Mechanisms of Sensitivity of B-Precursor, T-Lineage and BCR/ABL Positive Acute Lymphoblastic Leukemia (ALL) to the Glycolytic Inhibitor 2-Deoxy-D-Glucose (2-DG).

Abstract 3081

Poster Board III-18

Novel treatment strategies are needed for patients with ALL diagnosed with resistant phenotypes or after relapse. The glucose analogue 2-deoxy-D-glucose (2-DG) is a glycolytic inhibitor that induces growth arrest and cell death by inhibiting the key glycolytic enzymes phosphoglucose isomerase (PGI) and hexokinase (HK). Cancer cells and hypoxic cells are more sensitive to 2-DG due to their reliance on glycolysis for ATP generation. In this study, we evaluated the antileukemic activity of 2-DG in T- and Bp-ALL subtypes characterized by non-random translocations. The cytotoxicity of 2-DG was determined on CCRF-CEM (PTEN mutant T-ALL), NALM6 (Bp-ALL), REH (TEL/AML1+ Bp-ALL), and the BCR/ABL+ ALL cell lines SupB15 and TOM1. Cells were treated with 4mM 2-DG for 72 h under normoxic vs. hypoxic (0.5% O₂) conditions using a hypoxia box chamber (BioSpherix), and growth arrest and cell death were assessed. 2-DG induced 2-13 fold higher cell death in all ALL cells tested under normal O₂ conditions compared to hypoxia. Similarly, growth inhibition was greater under normoxia. Both BCR/ABL+ ALL cell lines TOM1 and SupB15 were among the most sensitive, while the PTEN mutant CCRF-CEM (T-ALL) cell model was the least. To analyze the mechanisms of higher sensitivity of ALL cells under normoxia, we first used mannose to assess the role of glycosylation in 2-DG induced ALL cell death. Mannose reversed 2-DG induced cell death in CCRF-CEM cells but only partially in NALM6 cells, indicating inhibition of glycosylation mainly mediated death in CCRF-CEM cells, while inhibition of both glycolysis and glycosylation mediated cell death in NALM6 cells. To further assess other mechanisms leading to cell death in ALL, we evaluated mitochondrial integrity in representative Bp- and T-ALL models by determining cellular respiration with a Clark electrode (Hansatech Instruments). Our data show that cellular respiration in NALM6 was 50% lower compared to CCRF-CEM cells, suggesting that an intrinsic mitochondrial dysfunction was also responsible for 2-DG induced cell death in NALM6 cells. We then evaluated the observed higher sensitivity of BCR/ABL+ ALL cells to 2-DG. The AMPK and PI3K/Akt/mTOR pathways regulate protein, fatty acid and glucose metabolism, and key glycolytic enzymes are known to be upregulated in BCR/ABL+ cells. Therefore, we determined changes on AMPK and PI3K/Akt/mTOR signaling following exposure to 2-DG in SupB15 (BCR/ABL+) and in CCRF-CEM cells, the least sensitive ALL model examined. Western immunoblotting showed that 2-DG led to upregulation of p-AMPK (Thr172) and downregulation of p-mTOR (S2448) and p-p70S6K (Thr389). In both cell lines, 2-DG also led to cell death by triggering an unfolded protein response (UPR) evidenced by CHOP expression and PARP cleavage. We previously demonstrated that activation of AMPK leads to upregulation of Akt as a compensatory survival mechanism in ALL cells (Mol Cancer 6: 46, 2007). On this basis, we then tested induction of cell death by the combination of 2-DG (4 mM) plus Akt inhibitor X (AIX) (12 μM) in CCRF-CEM vs. SupB15 cells under normoxic conditions. Again, SupB15 cells were more sensitive to 2-DG, while AIX alone at low dose (12 μM) had minimal effect on either cell line. When both drugs were used in combination at these same concentrations, significant synergism was seen in CCRF-CEM cells but not in SupB15 cells. Analysis of AMPK, mTOR and Akt in CCRF-CEM cells treated with the combination of 2-DG plus AIX showed higher p-AMPK activation and mTOR downregulation compared to 2-DG alone, while these signaling changes were lower in SupB15 cells. Consistent with these findings, this combination led to higher induction of UPR and PARP cleavage in CCRF-CEM vs. SupB15 cells. We conclude that the greater sensitivity of ALL cells to 2-DG under normoxia is due to concomitant inhibition of glycolysis and/or glycosylation, an inherent mitochondrial dysfunction in some phenotypes, and 2-DG induced changes in AMPK and PI3K/Akt/mTOR signaling leading to the induction of UPR. Concomitant Akt inhibition sensitizes the relatively 2-DG resistant CCRF-CEM cells to death with 2-DG. Chemotherapy resistant BCR/ABL+ ALL cells exhibit significant sensitivity to 2-DG, likely due to their increased reliance on glycolysis for ATP generation. On this basis, we propose that glycolytic inhibitors represent a promising novel therapeutic strategy for ALL, particularly for BCR/ABL+ ALL.