Metabolomic profiling of drug responses in acute myeloid leukaemia cell lines

Abstract

Combined bezafibrate (BEZ) and medroxyprogesterone acetate (MPA) exert unexpected antileukaemic activities against acute myeloid leukaemia (AML) and these activities are associated with the generation of reactive oxygen species (ROS) within the tumor cells. Although the generation of ROS by these drugs is supported by preceding studies including our own, the interrelationship between the cellular effects of the drugs and ROS generation is not well understood. Here we report the use of NMR metabolomic profiling to further study the effect of BEZ and MPA on three AML cell lines and to shed light on the underlying mechanism of action. For this we focused on drug effects induced during the initial 24 hours of treatment prior to the onset of overt cellular responses and examined these in the context of basal differences in metabolic profiles between the cell lines. Despite their ultimately profound cellular effects, the early changes in metabolic profiles engendered by these drugs were less pronounced than the constitutive metabolic differences between cell types. Nonetheless, drug treatments engendered common metabolic changes, most markedly in the response to the combination of BEZ and MPA. These responses included changes to TCA cycle intermediates consistent with recently identified chemical actions of ROS. Notable amongst these was the conversion of alpha-ketoglutarate to succinate which was recapitulated by the treatment of cell extracts with exogenous hydrogen peroxide. These findings indicate that the actions of combined BEZ and MPA against AML cells are indeed mediated downstream of the generation of ROS rather than some hitherto unsuspected mechanism. Moreover, our findings demonstrate that metabolite profiles represent highly sensitive markers for genomic differences between cells and their responses to external stimuli. This opens new perspectives to use metabolic profiling as a tool to study the rational redeployment of drugs in new disease settings.

Figure 1. Phenotypic and metabolic differences among AML cell lines (K562, HL-60 and KG1a).

(a) Cell morphology (Jenner-Giemsa stained cytospins) and fluorescence flow cytometry analysis for apoptosis (Annexin V and propidium iodide double staining) and differentiation (expression of the erythroid antigen Glycophorin A in K562 and the myeloid differentiation antigen CD11b in HL-60 and KG1a cells; sample results are compared to an isotype, grey-shaded); typical features of healthy K562, KG1a and HL-60 cells are observed (b) ¹H-NMR spectra (expanded between 0.5–4.5 ppm and between 1.95–2.2 ppm in the small insert) collected on AML extracts of HL-60, KG1a and K562 cells (12 replicates per cell line) and (c) scores plot (PC1 vs PC2) obtained from the PCA of the NMR spectra of 12 datasets per cell line.

Figure 2. Effect of treatment with MPA, BEZ and the combination of both agents on AML cell lines (K562, HL60 and KG1a).

(a) Cell morphology and flow cytometry analysis for apoptosis and differentiation are shown in K562, HL-60 and KG1a cells after 24 hrs and 96 hrs of treatment with the combination of MPA and BEZ. In these AML cell lines, neither apoptosis nor differentiation signatures are observed after a 24 hrs treatment with MPA and BEZ in combination. Prominent features of apoptosis and differentiation develop after 96 hours of treatment. (b) PCA scores plot obtained from the analysis of the NMR spectra of all the AML cell lines after 24 hrs of treatment. Solvent control (red), MPA (blue), BEZ (black), and MPA+BEZ (green) treatments are included, with 12 replicates per treatment per cell line.

Figure 3. Principal component analysis (PCA) scores plots (PC1 vs PC2) including the solvent control (red symbols) and the 3 drug treatments (MPA: blue, BEZ: black, and MPA+BEZ: green symbols) for K562, HL-60, and KG1a cell lines.

The metabolic profile of all the cell lines is significantly affected by both the individual and the combined treatments with MPA and BEZ.

Figure 4. Section of the loadings plots (0.7–3.3 ppm) obtained from the pairwise PCA of solvent control versus MPA (blue line), BEZ (red line) and MPA+BEZ (green line) for K562, HL-60, and KG1a cells.

Figure 5. Schematic representation of the metabolic pathways showing the most relevant metabolic changes induced by MPA+BEZ drug treatment for the individual AML cell lines (HL-60 and KG1a).

Metabolites in green/red have increased/decreased concentrations upon combined MPA+BEZ treatment.

Figure 6. Section of ¹H-NMR spectra (expanded between 2.3–2.67 ppm) of a de-proteinized extract of untreated KG1a cells (blue line) and after treatment with 0.2 mM (red line) and 0.8 mM (green line) H₂O₂.