High Metabolic Dependence on Oxidative Phosphorylation Drives Sensitivity to Metformin Treatment in MLL/AF9 Acute Myeloid Leukemia

Abstract

Acute myeloid leukemia (AML) is a group of hematological cancers with metabolic heterogeneity. Oxidative phosphorylation (OXPHOS) has been reported to play an important role in the function of leukemic stem cells and chemotherapy-resistant cells and are associated with inferior prognosis in AML patients. However, the relationship between metabolic phenotype and genetic mutations are yet to be explored. In the present study, we demonstrate that AML cell lines have high metabolic heterogeneity, and AML cells with MLL/AF9 have upregulated mitochondrial activity and mainly depend on OXPHOS for energy production. Furthermore, we show that metformin repressed the proliferation of MLL/AF9 AML cells by inhibiting mitochondrial respiration. Together, this study demonstrates that AML cells with an MLL/AF9 genotype have a high dependency on OXPHOS and could be therapeutically targeted by metformin.

Keywords: MLL/AF9; OXPHOS; heterogeneity; metformin.

Figure 1

Metabolic pathway dependence of human AML cell lines. (A) Mitochondrial respiration and glycolysis were determined in various AML cell lines by Seahorse XFe96 Extracellular Flux Analyzer. Basal and maximum oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured, and metabolic pathway dependence was obtained as the percent ratio of OCR and ECAR. Results are expressed as mean ± standard deviation, n = 3. (B) AML cell lines used in the experiment and their major genetic variations. Bold letters are a common mutation of THP1 and MOLM13 (*** p < 0.001).

Figure 2

Metabolic phenotype of MLL/AF9 AML cells. Schematic outline of results obtained with the ex vivo (A) and in vivo (D) murine MLL/AF9 AML model. The experimental details are described in Materials and Methods. Seahorse Mito Stress Test and Glycolysis Stress Test were performed in murine HPCs transduced with empty vector (EV) or MLL/AF9 vector (B), and c-kit+/GFP+ blast cells from MLL/AF9 AML mice or c-kit+ HPCs from control mice (E). Mitochondrial respiration, glycolysis and metabolic pathway dependence were calculated accordingly in murine HPCs transduced with EV or MLL/AF9 vector (C), and in c-kit+/GFP+ blast cells from MLL/AF9 AML mice or c-kit+ HPCs from control mice (F). (G) Mitochondrial membrane potential (MMP), mitochondrial number (mito mass) and MMP/mito mass ratio were determined by flow cytometry in c-kit+/GFP+ blast cells from MLL/AF9 AML mice or c-kit+ HPCs from control mice. n = 4 mice; three independent experiments. (H) Mitochondria number was determined by mtDNA in K562TRBSR and murine HPC transduced with EV or MLL/AF9. (I) NRF1 and PGC1α expression levels were determined by western blot in K562TRBSR and murine HPC transduced with EV or MLL/AF9; β-actin expression served as a loading control (original western blots can be found at Supplementary File S1). All data are expressed as the mean ± standard deviation. * p < 0.05; ** p < 0.01; *** p < 0.001 (Student’s t-test).

Figure 3

Evaluation of metformin-induced effects in MLL/AF9 AML. (A) The effect of metformin on the proliferation of various AML cell lines was determined after treating with 5 mM metformin for 72 h with or without Ara-C. Murine HPCs (B) or K562TRBSR cells (C) were transduced with EV or MLL/AF9 vector, and the proliferation rates were evaluated after 72 h treatment with metformin. (D) c-kit+/GFP+ blast cells from MLL/AF9 AML mice or c-kit+ HPCs from control mice were treated with metformin for 72 h, and proliferation were determined. (n = 3 mice; three independent experiments). Murine HPCs (E) or K562TRBSR cells (F) transduced with EV or MLL/AF9 vector were treated with rotenone for 48 h, and cell numbers were determined. (G) Murine HPCs transduced with EV or MLL/AF9 gene were treated with metformin for 24 h, and the effect of metformin on mitochondrial respiration and glycolysis was measured by the Seahorse Flux analyzer. All data are expressed as the mean ± standard deviation. * p < 0.01; ** p < 0.01; *** p < 0.001 (Student’s t-test).