EVI1 Protein Research Articles

Targeting Super-Enhancers Driving EVI1 Expression in Leukemia By Inhibition of p300/CBP

Introduction One of the most aggressive types of acute myeloid leukemia (AML) is caused by aberrant activation of EVI1 as a result of the hijacking of enhancers repositioned by structural rearrangements. One of these enhancers, the MYC-blood enhancer cluster ( MYC-BENC) (Bahr et al. 2018) activates EVI1 in AML patients with t(3;8). MYC-BENC consists of several enhancer modules, of which only one is essential for activating EVI1: deletion of this element abrogates MYC-BENC to EVI1 promoter interaction as well as transcription of EVI1 (Ottema et al. 2021). Approach Given the central role of acetyltransferases p300 and CBP in enhancer activation, in particular super-enhancers such as MYC-BENC, we wondered whether we could specifically inhibit MYC-BENC driven EVI1 transcription using p300/CBP inhibitors. We studied this in a previously reported t(3;8) K562 model (Ottema et al. 2021), in which we introduced GFP 3' of EVI1 as a readout for MYC-BENC-driven EVI1 expression. Results ChIP-seq in t(3;8) cells showed that p300 binds to the promoter of EVI1 and to MYC-BENC, whereas CBP only binds to MYC-BENC (Fig. A). Following rapid p300/CBP degradation (3 hours) with the heterobifunctional p300/CBP degrader dCBP-1 (Vannam et al. 2021), we found a severe reduction of nascent EVI1 (and MYC) transcripts (SLAM-seq), as well as of EVI1 protein levels, i.e. by EVI1-eGFP, intracellular anti-EVI1-PE staining (Fig. B) and western blot.. Importantly, p300/CBP degradation was accompanied by loss of H3K27ac at MYC-BENC, but not at the promoter of EVI1 (Fig. A). Thus, the activation of EVI1 in t(3;8) AML appears strongly dependent on the presence of p300/CBP at MYC-BENC super-enhancer. Importantly, in healthy HSPCs, we observed that EVI1 expression was not strongly downregulated upon p300/CBP degradation (Fig. B). These data suggest that MYC-BENC driven EVI1 transcription in AML can be targeted, while leaving EVI1 transcription in normal HSPCs unaffected. We next studied whether loss of enhancer activity was dependent on functional domains within p300/CBP using specific inhibitors of either the acetyltransferase domain (A485) or bromodomain (GNE781). Treatment of t(3;8) EVI1-GFP cells with single inhibitors had a mild effect on EVI1 expression, but simultaneous A485/GNE781 treatment showed strong synergy in reducing EVI1 levels (Fig. B). Dual inhibition also led to a profound loss of H3K27ac signal at the MYC-BENC locus, but not at the EVI1 promoter (Fig. A). Thus, both domains in CBP/p300 are required to activate EVI1 expression via the hijacked MYC-BENC enhancer. As with dCBP-1, we found that EVI1 expression levels in healthy HSPCs were not affected by A485 and GNE781 treatment (Fig. B). We hypothesized that A485/GNE781 treatment would only cause loss of p300/CBP activity, but p300 ChIP-sequencing showed that A485/GNE781 treatment caused strong loss of p300 binding at the enhancer while the total protein levels of p300 and CBP were not affected. p300 binding at the EVI1 promoter was not decreased by A485/GNE781 treatment. In line with this, we observed that genome-wide p300 binding at enhancers was reduced at 15939 out of 41160 (39%) putative enhancers upon A485/GNE781 exposure, whereas dual inhibitor treatment only affected p300 binding at 429 out of 12452 promoters (3.5%) (Fig. A), suggesting a high selectivity of p300 inhibitors for long distant regulatory elements. Conclusion In summary, EVI1 is sensitive to pharmacological p300/CBP inhibition when activated by the hijacked MYC-BENC enhancer, but not in normal HSPCs. Globally, the sensitivity of genomic regions to bromo- or acetyltransferase inhibition is highly locus-dependent and is mainly restricted to enhancers. Our findings demonstrate that aberrantly controlled expression of oncogenes by hijacked enhancers in leukemia can be pharmacologically targeted, pointing to a potential therapeutic avenue for patients who are refractory to current treatment regimens.

Preclinical Efficacy of Novel Drug Combination Against AML with 3q26 Lesions and EVI1 Overexpression

