ALBERT

Description: Nucleophosmin (NPM1) is a multi-functional ubiquitous phosphoprotein that shuttles between the nucleolus and cytoplasm. Located on chromosome 5q35 NPM1 is involved in the development of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) both as a chimeric fusion partner and as a putative key haploinsufficient tumor suppressor. Heterozygous NPMc+ mutations have been identified in 30% of AML, usually with normal karyotype. Such mutations create new nuclear export signals and disrupt the normal nucleolar localization signal, resulting in re-localization of both mutant and heterodimeric wild-type (WT) NPM protein from the nucleolus to the cytoplasm. The zebrafish is a model organism ideally suited to genotypic and phenotypic analysis of myelopoiesis and leukemogenesis, with a proven track record for facilitating the discovery of novel pathogenetic pathways. Using in silico analysis we identified two homologues (a common finding in zebrafish due to genome duplication during piscine evolution) of the human nucleophosmin gene in zebrafish. These two genes have been designated znpm1a and znpm1b. znpm1a is annotated by the National Center for Biotechnology (NCBI) while znpm1b is a known protein-coding region located via blast search of the human NPM1 amino acid sequence at www.ensembl.org/Danio_rerio/index.html. Whilst znpm1b exhibits slightly less identity to human NPM1 (47%) than znpm1a (64%) it demonstrates clear synteny with human chromosome 5q35 and mouse chromosome 11 (Figure 1). We confirmed expression of both znpm1a and znpm1b in embryonic tissue and adult hematopoietic tissue of the major lineages by RT-PCR of Green Fluorescent Protein (GFP)-sorted cells in pu.1-GFP transgenic zebrafish embryos and in adult zebrafish kidney cells (sorted by forward and side scatter charactersistics). Morpholinos (stable, synthesized antisense oligonucleotides that specifically block gene expression when injected into embryos at the one-cell stage) were designed to inhibit znpm1a or 1b and injected into zebrafish embryos at the 1–4 cells stage to assess the effect of knockdown of znpm1a and 1b alone and in combination on hematopoiesis. Whole-mount in situ hybridization of 48 hours post-fertilization(hpf) injected embryos demonstrated a 50% reduction in the expression of myeloperoxidase (mpo) and a similar reduction in alpha globin (α-globin) expression as markers of myelo- and erythropoiesis. To investigate the mechanism of the reduction in hematopoietic cells we injected the znpm1a and 1b morpholinos into zebrafish carrying mutated p53 and observed partial rescue of the hematopoietic phenotype suggesting that loss of npm1 in zebrafish activates p53 dependent cell cycle arrest, senescence or cell death. Thus zebrafish npm1 proteins are required for normal hematopoiesis consistent with the role for NPM1 as a tumor suppressor in AML/MDS with loss of all or part of chromosome 5. Future studies using this model will address which pathways are disrupted by the loss of npm1 and thus may contribute to the pathogenesis of human AML/MDS and facilitate identification of potential therapeutic targets. Figure 1. Zebrafish znpm1b exhibits synteny with human chromosome 5q35 and mouse chromosome 11. Figure 1. Zebrafish znpm1b exhibits synteny with human chromosome 5q35 and mouse chromosome 11.

