Prognostic significance of the EVI1 gene expression in patients with acute myeloid leukemia: a meta-analysis.

Ecotropic virus integration site-1 (EVI1) is frequently expressed in patients with acute myeloid leukemia (AML). Many studies have reported the potential poor prognostic impact of EVI1 higher expression (EVI1H) in the AML patients; however, the conclusions previously reported have not been fully assessed and are still controversial. Therefore, we performed a meta-analysis to evaluate the prognostic significance of EVI1H in patients with AML. The primary endpoint was overall survival (OS), and the event-free survival (EFS) was selected as the secondary endpoint. We extracted the hazard ratio (HR) and their 95% confidence interval (CI) for the OS and EFS from the multivariate COX proportional hazard models. A total of 4767 AML patients from 11 studies up to 23 February 2019 were subjected to our meta-analysis. Pooled HRs suggested that EVI1H had an adverse impact on OS (HR = 1.52, 95%CI 1.24-1.86) and EFS (HR = 1.41, 95%CI 1.14-1.74) in AML patients. EVI1H was also associated with a shorter OS (HR = 1.73, 95%CI 1.43-2.11) and EFS (HR = 1.17, 95%CI 1.05-1.31) in AML patients with the intermediate cytogenetic risk (ICR) according to the National Comprehensive Cancer Network (NCCN), European leukemia network (ELN), or International System for Human Cytogenetic Nomenclature (ISCN). Furthermore, EVI1H appeared to be a poor prognosis indicator in patients with normal cytogenetics (NC) (HR for OS:2.01, 95%CI 1.32-3.05; HR for EFS 1.54, 95%CI 1.09-2.17) and young patients (HR for OS 1.30, 95%CI 1.09-1.55), respectively. This meta-analysis indicates EVI1H has an independent and significantly adverse prognostic impact on AML patients in the entire population, and this conclusion same applies to some subgroups like AML patients with ICR, NC, and young AML patients.

Identification of novel MECOM gene fusion and personalized therapeutic targets through integrative clinical sequencing in secondary acute myeloid leukemia in a patient with severe congenital neutropenia: a case report and literature review.

Severe congenital neutropenia (SCN) is a rare hematologic disorder characterized by defective myelopoiesis and a high incidence of malignant transformation to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). SCN patients who develop MDS/AML have excessive toxicities to traditional chemotherapy, and safer therapies are needed to improve overall survival in this population. In this report, we outline the use of a prospective integrative clinical sequencing trial (PEDS-MIONCOSEQ) in a patient with SCN and AML to help identify oncogenic targets for less toxic agents. Integrative sequencing identified two somatic cis-mutations in the colony stimulating factor 3 receptor (CSF3R) gene, a p.T640N mutation in the transmembrane region and a p.Q768* truncation mutation in the cytoplasmic domain. A somatic mutation p.H105Y, in the runt homology domain (RHD) of runt-related transcription factor 1 (RUNX1), was also identified. In addition, sequencing discovered a unique in-frame EIF4A2-MECOM (MDS1 and ectopic viral integration site 1 complex) chromosomal translocation with high MECOM expression. His mutations in CSF3R served as potential targets for tyrosine kinase inhibition and therefore provided an avenue to avoid more harmful therapy. This study highlights the utility of integrative clinical sequencing in SCN patients who develop leukemia and outlines a strategy on how to approach these patients in a future clinical sequencing trial to improve historically poor outcomes. A thorough review of leukemia in SCN and the role of CSF3R mutations in oncologic therapy are provided to support a new strategy on how to approach MDS/AML in SCN.

Prominent Oncogenic Roles of EVI1 in Breast Carcinoma.

