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PLoS One. 2013; 8(8): e74420.
Published online 2013 August 23. doi:  10.1371/journal.pone.0074420
PMCID: PMC3751848

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

Ramiro Garzon, Editor


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.


Acute Lymphoblastic Leukemia (ALL), the most common malignancy in children and adolescents, remains the number one cause of cancer-related death for patients under the age of twenty [1]. Despite significant overall improvements in cure rates, outcome remains poor for patients with resistant phenotypes or after relapse, and long-term treatment-related morbidity can be significant for survivors of childhood ALL [2]. Consequently, novel and less toxic treatment strategies are needed to improve cure rates and decrease long-term sequelae for these patients. We identified the AMP activated protein kinase (AMPK), a regulator of energy homeostasis in eukaryotic cells [3], as a target for ALL therapy due to its effects on cell growth and cell cycle regulation, as well as its crosstalk with critical metabolic and oncogenic pathways [4]. AMPK is a heterotrimeric complex composed of a catalytic α subunit and two regulatory subunits (β and γ) [5]. AMPK is activated by metabolic stressors that deplete ATP and increase AMP, and by upstream kinases [6] that induce its phosphorylation at Thr172 [7]. Activated AMPK down-regulates processes that consume ATP (cell growth and protein synthesis) and activate pathways responsible for the generation of energy such as glycolysis and fatty acid oxidation [8].

The biguanide drug metformin (N,N-Dimethylimidodicarbonimidic diamide), currently used for treatment of diabetes [9], is known to activate AMPK. Metformin has been shown to induce metabolic stress by various mechanisms including inhibition of AMP deaminase [10] and the mitochondrial respiration chain complex 1 [11], both of which lower the ATP: AMP ratio leading to AMPK activation. Although metformin is linked to lower incidence of cancer and induction of cell death in various solid tumor types [1214], its mechanism of cell death has not been fully investigated in leukemia. We and others have reported that AMPK can act as a physiological suppressor of the unfolded protein response (UPR) following exposure to AMPK activators such as AICAR [15,16], metformin [17,18], or the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) [19]. This homeostatic mechanism is triggered in response to the accumulation of unfolded/misfolded proteins in the ER lumen [20]. The UPR is mediated via three ER transmembrane receptors: protein kinase dsRNA-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1) [21]. These receptors are activated upon dissociation from the main ER chaperone protein GRP78 to fully engage the UPR function, which encompasses blocking of protein synthesis (via phosphorylation of eIF2α), activation of proteasomal protein degradation, and transcriptional induction of ER chaperone genes (GRP78 and GRP94) as well as the pro-apoptotic transcription factor CHOP (CCAAT/enhancer binding protein homologous) [22]. In addition, GRP78 functions to suppress pro-apoptotic pathways of the UPR via activation of Akt and Erk signaling [23,24]. During sustained ER stress, the pro-apoptotic arm of the UPR activates IRE1α, CHOP, caspases, the apoptotic signaling-kinase-1 (ASK1) and its downstream target c-Jun NH2-terminal kinase (JNK) [25,26]. Therefore, both a functional anti-apoptotic and pro-apoptotic arm are ascribed to the UPR [27].

In mammalian cells, protein translation is mainly regulated by the mammalian target of rapamycin (mTOR), which phosphorylates among others the two key protein translation regulators p70S6K and 4-EBP1 [28]. Phosphorylation of the latter promotes its dissociation from the translational regulator eukaryotic initiation factor 4E allowing cap-dependent translation [29]. Recently, PIM kinases have been shown to regulate cell growth, energy metabolism, and programmed cell death through interactions with 4-EBP1 [30], AMPK [31], and BAD [32], respectively. The PIM kinase family consists of three oncogene-encoded serine/threonine kinases (PIM-1/2/3) [33] that are frequently overexpressed in human hematological and solid malignancies [34]. In most hematopoietic malignancies, PIM expression has been correlated with poor prognosis, underscoring the clinical significance of PIM kinases in cancer biology [35].

In this study, we used pharmacological and genetic approaches to investigate the mechanism of cell death induced by metformin in ALL. Our data indicate that metformin activates AMPK and induces apoptosis by triggering ER stress and preventing ALL cells from engaging the UPR in order to effectively buffer an increased proteotoxic load due to the accumulation of unfolded proteins in the ER lumen. Thus, metformin triggers the UPR-mediated apoptotic pathway via an AMPK-dependent mechanism.

Material and Methods

Ethics statement

This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the University of Miami Human Subject Research Office (HSRO) (IRB protocol # 20110555). Primary ALL samples were obtained from patients with ALL at the University of Miami following Institutional Review Board (IRB) approved written informed consent by the University of Miami Human Subject Research Office (HSRO).

