Publications by authors named "Martina Konantz"

29 Publications

  • Page 1 of 1

iPSC modeling of stage-specific leukemogenesis reveals BAALC as a key oncogene in severe congenital neutropenia.

Cell Stem Cell 2021 05 23;28(5):906-922.e6. Epub 2021 Apr 23.

Department of Oncology, Hematology, Immunology, and Rheumatology, University Hospital Tuebingen, 72074 Tuebingen, Germany. Electronic address:

Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome that can evolve to acute myeloid leukemia (AML). Mutations in CSF3R and RUNX1 are frequently observed in CN patients, although how they drive the transition from CN to AML (CN/AML) is unclear. Here we establish a model of stepwise leukemogenesis in CN/AML using CRISPR-Cas9 gene editing of CN patient-derived iPSCs. We identified BAALC upregulation and resultant phosphorylation of MK2a as a key leukemogenic event. BAALC deletion or treatment with CMPD1, a selective inhibitor of MK2a phosphorylation, blocked proliferation and induced differentiation of primary CN/AML blasts and CN/AML iPSC-derived hematopoietic stem and progenitor cells (HSPCs) without affecting healthy donor or CN iPSC-derived HSPCs. Beyond detailing a useful method for future investigation of stepwise leukemogenesis, this study suggests that targeting BAALC and/or MK2a phosphorylation may prevent leukemogenic transformation or eliminate AML blasts in CN/AML and RUNX1 mutant BAALC(hi) de novo AML.
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http://dx.doi.org/10.1016/j.stem.2021.03.023DOI Listing
May 2021

Dimethyl fumarate induces ferroptosis and impairs NF-κB/STAT3 signaling in DLBCL.

Blood 2021 Sep;138(10):871-884

Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany.

Despite the development of novel targeted drugs, the molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) still poses a substantial therapeutic challenge. DLBCL can be classified into at least 2 major subtypes (germinal center B cell [GCB]-like and activated B cell [ABC]-like DLBCL), each characterized by specific gene expression profiles and mutation patterns. Here we demonstrate a broad antitumor effect of dimethyl fumarate (DMF) on both DLBCL subtypes, which is mediated by the induction of ferroptosis, a form of cell death driven by the peroxidation of phospholipids. As a result of the high expression of arachidonate 5-lipoxygenase in concert with low glutathione and glutathione peroxidase 4 levels, DMF induces lipid peroxidation and thus ferroptosis, particularly in GCB DLBCL. In ABC DLBCL cells, which are addicted to NF-κB and STAT3 survival signaling, DMF treatment efficiently inhibits the activity of the IKK complex and Janus kinases. Interestingly, the BCL-2-specific BH3 mimetic ABT-199 and an inhibitor of ferroptosis suppressor protein 1 synergize with DMF in inducing cell death in DLBCL. Collectively, our findings identify the clinically approved drug DMF as a promising novel therapeutic option in the treatment of both GCB and ABC DLBCLs.
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http://dx.doi.org/10.1182/blood.2020009404DOI Listing
September 2021

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia.

J Vis Exp 2021 02 21(168). Epub 2021 Feb 21.

Department of Biomedicine, University Hospital Basel and University of Basel; Division for Hematology, University Hospital Basel and University of Basel; Department of Internal Medicine, Hematology and Oncology, University Hospital Tübingen;

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties (so called leukemic stem cells, LSCs) from more differentiated counterpart leukemic cells that lack disease initiation potential although they carry similar leukemia specific genetic mutations. NKG2DL are biochemically highly diverse MHC class I-like self-molecules. Healthy cells in homeostatic conditions generally do not express NKG2DL on the cell surface. Instead, expression of these ligands is induced upon exposure to cellular stress (e.g., oncogenic transformation or infectious stimuli) to trigger elimination of damaged cells via lysis through NKG2D-receptor-expressing immune cells such as natural killer (NK) cells. Interestingly, NKG2DL surface expression is selectively suppressed in LSC subpopulations, allowing these cells to evade NKG2D-mediated immune surveillance. Here, we present a side-by-side analysis of two different flow cytometry methods that allow the investigation of NKG2DL surface expression on cancer cells i.e., a method involving pan-ligand recognition and a method involving staining with multiple antibodies against single ligands. These methods can be used to separate viable NKG2DL negative cellular subpopulations with putative cancer stem cell properties from NKG2DL positive non-LSC.
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http://dx.doi.org/10.3791/61803DOI Listing
February 2021

Biodegradable Harmonophores for Targeted High-Resolution Tumor Imaging.

ACS Nano 2021 03 25;15(3):4144-4154. Epub 2021 Feb 25.

Department of Biosystems Science and Engineering (D-BSSE), Eidgenössische Technische Hochschule (ETH) Zurich, 4058 Basel, Switzerland.

