Joanna DeSalvo

Metformin Induces Apoptosis through AMPK-Dependent Inhibition of UPR Signaling in ALL Lymphoblasts

The outcome of patients with resistant phenotypes of acute lymphoblastic leukemia (ALL) or those who relapse remains poor. We investigated the mechanism of cell death induced by metformin in Bp- and T-ALL cell models and primary cells, and show that metformin effectively induces apoptosis in ALL cells. Metformin activated AMPK, down-regulated the unfolded protein response (UPR) demonstrated by significant decrease in the main UPR regulator GRP78, and led to UPR-mediated cell death via up-regulation of the ER stress/UPR cell death mediators IRE1α and CHOP. Using shRNA, we demonstrate that metformin-induced apoptosis is AMPK-dependent since AMPK knock-down rescued ALL cells, which correlated with down-regulation of IRE1α and CHOP and restoration of the UPR/GRP78 function. Additionally rapamycin, a known inhibitor of mTOR-dependent protein synthesis, rescued cells from metformin-induced apoptosis and down-regulated CHOP expression. Finally, metformin induced PIM-2 kinase activity and co-treatment of ALL cells with a PIM-1/2 kinase inhibitor plus metformin synergistically increased cell death, suggesting a buffering role for PIM-2 in metformin’s cytotoxicity. Similar synergism was seen with agents targeting Akt in combination with metformin, supporting our original postulate that AMPK and Akt exert opposite regulatory roles on UPR activity in ALL. Taken together, our data indicate that metformin induces ALL cell death by triggering ER and proteotoxic stress and simultaneously down-regulating the physiologic UPR response responsible for effectively buffering proteotoxic stress. Our findings provide evidence for a role of metformin in ALL therapy and support strategies targeting synthetic lethal interactions with Akt and PIM kinases as suitable for future consideration for clinical translation in ALL.

Inhibition of Akt Potentiates 2-DG–Induced Apoptosis via Downregulation of UPR in Acute Lymphoblastic Leukemia

The ability to pair the regulation of metabolism and cellular energetics with oncogenes and tumor suppressor genes provides cancer cells with a growth and survival advantage over normal cells. We investigated the mechanism of cell death induced by 2-deoxy-d-glucose (2-DG), a sugar analog with dual activity of inhibiting glycolysis and N-linked glycosylation, in acute lymphoblastic leukemia (ALL). We found that, unlike most other cancer phenotypes in which 2-DG only inhibits cell proliferation under normoxic conditions, ALL lymphoblasts undergo apoptosis. Bp-ALL cell lines and primary cells exhibited sensitivity to 2-DG, whereas T-ALL cells were relatively resistant, revealing phenotypic differences within ALL subtypes. Cotreatment with d-mannose, a sugar essential for N-linked glycosylation, rescues 2-DG–treated ALL cells, indicating that inhibition of N-linked glycosylation and induction of ER stress and the unfolded protein response (UPR) is the predominant mechanism of 2-DGs cytotoxicity in ALL. 2-DG–treated ALL cells exhibit upregulation of P-AMPK, P-Akt, and induction of ER stress/UPR markers (IRE1α, GRP78, P-eIF2α, and CHOP), which correlate with PARP cleavage and apoptosis. In addition, we find that pharmacologic and genetic Akt inhibition upregulates P-AMPK, downregulates UPR, and sensitizes ALL cells to remarkably low doses of 2-DG (0.5 mmol/L), inducing 85% cell death and overcoming the relative resistance of T-ALL. In contrast, AMPK knockdown rescues ALL cells by upregulating the prosurvival UPR signaling. Therefore, 2-DG induces ALL cell death under normoxia by inducing ER stress, and AKT and AMPK, traditionally thought to operate predominantly on the glycolytic pathway, differentially regulate UPR activity to determine cell death or survival. Mol Cancer Res; 10(7); 969–78. ©2012 AACR.

Mcl-1 downregulation leads to the heightened sensitivity exhibited by BCR-ABL positive ALL to induction of energy and ER-stress

BCR-ABL positive (+) acute lymphoblastic leukemia (ALL) accounts for ∼30% of cases of ALL. We recently demonstrated that 2-deoxy-d-glucose (2-DG), a dual energy (glycolysis inhibition) and ER-stress (N-linked-glycosylation inhibition) inducer, leads to cell death in ALL via ER-stress/UPR-mediated apoptosis. Among ALL subtypes, BCR-ABL+ ALL cells exhibited the highest sensitivity to 2-DG suggesting BCR-ABL expression may be linked to this increased vulnerability. To confirm the role of BCR-ABL, we constructed a NALM6/BCR-ABL stable cell line and found significant increase in 2-DG-induced apoptosis compared to control. We found that Mcl-1 was downregulated by agents inducing ER-stress and Mcl-1 levels correlated with ALL sensitivity. In addition, we showed that Mcl-1 expression is positively regulated by the MEK/ERK pathway, dependent on BCR-ABL, and further downregulated by combining ER-stressors with TKIs. We determined that energy/ER stressors led to translational repression of Mcl-1 via the AMPK/mTOR and UPR/PERK/eIF2α pathways. Taken together, our data indicate that BCR-ABL+ ALL exhibits heightened sensitivity to induction of energy and ER-stress through inhibition of the MEK/ERK pathway, and translational repression of Mcl-1 expression via AMPK/mTOR and UPR/PERK/eIF2α pathways. This study supports further consideration of strategies combining energy/ER-stress inducers with BCR-ABL TKIs for future clinical translation in BCR-ABL+ ALL patients.

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

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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