Cell culture and reagents

The ALL cell lines CCRF-CEM (T-ALL), Jurkat (T-ALL), and REH (B precursor ALL, Bp-ALL, t(12;21)/TEL-AML1) were obtained from the American Type Culture Collection (ATCC;, Manassas, VA). NALM6 (Bp-ALL) cell line was obtained from the Leibniz Institute Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ,, Braunschweig, Germany). These cell lines were maintained in RPMI-1640 medium (Cellgro, Manassas, VA) supplemented with 10% heat inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, MO) and antibiotics (penicillin, 100 I.U. /ml; streptomycin, 100 µg/ml) (Cellgro) at 37°C under an atmosphere of 5% CO2. Primary ALL cells were co-cultured using a human bone marrow stromal cell feeder layer as described previously [19]. Metformin and tunicamycin were purchased from Sigma-Aldrich, rapamycin from LC Laboratories (Woburn, MA), and perifosine from Selleck Chemicals (Houston, TX). The Akt Inhibitor X and the PIM-1/2 kinase inhibitor V were obtained from Calbiochem (Gibbstown, NJ).

Cell viability and apoptosis assays

Logarithmic cells were exposed to the indicated drugs for up to 72 h. At specified time points, cell proliferation and cell death were determined using the Vi-Cell XR system (trypan blue exclusion) (Beckman Coulter, Brea, CA) and expressed as a percentage relative to control values or a percentage of dead cells in the population (mean ±SEM, n=3), respectively. Apoptosis was determined by flow cytometric analysis of Annexin V-FITC/propidium iodide (PI) staining as described previously [36]. The Annexin V and PI data were combined and expressed as a percentage of apoptosis (mean ± SEM, n=3). Synergism was determined by calculating the combination index (CI) using the following equation: CI = [(D1-combination/D1-single) + (D2-combination/D2-single)] as described by Chou [37]. Statistical significance in cell growth or apoptosis data following drug treatments was assessed by one-way ANOVA followed by the Newman-Keuls multiple comparison tests or by unpaired Student’s t test using GraphPad PRISM (San Diego, CA).

Construction of stable shRNA expressing cell lines

To down-regulate the expression of AMPK1α, we used a pool of lentiviral particles containing three target-specific constructs that encode specific shRNAs against AMPKα1 (shAMPK) (sc-296730V; Santa Cruz Biotechnology, Santa Cruz, CA). Control lentiviral shRNAs (shCTRL, sc-108080) were obtained from Santa Cruz. Stable CCRF-CEM cells expressing shAMPK and shCTRL were generated by transduction using lentiviral stock as described elsewhere [19].

Protein extracts and Western immunoblots

Cells were sonicated in 1X RIPA (Thermo Scientific, Rockford, IL) containing Halt™ protease and phosphatase inhibitor cocktails (Thermo Scientific). Western blots were carried out as described elsewhere [15]. The primary antibodies and the secondary HRP-conjugated antibody were obtained from Cell Signaling (Beverly, MA).


Metformin induces growth inhibition and cell death by altering AMPK, Akt/mTOR and UPR signaling pathways in ALL cell models and primary cells

To evaluate the clinical relevance of metformin in ALL, we determined its effects on ALL cell growth and death. The ALL cell models CCRF-CEM (T-ALL) and NALM6 (Bp-ALL) were treated with metformin and effects on cell proliferation and apoptosis determined at 48 h. As shown in Figure 1, metformin (1-5 mM) induced cell growth arrest (Figure 1A) and apoptosis (Figure 1B) in both CCRF-CEM and NALM6 cell lines. Similar data were obtained with other representative T-ALL (Jurkat) and Bp-ALL (REH) models and primary T- and Bp-ALL cells from children with ALL (Figure 1C), indicating that metformin induces cells death in both T- and Bp-ALL primary and cell line models.

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

To understand the mechanism of cell death induced by metformin in T- and Bp-ALL cells, we examined the expression of critical signaling proteins associated with energy metabolism and cell proliferation pathways in CCRF-CEM and NALM6 cell lines treated with metformin. We found that metformin induced phosphorylation/activation of p-AMPK (T172), its downstream target p-ACC (S79), and phosphorylation/activation of Akt at Ser473 (Figure 2A). The activation of AMPK resulted in down-regulation of p-mTOR (S2448) and its target p-p70S6K (T389). These changes are consistent with those we previously reported with other AMPK activators [36].

