Linking the Glycolytic Enzyme HK3 to Neutrophil Differentiation of APL Cells Via PU.1

Abstract

Abstract 2425 Objectives:Identification and characterization of hexokinase 3 (HK3), as novel transcriptional target of the myeloid master regulator PU.1, with a role in APL cell differentiation and survival. Materials:HK3 and PU.1 quantitative RT-PCR in primary AML patient samples (n=170) and granulocytes isolated from healthy donors (n=20). Gene expression profiling in PU.1-knockout and PU.1-restored myeloid cell lines. Generation of NB4 and HT93 PU.1 as well as HK3 knockdown cell lines. ATRA-induced differentiation of these cell lines was assessed by CD11b and CEBPE expression. In vivo binding of PU.1 and PML-RARA to the HK3 promoter was shown by ChIP assays. Viability of NB4 cell lines was assessed by Alamar Blue staining. Results:We identified HK3 as putative transcriptional target of PU.1 by gene expression profiling in PU.1-knockout and PU.1-restored myeloid cell lines. Unlike the other three hexokinase family members (HK1, HK2 and HK4), HK3 expression is limited to hematopoietic cells, where it is mainly expressed in the myeloid lineage. We found 50-times lower HK3 mRNA levels in primary non-APL patients as compared to granulocytes from healthy donors. Most interestingly, we found that HK3 expression in APL patients with the t(15;17) translocation was 400-times lower (p<0.0001) than compared to granulocytes. Given the particularly low HK3 and PU.1 expression in APL patients, we decided to investigate the role of HK3 and its possible regulation by PU.1 during all-trans retinoic acid (ATRA)-induced neutrophil differentiation of NB4 and HT93 APL cell lines. Firstly, we observed a 5500- and 1100-fold increase of HK3 mRNA levels during neutrophil differentiation of NB4 and HT93, respectively. This was paralleled by markedly increased HK3 protein levels. No increased HK3 levels were observed in ATRA-resistant NB4-R2 cell lines. HK1 and HK2 mRNA expression was not markedly increased during neutrophil differentiation pointing to a particular role for HK3 in neutrophil differentiation. Regulation of the liver specific HK4 was not investigated. Next, we observed significantly decreased HK3 expression, both at the mRNA and protein levels, in all PU.1 knockdown cell lines upon ATRA-induced differentiation. In vivo PU.1 binding to three different regions of the HK3 promoter was found. Given our findings in primary APL patients, we decided to test if PML-RARa binds to the HK3 promoter, and indeed, PML-RARA binds to the same promoter regions as PU.1. Knocking down HK3 in APL cell lines resulted in significantly reduced neutrophil differentiation as measured by CD11b and CEBPE expression. Also, we observed that NB4 HK3 knockdown cell lines treated with ATRA were less viable compared to control cells. Conclusions:Our results strongly suggest that HK3 is a novel PU.1 transcriptional target functionally involved in neutrophil differentiation and cell viability of APL cells. Disclosures:No relevant conflicts of interest to declare.