Elspeth Payne

Endothelial von Willebrand factor regulates angiogenesis

The regulation of blood vessel formation is of fundamental importance to many physiological processes, and angiogenesis is a major area for novel therapeutic approaches to diseases from ischemia to cancer. A poorly understood clinical manifestation of pathological angiogenesis is angiodysplasia, vascular malformations that cause severe gastrointestinal bleeding. Angiodysplasia can be associated with von Willebrand disease (VWD), the most common bleeding disorder in man. VWD is caused by a defect or deficiency in von Willebrand factor (VWF), a glycoprotein essential for normal hemostasis that is involved in inflammation. We hypothesized that VWF regulates angiogenesis. Inhibition of VWF expression by short interfering RNA (siRNA) in endothelial cells (ECs) caused increased in vitro angiogenesis and increased vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2)-dependent proliferation and migration, coupled to decreased integrin αvβ3 levels and increased angiopoietin (Ang)-2 release. ECs expanded from blood-derived endothelial progenitor cells of VWD patients confirmed these results. Finally, 2 different approaches, in situ and in vivo, showed increased vascularization in VWF-deficient mice. We therefore identify a new function of VWF in ECs, which confirms VWF as a protein with multiple vascular roles and defines a novel link between hemostasis and angiogenesis. These results may have important consequences for the management of VWD, with potential therapeutic implications for vascular diseases.

L-Leucine improves the anemia and developmental defects associated with Diamond-Blackfan anemia and del(5q) MDS by activating the mTOR pathway.

Haploinsufficiency of ribosomal proteins (RPs) has been proposed to be the common basis for the anemia observed in Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome with loss of chromosome 5q [del(5q) MDS]. We have modeled DBA and del(5q) MDS in zebrafish using antisense morpholinos to rps19 and rps14, respectively, and have demonstrated that, as in humans, haploinsufficient levels of these proteins lead to a profound anemia. To address the hypothesis that RP loss results in impaired mRNA translation, we treated Rps19 and Rps14-deficient embryos with the amino acid L-leucine, a known activator of mRNA translation. This resulted in a striking improvement of the anemia associated with RP loss. We confirmed our findings in primary human CD34⁺ cells, after shRNA knockdown of RPS19 and RPS14. Furthermore, we showed that loss of Rps19 or Rps14 activates the mTOR pathway, and this is accentuated by L-leucine in both Rps19 and Rps14 morphants. This effect could be abrogated by rapamycin suggesting that mTOR signaling may be responsible for the improvement in anemia associated with L-leucine. Our studies support the rationale for ongoing clinical trials of L-leucine as a therapeutic agent for DBA, and potentially for patients with del(5q) MDS.

Pten mediates Myc oncogene dependence in a conditional zebrafish model of T cell acute lymphoblastic leukemia.

Loss-of-function mutations in pten genes, or expression of a constitutively active version of Akt2, render T-ALL cell survival and disease progression independent of Myc.

NUP98-HOXA9-transgenic zebrafish develop a myeloproliferative neoplasm and provide new insight into mechanisms of myeloid leukaemogenesis.

NUP98‐HOXA9 [t(7;11) (p15;p15)] is associated with inferior prognosis in de novo and treatment‐related acute myeloid leukaemia (AML) and contributes to blast crisis in chronic myeloid leukaemia (CML). We have engineered an inducible transgenic zebrafish harbouring human NUP98‐HOXA9 under the zebrafish spi1(pu.1) promoter. NUP98‐HOXA9 perturbed zebrafish embryonic haematopoiesis, with upregulated spi1expression at the expense of gata1a. Markers associated with more differentiated myeloid cells, lcp1, lyz, and mpx were also elevated, but to a lesser extent than spi1, suggesting differentiation of early myeloid progenitors may be impaired by NUP98‐HOXA9. Following irradiation, NUP98‐HOXA9‐expressing embryos showed increased numbers of cells in G2‐M transition compared to controls and absence of a normal apoptotic response, which may result from an upregulation of bcl2. These data suggest NUP98‐HOXA9‐induced oncogenesis may result from a combination of defects in haematopoiesis and an aberrant response to DNA damage. Importantly, 23% of adult NUP98‐HOXA9‐transgenic fish developed a myeloproliferative neoplasm (MPN) at 19–23 months of age. In summary, we have identified an embryonic haematopoietic phenotype in a transgenic zebrafish line that subsequently develops MPN. This tool provides a unique opportunity for high‐throughput in vivo chemical modifier screens to identify novel therapeutic agents in high risk AML.

Expression of the cytoplasmic NPM1 mutant (NPMc+) causes the expansion of hematopoietic cells in zebrafish

Mutations in the human nucleophosmin (NPM1) gene are the most frequent genetic alteration in adult acute myeloid leukemias (AMLs) and result in aberrant cytoplasmic translocation of this nucleolar phosphoprotein (NPMc+). However, underlying mechanisms leading to leukemogenesis remain unknown. To address this issue, we took advantage of the zebrafish model organism, which expresses 2 genes orthologous to human NPM1, referred to as npm1a and npm1b. Both genes are ubiquitously expressed, and their knockdown produces a reduction in myeloid cell numbers that is specifically rescued by NPM1 expression. In zebrafish, wild-type human NPM1 is nucleolar while NPMc+ is cytoplasmic, as in human AML, and both interact with endogenous zebrafish Npm1a and Npm1b. Forced NPMc+ expression in zebrafish causes an increase in pu.1(+) primitive early myeloid cells. A more marked perturbation of myelopoiesis occurs in p53(m/m) embryos expressing NPMc+, where mpx(+) and csf1r(+) cell numbers are also expanded. Importantly, NPMc+ expression results in increased numbers of definitive hematopoietic cells, including erythromyeloid progenitors in the posterior blood island and c-myb/cd41(+) cells in the ventral wall of the aorta. These results are likely to be relevant to human NPMc+ AML, where the observed NPMc+ multilineage expression pattern implies transformation of a multipotent stem or progenitor cell.