The EVI1 gene is located at 3q26.2 in the MECOM locus and encodes for a zinc finger transcription factor (TF). EVI1 protein is expressed in leukemia stem-progenitor cells, supporting their self-renewal and blocking differentiation. As a result of the chromosome translocation t(3;3) or inv(3) at 3q26, the distal enhancer of the TF and tumor suppressor GATA2 is dislocated and repositioned to induce expression of EVI1, while concomitantly repressing GATA2. EVI1 overexpression results in therapy refractoriness and inferior relapse-free and overall survival in AML. Previous reports highlighted that targeting BRD4 with BET inhibitor has activity against AML cells with inv(3)/t(3;3). Among the targets of EVI1 are the pro-growth and pro-survival proteins c-Myc and Bcl-xL, expressions of which are also regulated by enhancer activity that is undermined by BRD4 inhibition. We have recently reported that the nuclear TBL1/R1-β-catenin-TCF7L2 complex transcriptionally regulates c-Myc, cyclin D and Survivin and sustains AML stem cells, such that the undermining of this transcriptional axis by the TBL1/R1-β-catenin-binding disruptor tegavivint (BC-2059, Iterion) represses MYC, cyclin D1 and Survivin and inhibits growth and survival of AML stem-progenitor cells. Based on these observations, we interrogated the pre-clinical efficacy of pan-BET inhibitor OTX015 and/or tegavivint (TV) against AML cells with inv(3)/t(3;3) and overexpression of EVI1. Treatment with BET inhibitor OTX015 dose-dependently (100 to 1000 nM) induced apoptosis of AML cell lines (UCSD-AML1, OCI-AML20, MUTZ-3 and HNT-34) and patient-derived (PD) AML cells (AML191 and AML194) with inv(3)/t(3;3) and with or without monosomy 7. OTX015 treatment was associated with repression of c-Myc, c-Myb, CDK4, MECOM, and MCL1 as well as induction of HEXIM1 and cleaved PARP levels. We also determined that treatment with TV (10-100 nM) dose-dependently induced apoptosis in AML cell lines and PD AML cells with 3q26.2 lesions with/without monosomy 7. Furthermore, TV treatment resulted in attenuation of protein levels of EVI1, TCF7L2, c-Myc, c-Myb, RUNX1, CEBPα, c-KIT, BCL2, Bcl-xL and MCL1, while inducing p21, CD11b, BIM and cleaved PARP levels. Next, utilizing H3K27Ac antibody Hi-ChIP (Arima Hi-ChIP), we interrogated the active gene regulatory interactions through a combined Hi-C and ChIP workflow, followed by Tn5 transposase-mediated on-bead library construction and NGS. We observed that in untreated AML191 cells numerous loop interactions were present between the super-enhancers and promoters respectively of MYC and BCL2. Following TV treatment (100 nM for 16 hours), differential loop calling revealed a loss of these interactions. RNA-Seq and gene set enrichment analysis in AML cells showed that TV treatment caused log2 fold-enrichment of gene sets of TGFβ, inflammatory response, TNFα and interferon signaling, and apoptosis signaling, but negative enrichment of gene sets of MYC, E2F, DNA replication/repair, as well as showed reduction in expression of WNT targets and 17-gene stemness score. Confocal microscopy showed that TV treatment disrupted co-localization EVI1 and β-catenin with TBL1, also confirmed by Proximity Ligation Assay. Mass cytometry (CyTOF) analysis confirmed that TV treatment attenuated EVI1, c-Myc, RUNX1, β-catenin, TBL1/R1, Bcl-xL, BCL2, MCL1 and Ki67 but augmented protein levels of APC and cleaved PARP in the phenotypically characterized AML stem cells (with high expression of CLEC12A, CD123, CD244, CD99) harboring inv(3) and EVI1 overexpression. Co-treatment with TV and OTX015 or the BCL2 inhibitor venetoclax synergistically induced in vitro apoptosis (determined by SynergyFinder 2.0) in the AML cell lines and the PD AML cells. Notably, in the flank-implanted PD AML191 or tail vein AML242 models in NSG mice, treatment with TV and OTX015 for 2 to 5 weeks, compared to vehicle control or each drug alone, significantly reduced AML growth (p < 0.01). Furthermore, in the tail-vein infused and engrafted PD AML191 PDX model, treatment with TV and OTX015 or venetoclax for 6 weeks, as compared to vehicle control or each drug alone, significantly improved overall survival of the NSG mice (p < 0.01), without inducing any toxicity. These findings highlight the promising pre-clinical efficacy of novel combinations of TV and BET protein or venetoclax against AML models harboring 3q26 lesions and EVI1 overexpression.

Oncogene EVI1 Drives Acute Myeloid Leukemia Via a Targetable EVI1-CtBP1/2 Interaction and By Repressing Myeloid Regulator CEBPA

