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. 2021 Feb 5:10:621458.
doi: 10.3389/fonc.2020.621458. eCollection 2020.

Targeting Lactate Metabolism by Inhibiting MCT1 or MCT4 Impairs Leukemic Cell Proliferation, Induces Two Different Related Death-Pathways and Increases Chemotherapeutic Sensitivity of Acute Myeloid Leukemia Cells

Affiliations

Targeting Lactate Metabolism by Inhibiting MCT1 or MCT4 Impairs Leukemic Cell Proliferation, Induces Two Different Related Death-Pathways and Increases Chemotherapeutic Sensitivity of Acute Myeloid Leukemia Cells

Ernestina Saulle et al. Front Oncol. .

Abstract

Metabolism in acute myeloid leukemia (AML) cells is dependent primarily on oxidative phosphorylation. However, in order to sustain their high proliferation rate and metabolic demand, leukemic blasts use a number of metabolic strategies, including glycolytic metabolism. Understanding whether monocarboxylate transporters MCT1 and MCT4, which remove the excess of lactate produced by cancer cells, represent new hematological targets, and whether their respective inhibitors, AR-C155858 and syrosingopine, can be useful in leukemia therapy, may reveal a novel treatment strategy for patients with AML. We analyzed MCT1 and MCT4 expression and function in hematopoietic progenitor cells from healthy cord blood, in several leukemic cell lines and in primary leukemic blasts from patients with AML, and investigated the effects of AR-C155858 and syrosingopine, used alone or in combination with arabinosylcytosine, on leukemic cell proliferation. We found an inverse correlation between MCT1 and MCT4 expression levels in leukemic cells, and showed that MCT4 overexpression is associated with poor prognosis in AML patients. We also found that AR-C155858 and syrosingopine inhibit leukemic cell proliferation by activating two different cell-death related pathways, i.e., necrosis for AR-C155858 treatment and autophagy for syrosingopine, and showed that AR-C155858 and syrosingopine exert an anti-proliferative effect, additive to chemotherapy, by enhancing leukemic cells sensitivity to chemotherapeutic agents. Altogether, our study shows that inhibition of MCT1 or MCT4 impairs leukemic cell proliferation, suggesting that targeting lactate metabolism may be a new therapeutic strategy for AML, and points to MCT4 as a potential therapeutic target in AML patients and to syrosingopine as a new anti-proliferative drug and inducer of autophagy to be used in combination with conventional chemotherapeutic agents in AML treatment.

