Ecotropic Viral Integration Site-1 Gene Research Articles

Orthogonal proteogenomic analysis identifies the druggable PA2G4-MYC axis in 3q26 AML

The overexpression of the ecotropic viral integration site-1 gene (EVI1/MECOM) marks the most lethal acute myeloid leukemia (AML) subgroup carrying chromosome 3q26 abnormalities. By taking advantage of the intersectionality of high-throughput cell-based and gene expression screens selective and pan-histone deacetylase inhibitors (HDACis) emerge as potent repressors of EVI1. To understand the mechanism driving on-target anti-leukemia activity of this compound class, here we dissect the expression dynamics of the bone marrow leukemia cells of patients treated with HDACi and reconstitute the EVI1 chromatin-associated co-transcriptional complex merging on the role of proliferation-associated 2G4 (PA2G4) protein. PA2G4 overexpression rescues AML cells from the inhibitory effects of HDACis, while genetic and small molecule inhibition of PA2G4 abrogates EVI1 in 3q26 AML cells, including in patient-derived leukemia xenografts. This study positions PA2G4 at the crosstalk of the EVI1 leukemogenic signal for developing new therapeutics and urges the use of HDACis-based combination therapies in patients with 3q26 AML.

Identification of ZFP91 As the Master Transcription Factor for EVI1 Gene Expression

Ecotropic viral integration site 1 (EVI1) is essential for hematopoiesis, particularly for the maintenance of hematopoietic stem cells. Aberrant expression of EVI1 is widely known to be an independent poor prognostic factor in acute myeloid leukemia (AML). Previous studies have shown that enhancer hijacking or gene fusion caused by chromosomal translocation or inversion involving 3q26 leads to EVI1 overexpression. Intriguingly, however, aberrant EVI1 expression is seen in many cases of AML without 3q26 rearrangement. The prognosis of these cases is similarly dismal, but the mechanism that causes aberrant EVI1 expression in the absence of 3q26 chromosomal abnormalities is largely unknown. To identify key regulatory factors essential for EVI1 expression in AML, we performed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) knockout screen. We knocked in the green fluorescent protein (GFP) coding sequence at the EVI1 locus in the human myeloid leukemia cell line K562, which expresses high levels of EVI1 without a 3q26 chromosomal aberration. After transduction of a genome-wide CRISPR/Cas9 knockout library, transduced cells were separated into GFP High and GFP Low subpopulations by flow cytometry. By sequencing the uniquely attached barcodes, we estimated the effect of gene knockout for each gene. Genes counted more frequently in the GFP Low fraction were listed as candidates for activators of EVI1. The top 13 genes, including three apparent positive controls such as EVI1 and two ribosome-related genes, had a false discovery rate (FDR) of less than 1%. We validated these candidate genes in K562 and two other EVI1-overexpressing AML cell lines by transducing individual single guide RNAs targeting these genes. These analyses identified zinc finger protein 91 (ZFP91) as a potential regulatory factor that significantly upregulates EVI1 expression common to the three independent myeloid leukemia cell lines. ZFP91 is an atypical E3 ubiquitin ligase, a DNA-binding protein that functions as a transcription factor. ZFP91 promotes cell growth and inhibits apoptosis in AML and has been shown in previous studies to activate multiple oncogenic pathways, including nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and hypoxia-inducible factor-1α (HIF-1α). We demonstrated that knockout of ZFP91 in K562 substantially reduced EVI1 protein expression to undetectable levels by Western blotting (Figure 1). EVI1 mRNA expression also decreased dramatically after ZFP91 knockout. These results suggest that ZFP91 may play an indispensable role in EVI1 transcription. We performed chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) targeting the EVI1 promoter in K562 and demonstrated that ZFP91 knockout clones showed approximately 10-fold weaker signals for active histone modifications (H3K4me3 and H3K27ac) at the EVI1 promoter (Figure 2). In addition, chromatin immunoprecipitation sequencing (ChIP-seq) for ZFP91 in K562 revealed the presence of a distinct peak as well as the ZFP91-specific motif sequence within the EVI1 promoter region. These data strongly suggest that ZFP91 functions as an essential transcription factor for EVI1 gene overexpression in some EVI1-associated myeloid leukemias. We confirmed that ZFP91 knockout in K562 inhibited cell proliferation, which is consistent with previous studies in other AML cell lines. Based on these results, we believe that ZFP91 has the potential to serve as a therapeutic target in EVI1-associated AML. To conclude, we have identified the transcription factor ZFP91 as a novel prominent activator of EVI1 expression in some EVI1-associated AML. Our study suggests that ZFP91 may be the key to unraveling the pathogenesis of EVI1-associated AML and an attractive therapeutic target in EVI1-associated AML.