Description: The t(12;21)(p13;q22) chromosomal translocation resulting in the TEL-AML1 fusion gene is the most common translocation in childhood B cell precursor acute lymphoblastic leukemia (BCP-ALL) and is found in approximately 25% of cases. The translocation arises in utero, and can be found in approximately 1% of newborn infants. However, less than 1% of these children develop leukaemia and this translocation is not observed in adult ALL. This suggests that the cell of origin resulting in the eventual transformation to BCP-ALL is present and disrupted early during development but does not represent a potent oncoprotein. Further evidence for this is provided by animal models show that TEL-AML1 alone is necessary but not sufficient to trigger overt ALL. To further delineate the effects of TEL-AML1 during developmental haematopoiesis we utilized a zebrafish model, Tg(ZβA:hTEL-AML1-EGFP), that expresses human TEL-AML1 (hTEL-AML1) under the control of zebrafish β-actin gene promoter. Previous studies have shown that approximately 2% of these fish develop B-cell leukemia with a long latency. Primitive erythropoiesis was studied using whole mount in situ hybridisation (WISH) in Tg(ZβA:EGFP-hTEL-AML1+/-) and siblings. The expression pattern of ikarosand gata1a showed that primitive erythroid cells develop normally in Tg(ZβA:EGFP-hTEL-AML1+/-). The development of erythroid cells at later stages was studied using o-dianisidine staining, to assess the haemoglobinization of erythrocytes. Our results suggest normal differentiation of erythroblasts into erythrocytes in Tg(ZβA:EGFP-hTEL-AML1+/-) embryos and normal haemoglobinization of erythrocytes. We next examined myelopoiesis in Tg(ZβA:EGFP-hTEL-AML1)using WISH for cebpα and lcp1, and Sudan Black (SB) staining to label mature granulocytes. Primitive myelopoiesis was unperturbed by hTEL-AML1 at 18hpf, however we observed an increased number of SB positive cells commencing at 2 days post fertilization (dpf). SB positive cells continued to increase in number until 4dpf when the difference became less pronounced and was gone by 7dpf (Figure 1). SB positive myeloid cells at these stages are most likely to be derived from the earliest definitive hematopoietic precursors, such as the transient bi-potent erythro-myeloid progenitor (EMP) because primitive myeloid cells are predominantly macrophage-like cells that do not express SB. By 4dpf when definitive hematopoietic stem and progenitor cell (HSPC) derived myelopoiesis is dominant, the observed difference in mature myeloid cell numbers has resolved. The earliest lymphoid cells in zebrafish embryos are thymocytes. Thymocytes were visualized using WISH for gata3 (GATA binding protein 3), lck (T-cell specific tyrosine kinase) and rag1 (recombination activating gene 1). Expression of all three markers was unpertubed indicating that early T cell development is not affected by hTEL-AML1 fusion protein expression. B lymphoid cells have not been convincingly observed in zebrafish until 3 weeks post fertilization despite evidence of VDJ rearrangements in whole embryos from 4dpf. We attempted to identify evidence of B lymphoid cells using WISH for pax5 and cd79b, however we were not able to identify any cells before 10dpf. This supports the observation that B cells do not arise until later in development, or are too low in expression level or few in number to identify by WISH. In order to more closely recapitulate aspects of TEL-AML1 leukemia in humans, where one the remaining endogenouscopy of TEL is frequently lost at diagnosis of BCP-ALL, we have generated a knockout of zebrafish etv6 using CRISPR/Cas9. 4 Etv6 guides targeting zebrafish etv6 were more than 90% efficient at cleaving their DNA target. F0 animals with this mutational burden survived to adulthood with no increase in mortality. By contrast F0 Tg(ZβA:EGFP-hTEL-AML1+/-) injected with etv6 crispr had an increased mortality and showed a variety of developmental abnormalities. Assessment of their haematopoietic compartment is ongoing. In summary we show that early myelopoiesis is disrupted during development in a zebrafish transgenic model expressing human TELAML1, and that this occurs prior to the stage at which definitive pluripotent HSPC-derived myelopoiesis is dominant. This supports the hypothesis that effects of TELAML1 during developmental haematopoiesis underpin its oncogenic potential during childhood. Disclosures No relevant conflicts of interest to declare.

