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

An MLL-dependent network sustains hematopoiesis

The histone methyltransferase Mixed Lineage Leukemia (MLL) is essential to maintain hematopoietic stem cells and is a leukemia protooncogene. Although clustered homeobox genes are well-characterized targets of MLL and MLL fusion oncoproteins, the range of Mll-regulated genes in normal hematopoietic cells remains unknown. Here, we identify and characterize part of the Mll-dependent transcriptional network in hematopoietic stem cells with an integrated approach by using conditional loss-of-function models, genomewide expression analyses, chromatin immunoprecipitation, and functional rescue assays. The Mll-dependent transcriptional network extends well beyond the previously appreciated Hox targets, is comprised of many characterized regulators of self-renewal, and contains target genes that are both dependent and independent of the MLL cofactor, Menin. Interestingly, PR-domain containing 16 emerged as a target gene that is uniquely effective at partially rescuing Mll-deficient hematopoietic stem and progenitor cells. This work highlights the tissue-specific nature of regulatory networks under the control of MLL/Trithorax family members and provides insight into the distinctions between the participation of MLL in normal hematopoiesis and in leukemia

EVI1 overexpression in distinct subtypes of pediatric acute myeloid leukemia

Item does not contain fulltextOverexpression of the ecotropic virus integration-1 (EVI1) gene (EVI1+), localized at chromosome 3q26, is associated with adverse outcome in adult acute myeloid leukemia (AML). In pediatric AML, 3q26 abnormalities are rare, and the role of EVI1 is unknown. We studied 228 pediatric AML samples for EVI1+ using gene expression profiling and RQ-PCR. EVI1+ was found in 20/213 (9%) of children with de novo AML, and in 4/8 with secondary AML. It was predominantly found in MLL-rearranged AML (13/47), monosomy 7 (2/3), or FAB M6/7 (6/10), and mutually exclusive with core-binding factor AML, t(15;17), and NPM1 mutations. Fluorescent in situ hybridization (FISH) was performed to detect cryptic 3q26 abnormalities. However, none of the EVI1+ patients harbored structural 3q26 alterations. Although significant differences in 4 years pEFS for EVI1+ and EVI1- pediatric AML were observed (28%+/-11 vs 44%+/-4, P=0.04), multivariate analysis did not identify EVI1+ as an independent prognostic factor. We conclude that EVI1+ can be found in approximately 10% of pediatric AML. Although EVI1+ was not an independent prognostic factor, it was predominantly found in subtypes of pediatric AML that are related with an intermediate to unfavorable prognosis. Further research should explain the role of EVI1+ in disease biology in these cases. Remarkably, no 3q26 abnormalities were identified in EVI1+ pediatric AML.1 mei 201