Overexpression of the EVI1 oncogene is associated typically with aggressive myeloid leukemia, but is also detectable in breast carcinoma where its contributions are unexplored. Analyzing a tissue microarray of 608 breast carcinoma patient specimens, we documented EVI1 overexpression in both estrogen receptor-positive (ER+) and estrogen receptor-negative (ER-) breast carcinomas. Here, we report prognostic relevance of EVI1 overexpression in triple-negative breast carcinoma but not in the HER2-positive breast carcinoma subset. In human breast cancer cells, EVI1 silencing reduced proliferation, apoptosis resistance, and tumorigenicity, effects rescued by estrogen supplementation in ER+ breast carcinoma cells. Estrogen addition restored ERK phosphorylation in EVI1-silenced cells, suggesting that EVI1 and estradiol signaling merge in MAPK activation. Conversely, EVI1 silencing had no effect on constitutive ERK activity in HER2+ breast carcinoma cells. Microarray analyses revealed G-protein-coupled receptor (GPR) signaling as a prominent EVI1 effector mechanism in breast carcinoma. Among others, the GPR54-ligand KISS1 was identified as a direct transcriptional target of EVI1, which together with other EVI1-dependent cell motility factors such as RHOJ regulated breast carcinoma cell migration. Overall, our results establish the oncogenic contributions of EVI1 in ER- and HER2-negative subsets of breast cancer. Cancer Res; 77(8); 2148-60. ©2017 AACR.

Stem cell-like transcriptional reprogramming mediates metastatic resistance to mTOR inhibition.

Inhibitors of the mechanistic target of rapamycin (mTOR) are currently used to treat advanced metastatic breast cancer. However, whether an aggressive phenotype is sustained through adaptation or resistance to mTOR inhibition remains unknown. Here, complementary studies in human tumors, cancer models and cell lines reveal transcriptional reprogramming that supports metastasis in response to mTOR inhibition. This cancer feature is driven by EVI1 and SOX9. EVI1 functionally cooperates with and positively regulates SOX9, and promotes the transcriptional upregulation of key mTOR pathway components (REHB and RAPTOR) and of lung metastasis mediators (FSCN1 and SPARC). The expression of EVI1 and SOX9 is associated with stem cell-like and metastasis signatures, and their depletion impairs the metastatic potential of breast cancer cells. These results establish the mechanistic link between resistance to mTOR inhibition and cancer metastatic potential, thus enhancing our understanding of mTOR targeting failure.

Suppression of Evi1 promotes the osteogenic differentiation and inhibits the adipogenic differentiation of bone marrow-derived mesenchymal stem cells in vitro.

Osteoporosis (OP) is considered a complex disease with a strong genetic impact, mainly affecting post-menopausal women and is also a common cause of fracture. Elucidating the molecular mechanisms that regulate the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) is crucial to developing treatment strategies to combat OP. In the present study, we found that ectopic viral integration site­1 (Evi1) was highly expressed during the process of adipogenesis of rat BMSCs. Notably, Evi1 levels markedly increased on day 3 of adipogenic differentiation following the addition of adipogenic induction supplements. In addition, we interfered with the expression of the Evi1 gene in the adipogenesis of BMSCs by supplementing adenoviral plasmids and measured the expression levels of bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN), peroxisome proliferator­activated receptor γ2 (PPARγ2) and lipoprotein lipase (LPL) by RT-qPCR and western blot analysis. The mRNA and protein levels of osteogenic and adipogenic markers in the BMSCs were up­ and downregulated, respectively following the silencing of siEvi1. Our experimental results substantiate that the suppression of Evi1 in BMSCs by RNA interference inhibits adipogenic differentiation, while it promotes osteogenic differentiation. The results from our study demonstrated that the Evi1 gene may be targeted as a therapeutic strategy for promoting bone formation.

Zebrafish nephrogenesis is regulated by interactions between retinoic acid, mecom, and Notch signaling.