Description: MDS with isolated del(5q) (MDS5q) is a defined entity in the WHO classification for myeloid malignanies owing to its unique morphological and biological phenotype and striking responseslenalidamide. Nonetheless approximately 1/3 of patients with MDS5q fail to achieve tansfusion independance and dose limiting toxicity is common. Furthermore 3-year survival remains poor at around 50%, and this disease remains incurable without bone marrow transplantation. Thus there is an unmet need to identify additional therapies for MDS5q. Ribosomal protein gene 14 (RPS14), located on 5q, has been shown to be the likely genetic determinant of anemia in patients with MDS5q. Importantly, loss of 5q has been shown to be an initiating event in the development of MDS in these patients. We have previously shown that Rps14-deficient zebrafish exhibit a robust anemia during development permitting in vivo assessment of an MDS-like phenotype in these animals. We utilized Rps14 deficient embryos in drug screen to identify compounds that alleviate the anemia in this model. Rps14 was knocked down using antisense oligonucleotides directed against the splice donor site of the 2nd exon (first coding exon). At1 day post fertilization (dpf) embryos were plated in a 96 well plate, 2-3 embryos per well and exposed to compounds from the Spectrum library of known bioactive drugs at 5µM and 20µM in duplicate plates. L-Leucine was utilized as a positive control. At 4dpf embryos were assessed visually in vivo by 2 independent observers for anemia and developmental effects. They were then stained with o-dianisidine to assesshemoglobinization. Hits were defined as compounds that scored in at least 2 wells with a minimum of one from each observer. Our primary hit was validated with fresh compound and dose titration (Figure 1A,B). This compound targets TLR signaling which has recently been implicated in the pathogenesis of MDS5q in murine models. We went on to utilize an additional drug targeting the same receptor and observed similar improvements inhemoglobinization, validating this pathway as a putative therapeutic route for MDS5q. To further confirm our findings we generated a stable mutant line carrying a heritable mutation in zebrafish Rps14 using genome editing with Transcription activator-like effector nuclease (TALENS). These mutants have an 11bp complex indelin Exon 2 (Rps14E8fs). Rps14 E8fs/E8fs embryos show profound developmental anomalies including a marked anemia in keeping with other ribosomal protein mutants and are embryonic lethal by 5dpf. The majority of Rps14 E8fs/+ heterozygotes were indistinguishable from their WT siblings in early development and no overt abnormality in hemoglobinization could be detected by o-dianisidine staining. By contrast heterozygous adults were smaller than their siblings, weighed less and flow cytometry of whole kidney marrow demonstrated pancytopenia. As expected the features were most marked in the erythroid lineage highlighted using Tg(globin:eGFP);Rps14E8fs/+ animals. Rps14E8fs/+ adults also showed reduced hemoglobin levels in their peripheral blood although this was modest compared to the marrow features (Figure 1C). As all lineages appeared to be affected in Rps14E8fs/+ we went on to address myeloid development during development. Using Sudan Black (SB) staining Rps14E8fs/E8fs and Rps14E8fs/+ showed reduced numbers of mature myeloid cells from 2dpf. To further assess the effects of Rps14E8fs/+ in embryonic erythropoiesis we used Tg(Gata1:dsRed); Rps14E8fs/+ embryos and subjected them to flow cytometry. Rps14E8fs/+ at 3dpf showed reduced numbers of dsRed expressing erythroid cells compared to their siblings. Increased numbers of dsRed positive cells were observed when embryos were treated with L-leucine. Finally we treated embryos with our hit compound. Rps14E8fs/+ embryos showed increased numbers of dsRed expressing cells compared to Rps14+/+ siblings confirming our findings from Rps14morphants. In summary we have identified a putative new therapeutic compound that can increase erythroid cell numbers using an in vivo model of MDS5q anemia. We also describe the features of heterozygous Rps14E8fs/+ mutants that recapitulate features of MDS during developmental and adult hematopoiesis providing a novel platform for drug screen and second hit analyses in future studies. Our ongoing work is validating these compounds in patient cells from individuals with MDS5q. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: A comprehensive understanding of the genes and pathways regulating hematopoiesis is needed to identify genes causally related to bone marrow failure syndromes, myelodysplastic syndromes, and hematopoietic neoplasms. To identify novel genes involved in hematopoiesis, we performed an ethyl-nitrosourea mutagenesis screen in zebrafish (Danio rerio) to search for mutants with defective definitive hematopoiesis. We report the recovery and analysis of the grechetto mutant, which harbors an inactivating mutation in cleavage and polyadenylation specificity factor 1 (cpsf1), a gene ubiquitously expressed and required for 3′ untranslated region processing of a subset of pre-mRNAs. grechetto mutants undergo normal primitive hematopoiesis and specify appropriate numbers of definitive HSCs at 36 hours postfertilization. However, when HSCs migrate to the caudal hematopoietic tissue at 3 days postfertilization, their numbers start decreasing as a result of apoptotic cell death. Consistent with Cpsf1 function, c-myb:EGFP+ cells in grechetto mutants also show defective polyadenylation of snrnp70, a gene required for HSC development. By 5 days postfertilization, definitive hematopoiesis is compromised and severely decreased blood cell numbers are observed across the myeloid, erythroid, and lymphoid cell lineages. These studies show that cpsf1 is essential for HSC survival and differentiation in caudal hematopoietic tissue.

