Data availability statement. All data generated are included in the published article and in the Supplementary Information. Gene expression data that support the findings of this study have been deposited in the Gene Expression Omnibus under accession numbers GSE127200 and 127959. All data are also available from the authors on reasonable request.

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.

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 in vivo 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.

Heterozygous de novo missense variants of SRP54 were recently identified in patients with congenital neutropenia (CN), displaying symptoms overlapping with Shwachman-Diamond-Syndrome (SDS).1 Here, we investigate srp54 KO zebrafish as the first in vivo model of SRP54 deficiency. srp54-/- zebrafish are embryonically lethal and display, next to severe neutropenia, multi-systemic developmental defects. In contrast, srp54+/- zebrafish are viable, fertile and only show mild neutropenia. Interestingly, injection of human SRP54 mRNAs carrying 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 variable phenotypes observed in patients may be due to mutation-specific dominant negative effects on the functionality of the residual wildtype SRP54 protein. Consistently, overexpression of mutated SRP54 also induced neutropenia in wildtype fish and impaired granulocytic maturation of human promyelocytic HL-60 cells as well as of healthy cord-blood derived CD34+ HSPCs. Mechanistically, srp54 mutant fish and human cells show impaired unconventional splicing of the transcription factor X-box binding protein 1 (Xbp1). Vice-versa, 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 SRP54 loss. The heterogenic phenotypes observed in patients, ranging from mild CN to SDS-like disease, may be due to different dominant negative effects of mutated SRP54 proteins on downstream XBP1 splicing, which represents a potential therapeutic target.

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.

BACKGROUND: Arrhythmogenic cardiomyopathy (ACM) is characterized by progressive loss of cardiomyocytes with fibrofatty tissue replacement, systolic dysfunction, and life-threatening arrhythmias. A substantial proportion of ACM is caused by mutations in genes of the desmosomal cell–cell adhesion complex, but the underlying mechanisms are not well understood. Treatment options are for symptoms. We investigated the relevance of defective desmosomal adhesion for ACM development and progression. METHODS: We mutated the binding site of DSG2 (desmoglein-2), a crucial desmosomal adhesion molecule in cardiomyocytes. This DSG2-W2A mutation abrogates the tryptophan swap, a central interaction mechanism of DSG2 on the basis of structural data. Impaired adhesive function of DSG2-W2A was confirmed by cell–cell dissociation assays and force spectroscopy measurements by atomic force microscopy. The DSG2-W2A knock-in mouse model was analyzed by echocardiography, ECG, and histologic and biomolecular techniques including RNA sequencing and transmission electron and superresolution microscopy. The results were compared with ACM patient samples, and their relevance was confirmed in vivo and in cardiac slice cultures by inhibitor studies applying the small molecule EMD527040 or an inhibitory integrin-αVβ6 antibody. RESULTS: The DSG2-W2A mutation impaired binding on a molecular level and compromised intercellular adhesive function. Mice bearing this mutation develop a severe cardiac phenotype recalling the characteristics of ACM, including cardiac fibrosis, impaired systolic function, and arrhythmia. A comparison of the transcriptome of mutant mice with ACM patient data suggested deregulated integrin-αVβ6 and subsequent transforming growth factor–β signaling as driver of cardiac fibrosis. Blocking integrin-αVβ6 led to reduced expression of profibrotic markers and reduced fibrosis formation in mutant animals in vivo. CONCLUSIONS: We show that disruption of desmosomal adhesion is sufficient to induce a phenotype that fulfils the clinical criteria to establish the diagnosis of ACM, confirming the dysfunctional adhesion hypothesis. Deregulation of integrin-αVβ6 and transforming growth factor–β signaling was identified as a central step toward fibrosis. A pilot in vivo drug test revealed this pathway as a promising target to ameliorate fibrosis. This highlights the value of this model to discern mechanisms of cardiac fibrosis and to identify and test novel treatment options for ACM.

