Dissecting the role of the ETV6 transcriptional regulator in development and disease

This study studied the ETV6 gene using zebrafish as a model. The work demonstrated that ETV6 was important for the development of both white and red blood cells. It also characterized the pathogenic effects of mutant forms of ETV6 found in various leukemias and other blood disorders

ETV6 (TEL1) regulates embryonic hematopoiesis in zebrafish

Chromosomal translocations involving fusions of the human ETV6 (TEL1) gene occur frequently in hematologic malignancies. However, a detailed understanding of the normal function of ETV6 remains incomplete. This study has employed zebrafish as a relevant model to investigate the role of ETV6 during embryonic hematopoiesis. Zebrafish possessed a single conserved etv6 ortholog that was expressed from 12 hpf in the lateral plate mesoderm, and later in hematopoietic, vascular and other tissues. Morpholino-mediated gene knockdown of etv6 revealed the complex contribution of this gene toward embryonic hematopoiesis. During primitive hematopoiesis, etv6 knockdown resulted in reduced levels of progenitor cells, erythrocyte and macrophage populations, but increased numbers of incompletely differentiated heterophils. Definitive hematopoiesis was also perturbed, with etv6 knockdown leading to decreased erythrocytes and myeloid cells, but enhanced lymphopoiesis. This study suggests that ETV6 plays a broader and more complex role in early hematopoiesis than previously thought, impacting on the development of multiple lineages. © 2015 Ferrata Storti Foundation

Alternative TEL-JAK2 fusions associated with T-cell acute lymphoblastic leukemia and atypical chronic myelogenous leukemia dissected in zebrafish

Background Chromosomal translocations resulting in alternative fusions of the human TEL (ETV6) and JAK2 genes have been observed in cases of acute lymphoblastic leukemia and chronic myelogenous leukemia, but a full understanding of their role in disease etiology has remained elusive. In this study potential differences between these alternative TEL-JAK2 fusions, including their lineage specificity, were investigated.Design and Methods TEL-JAK2 fusion types derived from both T-cell acute lymphoblastic leukemia and atypical chronic myelogenous leukemia were generated using the corresponding zebrafish tel and jak2a genes and placed under the control of either the white blood cell-specific spi1 promoter or the ubiquitously-expressed cytomegalovirus promoter. These constructs were injected into zebrafish embryos and their effects on hematopoiesis examined using a range of molecular approaches. In addition, the functional properties of the alternative fusions were investigated in vitro.Results Injection of the T-cell acute lymphoblastic leukemia-derived tel-jak2a significantly perturbed lymphopoiesis with a lesser effect on myelopoiesis in zebrafish embryos. In contrast, injection of the atypical chronic myelogenous leukemia-derived tel-jak2a resulted in significant perturbation of the myeloid compartment. These phenotypes were observed regardless of whether expressed in a white blood cell-specific or ubiquitous manner, with no overt cellular proliferation outside of the hematopoietic cells. Functional studies revealed subtle differences between the alternative forms, with the acute lymphoblastic leukemia variant showing higher activity, but reduced downstream signal transducer and activator of transcription activation and decreased sensitivity to JAK2 inhibition. JAK2 activity was required to mediate the effects of both variants on zebrafish hematopoiesis.Conclusions This study indicates that the molecular structure of alternative TEL-JAK2 fusions likely contributes to the etiology of disease. The data further suggest that this class of oncogene exerts its effects in a cell lineage-specific manner, which may be due to differences in downstream signaling.<br /

Zebrafish as a model for leukemia and other hematopoietic disorders

Zebrafish is an established model for the study of vertebrate development, and is especially amenable for investigating hematopoiesis, where there is strong conservation of key lineages, genes, and developmental processes with humans. Over recent years, zebrafish has been increasingly utilized as a model for a range of human hematopoietic diseases, including malignancies. This review provides an overview of zebrafish hematopoiesis and describes its application as a model of leukemia and other hematopoietic disorders

Association of FXII 5’UTR 46C>T polymorphism with FXII activity and risk of thrombotic disease

