 1). [Display omitted] In multivariable analysis in our set, none of the analyzed clinical or biological parameters were significantly associated with OS or EFS. However, in multivariable analysis cytogenetics (intermediate vs. adverse) associated with OS by trend (P=.12); while EVI1 expression status (P=.14), cytogenetics (intermediate vs adverse; P=.11) and remission status at the time point of RIC-HCT (CR vs all other; P=.10) associated with EFS by trend.In conclusion, the presence of EVI1 expression associated with worse outcome in RIC-HCT treated AML pts. Pretreatment EVI1 expression may refine the risk stratification for AML pts undergoing RIC-HCT. Disclosures:No relevant conflicts of interest to declare.

Maintenance of the hematopoietic stem cell pool in bone marrow niches by EVI1-regulated GPR56

Acute myeloid leukemia with high ecotropic viral integration site-1 expression (EVI1(high) AML) is classified as a refractory type of leukemia with a poor prognosis. To provide new insights into the prevention and treatment of this disease, we identified the high expression of EVI1-regulated G protein-coupled receptor 56 (GPR56), and the association of high cell adhesion and antiapoptotic activities in EVI1(high) AML cells. Knockdown of GPR56 expression decreased the cellular adhesion ability through inactivation of RhoA signaling, resulting in a reduction of cellular growth rates and enhanced apoptosis. Moreover, in Gpr56(-/-) mice, the number of hematopoietic stem cells (HSCs) was significantly decreased in the bone marrow (BM) and, conversely, was increased in the spleen, liver and peripheral blood. The number of Gpr56(-/-) HSC progenitors in the G0/G1-phase was significantly reduced and was associated with impaired cellular adhesion. Finally, the loss of GPR56 function resulted in a reduction of the in vivo repopulating ability of the HSCs. In conclusion, GPR56 may represent an important GPCR for the maintenance of HSCs by acting as a co-ordinator of interactions with the BM osteosteal niche; furthermore, this receptor has the potential to become a novel molecular target in EVI1(high) leukemia.

EVI1 splice variants modulate functional responses in ovarian cancer cells

Amplification of 3q26.2, found in many cancer lineages, is a frequent and early event in ovarian cancer. We previously defined the most frequent region of copy number increase at 3q26.2 to EVI1 (ecotropic viral integration site-1) and MDS1 (myelodysplastic syndrome 1) (aka MECOM), an observation recently confirmed by the cancer genome atlas (TCGA). MECOM is increased at the DNA, RNA, and protein level and likely contributes to patient outcome. Herein, we report that EVI1 is aberrantly spliced, generating multiple variants including a Del190–515 variant (equivalent to previously reported) expressed in >90% of advanced stage serous epithelial ovarian cancers. Although EVI1Del190–515 lacks ∼70% of exon 7, it binds CtBP1 as well as SMAD3, important mediators of TGFβ signaling, similar to wild type EVI1. This contrasts with EVI1 1–268 which failed to interact with CtBP1. Interestingly, the EVI1Del190–515 splice variant preferentially localizes to PML nuclear bodies compared to wild type and EVI1Del427–515. While wild type EVI1 efficiently repressed TGFβ-mediated AP-1 (activator protein-1) and plasminogen activator inhibitor-1 (PAI-1) promoters, EVI1Del190–515 elicited a slight increase in both promoter activities. Expression of EVI1 and EVI1Del427–515 (but not EVI1Del190–515) in OVCAR8 ovarian cancer cells increased cyclin E1 LMW expression and cell cycle progression. Furthermore, knockdown of specific EVI1 splice variants (both MDS1/EVI1 and EVI1Del190–515) markedly increased claudin-1 mRNA and protein expression in HEY ovarian and MDA-MB-231 breast cancer cells. Changes in claudin-1 were associated with alterations in specific epithelial–mesenchymal transition markers concurrent with reduced migratory potential. Collectively, EVI1 is frequently aberrantly spliced in ovarian cancer with specific forms eliciting altered functions which could potentially contribute to ovarian cancer pathophysiology.