Optical imaging probes have played a major role in detecting and monitoring a variety of diseases. In particular, nonlinear optical imaging probes, such as second harmonic generating (SHG) nanoprobes, hold great promise as clinical contrast agents, as they can be imaged with little background signal and unmatched long-term photostability. As their chemical composition often includes transition metals, the use of inorganic SHG nanoprobes can raise long-term health concerns. Ideally, contrast agents for biomedical applications should be degraded without any long-term toxicological consequences to the organism. Here, we developed biodegradable harmonophores (bioharmonophores) that consist of polymer-encapsulated, self-assembling peptides that generate a strong SHG signal. When functionalized with tumor cell surface markers, these reporters can target single cancer cells with high detection sensitivity in zebrafish embryos . Thus, bioharmonophores will enable an innovative approach to cancer treatment using targeted high-resolution optical imaging for diagnostics and therapy.
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http://dx.doi.org/10.1021/acsnano.0c10634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8023799PMC
March 2021

Acute Myeloid Leukemia Stem Cells: The Challenges of Phenotypic Heterogeneity.

Cancers (Basel) 2020 Dec 12;12(12). Epub 2020 Dec 12.

Department of Biomedicine, University of Basel and University Hospital Basel, Hebelstrasse 20, 4031 Basel, Switzerland.

Patients suffering from acute myeloid leukemia (AML) show highly heterogeneous clinical outcomes. Next to variabilities in patient-specific parameters influencing treatment decisions and outcome, this is due to differences in AML biology. In fact, different genetic drivers may transform variable cells of origin and co-exist with additional genetic lesions (e.g., as observed in clonal hematopoiesis) in a variety of leukemic (sub)clones. Moreover, AML cells are hierarchically organized and contain subpopulations of more immature cells called leukemic stem cells (LSC), which on the cellular level constitute the driver of the disease and may evolve during therapy. This genetic and hierarchical complexity results in a pronounced phenotypic variability, which is observed among AML cells of different patients as well as among the leukemic blasts of individual patients, at diagnosis and during the course of the disease. Here, we review the current knowledge on the heterogeneous landscape of AML surface markers with particular focus on those identifying LSC, and discuss why identification and targeting of this important cellular subpopulation in AML remains challenging.
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http://dx.doi.org/10.3390/cancers12123742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7764578PMC
December 2020

SRP54 mutations induce congenital neutropenia via dominant-negative effects on XBP1 splicing.

Blood 2021 03;137(10):1340-1352

Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland.

Heterozygous de novo missense variants of SRP54 were recently identified in patients with congenital neutropenia (CN) who display symptoms that overlap with Shwachman-Diamond syndrome (SDS). Here, we investigate srp54 knockout zebrafish as the first in vivo model of SRP54 deficiency. srp54-/- zebrafish experience embryonic lethality and display multisystemic developmental defects along with severe neutropenia. In contrast, srp54+/- zebrafish are viable, fertile, and show only mild neutropenia. Interestingly, injection of human SRP54 messenger RNAs (mRNAs) that carry mutations observed in patients (T115A, T117Δ, and G226E) aggravated neutropenia and induced pancreatic defects in srp54+/- fish, mimicking the corresponding human clinical phenotypes. These data suggest that the various phenotypes observed in patients may be a result of mutation-specific dominant-negative effects on the functionality of the residual wild-type SRP54 protein. Overexpression of mutated SRP54 also consistently induced neutropenia in wild-type fish and impaired the granulocytic maturation of human promyelocytic HL-60 cells and healthy cord blood-derived CD34+ hematopoietic stem and progenitor cells. Mechanistically, srp54-mutant fish and human cells show impaired unconventional splicing of the transcription factor X-box binding protein 1 (Xbp1). Moreover, xbp1 morphants recapitulate phenotypes observed in srp54 deficiency and, importantly, injection of spliced, but not unspliced, xbp1 mRNA rescues neutropenia in srp54+/- zebrafish. Together, these data indicate that SRP54 is critical for the development of various tissues, with neutrophils reacting most sensitively to the loss of SRP54. The heterogenic phenotypes observed in patients that range from mild CN to SDS-like disease may be the result of different dominant-negative effects of mutated SRP54 proteins on downstream XBP1 splicing, which represents a potential therapeutic target.
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http://dx.doi.org/10.1182/blood.2020008115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7994924PMC
March 2021

Collagen-rich omentum is a premetastatic niche for integrin α2-mediated peritoneal metastasis.

Elife 2020 10 7;9. Epub 2020 Oct 7.

Ovarian Cancer Research, Department of Biomedicine, University Hospital Basel and University of Basel, Basel, Switzerland.

The extracellular matrix (ECM) plays critical roles in tumor progression and metastasis. However, the contribution of ECM proteins to early metastatic onset in the peritoneal cavity remains unexplored. Here, we suggest a new route of metastasis through the interaction of integrin alpha 2 (ITGA2) with collagens enriched in the tumor coinciding with poor outcome in patients with ovarian cancer. Using multiple gene-edited cell lines and patient-derived samples, we demonstrate that ITGA2 triggers cancer cell adhesion to collagen, promotes cell migration, anoikis resistance, mesothelial clearance, and peritoneal metastasis in vitro and in vivo. Mechanistically, phosphoproteomics identify an ITGA2-dependent phosphorylation of focal adhesion kinase and mitogen-activated protein kinase pathway leading to enhanced oncogenic properties. Consequently, specific inhibition of ITGA2-mediated cancer cell-collagen interaction or targeting focal adhesion signaling may present an opportunity for therapeutic intervention of metastatic spread in ovarian cancer.
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http://dx.doi.org/10.7554/eLife.59442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541088PMC
October 2020

NCKAP1L defects lead to a novel syndrome combining immunodeficiency, lymphoproliferation, and hyperinflammation.

