Jeffim N. Kuznetsov

AMPK and Akt Determine Apoptotic Cell Death following Perturbations of One-Carbon Metabolism by Regulating ER Stress in Acute Lymphoblastic Leukemia

AICAr is a cell-permeable nucleotide that has been used in vivo and in vitro to activate AMPK. Our previous findings have shown that AICAr as a single agent induces dose- and time-dependent growth inhibition in acute lymphoblastic leukemia (ALL) cell lines. In addition, the combination of AICAr with antifolates [methotrexate (MTX) or pemetrexed] has been shown to further potentiate AMPK activation and to lead to greater cytotoxicity and growth inhibition in leukemia and other malignant cell types. Our data presented herein show that sustained endoplasmic reticulum (ER) stress is the predominant mechanism behind the synergistic induction of cell death by the combination of AICAr plus the inhibitor of one-carbon metabolism, MTX, in Bp- and T-ALL, as evidenced by induction of several unfolded protein response markers leading to apoptosis. We also show for the first time that AICAr in combination with MTX significantly induces Akt phosphorylation in ALL. Under these conditions, the concomitant inhibition of Akt, a cellular antagonist of AMPK, leads to further upregulation of AMPK activity and alleviates AICAr plus MTX-induced ER stress and apoptosis. Therefore, we also show that the concomitant activation of AMPK actually rescues the cells from AICAr plus MTX-induced ER stress and apoptosis. Our data suggest that the effects of AMPK activation on cell death or survival differ contextually depending on its signaling alterations with related oncogenic pathways and provide insight into the reported paradoxical proapoptotic versus prosurvival effects of AMPK activation. Mol Cancer Ther; 10(3); 437–47. ©2011 AACR.

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.

Epigenetic reprogramming and aberrant expression of PRAME are associated with increased metastatic risk in Class 1 and Class 2 uveal melanomas

Background We previously identified PRAME as a biomarker for metastatic risk in Class 1 uveal melanomas. In this study, we sought to define a threshold value for positive PRAME expression (PRAME+) in a large dataset, identify factors associated with PRAME expression, evaluate the prognostic value of PRAME in Class 2 uveal melanomas, and determine whether PRAME expression is associated with aberrant hypomethylation of the PRAME promoter. Results Among 678 samples analyzed by qPCR, 498 (73.5%) were PRAME- and 180 (26.5%) were PRAME+. Class 1 tumors were more likely to be PRAME-, whereas Class 2 tumors were more likely to be PRAME+ (P < 0.0001). PRAME expression was associated with shorter time to metastasis and melanoma specific mortality in Class 2 tumors (P = 0.01 and P = 0.02, respectively). In Class 1 tumors, PRAME expression was directly associated with SF3B1 mutations (P < 0.0001) and inversely associated with EIF1AX mutations (P = 0.004). PRAME expression was strongly associated with hypomethylation at 12 CpG sites near the PRAME promoter. MATERIALS AND METHODS Analyses included PRAME mRNA expression, Class 1 versus Class 2 status, chromosomal copy number, mutation status of BAP1, EIF1AX, GNA11, GNAQ and SF3B1, and genomic DNA methylation status. Analyses were performed on 555 de-identified samples from Castle Biosciences, 123 samples from our center, and 80 samples from the TCGA. Conclusions PRAME is aberrantly hypomethylated and activated in Class 1 and Class 2 uveal melanomas and is associated with increased metastatic risk in both classes. Since PRAME has been successfully targeted for immunotherapy, it may prove to be a companion prognostic biomarker.

Abstract 605: A novel role for AMPK as a molecular switch regulating the activation of Akt and RAS signaling in acute lymphoblastic leukemia