Overexpression of EVI1 (located at 3q26), as a result of 3q26 rearrangements, causes the development of one of the most aggressive, chemotherapy-resistant forms of AML. We hypothesize that insight into the function of EVI1 in AML will lead to the identification of targetable vulnerabilities for those patients. EVI1 encodes a DNA-binding zinc-finger protein that has a critical role in transcriptional regulation and maintenance of quiescent hematopoietic stem cells. Here, we aim to understand the mechanism by which EVI1 overexpression in bone marrow progenitors drives AML development. To identify its target genes, we made EVI1 rapidly degradable by endogenous fusion with an auxin-inducible degron (AID) tag in inv(3q21;3q26) rearranged MUTZ3 leukemia cells. Degradation of EVI1 strongly increased myeloid differentiation of MUTZ3 cells. Using metabolic labeling of newly produced RNA (SLAM-seq), we found 6 genes to be upregulated after 1.5 hrs of degradation. Out of those genes, CEBPA was also upregulated upon shRNA knockdown of EVI1 or after introducing inactivating mutations in the hijacked GATA2 enhancer that drives EVI1 expression. Those findings suggest persistent upregulation of CEBPA when EVI1 levels are reduced. In line with this, CEBPA expression is 1.83 fold (p=0.00024) lower in AML patients with 3q26 rearrangements (n=18) compared to non-3q26 rearranged cases (n=97). Interestingly, overexpression of CEBPA in MUTZ3 cells bypassed EVI1 driven repression and caused myeloid differentiation as well. We next addressed the question how EVI1 represses CEBPA transcription. The CEBPA locus contains 14 enhancers in a 100kb region around the gene body, among which the myeloid specific +42kb enhancer, essential for neutrophil development. EVI1 binds this +42kb enhancer of CEBPA as determined by chromatin immunoprecipitation sequencing (ChIP-seq) in 3q26-rearranged AML and MUTZ3 cells. After degradation of EVI1, H3K27Ac levels increased at this enhancer, indicating that EVI1 reduces activity of the +42kb CEBPA enhancer. Since EVI1 lacks any enzymatic domains that could modulate chromatin, we sought to further understand how EVI1 represses activity of the +42kb CEBPA enhancer. EVI1 protein immunoprecipitation followed by mass spectrometry revealed interaction with CtBP1/2, factors known to interact with repressors such as Polycomb proteins or HDACs. ChIP-seq showed a strong overlap between CtBP2 and EVI1 binding sites in MUTZ3 cells. Moreover, EVI1 elimination caused loss of CtBP2 binding to the chromatin including to the +42kb CEBPA enhancer. EVI1 harbors a PLDLS domain essential for binding to CtBP1/2. Mutation of this domain abolished CtBP2 binding and reduced transformation of mouse bone marrow progenitors by EVI1, as determined by in vitro replating assays. We reasoned we could exploit the high affinity of EVI1 for CtBP through this single domain by overexpressing a repeat of a small section of EVI1 containing only the CtBP-binding PLDLS-site. Overexpression of "4X-PLDLS competitor peptide" was able to sequester CtBP and thereby prevent it's binding to EVI1. The competitor peptide was able to abolish in vitro replating capacity and re-enabled differentiation of mouse bone marrow progenitors transformed with EVI1. In line with this, transduction of the competitor peptide into MUTZ3 cells led to upregulation of CEBPA as well as growth inhibition. Furthermore, in vivo leukemia outgrowth in a xenotransplant model of 3q26 rearranged AML cell lines MUTZ3 and SB1690CB was fully abolished by the competitor peptide, but not by a mutant control peptide. In conclusion, EVI1 is a repressor of active chromatin regions, including CEBPA via the +42kb myeloid specific enhancer. Repression by EVI1 is dependent on its interaction with CtBP1/2, proteins that recruit repressors of transcription via an essential . A PLDLS domain in EVI1 is essential for this interaction. The competitor peptide experiments point to exploiting this interaction with repressive molecules as a potential therapeutic target in AML with overexpression of EVI1.

Expression of Evi-1 Gene in Leukaemia: Diagnostic and Prognostic Perspective

Leukaemias are malignant neoplasms characterized by disorderly, purposeless proliferation of white blood cells with abundance of one cell type. The exact aetiology of leukaemias is still yet to be fully understood. However, it is generally believed that neoplasm is caused by genetic mutation, chromosomal translocation, or activation of certain oncogenes. There are also nuclear oncogenes which are also vital genes in normal cell proliferation and differentiation, often being pivotal genes in developmental and cell cycle regulation. They are also important in cancer progression. Ecotropic viral integration site (EVI-1), a nuclear oncogene has been implicated in the progression of some leukaemias. EVI-1 gene is located on the human chromosome 3 band q24-q28 and spans over 100 kb. The EVI-1 gene encodes a 145 kDa protein of the zinc-finger family which is an essential transcription factor for appropriate murine and human development and is also associated with some leukaemias, following ectopic expression. EVI-1 protein is divided into two main regions: The N-terminal region that contains zinc finger domains (ZFi) and C-terminal region containing three zinc finger domains (ZFii) and a sequence of acidic amino acid. This review summarizes the biological, leukaemogenetic/oncogenic roles and biochemical properties of EVI-1. It further discusses the diagnostic and prognostic implication of EVI-1 in some leukaemias, encouraging incorporation of routine assay of EVI-1 in diagnosis and prognostic monitoring of leukaemias.

Arsenite suppresses the transcriptional activity of EVI1 through the binding to CCHC-type Zn finger domain

Transcription factor EVI1 is essential for normal hematopoiesis in embryos but is aberrantly elevated in bone marrow cells of myelodysplastic syndrome (MDS) patients. EVI1 and its downstream GATA-2 appear to be a possible therapeutic target of MDS. Here we found that treatment of EVI1-expressing K562 cells with arsenite (As(III)) reduced the mRNA and protein levels of EVI1 and GATA-2. A gel shift assay using the nuclear extract of K562 cells showed that As(III) suppressed the DNA-binding activity of EVI1. The DNA-binding activity of the recombinant EVI1 protein was also suppressed by As(III) but was recovered by excess amounts of dithiothreitol, suggesting the involvement of cysteine residues of EVI1. Since the 7th Zn finger domain of EVI1, having a motif of CCHC, is known to be involved in DNA-binding, the synthetic peptide of 7th Zn finger domain was reacted with As(III) and subjected to MALDI-TOF-MS analysis. The results showed that As(III) binds to this peptide via three cysteine residues. As(III)-induced reduction of the DNA-binding activity of the recombinant EVI1 was abolished by the mutations of each of three cysteine residues to alanine in the 7th Zn finger domain. These results demonstrate that As(III) causes the down-regulation of EVI1 and GATA-2 by inhibiting the transcriptional activity of EVI1 through the binding to the cysteine residues of CCHC-type Zn finger domain.