J Exp Med 2020 12;217(12)

Department of Pediatrics, King Abdulaziz Medical City, King Abdullah Specialized Children's Hospital, Riyadh, Saudi Arabia.

The Nck-associated protein 1-like (NCKAP1L) gene, alternatively called hematopoietic protein 1 (HEM-1), encodes a hematopoietic lineage-specific regulator of the actin cytoskeleton. Nckap1l-deficient mice have anomalies in lymphocyte development, phagocytosis, and neutrophil migration. Here we report, for the first time, NCKAP1L deficiency cases in humans. In two unrelated patients of Middle Eastern origin, recessive mutations in NCKAP1L abolishing protein expression led to immunodeficiency, lymphoproliferation, and hyperinflammation with features of hemophagocytic lymphohistiocytosis. Immunophenotyping showed an inverted CD4/CD8 ratio with a major shift of both CD4+ and CD8+ cells toward memory compartments, in line with combined RNA-seq/proteomics analyses revealing a T cell exhaustion signature. Consistent with the core function of NCKAP1L in the reorganization of the actin cytoskeleton, patients' T cells displayed impaired early activation, immune synapse morphology, and leading edge formation. Moreover, knockdown of nckap1l in zebrafish led to defects in neutrophil migration. Hence, NCKAP1L mutations lead to broad immune dysregulation in humans, which could be classified within actinopathies.
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http://dx.doi.org/10.1084/jem.20192275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526481PMC
December 2020

Oncogenic Kras causes myeloproliferation via NLRP3 inflammasome activation.

Nat Commun 2020 04 3;11(1):1659. Epub 2020 Apr 3.

Department of Medicine I, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany.

Oncogenic Ras mutations occur in various leukemias. It was unclear if, besides the direct transforming effect via constant RAS/MEK/ERK signaling, an inflammation-related effect of KRAS contributes to the disease. Here, we identify a functional link between oncogenic Kras and NLRP3 inflammasome activation in murine and human cells. Mice expressing active Kras in the hematopoietic system developed myeloproliferation and cytopenia, which is reversed in Kras mice lacking NLRP3 in the hematopoietic system. Therapeutic IL-1-receptor blockade or NLRP3-inhibition reduces myeloproliferation and improves hematopoiesis. Mechanistically, Kras-RAC1 activation induces reactive oxygen species (ROS) production causing NLRP3 inflammasome-activation. In agreement with our observations in mice, patient-derived myeloid leukemia cells exhibit KRAS/RAC1/ROS/NLRP3/IL-1β axis activity. Our findings indicate that oncogenic KRAS not only act via its canonical oncogenic driver function, but also enhances the activation of the pro-inflammatory RAC1/ROS/NLRP3/IL-1β axis. This paves the way for a therapeutic approach based on immune modulation via NLRP3 blockade in KRAS-mutant myeloid malignancies.
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http://dx.doi.org/10.1038/s41467-020-15497-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7125138PMC
April 2020

Modeling hematopoietic disorders in zebrafish.

Dis Model Mech 2019 09 6;12(9). Epub 2019 Sep 6.

Department of Biomedicine, University of Basel and University Hospital Basel, Basel 4031, Switzerland.

Zebrafish offer a powerful vertebrate model for studies of development and disease. The major advantages of this model include the possibilities of conducting reverse and forward genetic screens and of observing cellular processes by imaging of single cells. Moreover, pathways regulating blood development are highly conserved between zebrafish and mammals, and several discoveries made in fish were later translated to murine and human models. This review and accompanying poster provide an overview of zebrafish hematopoiesis and discuss the existing zebrafish models of blood disorders, such as myeloid and lymphoid malignancies, bone marrow failure syndromes and immunodeficiencies, with a focus on how these models were generated and how they can be applied for translational research.
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http://dx.doi.org/10.1242/dmm.040360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6765189PMC
September 2019

Stress and catecholamines modulate the bone marrow microenvironment to promote tumorigenesis.

Cell Stress 2019 Jun 4;3(7):221-235. Epub 2019 Jun 4.

Department of Biomedicine, University Hospital and University of Basel, Basel, Switzerland.