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

Since we have previously showed that AMPK suppresses the UPR in ALL [19], and others had reported that metformin can inhibit the UPR in normal renal tubular epithelial cells and certain carcinomas [38,39], we determined the effects of metformin on the expression of UPR markers in cell lines and primary ALL cells. Western blot analysis of CCRF-CEM and NALM6 cells treated with metformin revealed a significant decrease in GRP78 expression (Figure 2B), indicating that metformin alters the regulation of the UPR. In addition, we detected increased expression of IRE1α, ATF6, and of the known ER stress/UPR-mediated cell death marker CHOP [40] in ALL cells treated with metformin (Figure 2B). Our results indicate that metformin induces ER stress/UPR mediated cell death and suggests the mechanism of apoptotic death may be triggered by the inability of ALL cells to adequately respond to ER and/or proteotoxic stress through the UPR. Similar signaling alterations were observed in metformin-treated representative primary T- and Bp-ALL cells, highlighting the clinical relevance of this putative mechanism of action (Figure 2C). To examine if the effect of metformin on GRP78 down-regulation represented a direct effect and was not simply a correlative finding, we co-treated CCRF-CEM and NALM6 cells with metformin and tunicamycin, a known ER stress inducer [41]. Tunicamycin activates GRP78 via inhibition of N-linked glycosylation which triggers ER stress/UPR due to the accumulation of misfolded proteins in the ER. As expected, tunicamycin alone strongly induced GRP78 expression in ALL cells (Figure 2D), whereas we found that metformin alone significantly reduced the level of GRP78. More important, when both drugs were used in combination, GRP78 expression was significantly decreased compared to cells treated with tunicamycin alone (Figure 2D), confirming a cause and effect relationship between metformin and down-regulation of GRP78 expression and UPR function in ALL cells.

Down-regulation of AMPK rescues ALL cells from metformin-induced ER stress/UPR-mediated cell death by de-repressing the UPR and interrupting protein synthesis

The described role of AMPK as suppressor of the UPR [16,19] coupled with the observed activation of AMPK in cells treated with metformin, led us to investigate AMPK’s role in the mechanism of ALL cell death induced by metformin. For this purpose, stable CCRF-CEM cells expressing either shRNA targeting AMPKα1 (shAMPK) or control shRNA (shCTRL) were treated with metformin and assayed for apoptosis at 48 h. AMPK knockdown by shRNA rescued CCRF-CEM cells from metformin-induced cell death as compared to control CCRF-CEM/shCTRL cells (Figure 3A; CCRF-CEM/shAMPK (10.7%) vs. CCRF-CEM/shCTRL (56.4%) cell death, p<0.001). As expected, shAMPK expressing cells expressed significantly lower total AMPK compared to shCTRL cells, and exhibited down-regulation of p-ACC (S79) expression confirming a functional down-regulation of AMPK signaling (Figure 3B). Signaling changes in shCTRL cells mimicked those found in wild type cell lines and primary ALL cells treated with metformin (Figure 2). Down-regulation of AMPK in metformin treated cells resulted in increased expression of GRP78 indicating ALL cells regain the ability to engage the UPR to buffer metformin’s cytotoxicity. More important, the expression of IRE1α and CHOP, two UPR markers associated with apoptosis [27], were down-regulated in shAMPK expressing cells, which correlated with the rescue phenotype (Figure 3B). These data confirm our previous data describing for the first time the role for AMPK as a negative regulator of the UPR in ALL cells [15,19]. Further Western blots revealed that AMPK knockdown led to phosphorylation/inhibition of eIF2α (S51) and dephosphorylation/activation of the negative regulator of CAP-dependent protein translation 4EBP1 (T70), suggesting that a concomitant decrease in protein synthesis also contributed to decreased metformin-induced cell death in AMPK knockdown cells (Figure 3B). Taken together, our data indicate that AMPK knockdown rescues ALL cells from metformin-induced cell death by restoring the UPR/GRP78 function, down-regulating UPR-mediated apoptotic factors, and interrupting protein synthesis.

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

To further assess whether the rate of protein synthesis is also an important determinant of metformin-induced cell death, we used a pharmacological approach to inhibit mTOR-dependent protein synthesis with rapamycin [42]. As shown in Figure 3C, inhibition of mTOR with rapamycin significantly decreased the sensitivity of ALL cells to metformin and, these effects correlated with decreased phosphorylation (activation) of the negative regulator of protein synthesis 4EBP1 (T70), suggesting a decrease in protein synthesis (Figure 3C). In addition, these effects in rapamycin-treated ALL cells were associated with down-regulation of the UPR apoptotic markers IRE1α and CHOP. Consequently, the AMPK knockdown data plus these findings in rapamycin-treated ALL cells support that the mechanism of metformin-induced cell death is dependent on AMPK activation, and that AMPK’s suppression of the UPR plus failure to down-regulate protein synthesis in metformin treated ALL cells undergoing proteotoxic/ER stress leads to apoptotic cell death.

Metformin induces expression of PIM-2 and Akt as a compensatory survival mechanism in ALL

