Hexokinase Proteins Impart Distinct Functions in Myeloid Development and Cell Death
Authors: Seiler Kristina , Minder Petra , Mashimo Iris , Humbert Magali , Simpson Elizabeth , Tschan Mario P. , Torbett Bruce E. 
Published: October 2019

Abstract
Many key oncogenic pathways converge to adapt tumor cell metabolism to requirements of cancer cells. Aberrant proliferation that is frequently associated with cancer cells is also linked to an adjustment of metabolism in order to fuel cell growth and division. Cancer cells prefer utilizing glycolysis for energy production and providing essential building blocks for a variety of macromolecules. Hexokinases (HKs) are rate-limiting enzymes that catalyze the first and irreversible step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. Four HK isoforms are expressed in mammalian cells, HK1, HK2, HK3 and HK4 (also known as glucokinase). HKs promote and sustain a concentration gradient that facilitates glucose entry, which ensures the initiation of glucose dependent pathways. In general, HKs have a cytoprotective role that was highlighted by enhanced sensitivity of cancer cells to drugs when HKs were inhibited. Previously we have reported that HK3 was transcriptionally regulated by PU.1 (SPI-1) in myeloid cells. Further, HK3 expression was significantly reduced in patient acute myeloid leukemia (AML) cells, particularly in acute promyelocytic leukemia (APL) cells expressing the PML-RARA oncofusion protein.
We now report on the expression and regulatory function of HKs, particularly HK3, during myeloid differentiation and granulocyte associated cell death. First, we analyzed mRNA HK levels in human CD34+ hematopoietic progenitors cells differentiated towards granulocytes or macrophages by qPCR. Interestingly, while HK1 and HK2 levels remain stable during all stages of myeloid differentiation, HK3 mRNA levels significantly increased. The same pattern of HK mRNA expression was seen in NB4 APL and HL60 AML cell lines differentiated towards granulocytes and monocytes using all-trans retinoic acid (ATRA) and vitamin D3, respectively.
To determine a specific role for HK1-3 function in myeloid cells, HK1-3 knockdowns (KD) and knockouts (KO) in NB4 and HL60 cell lines, using shRNA or gRNAs (Cas9/CRISPR technology), respectively, were generated. NB4 HK KD AML cells were tested for their differentiation upon ATRA treatment. Knockdown of HKs generally resulted in a decreased differentiation response of about 20% as assessed by the differentiation marker CD11b. We next determined energy metabolism of HK altered KD and KO cells, relative to parental cells, using a seahorse analyzer. While lowering HK1 or HK3 levels in NB4 and HL60 AML cells did not affect glycolytic capacity at steady state, HK2 inhibition significantly reduced steady state glycolytic capacity. In contrast, knocking down or knocking out HK3 resulted in a higher sensitivity to ATRA-induced cell death during differentiation, which was coupled with higher glycolytic capacity.
Together, our findings suggest that HK2 has an important role in steady state metabolism of AML cells while HK3 appears to be a metabolic switch for cell survival during myeloid differentiation.

Disclosures
No relevant conflicts of interest to declare.