EVI1 in Acute Myeloid Leukemia

To diagnose patients with acute myeloid leukemia (AML) in an optimal manner, the combined application of conventional and modern cytogenetics with state-of-the-art molecular diagnostics is a requirement. Although at present, the WHO accurately classifies an array of human AML patients based on karyotyping combined with molecular diagnostic procedures, insight into the molecular defects of human AML is still increasing. As a result of that, the classifi cation of AML will be approved in the upcoming years. The focus of this thesis was to increase our understanding of specific subtypes of human leukemia. We focused on AMLs with chromosome 3q rearrangements, in particular on patients with an inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1 (shortly: (inv(3)/t(3;3)), frequently associated with ab

EVI1 activation in blast crisis CML due to juxtaposition to the rare 17q22 partner region as part of a 4-way variant translocation t(9;22)

<p>Abstract</p> <p>Background</p> <p>Variant translocations t(9;22) occur in 5 to 10% of newly diagnosed CMLs and additional genetic changes are present in 60–80% of patients in blast crisis (BC). Here, we report on a CML patient in blast crisis presenting with a four-way variant t(9;22) rearrangement involving the <it>EVI1 </it>locus.</p> <p>Methods</p> <p>Dual-colour Fluorescence In Situ Hybridisation was performed to unravel the different cytogenetic aberrations. Expression levels of <it>EVI1 </it>and <it>BCR/ABL1 </it>were investigated using real-time quantitative RT-PCR.</p> <p>Results</p> <p>In this paper we identified a patient with a complex 4-way t(3;9;17;22) which, in addition to <it>BCR/ABL1 </it>gene fusion, also resulted in <it>EVI1 </it>rearrangement and overexpression.</p> <p>Conclusion</p> <p>This report illustrates how a variant t(9;22) translocation can specifically target a second oncogene most likely contributing to the more aggressive phenotype of the disease. Molecular analysis of such variants is thus warranted to understand the phenotypic consequences and to open the way for combined molecular therapies in order to tackle the secondary oncogenic effect which is unresponsive to imatinib treatment.</p

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

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

The Increased Expression of Integrin α6 (ITGA6) Enhances Drug Resistance in EVI1high Leukemia

Ecotropic viral integration site-1 (EVI1) is one of the candidate oncogenes for human acute myeloid leukemia (AML) with chromosomal alterations at 3q26. High EVI1 expression (EVI1high) is a risk factor for AML with poor outcome. Using DNA microarray analysis, we previously identified that integrin α6 (ITGA6) was upregulated over 10-fold in EVI1high leukemia cells. In this study, we determined whether the increased expression of ITGA6 is associated with drug-resistance and increased cell adhesion, resulting in poor prognosis. To this end, we first confirmed the expression pattern of a series of integrin genes using semi-quantitative PCR and fluorescence-activated cell sorter (FACS) analysis and determined the cell adhesion ability in EVI1high leukemia cells. We found that the adhesion ability of EVI1high leukemia cells to laminin increased with the increased expression of ITGA6 and integrin β4 (ITGB4). The introduction of small-hairpin RNA against EVI1 (shEVI1) into EVI1high leukemia cells reduced the cell adhesion ability and downregulated the expression of ITGA6 and ITGB4. In addition, the overexpression of EVI1 in EVI1low leukemia cells enhanced their cell adhesion ability and increased the expression of ITGA6 and ITGB4. In a subsequent experiment, the introduction of shRNA against ITGA6 or ITGB4 into EVI1high AML cells downregulated their cell adhesion ability; however, the EVI1high AML cells transfected with shRNA against ITGA6 could not be maintained in culture. Moreover, treating EVI1high leukemia cells with neutralizing antibodies against ITGA6 or ITGB4 resulted in an enhanced responsiveness to anti-cancer drugs and a reduction of their cell adhesion ability. The expression of ITGA6 is significantly elevated in cells from relapsed and EVI1high AML cases; therefore, ITGA6 might represent an important therapeutic target for both refractory and EVI1high AML

The Oncoprotein EVI1 and the DNA Methyltransferase Dnmt3 Co-Operate in Binding and De Novo Methylation of Target DNA

EVI1 has pleiotropic functions during murine embryogenesis and its targeted disruption leads to prenatal death by severely affecting the development of virtually all embryonic organs. However, its functions in adult tissues are still unclear. When inappropriately expressed, EVI1 becomes one of the most aggressive oncogenes associated with human hematopoietic and solid cancers. The mechanisms by which EVI1 transforms normal cells are unknown, but we showed recently that EVI1 indirectly upregulates self-renewal and cell-cycling genes by inappropriate methylation of CpG dinucleotides in the regulatory regions of microRNA-124-3 (miR-124-3), leading to the repression of this small gene that controls normal differentiation and cell cycling of somatic cells. We used the regulatory regions of miR-124-3 as a read-out system to investigate how EVI1 induces de novo methylation of DNA. Here we show that EVI1 physically interacts with DNA methyltransferases 3a and 3b (Dnmt3a/b), which are the only de novo DNA methyltransferases identified to date in mouse and man, and that it forms an enzymatically active protein complex that induces de novo DNA methylation in vitro. This protein complex targets and binds to a precise region of miR-124-3 that is necessary for repression of a reporter gene by EVI1. Based on our findings, we propose that in cooperation with Dnmt3a/b EVI1 regulates the methylation of DNA as a sequence-specific mediator of de novo DNA methylation and that inappropriate EVI1 expression contributes to carcinogenesis through improper DNA methylation

Identifying Resistance Mechanisms against Five Tyrosine Kinase Inhibitors Targeting the ERBB/RAS Pathway in 45 Cancer Cell Lines