High vascularization and locally secreted factors make the bone marrow (BM) microenvironment particularly hospitable for tumor cells and bones to a preferred metastatic site for disseminated cancer cells of different origins. Cancer cell homing and proliferation in the BM are amongst other regulated by complex interactions with BM niche cells (e.g. osteoblasts, endothelial cells and mesenchymal stromal cells (MSCs)), resident hematopoietic stem and progenitor cells (HSPCs) and pro-angiogenic cytokines leading to enhanced BM microvessel densities during malignant progression. Stress and catecholamine neurotransmitters released in response to activation of the sympathetic nervous system (SNS) reportedly modulate various BM cells and may thereby influence cancer progression. Here we review the role of catecholamines during tumorigenesis with particular focus on pro-tumorigenic effects mediated by the BM niche.
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http://dx.doi.org/10.15698/cst2019.07.192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6612892PMC
June 2019

Absence of NKG2D ligands defines leukaemia stem cells and mediates their immune evasion.

Nature 2019 08 17;572(7768):254-259. Epub 2019 Jul 17.

Department of Biomedicine, University of Basel and University Hospital Basel, Basel, Switzerland.

Patients with acute myeloid leukaemia (AML) often achieve remission after therapy, but subsequently die of relapse that is driven by chemotherapy-resistant leukaemic stem cells (LSCs). LSCs are defined by their capacity to initiate leukaemia in immunocompromised mice. However, this precludes analyses of their interaction with lymphocytes as components of anti-tumour immunity, which LSCs must escape to induce cancer. Here we demonstrate that stemness and immune evasion are closely intertwined in AML. Using xenografts of human AML as well as syngeneic mouse models of leukaemia, we show that ligands of the danger detector NKG2D-a critical mediator of anti-tumour immunity by cytotoxic lymphocytes, such as NK cells-are generally expressed on bulk AML cells but not on LSCs. AML cells with LSC properties can be isolated by their lack of expression of NKG2D ligands (NKG2DLs) in both CD34-expressing and non-CD34-expressing cases of AML. AML cells that express NKG2DLs are cleared by NK cells, whereas NKG2DL-negative leukaemic cells isolated from the same individual escape cell killing by NK cells. These NKG2DL-negative AML cells show an immature morphology, display molecular and functional stemness characteristics, and can initiate serially re-transplantable leukaemia and survive chemotherapy in patient-derived xenotransplant models. Mechanistically, poly-ADP-ribose polymerase 1 (PARP1) represses expression of NKG2DLs. Genetic or pharmacologic inhibition of PARP1 induces NKG2DLs on the LSC surface but not on healthy or pre-leukaemic cells. Treatment with PARP1 inhibitors, followed by transfer of polyclonal NK cells, suppresses leukaemogenesis in patient-derived xenotransplant models. In summary, our data link the LSC concept to immune escape and provide a strong rationale for targeting therapy-resistant LSCs by PARP1 inhibition, which renders them amenable to control by NK cells in vivo.
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http://dx.doi.org/10.1038/s41586-019-1410-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934414PMC
August 2019

Zebrafish Xenografts for the In Vivo Analysis of Healthy and Malignant Human Hematopoietic Cells.

Methods Mol Biol 2019 ;2017:205-217

Department of Biomedicine, University of Basel and University Hospital Basel, Basel, Switzerland.

The zebrafish is a powerful vertebrate model for genetic studies on embryonic development and organogenesis. In the last decades, zebrafish were furthermore increasingly used for disease modeling and investigation of cancer biology. Zebrafish are particularly used for mutagenesis and small molecule screens, as well as for live imaging assays that provide unique opportunities to monitor cell behavior, both on a single cell and whole organism level in real time. Zebrafish have been also used for in vivo investigations of human cells transplanted into embryos or adult animals; this zebrafish xenograft model can be considered as an intermediate assay between in vitro techniques and more time-consuming and expensive mammalian models.Here, we present a protocol for transplantation of healthy and malignant human hematopoietic cells into larval zebrafish; transplantation into adult zebrafish and possible advantages and limitations of the zebrafish compared to murine xenograft models are discussed.
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http://dx.doi.org/10.1007/978-1-4939-9574-5_16DOI Listing
March 2020

Transition of Mesenchymal and Epithelial Cancer Cells Depends on α1-4 Galactosyltransferase-Mediated Glycosphingolipids.

Cancer Res 2018 06 23;78(11):2952-2965. Epub 2018 Mar 23.

Ovarian Cancer Research Program, Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland.

The reversible transitions of cancer cells between epithelial and mesenchymal states comprise cellular and molecular processes essential for local tumor growth and respective dissemination. We report here that globoside glycosphingolipid (GSL) glycosyltransferase-encoding genes are elevated in epithelial cells and correlate with characteristic EMT signatures predictive of disease outcome. Depletion of globosides through CRISPR--mediated deletion of the key enzyme A4GALT induces EMT, enhances chemoresistance, and increased CD24/CD44 cells. The cholera toxin-induced mesenchymal-to-epithelial transition occurred only in cells with functional A4GALT. Cells undergoing EMT lost E-cadherin expression through epigenetic silencing at the promoter region of However, in cells, demethylation was able to rescue E-cadherin-mediated cell-cell adhesion only in the presence of exogenous A4GALT. Overall, our data suggest another class of biomolecules vital for epithelial cancer cells and for maintaining cell integrity and function. This study highlights the essential role of glycosphingolipids in the maintenance of epithelial cancer cell properties. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-2223DOI Listing
June 2018

In Vitro Tumorigenic Assay: The Tumor Spheres Assay.

