Eric Bindels

Aberrant DNA hypermethylation signature in acute myeloid leukemia directed by EVI1

DNA methylation patterns are frequently dysregulated in cancer, although little is known of the mechanisms through which specific gene sets become aberrantly methylated. The ecotropic viral integration site 1 (EVI1) locus encodes a DNA binding zinc-finger transcription factor that is aberrantly expressed in a subset of acute myeloid leukemia (AML) patients with poor outcome. We find that the promoter DNA methylation signature of EVI1 AML blast cells differs from those of normal CD34(+) bone marrow cells and other AMLs. This signature contained 294 differentially methylated genes, of which 238 (81%) were coordinately hypermethylated. An unbiased motif analysis revealed an overrepresentation of EVI1 binding sites among these aberrantly hypermethylated loci. EVI1 was capable of binding to these promoters in 2 different EVI1-expressing cell lines, whereas no binding was observed in an EVI1-negative cell line. Furthermore, EVI1 was observed to interact with DNA methyl transferases 3A and 3B. Among the EVI1 AML cases, 2 subgroups were recognized, of which 1 contained AMLs with many more methylated genes, which was associated with significantly higher levels of EVI1 than in the cases of the other subgroup. Our data point to a role for EVI1 in directing aberrant promoter DNA methylation patterning in EVI1 AMLs.

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.

Deficiency of the ribosome biogenesis gene Sbds in hematopoietic stem and progenitor cells causes neutropenia in mice by attenuating lineage progression in myelocytes.

Shwachman-Diamond syndrome is a congenital bone marrow failure disorder characterized by debilitating neutropenia. The disease is associated with loss-of-function mutations in the SBDS gene, implicated in ribosome biogenesis, but the cellular and molecular events driving cell specific phenotypes in ribosomopathies remain poorly defined. Here, we established what is to our knowledge the first mammalian model of neutropenia in Shwachman-Diamond syndrome through targeted downregulation of Sbds in hematopoietic stem and progenitor cells expressing the myeloid transcription factor CCAAT/enhancer binding protein α (Cebpa). Sbds deficiency in the myeloid lineage specifically affected myelocytes and their downstream progeny while, unexpectedly, it was well tolerated by rapidly cycling hematopoietic progenitor cells. Molecular insights provided by massive parallel sequencing supported cellular observations of impaired cell cycle exit and formation of secondary granules associated with the defect of myeloid lineage progression in myelocytes. Mechanistically, Sbds deficiency activated the p53 tumor suppressor pathway and induced apoptosis in these cells. Collectively, the data reveal a previously unanticipated, selective dependency of myelocytes and downstream progeny, but not rapidly cycling progenitors, on this ubiquitous ribosome biogenesis protein, thus providing a cellular basis for the understanding of myeloid lineage biased defects in Shwachman-Diamond syndrome.

Influence of the microenvironment on invasiveness of human bladder carcinoma cell lines

Abstract To investigate the importance of the microenvironment in bladder cancer invasion, a panel of six bladder carcinoma cell lines (SD, RT112, JON, 1207, T24, and J82) was tested in both in vitro and in vivo invasion assays. Furthermore, invasiveness was correlated with the expression of components of the E-cadherin–catenin complex. The E-cadherin-negative cell lines, T24 and J82, displayed a high in vitro invasive capacity, whereas the E-cadherin-positive cell lines, SD and JON, completely lacked in vitro invasive capacity. In contrast, in vivo invasion was noted for all cell lines, with the exception of cell line JON. Most notably, SD formed highly invasive tumors in vivo. The in vivo invasiveness of the E-cadherin-positive bladder carcinoma cell lines was associated with a heterogeneous expression of the E-cadherin–catenin complex. The discrepancy between in vitro and in vivo invasive behavior implies that, in vivo, the microenvironment plays an important role in the establishment of the invasive phenotype. In addition, it was found that orthotopic xenografting of 1207 and T24 bladder carcinoma cells resulted in site-specific tumor take and an enhanced tumor outgrowth and invasiveness, respectively, compared with heterotopic (i.e., subcutaneous) inoculation. We conclude that the site-specific growth and invasion of the bladder carcinoma cell lines in vivo and the observed assay specific invasion (in vitro vs in vivo) points to an effect of the local (bladder) microenvironment on tumor cell behavior.

