Suppressed Expression of ASCT1 Contributes to the Maintenance of Leukemia Stemness Via the Enhancement of Antioxidant Capacity

Abstract

Introduction: Acute myeloid leukemia (AML) is one of the most common hematological malignancies derived from self-renewing leukemic stem cells (LSCs). LSCs represent a small cell fraction harboring the self-renewal capacity and potential to give rise to mature leukemic cells and play a central role in the relapse of AML. In general, LSCs can be enriched within reactive oxygen species (ROS) low fraction. Glutathione (GSH), a major antioxidant small molecule, plays a central role in the maintenance of lower ROS levels; however, the molecular mechanisms of GSH maintenance in LSCs remain unclear. Result: First, we evaluated the intracellular GSH levels in the CD34+ immature fraction, in which LSCs can be enriched and CD34- mature AML cells from the identical patients (n=#). CD34+AML cells contained significantly higher levels of GSH. 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 cysteine/cystine transporters. The expression levels of ASC1 and xCT were not significantly different among the three cellular fractions of AML: the most immature CD34+CD38-, CD34+CD38+, and more differentiated CD34- cells. However, we found that CD34+CD38- LSCs completely lacked the expression of ASCT1 in all the cases examined (n=#). The expression of ASCT1 increased along with cellular differentiation; more differentiated CD34- AML cells expressed the higher ASCT1 expression than the most immature CD34+CD38- cells. We, therefore, hypothesized that the different expression pattern of ASCT1 might be associated with higher GSH levels in LSCs through the regulation of cellular cysteine levels. ASCT1 is known to transport neutral amino acids (alanine, serine, cysteine, threonine), no role for ASCT1 in leukemia has ever been investigated. We found that ASCT1 was highly expressed in M4/M5 monocytic leukemias according to the FAB classification. Analysis of beatAML RNA-seq data showed that patients with low ASCT1 expression had a worse prognosis. We found that primary AML cells with the higher ASCT1 expression had reduced intracellular GSH as compared to those with lower ASCT1 expression (n=#), suggesting the association of ASCT1 expression and GSH levels in primary AML cells. To test this hypothesis, we knocked down (KD) the expression of ASCT1 using shRNAs in cell lines. KD of ASCT1 induced an increase in intracellular GSH compared to control cells, suggesting that ASCT1 decreases intracellular GSH by transporting cysteine extracellularly. To clarify the function of ASCT1 in human AML-LSCs in vivo, we performed xenotransplantation experiments. The identical number of FACS-purified ASCT1+ and ASCT- AML cells from the identical patients were transplanted into the irradiated NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. We found that ASCT1- AML cells efficiently reconstituted human AML in vivo, whereas ASCT1+ AML cells completely failed in the engraftment in vivo in all the cases tested, suggesting that LSCs exclusively reside within ASCT1- AML cellular fraction. These results collectively suggest that LSCs maintain low ASCT1 expression, which is one of the molecular machineries to keep intracellular GSH high. Furthermore, only the ASCT1- fraction reconstitute AML in immunodeficient mice, indicating that suppression of ASCT1 is important for the maintenance of leukemia stemness.