It has been shown that AMPK activity is regulated by multiple kinases such as LKB1 [43], PIM kinases [31], and CAMKK2 [44]. Among these, PIM kinases have been reported to regulate both AMPK and protein synthesis [45], raising the possibility that PIM kinases may interact with AMPK and play a role in the mechanism of metformin-induced cell death. To test this hypothesis, we examined the expression of PIM-2 in metformin-treated CCRF-CEM and NALM6 cells. Figure 4 shows that the expression of PIM-2 at 72 h was increased in both ALL cell lines treated with metformin, and correlated with increased phosphorylation of BAD at Ser112, indicating inhibition of the pro-apoptotic activity of BAD to promote cell survival [46]. The fact that BAD is a known downstream target of PIM-2 [32] suggests that PIM-2 is expressed to block the apoptotic events triggered by metformin. More important, we found that level of p-AMPK (T172) inversely correlated with level of PIM-2 expression resulting in inhibition of p-AMPK activity following PIM-2 up-regulation. Our findings are consistent with the described interaction between PIM kinases and AMPK in leukemia cells [31] and we hypothesized that up-regulation of PIM kinases represents a compensatory survival mechanism that is expressed in response to metformin’s cytotoxicity in ALL cells. To test the pro-survival role of PIM kinases, we evaluated the effects of a PIM-1/2 kinase inhibitor in metformin-treated ALL cells. Figure 5A demonstrates that inhibition of PIM-1/2 kinase activity using the PIM-1/2 kinase inhibitor V (PKI) synergized with metformin in inducing cell death in CCRF-CEM and NALM6 cells with CI values of 0.27 and 0.28, respectively. More important, these effects were associated with increase expression of p-ACC, a marker for AMPK activity, and down-regulation of GRP78, which were greater for the combination of metformin plus PKI (Figure 5B). These data suggest that PIM kinase activity can modulate metformin-induced cell death through its known cross-talk with AMPK which can influence the ability of ALL cells to engage the UPR in response to metformin-induced ER and proteotoxic stress. The observed changes in GRP78 expression following co-treatment with metformin and inhibitors of PIM-1/2 further support that metformin induces cell death by ER stress/UPR mediated mechanism(s) in ALL by preventing ALL cells from engaging the UPR to effectively buffer ER/proteotoxic stress.

Figure 4
Metformin induces expression of PIM-2 in ALL cells.
Figure 5
Inhibition of PIM-2 and Akt kinases synergistically sensitizes ALL cells to metformin.

We previously demonstrated that whereas AMPK negatively regulates the UPR, Akt up-regulates it allowing cells to better cope with ER and proteotoxic stressors [15,19]. On this basis, we used the Akt inhibitors perifosine and Akt inhibitor X (AIX), and determined their effects on metformin-induced apoptosis. NALM6 cells were treated with metformin, perifosine, AIX, or the combination of either Akt inhibitor plus metformin. Figure 5C shows that inhibition of Akt with both Akt inhibitors sensitized NALM6 cells to metformin-induced cell death. Indeed, these agents led to 40% (metformin + AIX) and 52% (metformin + perifosine) greater apoptotic death as compared to each agent alone (AIX, 4.8%; perifosine, 35.7% cell death, p<0.001), and these interactions were synergistic (CI=0.19 for metformin + AIX; CI=0.21 for metformin + perifosine). Immunoblots of NALM6 cells treated with AIX plus metformin showed significant down-regulation of p-Akt (S473) and increased in functional AMPK signaling (up-regulation of p-ACC (S79)) leading to down-regulation of the UPR markers GRP78 and IRE1α (Figure 5D). The magnitude of these changes was greater for the combination. These changes also correlated with greater increase in expression of p-eIF2α (S51) and CHOP, again supporting the premise that metformin’s induction of cell death is through a UPR-mediated mechanism.


Metformin, one of the most widely prescribed anti-diabetic drugs, has recently received significant attention as an antineoplastic agent due to its cytotoxic effects in a variety of solid tumor cancer cell lines including prostate, colorectal, lung, pancreatic, and breast [12]. Its anti-cancer effect is supported by epidemiological studies that show a decrease in cancer incidence in metformin-treated patients [14]. Although its mechanism of action in hepatic glucose metabolism is well documented, little is known about metformin’s mechanism of action in cancer cells, particularly in leukemia. It has also been proposed that most of metformin’s cytotoxic effects are mediated through activation of the AMPK pathway, the main regulator of cellular energy homeostasis [3]. Here, we show for the first time that metformin induced cell growth arrest and apoptosis in ALL cell models and primary cells occurs through induction of the ER stress/UPR-mediated cell death pathway, and that this effect is AMPK-dependent. In this context through AMPK’s effect as a negative regulator of the UPR, metformin prevents ALL cells from effectively engaging the UPR to overcome ER and proteotoxic stress-induced irreversible cellular damage leading to apoptotic death.

The notion that metformin is capable of inhibiting the UPR was first reported by Saito et al. [39], who found that antidiabetic biguanides could inhibit GRP78 activity during glucose deprivation. Our data not only confirm that metformin down-regulates GRP78 but also demonstrate that metformin induces stress in the ER lumen evidenced by activation of ATF6, IRE1α, and CHOP in metformin-treated ALL cells. It has been reported that metformin reduces the ATP/AMP ratio by targeting complex 1 of the respiratory chain [11,47]. Therefore, metformin-induced ER stress is likely triggered by accumulation of unfolded/misfolded proteins in the ER as a consequence of ATP depletion in a manner akin to glucose deprivation [48]. In various normal cell types including cardiomyocytes [17] and bovine aortic endothelial cells [18], the activation of AMPK by AICAR or metformin were found to be tissue protective via AMPK-dependent suppression of the UPR. Others have reported that the tissue protective effects of metformin in renal tubular epithelial cells were AMPK-independent [38]. In contrast, we previously demonstrated that AMPK activation by AICAR, methotrexate, or 2-DG led to inhibition of the UPR and cytotoxicity in ALL cells [15,19]. We now report that metformin’s induction of apoptosis in ALL cells is AMPK-dependent and occurs via a UPR-mediated mechanism. Therefore, tissue specificity appears to exist in the mechanism by which certain AMPK activators suppress the UPR and in addition, whereas AMPK suppression of the UPR may be beneficial to normal tissues, it induces cell death in several cancer phenotypes.

