Biology subjects

Hoogenboezem, R.

Publications and source records attributed to Hoogenboezem, R..

11 recordsLinked to original sources

bioRxiv2025

Single-Cell Roadmap of Early Hemato-Endothelial Development: Functions of Atf3, Zfp711 and Bcl6b

Hematopoiesis is the process of producing blood cells. In mammalian embryos, hematopoiesis occurs in three consecutive overlapping waves (Neo et al. 2021; Dzierzak and Bigas 2018) that are regulated by transcription factors (TFs) and signaling proteins. We investigated the functions of three relatively poorly studied TFs in early embryonic hematopoietic development at single-cell resolution: Activating transcription factor 3 (Atf3), Zinc finger protein 711 (Zfp711), and B cell CLL/lymphoma 6, member B (Bcl6b). These TFs are upregulated early in development when hematopoietic and endothelial lineages separate from cardiac and other mesodermal lineages. We combined multiplexed single-cell RNA sequencing (scRNA-seq) and cell identity analysis using Flow Cytometric Analysis (FCA) with TF knockouts (KO) in in-vitro differentiating mouse embryonic stem cells (mESCs) to dissect the function of these TFs in lineage induction, specification, and separation. The Atf3-KO showed an increase of distinct mesodermal subpopulations, but a decrease of endothelial and erythro-myeloid progenitors (EMPs) by downregulation of important genes (i.e. Runx1, Mafb, Egr1, Jun, Jund, Fos, Batf3, and Zf608) that likely explains the effects on EMPs and the increased expression of interferon-related genes. In Zfp711-KO cells, the number of blood progenitor cells and erythroid cells increased, while the number of endothelial cells decreased. Furthermore, Hoxa expressing mesoderm decreased, while Hoxb expressing mesoderm increased. In contrast, the Bcl6b-KO had no observable effects on early hematopoiesis. In conclusion, we report the function of these three TFs function at different stages of hemato-endothelial lineage specification. HIGHLIGHTSO_LICombined scRNA-seq and FCA on KO mESCs in vitro differentiation enabled unbiased identification of the respective TF functions during specific stages of hematoendothelial lineage specification. C_LIO_LIAtf3-KO: Increased abundance of mesodermal lineages and decreased endothelial lineages and EMPs. - Downregulation of key TF-encoding genes (e.g., Runx1, Mafb, Egr1, Jun, Jund, Fos, Batf3, Zf608) explain the effects on EMPs. - Generation of a distinct mesodermal subpopulation with high expression of sets of interferon and antiviral-related genes. - Upregulation of interferon and anti-viral-related genes, indicating Atf3 acts as a negative regulator of these genes. C_LIO_LIZfp711-KO: Increased numbers of blood progenitor and erythroid cells. Decreased numbers of endothelial cells. - Shift in mesodermal populations: Hoxa expressing mesoderm decreased, and Hoxb expressing mesoderm increased. - Hoxa9, Hoxa10, Hoxa13, Jun, Jund and Atf3 amongst the downregulated genes. The Atf3 promoter has a potential Zfp711-binding site. - Increased expression in a given Endothelium subtype in endocardium in vivo and in vitro. C_LIO_LIBcl6b-KO: No observable impact on early hematopoiesis. C_LI

developmental biology↗

bioRxiv2024

Hematological phenotypes in GATA2 deficiency syndrome arise from secondary injuries and maladaptation to proliferation

The GATA2 transcription factor is a pivotal regulator of hematopoiesis. Disruptions in the GATA2 gene drive severe hematologic abnormalities and are associated with an increased risk of myelodysplastic syndromes and acute myeloid leukemia; however, the mechanisms underlying the pathophysiology of GATA2 deficiency remain still unclear. We developed two different mouse models that are based on serial and limiting donor cell transplantation of (aged) GATA2 haploinsufficient cells and mirror the symptoms of GATA2 deficiency. Similar to what has been observed in patients, our models show that GATA2 haploinsufficiency leads to B lymphopenia, monocytopenia, lethal bone marrow failure (BMF), myelodysplasia and leukemia. Leukemia arises exclusively as a result of BMF, driven by somatic aberrations and accompanied by increased Myc target expression and genomic instability. These findings were confirmed in human GATA2+/- K562 cell lines showing defects in cytokinesis and are in line with the fact that monosomy 7 and trisomy 8 are frequent events in patients with MDS. Key pointsO_LIIn a mouse model for GATA2 deficiency, leukemia emerges from bone marrow failure C_LIO_LIMaladaptation to proliferative signals and chromosomal segregation defects contribute to the hematological phenotypes in GATA2 deficiency C_LI

cell biology↗

bioRxiv2023

Oncogene EVI1 Drives Acute Myeloid Leukemia Via a Targetable Interaction with CTBP2