Massive parallel RNA sequencing of highly purified mesenchymal elements in low-risk MDS reveals tissue-context-dependent activation of inflammatory programs

Massive parallel RNA sequencing of highly purified mesenchymal elements in low-risk MDS reveals tissue-context-dependent activation of inflammatory programs

Myeloid Transforming Protein Evi1 Interacts with Methyl-CpG Binding Domain Protein 3 and Inhibits in Vitro Histone Deacetylation by Mbd3/Mi-2/NuRD†

The ecotropic viral integration site 1 ( Evi1) gene encodes a putative transcription regulator, which is aberrantly expressed in acute myeloid leukemias (AML) with chromosomal abnormalities involving the 3q26 locus. Repression and activation of transcriptional control have been reported, but it is currently unclear how Evi1 may evoke these opposing effects. Using a yeast two-hybrid screen, we identified a novel binding partner of Evi1, i.e., methyl binding domain 3b (Mbd3b) protein, a member of the Mi-2/NuRD histone deacetylase complex. Applying in vitro and in vivo assays, we found that Evi1 interacts with Mbd3b but not with other MBD family members Mbd1, -2, and -4 or MeCP2. We show that interaction of Evi1 with Mbd3 requires 40 amino acids that are adjacent and downstream of the methyl binding domain (MBD). We further demonstrate that the first three zinc fingers of Evi1 are needed for Mbd3 interaction. Evi1 acts as a transcriptional repressor when recruited to an active promoter, yet when present in the Mi-2/NuRD complex through Mbd3 interaction, it inhibits the histone deacetylation function of this multiprotein structure. Our data may in part explain how Evi1 could act as a repressor as well as an activator of transcription.

Activation of NF-κB driven inflammatory programs in mesenchymal elements attenuates hematopoiesis in low-risk myelodysplastic syndromes

HighlightsActivation of NF-κB signaling in mesenchymal cells is common in LR-MDS.Activation of NF-κB in mesenchymal cells leads to transcriptional overexpression of inflammatory factors including negative regulators of hematopoiesis.Activation of NF-κB attenuates HSPC numbers and function ex vivo.

Abstract 5023: Multiple myeloma with a clonal del17p aberration is characterized by somatic TP53 mutations, which negatively affect prognosis in this cytogenetic subgroup

Background Multiple myeloma (MM) is a malignant plasma cell disorder, in which at diagnosis circa 10% of tumors are characterized by a deletion of chromosome 17p (del17p). Previously, others and we reported that this aberration is associated with a significantly inferior prognosis, classifying it as high-risk disease. As TP53 is located on chromosome 17p (Chr17p), most research has thus far been focused on this gene. However, only in a minority of del17p MM patients a mutation could be detected (Lode et al. - Haematologica 2010, Kortum et al. - Br J Haematol 2015). We therefore hypothesized that other relevant mutations might be present in TP53, its surrounding genes, or disease relevant pathways. Aim With this study, we sought to identify commonly mutated genes in del17p MM, which could explain its aggressive clinical behavior. Material and Methods We obtained high molecular DNA from CD138+ purified MM cells and matched peripheral blood from patients with a del17p in ≥50% of plasma cells, as detected with fluorescent in situ hybridization (FISH). Libraries were generated using a custom capture of 111 Mb (SeqCap EZ Exome Plus, Nimblegen), comprising the whole exome, Chr17p and the IgH, Igk, IgL and MYC regions. Paired-end sequencing was performed on a HiSeq2500 platform, followed by data-analysis using an in-house bioinformatics pipeline at Erasmus MC. Results Matched tumor and germline DNA was sequenced from 54 patients, with sequential tumor samples available from 12 patients. This resulted in an average coverage of 88x and overall coverage of 94%. Of 44 del17p patients, tumor DNA was obtained before treatment start. Clinical data were collected from 40/44 patients, with a median follow-up time of 18 months. We found TP53 to be mutated most frequently. 31/54 (57%) patients had a somatic nonsynonymous mutation, insertion or deletion (71% missense, 19% stopgain/frameshift, 10% splicing). Although no hotspot mutations were detected, most TP53 mutations resided in the DNA binding domain. Of note, most TP53wt/- patients had no clonal somatic nonsynonymous mutations in other p53 pathway genes, or on Chr17p. To assess the prognostic impact of TP53 mutations in del17p MM, we focused on the 40 chemotherapy-naive patients and stratified for age ≤65 years (n = 25) and >65 years (n = 15). TP53mut/- patients had a significantly worse overall survival (OS) than TP53wt/- patients (p = 0.002). Strikingly, TP53 missense mutations showed the worst OS (p Conclusions (1) In contrast with previous reports, we find TP53 to be somatically mutated in the majority of MM patients with a clonal del17p aberration. (2) Particularly TP53 missense mutations have a significantly negative impact on both PFS and OS in del17p MM patients. This study was supported by the Plaisier Foundation, Erasmus MC Grant and the Dutch CTMM Project. Citation Format: Davine Hofste op Bruinink, Remco Hoogenboezem, Eric Bindels, Mathijs Sanders, Claudia Erpelinck - Verschueren, Paulina van Strien, Jasper Koenders, Kristine Misund, Berna Beverloo, Bronno van der Holt, Ivo Touw, Anders Waage, Herve Avet-Loiseau, Pieter Sonneveld. Multiple myeloma with a clonal del17p aberration is characterized by somatic TP53 mutations, which negatively affect prognosis in this cytogenetic subgroup. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5023.