Herein, we hypothesized that concomitant persistence of metformin-induced ER stress via ATP depletion and inhibition of GRP78 following treatment with metformin leads to ALL cell death demonstrated by the increased expression of the UPR-mediated apoptotic factors IRE1α, and CHOP [25]. It has been shown that down-regulation of GRP78 expression is sufficient to induce apoptosis and plays a critical role in physiologic and pathologic stress coping mechanisms used by a variety of cell types [24]. This report is the first to elucidate the role of UPR in metformin-induced cell death in ALL and in that context it identifies AMPK as an essential regulator of this mechanism. Indeed, down-regulation of AMPK using shRNA completely abrogated metformin-induced apoptosis in ALL, and more important correlated with increased GRP78 expression and down-regulation of the UPR-mediated apoptotic factors IRE1α and CHOP. Mechanistically, this report provides further evidence and confirms our previous findings showing that AMPK acts as a physiological suppressor of the UPR whereas Akt up-regulates the UPR in ALL cells [15,19]. Therefore, our recent body of work demonstrates that in ALL cells undergoing metabolic and energetic stress, the UPR is regulated via crosstalk between AMPK and Akt, and these interactions determine the fate (i.e. death vs. survival) of ALL cells under conditions of metabolic and/or proteotoxic stress.

In our ALL cell models, the mechanisms responsible for the regulation of protein synthesis also appear altered following exposure to metformin. Our data showed that down-regulation of AMPK led to increased phosphorylation of p-eIF2α and dephosphorylation of p-4EBP1, both events being associated with inhibition of protein synthesis. Therefore, shRNA knockdown of AMPK rescued ALL cells through inhibition of protein synthesis coupled with up-regulation of the UPR (increased GRP78 expression). In support for this mechanism is the rescue we observed following co-treatment with the mTOR inhibitor rapamycin, a known inhibitor of CAP-dependent protein synthesis, which resulted in decreased IRE1α and CHOP expression. The relationship between protein translation and UPR activation was recently highlighted by Matsuo et al. [49], which showed that hyperactivation of 4EBP1 prevented UPR activation. Our findings do contrast with the recent report by Grimaldi et al. [50] proposing that metformin induces cell death by blocking the catalytic activity of mTORC1 and repressing mRNA translation, although these investigators did not assess the role of the UPR in their model. Based on our data, we propose that it is the balance between proteotoxic stress and UPR activity that results in either death or survival from metformin’s cytotoxicity in ALL cells.

To our knowledge, the induction of PIM kinase activity by metformin had not been reported. Herein, we interpreted that metformin induced PIM-2 kinase expression is a compensatory survival mechanism due to its anti-apoptotic role via down-regulation of BAD [32]. Further, it has been demonstrated that PIM kinases can negatively regulate AMPK activity [31] and promote hematopoietic cell growth and survival [45]. The synergistic effects observed between metformin and PIM kinase inhibition support our postulate that the increase in PIM-2 expression represents a buffering response to metformin. More important, this combination led to synergistic induction of cell death. Based on our findings, we propose a new role for PIM-2 kinase as capable of modulating UPR via down-regulation of AMPK activity. We also found that targeting Akt, another survival kinase capable of modulating AMPK activity through phosphorylation of Ser485 [51], resulted in significant synergistic cell death when combined with metformin. Our data confirm our report demonstrating antagonistic roles for Akt and AMPK in modulating the UPR [19]. Based on data presented herein, we propose a model for metformin-induced cell death in ALL cells via ER stress/UPR-mediated apoptosis in which metformin, by decreasing the ATP/AMP ratio promotes i) ATP depletion and unfolded protein accumulation in the ER lumen resulting in proteotoxic and ER stress, and ii) AMPK activation which leads to suppression of the UPR (down-regulation of GRP78), leading to UPR-mediated apoptotic cell death (Figure 6). In support of this proposed model we present evidence that down-regulation of AMPK rescues ALL cells from metformin-induced apoptosis by interrupting protein synthesis and increasing UPR activity, which would reduce the load of unfolded proteins in the ER and restore the ability of the cells to cope with the ER/proteotoxic stress.

