Ecotropic Viral Integration Site 1 Expression 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 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.

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

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

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

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

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

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

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

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

Low EVI1 expression at diagnosis predicted poor outcomes in pediatric Ph-negative B cell precursor acute lymphoblastic leukemia patients

Abnormally high ecotropic viral integration site 1 (EVI1) expression has been recognized as a poor prognostic factor in acute myeloid leukemia patients. However, its prognostic impact in B cell precursor acute lymphoblastic leukemia (BCP-ALL) remains unknown. A total of 176 pediatric Ph-negative BCP-ALL patients who received at least 1 course of chemotherapy and received chemotherapy only during follow-up were retrospectively tested for EVI1 transcript levels by real-time quantitative PCR at diagnosis, and survival analysis was performed. Clinical and EVI1 expression data of 129 pediatric BCP-ALL patients were downloaded from therapeutically applicable research to generate effective treatments (TARGET) database for validation. In our cohort, the median EVI1 transcript level was 0.33% (range, 0.0068–136.2%), and 0.10% was determined to be the optimal cutoff value for patient grouping by receiver operating characteristic curve analysis. Low EVI1 expression (<0.10%) was significantly related to lower 5-year relapse-free survival (RFS) and overall survival (OS) rates (P = 0.017 and 0.018, respectively). Multivariate analysis showed that EVI1 expression <0.10% was an independent adverse prognostic factor for RFS and OS. TARGET data showed that low EVI1 expression tended to be related to a lower 5-year OS rate (P = 0.066). In conclusion, low EVI1 expression at diagnosis could predict poor outcomes in pediatric Ph-negative BCP-ALL patients receiving chemotherapy. Supplemental data for this article is available online at https://doi.org/10.1080/08880018.2021.1939818 .

GFI1 Is a Downstream Target of EVI1 in Normal Hematopoiesis

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

Role of miR-96/EVI1/miR-449a Axis in the Nasopharyngeal Carcinoma Cell Migration and Tumor Sphere Formation.

The Wnt signaling pathway is one of the major signaling pathways used by cancer stem cells (CSC). Ecotropic Viral Integration Site 1 (EVI1) has recently been shown to regulate oncogenic development of tumor cells by interacting with multiple signaling pathways, including the Wnt signaling. In the present study, we found that the Wnt modulator ICG-001 could inhibit the expression of EVI1 in nasopharyngeal carcinoma (NPC) cells. Results from loss-of-function and gain-of-function studies revealed that EVI1 expression positively regulated both NPC cell migration and growth of CSC-enriched tumor spheres. Subsequent studies indicated ICG-001 inhibited EVI1 expression via upregulated expression of miR-96. Results from EVI1 3′UTR luciferase reporter assay confirmed that EVI1 is a direct target of miR-96. Further mechanistic studies revealed that ICG-001, overexpression of miR-96, or knockdown of EVI1 expression could restore the expression of miR-449a. The suppressive effect of miR-449a on the cell migration and tumor sphere formation was confirmed in NPC cells. Taken together, the miR-96/EVI1/miR-449a axis is a novel pathway involved in ICG-001-mediated inhibition of NPC cell migration and growth of the tumor spheres.

EVI1 as a Marker for Lymph Node Metastasis in HNSCC.

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

Downregulation of EVI1 Expression Inhibits Cell Proliferation and Induces Apoptosis in Hilar Cholangiocarcinoma via the PTEN/AKT Signalling Pathway.

Aims: Hilar cholangiocarcinoma (HCCA) is a tumour with high malignancy, low surgical resection potential, and a poor prognosis. Ecotropic Viral Integration site 1 (EVI1) is a transcriptional regulator that has been proven to be associated with tumourigenesis and progression in many human solid tumours. However, the expression of EVI1 and its role in HCCA progression remain unclear. The aim of this study was to clarify the association between EVI1 expression and clinical outcomes in patients with HCCA.Methods: The expression of EVI1 in HCCA tissue samples and cell lines was examined by quantitative real-time PCR (qRT-PCR), Western blotting, and immunohistochemistry (IHC). Kaplan-Meier analysis was used for survival analysis. A log-rank test was performed for univariate analysis of survival, and a Cox regression model was utilized for multivariate analysis of survival. Cell proliferation was measured by cell counting kit-8 (CCK-8), colony formation, and 5-ethynyl-2'-deoxyuridine (EdU) assays. The cell cycle was evaluated by flow cytometry. Cell apoptosis was detected by flow cytometry and a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) assay. In vivo tumour growth was observed for xenografts in nude mice.Results: EVI1 expression was upregulated in HCCA tissue samples and correlated with a poor prognosis. In clinical specimens, the expression of EVI1 correlated with tumour histological grade and tumour size. Knocking down EVI1 expression reduced HCCA cell proliferation, blocked cell cycle progression, and promoted apoptosis in vitro and in vivo. Furthermore, we found that EVI1 could regulate the AKT signalling pathway by regulating PTEN levels in HCCA.Conclusion: Our data revealed that EVI1 played important roles in HCCA tumourigenesis and development. Our findings suggest that EVI1 may be a potentially useful therapeutic target in HCCA.

