Ecotropic Viral Integration Site 1 Overexpression Research Articles

Actionable Findings from an Unbiased Drug Screen for Novel Single Agent and Combination Therapies Against AML with Mecom Re-Arrangement

EVI1 (Ecotropic Viral Integration Site 1) is a zinc finger transcriptional regulator encoded from the MECOM (MDS1 and EVI1 Complex) locus at chromosome 3q26.2. Aberrant EVI1 overexpression is observed in approximately 10% of de novo AML, of which approximately half are due to chromosomal rearrangement involving either inv3(q21;q26.2) or t(3;3)(q21;q26.2), where the distal GATA2 hematopoietic enhancer is relocated and drives EVI1 expression. In addition to the resulting haploinsufficiency of GATA2, monosomy 7 and activating mutations in the RAS pathway and SF3B1 commonly co-occur in 3q26.2-rearranged (r) AML. AML with EVI1 overexpression generally have a stem-like phenotype, are refractory to current therapy options and exhibit poor overall survival. Among the targets of EVI1 are the ERG, MYC, KIT, Bcl-xL and MPL genes. GATA2 repression, coupled with increased EVI1 activity, promotes AML progression and an aggressive phenotype in 3q26.2-rearranged AML. Therefore, there is a glaring need to develop and test novel targeted therapies with improved efficacy in repressing EVI1 and improving survival of AML with EVI1 overexpression. To identify druggable vulnerabilities in 3q26.2-r AML, we conducted an unbiased high-throughput drug screen, utilizing a library of 2480 oncology-focused, mechanistically annotated drugs (referred as NCATS Mechanism Interrogation Plates or-MIPE 5.0) (Cancer Cell. 2021; 39:566-579), against seven AML cell lines, i.e., four with 3q26.2-r: UCSD-AML1, HNT-34, AML191 and AML194, and three non-3q26.2-r AML: SET-2, MV4-11 and OCI-AML3. The library exploits mechanistic redundancy by including multiple inhibitors against well-validated oncology targets, while simultaneously encompassing mechanistic diversity, altogether targeting more than 850 distinct mechanisms of action. We exploited this redundancy to identify druggable, target-level dependencies in 3q26.2-r AML, both in terms of absolute potency and relative activity as compared to other AML subtypes. BRD4 was identified as an absolute dependency in 3q26.2-r AMLs. This was consistent with previous reports that BET inhibitors (e.g., OTX015, mivebresib or ABBV-075 and JQ1) are effective against 3q26.2-r AML cell lines, patient-derived (PD) AML cells and PDX models. Comparison of 3q26.2-r versus other AML cell lines also identified XIAP, mTOR, PIK3CA and Bcl-xL as druggable vulnerabilities in 3q26.2-r AML. In follow-up experiments, XIAP/cIAPs inhibitors birinapant (10-1000 nM) or SM-164 (30-1000 nM), chosen based on the MIPE screen outcomes, induced significantly more dose-dependent apoptosis in 3q26.2-r versus the other AML cell lines. This was associated on Western analyses with decline in XIAP, c-IAP1/2, p-ERK1/2, MCL1 and Bcl-xL, but increased protein levels of cleaved caspase-3 and PARP in UCSD-AML1 and AML191 cells. Mivebresib (50-250 nM) or OTX015 (100-500 nM) also induced dose-dependent apoptosis, and reduced EVI1, c-Myc, c-Myb, XIAP, c-IAP1/2, CDK4/6 and Bcl-xL while increasing protein levels of HEXIM1 and cleaved PARP. Consistent with this, co-treatment with birinapant with mivebresib was synergistically lethal in UCSD-AML1 and AML191 cells, with Delta Synergy scores > 1.0 calculated by the ZIP method. Treatment with the dual mTOR/PIK3CA inhibitor NVP-BGT226 (1-30 nM) or navitoclax or Bcl-xL-specific BH3 mimetic A-1155463 also exerted lethality and synergistically induced apoptosis with mivebresib in AML cells with inv3/t(3;3). We previously reported that the TBL1/β-catenin antagonist tegavivint induced apoptosis in 3q26.2-r PD AML cells and PDX models. Co-treatment with birinapant and tegavivint also synergistically induced apoptosis in 3q26.2-r AML cells. Finally, in the xenograft model of AML194-Luc cells in NSG mice, monotherapy with birinapant (30 mg/kg/t.i.w, Intraperitoneal), vs vehicle control, significantly reduced the AML burden, without causing toxicity. Additionally, compared to each drug or vehicle control, co-treatment with birinapant and the BETi OTX015 (30 mg/kg/day, by oral gavage) was more effective in reducing AML burden in the xenograft model. These findings demonstrate promising preclinical activity of IAP protein inhibition against the cellular models of AML with inv3/t(3;3) with EVI1 overexpression, supporting the rationale to further evaluate in vivo efficacy of birinapant and/or BETi-based combinations against this AML sub-type.