Orthogonal Proteogenomic Approaches Identify the Druggable PA2G4-MYC Axis in 3q26 AML

Despite next-generation technologies improved our understanding of the complexity of high-risk acute myeloid leukemia (AML), effective targeted therapies are delayed due to the prevalence of major oncogenic disease drivers such as RAS, RUNX1, TP53. In some cases, the integration of orthogonal omics led to the identification or repurposing of drugs for "undruggable" transcription factors (TFs), prospecting a benefit for patients with the most aggressive leukemia subtypes. Here, we sought to discover how to target the Ecotropic Viral Integration Site-1 gene (EVI1/MECOM), the most lethal oncogenic TF in AML carrying chromosome 3q26 abnormalities. To this end, we intersected phenotypic, gene expression-based and proteomic high-throughput approaches for small molecule and target identification. We screened a collection of 5294 compounds enriched for Food and Drug Administration (FDA) approved or bioactive drugs at 1 µM and identified those that caused more than 95% reduction of cell viability compared to vehicle control in MOLM1 and UCSD/AML1 cell lines carrying 3q26 rearrangments (EVI1high). We then selected a set of sentinel genes that defined an EVI1 "on" and "off" transcriptional signature based on the TF1 EVI1high cell line treated with an EVI1-directed siRNA or control, and used the Connectivity Map to identify inducers of an EVI1 "off" state. Histone deacetylase inhibitors (HDACi) scored as the top class of compounds able to repress leukemia proliferation by suppressing the EVI1 signature. We focused on the compounds with pan (AR-42, belinostat) or selective (entinostat) HDAC inhibitory properties. These molecules induced apoptosis and decreased the levels of EVI1 in AML cell lines and in primary EVI1high blast cells. Interestingly, the expression of EVI1 sensitizes cells to HDAC inhibition both in an inducible EVI1 cell model and in EVI1high compared to EVI1low AML cell lines. Primary EVI1high blasts were also more sensitive to HDAC inhibition compared to those from other AML subtypes, while equally resistant to chemotherapy agents. We translated these results in a "N-of-1" clinical trial proposing entinostat in association with azacytidine to patients with relapse/refractory 3q26 AML based on a compassionate use program for this regimen. Pharmacodynamic modulation of EVI1 was assessed at 6 hours (T6) after entinostat administration in patients and in matched patient-derived AML xenografts (PDLX). EVI1 expression markedly decreased in circulating blasts collected at T6 compared to T0 (before treatment), and EVI1 depletion paralleled clinical and hematologic improvements at follow-up evaluations. This effect was not observed in patients treated with cytarabine. Longitudinal single-cell RNA sequencing (scRNASeq) of bone marrow AML cells collected before and after patients' treatment cycle revealed an inhibitory effect on Myc target genes in the leukemic clone. This effect was also observed in cell lines treated with AR-42, belinostat and entinostat suggesting that different HDACi regulates the Evi1-Myc axis in 3q26 AML. Not surprisingly, HDACi mediated or genetic silencing of EVI1 led to a decrement of MYC in EVI1high AML models. To dissect the EVI1 co-transcriptional complex and explain for the modulation of Myc signaling in EVI1high AML, we performed rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME). Sixty-nine proteins co-immunoprecipitated with endogenous EVI1 both in cell lines and in 3q26 PDLX models. Eleven of these EVI1 interactors were targets of Myc signaling, including PA2G4, a RNA-binding protein that was markedly modulated by HDACi. Importantly, PA2G4 silencing or inhibition (WS6) depletes MYC at transcriptional and protein levels recapitulating the phenotypic effect seen with HDACi in EVI1high cell models. Consequently, WS6 treatment reduced the growth of AML cells in PDLX models in vivo by suppressing an EV1-MYC signature at single-cell leukemia level without inducing widespread toxicities. In contrast, PA2G4 overexpression rescued EVIhigh AML cell lines from the effects induced by HDACi inhibition. These data support the evidence that PA2G4 is a mediator of Evi1-Myc axis in AML. In conclusion, our work positions PA2G4 at the crosstalk of the Evi1-Myc leukemogenic signal for developing new therapeutics and, at this interesting time, urges upfront HDACi-based combination therapies in patients with 3q26 AML.