Methods Mol Biol 2018 ;1692:77-87

Departement of Biomedicine, University Hospital Basel and University of Basel, Hebelstrasse 20, 4031, Basel, Switzerland.

Cancer stem cells (CSCs) are a subpopulation of cells within cancer tissues that are thought to mediate tumor initiation. CSCs are furthermore considered the cause of tumor progression and recurrence after conventional therapies, based on their enhanced therapy resistance properties. A method commonly used to assess CSC potential in vitro is the so-called tumor spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors. Tumor spheres assays have been used in cancer research as an intermediate in vitro cell culture model to be explored before performing more laborious in vivo tumor xenograft assays.
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http://dx.doi.org/10.1007/978-1-4939-7401-6_7DOI Listing
June 2018

Mutations in signal recognition particle SRP54 cause syndromic neutropenia with Shwachman-Diamond-like features.

J Clin Invest 2017 Nov 3;127(11):4090-4103. Epub 2017 Oct 3.

Division of Pediatric Hematology, Hacettepe University Medical Faculty, Sihhiye, Ankara, Turkey.

Shwachman-Diamond syndrome (SDS) (OMIM #260400) is a rare inherited bone marrow failure syndrome (IBMFS) that is primarily characterized by neutropenia and exocrine pancreatic insufficiency. Seventy-five to ninety percent of patients have compound heterozygous loss-of-function mutations in the Shwachman-Bodian-Diamond syndrome (sbds) gene. Using trio whole-exome sequencing (WES) in an sbds-negative SDS family and candidate gene sequencing in additional SBDS-negative SDS cases or molecularly undiagnosed IBMFS cases, we identified 3 independent patients, each of whom carried a de novo missense variant in srp54 (encoding signal recognition particle 54 kDa). These 3 patients shared congenital neutropenia linked with various other SDS phenotypes. 3D protein modeling revealed that the 3 variants affect highly conserved amino acids within the GTPase domain of the protein that are critical for GTP and receptor binding. Indeed, we observed that the GTPase activity of the mutated proteins was impaired. The level of SRP54 mRNA in the bone marrow was 3.6-fold lower in patients with SRP54-mutations than in healthy controls. Profound reductions in neutrophil counts and chemotaxis as well as a diminished exocrine pancreas size in a SRP54-knockdown zebrafish model faithfully recapitulated the human phenotype. In conclusion, autosomal dominant mutations in SRP54, a key member of the cotranslation protein-targeting pathway, lead to syndromic neutropenia with a Shwachman-Diamond-like phenotype.
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http://dx.doi.org/10.1172/JCI92876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5663364PMC
November 2017

Prominent Oncogenic Roles of EVI1 in Breast Carcinoma.

Cancer Res 2017 04 16;77(8):2148-2160. Epub 2017 Feb 16.

Department of Biomedicine, University of Basel and University Hospital Basel, Basel, Switzerland.

Overexpression of the EVI1 oncogene is associated typically with aggressive myeloid leukemia, but is also detectable in breast carcinoma where its contributions are unexplored. Analyzing a tissue microarray of 608 breast carcinoma patient specimens, we documented EVI1 overexpression in both estrogen receptor-positive (ER) and estrogen receptor-negative (ER) breast carcinomas. Here, we report prognostic relevance of EVI1 overexpression in triple-negative breast carcinoma but not in the HER2-positive breast carcinoma subset. In human breast cancer cells, EVI1 silencing reduced proliferation, apoptosis resistance, and tumorigenicity, effects rescued by estrogen supplementation in ER breast carcinoma cells. Estrogen addition restored ERK phosphorylation in EVI1-silenced cells, suggesting that EVI1 and estradiol signaling merge in MAPK activation. Conversely, EVI1 silencing had no effect on constitutive ERK activity in HER2 breast carcinoma cells. Microarray analyses revealed G-protein-coupled receptor (GPR) signaling as a prominent EVI1 effector mechanism in breast carcinoma. Among others, the GPR54-ligand KISS1 was identified as a direct transcriptional target of EVI1, which together with other EVI1-dependent cell motility factors such as RHOJ regulated breast carcinoma cell migration. Overall, our results establish the oncogenic contributions of EVI1 in ER- and HER2-negative subsets of breast cancer. .
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http://dx.doi.org/10.1158/0008-5472.CAN-16-0593DOI Listing
April 2017

Long-term observation reveals high-frequency engraftment of human acute myeloid leukemia in immunodeficient mice.

Haematologica 2017 05 9;102(5):854-864. Epub 2017 Feb 9.

