Role of Hexokinase 2 and 3 in Energy Metabolism, Myeloid Differentiation and Cancer Development

Abstract

IntroductionTight control of metabolic processes is crucial for normal hematopoietic cell development, proliferation and cancer cell expansion. These processes all require variable bioenergetic demands, which are in part met by an upregulation of glycolytic activity. Hexokinases (HK1, 2, 3 and 4) catalyze the first and irreversible step of glycolysis. Recent evidence implicates a tumor promoting role for HK2 in KRas‐driven lung and ErbB2‐driven breast cancer. In contrast to the ubiquitously expressed HK1 and 2 in mammalian cells, HK3 is predominantly expressed in hematopoietic cells. We have identified HK3 as a transcriptional target of the hematopoietic‐specific transcription factor, PU.1 (SPI‐1). Moreover, HK3 is downregulated in acute myeloid leukemia (AML) patient samples and upregulated during terminal differentiation of selected acute myeloid leukemia cell lines concomitant with PU.1 upregulation.Methods and ResultsIn primary human CD34+ hematopoietic stem and early progenitor cells (HSPCs), we found HK1‐3 to be present at similar levels. While HK1 and HK2 mRNA levels remained unchanged during HSPC myeloid differentiation, HK3 mRNA levels significantly increased. Given the oncogenic role of HK2 and our findings of HK3 in AML cell lines, we next evaluated whether unique roles exist for HK2 and 3 in myeloid cells. Using shRNA or CRISPR/CAS9 technology, we generated HK2 and HK3 knockdowns (KD) and knockouts (KO) in HL60 and NB4 AML cell lines. Assessment of glycolytic and oxidative metabolism in HK altered cells revealed that loss of HK3 had no effect on steady state glycolytic activity, while loss of HK2 drastically reduced steady state glycolysis. In contrast, loss of HK3 expression resulted in increased cell death during all‐trans retinoic acid (ATRA) induced differentiation of HL60 (86±2% vs 36±3% viable cells at day 4, p<0.0001). Furthermore, investigation of HK3 subcellular localization point towards both cytosolic and nuclear localization for ectopically expressed HK3 in HL60 cells by western blot analysis. Mass spectrometric (MS) analysis of HL60 HK2 KO lines identified upregulation of proteins involved in key signaling pathways (Wnt, EGF and PDGF), as well as macromolecular metabolic processes, Fig. 1A. In contrast, HK3 KO HL60 cells showed upregulation of proteins involved in cellular homeostatic processes (ubiquitin proteasome pathway) and cancer‐related signaling pathways (Wnt, Ras) (p‐value<0.05), Fig. 1B.DiscussionTogether our findings implicate HK2 as the major glycolytically active isoform of hexokinases in myeloid cell lines, whereas HK3 exerts non‐canonical regulatory functions impacting normal myeloid differentiation and cancer regulation. Investigations are underway characterizing the metabolic phenotype of HK KO AML cells as well as defining the precise roles of HK2 and HK3 in normal HSPC differentiation, proliferation and metabolic activity.Support or Funding InformationSwiss Cancer ResearchPanther Pathway analyses of significantly upregulated proteins identified by MS analyses upon loss of either HK2 (A) or HK3 (B) in HL60 cell lines evaluated at steady state.Figure 1