AKT inhibition sensitizes EVI1 expressing colon cancer cells to irinotecan therapy by regulating the Akt/mTOR axis.

Ecotropic viral integration site 1 (EVI1) is an oncogenic transcription factor that has been attributed to chemotherapy resistance in different cancers. As yet, however, its role in colon cancer drug resistance is not completely understood. Here, we set out to investigate the functional and therapeutic relevance of EVI1 in colon cancer drug resistance. The EVI1 gene was knocked down in colon cancer cells that were subsequently tested for susceptibility to irinotecan using in vitro assays and in vivo subcutaneous mouse colon cancer models. The effect of EVI1 knockdown on the AKT-mTOR signaling pathway was assessed using cell line models, immunohistochemistry and bioinformatics tools. The anti-proliferative activity of AKT inhibitor GSK690693 and its combination with irinotecan was tested in colon cancer cell line models (2D and 3D). Finally, the therapeutic efficacy of GSK690693 and its combination with irinotecan was evaluated in xenografted EVI1 expressing colon cancer mouse models. We found that EVI1 knockdown decreased cancer stem cell-like properties and improved irinotecan responses in both cell line and subcutaneous mouse models. In addition, we found that EVI1 downregulation resulted in inhibition of AKT/mTOR signaling and RICTOR expression. Knocking down RICTOR expression increased the cytotoxic effects of irinotecan in EVI1 downregulated colon cancer cells. Co-treatment with irinotecan and ATP-competitive AKT inhibitor GSK690693 significantly reduced colon cancer cell survival and tumor progression rates. Inhibition of the AKT signaling cascade by GSK690693 may serve as an alternative to improve the irinotecan response in EVI1-expressing colon cancer cells.

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.

EVI1 activates tumor-promoting transcriptional enhancers in pancreatic cancer.

Cancer cells utilize epigenetic alterations to acquire autonomous capabilities for tumor maintenance. Here, we show that pancreatic ductal adenocarcinoma (PDA) cells utilize super-enhancers (SEs) to activate the transcription factor EVI1 (ecotropic viral integration site 1) gene, resulting in activation of an EVI1-dependent transcription program conferring PDA tumorigenesis. Our data indicate that SE is the vital cis-acting element to maintain aberrant EVI1 transcription in PDA cells. Consistent with disease progression and inferior survival outcomes of PDA patients, we further show that EVI1 upregulation is a major cause of aggressive tumor phenotypes. Specifically, EVI1 promotes anchorage-independent growth and motility in vitro and enhances tumor propagation in vivo. Mechanistically, EVI1-dependent activation of tumor-promoting gene expression programs through the stepwise configuration of the active enhancer chromatin attributes to these phenotypes. In sum, our findings support the premise that EVI1 is a crucial driver of oncogenic transcription programs in PDA cells. Further, we emphasize the instructive role of epigenetic aberrancy in establishing PDA tumorigenesis.

Role of Arsenic Trioxide in EVI-1 Apoptosis in Patients with Acute Myeloid Leukaemia