Description: Introduction: Phase I clinical trials are primarily designed to assess the safety and toxicity of a new agent and determine the recommended dose. Such trials are challenging as patients typically have multiply relapsed/ refractory disease and have received all standard therapies. Many have a poor prognosis and would otherwise receive supportive/ palliative treatment. However, the advent of molecularly targeted therapies and immunotherapies has challenged this paradigm. The outcomes of phase 1 trials in solid tumours have been reported, but not in haematological malignancies. Here we report the phase I/II trials experience from a dedicated trials unit within a large UK Haematology centre. Methods: This was a retrospective review of patients with haematological malignancies sequentially treated in Phase I/II clinical trials at the NIHR/ UCLH Clinical Research Facility, London, UK. Patients met the relevant eligibility criteria for each study and received at least one dose of study medication. Adverse events were graded by CTCAE 4.0, response assessments as per trial protocol. Univariate and multivariate analysis was performed upon demographic, clinical and haematological/ biochemical parameters to investigate predictive markers of outcome. Kaplan Meier method was used for survival analysis and censored for those proceeding to ASCT. Results: 69 patients were enrolled onto 11 trials between March 2012 and July 2016. 6 were phase I studies (four first-in-human); 4 phase I/II studies; and one phase II study with a dose finding phase. All involved molecularly or immunologically targeted therapies. Median age was 60.3 yrs (range 32.5-80.2). The median time from diagnosis to trial treatment was 42.8 months (range 1.9-202.5). Disease types included multiple myeloma (MM) (n=41), non-Hodgkin's lymphoma (NHL) (n=16: DLBCL 12, FL 2, WM 1, MCL 1), AML (n=10) and myelofibrosis (n=2). Patients had a median of 3 (range 0-8) prior lines of therapy and 29 (42%) had at least 4 prior lines. 28/68 (41.2%) were refractory to the last treatment and 1 patient with newly diagnosed MM was enrolled. A median of 4 (range 0.3-25) cycles of treatment were completed with a median duration of treatment of 3.7 months (range: 0.2-24.4). Treatment was discontinued due to disease progression (36, 62.1%), toxicity (8, 13.8%), proceeding to ASCT (11, 19%), patient choice (2, 3.4%), completed treatment (1, 1.7%). 11 patients are on-going. Following trial discontinuation, 40 (71.4%) received further treatment (clinical trials (n=5), standard therapy (n=24), ASCT (n=11)), 2 (3.6%) were managed expectantly and 14 (25%) palliated. 17 (25%) developed grade 3-4 AEs. 30.9% had dose interruptions for 7 or more days, and 77.9% maintained their planned dose throughout. For those completing 1 or more cycle (n=61, 57 were evaluable), the clinical benefit rate (SD or more) was 68.4% (SD 5 (8.8%), MR 7 (12.3%), PR 15 (26.3%), VGPR 10 (17.5%), CR 2 (3.5%)) with an overall response rate (PR or more) of 47.4%. 18 (31.6%) were refractory to trial treatment. With a median follow-up of 9.6 months (range: 0.2-44.6), the median progression free survival (PFS) was 10.0 months (95%CI: 0.9-19.1 months). The median overall survival was 31.1 months (95%CI: 20.4-41.8) The 90 day mortality rate was 12.8%. In univariate analysis, LDH and disease types were significant for PFS and OS. However, in multivariate analysis albumin and disease type were independent predictors of OS but not PFS. Serum albumen of 〉35g/L was associated with an improved OS (HR 0.21, 95%CI 0.06-0.82, p=0.024). Age, performance status, Hb, LDH were not predictive in multivariate analysis. MM was associated with a better PFS and OS over other disease types (see table). Conclusions: These data demonstrate the wide range of PFS and OS to phase I/II studies according to disease type. Patients with MM had better outcomes and were more likely to access subsequent treatments. Most NHL patients had refractory