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

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

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

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

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.

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.

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

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

EVI-1 modulates arsenic trioxide induced apoptosis through JNK signalling pathway in leukemia cells

High expression of the oncogene ecotropic viral integration site-1 (EVI-1) is an independent negative prognostic indicator of survival in leukemia patients. This study aimed to examine the effects of arsenic trioxide (ATO) on EVI-1 in acute myeloid leukemia (AML). Mononuclear cells were isolated from the bone marrow and peripheral blood of AML patients and healthy donors. EVI-1 expression in hematopoietic cells was evaluated by RT-qPCR and Western blot analysis. EVI-1 was highly expressed in both primary AML and leukemia cell lines (THP-1 and K562). ATO down-regulated EVI-1 mRNA in zebrafish in vivo as well as in primary leukemia cells and THP-1 and K562 cells in vitro. Additionally, ATO treatment induced apoptosis, down-regulated both EVI-1 mRNA and oncoprotein expression, increased the expression of pro-apoptosis proteins, and decreased the expression of anti-apoptotic proteins in leukemia cells in vitro. EVI-1 expression in leukemia cells (THP-1 and K562) transduced with EVI-1 siRNA was significantly reduced. Silencing EVI-1 had a significant effect on the activation of the JNK pathway and the induction of leukemia cell apoptosis. ATO may downregulate EVI-1 mRNA and oncoprotein levels and block the inhibitory effects of EVI-1 on the JNK pathway, which activates the JNK apoptotic pathway, thereby leading to the apoptosis of EVI-1 in AML patients.

Negative regulation of microRNA-9 by ecotropic viral integration site1 involved in the pathogenesis of acute myeloid leukemia

Objective To investigate the regulation mechanism of microRNA-9(miR-9)by ecotropic viral integration site1(EVI1)its impact on proliferation of AML cells and its role in the pathogenesis of myelogenous leukemia. Methods EVI1 was forced to express in Uocm1 cell lines by murine stem cell virus-EVI1(MSCV-EVI1) plasmid infection.EVI1 overexpressed Uocm1 cells were then treated with 0.1 μmol/L 5-aza-2′-deoxycytidine (5-AZA) dissolved in dimethyl sulfoxide (DMSO). The methylation level of miR-9 promoter was tested by DNA bisulfite sequencing technology.The cell cycle was observed by flow cytometry (FCM). The proliferation ability of the cells was detected by the colony forming assay in semi-solid Methylcellulose medium culture. Results EVI1 level was dramatically increased after being infected by MSCV-EVI1 plasmid.Forced expression of EVI1 in Uocm1 significantly downregulated miR-9 by inducing hypermethylation of miR-9 promoter.Relative expression level of miR-9 was lower in EVI1 overexpressed group(0.004±0.000)than that of the control group(0.006±0.001)(t=4.09, P<0.05). When EVI1 was overexpressed in Uocm1, the rate of G0/G1 cells decreased markedly(P<0.05), while rates of S phage and G2 phage increased significantly(all P<0.05). Seven days after 500 cells plated in semi-solid medium, EVI1 overexpressed Uocm1 cells gave rise to more colony (122.3±7.8) than Uocm1 cells infected with vector(45.7±6.1)(t=-13.44, P<0.01).0.1 μmol/L 5-AZA recovered miR-9 expression(P<0.01)by decreasing EVI1 induced hypermethylation of miR-9 promoter.G0/G1 phase cell proportion was(48.25±2.19)% in control group, while (65.90±2.90)% in 5-AZA group(t=-6.85, P<0.05).5-AZA group formed less colony (51.00±10.01)than the control group (123.40±8.12) (t=9.59, P<0.01), which indicated that 5-AZA inhibited cell proliferation by G0/G1 cell cycle retardation in EVI1 overexpressed uocm1 cells. Conclusions EVI1 may enhance proliferation ability of myeloid leukemia cells by downregulating miR-9 through inducing hypermethylation of miR-9 promotor, which plays a crucial role in the pathogenesis of AML.5-AZA may be an effective hypomethylating agent in the therapy of EVI1 high acute myeloid leukemia. Key words: Ecotropic viral integration site 1; MicroRNA-9; Acute myeloid leukemia; Pathogenesis; 5-aza-2′-deoxycytidine