Glucose Metabolism as a Potential Therapeutic Target in Cytarabine-Resistant Acute Myeloid Leukemia

Abstract

Altered glycolytic metabolism has been associated with chemoresistance in acute myeloid leukemia (AML). However, there are still aspects that need clarification, as well as how to explore these metabolic alterations in therapy. In the present study, we aimed to elucidate the role of glucose metabolism in the acquired resistance of AML cells to cytarabine (Ara-C) and to explore it as a therapeutic target. Resistance was induced by stepwise exposure of AML cells to increasing concentrations of Ara-C. Ara-C-resistant cells were characterized for their growth capacity, genetic alterations, metabolic profile, and sensitivity to different metabolic inhibitors. Ara-C-resistant AML cell lines, KG-1 Ara-R, and MOLM13 Ara-R presented different metabolic profiles. KG-1 Ara-R cells exhibited a more pronounced glycolytic phenotype than parental cells, with a weaker acute response to 3-bromopyruvate (3-BP) but higher sensitivity after 48 h. KG-1 Ara-R cells also display increased respiration rates and are more sensitive to phenformin than parental cells. On the other hand, MOLM13 Ara-R cells display a glucose metabolism profile similar to parental cells, as well as sensitivity to glycolytic inhibitors. These results indicate that acquired resistance to Ara-C in AML may involve metabolic adaptations, which can be explored therapeutically in the AML patient setting who developed resistance to therapy.

Keywords: 3-bromopyruvate; acute myeloid leukemia; chemoresistance; cytarabine; glucose metabolism; metabolic inhibitors; phenformin; seahorse.

Figure 1

Viability of AML cell lines in response to chemotherapy. Dose-response curves and IC₅₀ values for HL-60, NB-4, MOLM13, and KG-1 cell lines treated with (A) cytarabine (Ara-C) and (B) daunorubicin (DNR). Values are expressed as cell viability relative to vehicle-treated cells normalized to 100%. Values are given as mean ± SD. Two-way ANOVA followed by Sidak’s Multiple Comparison Test: *, p ≤ 0.05; ## p ≤ 0.01; ***, ### p ≤ 0.001. Comparing all cell lines with the KG-1 (*) or the MOLM13 (#) cell line. Results are from at least three independent experiments with two replicates each.

Figure 2

Dose–response curves and determination of the IC₅₀ values of cytarabine (Ara-C) in (A) KG-1 and (B) MOLM13 parental and resistant cell lines. Values are expressed as cell viability relative to vehicle-treated cells normalized to 100%. Values are given as mean ± SD. Two-way ANOVA followed by Sidak’s Multiple Comparison Test: *** p ≤ 0.001. Results are from at least three independent experiments with two replicates each.

Figure 3

Cell growth rates and doubling times of AML Ara-R resistant and parental cell lines. Cell growth curves of (A) KG-1 and KG-1 Ara-R and (B) MOLM13 and MOLM13 Ara-R were analyzed over multiple population doublings. 2.5 × 10⁴ cells were plated, and cells were counted every 24 h using Trypan blue dye. (C) Values of growth rates (µ) and doubling times (Td) were calculated from the respective line equations (Figure S2). Statistical significance was determined by two-way ANOVA followed by Sidak’s Multiple Comparison Test. * p ≤ 0.05; *** p ≤ 0.001. At least three independent experiments with three replicates were performed.

Figure 4

Lactate secretion and glucose consumption in parental and Ara-R cell lines. (A) Levels of lactate secretion and (B) extracellular glucose were evaluated at 4 h for KG-1, KG-1 Ara-R, MOLM13, and MOLM13 Ara-R cells. (C) Glucose consumption corresponds to the difference in glucose concentration between 0 h and 4 h of incubation (Figure S6). Results are presented as mean ± SD of at least three independent experiments. Statistical significance estimated by two-way ANOVA followed by Sidak’s Multiple Comparison Test.

Figure 5

Characterization of the glycolytic and respiratory profile in parental and Ara-R cell lines. Results of the Glycolytic rate (A) and Mito Stress (E) test in KG-1, KG-1 Ara-R, MOLM13, and MOLM13 Ara-R cells are presented as real-time measurements of glycolytic proton efflux rate (glycoPER) and oxygen consumption rate (OCR) normalized to cell number, respectively. (B) Basal glycolysis, (C) maximal glycolysis, (D) glycolytic reverse in %, (F) basal respiration, (G) maximal respiration, (H) spare respiratory capacity in %, (I) ratio of respiration to glycolysis, (J) ATP-linked respiration, (K) proton leak-linked respiration, (L) coupling efficiency in %. Values are given as mean ± SD. One-way ANOVA followed by Sidak’s Multiple Comparison Test. At least three independent measurements with 2 to 8 replicates were performed for each cell line. Treatments: 2.5 µM Oligomycin (Oligo.); 0.5 µM fluoro-carbonyl cyanide phenylhydrazone (FCCP); 0.5 µM Rotenone and Antimycin A (Rot/AA); 50 mM 2-Deoxy-d-glucose (2DG).

Figure 6

Effect of 3-bromopyruvate (3-BP) on glycolysis and respiration in parental and Ara-R cell lines. Results of the Glycolytic rate (A,I) and Mito Stress (E,M) test in (A–H)) KG-1, KG-1 Ara-R, (I–P) MOLM13, and MOLM13 Ara-R cells are presented as real-time measurements of glycolytic proton efflux rate (glycoPER) and oxygen consumption rate (OCR) normalized to cell number. (B,J) acute response of glycolysis to 3-BP, (C,K) glycolytic reserve in %, (D,L) ratio of respiration to glycolysis. (F,N) acute response of respiration to 3-BP, (G,O) spare respiratory capacity in %, (H,P) ATP-linked respiration. Values were given as mean ± SD. One-way ANOVA followed by Sidak’s Multiple Comparison Test. Two independent measurements with 2 to 6 replicates were performed for each cell line. Treatments: 2.5 µM Oligomycin (Oligo.); 0.5 µM fluorocarbonyl cyanide phenylhydrazone (FCCP); 0.5 µM Rotenone and Antimycin A (Rot/AA); 50 mM 2-Deoxy-d-glucose (2DG).

Figure 7

Effect of 3-bromopyruvate (3-BP) and phenformin on Ara-C-resistant and parental cell viability. Dose-response curve to generate IC₅₀ values of (A,C) KG-1 and KG-1 Ara-R cell lines and (B,D) MOLM13 and MOLM13 Ara-R cell lines in response to (A,B) 3-BP, and (C,D) phenformin. Values are expressed as cell viability relative to vehicle-treated cells normalized to 100%. Values are given as mean ± SD. Two-way ANOVA followed by Sidak’s Multiple Comparison Test. *** p < 0.001. At least three independent experiments with two replicates were performed.