Background:High expression of the ecotropic viral integration site-1 (EVI-1) is an independent negative prognostic indicator of survival in leukaemia patients. Zebrafish (Danio rerio) is a vertebrate animal model commonly used to examine haematopoiesis and myeloid malignancies. To clarify the molecular mechanisms of EVI-1, we previously introduced the human EVI-1 gene into embryonic zebrafish through a heat-shock promoter and established the stable germ-line Tg(EVI-1: HSE: EGFP) zebrafish (Shen et al, 2013). Arsenic trioxide (As2O3, ATO) is one of the effective anticancer drugs, especially for patients with leukaemia (Udupa et al, 2017). We thus aimed to explore the anticancer effects of ATO and the underlying functions associated with EVI-1 in an in vivo zebrafish model and in AML cells in vitro.Results:We determined EVI-1 expression in mononuclear cells isolated from the bone marrow and peripheral blood of AML patients and healthy donors by RT-qPCR and Western blot analysis. EVI-1 was highly expressed in primary AML (Fig 1A). Then, EVI-1 expression was detected in five leukaemia cell lines (K562, HL-60, U937, THP-1 and MV4-11) and normal PBMCs. Among these five leukaemia cell lines, THP-1 has the highest EVI-1 expression (Fig 1B and Fig1C).Primary acute monocytic leukaemia cells from one patient with high expression of EVI-1 were treated with ATO. We found ATO could significantly decrease EVI-1 mRNA (Fig 2A). Between the ATO-treated groups and the control group, the expression of EVI-1 were significantly reduced in the THP-1 cell line (Fig 2B). Next, we evaluated the EVI-1 expression in Tg(EVI-1: HSE; EGFP) transgenic zebrafish embryos over dose courses of ATO exposure (Fig 2C). Consistent with the results of our in vitro study, ATO decreased EVI-1 expression in a dose-dependent manner after 72 h (Fig 2C). Taken together, these results indicate that ATO is an inhibitor of EVI-1 expression both in vivo and in vitro.We investigate whether the reduction of THP-1 cells viability is due to apoptosis, THP-1 cells were incubated with 3 µM of ATO for 24 h, 48 h or 72 h. In the light microscopy images, THP-1 cells exhibited typical apoptotic characteristics (Fig 3A). The proportion of apoptotic cells was represented as early apoptotic cells (annexin V+/PI- staining, the lower right quadrant) plus late apoptotic cells (annexin V+/PI+ staining, the upper right quadrant) (Fig 3B). In cytometric analysis, ATO increased the percentage of apoptotic THP-1 cells in a dose- and time-dependent manner. We found that ATO increased the expressions of JNK, p-JNK, p-P53, PUMA, Bax, caspase-9 and caspase-3 (including cleaved caspase-9 and -3) but decreased the expressions of Bcl-2 and Bcl-xl (Fig 3C).To further verify the role of the JNK pathway in ATO-mediated THP-1 cell apoptosis, we examined if the inhibitor of JNK (SP600125) could reverse ATO-induced apoptosis in THP-1 cells. We found SP600125 not only decreased the pro-apoptotic effect of ATO in the THP-1 cell line (Fig 4A and Fig 4B) but also decreased the activation of the JNK-mediated apoptotic signalling pathway (Fig 4C). SP600125 silenced the activation of JNK by completely inhibiting the phosphorylation of JNK but had little effect on EVI-1 expression (Fig 4C).To test whether EVI-1 modulates apoptosis via the JNK signalling pathway, we transiently transfected THP-1 cells with EVI-1 siRNA which significantly reduced EVI-1 expression (Fig 5A). Silencing EVI-1 had a significant effect on the activation of the JNK pathway and the induction of THP-1 cell apoptosis (Fig 5B and Fig 5C).Conclusion:Our study demonstrated that the apoptotic pathway in THP-1 cells induced by ATO is closely associated with the oncogene EVI-1, the pro-apoptotic protein JNK, p-JNK, p-P53, PUMA, Bax, caspase-9 and caspase-3 (including cleaved caspase-9 and cleaved caspase-3), and the anti-apoptotic proteins Bcl-2 and Bcl-xL. ATO can downregulate EVI-1 mRNA and oncoprotein and block the repression of EVI-1 in the JNK pathway. Furthermore, the activated JNK signalling pathway regulated the expression level of apoptosis-associated proteins, including p-P53, PUMA, Bax, Bcl‐xL, Bcl‐2, Bax, caspase-9 and caspase-3(Fig 6). These findings may provide a novel theoretical basis for the development of personalized medical strategies for the treatment of EVI-1 positive AML patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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

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.