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Whole exome sequencing analyses are increasingly performed on patients presenting with suspected inherited disease but lacking classical mutations linked to presented phenotypes. Using whole-exome sequencing in SBDS-negative Shwachman-Diamond Syndrome (SDS) families, we recently identified three independent patients, each of whom carried a heterozygous de novo missense variant of SRP54 (encoding signal recognition particle 54 kDa). The SRP54 protein is a key component of the ribonucleoprotein complex that mediates the co-translational targeting of secretory and membrane proteins to the endoplasmic reticulum (ER). Whilst two of the identified patients were carrying nucleotide transversion in SRP54 (p.T115A and p.G226E), which manifested in typical SDS features like neutropenia and exocrine pancreatic insufficiency, the third patient was carrying a nucleotide deletion (p.T117Δ), which only manifested in mild neutropenia without additional SDS features (Carapito et al. 2017, JCI). Here, we describe a zebrafish knock-out (KO) mutant as the very first transgenic in vivo model of SRP54 deficiency, translate our previous findings into living organisms and propose disease-driving mechanisms. We show that homozygous srp54 mutant zebrafish are suffering not only from severe neutropenia as shown by flow cytometry and Whole-Mount-In-Situ Hybridization (WISH), but also from gross developmental defects leading to early embryonic lethality. In fact, srp54-/- zebrafish did not survive more than 72 hours post fertilization, indicating that complete loss of Srp54 is not compatible with life. Injection with wild-type human SRP54 mRNA induced transient restoration of SRP54 protein expression and slightly enhanced the survival of the homozygous mutants. However, long-term viability could not be restored, revealing that srp54 is not only critically required during early embryogenesis but also at later stages of development. Heterozygous siblings on the other hand are viable and display only mild neutropenia but no pancreas defects. Interestingly however, injection of mutant mRNAs of human SRP54 (p.T115A, p.T117Δ, p.226E) into heterozygous srp54 KO mutants aggravated the phenotype inducing more profound neutropenia and pancreas changes similar to those observed in classical SDS patients. Of note, these effects were more severe for the transversions p.T115A and p.G226E. Mutation p.T117Δ only caused a minor reduction in the number of neutrophils, without affecting the pancreas. To further investigate SRP54 driven neutrophil defects, we used lentiviral transduction to exogenously express human SRP54 mutant variants in promyelocytic HL-60 cells. When stimulating these cells to differentiate by ATRA treatment, we found significantly impaired morphologic differentiation and CD11b surface induction compared to control cells. The severity of these effects was again specific to the three different identified mutations, with p.T115A and p.G226E being more severe than p.T117Δ. These findings confirm the type-specific effects of SRP54 mutations and indicate that SRP54 defects interfere with neutrophil differentiation and thus ultimately lead to neutropenia. Collectively, we here describe a novel zebrafish disease model of SDS and congenital neutropenia founding on SRP54 as molecular driver. Our model demonstrates that at least one healthy allele of srp54 is pivotal for survival, which is in line with the findings in humans, where homozygous mutations in SRP54 have never been detected. We reveal that the phenotypic manifestation of heterozygous SRP54 mutations strongly depends on the type of mutation: while mutations likely causing a simple SRP54 loss of function (e.g. p.T117Δ) induce a rather mild phenotype characterized by moderate neutropenia only (analogous to the heterozygous fish mutant), more severe SDS-like phenotypes involve SRP54 mutations that exert dominant negative effects (e.g. p.T115A and p.G226E). Ultimately, we make use of the promyelocytic cell line HL-60 to propose neutrophil differentiation defects as the underlying cause of SRP54 driven neutropenia. At the time being, RNA sequencing and protein expression analyses are performed in our laboratory, which will add to the understanding of the mechanistical background of the neutrophilic differentiation blockage and eventually uncover novel treatment strategies for SRP54 deficiency. Disclosures No relevant conflicts of interest to declare.