University of Basel and University Hospital Basel, Department of Biomedicine, Switzerland

Repopulation of immunodeficient mice remains the primary method for functional assessment of human acute myeloid leukemia. Published data report engraftment in ~40-66% of cases, mostly of intermediate- or poor-risk subtypes. Here we report that extending follow-up beyond the standard analysis endpoints of 10 to 16 weeks after transplantation permitted leukemic engraftment from nearly every case of xenotransplanted acute myeloid leukemia (18/19, ~95%). Xenogeneic leukemic cells showed conserved immune pheno-types and genetic signatures when compared to corresponding pre-transplant cells and, furthermore, were able to induce leukemia in re-transplantation assays. Importantly, bone marrow biopsies taken at standardized time points failed to detect leukemic cells in 11/18 of cases that later showed robust engraftment (61%, termed "long-latency engrafters"), indicating that leukemic cells can persist over months at undetectable levels without losing disease-initiating properties. Cells from favorable-risk leukemia subtypes required longer to become detectable in NOD/SCID/IL2Rγ mice (27.5±9.4 weeks) than did cells from intermediate-risk (21.9±9.4 weeks, <0.01) or adverse-risk (17±7.6 weeks; <0.0001) subtypes, explaining why the engraftment of the first was missed with previous protocols. Mechanistically, leukemic cells engrafting after a prolonged latency showed inferior homing to the bone marrow. Finally, we applied our model to favorable-risk acute myeloid leukemia with inv(16); here, we showed that CD34 (but not CD34) blasts induced robust, long-latency engraftment and expressed enhanced levels of stem cell genes. In conclusion, we provide a model that allows mouse studies with a wide range of molecular subtypes of acute myeloid leukemia subtypes which were previously considered not able to engraft, thus enabling novel insights into leukemogenesis.
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http://dx.doi.org/10.3324/haematol.2016.153528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477604PMC
May 2017

Evi1 regulates Notch activation to induce zebrafish hematopoietic stem cell emergence.

EMBO J 2016 11 16;35(21):2315-2331. Epub 2016 Sep 16.

Department of Biomedicine, University Hospital Basel, Basel, Switzerland

During development, hematopoietic stem cells (HSCs) emerge from aortic endothelial cells (ECs) through an intermediate stage called hemogenic endothelium by a process known as endothelial-to-hematopoietic transition (EHT). While Notch signaling, including its upstream regulator Vegf, is known to regulate this process, the precise molecular control and temporal specificity of Notch activity remain unclear. Here, we identify the zebrafish transcriptional regulator evi1 as critically required for Notch-mediated EHT In vivo live imaging studies indicate that evi1 suppression impairs EC progression to hematopoietic fate and therefore HSC emergence. evi1 is expressed in ECs and induces these effects cell autonomously by activating Notch via pAKT Global or endothelial-specific induction of notch, vegf, or pAKT can restore endothelial Notch and HSC formations in evi1 morphants. Significantly, evi1 overexpression induces Notch independently of Vegf and rescues HSC numbers in embryos treated with a Vegf inhibitor. In sum, our results unravel evi1-pAKT as a novel molecular pathway that, in conjunction with the shh-vegf axis, is essential for activation of Notch signaling in VDA endothelial cells and their subsequent conversion to HSCs.
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http://dx.doi.org/10.15252/embj.201593454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5090218PMC
November 2016

Targeting DDR2 in head and neck squamous cell carcinoma with dasatinib.

Int J Cancer 2016 11 30;139(10):2359-69. Epub 2016 Jul 30.

Pathology of the University Medical Center Schleswig-Holstein, Campus Luebeck and the Research Center Borstel, Leibniz Center for Medicine and Biosciences, Luebeck and Borstel, Germany.

Squamous cell carcinoma of the head and neck (HNSCC) is the tenth most common tumor entity in men worldwide. Nevertheless therapeutic options are mostly limited to surgery and radio-chemotherapy resulting in 5-year survival rates of around 50%. Therefore new therapeutic options are urgently needed. During the last years, targeting of receptor tyrosine kinases has emerged as a promising strategy that can complement standard therapeutical approaches. Here, we aimed at investigating if the receptor tyrosine kinase DDR2 is a targetable structure in HNSCC. DDR2 expression was assessed on a large HNSCC cohort (554 patients) including primary tumors, lymph node metastases and recurrences and normal mucosa as control. Subsequently, DDR2 was stably overexpressed in two different cell lines (FaDu and HSC-3) using lentiviral technology. Different tumorigenic properties such as proliferation, migration, invasion, adhesion and anchorage independent growth were assessed with and without dasatinib treatment using in-vitro cell models and in-vivo zebrafish xenografts. DDR2 was overexpressed in all tumor tissues when compared to normal mucosa. DDR2 overexpression led to increased migration, invasion, adhesion and anchorage independent growth whereas proliferation remained unaltered. Upon dasatinib treatment migration, invasion and adhesion could be inhibited in-vitro and in-vivo whereas proliferation was unchanged. Our data suggest treatment with dasatinib as a promising new therapeutic option for patients suffering from DDR2 overexpressing HNSCC. Since dasatinib is already FDA-approved we propose to test this drug in clinical trials so that patients could directly benefit from this new treatment option.
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http://dx.doi.org/10.1002/ijc.30279DOI Listing
November 2016

Endothelial-to-hematopoietic transition: Notch-ing vessels into blood.