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.

EVI1-mediated Programming of Normal and Malignant Hematopoiesis.

Ecotropic viral integration site 1 (EVI1), encoded at the MECOM locus, is an oncogenic zinc finger transcription factor with diverse roles in normal and malignant cells, most extensively studied in the context of hematopoiesis. EVI1 interacts with other transcription factors in a context-dependent manner and regulates transcription and chromatin remodeling, thereby influencing the proliferation, differentiation, and survival of cells. Interestingly, it can act both as a transcriptional activator as well as a transcriptional repressor. EVI1 is expressed, and fulfills important functions, during the development of different tissues, including the nervous system and hematopoiesis, demonstrating a rigid spatial and temporal expression pattern. However, EVI1 is regularly overexpressed in a variety of cancer entities, including epithelial cancers such as ovarian and pancreatic cancer, as well as in hematologic malignancies like myeloid leukemias. Importantly, EVI1 overexpression is generally associated with a very poor clinical outcome and therapy-resistance. Thus, EVI1 is an interesting candidate to study to improve the prognosis and treatment of high-risk patients with "EVI1high" hematopoietic malignancies.

EVI1 Promotes Immune-Evasive Microenvironment Via Cyclin D1 in Acute Myeloid Leukemia

Inappropriate expression of Ecotropic viral integration site 1 (EVI1) has been associated with dismal clinical outcomes in acute myeloid leukemia (AML), while EVI1high (EVI1+) AML cells do not show clear resistance to cytotoxic agents in vitro. Since EVI1 is essential for normal hematopoietic stem cells (HSCs), we performed chromatin-immunoprecipitation (ChIP)- and RNA-sequencing of murine EVI1+ AML cells and normal hematopoietic cells to identify factors involved explicitly in AML, and thereby to elucidate molecular cues to refractory EVI1+ AML. We firstly employed a murine EVI1-transformed AML (EVI1-AML) model, where murine hematopoietic cells exogenously expressing 3×FLAG-tagged EVI1 were transplanted into syngeneic mice. Using EVI1-AML cells, we next performed ChIP coupled to next-generation sequencing (ChIP-seq) by anti-FLAG tag antibody and transcriptome analysis by RNA-seq. We further generated another murine EVI1+ AML model by retrovirally transducing the KMT2A-MLLT1 fusion gene into Lineage- Sca-1+ c-kit+ cells from EVI1-GFP knock-in mice, where Evi1-IRES-GFP allele was inserted into the Evi1 locus to enable real-time monitoring of Evi1 expression by GFP, followed by transplantation into lethally irradiated syngeneic mice. Intriguingly, GFPhigh L-GMPs from KMT2A-MLLT1 AML mice showed distinct expression profiles from GFPlow L-GMPs, including high NF-kB activity and low stem cell activity, along with the lower frequency of leukemia-initiating cells in GFPhigh fraction. Consistently, an exogenous expression of EVI1 in KMT2A-MLLT1 AML cells without Evi1 expression decreased colony-forming and leukemia-initiating activity, suggesting a differential role of EVI1 in AML from normal HSCs, where EVI1 is essential in maintaining stem cell function. Combined with functional screening by cell proliferation-related short hairpin RNAs (shRNAs), we identified that cyclin D1 (CCND1) was a downstream target, commonly implied in the above analyses, and a potential therapeutic vulnerability of EVI1-AML. EVI1 bound to the region 3.8 kb upstream of the transcription start site of Ccnd1, through which EVI1 demonstrated strong transcriptional