We have been pursuing two specific aims in this project in the last fiscal year. They are:
specific aim 1, Determining the roles of CBFB in HSC formation in zebrafish;
and specific aim 2, Studying familial platelet disorder and studying the role of RUNX1 in this disease using human iPSCs. CBFβand RUNX1 form a DNA-binding heterodimer and they are both required for definitive hematopoiesis at the stage of hematopoietic stem cells (HSCs). However, the exact role of CBFβin the development of HSCs remains unclear. To dissect the role of CBFβin the emergence and maintenance of HSCs we generated two zebrafish cbfb null mutants using zinc finger nuclease (ZFN) technology. Similar to our published runx1 mutant embryos, cbfb-/- embryos underwent primitive hematopoiesis, but lacked definitive hematopoiesis. Unlike the runx1 mutants, however, the emergence of HSCs in the AGM was unaffected in cbfb-/- embryos. Rather, the subsequent mobilization of the HSCs from AGM was blocked, as evidenced by the accumulation of runx1+ HSCs in the AGM and the concomitant absence of such cells in the caudal hematopoietic tissue (CHT). We found that cbfb was downstream of the Notch pathway during HSC development, since cbfb expression was expanded in Notch transgenic embryos but abrogated in the Notch-deficient mind bomb mutants. Moreover, embryos treated with Ro5-3335, the inhibitor of RUNX1-CBFβinteraction, phenocopied the hematopoietic defects in the cbfb-/- mutants. Overall our data suggest that CBFβand functional CBFβ-RUNX1 heterodimers are not required for the emergence of HSCs but are essential for the mobilization of HSCs during early definitive hematopoiesis. (Bresciani et al., Manuscript submitted) Heterozygous germline mutations in RUNX1 lead to familial platelet disorder (FPD), which is one of the first known haploinsufficiency diseases. Patients with this disorder have defective megakaryocytic development, low platelet counts, prolonged bleeding times, frequent bruises, and a high frequency (>35%) of developing AML at some point in their lifetime. The clinical manifestations of the disease underscore the critical role of RUNX1 in megakaryocyte differentiation and platelet function, in addition to its role in early hematopoiesis. Since it is the only known inherited disease caused by RUNX1 mutations, FPD is a good model to study RUNX1 function in human hematopoiesis. In addition, we hope our studies will eventually lead to better management of the FPD patients, especially in the form of cell therapy, which is potentially curative of the disease. Moreover, the approaches and reagents developed in this aim can be applicable to cell-based therapies of many other hematological diseases. Importantly, no animal models are available for FPD: Runx1 heterozygous knockout animals (both mouse and zebrafish) have no defects in megakaryocytic development and they do not develop leukemia. The induced pluripotent stem cell (iPSC) technology is one of the most important advances in biology and medicine in the first decade of the 21st century. The iPSCs have the potential to differentiate into any cell type of the human body, so they can be used to model many human diseases. Since no suitable animal models are available to study FPD, the hematopoietic defects in FPD can potentially be replicated or modeled in cell culture. We have established iPSC lines from skin fibroblasts of 2 FPD patients harboring a Y260X mutation in the RUNX1 gene. We demonstrate, in vitro, that the FPD iPSCs display defects in hematopoietic differentiation, particularly towards megakaryopoiesis. We then performed zinc finger nuclease (ZFN) mediated gene targeting to correct the mutation in one of the FPD iPSC lines. We could demonstrate that ZFN-mediated mutation correction rescued the FPD phenotype with increased number of CD41+CD42+ megakaryocytes. Our innovative approach to model FPD using iPSC lines with an ability to correct the mutation by gene targeting provide a unique tool to perform translational studies of FPD. We will perform molecular characterizations to understand the mechanisms through which RUNX1 regulates megakaryopoiesis and the underlying defects in the FPD iPSCs. (Kwon et al., Manuscript in preparation)

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National Human Genome Research Institute
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