Upregulation Of p-AMPK Research Articles

Axonal protection by combination of ripasudil and brimonidine with upregulation of p-AMPK in TNF-induced optic nerve degeneration

PurposeThe ROCK inhibitor ripasudil hydrochloride hydrate was shown to have axonal protective effects in TNF-induced optic nerve degeneration. The α2-adrenoreceptor agonist brimonidine was also shown to exert axonal protection. The current study aimed to elucidate whether additive axonal protection was achieved by the simultaneous injection of ripasudil and brimonidine and examine the association with AMPK activation.MethodsIntravitreal administration was performed in the following groups: PBS, TNF, or TNF with ripasudil, with brimonidine, or with a combination of ripasudil and brimonidine. Axon numbers were counted to evaluate the effects against axon loss. Immunoblot analysis was performed to examine phosphorylated AMPK expression in optic nerves, and immunohistochemical analysis was performed to evaluate the expression levels of p-AMPK and neurofilament in the optic nerve.ResultsBoth ripasudil alone or brimonidine alone resulted in significant neuroprotection against TNF-induced axon loss. The combination of ripasudil and brimonidine showed additive protective effects. Combined ripasudil and brimonidine plus TNF significantly upregulated p-AMPK levels in the optic nerve compared with the TNF groups. Immunohistochemical analysis revealed that p-AMPK is present in axons and enhanced by combination therapy.ConclusionThe combination of ripasudil and brimonidine may have additive protective effects compared with single-agent treatment alone. These protective effects may be at least partially associated with AMPK activation.

Gastrodin ameliorates postoperative cognitive dysfunction in aged mice by promoting nuclear translocation of Nrf2 through activation of AMPK

BackgroundPerioperative neurocognitive disorder (PND) remains a prevalent complication following anesthesia and surgery. Recent studies have revealed the therapeutic potential of gastrodin (GAS) in treating cognitive disturbances. This study delves deeper into the mechanisms through which GAS impacts PND. MethodsMale C57BL/6 mice (18 months old) underwent laparotomies and were administered GAS orally daily for three weeks preceding surgery and one week post-surgery. Thirty minutes before GAS administration, an intraperitoneal injection of Compound C was given. In vitro, H2O2-incubated SH-SY-5Y cells, with or without Nrf2-siRNA transfection, were set up and subjected to GAS or Compound C treatments. Cell viability was assessed via MTT assays, and apoptosis levels were assessed through flow cytometry. Cognitive function was evaluated using the Morris water maze, novel object recognition, and Y-maze tests. Oxidative stress markers, including MDA, SOD, GSH, GSH-px, and intracellular ROS (determined through immunofluorescence), were quantified. The expression of the genes Caspase3, Bax, Bcl-2, GST, and NQO1 was gauged using real-time RT-PCR. Brain, cortex and hippocampal pathologies were examined with hematoxylin-eosin (HE) and NeuN/TUNEL costaining. Finally, Nrf2 and p-AMPK were analyzed using Western blotting (WB) and immunofluorescence assays. ResultsGAS improved cognitive dysfunction in PND mice and reduced oxidative stress, neuro-apoptosis, and ROS levels both in vivo and in vitro experiment. In vivo, Immunofluorescence and Western blot outcomes indicated that postoperative p-AMPK and Nrf2 levels in the hippocampus were mitigated but were augmented by GAS. In vitro studies revealed GAS's protective effect against H2O2-induced oxidative stress and apoptosis and its upregulation of p-AMPK and Nrf2 in SH-SY-5Y cells. Notably, this protective effect was negated when Nrf2 siRNA was introduced. ELISA and PCR results highlighted the role of GAS in enhancing GST and NQO1 activity in both the mice hippocampus and SH-SY-5Y cells. Compound C, an AMPK inhibitor, both in vitro and in vivo, reversed the beneficial effects of GAS on Nuc-Nrf2/Cyt-Nrf2 expression and counteracted the positive influence of GAS on cognitive functions in PND mice. ConclusionGAS facilitates the nuclear translocation of Nrf2 via AMPK activation, offering a therapeutic avenue for alleviating postoperative cognitive impairments in mice, with a significant reduction in oxidative stress.