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

In summary, our studies demonstrate that the crosstalk between AMPK, Akt, PIM-2 kinase, and UPR signaling pathways determines metformin-induced cell death in ALL. We demonstrate for the first time that metformin induced apoptosis in ALL lymphoblasts occurs via UPR-mediated mechanisms which are entirely AMPK-dependent. We also provide further evidence for the roles of AMPK and Akt as regulators of the UPR in ALL. Finally, our data not only demonstrate the ability of metformin to induce significant cell death in ALL cell lines and primary cells supporting future translation into clinical trials, but also uncover strategies exploiting synthetic lethality by combining metformin and selective inhibitors of these pathways that may also be suitable for clinical translation in patients with ALL.

Funding Statement

This research was supported by grants from the Leukemia & Lymphoma Society (grant number 6168-09), and the Micah Batchelor Award to Julio C. Barredo. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


1. Siegel R, Naishadham D, Jemal A (2012) Cancer statistics, 2012. CA Cancer J Clin 62: 10-29.10.3322/caac.20138 PubMed: 22237781 [PubMed]
2. Pui CH, Mullighan CG, Evans WE, Relling MV (2012) Pediatric acute lymphoblastic leukemia: where are we going and how do we get there? Blood 120: 1165-1174.10.1182/blood-2012-05-378943 PubMed: 22730540 [PubMed]
3. Hardie DG (2011) AMP-activated protein kinase: a cellular energy sensor with a key role in metabolic disorders and in cancer. Biochem Soc Trans 39: 1-13.10.1042/BST0390001 PubMed: 21265739 [PubMed]
4. Sengupta TK, Leclerc GM, Hsieh-Kinser TT, Leclerc GJ, Singh I et al. (2007) Cytotoxic effect of 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) on childhood acute lymphoblastic leukemia (ALL) cells: implication for targeted therapy. Mol Cancer 6: 46.10.1186/1476-4598-6-46 PubMed: 17623090 [PMC free article] [PubMed]
5. Carling D, Mayer FV, Sanders MJ, Gamblin SJ (2011) AMP-activated protein kinase: nature’s energy sensor. Nat Chem Biol 7: 512-518.10.1038/nchembio.610 PubMed: 21769098 [PubMed]
6. Fay JR, Steele V, Crowell JA (2009) Energy homeostasis and cancer prevention: the AMP-activated protein kinase. Cancer Prev Res (Phila) 2: 301-309.10.1158/1940-6207.CAPR-08-0166 PubMed: 19336731 [PubMed]
7. Hawley SA, Boudeau J, Reid JL, Mustard KJ, Udd L et al. (2003) Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade. J Biol 2: 28.10.1186/1475-4924-2-28 PubMed: 14511394 [PMC free article] [PubMed]
8. Steinberg GR, Kemp BE (2009) AMPK in Health and Disease. Physiol Rev 89: 1025-1078.10.1152/physrev.00011.2008 PubMed: 19584320 [PubMed]
9. Bolen S, Feldman L, Vassy J, Wilson L, Yeh HC et al. (2007) Systematic review: comparative effectiveness and safety of oral medications for type 2 diabetes mellitus. Ann Intern Med 147: 386-399.10.7326/0003-4819-147-6-200709180-00178 PubMed: 17638715 [PubMed]
10. Ouyang J, Parakhia RA, Ochs RS (2011) Metformin activates AMP kinase through inhibition of AMP deaminase. J Biol Chem 286: 1-11.10.1074/jbc.O110.000242 PubMed: 21059655 [PMC free article] [PubMed]
11. Owen MR, Doran E, Halestrap AP (2000) Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain. Biochem J 3483: 607-614.10.1042/0264-6021:3480607 PubMed: 10839993 [PubMed]
12. Kourelis TV, Siegel RD (2012) Metformin and cancer: new applications for an old drug. Med Oncol 29: 1314-1327.10.1007/s12032-011-9846-7 PubMed: 21301998 [PubMed]
13. Ben Sahra I, Le Marchand-Brustel Y, Tanti JF, Bost F (2010) Metformin in cancer therapy: a new perspective for an old antidiabetic drug? Mol Cancer Ther 9: 1092-1099.10.1158/1535-7163.MCT-09-1186 PubMed: 20442309 [PubMed]
14. Pollak M (2010) Metformin and other biguanides in oncology: advancing the research agenda. Cancer Prev Res (Phila) 3: 1060-1065.10.1158/1940-6207.CAPR-10-0175 PubMed: 20810670 [PMC free article] [PubMed]
15. Kuznetsov JN, Leclerc GJ, Leclerc GM, Barredo JC (2011) AMPK and Akt Determine Apoptotic Cell Death following Perturbations of One-Carbon Metabolism by Regulating ER Stress in Acute Lymphoblastic Leukemia. Mol Cancer Ther 10: 437-447.10.1158/1535-7163.MCT-10-0777 PubMed: 21262957 [PMC free article] [PubMed]