EVI1 as a Prognostic and Predictive Biomarker of Clear Cell Renal Cell Carcinoma.

The transcription factor EVI1 plays an oncogenic role in several types of neoplasms by promoting aggressive cancer features. EVI1 contributes to epigenetic regulation and transcriptional control, and its overexpression has been associated with enhanced PI3K-AKT-mTOR signaling in some settings. These observations raise the possibility that EVI1 influences the prognosis and everolimus-based therapy outcome of clear cell renal cell carcinoma (ccRCC). Here, gene expression and protein immunohistochemical studies of ccRCC show that EVI1 overexpression is associated with advanced disease features and with poorer outcome—particularly in the CC-e.3 subtype defined by The Cancer Genome Atlas. Overexpression of an oncogenic EVI1 isoform in RCC cell lines confers substantial resistance to everolimus. The EVI1 rs1344555 genetic variant is associated with poorer survival and greater progression of metastatic ccRCC patients treated with everolimus. This study leads us to propose that evaluation of EVI1 protein or gene expression, and of EVI1 genetic variants may help improve estimates of prognosis and the benefit of everolimus-based therapy in ccRCC.

MiRNA-206 Regulates EVI1 Gene Expression, Akt Signaling Pathway and Cell Biological Behavior in Gastric Cancer Cells

To investigate the impact of miRNA-206 on the transcriptional expression of EVI1 gene and activation of Akt/JNK signaling pathway in gastric cancer cells, and to provide a new idea for gene-targeted therapy of gastric cancer. The miRNA-206 transfection experiment was firstly used to verify the regulation of EVI1. The experiment was divided into three groups: miRNA-206 mimics (100 nM), miRNA-206 inhibitor (100 nM), miR-NC (100 nM), and transfected into gastric cancer cells sgc7901, Western blot. EVI1 protein expression was detected; then the signal transduction and biological behavior of the cells were verified by miRNA-206 lentiviral vector transfection experiments. The experiment was divided into three groups: pLB-miRNA-206 group, empty vector group and control group (sgc7901 cell group). miRNA-206 and EVI1 mRNA levels were detected by real-time fluorescence quantitative (RT-PCR), and p-Akt and p-JNK were detected by Western blot. Protein expression, cell proliferation was quantified by MTT assay, and the alteration of cell cycle were detected by flow cytometry. miRNA-206 may affect the cell proliferation and division cycle by targeting the regulation of EVI1 transcriptional gene expression and activation of Akt/JNK signaling pathway in gastric cancer cells, and it is expected to provide an important selection site for gene-targeted therapy of gastric cancer.

Erratum: EVI1 carboxy-terminal phosphorylation is ATM-mediated and sustains transcriptional modulation and self-renewal via enhanced CtBP1 association.

The transcriptional regulator EVI1 has an essential role in early hematopoiesis and development. However, aberrantly high expression of EVI1 has potent oncogenic properties and confers poor prognosis and chemo-resistance in leukemia and solid tumors. To investigate to what extent EVI1 function might be regulated by post-translational modifications we carried out mass spectrometry- and antibody-based analyses and uncovered an ATM-mediated double phosphorylation of EVI1 at the carboxy-terminal S858/S860 SQS motif. In the presence of genotoxic stress EVI1-WT (SQS), but not site mutated EVI1-AQA was able to maintain transcriptional patterns and transformation potency, while under standard conditions carboxy-terminal mutation had no effect. Maintenance of hematopoietic progenitor cell clonogenic potential was profoundly impaired with EVI1-AQA compared with EVI1-WT, in particular in the presence of genotoxic stress. Exploring mechanistic events underlying these observations, we showed that after genotoxic stress EVI1-WT, but not EVI1-AQA increased its level of association with its functionally essential interaction partner CtBP1, implying a role for ATM in regulating EVI1 protein interactions via phosphorylation. This aspect of EVI1 regulation is therapeutically relevant, as chemotherapy-induced genotoxicity might detrimentally sustain EVI1 function via stress response mediated phosphorylation, and ATM-inhibition might be of specific targeted benefit in EVI1-overexpressing malignancies.

EVI1 carboxy-terminal phosphorylation is ATM-mediated and sustains transcriptional modulation and self-renewal via enhanced CtBP1 association.

The transcriptional regulator EVI1 has an essential role in early hematopoiesis and development. However, aberrantly high expression of EVI1 has potent oncogenic properties and confers poor prognosis and chemo-resistance in leukemia and solid tumors. To investigate to what extent EVI1 function might be regulated by post-translational modifications we carried out mass spectrometry- and antibody-based analyses and uncovered an ATM-mediated double phosphorylation of EVI1 at the carboxy-terminal S858/S860 SQS motif. In the presence of genotoxic stress EVI1-WT (SQS), but not site mutated EVI1-AQA was able to maintain transcriptional patterns and transformation potency, while under standard conditions carboxy-terminal mutation had no effect. Maintenance of hematopoietic progenitor cell clonogenic potential was profoundly impaired with EVI1-AQA compared with EVI1-WT, in particular in the presence of genotoxic stress. Exploring mechanistic events underlying these observations, we showed that after genotoxic stress EVI1-WT, but not EVI1-AQA increased its level of association with its functionally essential interaction partner CtBP1, implying a role for ATM in regulating EVI1 protein interactions via phosphorylation. This aspect of EVI1 regulation is therapeutically relevant, as chemotherapy-induced genotoxicity might detrimentally sustain EVI1 function via stress response mediated phosphorylation, and ATM-inhibition might be of specific targeted benefit in EVI1-overexpressing malignancies.