activity in a luciferase reporter assay. Genetic silencing of Ccnd1 and pharmacologic inhibition of Cyclin D1 in EVI1- and EVI1+ KMT2A-MLLT1 AML cells negatively affected proliferation and colony-forming activity in vitro. Moreover, in vivo knockdown of Ccnd1 strikingly impaired the leukemogenic capacity of murine EVI1-AML models. It was surprising that transcriptomic analysis of EVI1-AML cells with Ccnd1 silencing showed the downregulation of genes involved in chemokine production and response to interferon instead of cell cycle-related genes. The expression of Cd274 (PD-L1) as well as Stat1, the primary signaling mediator for type I and II IFN, was decreased in EVI1-AML cells with Ccnd1 knockdown. EVI1 directly bound to the promoter region of Stat1 and activated its transcription in line with a previous report, and indirectly up-regulated STAT1 expression via Cyclin D1. Accordingly, infiltrating CD4 and CD8 T cells from EVI1-shCcnd1 AML mice demonstrated decreased exhaustion markers, including LAG3, PDCD1, and TIGIT. Silencing of Stat1 and Ifngr, the receptor for IFN-γ, impaired the leukemogenic capacity of EVI1-AML in vivo, with a decrease in T and NK cells with exhaustion phenotypes, while Ifnar, the receptor for IFN-α, was not involved in this process. Genetic inhibition of the IFN-γ-STAT1 axis in non-EVI1+ AML models did not affect AML development, nor the frequency of immune cells expressing exhaustion-related molecules, suggesting this axis is specifically important in EVI1+ AML. By using human AML datasets, overexpression of EVI1 and CCND1 were associated with IFN-γ signature, including up-regulation of STAT1 and CD274, and elevated expression of chemokines, with increased exhaustion molecules in T cells. These data collectively suggest that cyclin D1 is implicated in the development of EVI1-AML through the formation of IFN-γ signature and exhausted T cell phenotypes, which could be potentially targeted.

Clinical features and prognostic implications of ecotropic viral integration site 1 (EVI1) in childhood acute lymphoblastic leukemia

In contrast to the extensive knowledge on EVI1 in myeloid malignancies, few data are available on the EVI1 transcript in pediatric ALL. The purpose of this study was to examine the clinical and biological significance of EVI1 and validate its prognostic significance in pediatric patients with ALL. Here, we examined the clinical and biological significance of EVI1 expression, as measured by real-time polymerase chain reaction (PCR) in 837 children with newly diagnosed ALL treated on the National Protocol of Childhood Leukemia in China (NPCLC)-ALL-2008 protocol, and aimed to explore their prognostic significance in pediatric ALL patients. The EVI1 expression was detected in 27 of 837 (3.2%) patients. No statistically significant differences in prednisone response, complete remission (CR) rates and relapse rates were found between EVI1 overexpression (EVI1+) group and EVI1− group. Moreover, we found no significant difference in event-free survival (EFS) and overall survival (OS) between these two groups, also multivariate analysis did not identify EVI1+ as an independent prognostic factor. In the subgroup analysis, there was no difference in clinical outcome between EVI1+ and EVI1− patients in standard‑risk (SR), intermediate-risk (IR) and high-risk (HR) groups. In the minimal residual disease (MRD)<10−4 group, EVI1+ patients have significantly lower EFS and OS rates compared to EVI1− patients. Further large‑scale and well‑designed prospective studies are required to confirm the results in the future.