The zebrafish pronephros provides a conserved model to study kidney development, in particular to delineate the poorly understood processes of how nephron segment pattern and cell type choice are established. Zebrafish nephrons are divided into distinct epithelial regions that include a series of proximal and distal tubule segments, which are comprised of intercalated transporting epithelial cells and multiciliated cells (MCC). Previous studies have shown that retinoic acid (RA) regionalizes the renal progenitor field into proximal and distal domains and that Notch signaling later represses MCC differentiation, but further understanding of these pathways has remained unknown. The transcription factor mecom (mds1/evi1 complex) is broadly expressed in renal progenitors, and then subsequently marks the distal tubule. Here, we show that mecom is necessary to form the distal tubule and to restrict both proximal tubule formation and MCC fate choice. We found that mecom and RA have opposing roles in patterning discrete proximal and distal segments. Further, we discovered that RA is required for MCC formation, and that one mechanism by which RA promotes MCC fate choice is to inhibit mecom. Next, we determined the epistatic relationship between mecom and Notch signaling, which limits MCC fate choice by lateral inhibition. Abrogation of Notch signaling with the γ-secretase inhibitor DAPT revealed that Notch and mecom did not have additive effects in blocking MCC formation, suggesting that they function in the same pathway. Ectopic expression of the Notch signaling effector, Notch intracellular domain (NICD), rescued the expansion of MCCs in mecom morphants, indicating that mecom acts upstream to induce Notch signaling. These findings suggest a model in which mecom and RA arbitrate proximodistal segment domains, while MCC fate is modulated by a complex interplay in which RA inhibition of mecom, and mecom promotion of Notch, titrates MCC number. Taken together, our studies have revealed several essential and novel mechanisms that control pronephros development in the zebrafish.

Therapeutic advances in acute myeloid leukemia.

The choice of treatment approach and outcome in acute myeloid leukemia (AML) depends on the age of the patient. In younger patients, arbitrarily defined as being younger than 60 years, 70% to 80% enter complete disease remission with several anthracycline-based chemotherapy combinations. Consolidation with high-dose cytarabine or stem-cell transplantation in high-risk patients will restrict overall relapse to approximately 50%. A number of demographic features can predict the outcome of treatment including cytogenetics and an increasing list of molecular features (ie, FLT3, NPM1, MLL, WT1, CEBPalpha, EVI1). These are increasingly being used to direct postinduction therapy, but they are also molecular targets for a new generation of small molecule inhibitors that are in early development; however, randomized data have yet to emerge. In older patients who comprise the majority, which will increase with demographic change, the initial clinical decision to be made is whether the patient should receive an intensive or nonintensive approach. If the same anthracycline/cytarabine-based approach is deployed, the remission rate will be around 50%, but the risk of subsequent relapse is approximately 85% at 3 years. This difference from younger patients is explained partly by the ability of patients to tolerate effective therapy, and also the aggregation of several poor risk factors compared with the young. There remains a substantial proportion of patients older than 60 years who do not receive intensive chemotherapy. Their survival is approximately 4 months, but there is considerable interest in developing new treatments for this patient group, including novel nucleoside analogs and several other agents.

In vivo expansion of MDR1-transduced cells accompanied by a post-transplantation chemotherapy regimen with mitomycin C and methotrexate.

BACKGROUND: A recurrent breast cancer patient received high-dose chemotherapy, a transplant of multidrug resistance 1 (MDR1)-transduced cells and four different protocols of post-transplantation chemotherapy. We report the analysis of MDR1-transduced cells in this patient. METHODS: MDR1 transgene levels in the peripheral blood mononuclear cells of the patient were evaluated by polymerase chain reaction (PCR). Retroviral integration sites of the MDR1-transduced cells were identified by linear amplification-mediated (LAM)-PCR. RESULTS: Twelve days after transplantation, approximately 1% of the peripheral blood mononuclear cells were MDR1 transgene-positive. The transgene levels decreased quickly, and were at low levels until day 504. A remarkable increase in MDR1 transgene-positive cells was observed on day 532, during combination chemotherapy with mitomycin C and methotrexate. Using LAM-PCR, 31 MDR1-transduced clones were identified, and eight of these were long-life clones that survived for more than 500 days. Among the 31 clones, ten had a retroviral integration site near genes listed in the Retroviral Tagged Cancer Gene (RTCG) Database. Two long-life clones, N-30 and N-31, had retroviral integration sites within the MDS1-EVI1 locus. Another two long-life clones had integration sites close to PRDM16 or CUEDC1. CONCLUSIONS: These results suggest that MDR1-transduced cells were enriched in vivo by an MDR1 substrate, mitomycin C. The possible activation of EVI1 or other RTCGs by retroviral insertion may have affected the survival and persistence of a proportion of the transduced cells.

Expression and prognostic significance of different mRNA 5'-end variants of the oncogene EVI1 in 266 patients with de novo AML: EVI1 and MDS1/EVI1 overexpression both predict short remission duration.