16. Terai K, Hiramoto Y, Masaki M, Sugiyama S, Kuroda T et al. (2005) AMP-activated protein kinase protects cardiomyocytes against hypoxic injury through attenuation of endoplasmic reticulum stress. Mol Cell Biol 25: 9554-9575.10.1128/MCB.25.21.9554-9575.2005 PubMed: 16227605 [PMC free article] [PubMed]
17. Yeh CH, Chen TP, Wang YC, Lin YM, Fang SW (2010) AMP-activated protein kinase activation during cardioplegia-induced hypoxia/reoxygenation injury attenuates cardiomyocytic apoptosis via reduction of endoplasmic reticulum stress. Mediat Inflamm, 2010: 2010: 130636 PubMed: 21318153 [PMC free article] [PubMed]
18. Dong Y, Zhang M, Wang S, Liang B, Zhao Z et al. (2010) Activation of AMP-activated protein kinase inhibits oxidized LDL-triggered endoplasmic reticulum stress in vivo. Diabetes 59: 1386-1396.10.2337/db09-1637 PubMed: 20299472 [PMC free article] [PubMed]
19. DeSalvo J, Kuznetsov JN, Du J, Leclerc GM, Leclerc GJ et al. (2012) Inhibition of Akt potentiates 2-DG-induced apoptosis via downregulation of UPR in acute lymphoblastic leukemia. Mol Cancer Res 10: 969-978.10.1158/1541-7786.MCR-12-0125 PubMed: 22692960 [PubMed]
20. Woehlbier U, Hetz C (2011) Modulating stress responses by the UPRosome: a matter of life and death. Trends Biochem Sci 36: 329-337.10.1016/j.tibs.2011.03.001 PubMed: 21482118 [PubMed]
21. Kim I, Xu W, Reed JC (2008) Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nat Rev Drug Discov 7: 1013-1030.10.1038/nrd2755 PubMed: 19043451 [PubMed]
22. Boyce M, Yuan J (2006) Cellular response to endoplasmic reticulum stress: a matter of life or death. Cell Death Differ 13: 363-373.10.1038/sj.cdd.4401817 PubMed: 16397583 [PubMed]
23. Hu P, Han Z, Couvillon AD, Exton JH (2004) Critical role of endogenous Akt/IAPs and MEK1/ERK pathways in counteracting endoplasmic reticulum stress-induced cell death. J Biol Chem 279: 49420-49429.10.1074/jbc.M407700200 PubMed: 15339911 [PubMed]
24. Pfaffenbach KT, Lee AS (2011) The critical role of GRP78 in physiologic and pathologic stress. Curr Opin Cell Biol 23: 150-156.10.1016/ PubMed: 20970977 [PMC free article] [PubMed]
25. Shore GC, Papa FR, Oakes SA (2011) Signaling cell death from the endoplasmic reticulum stress response. Curr Opin Cell Biol 23: 143-149.10.1016/ PubMed: 21146390 [PMC free article] [PubMed]
26. Kim I, Shu CW, Xu W, Shiau CW, Grant D et al. (2009) Chemical biology investigation of cell death pathways activated by endoplasmic reticulum stress reveals cytoprotective modulators of ASK1. J Biol Chem 284: 1593-1603 PubMed: 19004820 [PMC free article] [PubMed]
27. Rutkowski DT, Kaufman RJ (2007) That which does not kill me makes me stronger: adapting to chronic ER stress. Trends Biochem Sci 32: 469-476.10.1016/j.tibs.2007.09.003 PubMed: 17920280 [PubMed]
28. Yecies JL, Manning BD (2011) Transcriptional control of cellular metabolism by mTOR signaling. Cancer Res 71: 2815-2820.10.1158/0008-5472.CAN-10-4158 PubMed: 21487041 [PMC free article] [PubMed]
29. Korets SB, Czok S, Blank SV, Curtin JP, Schneider RJ (2011) Targeting the mTOR/4E-BP pathway in endometrial cancer. Clin Cancer Res 17: 7518-7528.10.1158/1078-0432.CCR-11-1664 PubMed: 22142830 [PubMed]
30. Tamburini J, Green AS, Bardet V, Chapuis N, Park S et al. (2009) Protein synthesis is resistant to rapamycin and constitutes a promising therapeutic target in acute myeloid leukemia. Blood 114: 1618-1627.10.1182/blood-2008-10-184515 PubMed: 19458359 [PubMed]
31. Beharry Z, Mahajan S, Zemskova M, Lin YW, Tholanikunnel BG et al. (2011) The Pim protein kinases regulate energy metabolism and cell growth. Proc Natl Acad Sci U S A 108: 528-533.10.1073/pnas.1013214108 PubMed: 21187426 [PubMed]
32. Amaravadi R, Thompson CB (2005) The survival kinases Akt and Pim as potential pharmacological targets. J Clin Invest 115: 2618-2624.10.1172/JCI26273 PubMed: 16200194 [PMC free article] [PubMed]
