Publication Date:
2018-11-29
Description:
The TET2 gene is frequently mutated in pre-leukemic hematopoietic stem cells in human acute myeloid leukemia (AML) and encodes for an enzyme that catalyzes the conversion of DNA 5-methylcytosine to 5-hydroxymethylcytosine. Recent studies suggest that (i) the product of this reaction can be enhanced using high dose ascorbate, and (ii) formation of the substrate 5-methylcytosine can be blocked with azacitidine. To understand the mechanisms of TET2 mutation-driven leukemogenesis, we developed two CRISPR/Cas9 approaches to disrupt the TET2 gene in primary human CD34+ HSPCs to mimic TET2-mutated pre-leukemia. First, in "Hit & Run," we use Cas9 with two single-guide RNAs (sgRNAs) to disrupt the TET2 gene within exon 3 (average indel frequencies=94.3%). Second, we using homology directed repair (HDR) of Cas9-mediated dsDNA breaks to disrupt the TET2 gene within exon 7 by inserting a GFP expression cassette to generate in vivo traceable cells. Thus, we have developed a tractable and cell-traceable model that recapitulates TET2-mutated pre-leukemia and clonal hematopoiesis. First, we examined the effects of TET2 disruption on human erythroid differentiation in vitro by culturing bulk CD34+ cells for 10 days under conditions that promote erythroid differentiation. Both Hit & Run and HDR (GFP+) TET2 disruption decreased CD71+CD235+ erythroid differentiation compared to control cells. Exposure to high dose ascorbate partially rescued the erythroid defect in TET2-disrupted cells (Hit & Run, n=3 independent experiments, p
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
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