Rearrangements of chromosome band 3q26.2 lead to overexpression of the EVI1 gene and are associated with a poor prognosis in myeloid malignancies. EVI1 is also overexpressed in some cases without 3q26 rearrangements. To uncover its prognostic significance in this patient group, however, it may be necessary to distinguish among several known 5'-end variants of its mRNA. According to a recent report, overexpression of the transcript variant EVI1_1d was associated with shortened survival in acute myeloid leukemia (AML), but overexpression of MDS1/EVI1, whose protein product differs structurally and functionally from that of all other known EVI1 5'-end variants, was not. The aim of the present study was to determine, for the first time, the expression and prognostic significance of all known EVI1 5'-end variants in AML. Quantitative RT-PCR was used to measure the expression of EVI1_1a, EVI1_1b, EVI1_1d, EVI1_3L, and MDS1/EVI1 in 266 samples from patients with de novo AML. To correlate expression of the EVI1 5'-end variants with survival parameters, regression analyses were performed. 41/266 patients (15.4%) overexpressed at least one, but more often several or all, EVI1 transcript type(s). High expression of each of the EVI1 mRNA variants, including MDS1/EVI1, was significantly associated with shortened continuous complete remission in the total patient population as well as in the subgroups of patients with intermediate risk or normal cytogenetics. The present study therefore shows that high levels of each of the known EVI1 mRNA 5'-end variants represents an adverse prognostic factor in de novo AML without 3q26 rearrangements. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.

Evi-1 expression in Xenopus.

The Evi-1 gene was first identified as a site for viral integration in murine myeloid leukemia. Evi-1 is a zinc finger transcription factor that has been implicated in the development of myeloid neoplasia. In humans, disruption of the Evi-1 locus, by chromosomal rearrangements, is associated with myeloid leukemia and myelodyplastic syndromes. Here, we report the cloning and developmental pattern of expression of Xenopus Evi-1. xEvi-1 is expressed during oogenesis and during embryonic development. In situ hydridization reveals that xEvi-1 has a dynamic expression profile during early embryonic development. Expression of Evi-1 is detected by in situ hybridization in the pronephric tissue, the brain and in neural crest derivatives of the head and neck.

CtBP, an unconventional transcriptional corepressor in development and oncogenesis.

CtBP family proteins are conserved among vertebrates and invertebrates and function as transcriptional corepressors. They repress transcription in a histone deacetylase-dependent or -independent manner. CtBPs play important roles during development and oncogenesis. In this review, their unusual properties, the mechanisms of transcriptional repression, regulation, and their biological functions are discussed.

Identification of candidate target genes for EVI-1, a zinc finger oncoprotein, using a novel selection strategy.

We have sought to identify and isolate target genes for the zinc finger protein, EVI-1, which has been implicated in the genesis of myelogenous leukemia both in mouse and human. We have approached this with a two-step selection: we first selected for genomic fragments of mouse DNA that bind to the protein with high affinity; second, we employed cDNA hybrid selection to identify gene sequences contained within these fragments. We show that we have constructed a sublibrary of genomic fragments that contains a significant fraction of the EVI-1-binding sites in the mouse genome. Our data has allowed us to estimate that there are approximately 4300 binding sites per haploid genome in the mouse. We further demonstrate that by using cDNA hybrid selection, it is relatively straightforward to isolate cDNAs that correspond to genes embedded in the EVI-1-binding sublibrary. Several of these are novel, but are represented in databases of anonymous human or mouse cDNAs (expressed sequence tags). One selected gene is Itpr2, encoding the inositol trisphosphate type two receptor, which is transcriptionally regulated during myelopoiesis. Finally, using a chimeric EVI-1-VP16-fusion protein under the control of a tetracycline-regulated system, we have shown that this chimeric activator can directly regulate Itpr2.

Identification of translocational breakpoints within the intron region before the last coding exon (exon 12) of the EVI1 gene in two cases of CML-BC with inv(3)(q21q26).

