Relationship between mTOR-Mediated Upregulation of Glycolysis and Chemosensitivity of ALL Blasts.

Abstract

Cancer cells exhibit a high rate of glycolysis, which leads to a stringent dependence on extracellular glucose for maintaining intracellular ATP levels. Leukemic cells infiltrating bone marrow have been shown to be markedly hypoxic compared to normal bone marrow cells, and stimulation of glycolysis is thought to be an adaptive mechanism to hypoxia. The serine/threonine kinase AKT promotes continued cell growth and metabolism by increasing glucose uptake, stimulating glycolysis and ATP production, at least in part via mTOR-dependent stabilization of HIF1-α. The purpose of this study was to investigate the role of mTOR signaling in the regulation of glycolytic activity of leukemic cells growing under high glucose or hypoxic conditions. Culture of Jurkat cells or primary ALL lymphoblasts in hypoxia (1% O2) resulted in robust induction of HIF-1α protein level which was significantly higher in high glucose conditions. This induction was blocked by inhibition of mTOR signaling, in association with decreased HK-2 mRNA and Glut-1 mRNA/protein expression. Likewise, hypoxia induced lactic acid production, a surrogate marker of glycolysis, and this induction was prevented by RAD001 (p=0.01). This data suggest that PI3K/AKT/mTOR pathway critically controls HIF-1α-dependent upregulation of glucose transport and glycolysis under in leukemic cells growing under pro-glycolytic conditions. We next examined the relationship between enhanced glycolytic activity and chemosensitivity to Doxorubicin. Doxorubicin (25ng/ml) efficiently inhibited growth of Jurkat cells under normoxic conditions supplemented with 4 mM glucose (equivalent of 72mg/dl). In contrast, Doxorubicin was approximately 50% less effective when cells were cultured either in high (14mM, equivalent of 252mg/dl) glucose or under hypoxia with low- or high-glucose supplementation. Inhibition of mTOR signaling strikingly enhanced effects of Doxorubicin resulting in complete inhibition of growth, and this effect was apparent only under high glucose conditions. We next examined effects of RAD001 in samples from 11 patients' samples from newly diagnosed B-ALL patients growing under high/low glucose in hypoxic or normoxic conditions. RAD001 alone did not induce apoptosis in ALL cells (p>0.05). In contrast, inhibition of mTOR signaling significantly enhanced Doxorubicin-induced apoptosis in 8 samples, with highest sensitization observed in high glucose environment (specific apoptosis, Doxorubicin alone, 5.6%, RAD001 7.7%, RAD001+Doxorubicin 22.4%, p<0.02). In conclusion, blockade of mTOR-dependent HIF-1α and its downstream target Glut-1 efficiently promotes chemosensitivity of ALL cells under pro-glycolytic conditions. Hence, mTOR inhibition or blockade of HIF-1α-mediated glycolysis may play an important role in chemosensitization and improved outcomes in ALL.