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

Phosphorylation of the Leukemic Oncoprotein EVI1 on Serine 196 Modulates DNA Binding, Transcriptional Repression and Transforming Ability

The EVI1 (ecotropic viral integration site 1) gene at 3q26 codes for a transcriptional regulator with an essential role in haematopoiesis. Overexpression of EVI1 in acute myeloid leukaemia (AML) is frequently associated with 3q26 rearrangements and confers extremely poor prognosis. EVI1 mediates transcriptional regulation, signalling, and epigenetic modifications by interacting with DNA, proteins and protein complexes. To explore to what extent protein phosphorylation impacts on EVI1 functions, we analysed endogenous EVI1 protein from a high EVI1 expressing Fanconi anaemia (FA) derived AML cell line. Mass spectrometric analysis of immunoprecipitated EVI1 revealed phosphorylation at serine 196 (S196) in the sixth zinc finger of the N-terminal zinc finger domain. Mutated EVI1 with an aspartate substitution at serine 196 (S196D), which mimics serine phosphorylation of this site, exhibited reduced DNA-binding and transcriptional repression from a gene promotor selectively targeted by the N-terminal zinc finger domain. Forced expression of the S196D mutant significantly reduced EVI1 mediated transformation of Rat1 fibroblasts. While EVI1-mediated serial replating of murine haematopoietic progenitors was maintained by EVI1-S196D, this was associated with significantly higher Evi1-trancript levels compared with WT-EVI1 or EVI1-S196A, mimicking S196 non-phosphorylated EVI1. These data suggest that EVI1 function is modulated by phosphorylation of the first zinc finger domain.

Zinc finger transcription factor ecotropic viral integration site 1 is induced by all-trans retinoic acid (ATRA) and acts as a dual modulator of the ATRA response

Ecotropic viral integration site 1 (EVI1) plays important roles in leukaemia and development, and its expression is temporally and spatially highly restricted during the latter process. Nevertheless, the only physiological agent that to date has been shown to regulate transcription of this gene in mammalian cells is all-trans retinoic acid. Here we describe the identification of a retinoic acid response element that was located in the most distal of several alternative first exons of the human EVI1 gene and was constitutively bound by canonical retinoid receptors in NTERA-2 teratocarcinoma cells. Furthermore, it was the target of negative feedback by EVI1 on the induction of its own promoter by retinoic acid. This process required a previously described transcription repression domain of EVI1. Extending its role as a modulator of the retinoic acid response, EVI1 had the opposite effect on the RARβ retinoic acid response element, whose induction by all-trans retinoic acid it enhanced through a mechanism that involved almost all of its known functional domains. Augmentation of the retinoic acid response by EVI1 was also observed for the endogenous RARβ gene. Thus, we have established EVI1 as a novel type of modulator of the retinoic acid response, which can both enhance and repress induction by this agent in a promoter-specific manner.

Hematologic and Clinical Features of 3q21q26 Syndrome: Extremely Poor Prognosis and Association with Central Diabetes Insipidus

3q21q26 syndrome includes chromosomal abnormalities of inv(3)(q21q26), t(3;3) (q21;q26), and ins(3;3)(q26;q21q26). It causes hematological diseases by the leukemogenic mechanism that the enhancer of ribophorin I gene in 3q21 induces the transcription of ecotropic viral integration site-1 gene in 3q26. Recently, it has been proposed that the 3q21q26 syndrome may be preceded by diabetes insipidus (DI), particularly when combined with monosomy 7, and is a unique disease entity. From May 2001 to June 2006, a total of 5 patients with hematologic malignancy were found to have 3q21q26 syndrome and monosomy 7. Laboratory findings, clinical data, and association with DI were investigated. The rearrangement type of 3q21q26 was inv(3)(q21q26) in four patients and t(3;3)(q21; q26) in one. These patients' French American British types were AML M1, M2, M4 and M7, showing evident dysmegakaryopoiesis. Aberrant antigenic expressions of CD7 and CD56 were observed. The platelet count was relatively high as AML. All the five patients were refractory or in early relapse. Patient 5 was diagnosed with AML M7 20 days after being diagnosed with DI. While DI was well controlled with oral desmopressin, leukemia was refractory to chemotherapy. This study supports the recent opinion that 3q21q26 syndrome with monosomy 7 combined with DI is a disease of unique characteristics. In the relation between DI and monosomy 7 or 3q21q26 syndrome, there has been no explanation about how acquired abnormality of hematopoietic cells affects production of DDAVP by neurohormonal cells in hypothalamus. The mechanism needs further study, and this research should contribute to the understanding of genetic roles in leukemia appearing in different forms.