Keywords: AR-C155858; MCT1; MCT4; acute myeloid leukemia; autophagy; lactate metabolism; syrosingopine.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
MCT1 and MCT4 mRNA are inversely expressed during Mo and G differentiation of CD34+ HPCs. (A, B) qRT-PCR analysis of MCT1 and MCT4 mRNA expression during selective G and Mo proliferation and differentiation of CD34+ HPCs, as compared to NB4 and U937 leukemic cells; AU is for arbitrary units; the results of three independent experiments (mean ± SEM values) are shown; significance is *p <0.05; **p <0.01; ***p <0.001; ns is for no significant. (C) Western blot analysis of MCT1 and MCT4 protein expression during G and Mo proliferation and differentiation of CD34+ HPCs. Actin is shown as an internal control; one representative western blot experiment out of three is shown.
Figure 2
Figure 2
AR-C and SYRO treatment has no major significant effect on cell growth, differentiation and cell cycle progression of G and Mo differentiating HPCs. (A) Cell growth analysis during G and Mo differentiation of HPCs, in presence of AR-C or SYRO, used at 1 µM and added every 2 days in cultures, as compared to control (c) cells. (B) Clonogenic assays performed under G and Mo culture conditions with AR-C- or SYRO- treated HPCs, as compared to untreated G- and Mo- differentiating HPCs of control (c). (A, B) The results of three independent experiments (mean ± SEM values) are shown; significance is *p <0.05; ns is for no significant. (C) Morphological analysis at day 21 of the differentiation and maturation of G and Mo differentiating HPCs treated with AR-C or SYRO, as compared to control (c) HPCs, and stained with May-Grünwald-Giemsa. One representative experiment out of three is shown.
Figure 3
Figure 3
Overexpressed in AML, as compared to normal CD34+ HPCs, MCT1 and MCT4 mRNA expression is inversely correlated in AMLs. (A, B) qRT-PCR analysis of MCT1 and MCT4 mRNA expression in primary leukemic cells of AMLs pertaining from M0 to M5 subtypes of FAB classification, as compared to normal CD34+ HPCs. (A, B) The results of three independent experiments (mean ± SEM values) are shown; significance *, **, and *** are p <0.05, p <0.01, and p <0.001, respectively, ns is for no significant. (C, D) MCT1 and MCT4 mRNA expression data analysis from AML samples generated by TCGA Research Network. (E, F) Inverse correlation between MCT1 and MCT4 mRNA expression levels in AMLs, according data from: (E) our AML samples; (F) AML samples generated by TCGA Network. (A, B, E) AU is for arbitrary units. (C, D, F) RPKM is for Reads Per Kilobase of exon per Million mapped reads.
Figure 4
Figure 4
High expression of MCT4 is correlated with poor overall survival in AML patients, but not MCT1 expression. (A) Kaplan-Meier survival analysis in AMLs patients, based on MCT4 gene expression and stratified according to their MCT4 mRNA levels in two groups of patients : Low MCT4 (MCT4 ≤ -0.07 RPKM) and high MCT4 (MCT4 ≥ -0.07 RPKM), indicates that the kinetic of death is more rapid (within 80 months) among MCT4 high-patients, as compared with those with low MCT4 levels (p = 0.021). (B) Kaplan-Meier survival analysis in AMLs patients, based on MCT1 gene expression and stratified according to their MCT1 mRNA levels in two groups: Low (MCT1 ≤ -0.2 RPKM); High (MCT1 ≥ -0.2 RPKM), indicates that the kinetic of death is similar among MCT1 high-patients, as compared with those with low (p = 0.9422) MCT1 levels. (A, B) P-value is calculated by using the log-rank test. (A, B) RPKM is for Reads Per Kilobase of exon per Million mapped reads. (C) White blood count (WBC) number, at age diagnosis and proportion of patients with poor cytogenetics were comparable in both AML subgroups subdivided according to MCT4 expression level, ns is for no significant. (D) MCT4 mRNA levels were analyzed in AMLs stratified into three risk groups, poor, intermediate, and good/favorable, according to the European LeukemiaNet (ELN) risk classification. Significance *** is p <0.001. (E) Relationship between the most recurrent gene mutations observed in all AMLs and the level of MCT4 mRNA expression, according to TCGA dataset.
Figure 5
Figure 5
Dose response analysis of SYRO and AR-C treatment of U937 and MV4-11 cells. (A) Lactate Glo assays were performed to analyze intracellular and extracellular lactate levels in U937 and MV4-11 leukemic cells treated 2 h by SYRO, as compared to untreated cells of control (0). (B) Lactate Glo assays were performed to analyze intracellular and extracellular lactate levels in U937 and MV4-11 cells treated 2 h by AR-C, as compared to untreated cells of control (0). (A, B) The results of three independent experiments (mean ± SEM values) are shown; significance is *p <0.05; **p <0.01; ***p <0.001; ns is for not significant.
Figure 6
Figure 6