EVI1 Inhibits Apoptosis Induced by Antileukemic Drugs via Upregulation of CDKN1A/p21/WAF in Human Myeloid Cells

Overexpression of ecotropic viral integration site 1 (EVI1) is associated with aggressive disease in acute myeloid leukemia (AML). Despite of its clinical importance, little is known about the mechanism through which EVI1 confers resistance to antileukemic drugs. Here, we show that a human myeloid cell line constitutively overexpressing EVI1 after infection with a retroviral vector (U937_EVI1) was partially resistant to etoposide and daunorubicin as compared to empty vector infected control cells (U937_vec). Similarly, inducible expression of EVI1 in HL-60 cells decreased their sensitivity to daunorubicin. Gene expression microarray analyses of U937_EVI1 and U937_vec cells cultured in the absence or presence of etoposide showed that 77 and 419 genes were regulated by EVI1 and etoposide, respectively. Notably, mRNA levels of 26 of these genes were altered by both stimuli, indicating that EVI1 regulated genes were strongly enriched among etoposide regulated genes and vice versa. One of the genes that were induced by both EVI1 and etoposide was CDKN1A/p21/WAF, which in addition to its function as a cell cycle regulator plays an important role in conferring chemotherapy resistance in various tumor types. Indeed, overexpression of CDKN1A in U937 cells mimicked the phenotype of EVI1 overexpression, similarly conferring partial resistance to antileukemic drugs.

Overexpression of EVI1 Causes Genomic Instability and Cell Cycle Arrest in Hematopoietic Cells.

Abstract 2398Deregulated expression of the zinc finger transcription factor ecotropic viral integration site 1 (EVI1) is an independent poor prognostic marker for patients with acute myeloid leukemia. Moreover, we have recently shown that in clinical gene therapy of chronic granulomatous disease (cGD) the activation of this gene locus by strong promoter and enhancer elements within the gamma-retroviral vector LTR has led to clonal dominance and malignant transformation. Physiologically, EVI1 is essential for embryonic development and in regulating hematopoietic stem cell self-renewal. As very little is known about its molecular mechanism driving hematopoietic transformation towards leukemia, we aim to systematically analyze the role of deregulated EVI1 expression and its larger splice form MDS1/EVI1 in hematopoiesis.Lentiviral vector particles encoding for EVI1 (E) or MDS1/EVI1 (ME) and eGFP as marker protein were produced to stably overexpress the transgenes. Transgene expression was verified in myeloid HL60 cells by western blotting and immunofluorescence staining. Analysis of growth kinetics of human HL60 cells in suspension cultures revealed 1.5 – 3 folds lower cell counts of ME and E expressing cells as compared to eGFP control vector transduced cells. We further analyzed the expression of transferrin receptor CD71 which is mainly expressed on proliferating cells to promote iron uptake. Both, E and ME transduced cells, down-regulated CD71 during seven days in culture as compared to eGFP-transduced control cells. 64.2 ± 0.4% of ME cells and 31.1 ± 1.6% of E cells expressed CD71 compared to untransduced (97.4 ± 0.0%) and control vector cells (94.8 ± 0.8%) (p<0.001). For further analyzing the transgene effect on cell cycle activity, 3 populations with different intensity of transgene expression (negative, intermediate and high eGFP+ cells) were isolated. With raising ME expression a 5-fold decrease in the percentage of cells in sub-G1 phase but a 1.3-fold increase in the percentage of cells in G1/G0 phase of the cell cycle was detected. In the highly EVI1 expressing fraction 91.4% arrested in G1/G0 phase of the cell cycle (50.4% in G1/G0 phase in eGFP− E cells). Moreover, almost 1/3 of these transcription factor expressing cells (29.8 ± 8.3% of ME and 27.2 ± 1.6% of EVI1 positive cells) could be detected in G0 phase as compared to 5.0 ± 1.3% of control vector transduced cells. In human CD34+ hematopoietic cells, E and ME overexpression led to a decrease of eGFP+ cells from 8% at day 3 after transduction to 0.5 – 2.5% at day 14 in suspension culture. In contrast, the proportion of eGFP+ human primary cells remained stable for the time period analyzed after transduction with the control vector.We then asked if the cell cycle arrest in G0/G1 is associated with genetic instability, as patients with insertional activation of EVI1 developed a myelodysplastic syndrome with monosomy 7. Analyzing global gene expression comparing mock and eGFP control vector transduced cells with EVI1 expressing hematopoietic cells revealed more than 2000 differentially regulated genes. Genes involved in cell cycle regulation, recombination, replication and DNA repair were significantly downregulated upon EVI1 expression compared to control cells. For further studying DNA damage repair capacity, we irradiated EVI1- and eGFP-control vector transduced cells. Staining of γ-H2AX, an indirect marker for DNA double strand breaks (DSB), revealed that EVI1+ γ-H2AX+ cells were enriched almost 2-fold in G0/G1 phase of the cell cycle as compared to control vector transduced cells. The number of DSB positive cells decreased within 6 hours without apoptosis indicating that most of the double-strand breaks were repaired by non-homologous end-joining.In summary, our data show that EVI1 overexpression causes G0 cell cycle arrest of hematopoietic cells possibly associated with genetic instability. DSB repair in EVI1+ cells may subsequently lead to the accumulation of additional mutations. Systematic investigation of EVI1 and MDS1/EVI1 overexpression in human hematopoietic cells will gain insights into mechanisms leading to clonal dominance and malignant transformation. Disclosures:No relevant conflicts of interest to declare.