Acute myeloid leukemia (AML) driven by the activation of EVI1 due to chromosome 3q26/MECOM rearrangements is incurable. Since transcription factors like EVI1 are notoriously hard to target, insight into the mechanism by which EVI1 drives myeloid transformation could provide alternative avenues for therapy. Applying protein folding predictions combined with proteomics technologies, we demonstrate that interaction with CTBP1 and CTBP2 via a single PLDLS motif in EVI1 is indispensable for leukemic transformation. Furthermore, we show that a 4x PLDLS repeat construct outcompetes binding of EVI1 to CTBP1 and CTBP2 and thereby inhibits proliferation of 3q26/MECOM rearranged AML both in in vitro and in xenotransplant models. This proof-of-concept study opens the possibility to therapeutically target one of the most incurable forms of AML with specific EVI1-CTBP inhibitors. This has important implications for other tumour types with aberrant expression of EVI1 as well as for cancers transformed by distinct CTBP-dependent oncogenic transcription factors.

cancer biology↗

bioRxiv2023

Single-cell transcriptomic analysis of NK cell dynamics in myeloma patients reveal persistent reduction of cytotoxic NK cells from diagnosis to relapse

Natural killer (NK) cells mediate the cytotoxic immune response against multiple myeloma and are important effector cells in immune therapies through antibody-dependent cellular cytotoxicity. Here, we used single-cell transcriptomics, flow cytometry and functional assays to investigate the bone marrow NK cell compartment of myeloma patients at diagnosis, during treatment and after relapse. The bone marrow of myeloma patients is characterized by a reduction in conventional cytotoxic NK cells that persists throughout treatment. We show in 20% of newly diagnosed myeloma patients that an altered balance between cytotoxic and cytokine-producing NK cells translates into a reduced cytotoxic ability in response to therapeutic antibodies. The relative loss of cytotoxic NK cells persists at relapse and is accompanied by an expansion of IFN-responsive NK cells. These findings reveal previously unappreciated alterations in bone marrow NK cell composition and highlight the importance of understanding the bone marrow immune system in patients receiving immunotherapies. Statement of significanceThe bone marrow of multiple myeloma patients is characterized by a persistent reduction in cytotoxic CD56dim NK cells, accompanied by inferior in vitro responses to therapeutic antibodies at diagnosis and an increase in IFN-responsive NK cells at relapse. These findings highlight the importance of understanding the BM microenvironment in multiple myeloma patients receiving immunotherapies.

immunology↗

bioRxiv2023

Deconvolution Of Hematopoietic Stem/Progenitor Cell Signaling Predicts Inflammatory Niche Remodeling To Be A Determinant Of Tissue Failure And Outcome In Human AML

Cancer initiation is orchestrated by interplay between tumor-initiating cells and their stromal/immune environment. Here, by adapted scRNAsequencing, we decipher the predicted signaling between tissue-resident hematopoietic stem/progenitor cells (HSPCs) and their neoplastic counterparts with their native niches in the human bone marrow. LEPR+ stromal cells are identified as central regulators of hematopoiesis through predicted interactions with all cells in the marrow. Inflammatory niche remodeling and the resulting deprivation of critical HSPC regulatory factors is predicted to repress distinct high-output HSC subsets in NPM1-mutated AML, with relative resistance of clonal cells. Stromal gene signatures reflective of niche remodeling are associated with reduced relapse rates and favorable outcome after chemotherapy, across all genetic risk categories. Elucidation of the intercellular signaling defining human AML, thus, predicts that inflammatory remodeling of stem cell niches drives tissue repression and clonal selection, but may pose a vulnerability for relapse-initiating cells in the context of chemotherapeutic treatment. statement of significanceTumor-promoting inflammation is considered an enabling characteristic of tumorigenesis, but mechanisms remain incompletely understood. By deciphering the predicted signaling between tissue-resident stem cells and their neoplastic counterparts with their environment, we identify inflammatory remodeling of stromal niches as a determinant of normal tissue repression and clinical outcome in human AML. Key pointsO_LIA comprehensive taxonomy of the predicted interactions between LEPR+ stromal niches, HSPCs and adaptive/innate immune cells in the human NBM. C_LIO_LIInflammation-associated decline of stromal niches in AML represses residual normal hematopoiesis with relative resistance of leukemic cells. C_LIO_LIInflammatory decline of stromal niches is associated with reduced relapse risk and favorable outcome. C_LI

