Publications by authors named "Eric Bindels"

34 Publications

Allele-specific expression of GATA2 due to epigenetic dysregulation in CEBPA double mutant AML.

Blood 2021 Apr 8. Epub 2021 Apr 8.

Erasmus University Medical Center, Rotterdam, Netherlands.

Transcriptional deregulation is a central event in the development of acute myeloid leukemia (AML). To identify potential disturbances in gene regulation, we conducted an unbiased screen of allele-specific expression (ASE) in 209 AML cases. The gene encoding GATA binding protein 2 (GATA2) displayed ASE more often than any other myeloid or cancer-related gene. GATA2 ASE was strongly associated with CEBPA double mutations (CEBPA DM), with 95% of cases presenting GATA2 ASE. In CEBPA DM AML with GATA2 mutations, the mutated allele was preferentially expressed. We found that GATA2 ASE is a somatic event lost in complete remission, supporting the notion that it plays a role in CEBPA DM AML. Acquisition of GATA2 ASE involved silencing of one allele via promoter methylation and concurrent overactivation of the other allele, thereby preserving expression levels. Notably, promoter methylation was also lost in remission together with GATA2 ASE. In summary, we propose that GATA2 ASE is acquired by epigenetic mechanisms and is a prerequisite for the development of AML with CEBPA DM. This finding constitutes a novel example of an epigenetic hit cooperating with a genetic hit in the pathogenesis of AML.
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http://dx.doi.org/10.1182/blood.2020009244DOI Listing
April 2021

Extracellular Vesicles Derived From Adult and Fetal Bone Marrow Mesenchymal Stromal Cells Differentially Promote Expansion of Hematopoietic Stem and Progenitor Cells.

Front Bioeng Biotechnol 2021 25;9:640419. Epub 2021 Feb 25.

Department of Internal Medicine, Erasmus MC, University Medical Center, Rotterdam, Netherlands.

Recently, we and others have illustrated that extracellular vesicles (EVs) have the potential to support hematopoietic stem and progenitor cell (HSPC) expansion; however, the mechanism and processes responsible for the intercellular communication by EVs are still unknown. In the current study, we investigate whether primary human bone marrow derived mesenchymal stromal cells (BMSC) EVs isolated from two different origins, fetal (fEV) and adult (aEV) tissue, can increase the relative low number of HSPCs found in umbilical cord blood (UCB) and which EV-derived components are responsible for HSPC expansion. Interestingly, aEVs and to a lesser extent fEVs, showed supportive expansion capacity of UCB-HSPCs. Taking advantage of the two BMSC sources with different supportive effects, we analyzed the EV cargo and investigated how gene expression is modulated in HSPCs after incubation with aEVs and fEVs. Proteomics analyses of the protein cargo composition of the supportive aEV vs. the less-supportive fEV identified 90% of the Top100 exosome proteins present in the ExoCarta database. Gene Ontology (GO) analyses illustrated that the proteins overrepresented in aEVs were annotated to oxidation-reduction process, mitochondrial ATP synthesis coupled proton transport, or protein folding. In contrast, the proteins overrepresented in fEVs were annotated to extracellular matrix organization positive regulation of cell migration or transforming growth factor beta receptor (TGFBR) signaling pathway. Small RNA sequencing identified different molecular signatures between aEVs and fEVs. Interestingly, the microRNA cluster miR-99b/let-7e/miR-125a, previously identified to increase the number of HSPCs by targeting multiple pro-apoptotic genes, was highly and significantly enriched in aEVs. Although we identified significant differences in the supportive effects of aEVs and fEVs, RNAseq analyses of the 24 h treated HSPCs indicated that a limited set of genes was differentially regulated when compared to cells that were treated with cytokines only. Together, our study provides novel insights into the complex biological role of EVs and illustrates that aEVs and fEVs differentially support expansion capacity of UCB-HSPCs. Together opening new means for the application of EVs in the discovery of therapeutics for more efficient HSPC expansion.
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http://dx.doi.org/10.3389/fbioe.2021.640419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7947881PMC
February 2021

The miR-200c/141-ZEB2-TGFβ axis is aberrant in human T-cell prolymphocytic leukemia.

Haematologica 2021 Feb 18. Epub 2021 Feb 18.

Department of Immunology, Erasmus University Medical Center, Rotterdam.

T cell prolymphocytic leukemia (T-PLL) is mostly characterized by aberrant expansion of small to medium sized pro-lymphocytes with a mature post-thymic phenotype, high aggressiveness of the disease and poor prognosis. However, T-PLL is more heterogeneous with a wide-range of clinical, morphological, and molecular features, which occasionally impedes the diagnosis. We hypothesized that T-PLL consists of phenotypic and/or genotypic subgroups that may explain the heterogeneity of the disease. Multi-dimensional immuno-phenotyping and gene expression profiling did not reveal clear T-PLL subgroups, whereas no clear T cell receptor alpha (TRA) or beta (TRB) CDR3 skewing was observed between different T-PLL cases. We revealed that the expression of miRNAs is aberrant and often heterogeneous in T-PLL. We identified 35 miRNAs that were aberrantly expressed in T-PLL with miR-200c/141 as the most differentially expressed cluster. High miR-200c/141 and miR-181a/181b expression was significantly correlated with increased white blood cell counts and poor survival. Furthermore, we found that overexpression of miR-200c/141 correlated with downregulation of their targets ZEB2 and TGFβR3 and aberrant TGF-β1-induced phosphorylated SMAD2 (p-SMAD2) and p-SMAD3, indicating that the TGFβ pathway is affected in T-PLL. Our results thus highlight the potential role for aberrantly expressed oncogenic miRNAs in T-PLL and pave the way for new diagnostic targets in this disease.
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http://dx.doi.org/10.3324/haematol.2020.263756DOI Listing
February 2021

Endothelium-derived stromal cells contribute to hematopoietic bone marrow niche formation.