Ann N Y Acad Sci 2016 04 25;1370(1):97-108. Epub 2016 Mar 25.

Department of Biomedicine, University Hospital Basel, Basel, Switzerland.

During development, hematopoietic stem cells (HSCs) are formed in a temporally and spatially restricted manner, arising from specialized endothelial cells (ECs) in the ventral wall of the dorsal aorta within the evolutionary conserved aorta-gonad-mesonephros region. Our understanding of the processes regulating the birth of HSCs from ECs has been recently advanced by comprehensive molecular analyses of developing murine hematopoietic cell populations complemented by studies in the zebrafish model, with the latter offering unique advantages for genetic studies and direct in vivo visualization of HSC emergence. Here, we provide a concise review of the current knowledge and recent advances regarding the cellular origin and molecular regulation of HSC development, with particular focus on the process of endothelial-to-hematopoietic transition and its primary regulator, the Notch signaling pathway.
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http://dx.doi.org/10.1111/nyas.13030DOI Listing
April 2016

Molecular and functional interactions between AKT and SOX2 in breast carcinoma.

Oncotarget 2015 Dec;6(41):43540-56

Department of Biomedicine, University Hospital Basel, Basel, Switzerland.

The transcription factor SOX2 is a key regulator of pluripotency in embryonic stem cells and plays important roles in early organogenesis. Recently, SOX2 expression was documented in various cancers and suggested as a cancer stem cell (CSC) marker. Here we identify the Ser/Thr-kinase AKT as an upstream regulator of SOX2 protein turnover in breast carcinoma (BC). SOX2 and pAKT are co-expressed and co-regulated in breast CSCs and depletion of either reduces clonogenicity. Ectopic SOX2 expression restores clonogenicity and in vivo tumorigenicity of AKT-inhibited cells, suggesting that SOX2 acts as a functional downstream AKT target. Mechanistically, we show that AKT physically interacts with the SOX2 protein to modulate its subcellular distribution. AKT kinase inhibition results in enhanced cytoplasmic retention of SOX2, presumably via impaired nuclear import, and in successive cytoplasmic proteasomal degradation of the protein. In line, blockade of either nuclear transport or proteasomal degradation rescues SOX2 expression in AKT-inhibited BC cells. Finally, AKT inhibitors efficiently suppress the growth of SOX2-expressing putative cancer stem cells, whereas conventional chemotherapeutics select for this population. Together, our results suggest the AKT/SOX2 molecular axis as a regulator of BC clonogenicity and AKT inhibitors as promising drugs for the treatment of SOX2-positive BC.
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http://dx.doi.org/10.18632/oncotarget.6183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4791249PMC
December 2015

The zebrafish reference genome sequence and its relationship to the human genome.

Nature 2013 Apr 17;496(7446):498-503. Epub 2013 Apr 17.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

Zebrafish have become a popular organism for the study of vertebrate gene function. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.
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http://dx.doi.org/10.1038/nature12111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3703927PMC
April 2013

Slc45a2 and V-ATPase are regulators of melanosomal pH homeostasis in zebrafish, providing a mechanism for human pigment evolution and disease.

Pigment Cell Melanoma Res 2013 Mar 20;26(2):205-17. Epub 2012 Dec 20.

Max Planck Institute for Developmental Biology, Tübingen, Germany.

We present here the positional cloning of the Danio rerio albino mutant and show that the affected gene encodes Slc45a2. The human orthologous gene has previously been shown to be involved in human skin color variation, and mutations therein have been implicated in the disease OCA4. Through ultrastructural analysis of the melanosomes in albino alleles as well as the tyrosinase-deficient mutant sandy, we add new insights into the role of Slc45a2 in the production of melanin. To gain further understanding of the role of Slc45a2 and its possible interactions with other proteins involved in melanization, we further analyzed the role of the V-ATPase as a melanosomal acidifier. We show that it is possible to rescue the melanization potential of the albino melanosomes through genetic and chemical inhibition of V-ATPase, thereby increasing internal melanosome pH.
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http://dx.doi.org/10.1111/pcmr.12053DOI Listing
March 2013

The transcriptional landscape of hematopoietic stem cell ontogeny.

Cell Stem Cell 2012 Nov;11(5):701-14

Division of Pediatric Hematology/Oncology, Children's Hospital Boston and Harvard Medical School, Harvard Stem Cell Institute, Boston, MA 02115, USA.

Transcriptome analysis of adult hematopoietic stem cells (HSCs) and their progeny has revealed mechanisms of blood differentiation and leukemogenesis, but a similar analysis of HSC development is lacking. Here, we acquired the transcriptomes of developing HSCs purified from >2,500 murine embryos and adult mice. We found that embryonic hematopoietic elements clustered into three distinct transcriptional states characteristic of the definitive yolk sac, HSCs undergoing specification, and definitive HSCs. We applied a network-biology-based analysis to reconstruct the gene regulatory networks of sequential stages of HSC development and functionally validated candidate transcriptional regulators of HSC ontogeny by morpholino-mediated knockdown in zebrafish embryos. Moreover, we found that HSCs from in vitro differentiated embryonic stem cells closely resemble definitive HSCs, yet lack a Notch-signaling signature, likely accounting for their defective lymphopoiesis. Our analysis and web resource will enhance efforts to identify regulators of HSC ontogeny and facilitate the engineering of hematopoietic specification.
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http://dx.doi.org/10.1016/j.stem.2012.07.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3545475PMC
November 2012

Zebrafish xenografts as a tool for in vivo studies on human cancer.

