PS1014 GLYCOLYSIS VERSUS OXIDATIVE PHOSPHORYLATION – POTENTIAL THERAPEUTIC TARGETS IN AML
Authors: Lapa B. , Jorge J. , Alves R.S. , Pires A.S. , Abrantes A.M. , Coucelo M. , Abrunhosa A. , Botelho M.F. , Ribeiro M.L. , Nascimento‐Costa J.M. , Gonçalves A. , Sarmento‐Ribeiro A.B. 
Published: June 2019

Background:

Cancer cells alter their metabolism to sustain high levels of growth and proliferation. One of the most common alterations is the metabolic shift of cancer cells from oxidative phosphorylation (OXPHOS) to glycolysis, regardless of oxygen presence (aerobic glycolysis), resulting in lower ATP production. This alteration is associated with up‐regulation of glucose transporters (GLUTs) and glycolytic enzymes, such as hexokinases, and enhanced lactate fermentation. This shift is mainly regulated by HIF‐1, p53 and MYC. Hence, metabolism constitute a new target in acute myeloid leukemia (AML), where mutations in metabolism [isocitrate dehydrogenase 1/2 (IDH1/2)] were already describe. AML is a heterogeneous hematologic neoplasia characterized by myeloid differentiation arrest and uncontrolled proliferation.

Aims:

This study aimed to metabolically characterize AML cell lines and assess the potential of glycolysis and OXPHOS metabolism as a therapeutic target in AML using in vitro models.

Methods:

Six AML cell lines of different subtypes and with different genetic backgrounds were used (HEL, HL‐60, K‐562, KG‐1, NB‐4 and THP‐1). The presence of IDH1/2 exon 4 mutations was assessed using Sanger sequencing. Intracellular peroxides, superoxide anion and mitochondrial membrane potential levels were measured by fluorimetry using DCFH2‐DA, DHE and JC‐1 probes, respectively. Cells were incubated in presence or absence of 2‐deoxy‐D‐glucose (2‐DG; glycolysis inhibitor) or oligomycin complex (OXPHOS inhibitor). Metabolic activity was assessed by resazurin assay and cell death by flow cytometry (FC) using Annexin V and 7‐AAD double staining. Cell cycle was also assessed by FC, using propidium iodide/RNase. Glucose intake was measured by 18F‐FDG uptake and expression levels of 8 SLC2A, 2 hexokinases and HIF‐1α were assessed using RT‐PCR.

Results:

NB‐4 cell line has the highest 18F‐FDG uptake, whereas HL‐60 and THP‐1 cells showed the lowest. HEL, K‐562 and KG‐1 had similar 18F‐FDG uptake. THP‐1 cells expressed the 8 tested SLC2A genes. NB‐4 cell line also had the highest peroxide and superoxide anion levels. K‐562 and KG‐1 cells had similar ROS levels and IDH1 genotype (single nucleotide variant rs11554137 in heterozygosity). Glycolysis inhibition reduced metabolic activity in a dose‐ and cell line dependent manner and HEL and KG‐1 were the most sensitive cell lines to 2‐DG. OXPHOS inhibition led to a decrease in metabolic activity in a cell line‐dependent manner. K‐562 cells were the most sensitive to this inhibition and THP‐1 cells were the most resistant to both compounds (2‐DG and oligomycim). These inhibitors induce cell death by apoptosis and cell cycle arrest in G0/G1 phase. 2‐DG induced a more evident cytotoxic effect on K‐562 and NB‐4 and a cytostatic effect on KG‐1 and THP‐1 cells. Conversely, oligomycin had a cytotoxic effect on K‐562 and KG‐1 cells and a cytostatic effect on NB‐4, THP‐1 and K 562. Moreover, 2‐DG decreased 18F‐FDG uptake, mainly in KG‐1 and NB‐4 cell lines, inducing up‐regulation of SLC2A11 and down‐regulation of HK1 gene expression. Lastly, oligomycin increased 18F‐FDG uptake, particularly in K‐562 cells, with up‐regulation of SLC2A1 and HK2 gene expression.

Summary/Conclusion:

AML cell lines have different metabolic preferences, with HEL, KG‐1 and NB‐4 cells appearing to be more glycolytic, whereas K‐562 cells more dependent on OXPHOS. Contrary, THP‐1 cells seems to be OXPHOS independent. These cell lines seem to reprogram their metabolism in order to overcome OXPHOS inhibition, suggesting that glycolysis could be a better therapeutic target in AML.