Functions of Idh1 in Zebrafish Hematopoiesis and Its Relevance to Human Acute Myeloid Leukemia

This dissertation, "Functions of IDH1 in Zebrafish Hematopoiesis and Its Relevance to Human Acute Myeloid Leukemia" by Xiangguo, Shi, 石祥国, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Isocitrate dehydrogenase (IDH) is involved in tricarboxylic acid (TCA) cycle by converting isocitrate to α-ketoglutarate (α-KG) in physiological condition. Recently, recurrent mutations of the IDH1/2 have been identified in a variety of cancers, including up to 30% of cytogenetically normal acute myeloid leukemia (CN-AML). IDH1/2 mutations confer new metabolic function to the mutant enzymes that catalyze conversion of α-KG to 2-hydroxyglutarate (2-HG). The latter was thought to inhibit α-KG-dependent dioxygenases, including ten-eleven translocation 2 (TET2) and disrupt the epigenetic regulatory network that normally keeps leukemia initiation in check. Zebrafish has emerged as a model organism for the study of hematopoiesis and human diseases and zebrafish IDH1/2 (zidh1/2) are true orthologs of mammalian IDH1/2. In the present study, we investigated the role of zidh1 in embryonic and adult hematopoiesis and evaluated its pathogenetic and therapeutic relevance to human AML. In adult zebrafish, zidh1 was preferentially expressed in the kidney, the equivalence of human bone marrow. During early embryogenesis, it was first ubiquitously expressed and later around the regions of ventral wall of dorsal aorta (DA) and the caudal hematopoietic tissue (CHT), the sites of definitive hematopoiesis. Morpholino knock-down of zidh1 induced differentiation block of primitive myelopoiesis, characterized by an increase in gene expression associated with early myeloid progenitor (pu.1) and decrease in those associated with late myelomonocytic differentiation (l-plastin, mpeg1 and mpo). Definitive hematopoiesis was also reduced, as shown by the down-regulation of c-myb and runx1 along the ventral wall of DA and T-cell marker rag1 in the thymus. To validate our observations, we constructed a TALEN pair targeting exon 1 of zidh1 and injected the TALEN mRNAs into zygotic stage embryos. The efficiency of inducing small insertions or deletions (indels) was 98.71.5%. The mutant embryos exhibited defective primitive myelopoiesis and definitive hematopoiesis. Mutant adult fish exhibited an increase in hematopoietic precursor cells in the kidney marrow. Morpholino knock-down of zidh2 also resulted in a blockade of myeloid differentiation as evidenced by increased expression associated with early myeloid progenitor (pu.1) and decreased expression associated with late myelomonocytic differentiation (mpo and l-plastin). Definitive hematopoiesis was not affected. Co-injecting zidh2 mRNA with zidh1 morpholino could not rescue the hematopoietic defects of the latter, indicating non-redundant functions of zidh1 and zidh2. To examine whether the leukemogenic mechanisms of human IDH1-R132H is conserved in zebrafish, we microinjected human IDH1-R132H cDNA into zygotic stage embryos. There was significant increase in 2-HG, associated with increased expression of pu.1, l-plastin, mpeg1 and mpo. The response could be ameliorated by an IDH1-R132H specific inhibitor (AGI-5198). Overexpression of a zebrafish orthologue zidh1-R146H significantly increased 2-HG and primitive myelopoiesis but the responses were not inhibited by AGI-5198. The results provided important insights to the role of zidh in myelopoiesis and definitive hematopoiesis. We also developed a zebrafish model of IDH1-R132H that underscored the conserved leukemogenic mechanisms of IDH1 mutation and provid