Functional and translational studies of RUNX1 and CBFB in hematopoiesis

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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 and RUNX1 in the formation of hematopoietic stem cells (HSCs) in zebrafish; and specific aim 2, Studying the familial platelet disorder (FPD) and the role of RUNX1 in this disease using human induced pluripotent stem cells (iPSCs). In the first aim we study the role of CBFbeta and RUNX1 during early hematopoiesis in zebrafish. CBFbeta and RUNX1 form a DNA-binding heterodimer and are both required for HSC production. However, the exact role of CBFbeta in the production of HSCs remains unclear. We generated and characterized two zebrafish cbfb null mutants using zinc finger nuclease technology. The cbfb-/- embryos lacked definitive hematopoiesis. Unlike runx1 mutants in which HSCs are not formed, nascent, runx1+/c-myb+ HSCs were formed in cbfb-/- embryos. However, the nascent HSCs were not released from the aorta-gonad-mesonephros (AGM) region. Moreover, wild type embryos treated with an inhibitor of RUNX1-CBFbeta interaction, Ro5-3335, phenocopied the hematopoietic defects in cbfb-/- mutants, rather than those in runx1-/- mutants. Finally, we found that cbfb was downstream of the Notch pathway during HSC development. Our data suggest that runx1 and cbfb are required at two different steps during early HSC development. This research has been recently published in Blood. (Bresciani et al., Blood 124: 70, 2014). Moreover, we previously showed that zebrafish homozygous for an ENU-induced truncation mutation in runx1 (runx1W84X/W84X) were able to recover from a larval bloodless phase and develop adult definitive hematopoiesis, suggesting the formation of runx1-independent adult HSCs (Sood et al., Blood 115:2806, 2010). Therefore, in the last fiscal year we generated two new runx1 mutants using the TALEN technology in order to further investigate if RUNX1-independent pathway(s) exists for the formation of adult HSCs. In the second aim we are using the human iPSC model to study familial platelet disorder (FPD), a blood disease caused by heterozygous germline mutations in RUNX1, 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 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 derived induced pluripotent stem cells (iPSCs) from two patients in a family with FPD, and found that the FPD iPSCs display defects in megakaryocytic differentiation in vitro. We corrected the RUNX1 mutation in one FPD iPSC line through gene targeting, mediated by zinc finger nuclease technology, which led to normalization of megakaryopoiesis of the iPSCs in culture. Our results demonstrate successful in vitro modeling of FPD with patient-specific iPSCs and confirm that RUNX1 mutations are responsible for megakaryopoietic defects in FPD patients. These findings have just been accepted for publication in Blood (Connelly et al., Blood, e-pub ahead of print).