cancer biology↗

bioRxiv2023

An IL-1β driven neutrophil-stromal cell axis fosters a BAFF-rich microenvironment in multiple myeloma

The bone marrow permanently harbors high numbers of neutrophils, and a tumor-supportive bias of these cells could significantly impact bone marrow-confined malignancies. In multiple myeloma, the bone marrow is characterized by inflammatory stromal cells with the potential to influence neutrophils. We investigated myeloma-associated alterations in marrow neutrophils and the impact of stromal inflammation on neutrophil function. Mature neutrophils in myeloma marrow are activated and tumor-supportive, transcribing increased levels of IL-1{beta}, and myeloma cell survival factor BAFF. Interactions with inflammatory stromal cells can induce neutrophil activation, including BAFF secretion, in a STAT3-dependent manner and once activated, neutrophils gain the ability to reciprocally induce stromal activation. After first-line myeloid-depleting treatment, patient bone marrow retains residual stromal inflammation and newly-formed neutrophils are reactivated. Combined, we identify a neutrophil-stromal cell feed-forward loop driving tumor-supportive inflammation that persists after treatment and warrants novel strategies to target both stromal and immune microenvironments in multiple myeloma.

immunology↗

bioRxiv2023

Immune-mediated tumor control in the 5TGM1 transfer model of multiple myeloma

Multiple myeloma is a disease of malignant plasma cells residing in the bone marrow, where interactions with local immune cells are thought to contribute to disease pathobiology. However, since a multiple myeloma diagnosis is virtually always preceded by an asymptomatic precursor phase, identifying early alterations in the bone marrow micro-environment following occupation by multiple myeloma cells remains challenging. Here we used the 5TGM1 transfer model of murine myeloma in combination with myeloma-permissive KaLwRij mice and myeloma-resistant C57Bl/6 mice and hypothesized that differential sensitivity to myeloma in these HLA-identical mouse strains has an immunological basis and might allow for dissection of early immune responses to myeloma cells. Using flow cytometry and single-cell RNA sequencing we show that C57Bl/6 mice can restrain tumor growth for prolonged periods, associated with activation of cytotoxic immune responses that were absent from KaLwRij mice. Transcriptional analysis of immune cells and stromal cells identified a central role for IFN-signaling in tumor containment, and antibody-mediated neutralization of IFN{gamma} increased both incidence and outgrowth of multiple myeloma in C57Bl/6 mice. Together these findings highlight the ability of a fully functional immune system to control multiple myeloma progression in an IFN{gamma}-dependent manner and suggest that transfer of 5TGM1 cells into parental C57Bl/6 mice can serve as a faithful model to track anti-myeloma immune responses in immune competent and genetically modifiable mice.

immunology↗

bioRxiv2022

Gata2-regulated Gfi1b expression controls endothelial programming during endothelial-to-hematopoietic transition

The first hematopoietic stem cells (HSCs) are formed through endothelial-to-hematopoietic transition (EHT) events during embryonic development. The transcription factor GATA2 is a crucial regulator of EHT and HSC function throughout life. Because GATA2 haploinsufficiency patients have inborn mutations, prenatal defects are likely to have an influence on disease development. In mice, Gata2 haploinsufficiency (Gata2+/-) reduces the number and the functionality of embryonic hematopoietic stem and progenitor cells (HSPCs) generated through EHT. However, the embryonic HSPC pool is heterogeneous and the mechanisms underlying this defect in Gata2+/- embryos are unclear. Here, we investigated whether Gata2 haploinsufficiency selectively affects a cellular subset undergoing EHT. We show that Gata2+/- HSPCs initiate but cannot fully activate hematopoietic programming during EHT. In addition, due to reduced activity of the endothelial repressor Gfi1b, Gata2+/- HSPCs cannot repress the endothelial identity to complete maturation. Finally, we show that hematopoietic-specific induction of gfi1b can restore HSC production in gata2b-null (gata2b-/-) zebrafish embryos. This study illustrates pivotal roles of Gata2 on the regulation of transcriptional network governing HSPC identity throughout EHT. HighlightsO_LIMaturation of embryonic Gata2+/- HSPCs is disturbed due to aberrant endothelial gene expression and incomplete activation of hematopoietic transcriptional programming. C_LIO_LIGata2 activates Gfi1b to repress endothelial identity of embryonic HSPCs during maturation. C_LIO_LIHematopoietic-specific induction of gfi1b restores the number of embryonic HSCs in gata2b-/- zebrafish. C_LI