AR-C and SYRO are selective inhibitors of lactate metabolism in leukemic cells. (A, B) Intracellular/extracellular metabolome analysis by NMR spectroscopy in U937 and MV4-11 cells treated 24 h with AR-C and SYRO used at 5 µM, normalized to untreated cells. Fold increase of intracellular aqueous and lipid metabolites in SYRO- and AR-C-treated U937 (n = 3) and MV4-11 cells (n = 2) relative to untreated cells (reference value = 1) is shown. Lipid metabolites: DAG, Diacylglycerols; FA, Fat acids; MUFA, Monounsaturated FA; PC, Phosphatidylcholine; LysoPC, Lyso-phosphosphatidylcholine; PUFA, Polyunsaturated FA. Aqueous metabolites: AXP, (AMP+ADP+ATP); Glx, Glutamic acid + Glutamine; tCho, total choline = (free choline + glycerophosphocholine + phosphocholine); UDP-GLC-NAC, Uridine diphosphate N-acetylglucosamine; UXP, (UMP+UDP+UTP). (C, D) Lactate Glo assays were performed to analyze intra and extracellular lactate levels in U937 and MV4-11 cells treated 2 h with 10 µM of AR-C or SYRO, as compared to U937 and MV4-11 leukemic cells treated 2 h with AC-73 (10 µM) and to untreated cells of control (c). The results of three independent experiments (mean ± SEM values) are shown; significance * and ** are p <0.05 and p <0.01, respectively; ns is for not significant.
Figure 7
Figure 7
Effects of AR-C and SYRO on cell growth and viability of AML cell lines. (A, B) Dose response analysis of AR-C (A) and SYRO (B) treatment on U937 and MV4-11 leukemic cell growth, as compared to control cells (c). (C) Cell viability assays on leukemic cells treated: (left panel) with AR-C used at different concentrations for 3 days; (right panel) at different times with 1 µM AR-C, as compared to control cells (day 0). (D) Cell viability assays on leukemic cells treated: (left panel) with SYRO used at different concentrations for 3 days; (right panel) at different times with 1 µM SYRO, as compared to control cells (day 0). (C, D) Viability is presented as percentage viable cell relative to control. (A–D) The results of three independent experiments (mean ± SEM values) are shown; significance is *p <0.05; **p <0.01; ***p <0.001; ns is for not significant.
Figure 8
Figure 8
Effects of AR-C and SYRO on apoptosis and on the related cell-death pathways, necrosis, and autophagy. (A) Dose response analysis of AR-C treatment performed for 3 days on leukemic cell apoptosis, as compared to control (c) leukemic cell. (B) Western blot analysis of the necrosis related HMGB1 protein in U937 cells treated with AR-C used at different concentrations (left panel) and used at 1 µM in AML cell lines (right panel), as compared to untreated cells of control (–) cells. Quantification of HMGB1 proteins by densitometry analysis is indicated (AU is for arbitrary units). (C) Dose response analysis of SYRO treatment performed for 3 days on leukemic cell apoptosis, as compared to control (c) leukemic cells. (D) Western blot analysis of the autophagy related protein LC3 and its conversion from LC3-I to LC3-II form in U937 cells treated with SYRO used at different concentrations, and in MV4-11 cells treated with 1 µM SYRO, as compared to untreated leukemic cells (–). (E) Dose response analysis of SYRO treatment on the autophagy flux in U937 leukemic cells, as compared to control cells (c). (F) Induction of autophagy by SYRO treatment used at 1 µM for 3 days in several AML cell lines, as compared to control cells (c). (A, C) The results of three independent experiments (mean ± SEM values) are shown; significance is **p <0.01; ***p <0.001; NS is for not significant. Total apoptosis by annexin V/PI (%) detected by using flow cytometric apoptotic assays, is indicated. (B, D left panel) One representative western blot experiment out of three is shown; Actin is an internal control. (E, F) One representative experiment out of three is shown.
Figure 9
Figure 9
AR-C and SYRO by inhibiting lactate metabolism, decrease cell viability and potentiated cytotoxicity of Ara-C in AML cells. (A) Cell viability assays on U937 and MV4-11 leukemic cells, treated 24 h with AR-C used alone (1 µM), then in combination with Ara-C (AR-C (1µM) + Ara-C), as compared to treatment with single drug, AR-C (AR-C + 0.00 µM Ara-C). (B) Cell viability assays on U937 and MV4-11 leukemic cells, treated 24 h with SYRO used alone (1 µM), then in combination with Ara-C (SYRO (1µM) + Ara-C), as compared to treatment with single drug, SYRO (SYRO + 0.00 µM Ara-C). (C) Cell viability assays were performed on leukemic blasts obtained from 2 AML patients LEUK1 and LEUK2, treated in vitro by single drug AC-73, AR-C or SYRO used at 2.5 µM, as compared to control (c) leukemic blasts. (D) Cell viability assays performed on leukemic blasts of one AML patient LEUK 3, treated in vitro by AR-C or SYRO used at low dosage (1 µM) in combination with Ara-C (1µM), as compared to single treatment with AR-C, SYRO and Ara-C (1 µM) and to control (c) leukemic blasts. (A–D) The results of three independent experiments (mean ± SEM values) are shown; significance is *p <0.05; **p <0.01; ***p <0.001; ns is for no significant.

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