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL 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 identified AMP activated protein kinase (AMPK) as a potential target for ALL therapy due to its effects on cell proliferation and cell cycle regulation, as well as its crosstalk with critical metabolic and oncogenic pathways. We demonstrated that treatment of NALM6 and CCRF-CEM cells with the AMPK activators AICAR and metformin induced significant cell growth inhibition and apoptosis. Rescue experiments using the AMPK inhibitor compound-C (CC) failed to completely abrogate the cytotoxic effects induced by AICAR, attenuating AICARs apoptosis by 42% in CCRF-CEM and 45% in NALM6. Whereas when used alone, both CC and Ara-A induced significant apoptosis in ALL cells. Using the caspase inhibitors Z-VAD and Z-IETD, we demonstrated that activation of AMPK by AICAR induced cell death via both caspase-dependent and independent mechanisms, whereas inhibition of AMPK by CC induced apoptosis mainly via the intrinsic caspase-dependent pathway. To examine the effects of AMPK activation vs. inhibition on downstream signaling, we used Western blot analysis of key signaling factors associated with the PI3K/Akt/mTOR and RAS/RAF/ERK pathways in ALL cells. We found that AICAR and CC exerted opposite effects on PI3K/Akt and RAS pathway signaling. CC decreased P-Akt (Ser473) and activated the RAS pathway, whereas AICAR increased P-Akt, suggesting that AMPK may play a role as regulator of Akt and RAS activity. We showed that activation of AMPK and Akt by AICAR down-regulated the RAS pathway via phosphorylation of cRAF at Ser621 and Ser259, respectively. Inhibition of the RAS pathway with farnesylthiosalicylic acid (FTS) and U-0126 induced significant cell growth arrest and apoptosis in ALL cells (p<0.0001) and resulted in up-regulation of P-AMPK (Thr172) and P-Akt (Thr308). The phosphorylation of Akt at Thr308 was mediated by AMPK-dependent activation of IGF-1R/IRS-1, since down-regulation of AMPK using shRNA abolished the activation of both P-Akt (Thr308) and IGF-1R (Tyr1131). Inhibition of AMPK activity with either CC or shRNA sensitized ALL cells to RAS inhibition (p<0.001). In addition, co-targeting RAS pathway signaling plus either IGF-1R or Akt pathways resulted in significant cell growth inhibition and apoptosis in ALL cells (p<0.001), confirming the functional relevance of this feedback loop mechanism. Taken together, our results indicate that activation and inhibition of AMPK induce cell death in ALL cells through alternative signaling mechanisms impinging on the expression of critical cell proliferation pathways. The data presented here support a new role for AMPK as a molecular regulatory switch coordinating cellular survival responses by activating PI3K/Akt/mTOR and RAS/RAF/ERK oncogenic pathways in ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 605. doi:10.1158/1538-7445.AM2011-605

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

Abstract 2736: PIM2 is up-regulated in response to metformin-induced cell death triggered by ER stress/UPR in ALL lymphoblasts

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children, and despite significant overall improvements in cure rates, outcome for patients diagnosed with resistant phenotypes or those who relapse is dismal. We investigated the mechanism of cell death induced by metformin (MET) in ALL cell models. We showed that MET induced significant growth inhibition and apoptosis in CCRF-CEM (T-ALL) and NALM6 (Bp-ALL) cells. Western blots revealed that MET activated p-AMPK, p-ACC, p-Akt, and p-4EBP1. The latter suggested that regulation of protein translation may be an important determinant in MET-induced cell death. Indeed, inhibition of mTOR/protein translation with rapamycin (RAPA) rescued MET-induced cell death in ALL cells. In addition, knockdown of AMPK expression in ALL cells using shRNA (shAMPK) abrogated MET-induced apoptosis as compared to control cells expressing scramble shRNA (shCTRL), indicating that AMPK mediated MET9s cytotoxicity. Western blots demonstrated that shAMPK cells expressed lower level of total AMPK, p-p38MAPK, p-mTOR, and p-4EBP1 compared to shCTRL cells, indicating that AMPK and protein translation are critical in MET sensitivity. Indeed, pulse labeled 35S-methionine experiments demonstrated increase incorporation confirming the importance of protein synthesis is MET-induced cytotoxicity. More important, we uncovered that MET-induced apoptosis correlated with induction of ER stress as evidenced by up-regulation of IRE1α and CHOP although GRP78 decreased significantly. We further demonstrated that RAPA rescued MET-treated cells by relieving ER stress/UPR mediated cell death. We previously showed that the unfolded protein response (UPR) in ALL cells is regulated by the contextual crosstalk between AMPK and Akt (Mol Cancer Ther 10:437, 2011). Therefore, we evaluated the effects of co-targeting Akt and AMPK using the Akt inhibitor X/perifosine + MET and found these combinations to be synergistic. To further investigate the relationship between protein translation and ER stress/UPR in MET-induced cell death, we examined the role of PIM1/2 kinases. We found that expression of PIM2 was increased in MET-treated ALL with concomitant decreased in the expression of IRE1α, ATF6, and CHOP, suggesting that PIM2 maybe up-regulated as a compensatory survival mechanism aimed at relieving MET-induced ER stress/UPR mediated cell death. To test this hypothesis, we co-treated ALL cells with a PIM1/2 kinase inhibitor + MET and found that PIM2 inhibition synergistically sensitized ALL cells to MET (CI=0.28). Taken together, our data indicate that PIM2 plays a role in buffering cell death in MET-treated cells, and that regulation of protein translation modulates ER stress/UPR induced apoptosis in ALL cells. Consequently, our data support strategies targeting these synthetic lethal interactions as suitable for clinical translation in patients with ALL. 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 2736. doi:1538-7445.AM2012-2736