MECOM-associated syndrome: a heterogeneous inherited bone marrow failure syndrome with amegakaryocytic thrombocytopenia

AbstractHeterozygous mutations in MECOM (MDS1 and EVI1 complex locus) have been reported to be causative of a rare association of congenital amegakaryocytic thrombocytopenia and radioulnar synostosis. Here we report on 12 patients with congenital hypomegakaryocytic thrombocytopenia caused by MECOM mutations (including 10 novel mutations). The mutations affected different functional domains of the EVI1 protein. The spectrum of phenotypes was much broader than initially reported for the first 3 patients; we found familial as well as sporadic cases, and the clinical spectrum ranged from isolated radioulnar synostosis with no or mild hematological involvement to severe bone marrow failure without obvious skeletal abnormality. The clinical picture included radioulnar synostosis, bone marrow failure, clinodactyly, cardiac and renal malformations, B-cell deficiency, and presenile hearing loss. No single clinical manifestation was detected in all patients affected by MECOM mutations. Radioulnar synostosis and B-cell deficiency were observed only in patients with mutations affecting a short region in the C-terminal zinc finger domain of EVI1. We propose the term MECOM-associated syndrome for this heterogeneous hereditary disease and inclusion of MECOM sequencing in the diagnostic workup of congenital bone marrow failure.

Research process of ecotropic virus integration site-1 protein in hematologic malignancies

Ecotropic virus integration site (Evi)-1 gene is located on chromosome 3 (3q26.2) in the human genome, and it encodes Evi-1 protein which is transcription factors binding to specific DNA. Evi-1 protein is involved in the regulation of RNA transcription. Studies have found that Evi-1 protein expresses abnormally in a variety of hematological tumors and it is involved in regulating tumor cells differentiation, apoptosis, proliferation and related signal transduction pathways. This article reviews literatures on the basic biological characteristics of Evi-1 protein and its research progress in hematologic malignancies. Key words: Evi-1 gene; Hematologic neoplasms; Cell differentiation; Apoptosis; Molecular targeted therapy

Abstract A45: EVI1 – a novel oncogene in breast carcinoma

Abstract Ecotropic viral integration site 1 (EVI1) is a nuclear transcription factor essential for the maintenance of hematopoietic stem cells. Aberrant expression of EVI1 is found in ~10% of acute myeloid leukemia, where it associates with particularly aggressive disease. EVI1 is also expressed in solid tumors, but remains largely unstudied in this setting. A recent analysis of primary breast carcinoma (BC) samples suggests that enhanced EVI1 expression indicates poor outcome specifically in estrogen receptor (ER) negative tumors (Patel et al., 2011). Here, we explored expression, functional roles and molecular targets of EVI1 in human BC. EVI1 expression was analyzed by qRT-PCR and immunoblot analysis in 8 human BC cell lines and by immunohistochemistry in a cohort of 373 primary BC patient samples collected at the University Hospital Zurich. EVI1 was found to be expressed in most BC samples independently of their ER status. Long-term survival was influenced by EVI1 in ER-, but not ER+ patients, confirming previous results. To investigate the function of EVI1 in BC, cell lines with high and low endogenous EVI1 expression were transduced with EVI1 shRNA knockdown or inducible EVI1-expressing lentiviral particles. Growth, proliferation and cell cycle assays demonstrated that EVI1 promoted BC cell growth by accelerating cell cycle transition. Consistently, EVI1 knockdown BC cells showed reduced in vivo tumor growth compared to control cells in two xenotransplantation models (NSG mice and zebrafish embryo). Interestingly, estrogen supplementation rescued proliferation and tumorigenicity of EVI1-knockdown ER+ but not ER- BC cells, indicating that signaling through the ER pathway might override EVI1-dependent effects on proliferation. Conversely, EVI1-induced proliferation might be particularly important for tumors that are not regulated via the ER pathway, which is in line with its documented prognostic significance in such tumor types. In addition to its effects on proliferation, EVI1 was also found to inhibit BC cell apoptosis in response to both intrinsic and extrinsic stimuli, as previously shown by our group in acute lymphoblastic leukemia cells (Konantz et al, 2013). Moreover, in vitro migration assays showed impaired migration in EVI1 knockdown MDAMB231 cells suggesting that EVI1 regulates BC biology via different mechanisms. Currently, we are exploring the molecular mechanisms by which EVI1 mediates these different effects. Microarray analysis revealed molecules of the MAPK pathways, pro-apoptotic genes and, most interestingly, genes involved in GPCR signaling pathway (e.g. KISS1) as most significantly regulated by EVI1. Analysis of the KISS1 promoter revealed several potential EVI1-binding sites, and a direct association of EVI1 protein with one of these could be confirmed by CHIP. We are currently further exploring the relevance of this binding site for EVI1-mediated activation of KISS1 using luciferase KISS1 reporter assays and site-directed-mutagenesis. In ER- BC, KISS1 expression was reported to enhance cell motility and invasiveness and to reduce cell adhesion. Interestingly, the impaired migration observed in EVI1 knockdown MDAMB231 cells could be indeed rescued by stimulation with Kisspeptin. Neither EVI1 overexpression nor stimulation with Kisspeptin could influence the poorly migrating ER+ MCF7 cells. Taken together, our data identify EVI1 as a novel oncogene particularly important for ER- BC biology. Next to stimulation of BC cell proliferation and reduction of apoptosis sensitivity, we show that EVI1 stimulates ER- BC cell migration via activation of its direct molecular target KISS1. Citation Format: Hui Wang, Thorsten Schäfer, Martina Konantz, Selina Reich, Martin Braun, Sven Perner, Zsuzsanna Varga, Holger Moch, Lothar Kanz, Klaus Schulze-Osthoff, Claudia Lengerke. EVI1 – a novel oncogene in breast carcinoma. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A45.