Cell Stem Cell 2021 Apr 8;28(4):653-670.e11. Epub 2021 Feb 8.

Department of Hematology, Erasmus MC Cancer Institute, Rotterdam 3015 CN, the Netherlands. Electronic address:

Bone marrow stromal cells (BMSCs) play pivotal roles in tissue maintenance and regeneration. Their origins, however, remain incompletely understood. Here we identify rare LNGFR cells in human fetal and regenerative bone marrow that co-express endothelial and stromal markers. This endothelial subpopulation displays transcriptional reprogramming consistent with endothelial-to-mesenchymal transition (EndoMT) and can generate multipotent stromal cells that reconstitute the bone marrow (BM) niche upon transplantation. Single-cell transcriptomics and lineage tracing in mice confirm robust and sustained contributions of EndoMT to bone precursor and hematopoietic niche pools. Interleukin-33 (IL-33) is overexpressed in subsets of EndoMT cells and drives this conversion process through ST2 receptor signaling. These data reveal generation of tissue-forming BMSCs from mouse and human endothelial cells and may be instructive for approaches to human tissue regeneration.
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http://dx.doi.org/10.1016/j.stem.2021.01.006DOI Listing
April 2021

PML-controlled responses in severe congenital neutropenia with ELANE-misfolding mutations.

Blood Adv 2021 Feb;5(3):775-786

Department of Hematology.

Mutations in ELANE cause severe congenital neutropenia (SCN), but how they affect neutrophil production and contribute to leukemia predisposition is unknown. Neutropenia is alleviated by CSF3 (granulocyte colony-stimulating factor) therapy in most cases, but dose requirements vary between patients. Here, we show that CD34+CD45+ hematopoietic progenitor cells (HPCs) derived from induced pluripotent stem cell lines from patients with SCN that have mutations in ELANE (n = 2) or HAX1 (n = 1) display elevated levels of reactive oxygen species (ROS) relative to normal iPSC-derived HPCs. In patients with ELANE mutations causing misfolding of the neutrophil elastase (NE) protein, HPCs contained elevated numbers of promyelocyte leukemia protein nuclear bodies, a hallmark of acute oxidative stress. This was confirmed in primary bone marrow cells from 3 additional patients with ELANE-mutant SCN. Apart from responding to elevated ROS levels, PML controlled the metabolic state of these ELANE-mutant HPCs as well as the expression of ELANE, suggestive of a feed-forward mechanism of disease development. Both PML deletion and correction of the ELANE mutation restored CSF3 responses of these ELANE-mutant HPCs. These findings suggest that PML plays a crucial role in the disease course of ELANE-SCN characterized by NE misfolding, with potential implications for CSF3 therapy.
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http://dx.doi.org/10.1182/bloodadvances.2020003214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876869PMC
February 2021

Detection of Aneuploidy in Cerebrospinal Fluid from Patients with Breast Cancer can Improve Diagnosis of Leptomeningeal Metastases.

Clin Cancer Res 2021 Jan 29. Epub 2021 Jan 29.

Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, the Netherlands.

Purpose: Detection of leptomeningeal metastasis is hampered by limited sensitivities of currently used techniques: MRI and cytology of cerebrospinal fluid (CSF). Detection of cell-free tumor DNA in CSF has been proposed as a tumor-specific candidate to detect leptomeningeal metastasis at an earlier stage. The aim of this study was to investigate mutation and aneuploidy status in CSF-derived cell-free DNA (cfDNA) of patients with breast cancer with a clinical suspicion of leptomeningeal metastasis.

Experimental Design: cfDNA was isolated from stored remnant CSF and analyzed by targeted next-generation sequencing (NGS; = 30) and the modified fast aneuploidy screening test-sequencing system (mFAST-SeqS; = 121). The latter method employs selective amplification of long interspaced nuclear elements sequences that are present throughout the genome and allow for fast and cheap detection of aneuploidy. We compared these results with the gold standard to diagnose leptomeningeal metastasis: cytology.

Results: Leptomeningeal metastasis was cytology proven in 13 of 121 patients. Low DNA yields resulted in insufficient molecular coverage of NGS for the majority of samples (success rate, 8/30). The mFAST-SeqS method, successful in 112 of 121 (93%) samples, detected genome-wide aneuploidy in 24 patients. Ten of these patients had cytology-proven leptomeningeal metastasis; 8 additional patients were either concurrently diagnosed with central nervous system metastases by radiological means or developed these soon after the lumbar puncture. The remaining six cases were suspected of leptomeningeal metastasis, but could not be confirmed by cytology or imaging. Aneuploidy was associated with development of leptomeningeal metastasis and significantly worse overall survival.

