Abstract 4991: Akt and AMPK modulate UPR activity and determine ER stress-induced cell death in response to 2-DG in ALL

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Abstract Acute Lymphoblastic Leukemia (ALL) is the most common hematological malignancy and the main cause of cancer-related death in children. In search for novel treatment strategies, we investigated the mechanism of cell death induced by the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) in ALL cell models. We found that 2-DG induced significant cell death under normoxia in Bp-ALL and T-ALL cells. Similar treatments with 2-FDG, a more selective glycolytic inhibitor, failed to induce equivalent degrees of cell death in ALL, suggesting that 2-DG induces apoptosis through pathways independent of glycolysis, such as inhibition of N-linked glycosylation (N-LG). Co-treatment with mannose, a sugar essential for N-LG, rescued 2-DG induced cell death in ALL cells (p < 0.01), indicating that inhibition of N-LG leads to apoptosis in ALL. Western blot analysis of 2-DG treated cells showed sustained activation of P-AMPK (T172) and transient up-regulation of P-Akt (S473). More important, treatment with 2-DG resulted in up-regulation of the UPR markers GRP78, GRP94, IRE1α, P-eIF2α, and CHOP, and correlated with increased cleavage of PARP and apoptosis in CCRF-CEM (T-ALL) and NALM6 (Bp-ALL). Therefore, inhibition of the N-LG by 2-DG leads to ER stress/UPR mediated cell death in ALL. We then evaluated the role of Akt using the Akt inhibitor X (AIX) and shRNA against Akt (shAkt). Inhibition of Akt synergistically sensitized CCRF-CEM and NALM6 cells to 2-DG (CI = 0.15; p < 0.05 for AIX vs. each drug alone; p < 0.01 for shAkt vs. scramble shRNA). Western blots showed that P-AMPK was increased in shAkt vs. scramble shRNA expressing cells, whereas P-mTOR, IRE1α, GRP78, P-eIF2α, and CHOP were down-regulated. Again, mannose completely reversed 2-DG+AIX induced cytotoxicity (p < 0.0001, 7% apoptosis for 2DG+AIX+MAN vs. 85% for 2DG+AIX), and correlated with down-regulation of UPR markers, confirming that inhibition of N-LG is the main mechanism by which 2-DG induces apoptosis in ALL. Next, we examined the role of AMPK using shRNA and found that inhibition of AMPK rescued 2-DG+AIX induced cell death and growth inhibition (p < 0.001), and led to up-regulation of P-Akt, P-mTOR, and UPR markers, suggesting that functional UPR activity is critical for ALL cell survival following treatment with 2-DG. Indeed, inhibition of UPR via down-regulation of GRP78 increased cell death in both CCRF-CEM and NALM6 cells treated with 2-DG (p < 0.01). Taken together, we demonstrate that 2-DG induces ER-stress mediated cell death in ALL cells by inhibiting N-LG, and that the ability of ALL cells to effectively engage UPR determines ALL cell fate (survival vs. death). Our data also demonstrate that Akt and AMPK differentially modulate UPR activity in response to 2-DG in ALL. These findings provide a rationale to develop novel strategies for ALL therapy using glycolytic inhibitors alone or in combination with agents targeting these metabolic and oncogenic pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4991. doi:1538-7445.AM2012-4991