Abstract 5089: AMPK activation vs. inhibition induces apoptosis in acute lymphoblastic leukemia cells by differentially altering PI3K/Akt/mTOR or RAS/cRAF/MEK/Erk signaling

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Acute Lymphoblastic Leukemia (ALL) is the most common hematological malignancy and the main cause of cancer-related deaths in children. Therefore, search for novel treatment strategies is warranted. We identified AMP activated protein kinase (AMPK), a master regulator of bioenergetics, as a potential target for ALL therapy due to its effects on cell proliferation and cell cycle regulation, as well as its crosstalk with critical metabolic and oncogenic pathways. We demonstrated that treatment of NALM6 (Bp-ALL) and CEM (T-ALL) cells with AICAR, an AMPK activator, induced growth inhibition and apoptosis. Using metformin, another AMPK agonist, we found 40% growth inhibition, and up to five- and three-fold greater induction of apoptosis relative to controls in CEM and NALM6, respectively. Unexpectedly, rescue experiments with AMPK inhibitors Ara-A and compound-C (CC) failed to abrogate the cytotoxic effects induced by AICAR. When used alone, Ara-A induced 60% and 40% cell death in NALM6 and CEM cells, respectively, whereas CC induced 75- and 15-fold more apoptotic death relative to controls. To investigate the mechanism by which AMPK activation vs. inhibition induced apoptosis, we determined levels of P-AMPK (T172) and factors associated with the PI3K/Akt/mTOR and RAS/cRAF/Erk signaling pathways in NALM6 and CEM cells treated with either CC, AICAR, or in combination. Our data show that P-AMPK levels were decreased by CC and increased by AICAR. Additional Western blots demonstrated that these agents exerted opposite effects on Akt and RAS signaling. CC decreased P-Akt (S473) and activated the RAS pathway, while AICAR increased P-Akt. We showed that activation of Akt by AICAR down-regulated the RAS pathway via phosphorylation of cRAF (S259). P-mTOR (S2448) and P-4EBP1 (T70) exhibited a greater decrease in cells treated with CC + AICAR as compared to each agent alone. A significant decrease in P-Akt was also detected in cells treated with both agents vs. each drug alone. Together, our data indicate that AICAR and CC induce cell death in ALL cells by two different mechanisms mediated by AMPK: AICAR-activation of AMPK inhibited the RAS-dependent cell proliferation pathway, and CC-inhibition of AMPK by down-regulating the Akt cell survival pathway. These results suggest that alterations in AMPK signaling may regulate the cross-talk between the PI3K/Akt/mTOR and RAS/cRAF/Erk cascades and may dictate the fate of ALL cells by regulating apoptosis after exposure to agents targeting these pathways. Experiments co-targeting AMPK and Akt using AICAR and Akt-inhibitor X, respectively, induced synergistic growth inhibition in CEM (CI=0.90) and NALM6 (CI=0.85) cells compared to each drug alone. These findings provide a rationale for simultaneously targeting AMPK and key signaling factors associated with either PI3K/Akt/mTOR or RAS/cRAF/Erk pathways in ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5089.