Ann N Y Acad Sci 2012 Aug;1266:124-37

Department of Hematology and Oncology, University of Tübingen Medical Center II, Tübingen, Germany.

The zebrafish has become a powerful vertebrate model for genetic studies of embryonic development and organogenesis and increasingly for studies in cancer biology. Zebrafish facilitate the performance of reverse and forward genetic approaches, including mutagenesis and small molecule screens. Moreover, several studies report the feasibility of xenotransplanting human cells into zebrafish embryos and adult fish. This model provides a unique opportunity to monitor tumor-induced angiogenesis, invasiveness, and response to a range of treatments in vivo and in real time. Despite the high conservation of gene function between fish and humans, concern remains that potential differences in zebrafish tissue niches and/or missing microenvironmental cues could limit the relevance and translational utility of data obtained from zebrafish human cancer cell xenograft models. Here, we summarize current data on xenotransplantation of human cells into zebrafish, highlighting the advantages and limitations of this model in comparison to classical murine models of xenotransplantation.
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http://dx.doi.org/10.1111/j.1749-6632.2012.06575.xDOI Listing
August 2012

Interactions between Cdx genes and retinoic acid modulate early cardiogenesis.

Dev Biol 2011 Jun 3;354(1):134-42. Epub 2011 Apr 3.

Department of Hematology and Oncology, University of Tuebingen Medical Center II, Tuebingen, Germany.

Cdx transcription factors regulate embryonic positional identities and have crucial roles in anteroposterior patterning (AP) processes of all three germ layers. Previously we have shown that the zebrafish homologues cdx1a and cdx4 redundantly regulate posterior mesodermal derivatives inducing embryonic blood cell fate specification and patterning of the embryonic kidney. Here we hypothesize that cdx factors restrict formation of anterior mesodermal derivatives such as cardiac cells by imposing posterior identity to developing mesodermal cells. We show that ectopic expression of Cdx1 or Cdx4 applied during the brief window of mesoderm patterning in differentiating murine embryonic stem cell (ESC) strongly suppresses cardiac development as assayed by expression of cardiac genes and formation of embryoid bodies (EB) containing "beating" cell clusters. Conversely, in loss-of-function studies performed in cdx-deficient zebrafish embryos, we observed a dose-dependent expansion of tbx5a(+) anterior-lateral plate mesoderm giving rise to cardiac progenitors. However, further cardiac development of these mesodermal cells required additional suppression of the retinoic acid (RA) pathway, possibly due to differential activity of inhibitory RA signals in cdx mutants. Together, our data suggest that cdx proteins affect cardiogenesis by regulating the formation of cardiogenic mesoderm and together with the RA pathway control the early development of cardiac precursor cells.
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http://dx.doi.org/10.1016/j.ydbio.2011.03.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3502019PMC
June 2011

Large-scale mapping of mutations affecting zebrafish development.

BMC Genomics 2007 Jan 9;8:11. Epub 2007 Jan 9.

Department 3--Genetics, Max-Planck-Institut für Entwicklungsbiologie, Spemannstr, 35/III, 72076 Tübingen, Germany.

Background: Large-scale mutagenesis screens in the zebrafish employing the mutagen ENU have isolated several hundred mutant loci that represent putative developmental control genes. In order to realize the potential of such screens, systematic genetic mapping of the mutations is necessary. Here we report on a large-scale effort to map the mutations generated in mutagenesis screening at the Max Planck Institute for Developmental Biology by genome scanning with microsatellite markers.

Results: We have selected a set of microsatellite markers and developed methods and scoring criteria suitable for efficient, high-throughput genome scanning. We have used these methods to successfully obtain a rough map position for 319 mutant loci from the Tübingen I mutagenesis screen and subsequent screening of the mutant collection. For 277 of these the corresponding gene is not yet identified. Mapping was successful for 80 % of the tested loci. By comparing 21 mutation and gene positions of cloned mutations we have validated the correctness of our linkage group assignments and estimated the standard error of our map positions to be approximately 6 cM.

Conclusion: By obtaining rough map positions for over 300 zebrafish loci with developmental phenotypes, we have generated a dataset that will be useful not only for cloning of the affected genes, but also to suggest allelism of mutations with similar phenotypes that will be identified in future screens. Furthermore this work validates the usefulness of our methodology for rapid, systematic and inexpensive microsatellite mapping of zebrafish mutations.
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http://dx.doi.org/10.1186/1471-2164-8-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1781435PMC
January 2007
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