Zebrafish modelling of leukaemias

The use of animal models has revealed important mechanisms relevant to the development and treatment of human cancer. In recent years the zebrafish has emerged as an exciting new organism in which to model leukaemogenesis. The zebrafish model has distinct advantages over other animal models, most notably a capacity for forward genetic studies and rapid small molecule screens which can be used to dissect novel genetic pathways contributing to the development of leukaemia. Additionally, the high fecundity and optical clarity of the zebrafish make it an attractive organism in which to directly visualize the localization and development of normal and abnormal haematopoiesis in vivo. Until recently, targeting mutations to specific genes was technically difficult in the zebrafish, but new technology using chimeric zinc fingers to create targeted gene knockouts has made reverse genetic modelling possible and promises to deliver many new and exciting models. This review summarizes the benefits of using the zebrafish to study leukaemogenesis, reviews current zebrafish models of specific leukaemias, and gives an overview of future direction for the zebrafish in the study of cancer.

Ddx18 is essential for cell-cycle progression in zebrafish hematopoietic cells and is mutated in human AML.

In a zebrafish mutagenesis screen to identify genes essential for myelopoiesis, we identified an insertional allele hi1727, which disrupts the gene encoding RNA helicase dead-box 18 (Ddx18). Homozygous Ddx18 mutant embryos exhibit a profound loss of myeloid and erythroid cells along with cardiovascular abnormalities and reduced size. These mutants also display prominent apoptosis and a G1 cell-cycle arrest. Loss of p53, but not Bcl-xl overexpression, rescues myeloid cells to normal levels, suggesting that the hematopoietic defect is because of p53-dependent G1 cell-cycle arrest. We then sequenced primary samples from 262 patients with myeloid malignancies because genes essential for myelopoiesis are often mutated in human leukemias. We identified 4 nonsynonymous sequence variants (NSVs) of DDX18 in acute myeloid leukemia (AML) patient samples. RNA encoding wild-type DDX18 and 3 NSVs rescued the hematopoietic defect, indicating normal DDX18 activity. RNA encoding one mutation, DDX18-E76del, was unable to rescue hematopoiesis, and resulted in reduced myeloid cell numbers in ddx18(hi1727/+) embryos, indicating this NSV likely functions as a dominant-negative allele. These studies demonstrate the use of the zebrafish as a robust in vivo system for assessing the function of genes mutated in AML, which will become increasingly important as more sequence variants are identified by next-generation resequencing technologies.

L-Leucine improves the anaemia in models of Diamond Blackfan anaemia and the 5q- syndrome in a TP53-independent way

Haploinsufficiency of ribosomal proteins (RPs) and upregulation of the tumour suppressor TP53 have been shown to be the common basis for the anaemia observed in Diamond Blackfan anaemia and 5q‐ myelodysplastic syndrome. We previously demonstrated that treatment with L‐Leucine resulted in a marked improvement in anaemia in disease models. To determine if the L‐Leucine effect was Tp53‐dependent, we used antisense MOs to rps19 and rps14 in zebrafish; expression of tp53 and its downstream target cdkn1a remained elevated following L‐leucine treatment. We confirmed this observation in human CD34+ cells. L‐Leucine thus alleviates anaemia in RP‐deficient cells in a TP53‐independent manner.

Efficacy of recombinant activated factor VII in unselected patients with uncontrolled haemorrhage : a single centre experience

Recombinant activated factor VII (rFVIIa /Novoseven) has been used in a wide variety of circumstances as a treatment for uncontrolled bleeding. We present a retrospective report of the use of rFVIIa in 40 consecutive patients without inherited bleeding disorders in a single centre. Twenty-one (68%) of the 31 patients whose response to rFVIIa was documented showed a reduction or cessation in bleeding; in nine patients (29%) bleeding was unchanged and in one patient (3%) bleeding increased despite rFVIIa. One person suffered a thrombotic stroke after rFVIIa treatment. There were no other adverse events directly attributable to rFVIIa. Twenty-four patients (60%) died during the hospital admission in which the rFVIIa was administered. Twelve patients (30%) who received rFVIIa had bleeding secondary to haematological malignancy and 21 patients (53%) had bleeding complicating a surgical procedure. There were 11 deaths (92%) in the haematological malignancy group and 10 deaths (48%) in the surgical group. Patients with haematological malignancy received a significantly greater median number of doses of rFVIIa than patients with surgical bleeding complications (three versus one dose, P = 0.04). We conclude that rFVIIa can be used safely in uncontrolled haemorrhage and the majority of patients show a response.

Myelopoiesis and myeloid leukaemogenesis in the zebrafish.

Over the past ten years, studies using the zebrafish model have contributed to our understanding of vertebrate haematopoiesis, myelopoiesis, and myeloid leukaemogenesis. Novel insights into the conservation of haematopoietic lineages and improvements in our capacity to identify, isolate, and culture such haematopoietic cells continue to enhance our ability to use this simple organism to address disease biology. Coupled with the strengths of the zebrafish embryo to dissect developmental myelopoiesis and the continually expanding repertoire of models of myeloid malignancies, this versatile organism has established its niche as a valuable tool to address key questions in the field of myelopoiesis and myeloid leukaemogenesis. In this paper, we address the recent advances and future directions in the field of myelopoiesis and leukaemogenesis using the zebrafish system.