UC San Diego

Hexokinase (HK) 1, 2, and 3 are metabolic enzymes that catalyze the first step of glycolysis. Previous studies have found that HK3 is directly activated by the transcription factor PU.1 and may be involved in neutrophil differentiation. To examine the role of HK3 in myelopoiesis, the gene expression patterns of HK1, HK2, and HK3 during neutrophil and macrophage differentiation from CD34+ hematopoietic stem and progenitors cells (HSPCs) were investigated. Neutrophil differentiation of myeloid progenitors from cord blood (CB) as well as mobilized peripheral blood HSPCs showed an increase in HK3 gene expression, while HK1 and HK2 transcripts remained constant. Monocytes/macrophages differentiated from CB HSPCs also showed an increased expression of HK3 mRNA, whereas HK1 and HK2 transcripts were moderately expressed and remained constant. In order to investigate the role of HKs in myelopoiesis, human NB4 and HL-60 promyelocyte cell lines, which demonstrated similar patterns of HK1, HK2 and HK3 gene expression during ATRA-mediated neutrophil differentiation, were utilized to generate shRNA-mediated knockdowns of HK mRNAs. The generated HK shRNA cell lines showed marginal to low decreases in HK levels and also were inconclusive in regards to the extent of neutrophil differentiation. Therefore, I performed CRISPR-Cas9 gene editing of HK3 in NB4 cells. The generated gene-edited cell line had a heterogeneous population and therefore will require additional clonal selection for future experiments. Taken together, my results indicate that there is a prominent increase in HK3 gene expression during myeloid differentiation, suggesting that HK3 may play a role in myelopoiesis and/or neutrophil function.