RUNX1 and CBFB are not only important for leukemogenesis but they are also key regulators of normal hematopoiesis. These two genes are required during the earliest steps of hematopoietic stem cell formation and in subsequent stages of several blood lineages. Multiple studies suggest that dysregulation of the normal transcriptional program controlled by RUNX1 and CBFB is likely to be an important mechanism for leukemogenesis. Therefore, better understanding of the RUNX1/CBFB transcriptional program and the roles of RUNX1 and CBFB in normal hematopoiesis will lead to better understanding of the mechanisms for leukemogenesis. We have been pursuing two specific aims in this project in the last fiscal year. In the first specific aim, we have been studying the role of RUNX1 in the formation of hematopoietic stem cells (HSCs) in zebrafish. Runx1 null mouse embryos lack definitive hematopoiesis and cannot survive past E13. Zebrafish with a runx1 stop codon mutation (runx1W84X/W84X), however, were able to recover from a larval bloodless phase and develop to adults with multi-lineage hematopoiesis. In order to determine if a RUNX1-independent mechanism can support the generation of HSCs we have generated three new runx1-/- lines using TALEN and CRISPR-Cas9. Two runx1-/- lines carry deletions within the runt-homology domain (runx1del8/del8 and runx1del25/del25). The third mutation removes most of the coding region of runx1, from exon 3 to exon 8 (runx1del(e3-8)/del(e3-8)). All three runx1-/- lines failed to initiate definitive hematopoiesis at 2 dpf. However, 40% of the runx1-/- embryos developed into fertile adults with circulating blood cells of multi-lineages. mRNA-sequencing of adult kidney showed that the runx1-/- hematopoietic cells are very similar to WT cells, except for the thrombocyte lineage. Live confocal imaging revealed the presence of cd41-GFP+ precursors in the hematopoietic tissues of runx1- /- embryos and larvae, which may be responsible for the recovery of hematopoiesis. We used single-cell RNA sequencing to transcriptionally profile WT and runx1-/- cd41-GFP+ cells at 2.5 dpf and 16 dpf (at the time of normal HSC initiation and hematopoietic recovery in runx1-/- larvae, respectively). At 2.5 dpf, we identified a residual population of cd41-GFP+ cells in the runx1-/- embryos that expresses HSC markers. At 16 dpf, runx1-/- and WT cd41-GFP+ precursors segregated in different cell clusters. Interestingly, the cd41-GFP+ precursors in the runx1-/- larvae are highly active and a large cluster of erythroid-primed precursors can be identified, corroborating with our hypothesis that the residual cd41-GFP+ cells are responsible for recovering hematopoiesis. Further analysis allowed us to identify candidate transcription factors that could be responsible for driving runx1-independent hematopoiesis. Genetic complementation analysis confirmed at least one of the transcription factors to be responsible for runx1-independent hematopoiesis. A manuscript reporting these findings is under preparation. In the second aim, we are using genetic and genomic approaches to study familial platelet disorder with associated myeloid malignancy (FPDMM). FPDMM patients have platelet defects and a life-long risk of developing hematopoietic malignancies, with variable clinical presentation and disease penetrance among families with different germline mutations, and even between affected individuals within a single family. An autosomal dominant disease, FPDMM is caused by inherited mutations in the RUNX1 gene. FPDMM is a rare disease; so far only about 50 families with the disease have been reported. Consequently, the pathogenesis of FPDMM has not been studied extensively. The problem is compounded by the fact that existing animal models (mouse and zebrafish) do not recapitulate FPDMM clinical phenotypes. My group has been taking two different approaches to study FPDMM pathogenesis. In the first approach, we have been using induced pluripotent stem cells (iPSC) to study the hematopoietic defects in FPDMM patients; and in the second approach, we have been using genomic sequencing to determine if there are germline and somatic mutations in other genes that cooperate with RUNX1 mutation for myeloid malignancy in FPDMM patients. We have established iPSC lines from FPDMM patients of two families (with different RUNX1 mutations) and demonstrated that these patient-specific iPSCs had hematopoietic differentiation defects, which could be rescued by genome editing to correct the RUNX1 mutations in these iPSCs. In the second approach, we have initiated a longitudinal study to prospectively sequence blood and bone marrow samples from FPDMM patients to detect germline and somatic mutations and correlate the findings with clinical observations. This will be achieved through extensive collaborations with extramural colleagues, such as Lucy Godley at University of Chicago. In addition, we plan to set up a clinical program at the NIH Clinical Center dedicated to this longitudinal study.
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