high grade disease but still had a median OS of 17.9 months. Those with refractory AML/ myelofibrosis had a poor outcome, highlighting a clear unmet need. The PFS and OS for MM and NHL patients was encouraging. Serum albumin was predictive of OS across all disease groups. Whilst such parameters are not primary endpoints for early phase trials, these data indicate their potential clinical benefit. Stratification according to molecular profiles may further improve outcomes. Table Table. Figure Figure. Disclosures Yong: Autolus Ltd: Equity Ownership, Patents & Royalties: APRIL based chimeric antigen receptor; Janssen: Research Funding.

Description: The current model for the pathogenesis of AML involves a normal haematopoietic stem or progenitor cell (HSPC) acquiring successive genetic abnormalities leading to clonal expansion and malignant transformation. Next generation sequencing (NGS) has facilitated the identification of more than 250 recurrent genetic changes in AML, with each case possessing between 5-20. Only a fraction of these have been validated as driver events, and the epistatic relationships and cellular consequences of combined mutational genotypes remain largely unexplored. A unique insight into this is afforded by a subset of patients who have inherited a mutation that predisposes to the development of AML, so-called familial leukaemia with predisposition to AML (FPD-AML) since these mutations are unequivocally the initiating driver mutations. One such mutation occurs in the N-terminal domain of the transcription factor CEBPA. To further understand the role of N-terminal CEBPA mutations and co-operating secondary driver mutations we have used a TALEN pair to generate a zebrafish model carrying an analogous N-terminal frame-shift mutation in Cebpa (cebpaNterm). Initial analysis shows cebpaNterm/Nterm mutants have severe defects in developmental myelopoeisis. By 5 days post fertilisation (dpf), homozygous cebpaNterm/Nterm mutants show a complete absence of Sudan Black (SB) staining myeloid cells. cebpaNterm/+siblings display a minor delay in myeloid development, but no significant difference in numbers of SB staining cells at 5 dpf (Figure 1A,B). cebpaNterm/Nterm mutants are morphologically indistinguishable from their siblings during the first 5 days of life, however, they show diminished growth and survival thereafter. At 4 weeks, remaining juvenile cebpaNterm/Nterm mutants show markedly reduced length and weight compared to age matched controls (cebpa+/+and cebpaNterm/+) and are not viable beyond 8 weeks (Figure 1C). To facilitate assessment of HSPCs/thrombocytes and myeloid cells, we crossed cebpaNterm/+mutants to transgenic reporter lines expressing eGFP from the CD41 promoter (Tg(cd41:eGFP)) and mCherry (mCh) from the lysozyme C promoter (Tg(LyzC:mCherry)). Tg(LyzC:mCherry);cebpaNterm/Nterm fry can be distinguished from their siblings in vivo by the absence of mCherry expressing myeloid cells from 3.5dpf. Flow cytometric analysis in surviving juveniles at 32dpf demonstrated a continued absence of mCh expressing myeloid cells in both peripheral blood and whole kidney marrow. However, evaluation of cebpaNterm/Nterm mutant juveniles at 6 weeks showed evidence of increased numbers of mCh positive cells, compared to wild type and heterozygous sibling controls. Blood smears in homozygous mutants at this stage also revealed cells morphologically suggestive of primitive myeloid origin. Finally, epifluorescent microscopy of 4-6 week old fish demonstrated a widespread GFP dim fluorescence in the cebpaNterm/Nterm mutants. In contrast siblings displayed only GFP bright thrombocytes, suggesting an expansion of CD41-GFPdimexpressing HSPC-like cells in mutant animals. The occurrence of biallelic N-terminal CEBPA-mutations in human AML is however extremely rare. By contrast the most frequent "second hit" mutation occurs in the C-terminal DNA binding domain of the other allele of CEBPA. These mutations are