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 is an essential factor for HSC development. However, even though zebrafish with a runx1 stop codon mutation have defects in definitive hematopoiesis during embryogenesis, the runx1 mutant embryos can develop to fertile adults with blood cells of multi-lineages, raising the possibility that HSCs can emerge without RUNX1. We have generated three new zebrafish runx1 knockout lines with engineered deletions of the runx1 gene. We found all three had identical phenotype as observed in the initial runx1 mutant fish, further confirming the existence of a RUNX1-independent mechanism for the development of HSCs. We found that, in the absence of a functional runx1, a cd41-GFP+ population of hematopoietic precursors still emerge from the aorta-gonad-mesonephros (AGM) and can colonize the hematopoietic tissues of the mutant embryos. Single cell RNA sequencing of the wild type and mutant HSC/HSPC at embryonic and larval stages confirmed the presence of a population of cd41:GFP cells expressing HSC signature genes in the runx1 mutant embryos. At larval stages the runx1 mutant HSCs maintain their ability to generate erythroid and myeloid lineage progenitors but they present a different expression profile compared to the wild type. Our analysis shows that the master transcription factor gata2b is strongly upregulated in the runx1 mutant HSCs during the recovery of hematopoiesis and it is also upregulated in the kidney marrow of the surviving runx1 mutant adults. A germline mutation and morpholino knockdown of gata2b in the runx1 mutants were able to block the emergence of the cd41:GFP hematopoietic precursors and abolish the survival ability of the runx1 mutant fish. Interestingly in the Runx1 mutant mice Gata2 is upregulated and is the top upstream regulator responsible for the differentially expressed genes in the bone marrow. Taken together our results indicate that gata2b can drive HSC emergence in the absence of runx1 and suggest that the interplay between Runx1 and Gata2 is evolutionary conserved. A manuscript reporting these findings is under revision for publication in a scientific journal. In the second aim, we have been 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. It is believed that RUNX1 mutations themselves are not sufficient for leukemia development, acquisition of additional somatic mutations followed by clonal evolution are needed for progression to leukemia. FPDMM is a rare disease; so far only about 80 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. To address these issues for better understanding of the clinical course and underlying pathogenic mechanism of FPDMM, we launched a natural history study of FPDMM at the NIH Clinical Center in early 2019. https://clinicaltrials.gov/ct2/show/NCT03854318 https://clinicalstudies.info.nih.gov/ProtocolDetails.aspx?A_19-HG-0059.html%20InternalRUNX1 We started to admit patients in July and we have already seen more than 20 patients from 7 FPDMM families. For each patient we confirm the RUNX1 mutation and perform extensive clinical tests to catalog disease symptoms and signs. We collect peripheral blood and bone marrow samples for hematological tests as well as genomic sequencing tests. We focus our attentions on documenting clinical manifestations associated with FPDMM, including those outside of the hematopoietic system, and detecting secondary mutations and clonal hematopoiesis, which may be associated with progression to malignancies. We plan to prospectively sequence blood and bone marrow samples from these FPDMM patients to detect germline and somatic mutations and correlate the findings with clinical observations. We are encouraged by the relatively large volume of patient interests and anticipate a fairly large cohort of FPDMM in our natural history study in the near future. A parallel approach in the lab is to use genomic sequencing technologies to identify secondary mutations in existing samples from FPDMM patients through an international consortium led by Dr. Lucy Godley in University of Chicago. FPDMM patients have a life-long risk of developing MDS and AML, but currently there are no easy and reliable predictive tests. Dr. Godley has established collaborations with hematologists around the world to collect patient samples. We are performing next generation sequencing to detect mutations associated with leukemic transformation. We will follow with studies in cell culture and animal models to determine the functional significance of recurrently mutated genes on the phenotypes induced by RUNX1 deficiency. This study was recently awarded a Bench-to-Bedside grant by the NIH Office of Clinical Research.
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