Conclusions: Aneuploidy in CSF-derived cfDNA may provide a promising biomarker to improve timely detection of leptomeningeal metastasis.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-3954DOI Listing
January 2021

Causal integration of multi-omics data with prior knowledge to generate mechanistic hypotheses.

Mol Syst Biol 2021 01;17(1):e9730

Faculty of Medicine, and Heidelberg University Hospital, Institute for Computational Biomedicine, Heidelberg University, Heidelberg, Germany.

Multi-omics datasets can provide molecular insights beyond the sum of individual omics. Various tools have been recently developed to integrate such datasets, but there are limited strategies to systematically extract mechanistic hypotheses from them. Here, we present COSMOS (Causal Oriented Search of Multi-Omics Space), a method that integrates phosphoproteomics, transcriptomics, and metabolomics datasets. COSMOS combines extensive prior knowledge of signaling, metabolic, and gene regulatory networks with computational methods to estimate activities of transcription factors and kinases as well as network-level causal reasoning. COSMOS provides mechanistic hypotheses for experimental observations across multi-omics datasets. We applied COSMOS to a dataset comprising transcriptomics, phosphoproteomics, and metabolomics data from healthy and cancerous tissue from eleven clear cell renal cell carcinoma (ccRCC) patients. COSMOS was able to capture relevant crosstalks within and between multiple omics layers, such as known ccRCC drug targets. We expect that our freely available method will be broadly useful to extract mechanistic insights from multi-omics studies.
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http://dx.doi.org/10.15252/msb.20209730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7838823PMC
January 2021

Heterogeneous bone-marrow stromal progenitors drive myelofibrosis via a druggable alarmin axis.

Cell Stem Cell 2021 Apr 9;28(4):637-652.e8. Epub 2020 Dec 9.

Department of Hematology, Erasmus Medical Center, Rotterdam 3015GD, the Netherlands; Department of Cell Biology, Institute for Biomedical Engineering, Faculty of Medicine, RWTH Aachen University, Pauwelsstrasse 30, 52074 Aachen, Germany; Oncode Institute, Erasmus Medical Center, Rotterdam 3015GD, the Netherlands. Electronic address:

Functional contributions of individual cellular components of the bone-marrow microenvironment to myelofibrosis (MF) in patients with myeloproliferative neoplasms (MPNs) are incompletely understood. We aimed to generate a comprehensive map of the stroma in MPNs/MFs on a single-cell level in murine models and patient samples. Our analysis revealed two distinct mesenchymal stromal cell (MSC) subsets as pro-fibrotic cells. MSCs were functionally reprogrammed in a stage-dependent manner with loss of their progenitor status and initiation of differentiation in the pre-fibrotic and acquisition of a pro-fibrotic and inflammatory phenotype in the fibrotic stage. The expression of the alarmin complex S100A8/S100A9 in MSC marked disease progression toward the fibrotic phase in murine models and in patient stroma and plasma. Tasquinimod, a small-molecule inhibiting S100A8/S100A9 signaling, significantly ameliorated the MPN phenotype and fibrosis in JAK2V617F-mutated murine models, highlighting that S100A8/S100A9 is an attractive therapeutic target in MPNs.
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http://dx.doi.org/10.1016/j.stem.2020.11.004DOI Listing
April 2021

Malignant Transformation Involving Mutations Identified in a Leukemic Progression Model of Severe Congenital Neutropenia.

Cell Rep Med 2020 Aug 25;1(5):100074. Epub 2020 Aug 25.

Department of Hematology, Erasmus University Medical Center, Rotterdam 3015 CN, the Netherlands.

Severe congenital neutropenia (SCN) patients treated with CSF3/G-CSF to alleviate neutropenia frequently develop acute myeloid leukemia (AML). A common pattern of leukemic transformation involves the appearance of hematopoietic clones with CSF3 receptor () mutations in the neutropenic phase, followed by mutations in before AML becomes overt. To investigate how the combination of CSF3 therapy and and mutations contributes to AML development, we make use of mouse models, SCN-derived induced pluripotent stem cells (iPSCs), and SCN and SCN-AML patient samples. CSF3 provokes a hyper-proliferative state in / mutant hematopoietic progenitors but does not cause overt AML. Intriguingly, an additional acquired driver mutation in causes elevated CXXC4 and reduced TET2 protein levels in murine AML samples. Expression of multiple pro-inflammatory pathways is elevated in mouse AML and human SCN-AML, suggesting that inflammation driven by downregulation of TET2 activity is a critical step in the malignant transformation of SCN.
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http://dx.doi.org/10.1016/j.xcrm.2020.100074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7659587PMC
August 2020

Decoding myofibroblast origins in human kidney fibrosis.

Nature 2021 01 11;589(7841):281-286. Epub 2020 Nov 11.

Division of Nephrology and Clinical Immunology, RWTH Aachen University, Aachen, Germany.