EVI1 overexpression promotes ovarian cancer progression by regulating estrogen signaling

High-grade serous ovarian cancer (HGSOC) is characterized by TP53 mutation and somatic copy number alterations (SCNAs). Here we show that the oncogenic transcription factor EVI1 (ecotropic viral integration site-1) is amplified and overexpressed up to 30% of 1640 HGSOC cases in The Cancer Genome Atlas (TCGA). Functionally, EVI1 promotes proliferation/invasion in vitro and tumor growth of xenograft model in vivo. Importantly, we discover that EVI1 regulates estrogen signaling by directly activating ESR1 (estrogen receptor 1) transcription determined by the ChIP and luciferase assay. Interestingly, EVI1 and ESR1 share common regulatory targets as indicated by the analysis of ChIP-Seq data. EVI1 and ESR1 collaborate in the regulation of some estrogen receptor-regulated genes. Furthermore, EVI1 drives tumor aggressiveness partially by regulating estrogen signaling. Estrogen enhances the proliferation, invasion and xenograft growth of ovarian cancer cells. Importantly, estrogen can rescue the inhibition of proliferation, invasion and xenograft growth induced by silencing EVI1. These findings suggest that EVI1 functions as a novel regulator of the estrogen signaling network in ovarian cancer.

EVI‑1 acts as an oncogene and positively regulates calreticulin in breast cancer.

Ecotropic viral integration site-1 (EVI-1) is an important transcription factor involved in oncogenesis. Aberrant EVI-1 expression has been reported to be a characteristic of multiple types of malignancies; however, very little is known about how EVI-1 regulates breast cancer. Current knowledge of how target genes mediate the biological function of EVI-1 remains limited. In the present study, overexpression of EVI-1 promoted cell proliferation, migration, and invasion, and inhibited apoptosis in breast cancer. By contrast, silencing of EVI-1 inhibited cell proliferation, migration and invasion, and enhanced apoptosis in breast cancer. In addition, the results also revealed that the aberrant expression of EVI-1 regulates genes associated with the apoptotic pathway in breast cancer. Furthermore, EVI-1 was also likely to target the promoter region of calreticulin (CRT) in vitro. It was concluded that EVI-1 can affect epithelial mesenchymal transition-associated genes by regulating the expression of CRT in breast cancer. The results revealed that EVI-1 may be a potential effective therapeutic target in breast 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.

Primary acute myeloid leukemia cells with overexpression of EVI-1 are sensitive to all-trans retinoic acid

AbstractEnhanced expression of ecotropic viral integration site 1 (EVI-1) occurs in ∼10% of acute myeloid leukemia (AML) patients and is associated with a very poor disease outcome. Patients with EVI-1–positive AML have poor initial responses to chemotherapy and high relapse rates, indicating an urgent need for alternative treatment strategies improving clinical outcome for these patients. Because treatment of acute promyelocytic patients with all-trans retinoic acid (ATRA) has improved the survival of these patients substantially, we investigated whether ATRA might also be effective for the subgroup of AML patients with EVI-1 overexpression. Here, we show that a substantial part of the EVI-1–positive AML cases respond to ATRA by induction of differentiation and decreased clonogenic capacity of myeloid blasts. Most importantly, we demonstrate that in vivo treatment of primary EVI-1–positive AML with ATRA leads to a significant reduction in leukemic engraftment. Altogether, our results show that a considerable part of the EVI-1–positive primary AML cases are sensitive to ATRA, suggesting that combining ATRA with the currently used conventional chemotherapy might be a promising treatment strategy decreasing relapse rates and enhancing complete remissions in this poor prognostic subgroup of AML patients.

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

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.

EVI1, A Potential Regulator Of CEBPA; A Contributory Mechanism In Leukemogenesis?