Abstract LB-103: L1CAM-targeted delivery of siRNA using elastin-like polypeptide (ELP) nanoparticles inhibits the growth of human tumors implanted in mice

Abstract Small interfering RNA (siRNA) drugs provide ideal means for perturbing intracellular oncogenic targets. However, specific delivery of siRNA to tumors has proven to be difficult. An ELP was engineered with an N-terminal region that binds to L1 cell adhesion molecule (L1CAM), and a C-terminal region that binds siRNA. Upon binding of siRNA, the L1CAM targeted ELP (ELP-L) spontaneously assembled into a spherical nanocomplex with the siRNA protected within, and the CAM-binding region protruding from the surface. These nanoparticles were used to deliver siRNA to SKOV3 tumors formed following surgical implantation of the cells into the left ovary of mice. Two weeks after implantation, mice were dosed with vehicle, ELP-L-complexed control siRNA (siCON), or ELP-L-complexed siEVI1, an siRNA to the oncogenic transcription factor EVI1, that is known to enable cell proliferation and inhibits apoptosis in ovarian tumor cells. To initially assess biodistribution, a single dose of ELP-L-DY800-labeled siEVI1 (10 mg/kg, ip) localized into ovarian tumors as early as 4 h, and remained for 72 h, as measured by co-registration of near infrared fluorescence with tumor bioluminescence in live mice. The ELP-L-DY800-siEVI1 was eliminated via kidney and did not accumulate in heart, lung, spleen or liver showing specific localization to tumors. To evaluate efficacy, the mean flux of bioluminescence from ovarian tumors of ten mice receiving 14 doses of 10 mg/kg siEVI1 in ELP-L over 28 days decreased by 92% with respect to mice dosed with 5% dextrose. Immunoblot and qPCR results showed EVI1 protein and mRNA levels decreased more than 50% in mice dosed with siEVI1 compared to siCON or vehicle. There were no observable adverse effects in the mice throughout the dosing period. These results are consistent with an RNA interference mechanism and suggest that ELP-L effectively delivers siEVI1 to tumors with limited toxicity. Furthermore, EVI1 appears to be a good target for ovarian cancer therapy and silencing by siRNAs is an effective means of triggering the death of ovarian tumor cells. Citation Format: Thomas Primiano, Bey-Dih Chang, Jeremy D. Heidel. L1CAM-targeted delivery of siRNA using elastin-like polypeptide (ELP) nanoparticles inhibits the growth of human tumors implanted in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-103. doi:10.1158/1538-7445.AM2014-LB-103

The role of EVI1 in myeloid malignancies

The EVI1 oncogene at human chr 3q26 is rearranged and/or overexpressed in a subset of acute myeloid leukemias and myelodysplasias. The EVI1 protein is a 135 kDa transcriptional regulator with DNA-binding zinc finger domains. Here we provide a critical review of the current state of research into the molecular mechanisms by which this gene plays a role in myeloid malignancies. The major pertinent cellular effects are blocking myeloid differentiation and preventing cellular apoptosis, and several potential mechanisms for these phenomena have been identified. Evidence supports a role for EVI1 in inducing cellular quiescence, and this may contribute to the resistance to chemotherapy seen in patients with neoplasms that overexpress EVI1. Another isoform, MDS1-EVI1 (or PRDM3), encoded by the same locus as EVI1, harbors an N-terminal histone methyltransferase(HMT) domain; experimental findings indicate that this protein and its HMT activity are critical for the progression of a subset of AMLs, and this provides a potential target for therapeutic intervention.

Essential role of PR-domain protein MDS1-EVI1 in MLL-AF9 leukemia

AbstractA subgroup of leukemogenic mixed-lineage leukemia (MLL) fusion proteins (MFPs) including MLL-AF9 activates the Mecom locus and exhibits extremely poor clinical prognosis. Mecom encodes EVI1 and MDS1-EVI1 (ME) proteins via alternative transcription start sites; these differ by the presence of a PRDI-BF1-RIZ1 (PR) domain with histone methyltransferase activity in the ME isoform. Using an ME-deficient mouse, we show that ME is required for MLL-AF9–induced transformation both in vitro and in vivo. And, although Nup98-HOXA9, MEIS1-HOXA9, and E2A-Hlf could transform ME-deficient cells, both MLL-AF9 and MLL-ENL were ineffective, indicating that the ME requirement is specific to MLL fusion leukemia. Further, we show that the PR domain is essential for MFP-induced transformation. These studies clearly indicate an essential role of PR-domain protein ME in MFP leukemia, suggesting that ME may be a novel target for therapeutic intervention for this group of leukemias.