Kidney fibrosis is the hallmark of chronic kidney disease progression; however, at present no antifibrotic therapies exist. The origin, functional heterogeneity and regulation of scar-forming cells that occur during human kidney fibrosis remain poorly understood. Here, using single-cell RNA sequencing, we profiled the transcriptomes of cells from the proximal and non-proximal tubules of healthy and fibrotic human kidneys to map the entire human kidney. This analysis enabled us to map all matrix-producing cells at high resolution, and to identify distinct subpopulations of pericytes and fibroblasts as the main cellular sources of scar-forming myofibroblasts during human kidney fibrosis. We used genetic fate-tracing, time-course single-cell RNA sequencing and ATAC-seq (assay for transposase-accessible chromatin using sequencing) experiments in mice, and spatial transcriptomics in human kidney fibrosis, to shed light on the cellular origins and differentiation of human kidney myofibroblasts and their precursors at high resolution. Finally, we used this strategy to detect potential therapeutic targets, and identified NKD2 as a myofibroblast-specific target in human kidney fibrosis.
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http://dx.doi.org/10.1038/s41586-020-2941-1DOI Listing
January 2021

Only Hyperuricemia with Crystalluria, but not Asymptomatic Hyperuricemia, Drives Progression of Chronic Kidney Disease.

J Am Soc Nephrol 2020 12 16;31(12):2773-2792. Epub 2020 Sep 16.

Division of Nephrology, Department of Medicine IV, Ludwig-Maximilian's-University Hospital, Munich, Germany

Background: The roles of asymptomatic hyperuricemia or uric acid (UA) crystals in CKD progression are unknown. Hypotheses to explain links between UA deposition and progression of CKD include that () asymptomatic hyperuricemia does not promote CKD progression unless UA crystallizes in the kidney; () UA crystal granulomas may form due to pre-existing CKD; and () proinflammatory granuloma-related M1-like macrophages may drive UA crystal-induced CKD progression.

Methods: MALDI-FTICR mass spectrometry, immunohistochemistry, 3D confocal microscopy, and flow cytometry were used to characterize a novel mouse model of hyperuricemia and chronic UA crystal nephropathy with granulomatous nephritis. Interventional studies probed the role of crystal-induced inflammation and macrophages in the pathology of progressive CKD.

Results: Asymptomatic hyperuricemia alone did not cause CKD or drive the progression of aristolochic acid I-induced CKD. Only hyperuricemia with UA crystalluria due to urinary acidification caused tubular obstruction, inflammation, and interstitial fibrosis. UA crystal granulomas surrounded by proinflammatory M1-like macrophages developed late in this process of chronic UA crystal nephropathy and contributed to the progression of pre-existing CKD. Suppressing M1-like macrophages with adenosine attenuated granulomatous nephritis and the progressive decline in GFR. In contrast, inhibiting the JAK/STAT inflammatory pathway with tofacitinib was not renoprotective.

Conclusions: Asymptomatic hyperuricemia does not affect CKD progression unless UA crystallizes in the kidney. UA crystal granulomas develop late in chronic UA crystal nephropathy and contribute to CKD progression because UA crystals trigger M1-like macrophage-related interstitial inflammation and fibrosis. Targeting proinflammatory macrophages, but not JAK/STAT signaling, can attenuate granulomatous interstitial nephritis.
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http://dx.doi.org/10.1681/ASN.2020040523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790211PMC
December 2020

Human kidney organoids produce functional renin.

Kidney Int 2021 01 9;99(1):134-147. Epub 2020 Sep 9.

Department of Internal Medicine, Division of Nephrology and Transplantation, Erasmus MC, University Medical Center, Rotterdam, The Netherlands.

Renin production by the kidney is of vital importance for salt, volume, and blood pressure homeostasis. The lack of human models hampers investigation into the regulation of renin and its relevance for kidney physiology. To develop such a model, we used human induced pluripotent stem cell-derived kidney organoids to study the role of renin and the renin-angiotensin system in the kidney. Extensive characterization of the kidney organoids revealed kidney-specific cell populations consisting of podocytes, proximal and distal tubular cells, stromal cells and endothelial cells. We examined the presence of various components of the renin-angiotensin system such as angiotensin II receptors, angiotensinogen, and angiotensin-converting enzymes 1 and 2. We identified by single-cell sequencing, immunohistochemistry, and functional assays that cyclic AMP stimulation induces a subset of pericytes to increase the synthesis and secretion of enzymatically active renin. Renin production by the organoids was responsive to regulation by parathyroid hormone. Subcutaneously implanted kidney organoids in immunodeficient IL2Ry/-Rag2 mice were successfully vascularized, maintained tubular and glomerular structures, and retained capacity to produce renin two months after implantation. Thus, our results demonstrate that kidney organoids express renin and provide insights into the endocrine potential of human kidney organoids, which is important for regenerative medicine in the context of the endocrine system.
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http://dx.doi.org/10.1016/j.kint.2020.08.008DOI Listing
January 2021

Increased CXCL4 expression in hematopoietic cells links inflammation and progression of bone marrow fibrosis in MPN.

