Enhanced Expression of Glycolytic Enzymes and Succinate Dehydrogenase Complex Flavoprotein Subunit A by Mesothelin Promotes Glycolysis and Mitochondrial Respiration in Myeloblasts of Acute Myeloid Leukemia

Abstract

Acute myeloid leukemia (AML) is an aggressive malignancy characterized by rapid growth and uncontrolled proliferation of undifferentiated myeloid cells. Metabolic reprogramming is commonly observed in the bone marrow of AML patients, as leukemia cells require increased ATP supply to support disease progression. In this study, we examined the potential role of mesothelin as a metabolic modulator in myeloid cells in AML. Mesothelin is a well-known marker of solid tumors that promotes cancer cell proliferation and survival. We initially analyzed alterations in mesothelin expression in the myeloblast subpopulations, defined as SSC-Alow/CD45dim, obtained from the bone marrow of AML patients using flow cytometry. Our results showed overexpression of mesothelin in 34.8% of AML patients. Subsequently, metabolic changes in leukemia cells were evaluated by comparing the oxygen consumption rates (OCR) of bone marrow samples derived from adult AML patients. Notably, a higher OCR was observed in the mesothelin-positive compared to the mesothelin-low and non-expressing groups. Treatment with recombinant human mesothelin protein enhanced OCR and increased the mRNA expression of glycolytic enzymes and mitochondrial complex II in KG1α AML cells. Notably, siRNA targeting mesothelin in KG1α cells led to the reduction of glycolysis-related gene expression but had no effect on the mitochondrial complex gene. The collective results demonstrate that mesothelin induces metabolic changes in leukemia cells, facilitating the acquisition of a rapid supply of ATP for proliferation in AML. Therefore, the targeting of mesothelin presents a potentially promising approach to mitigating the progression of AML through the inhibition of glycolysis and mitochondrial respiration in myeloid cells.

Keywords: acute myeloid leukemia; glycolysis; mesothelin; oxygen consumption rate.

Figure 1

Expression of mesothelin in myeloblast subpopulation of AML patients at diagnosis and after induced therapy. (A) Gating strategy for AML bone marrow (2 × 10⁵ cells) based on CD45 expression. Myeloblast subsets are identified as CD45^dim/SSC-A^low. Mature lymphocyte subsets are identified as CD45^high/SSC-A^low. Histogram showing mesothelin surface expression in AML bone marrow at diagnosis (red), normal (gray area), and after treatment (blue) in the CD45^dim/SSC-A^low myeloblast subsets (Upper). Histogram showing mesothelin expression in AML and normal CD45^high/SSC-A^low subsets (Lower). (B) Percentage of mesothelin-positive cells in the CD45^dim/SSC-A^low myeloblast subsets calculated using Flowjo software (v10.9), represented as a bar graph. (C) Percentage of mesothelin-positive cells in the CD45^high/SSC-A^low population. (D) Mesothelin median fluorescence intensity (MFI) in CD45^dim/SSC-A^low myeloblast subsets. Values are presented as mean ± SD (bars) (* p < 0.05, vs. corresponding controls). (E) Paired t-test of BF and AF in mesothelin-positive cells percentage. (F) Paired t-test of BF and AF in mesothelin MFI (* p < 0.05). HC, Healthy control; BF, Before induced-therapy (IT); AF, after IT.

Figure 2

Enhanced oxygen consumption rate and extracellular acidification rate in the bone marrow of patients with mesothelin-high acute myeloid leukemia. (A) OCR of AML bone marrow (1 × 10⁵ cells/well) obtained at diagnosis, grouped by mesothelin expression levels, was measured after sequential injection of mitochondrial inhibitors, including oligomycin, CCCP, or rotenone. (B–I) Baseline OCR, ATP production, non-mitochondrial OCR, maximal respiration, spare respiratory capacity (SRC), SRC (%), coupling efficiency, and proton leak were calculated using WAVE software(v2.6). (J) ECAR was measured using the XFe96 analyzer simultaneously with OCR measurements. Values are presented as mean ± SD (bars) (* p < 0.05, ** p < 0.01 vs. corresponding controls). The black line indicates OCR or ECAR of the mesothelin-low group, while the blue line represents the mesothelin-high group.

Figure 3

Mesothelin treatment increased oxygen consumption rate and glycolysis in AML cells. (A) KG1α (2 × 10⁵ cells/well) cells incubated with different concentrations (0, 0.25, or 0.5 μg/mL) of rhMesothelin for 1 h; OCR was measured after sequential injection of mitochondrial inhibitors. (B–I) Values of OCR parameters were calculated using WAVE software, and the values are presented as mean ± SD (bars) (* p < 0.05, ** p < 0.01 vs. corresponding controls). (J) ECAR was measured with XFe96 analyzer along with OCR after mitochondrial inhibitor injection. The black line indicates OCR or ECAR of control KG1α cells, the blue line represents the 0.25 μg/mL rhMesothelin-treated group, and the red line represents the 0.5 μg/mL rhMesothelin-treated group.

Figure 4

Effects of recombinant human mesothelin treatment on glycolysis and mitochondrial complex gene in KG-1a acute myeloid leukemia cells. (A–I) KG1α cells were incubated in media containing rhMesothelin (0, 0.25, or 0.5 μg/mL) for 24 h. mRNA expression of glycolysis-related enzyme gene (A–D) and mitochondrial OXPHOS complex gene (E–I) was assessed using qPCR analysis. Values were normalized to GAPDH levels and presented as mean ± SD (bars) (* p < 0.05, ** p < 0.01 vs. corresponding controls).

Figure 5

Effects of mesothelin knockdown on the expression of glycolysis and mitochondrial complex gene mRNA in KG-1a cells and schematic representation illustrating the modulation of AML cell metabolism by mesothelin. A-J, KG1α cells were incubated in media containing siRNA targeting mesothelin for 72 h. mRNA expression level of human mesothelin (A), glycolysis-related gene (B–E), and mitochondrial OXPHOS complex gene (F–J) was determined using qPCR analysis. Values are normalized to GAPDH levels and presented as mean ± SD (bars) (* p < 0.05, ** p < 0.01 vs. corresponding controls). (K) Illustration of metabolic modulation by mesothelin in human AML. Red arrow represents upregulation.

Figure 6

Mesothelin induces mitochondrial antioxidant enzyme expression. (A−D), KG1α cells were incubated in media containing rhMesothelin (0.5 μg/mL). Cells were stained with MitoSOX^TM or H₂DCFDA, and fluorescence intensity was measured via flowcytometry. Mitochondrial ROS was determined by measuring relative cell number of MitoSOX^TM-stained cells and H₂DCFD-stained cells (A,B). Dashed line represents median value of relative cells number. (C,D) Median fluorescence intensity (MFI) values are presented as a mean ± SD (bars) (** p < 0.01 vs. corresponding controls). (E–J), mRNA expression level of human antioxidant enzyme genes was determined using qPCR analysis. Values are normalized to GAPDH level and presented as mean ± SD (bars) (* p < 0.05, ** p < 0.01 vs. corresponding controls).