We have previously shown that the EVI1 gene at chromosome 3q26 is transcriptionally activated by chromosomal rearrangements in acute myelogenous leukemias (AMLs) with inv(3)(q21q26) or t(3;3)(q21;q26). The breakpoints in t(3;3) cases were 15 to 330 kb upstream of the EVI1 gene, while those in inv(3) cases were 150 to 200 kb downstream and outside of the EVI1 coding region. The EVI1 gene is also activated in chronic myelogenous leukemia-blastic crisis (CML-BC) with inv(3)(q21q26); however, the molecular mechanism of EVI1 activation in CML-BC is still unclear. In this paper, we have analyzed chromosomal rearrangements in two leukemia cell lines derived from CML-BC with inv(3)(q21q26) and have identified the breakpoints within the EVI1 coding area. The EVI1 gene spans over 100 kb with 12 exons (10 coding exons), and the chromosomal breakpoints are clustered in the intron region before the last coding exon (exon 12). The nucleotide sequence of EVI1 cDNA clones from MOLM-1 cells was truncated at exon 11, and a novel sequence of 681 nucleotides was added at the 3' end of the EVI1 transcripts. The novel sequence was derived from a readthrough intron sequence 3' to the coding exon 11 adjacent to the breakpoint. The breakpoints at 3q21 were within the breakpoint cluster area downstream of the ribophorin I gene, suggesting that the activation mechanism of the EVI1 gene in CMLs with inv(3) is the same as that in AMLs with inv(3). These results indicate that expression of a truncated EVI1 gene is an important factor in the progression of CML.

Structurally altered Evi-1 protein generated in the 3q21q26 syndrome.

Overexpression of the Evi-1 gene appears to be a consistent feature of the 3q21q26 syndrome, an association of myeloid leukemias/myelodysplastic syndrome with a specific chromosomal aberration involving both 3q21 and 3q26, such as t(3;3)(q21;q26) or inv(3)(q21q26). The rearrangement in 3q26 has been reported to occur near the Evi-1 locus, implicating that it is the critical gene deregulated in the 3q21q26 syndrome. Here we present a structural abnormality of Evi-1 protein in a case with the 3q21q26 syndrome. In this case carrying typical inv(3)(q21q26), the 3q26 breakpoint is located within an intron of the Evi-1 gene, and resulted in overexpression of normally unexpressed, an aberrant form of Evi-1 protein, in which the C-terminal 44 amino acids of wild-type Evi-1 protein were truncated and replaced by five amino acids. The truncated Evi-1 protein is shown to increase AP1 activity when expressed in NIH3T3 cells as its wild-type counterpart. We also show that the origin of this peculiar type of rearrangement of the Evi-1 gene is not an artifact during establishment of the cell line, but is the event that occurred in the primary leukemic cells. Our results strongly support that the primary target for the 3q21q26 syndrome is the Evi-1 gene, and provide the first evidence that the structurally altered Evi-1 gene may be involved in the 3q21q26 syndrome.

Intergenic splicing of MDS1 and EVI1 occurs in normal tissues as well as in myeloid leukemia and produces a new member of the PR domain family.

The EVI1 gene, located at chromosome band 3q26, is overexpressed in some myeloid leukemia patients with breakpoints either 5' of the gene in the t(3;3)(q21;q26) or 3' of the gene in the inv(3)(q21q26). EVI1 is also expressed as part of a fusion transcript with the transcription factor AML1 in the t(3;21)(q26;q22), associated with myeloid leukemia. In cells with t(3;21), additional fusion transcripts are AML1-MDS1 and AML1-MDS1-EVI1. MDS1 is located at 3q26 170-400 kb upstream (telomeric) of EVI1 in the chromosomal region in which some of the breakpoints 5' of EVI1 have been mapped. MDS1 has been identified as a single gene as well as a previously unreported exon(s) of EVI1 We have analyzed the relationship between MDS1 and EVI1 to determine whether they are two separate genes. In this report, we present evidence indicating that MDS1 exists in normal tissues both as a unique transcript and as a normal fusion transcript with EVI1, with an additional 188 codons at the 5' end of the previously reported EVI1 open reading frame. This additional region has about 40% homology at the amino acid level with the PR domain of the retinoblastoma-interacting zinc-finger protein RIZ. These results are important in view of the fact that EVI1 and MDS1 are involved in leukemia associated with chromosomal translocation breakpoints in the region between these genes.