Blood 2020 10;136(18):2051-2064

Department of Hematology, Erasmus Medical Center Cancer Institute, Rotterdam, The Netherlands.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) that leads to progressive bone marrow (BM) fibrosis. Although the cellular mutations involved in the pathogenesis of PMF have been extensively investigated, the sequential events that drive stromal activation and fibrosis by hematopoietic-stromal cross-talk remain elusive. Using an unbiased approach and validation in patients with MPN, we determined that the differential spatial expression of the chemokine CXCL4/platelet factor-4 marks the progression of fibrosis. We show that the absence of hematopoietic CXCL4 ameliorates the MPN phenotype, reduces stromal cell activation and BM fibrosis, and decreases the activation of profibrotic pathways in megakaryocytes, inflammation in fibrosis-driving cells, and JAK/STAT activation in both megakaryocytes and stromal cells in 3 murine PMF models. Our data indicate that higher CXCL4 expression in MPN has profibrotic effects and is a mediator of the characteristic inflammation. Therefore, targeting CXCL4 might be a promising strategy to reduce inflammation in PMF.
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http://dx.doi.org/10.1182/blood.2019004095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7678060PMC
October 2020

Rapid in vitro generation of bona fide exhausted CD8+ T cells is accompanied by Tcf7 promotor methylation.

PLoS Pathog 2020 06 24;16(6):e1008555. Epub 2020 Jun 24.

Department of Immunology, Erasmus University Medical Center, Rotterdam, The Netherlands.

Exhaustion is a dysfunctional state of cytotoxic CD8+ T cells (CTL) observed in chronic infection and cancer. Current in vivo models of CTL exhaustion using chronic viral infections or cancer yield very few exhausted CTL, limiting the analysis that can be done on these cells. Establishing an in vitro system that rapidly induces CTL exhaustion would therefore greatly facilitate the study of this phenotype, identify the truly exhaustion-associated changes and allow the testing of novel approaches to reverse or prevent exhaustion. Here we show that repeat stimulation of purified TCR transgenic OT-I CTL with their specific peptide induces all the functional (reduced cytokine production and polyfunctionality, decreased in vivo expansion capacity) and phenotypic (increased inhibitory receptors expression and transcription factor changes) characteristics of exhaustion. Importantly, in vitro exhausted cells shared the transcriptomic characteristics of the gold standard of exhaustion, CTL from LCMV cl13 infections. Gene expression of both in vitro and in vivo exhausted CTL was distinct from T cell anergy. Using this system, we show that Tcf7 promoter DNA methylation contributes to TCF1 downregulation in exhausted CTL. Thus this novel in vitro system can be used to identify genes and signaling pathways involved in exhaustion and will facilitate the screening of reagents that prevent/reverse CTL exhaustion.
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http://dx.doi.org/10.1371/journal.ppat.1008555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340326PMC
June 2020

Atypical 3q26/MECOM rearrangements genocopy inv(3)/t(3;3) in acute myeloid leukemia.

Blood 2020 07;136(2):224-234

Department of Hematology.

Acute myeloid leukemia (AML) with inv(3)/t(3;3)(q21q26) is a distinct World Health Organization recognized entity, characterized by its aggressive course and poor prognosis. In this subtype of AML, the translocation of a GATA2 enhancer (3q21) to MECOM (3q26) results in overexpression of the MECOM isoform EVI1 and monoallelic expression of GATA2 from the unaffected allele. The full-length MECOM transcript, MDS1-EVI1, is not expressed as the result of the 3q26 rearrangement. Besides the classical inv(3)/t(3;3), a number of other 3q26/MECOM rearrangements with poor treatment response have been reported in AML. Here, we demonstrate, in a group of 33 AML patients with atypical 3q26 rearrangements, MECOM involvement with EVI1 overexpression but no or low MDS1-EVI1 levels. Moreover, the 3q26 translocations in these AML patients often involve superenhancers of genes active in myeloid development (eg, CD164, PROM1, CDK6, or MYC). In >50% of these cases, allele-specific GATA2 expression was observed, either by copy-number loss or by an unexplained allelic imbalance. Altogether, atypical 3q26 recapitulate the main leukemic mechanism of inv(3)/t(3;3) AML, namely EVI1 overexpression driven by enhancer hijacking, absent MDS1-EVI1 expression and potential GATA2 involvement. Therefore, we conclude that both atypical 3q26/MECOM and inv(3)/t(3;3) can be classified as a single entity of 3q26-rearranged AMLs. Routine analyses determining MECOM rearrangements and EVI1 and MDS1-EVI1 expression are required to recognize 3q-rearranged AML cases.
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http://dx.doi.org/10.1182/blood.2019003701DOI Listing
July 2020

Identification of osteolineage cell-derived extracellular vesicle cargo implicated in hematopoietic support.

FASEB J 2020 04 21;34(4):5435-5452. Epub 2020 Feb 21.

Department of Hematology, Erasmus University Medical Center, Rotterdam, the Netherlands.

Osteolineage cell-derived extracellular vesicles (EVs) play a regulatory role in hematopoiesis and have been shown to promote the ex vivo expansion of human hematopoietic stem and progenitor cells (HSPCs). Here, we demonstrate that EVs from different human osteolineage sources do not have the same HSPC expansion promoting potential. Comparison of stimulatory and non-stimulatory osteolineage EVs by next-generation sequencing and mass spectrometry analyses revealed distinct microRNA and protein signatures identifying EV-derived candidate regulators of ex vivo HSPC expansion. Accordingly, the treatment of umbilical cord blood-derived CD34 HSPCs with stimulatory EVs-altered HSPC transcriptome, including genes with known roles in cell proliferation. An integrative bioinformatics approach, which connects the HSPC gene expression data with the candidate cargo in stimulatory EVs, delineated the potentially targeted biological functions and pathways during hematopoietic cell expansion and development. In conclusion, our study gives novel insights into the complex biological role of EVs in osteolineage cell-HSPC crosstalk and promotes the utility of EVs and their cargo as therapeutic agents in regenerative medicine.
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http://dx.doi.org/10.1096/fj.201902610RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7136136PMC
April 2020

Temporal autoregulation during human PU.1 locus SubTAD formation.