Deletion of the 3q26 region including the EVI1 and MDS1 genes in a neonate with congenital thrombocytopenia and subsequent aplastic anaemia

BackgroundGene-targeting studies in mice have revealed a key role for EVI1 protein in the maintenance of haematopoiesis, and argue in favour of a gene dosage requirement for EVI1 in the...

CML cells expressing the TEL/MDS1/EVI1 fusion are resistant to imatinib-induced apoptosis through inhibition of BAD, but are resensitized with ABT-737

Chronic myeloid leukemia is the first disease in which the potential of molecular targeted therapy with tyrosine kinase inhibitors (TKIs) was realized. Despite this success, a proportion of patients, particularly with advanced disease, are, or become, resistant to this treatment. Overcoming resistance and uncovering the underlying mechanisms is vital for further improvement of clinical outcomes. Here we report the identification, development, and characterization of a novel chronic myeloid leukemia cell line carrying the additional chromosomal aberration t(3;12)(q26;p13) resulting in expression of the TEL/MDS1/EVI1 fusion protein, which is resistant to TKIs. Resistance to TKIs was overcome by the co-administration of the BH3-mimetic, ABT-737. In addition, application of EVI1-specific small interfering RNA decreased expression of the TEL/MDS1/EVI1 fusion, reduced resistance to imatinib, and increased sensitivity to ABT-737. Subsequent studies revealed a role for the BH3-only protein BAD, probably via a phosphoinositide 3-kinase/AKT-dependent pathway, as pharmacological inhibition of AKT could also resensitize cells to death from TKIs. These findings indicate a novel pathway of TKI resistance regulated by EVI1 proteins and provide a promising means for overcoming resistance in chronic myeloid leukemia and other hematological malignancies displaying EVI1 overexpression.

Abstract 1696: Targeting EVI1-overexpressing AML with darinaparsin

Abstract Activation of EVI1, due to chromosomal translocation, inversion or transcriptional upregulation, occurs in 8-10% of AML and results in both activation and repression of specific gene sets. Importantly, the tumor suppressor PTEN is repressed by EVI1. Arsenic trioxide targets EVI1 for degradation, suggesting that arsenicals may be effective in treating EVI1-positive malignancies. Indeed, MDS patients with EVI1 deregulation had greater benefit from the combination of arsenic trioxide (inorganic arsenic) and thalidomide than patients without EVI1 deregulation. Here, we describe the use of a novel organic arsenical, darinaparsin, to treat a patient with EVI1-overexpressing AML. A 36 year old woman was diagnosed with AML inv(3)(q21q26.2) where two copies of inv(3) were detected. Her initial treatment included two induction regimens followed by an allogeneic stem cell transplant. She had a complete remission lasting 5 years after which time her AML relapsed. After three high dose regimens and another investigational combination therapy failed to induce remission (&amp;gt;80% blasts in the bone marrow), we treated the patient's peripheral blasts ex vivo and found that darinaparsin induced significantly more cell death than arsenic trioxide. Thus, the patient started darinaparsin (300 mg/m2 IV over 60 minutes for 5 days every 21 days). Within 10 hours of receiving her first dose, her fever and night sweats had resolved. Her performance status and appetite improved, and she was discharged home 2 days after her last dose. Unlike previous drug regimens, darinaparsin allowed the patient to enjoy a good quality of life for more than 30 days with an ECOG performance status of 1. Unfortunately, while darinaparsin stabilized her peripheral white blood cell counts, the patient died of extramedullary manifestations and complications of her AML, 12 days after receiving a second cycle of darinaparsin. Darinaparsin decreased her peripheral white blood cell counts during the five days of treatment, which was followed by an additional decrease in the white blood cell count when serum arsenic levels were low to undetectable. We observed visible nuclear and cytoplasmic blebbing consistent with apoptosis. Despite the anti-tumor activity, neither EVI1 protein levels nor the transcriptional repression activity as measured by PTEN mRNA expression were altered within the first 72 hours of darinaparsin treatment. cDNA microarray expression profiling showed a dramatic change in gene expression of her tumor between the first and second cycle, including a significant increase in the pro-survival NF-κB pathway. NF-κB family member (i.e. NFKB1, 2, IA, IB, IE, RELA, TANK, TRAF1 and TRAF2) mRNA expression increased 2-8 fold following the first cycle of darinaparsin. Based on our clinical observations and correlative studies, future studies will investigate a link between EVI1 overexpression, NF-κB signaling and darinaparsin sensitivity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1696. doi:1538-7445.AM2012-1696

Physical Interaction of the Proto-Oncogene EVI1 with Hypermethylated in Cancer 1 (HIC1) Deregulates EVI1 Mediated Apoptosis in Cancer Cells

