Metformin induces apoptosis through AMPK-dependent inhibition of UPR signaling in ALL lymphoblasts

Abstract

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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Metformin induces cell growth arrest and apoptosis in ALL cell lines.

Growth inhibition (A) and apoptosis (B) in CCRF-CEM and NALM6 cells treated with metformin (MET, 1, 2, and 5 mM) for 48 h. Apoptosis (C) in T-ALL (CCRF-CEM, Jurkat, and primary T-ALL) and Bp-ALL (NALM6, REH, and primary Bp-ALL) cells treated with metformin (MET, 5 mM) for 48 h. Growth inhibition was expressed relative to control values (mean ±SEM, n = 3). * and # denote p < 0.001 and p < 0.0001 for metformin vs. CTRL, respectively.

Figure 2. Metformin activates AMPK, Akt, and UPR signaling pathway proteins in ALL primary and cell line models.

A) Western blot analysis of proteins associated with AMPK, and Akt/mTOR signaling pathways in CCRF-CEM and NALM6 cells treated with metformin (MET, 2.5 and 5.0 mM) for 48 h. The density value of each band was normalized to β-actin level and expressed relative to control (shown as fold induction). B) Immunoblotting of UPR signaling factors in CCRF-CEM and NALM6 cells treated with metformin (MET, 2.5 and 5.0 mM) for 48 h. C) Western blots of AMPK, Akt/mTOR and UPR signaling proteins in representative sample of primary T- and Bp-ALL cells treated with metformin (MET, 10 mM) for 24 h. D) Western blot analysis of GRP78 expression in CCRF-CEM and NALM6 cells treated with metformin (MET, 10mM) and tunicamycin (TUN, 2.5 µg/ml for NALM6; 5.0 µg/ml for CCRF-CEM), either alone or in combination for 48 h.

Figure 3. Inhibition of mTOR-dependent protein synthesis reverses metformin-induced cell death.

A) Apoptosis in CCRF-CEM cells expressing either scramble shRNA (shCTRL) or shRNA against AMPKα1 (shAMPK) treated with metformin (MET, 10 mM) for 48 h. B) Immunoblotting of AMPK, Akt/mTOR, and ER stress/UPR signaling pathway proteins in the cells described in (A). C) Cell death (upper panel) in CCRF-CEM and NALM6 cells treated with metformin (MET, 5 and 10 mM) and rapamycin (RAPA; 0.1 µg/mL), either alone or in combination for 48 h. A statistical value of p < 0.01 was obtained for MET + RAPA vs. either control or each agent alone. The cell death was expressed as a percentage (%) of cells in the population (mean ±SEM, n = 3). Western blot analysis (lower panel) of p-4EBP1 (T70), IRE1α, and CHOP expression in the CCRF-CEM and NALM6 cells treated with metformin plus rapamycin described in the upper panel.

Figure 4. Metformin induces expression of PIM-2 in ALL cells.

Western blot analysis of PIM-2, p-BAD (S112) and p-AMPK (T172) expression in CCRF-CEM and NALM6 cells treated with metformin (MET, 5 and 10 mM) for 72 h at 37°C.

Figure 5. Inhibition of PIM-2 and Akt kinases synergistically sensitizes ALL cells to metformin.

A) Cell death in CCRF-CEM and NALM6 cells treated with metformin (MET, 4 mM) and the PIM-1/2 kinase inhibitor V (PKI; 80 µM), either alone or in combination for 72 h at 37°C. The CI values of 0.27 and 0.28 indicate synergism. B) Immunoblotting of p-ACC (S79), GRP78, and PIM-2 expression in the CCRF-CEM and NALM6 cells treated with MET plus PKI described in (A). C) Cell death in NALM6 cells treated with metformin (MET, 5.0 mM) and Akt inhibitor X (AIX; 5 µM) or perifosine (PER; 6 µM), either alone or in combination for 72 h at 37°C. CI values of 0.19 (MET + AIX) and 0.21 (MET + PER) indicate synergism. The cell death values were expressed as a percentage (%) of cells in the population (mean ±SEM, n = 3). D) Immunoblotting of AMPK/ACC, Akt/mTOR, and UPR signaling pathway proteins in the NALM6 cells treated with MET (5.0 mM) plus AIX (5 µM) described in (C).

Figure 6. Proposed mechanism of action for metformin in ALL cells.

Metformin induces metabolic stress by decreasing the ATP: AMP ratio, which leads to activation of AMPK, and increased level of unfolded/misfolded proteins in the ER lumen. The inability of ALL cells to engage the UPR caused by AMPK-dependent down-regulation of GRP78 leads to ER stress/UPR mediated cell death. The survival PIM and Akt kinases are expressed as compensatory survival mechanisms in response to metformin’s cytotoxicity to down-regulate AMPK allowing cells to effectively engage UPR and process the ER stress.