universally in frame deletions or insertions, suggesting retained dimerization capability but abrogation of DNA binding. We have generated a knock-in C-terminal model of the most commonly observed human mutation in the orthologous zebrafish sequence using TALENs and plasmid mediated homology directed repair. This donor plasmid adds 3bp encoding an additional leucine within the C-terminal zipper domain. MISEQ analysis of F0 putative founder eggs and sperm has shown a 2-5% germline transmission rate of the knockin allele. In addition to CEBPA C-terminal mutations, a number of other recurrently mutated genes have been found in CEBPA mutated AML. Our ongoing work will assess the effects of combined N and C terminal cebpa mutation, along with CRISPR-targeting of other common "second hits" including cohesion proteins, WT1, GATA2 and TET2 to define their role in leukaemogenesis in CEBPA-mutated AML. Our ongoing experiments are also defining the self-renewal capacity of myeloid and HSPCs derived from 4-6 week old cebpaNterm/Nterm mutants using rag2E450fsknockout transplantation assays. Disclosures No relevant conflicts of interest to declare.

Description: 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 ddx18hi1727/+ 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.

Description: Diamond-Blackfan Anemia (DBA) is a congenital bone marrow failure syndrome that manifests as a profound macrocytic anemia and classically presents within the first year of life. Heterozygous mutations in, or genomic loss of one of several Ribosomal Protein (RP) genes have been identified in over 50% of DBA patients, most commonly RPS19, accounting for 25% of all cases. DBA shares a similar erythroid phenotype to the 5q- subtype of myelodysplastic syndrome in which anemia is thought to arise from heterozygous loss of RPS14. Anemia in these conditions is at least partially due to p53-mediated apoptosis and cell cycle arrest of erythroid progenitors. To further study the role of p53 in the pathogenesis of DBA and 5q- syndrome, we employed genome editing tools to generate stable Rps14 and Rps19 knockout zebrafish lines. We generated Transcription Activator-Like Effector Nucleases (TALENs) targeting exon 1 of rps19 and exon 1 of rps14 as well as Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR) single guide RNAs (sgRNA) targeting exon 2 of rps19. TALENs or CRISPRs were injected into p53m214k/m214k zebrafish embryos at the single-cell stage. This zebrafish line carries a mutated p53 that is insensitive to DNA damage and hence prone to tumor formation. rps19 CRISPR sgRNAs were injected with mRNAs encoding Cas9, Cas9D10A nickase, and a ssDNA guide with a human DBA mutation. For each cohort of embryos injected, genomic DNA analysis from 20 phenotypically normal embryos from each clutch was screened to determine the efficacy of cleavage by TALEN and CRISPR using MiSeq. Mutations were identified in 30% (rps14 TALEN) 29% (rps19 TALEN), 27% (rps19 Crispr Cas9) and 12% (rps19 Crispr Cas9D10A) of reads. None of the rps19Crispr Cas9D10A carried the ssDNA guide mutation, rather single nucleotide variants and indels similar to those observed with Cas9. The remaining embryos from each F0 clutch were raised in order to generate stable mutant lines in the F1 generation; however, early, overt tumor growth was noted in all RP injected lines. Tumors were observed from 4 months post fertilization compared with 9 months for uninjected controls. F0 RP mosaic fish continued to develop tumors earlier than uninjected counterparts. At 10 months of age tumor development was statistically significantly higher in rps19 and rps14 TALEN and rps19 Cas9D10A and trended towards significance in rps19 Cas9 injected fish. Overall survival was significantly reduced in each of the cohorts compared to p53m214k/m214k uninjected controls (p

Description: 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.

Description: 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.