Blood 2018 12 12;132(25):2643-2655. Epub 2018 Oct 12.

Institute of Molecular Tumor Biology and.

Epigenetic control of gene expression occurs within discrete spatial chromosomal units called topologically associating domains (TADs), but the exact spatial requirements of most genes are unknown; this is of particular interest for genes involved in cancer. We therefore applied high-resolution chromosomal conformation capture sequencing to map the three-dimensional (3D) organization of the human locus encoding the key myeloid transcription factor PU.1 in healthy monocytes and acute myeloid leukemia (AML) cells. We identified a dynamic ∼75-kb unit (SubTAD) as the genomic region in which spatial interactions between PU.1 gene regulatory elements occur during myeloid differentiation and are interrupted in AML. Within this SubTAD, proper initiation of the spatial chromosomal interactions requires PU.1 autoregulation and recruitment of the chromatin-adaptor protein LDB1 (LIM domain-binding protein 1). However, once these spatial interactions have occurred, LDB1 stabilizes them independently of PU.1 autoregulation. Thus, our data support that PU.1 autoregulates its expression in a "hit-and-run" manner by initiating stable chromosomal loops that result in a transcriptionally active chromatin architecture.
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http://dx.doi.org/10.1182/blood-2018-02-834721DOI Listing
December 2018

MBD4 guards against methylation damage and germ line deficiency predisposes to clonal hematopoiesis and early-onset AML.

Blood 2018 10 26;132(14):1526-1534. Epub 2018 Jul 26.

Division of Cancer and Haematology, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.

The tendency of 5-methylcytosine (5mC) to undergo spontaneous deamination has had a major role in shaping the human genome, and this methylation damage remains the primary source of somatic mutations that accumulate with age. How 5mC deamination contributes to cancer risk in different tissues remains unclear. Genomic profiling of 3 early-onset acute myeloid leukemias (AMLs) identified germ line loss of MBD4 as an initiator of 5mC-dependent hypermutation. MBD4-deficient AMLs display a 33-fold higher mutation burden than AML generally, with >95% being C>T in the context of a CG dinucleotide. This distinctive signature was also observed in sporadic cancers that acquired biallelic mutations in and in knockout mice. Sequential sampling of germ line cases demonstrated repeated expansion of blood cell progenitors with pathogenic mutations in , a key driver gene for both clonal hematopoiesis and AML. Our findings reveal genetic and epigenetic factors that shape the mutagenic influence of 5mC. Within blood cells, this links methylation damage to the driver landscape of clonal hematopoiesis and reveals a conserved path to leukemia. Germ line MBD4 deficiency enhances cancer susceptibility and predisposes to AML.
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http://dx.doi.org/10.1182/blood-2018-05-852566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6172562PMC
October 2018

Characterization of Endothelial Cells Associated with Hematopoietic Niche Formation in Humans Identifies IL-33 As an Anabolic Factor.

Cell Rep 2018 01;22(3):666-678

Department of Hematology, Erasmus MC Cancer Institute, Rotterdam 3015CN, the Netherlands. Electronic address:

Bone marrow formation requires an orchestrated interplay between osteogenesis, angiogenesis, and hematopoiesis that is thought to be mediated by endothelial cells. The nature of the endothelial cells and the molecular mechanisms underlying these events remain unclear in humans. Here, we identify a subset of endoglin-expressing endothelial cells enriched in human bone marrow during fetal ontogeny and upon regeneration after chemotherapeutic injury. Comprehensive transcriptional characterization by massive parallel RNA sequencing of these cells reveals a phenotypic and molecular similarity to murine type H endothelium and activation of angiocrine factors implicated in hematopoiesis, osteogenesis, and angiogenesis. Interleukin-33 (IL-33) was significantly overexpressed in these endothelial cells and promoted the expansion of distinct subsets of hematopoietic precursor cells, endothelial cells, as well as osteogenic differentiation. The identification and molecular characterization of these human regeneration-associated endothelial cells is thus anticipated to instruct the discovery of angiocrine factors driving bone marrow formation and recovery after injury.
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http://dx.doi.org/10.1016/j.celrep.2017.12.070DOI Listing
January 2018

Gli1 Mesenchymal Stromal Cells Are a Key Driver of Bone Marrow Fibrosis and an Important Cellular Therapeutic Target.

Cell Stem Cell 2017 06 27;20(6):785-800.e8. Epub 2017 Apr 27.