33. Mikkers H, Nawijn M, Allen J, Brouwers C, Verhoeven E et al. (2004) Mice deficient for all PIM kinases display reduced body size and impaired responses to hematopoietic growth factors. Mol Cell Biol 24: 6104-6115.10.1128/MCB.24.13.6104-6115.2004 PubMed: 15199164 [PMC free article] [PubMed]
34. Cohen AM, Grinblat B, Bessler H, Kristt D, Kremer A et al. (2004) Increased expression of the hPim-2 gene in human chronic lymphocytic leukemia and non-Hodgkin lymphoma. Leuk Lymphoma 45: 951-955.10.1080/10428190310001641251 PubMed: 15291354 [PubMed]
35. Nawijn MC, Alendar A, Berns A (2011) For better or for worse: the role of Pim oncogenes in tumorigenesis. Nat Rev Cancer 11: 23-34.10.1038/nrc2986 PubMed: 21150935 [PubMed]
36. Leclerc GM, Leclerc GJ, Fu G, Barredo JC (2010) AMPK-induced activation of Akt by AICAR is mediated by IGF-1R dependent and independent mechanisms in acute lymphoblastic leukemia. J Mol Signal 5: 15.10.1186/1750-2187-5-15 PubMed: 20863384 [PMC free article] [PubMed]
37. Chou TC (2006) Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 58: 621-681.10.1124/pr.58.3.10 PubMed: 16968952 [PubMed]
38. Thériault JR, Palmer HJ, Pittman DD (2011) Inhibition of the Unfolded Protein Response by metformin in renal proximal tubular epithelial cells. Biochem Biophys Res Commun 409: 500-505.10.1016/j.bbrc.2011.05.034 PubMed: 21600878 [PubMed]
39. Saito S, Furuno A, Sakurai J, Sakamoto A, Park HR et al. (2009) Chemical genomics identifies the unfolded protein response as a target for selective cancer cell killing during glucose deprivation. Cancer Res 69: 4225-4234.10.1158/0008-5472.CAN-08-2689 PubMed: 19435925 [PubMed]
40. Oyadomari S, Mori M (2004) Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ 11: 381-389.10.1038/sj.cdd.4401373 PubMed: 14685163 [PubMed]
41. Schönthal AH (2013) Pharmacological targeting of endoplasmic reticulum stress signaling in cancer. Biochem Pharmacol 85: 653-666.10.1016/j.bcp.2012.09.012 PubMed: 23000916 [PubMed]
42. Benjamin D, Colombi M, Moroni C, Hall MN (2011) Rapamycin passes the torch: a new generation of mTOR inhibitors. Nat Rev Drug Discov 10: 868-880.10.1038/nrd3531 PubMed: 22037041 [PubMed]
43. Woods A, Johnstone SR, Dickerson K, Leiper FC, Fryer LG et al. (2003) LKB1 is the upstream kinase in the AMP-activated protein kinase cascade. Curr Biol 13: 2004-2008.10.1016/j.cub.2003.10.031 PubMed: 14614828 [PubMed]
44. Hawley SA, Pan DA, Mustard KJ, Ross L, Bain J et al. (2005) Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase. Cell Metab 2: 9-19.10.1016/j.cmet.2005.05.009 PubMed: 16054095 [PubMed]
45. Hammerman PS, Fox CJ, Birnbaum MJ, Thompson CB (2005) Pim and Akt oncogenes are independent regulators of hematopoietic cell growth and survival. Blood 105: 4477-4483.10.1182/blood-2004-09-3706 PubMed: 15705789 [PubMed]
46. Zha J, Harada H, Yang E, Jockel J, Korsmeyer SJ (1996) Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X. Cell Volumes L: 87: 619-628 [PubMed]
47. El-Mir MY, Nogueira V, Fontaine E, Avéret N, Rigoulet M et al. (2000) Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I. J Biol Chem 275: 223-228.10.1074/jbc.275.1.223 PubMed: 10617608 [PubMed]
48. Xu C, Bailly-Maitre B, Reed JC (2005) Endoplasmic reticulum stress: cell life and death decisions. J Clin Invest 115: 2656-2664.10.1172/JCI26373 PubMed: 16200199 [PMC free article] [PubMed]
49. Matsuo J, Tsukumo Y, Saito S, Tsukahara S, Sakurai J et al. (2012) Hyperactivation of 4E-binding protein 1 as a mediator of biguanide-induced cytotoxicity during glucose deprivation. Mol Cancer Ther 11: 1082-1091.10.1158/1535-7163.MCT-11-0871 PubMed: 22402126 [PubMed]
50. Grimaldi C, Chiarini F, Tabellini G, Ricci F, Tazzari PL et al. (2012) AMP-dependent kinase/mammalian target of rapamycin complex 1 signaling in T-cell acute lymphoblastic leukemia: therapeutic implications. Leukemia 26: 91-100.10.1038/leu.2011.269 PubMed: 21968881 [PubMed]
51. Kovacic S, Soltys CL, Barr AJ, Shiojima I, Walsh K et al. (2003) Akt activity negatively regulates phosphorylation of AMP-activated protein kinase in the heart. J Biol Chem 278: 39422-39427.10.1074/jbc.M305371200 PubMed: 12890675 [PubMed]

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