Because of the low overall response rates of 10-47% to targeted cancer therapeutics, there is an increasing need for predictive biomarkers. We aimed to identify genes predicting response to five already approved tyrosine kinase inhibitors. We tested 45 cancer cell lines for sensitivity to sunitinib, erlotinib, lapatinib, sorafenib and gefitinib at the clinically administered doses. A resistance matrix was determined, and gene expression profiles of the subsets of resistant vs. sensitive cell lines were compared. Triplicate gene expression signatures were obtained from the caArray project. Significance analysis of microarrays and rank products were applied for feature selection. Ninety-five genes were also measured by RT-PCR. In case of four sunitinib resistance associated genes, the results were validated in clinical samples by immunohistochemistry. A list of 63 top genes associated with resistance against the five tyrosine kinase inhibitors was identified. Quantitative RT-PCR analysis confirmed 45 of 63 genes identified by microarray analysis. Only two genes (ANXA3 and RAB25) were related to sensitivity against more than three inhibitors. The immunohistochemical analysis of sunitinib-treated metastatic renal cell carcinomas confirmed the correlation between RAB17, LGALS8, and EPCAM and overall survival. In summary, we determined predictive biomarkers for five tyrosine kinase inhibitors, and validated sunitinib resistance biomarkers by immunohistochemistry in an independent patient cohort. © 2013 Pénzváltó et al

The creatine kinase pathway is a metabolic vulnerability in EVI1-positive acute myeloid leukemia

Expression of the MECOM (also known as EVI1) proto-oncogene is deregulated by chromosomal translocations in some cases of acute myeloid leukemia (AML) and is associated with poor clinical outcome. Here, through transcriptomic and metabolomic profiling of hematopoietic cells, we reveal that EVI1 overexpression alters cellular metabolism. A screen using pooled short hairpin RNAs (shRNAs) identified the ATP-buffering, mitochondrial creatine kinase CKMT1 as necessary for survival of EVI1-expressing cells in subjects with EVI1-positive AML. EVI1 promotes CKMT1 expression by repressing the myeloid differentiation regulator RUNX1. Suppression of arginine-creatine metabolism by CKMT1-directed shRNAs or by the small molecule cyclocreatine selectively decreased the viability, promoted the cell cycle arrest and apoptosis of human EVI1-positive cell lines, and prolonged survival in both orthotopic xenograft models and mouse models of primary AML. CKMT1 inhibition altered mitochondrial respiration and ATP production, an effect that was abrogated by phosphocreatine-mediated reactivation of the arginine-creatine pathway. Targeting CKMT1 is thus a promising therapeutic strategy for this EVI1-driven AML subtype that is highly resistant to current treatment regimens. Keywords: AML; RUNX1; CKMT1; cyclocreatine; arginine metabolismNational Cancer Institute (U.S.) (NIH 1R35 CA210030-01)Stand Up To CancerBridge ProjectNational Cancer Institute (U.S.) (David H. Koch Institute for Integrative Cancer Research at MIT. Grant P30-CA14051

Concordant Gene Expression in Leukemia Cells and Normal Leukocytes Is Associated with Germline cis-SNPs

The degree to which gene expression covaries between different primary tissues within an individual is not well defined. We hypothesized that expression that is concordant across tissues is more likely influenced by genetic variability than gene expression which is discordant between tissues. We quantified expression of 11,873 genes in paired samples of primary leukemia cells and normal leukocytes from 92 patients with acute lymphoblastic leukemia (ALL). Genetic variation at >500,000 single nucleotide polymorphisms (SNPs) was also assessed. The expression of only 176/11,783 (1.5%) genes was correlated (p<0.008, FDR = 25%) in the two tissue types, but expression of a high proportion (20 of these 176 genes) was significantly related to cis-SNP genotypes (adjusted p<0.05). In an independent set of 134 patients with ALL, 14 of these 20 genes were validated as having expression related to cis-SNPs, as were 9 of 20 genes in a second validation set of HapMap cell lines. Genes whose expression was concordant among tissue types were more likely to be associated with germline cis-SNPs than genes with discordant expression in these tissues; genes affected were involved in housekeeping functions (GSTM2, GAPDH and NCOR1) and purine metabolism

Telomeric DNA induces apoptosis and senescence of human breast carcinoma cells

INTRODUCTION: Cancer is a leading cause of death in Americans. We have identified an inducible cancer avoidance mechanism in cells that reduces mutation rate, reduces and delays carcinogenesis after carcinogen exposure, and induces apoptosis and/or senescence of already transformed cells by simultaneously activating multiple overlapping and redundant DNA damage response pathways. METHODS: The human breast carcinoma cell line MCF-7, the adriamycin-resistant MCF-7 (Adr/MCF-7) cell line, as well as normal human mammary epithelial (NME) cells were treated with DNA oligonucleotides homologous to the telomere 3' overhang (T-oligos). SCID mice received intravenous injections of MCF-7 cells followed by intravenous administration of T-oligos. RESULTS: Acting through ataxia telangiectasia mutated (ATM) and its downstream effectors, T-oligos induced apoptosis and senescence of MCF-7 cells but not NME cells, in which these signaling pathways were induced to a far lesser extent. In MCF-7 cells, experimental telomere loop disruption caused identical responses, consistent with the hypothesis that T-oligos act by mimicking telomere overhang exposure. In vivo, T-oligos greatly prolonged survival of SCID mice following intravenous injection of human breast carcinoma cells. CONCLUSION: By inducing DNA damage-like responses in MCF-7 cells, T-oligos provide insight into innate cancer avoidance mechanisms and may offer a novel approach to treatment of breast cancer and other malignancies