Division of Nephrology and Clinical Immunology, RWTH Aachen University, 52074 Aachen, Germany. Electronic address:

Bone marrow fibrosis (BMF) develops in various hematological and non-hematological conditions and is a central pathological feature of myelofibrosis. Effective cell-targeted therapeutics are needed, but the cellular origin of BMF remains elusive. Here, we show using genetic fate tracing in two murine models of BMF that Gli1 mesenchymal stromal cells (MSCs) are recruited from the endosteal and perivascular niche to become fibrosis-driving myofibroblasts in the bone marrow. Genetic ablation of Gli1 cells abolished BMF and rescued bone marrow failure. Pharmacological targeting of Gli proteins with GANT61 inhibited Gli1 cell expansion and myofibroblast differentiation and attenuated fibrosis severity. The same pathway is also active in human BMF, and Gli1 expression in BMF significantly correlates with the severity of the disease. In addition, GANT61 treatment reduced the myofibroblastic phenotype of human MSCs isolated from patients with BMF, suggesting that targeting of Gli proteins could be a relevant therapeutic strategy.
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http://dx.doi.org/10.1016/j.stem.2017.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6485654PMC
June 2017

Mesenchymal Inflammation Drives Genotoxic Stress in Hematopoietic Stem Cells and Predicts Disease Evolution in Human Pre-leukemia.

Cell Stem Cell 2016 11 22;19(5):613-627. Epub 2016 Sep 22.

Department of Hematology, Erasmus MC Cancer Institute, Rotterdam 3015CN, the Netherlands. Electronic address:

Mesenchymal niche cells may drive tissue failure and malignant transformation in the hematopoietic system, but the underlying molecular mechanisms and relevance to human disease remain poorly defined. Here, we show that perturbation of mesenchymal cells in a mouse model of the pre-leukemic disorder Shwachman-Diamond syndrome (SDS) induces mitochondrial dysfunction, oxidative stress, and activation of DNA damage responses in hematopoietic stem and progenitor cells. Massive parallel RNA sequencing of highly purified mesenchymal cells in the SDS mouse model and a range of human pre-leukemic syndromes identified p53-S100A8/9-TLR inflammatory signaling as a common driving mechanism of genotoxic stress. Transcriptional activation of this signaling axis in the mesenchymal niche predicted leukemic evolution and progression-free survival in myelodysplastic syndrome (MDS), the principal leukemia predisposition syndrome. Collectively, our findings identify mesenchymal niche-induced genotoxic stress in heterotypic stem and progenitor cells through inflammatory signaling as a targetable determinant of disease outcome in human pre-leukemia.
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http://dx.doi.org/10.1016/j.stem.2016.08.021DOI Listing
November 2016

An autonomous CEBPA enhancer specific for myeloid-lineage priming and neutrophilic differentiation.

Blood 2016 06 10;127(24):2991-3003. Epub 2016 Mar 10.

Department of Hematology.

Neutrophilic differentiation is dependent on CCAAT enhancer-binding protein α (C/EBPα), a transcription factor expressed in multiple organs including the bone marrow. Using functional genomic technologies in combination with clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 genome editing and in vivo mouse modeling, we show that CEBPA is located in a 170-kb topological-associated domain that contains 14 potential enhancers. Of these, 1 enhancer located +42 kb from CEBPA is active and engages with the CEBPA promoter in myeloid cells only. Germ line deletion of the homologous enhancer in mice in vivo reduces Cebpa levels exclusively in hematopoietic stem cells (HSCs) and myeloid-primed progenitor cells leading to severe defects in the granulocytic lineage, without affecting any other Cebpa-expressing organ studied. The enhancer-deleted progenitor cells lose their myeloid transcription program and are blocked in differentiation. Deletion of the enhancer also causes loss of HSC maintenance. We conclude that a single +42-kb enhancer is essential for CEBPA expression in myeloid cells only.
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http://dx.doi.org/10.1182/blood-2016-01-695759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5043424PMC
June 2016

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

Haematologica 2015 Oct 16;100(10):1285-93. Epub 2015 Jul 16.

Department of Hematology, Erasmus Medical Center Cancer Institute, Rotterdam, the Netherlands

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.
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http://dx.doi.org/10.3324/haematol.2015.131573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4591760PMC
October 2015

Mutational spectrum of myeloid malignancies with inv(3)/t(3;3) reveals a predominant involvement of RAS/RTK signaling pathways.

Blood 2015 Jan 7;125(1):133-9. Epub 2014 Nov 7.

Department of Hematology, Erasmus University Medical Center, Rotterdam, The Netherlands;

Myeloid malignancies bearing chromosomal inv(3)/t(3;3) abnormalities are among the most therapy-resistant leukemias. Deregulated expression of EVI1 is the molecular hallmark of this disease; however, the genome-wide spectrum of cooperating mutations in this disease subset has not been systematically elucidated. Here, we show that 98% of inv(3)/t(3;3) myeloid malignancies harbor mutations in genes activating RAS/receptor tyrosine kinase (RTK) signaling pathways. In addition, hemizygous mutations in GATA2, as well as heterozygous alterations in RUNX1, SF3B1, and genes encoding epigenetic modifiers, frequently co-occur with the inv(3)/t(3;3) aberration. Notably, neither mutational patterns nor gene expression profiles differ across inv(3)/t(3;3) acute myeloid leukemia, chronic myeloid leukemia, and myelodysplastic syndrome cases, suggesting recognition of inv(3)/t(3;3) myeloid malignancies as a single disease entity irrespective of blast count. The high incidence of activating RAS/RTK signaling mutations may provide a target for a rational treatment strategy in this high-risk patient group.
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http://dx.doi.org/10.1182/blood-2014-07-591461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4334729PMC
January 2015

A single oncogenic enhancer rearrangement causes concomitant EVI1 and GATA2 deregulation in leukemia.