Objective: Thrombotic diseases are caused by genetic and environmental factors. There are a number of well-characterized genetic defects that lead to increased risk of thrombosis. Results from previous studies have indicated that FXII is involved in the pathogenesis of thrombophilic diseases. However, the results in this regard are highly controversial. One of the most important determinants of Plasma FXII level is 46CgT polymorphism in the FXII gene. In the present study, the risk of thrombophilic diseases related to this polymorphism was investigated in a case-control study. Material and Methods: One hundred and sixty subjects were studied: 120 patients diagnosed with thrombophilia (96 venous thromboembolism, 24 arterial thrombosis), and 40 age-gender-matched controls. For each subject, FXII activity level was measured by a one-step clotting assay with FXII-deficient plasma, and 46CγT polymorphism was genotyped using a restriction fragment length polymorphism (RFLP) method. Results: In this study, the previous observation that individuals with different genotypes for the 46 CγT polymorphism show significant differences in FXII activity levels was confirmed. Most importantly, FXII activity ≤68% was associated with an increased risk of venous thrombosis with an adjusted odds ratio (OR) of 4.7 (95% confidence interval [CI]: 1.03-21.1, p=0.04). However, it was not a risk factor for arterial thrombosis with adjusted OR of 5 (95% CI: 0.91-27.1, p=0.09). In CT and TT genotype, the adjusted ORs were 2 (95% CI: 0.9-4.4, p=0.11) and 2.3 (95% CI: 0.45-11, p=0.48), respectively, for patients with venous thrombosis compared with the controls. Similarly, the adjusted ORs in arterial thrombosis were 1.2 (95% CI: 0.4-3.6, p=0.76) for CT and 1.8 (95% CI: 0.2-14.9, p=0.59) for TT genotype. Thus, we did not find any association of the mutated T allele in the heterozygous or homozygous state with an increased risk of venous or arterial thrombosis. Conclusion: Lower FXII activity is not a risk factor; rather, it simply represents a risk marker for thrombosis

Cancer cell CCL5 mediates bone marrow independent angiogenesis in breast cancer

It has recently been suggested that the chemokine receptor (CCR5) is required for bone marrow (BM) derived endothelial progenitor cell (EPC) mediated angiogenesis. Here we show that suppression of either cancer cell produced CCL5, or host CCR5 leads to distinctive vascular and tumor growth defects in breast cancer. Surprisingly, CCR5 restoration in the BM alone was not sufficient to rescue the wild type phenotype, suggesting that impaired tumor growth associated with inhibiting CCL5/CCR5 is not due to defects in EPC biology. Instead, to promote angiogenesis cancer cell CCL5 may signal directly to endothelium in the tumor-stroma. In support of this hypothesis, we have also shown: (i) that endothelial cell CCR5 levels increases in response to tumor-conditioned media; (ii) that the amount of CCR5+ tumor vasculature correlates with invasive grade; and (iii) that inhibition of CCL5/CCR5 signaling impairs endothelial cell migration, associated with a decrease in activation of mTOR/AKT pathway members. Finally, we show that treatment with CCR5 antagonist results in less vasculature, impaired tumor growth, reduced metastases and improved survival. Taken as a whole, this work demonstrates that directly inhibiting CCR5 expressing vasculature constitutes a novel strategy for inhibiting angiogenesis and blocking metastatic progression in breast cancer

Conserved IL-2Rγc signaling mediates lymphopoiesis in Zebrafish

The IL-2 receptor &gamma; common (IL-2R&gamma;c) chain is the shared subunit of the receptors for the IL-2 family of cytokines, which mediate signaling through JAK3 and various downstream pathways to regulate lymphopoiesis. Inactivating mutations in human IL-2R&gamma;c result in SCID, a primary immunodeficiency characterized by greatly reduced numbers of lymphocytes. This study used bioinformatics, expression analysis, gene ablation, and specific pharmacologic inhibitors to investigate the function of two putative zebrafish IL-2R&gamma;c paralogs, il-2r&gamma;c.a and il-2r&gamma;c.b, and downstream signaling components during early lymphopoiesis. Expression of il-2r&gamma;c.a commenced at 16 h post fertilization (hpf) and rose steadily from 4-6 d postfertilization (dpf) in the developing thymus, with il-2r&gamma;c.a expression also confirmed in adult T and B lymphocytes. Transcripts of il-2r&gamma;c.b were first observed from 8 hpf, but waned from 16 hpf before reaching maximal expression at 6 dpf, but this was not evident in the thymus. Knockdown of il-2r&gamma;c.a, but not il-2r&gamma;c.b, substantially reduced embryonic lymphopoiesis without affecting other aspects of hematopoiesis. Specific targeting of zebrafish Jak3 exerted a similar effect on lymphopoiesis, whereas ablation of zebrafish Stat5.1 and pharmacologic inhibition of PI3K and MEK also produced significant but smaller effects. Ablation of il-2r&gamma;c.a was further demonstrated to lead to an absence of mature T cells, but not B cells in juvenile fish. These results indicate that conserved IL-2R&gamma;c signaling via JAK3 plays a key role during early zebrafish lymphopoiesis, which can be potentially targeted to generate a zebrafish model of human SCID