Identification of ASCT1 As a Candidate Molecule Enhancing Antioxidant Activity in Primary Human AML Cells

Introduction:

Recent studies have shown that the specific alteration of metabolic pathways are involved in the regulation of function of normal hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs) in acute myeloid leukemia (AML). However, little is known about the features of metabolic activity in human HSCs and LSCs. To reveal the metabolic pathway alterations in primary AML cells, we performed the comprehensive metabolome analysis by comparing normal human CD34+ hematopoietic stem/progenitor cells (HSPCs)(n=5) and CD34+ primitive AML cells containing LSCs (n=16) using highly sensitive CE-tandem mass spectrometry.

Method:

Metabolome analysis

Metabolites were extracted from primitive CD34+ AML cells (n=16) and normal CD34+ bone marrow cells (n=4) and cord blood cells (n=1). Metabolome analysis was conducted by the C-SCOPE package of HMT using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) for cation analysis, and capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) for anion analysis. 116 metabolites were targeted for analysis in this study.

Oxygen consumption rates and extracellular acidification rate

O₂ consumption rates (OCR) and extracellular acidification rate (ECAR) were measured by the Seahorse XF96 extracellular flux analyzer. Three replicate wells of 400,000 leukemic or normal cells per well were seeded in 96-well XF96 well plates coated with BD Cell-Tak (BD Biosciences) in serum-free unbuffered DMEM. Analyses were performed both at basal conditions and after injection of OLI (1 mg/ml), FCCP (1 mM), Antimycin A (5 mM).

Result:

We detected 101 metabolites involved in central carbon and energy metabolism. In glucose metabolism, the level of lactate, an end-product of aerobic glycolysis, were lower in CD34+ AML cells than normal HSPCs, whereas the level of pyruvate, a precursor of lactate, was not different. Thus, CD34+ AML cells had a significantly high pyruvate/lactate ratio as compared to normal HSPCs, suggesting that aerobic respiration is preferentially utilized in CD34+ AML cells. To confirm this observation, we directly measured the O₂ consumption rate (OCR) and extracellular acidification rate (ECAR) of CD34+ AML cells (n=4) and normal HSPCs (n=5) by XF96 extracellular flux analyzer. OCR reflects the activity of aerobic respiration, whereas ECAR reflects lactate generation and correlates with anaerobic respiration activity. Therefore, OCR/ECAR ratio is a good index for the discrimination of aerobic and anaerobic respiration pattern. We found that the OCR/ECAR ratio of CD34+ primitive AML cells was significantly high as compared to that of HSPCs, suggesting that CD34+ AML cells predominantly utilized aerobic respiration.

Although the aerobic respiration resulted in the production of reactive oxygen species (ROS), the intracellular ROS level was not different between CD34+ AML (n=7) cells and normal HSPCs (n=3), suggesting that the antioxidant activity should be strongly enhanced in CD34+ AML cells. Consistent with the observation, we found that CD34+ AML cells had a much higher level of glutathione (GSH), a primary intracellular antioxidant, than normal HSPCs.

To clarify the molecular mechanisms how CD34+ primitive AML cells could maintain high GSH level, we analyzed the expression of cysteine transporters, because cysteine uptake is the rate-limiting step of GSH synthesis. In human, three amino acid transporters including ASCT1, ASC1 and xCT are known as cystine/cysteine transporters. Interestingly, ASCT1 was significantly highly expressed in CD34+ AML cells (n=10) as compared to normal CD34+ HSPCs (n=3). Of note, normal CD34+CD38- HSCs completely lacked ASCT1 expression, whereas CD34+CD38-LSCs expressed at a high level, indicating the possibility that the high expression of ASCT1 should be a LSC specific machinery for enhanced GSH synthesis.

Thus, human AML cells predominantly utilize aerobic respiration that is supported by a high level of GSH, and AML specific ASCT1 expression presumably contributes to the high level of GSH. These data suggest that ASCT1 should be a promising molecule to specifically target AML stem/progenitor cells.