developmental biology↗

bioRxiv2021

Gata2b haploinsufficiency causes aberrant transcriptional signatures in HSPCs resulting in myeloid and erythroid dysplasia in zebrafish

The transcription factor GATA2 has pivotal roles in hematopoiesis. Germline GATA2 mutations in patients result in GATA2 haploinsufficiency syndrome characterized by immunodeficiency, bone marrow failure, and predispositions to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Clinical symptoms in GATA2 patients are diverse and mechanisms driving GATA2 related phenotypes largely unknown. To explore the impact of GATA2 haploinsufficiency on hematopoiesis, we generated a zebrafish model carrying a heterozygous mutation of gata2b (gata2b+/-), an orthologue of GATA2. Morphological analysis revealed myeloid and erythroid dysplasia in gata2b+/- kidney marrow (KM). single nucleus (sn)-ATAC-seq showed that the co-accessibility between the transcription start site (TSS) and a +3.5-4.1kb enhancer was more robust in gata2b+/- zebrafish HSPCs compared to wild type, increasing gata2b expression. This is suggestive of an auto-regulatory feedback mechanism, where gata2b expression remains at sufficient levels after the loss of a single allele to maintain the HSPC pool. As a result, gata2b+/- chromatin is also more accessible in the erythroid and myeloid lineage, causing several defects. scRNA-seq data revealed a differentiation delay in erythroid progenitors, hallmarked by downregulation of intrinsic signals like cytoskeletal transcripts, aberrant proliferative signatures, and downregulation of Gata1a, a master regulator of erythropoiesis, likely preceding erythroid dysplasia. This shows that the cell intrinsic compensatory mechanisms for the maintenance of normal levels of Gata2b to maintain HSPC integrity result in aberrant lineage differentiation and a preleukemia syndrome.

cancer biology↗

bioRxiv2020

Spatial multi-omic map of human myocardial infarction

Myocardial infarction is a leading cause of mortality. While advances in the acute treatment have been made, the late-stage mortality is still high, driven by an incomplete understanding of cardiac remodeling processes1,2. Here we used single-cell gene expression, chromatin accessibility and spatial transcriptomic profiling of different physiological zones and timepoints of human myocardial infarction and human control myocardium to generate an integrative high-resolution map of cardiac remodeling. This approach allowed us to increase spatial resolution of cell-type composition and provide spatially resolved insights into the cardiac transcriptome and epigenome with identification of distinct cellular zones of injury, repair and remodeling. We here identified and validated mechanisms of fibroblast to myofibroblast differentiation that drive cardiac fibrosis. Our study provides an integrative molecular map of human myocardial infarction and represents a reference to advance mechanistic and therapeutic studies of cardiac disease.

systems biology↗

bioRxiv2019

Single cell transcriptome analysis reveals an essential role for Gata2b in hematopoietic lineage decisions in zebrafish

Hematopoietic stem cells (HSCs) are tightly controlled to keep a balance between myeloid and lymphoid cell differentiation. Gata2 is a pivotal hematopoietic transcription factor required for HSC generation and maintenance. We generated a zebrafish mutant for the mammalian Gata2 orthologue, gata2b. We found that in adult zebrafish, gata2b is required for both neutrophilic- and monocytic lineage differentiation. Single cell transcriptome analysis revealed that the myeloid defect present in Gata2b deficient zebrafish arise in the most immature hematopoietic stem and progenitor cell (HSPC) compartment and that this population is instead committed towards the lymphoid and erythroid lineage. Taken together, we find that Gata2b is vital for the fate choice between the myeloid and lymphoid lineages.

immunology↗