ALBERT

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: 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.