Cell 2014 Apr 3;157(2):369-381. Epub 2014 Apr 3.

Department of Hematology, Erasmus University Medical Center, Rotterdam, 3015 GE, the Netherlands. Electronic address:

Chromosomal rearrangements without gene fusions have been implicated in leukemogenesis by causing deregulation of proto-oncogenes via relocation of cryptic regulatory DNA elements. AML with inv(3)/t(3;3) is associated with aberrant expression of the stem-cell regulator EVI1. Applying functional genomics and genome-engineering, we demonstrate that both 3q rearrangements reposition a distal GATA2 enhancer to ectopically activate EVI1 and simultaneously confer GATA2 functional haploinsufficiency, previously identified as the cause of sporadic familial AML/MDS and MonoMac/Emberger syndromes. Genomic excision of the ectopic enhancer restored EVI1 silencing and led to growth inhibition and differentiation of AML cells, which could be replicated by pharmacologic BET inhibition. Our data show that structural rearrangements involving the chromosomal repositioning of a single enhancer can cause deregulation of two unrelated distal genes, with cancer as the outcome.
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http://dx.doi.org/10.1016/j.cell.2014.02.019DOI Listing
April 2014

Two splice-factor mutant leukemia subgroups uncovered at the boundaries of MDS and AML using combined gene expression and DNA-methylation profiling.

Blood 2014 May 25;123(21):3327-35. Epub 2014 Mar 25.

Department of Hematology, and Clinical Trial Center, Erasmus University Medical Center, Rotterdam, The Netherlands;

Mutations in splice factor (SF) genes occur more frequently in myelodysplastic syndromes (MDS) than in acute myeloid leukemias (AML). We sequenced complementary DNA from bone marrow of 47 refractory anemia with excess blasts (RAEB) patients, 29 AML cases with low marrow blast cell count, and 325 other AML patients and determined the presence of SF-hotspot mutations in SF3B1, U2AF35, and SRSF2. SF mutations were found in 10 RAEB, 12 AML cases with low marrow blast cell count, and 25 other AML cases. Our study provides evidence that SF-mutant RAEB and SF-mutant AML are clinically, cytologically, and molecularly highly similar. An integrated analysis of genomewide messenger RNA (mRNA) expression profiling and DNA-methylation profiling data revealed 2 unique patient clusters highly enriched for SF-mutant RAEB/AML. The combined genomewide mRNA expression profiling/DNA-methylation profiling signatures revealed 1 SF-mutant patient cluster with an erythroid signature. The other SF-mutant patient cluster was enriched for NRAS/KRAS mutations and showed an inferior survival. We conclude that SF-mutant RAEB/AML constitutes a related disorder overriding the artificial separation between AML and MDS, and that SF-mutant RAEB/AML is composed of 2 molecularly and clinically distinct subgroups. We conclude that SF-mutant disorders should be considered as myeloid malignancies that transcend the boundaries of AML and MDS.
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http://dx.doi.org/10.1182/blood-2013-07-512855DOI Listing
May 2014

Phosphorylation of the leukemic oncoprotein EVI1 on serine 196 modulates DNA binding, transcriptional repression and transforming ability.

PLoS One 2013 12;8(6):e66510. Epub 2013 Jun 12.

Stem Cell and Leukaemia Proteomics Laboratory, University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066510PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3680417PMC
January 2014

EVI1 is critical for the pathogenesis of a subset of MLL-AF9-rearranged AMLs.

Blood 2012 Jun 2;119(24):5838-49. Epub 2012 May 2.

Department of Hematology, Erasmus University Medical Center, Rotterdam, The Netherlands.

The proto-oncogene EVI1 (ecotropic viral integration site-1), located on chromosome band 3q26, is aberrantly expressed in human acute myeloid leukemia (AML) with 3q26 rearrangements. In the current study, we showed, in a large AML cohort carrying 11q23 translocations, that ∼ 43% of all mixed lineage leukemia (MLL)-rearranged leukemias are EVI1(pos). High EVI1 expression occurs in AMLs expressing the MLL-AF6, -AF9, -AF10, -ENL, or -ELL fusion genes. In addition, we present evidence that EVI1(pos) MLL-rearranged AMLs differ molecularly, morphologically, and immunophenotypically from EVI1(neg) MLL-rearranged leukemias. In mouse bone marrow cells transduced with MLL-AF9, we show that MLL-AF9 fusion protein maintains Evi1 expression on transformation of Evi1(pos) HSCs. MLL-AF9 does not activate Evi1 expression in MLL-AF9-transformed granulocyte macrophage progenitors (GMPs) that were initially Evi1(neg). Moreover, shRNA-mediated knockdown of Evi1 in an Evi1(pos) MLL-AF9 mouse model inhibits leukemia growth both in vitro and in vivo, suggesting that Evi1 provides a growth-promoting signal. Using the Evi1(pos) MLL-AF9 mouse leukemia model, we demonstrate increased sensitivity to chemotherapeutic agents on reduction of Evi1 expression. We conclude that EVI1 is a critical player in tumor growth in a subset of MLL-rearranged AMLs.
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http://dx.doi.org/10.1182/blood-2011-11-393827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3382941PMC
June 2012