EVI-1 modulates leukemogenic potential and apoptosis sensitivity in human acute lymphoblastic leukemia

The 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 in lymphoid malignancies such as acute lymphoblastic leukemia (ALL) and a subset of chronic lymphocytic leukemia. Expression data from pediatric ALL further suggest that high EVI-1 levels are associated with poor prognosis. Suppression of EVI-1 expression by RNA interference reduces cell growth and enhances apoptosis sensitivity in response to various stimuli in lymphoblastic leukemia cells. At the molecular level, EVI-1 modulates expression of several apoptosis-related genes (such as BCL2, BCL-x, XIAP, NOXA, PUMA, TRAIL-R1). Furthermore, EVI-1 knockdown strongly impairs in vivo engraftment of lymphoblastic leukemia cells upon transplantation in immune-permissive NOD/SCID/IL2Rγ(null) mice, conferring a survival benefit when compared with mice transplanted with control cells. Thus, our data show that EVI-1 is expressed not only in myeloid but also in lymphoid leukemias, and contributes to the leukemogenic potential and apoptosis resistance of ALL cells.

EVI1 acts as an inducible negative-feedback regulator of NF-κB by inhibiting p65 acetylation.

Inflammation is a hallmark of many important human diseases. Appropriate inflammation is critical for host defense; however, an overactive response is detrimental to the host. Thus, inflammation must be tightly regulated. The molecular mechanisms underlying the tight regulation of inflammation remain largely unknown. Ecotropic viral integration site 1 (EVI1), a proto-oncogene and zinc finger transcription factor, plays important roles in normal development and leukemogenesis. However, its role in regulating NF-κB-dependent inflammation remains unknown. In this article, we show that EVI1 negatively regulates nontypeable Haemophilus influenzae- and TNF-α-induced NF-κB-dependent inflammation in vitro and in vivo. EVI1 directly binds to the NF-κB p65 subunit and inhibits its acetylation at lysine 310, thereby inhibiting its DNA-binding activity. Moreover, expression of EVI1 itself is induced by nontypeable Haemophilus influenzae and TNF-α in an NF-κB-dependent manner, thereby unveiling a novel inducible negative feedback loop to tightly control NF-κB-dependent inflammation. Thus, our study provides important insights into the novel role for EVI1 in negatively regulating NF-κB-dependent inflammation, and it may also shed light on the future development of novel anti-inflammatory strategies.

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.