Ecotropic Viral Integration Site 1 (EVI1) is a zinc finger oncoprotein implicated in a subtype of acute myeloid leukemia associated with poor prognosis. Leukemic cells overexpressing EVI1 display a block in myeloid differentiation and resistance to apoptosis, both of which are reversed with EVI1 shRNA knockdown. Previous mutagenesis studies have shown that DNA binding via the first set of zinc fingers (ZF1) is critical for leukemic transformation by EVI1. The mediators between EVI1 DNA-binding and leukemogenesis are mostly unknown. Using chromatin immunoprecipitation and sequencing (ChIP-Seq) in myeloid leukemic cells, we have recently identified an evolutionarily conserved EVI1 binding site 35 kilobases downstream of Cebpa, encoding the CCAAT enhancer-binding protein alpha, a master regulator of early granulopoiesis. In order to obtain a better understanding of the potential regulatory role of this region in leukemogenesis, we used genome database analysis to see what other transcription factors or epigenetic marks are present. ChIP-Seq data deposited in the UCSC databank indicates the presence of CTCF, GATA2 and CEBPβ, as well as TAL1, p300, and GATA1 in leukemic cells, the latter three of which are known to bind together in a complex. This data suggests that the Cebpa +35 region serves an important role in regulating hematopoietic development and Cebpa transcription. In order to characterize the binding of EVI1 ZF1 to the Cebpa +35 site, we performed methylation interference assays and oligonucleotide mutagenesis assays. EVI1 ZF1 binds within this +35kb control region to the sequence TGACAGTGACGG with 133% affinity relative to the canonical motif. In an effort to assess any biological effects linked to EVI1 binding at the Cebpa +35 motif, we conducted EVI1 overexpression studies in the EML hematopoietic progenitor cell line. While control EML cells exhibited marked upregulation of Cebpa upon induction, EVI1-transduced cells displayed a significant suppression of Cebpa transcript. To determine if this suppression requires DNA binding, we tested a mutant of EVI1 that is defective in DNA binding (EVI1R205N); this was unable to suppress Cebpa induction. To test whether EVI1 can bind to the +35 KB site, we performed ChIP-PCR, which indeed showed EVI1 occupancy at the +35 KB site. In conclusion, we have shown evidence that EVI1 suppresses Cebpa transcription in early hematopoietic progenitor cells, and that the EVI1 binding site at Cebpa +35 serves an important function in myeloid maturation. This regulatory node represents a possible therapeutic target for the treatment of acute myeloid leukemia. Disclosures:No relevant conflicts of interest to declare.

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.

EVI1 oncogene promotes KRAS pathway through suppression of microRNA-96 in pancreatic carcinogenesis

Despite frequent KRAS mutation, the early molecular mechanisms of pancreatic ductal adenocarcinoma (PDAC) development have not been fully elucidated. By tracking a potential regulator of another feature of PDAC precursors, acquisition of foregut or gastric epithelial gene signature, we herein report that aberrant overexpression of ecotropic viral integration site 1 (EVI1) oncoprotein, which is usually absent in normal pancreatic duct, is a widespread marker across the full spectrum of human PDAC precursors and PDAC. In pancreatic cancer cells, EVI1 depletion caused remarkable inhibition of cell growth and migration, indicating its oncogenic roles. Importantly, we found that EVI1 upregulated KRAS expression through suppression of a potent KRAS suppressor, miR-96, in pancreatic cancer cells. Collectively, the present findings suggest that EVI1 overexpression and KRAS mutation converge on activation of the KRAS pathway in early phases of pancreatic carcinogenesis and propose EVI1 and/or miR-96 as early markers and therapeutic targets in this dismal disease.

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.

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.

Deregulated EVI1 Expression Leads to Genomic Instability and G1 Cell Cycle Arrest