Abstract 2449The ecotropic viral integration site I (EVII) gene has attracted considerable attention in recent years because of its reported involvement in chronic myeloid leukemia, high risk myelodysplastic syndrome and a very high percentage of acute myeloid leukemias. The most recent World Health Organization classification has designated AML with inv (3) or t(3;3) and associated RPN1/EVI1 fusion, as a distinct AML subgroup associated with an unfavorable prognosis. Studies also indicate that EVI1 may be overexpressed in a subset of human colon cancers, and that EVI1 might affect disease progression and/or sensitivity to chemotherapy. The presence of zinc finger domains that are able to bind to specific sequences of DNA suggests that EVI1 is a transcriptional regulator; however, few target genes of EVI1 have been functionally identified. In the present study, we demonstrate by co-immunoprecipitation assay that the proximal zinc finger domain of EVI1 binds to HIC1, which abolishes the ability of EVI1 to induce expression of Bcl-xL. HIC1, is an epigenetically regulated transcriptional repressor that functionally cooperates with p53 to suppress age dependent development of cancer in mice. Inactivation of HIC1 results in up regulation of SIRT1, which deacetylate and inactivates p53; allowing cells to bypass apoptosis and DNA damage survival., Our study also demonstrates via indirect immuno fluorescence analysis that EVI1 and HIC1 proteins are colocalized in speckles in the nucleus. We recently reported that EVI1 not only binds the Bcl-xL promoter but also up regulates its transcriptional activity and protein expression. We now demonstrate by EMSA and luciferase assays that physical interaction between EVI1 and HIC1 releases the binding of EVI1 from the Bcl-xL promoter, and hence promotes apoptosis. Thus by directly binding to EVI1, HIC1 displaces EVI1 from its DNA consensus site leading to down regulation of Bcl-xL. Also we observed that posttranslational modification of EVI1, mainly acetylation plays a critical role in this EVI1-HIC1 protein-protein interaction. Further elucidation of the counteracting effects of HIC1 and EVI1 on Bcl-xL expression may provide new insights in EVI1 mediated disease progression. Disclosures:No relevant conflicts of interest to declare.

Targeting EVI1-Overexpressing AML with Darinaparsin

Abstract 4269Ecotropic viral integration site 1 (EVI1) is a critical oncogene driving a subset of myeloid malignancies and overexpression of EVI1 predicts an extremely poor prognosis in both adult and pediatric AML patient populations. Activation of EVI1, due to chromosomal translocation, inversion or transcriptional upregulation, occurs in 8–10% of AML and results in both activation and repression of specific gene sets. Importantly, the tumor suppressor PTEN is repressed by EVI1. Arsenic trioxide targets EVI1 for degradation, suggesting that arsenicals may be effective in treating EVI1-positive malignancies. Indeed, MDS patients with EVI1 deregulation had greater benefit from the combination of arsenic trioxide (inorganic arsenic) and thalidomide than patients without EVI1 deregulation. Here, we describe the use of a novel arsenical, darinaparsin (an organic form of arsenic), to treat a patient with EVI1-overexpressing AML, as a 10th line of chemotherapy.A 36 year old woman was diagnosed with AML inv(3)(q21q26.2) where two copies of inv(3) were detected. Her initial treatment included two induction regimens followed by an allogeneic stem cell transplant. She had complete remission lasting 5 years after which time her AML relapsed. Three high dose regimens, including VP16 (4g) and cyclophosphamide (2.7g), failed to induce another remission (>80% blasts in the bone marrow). She was offered an investigational combination therapy of ribavirin and low dose cytarabine but after one month, her peripheral blast count rose to >60 and was not responding to hydrea (8g) or mitoxantrone (20mg). At this time, we compared the anti-tumor activity of arsenic trioxide and darinaparsin on the patient's peripheral blasts in vitro and found that darinaparsin induced significantly more cell death than arsenic trioxide. Thus, the patient started darinaparsin (300 mg/m2 IV over 60 minutes for 5 days every 21 days). Within 10 hours of receiving her first dose, her fever and night sweats had resolved. She regained her energy and appetite and was discharged home 2 days after her last dose. Unlike previous drug regimens, darinaparsin allowed the patient to enjoy a good quality of life for more than 30 days with an ECOG performance status of 1. Unfortunately, while darinaparsin stabilized her peripheral white blood cell counts, the patient died of extramedullary manifestations and complications of her AML, 36 days after receiving the first dose of darinaparsin.Darinaparsin decreased her peripheral white blood cell counts during the five days of treatment, which was followed by an additional decrease in the white blood cell count when serum arsenic levels were low to undetectable. We observed visible nuclear and cytoplasmic blebbing consistent with cells undergoing apoptosis. Although EVI1 protein levels were difficult to measure consistently, we analyzed the transcriptional repression activity of EVI1 by measuring PTEN mRNA expression. Intriguingly, we found a significant increase in PTEN mRNA in leukemic blasts following each course of darinaparsin, supporting the hypothesis that darinaparsin may target EVI1 or its activity. We utilized cDNA microarray expression profiling and found that gene expression of her tumor changed dramatically between the first and second cycle, including a significant increase in the pro-survival NF-κB pathway. NF-κB family member (i.e. NFKB1, 2, IA, IB, IE, RELA, TANK, TRAF1 and TRAF2) mRNA expression increased 2–8 fold following the first cycle of darinaparsin. Based on these data, we conclude that darinaparsin should be further explored as a treatment for EVI1-overexpressing myeloid malignancies using the transcriptional repression activity of EVI1 by measuring PTEN mRNA expression as a biomarker for identification of patients likely to respond to therapy. Disclosures:Mann:ZIOPHARM Oncology Inc: Research Funding. Morgan:ZIOPHARM Oncology Inc: Employment. Miller:ZIOPHARM Oncology Inc: Research Funding.