Abstract 2431Insertional activation of the zinc finger transcription factor ecotropic viral integration site 1 (EVI1) by promoter and enhancer elements within the gamma-retroviral vector LTR has led to clonal dominance and malignant transformation in clinical gene therapy of chronic granulomatous disease (cGD). EVI1 has been shown to inhibit stress induced cell death or TGFβ signaling and is essential for embryonic development. Cytogenetic rearrangements leading to the activation of the gene locus are a marker for poor prognosis in myeloid malignancies. Very little is known about its larger splice variant MDS1/EVI1 in normal or malignant hematopoiesis. We aim to systematically analyze the role of deregulated EVI1 and MDS1/EVI1 expression in hematopoiesis to further elucidate the stepwise progression of clonal selection up to malignant transformation.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. Analysis of growth kinetics revealed a 1.5 – 3 fold lower proliferation of ME and E expressing cells as compared to eGFP control vector transduced cells. Additional analysis on cell cycle distribution revealed that in both, E and ME transduced cells, a higher percentage of cells could be detected in the G1/G0 phase of the cell cycle (52.5 ± 1.6% in ME cells and 55.2 ± 1.8% in E cells) compared to untransduced (48.9 ± 1.6%) and control vector cells (47.7 ± 0.3%) (p<0.01). Consequently, a 1.3 – 1.4 fold lower proportion of E and ME cells was observed in the S/G2/M phases compared to control cells. 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 of cells in sub-G1 phase but a 1.3 fold increase of cells in G1/G0 phase 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). In line with this, we observed a decrease of eGFP+ E and ME transduced human hematopoietic CD34+ cells from 8% at day 3 after transduction to 0.5 – 2.5% at day 14, respectively. In contrast, the proportion of eGFP+ human primary cells remained stable for the time period analyzed after transduction with the control vector. To investigate E and ME overexpression in vivo, hematopoietic stem and progenitor cells were isolated from murine bone marrow, transduced with the lentiviral vectors (LV) and transplanted into lethally irradiated recipient mice. Although all recipients showed donor cell engraftment, eGFP expression dropped from 47.7 – 49% at 4 to 7 weeks to 0.9 – 4.4% at 40 weeks after transplantation (BMT). Interestingly, EVI1 transplanted mice showed a significantly lower thrombocyte recovery (2.2 fold; p<0.05) within the first 13 weeks after BMT in comparison to untransduced, control vector or ME transplanted mice. We then asked if the cell cycle arrest in G1 is associated with genetic instability, as patients with insertional activation of EVI1 developed a myelodysplastic syndrome with monosomy 7. E and ME transduced human fibroblasts showed a significant higher percentage of cells with abnormal centrosome numbers as compared to controls (p<0.05). In addition, staining of γ-H2AX, an indirect marker for double strand breaks (DSB), in E and ME transduced HL60 cells revealed that EVI1+ γ-H2AX+ cells were 2-fold enriched in G1 as compared to control vector transduced cells.In summary, our data show that EVI1 overexpression causes G1 cell cycle arrest of hematopoietic cells that may be caused by genetic instability or inefficient DSB repair. No sign for clonal selection could be detected, neither in vitro nor in vivo, but EVI1 transplanted mice showed a delay in thrombocyte recovery. Systematic investigation of EVI1 and MDS1/EVI1 overexpression in human hematopoietic cells will help us to gain insights into regulatory processes of hematopoietic stem cells and mechanisms leading to dominant clones in gene modified hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Frequent EVI1 translocations in myeloid blast crisis CML that evolves through tyrosine kinase inhibitors

Clinical variables associated with ecotropic viral integration site 1 (EVI1) translocations were evaluated in 42 consecutive chronic myeloid leukemia (CML) patients in myeloid blast crisis (MBC). Translocations were confirmed with fluorescence in situ hybridization, and Western blot analysis demonstrated EVI1 expression. Translocations of EVI1 were present in 3 of 24 (12%) patients whose disease evolved MBC before tyrosine kinase inhibitor (TKI) exposure, and 7 of 18 (39%) patients who had received one or more TKIs. Univariate analysis showed that prior TKI therapy was the only clinical variable that was significantly associated with EVI1 translocation (P = 0.047). TKI-resistant BCR-ABL1 mutations were present in 71% of MBC patients with EVI1 translocations at the time of disease progression. These observations suggest that EVI1 overexpression collaborates with BCR-ABL1 in the evolution of TKI-resistant MBC. Inhibition of c-ABL kinase-mediated DNA double-strand repair by TKIs may predispose to EVI1 translocation in this setting.