Axonal Protection by Oral Nicotinamide Riboside Treatment with Upregulated AMPK Phosphorylation in a Rat Glaucomatous Degeneration Model.

Nicotinamide riboside (NR), a precursor of nicotinamide adenine dinucleotide (NAD+), has been studied to support human health against metabolic stress, cardiovascular disease, and neurodegenerative disease. In the present study, we investigated the effects of oral NR on axonal damage in a rat ocular hypertension model. Intraocular pressure (IOP) elevation was induced by laser irradiation and then the rats received oral NR of 1000 mg/kg/day daily. IOP elevation was seen 7, 14, and 21 days after laser irradiation compared with the controls. We confirmed that oral NR administration significantly increased NAD+ levels in the retina. After 3-week oral administration of NR, morphometric analysis of optic nerve cross-sections showed that the number of axons was protected compared with that in the untreated ocular hypertension group. Oral NR administration significantly prevented retinal ganglion cell (RGC) fiber loss in retinal flat mounts, as shown by neurofilament immunostaining. Immunoblotting samples from the optic nerves showed that oral NR administration augmented the phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) level in rats with and without ocular hypertension induction. Immunohistochemical analysis showed that some p-AMPK-immunopositive fibers were colocalized with neurofilament immunoreactivity in the control group, and oral NR administration enhanced p-AMPK immunopositivity. Our findings suggest that oral NR administration protects against glaucomatous RGC axonal degeneration with the possible upregulation of p-AMPK.

TRIM38 suppresses migration, invasion, metastasis, and proliferation in non-small cell lung cancer (NSCLC) via regulating the AMPK/NF-κB/NLRP3 pathway.

Accumulating data have revealed the pivotal function of tripartite motif protein 38 (TRIM38) in tumors. In view of this, this investigation aims to explore the function and potential mechanism of TRIM38 in non-small cell lung cancer (NSCLC). A xenotypic tumor model was established in vivo by subcutaneously injecting NSCLC cells (2 × 106 cells) in tail vein of each mouse. Relative expression of TRIM38 mRNA was detected via quantitative real-time polymerase chain reaction (qRT-PCR). For exploring the role of TRIM38 in vivo and in vitro, mice or NSCLC cells were divided into two groups: the vector group and the TRIM38 overexpression group. Also, protein expression levels of TRIM38, Vimentin, E-cadherin, and N-cadherin were determined using western blotting and immunohistochemistry staining. Tumor nodules of mouse lung tissues were assessed via performing H&E staining. Moreover, proliferation of NSCLC cells was evaluated through colony formation and CCK-8 assays. Further, migration and invasion of NSCLC cells were assessed through wound healing and transwell assays. Protein levels of pathway-related proteins including p-p65, p65, IκB, p-IκB, p-AMPK, AMPK, and NLRP3 were examined through western blotting analysis. Tumor lung tissues of mice and NSCLC cells showed low protein and mRNA expression of TRIM38. Functionally, up-regulation of TRIM38 reduced the number of tumor nodules and suppressed epithelial-to-mesenchymal transition (EMT) in lung tissues of mice. Furthermore, up-regulation of TRIM38 in NSCLC cells inhibited migration, invasion, EMT, and proliferation. With respect to the mechanism, in vivo experiments, the inhibitory effects of TRIM38 overexpression on tumor nodules, and EMT were reversed by AMPK inhibitor. In vitro experiments, TRIM38 overexpression caused down-regulation of p-IκB and p-p65 as well as up-regulation of p-AMPK. The inhibitory effects of TRIM38 overexpression on migration, proliferation, invasion, and EMT of NSCLC cells were reversed by overexpression of NLRP3. Concurrently, AMPK inhibitor enhanced the TRIM38-overexpressed NSCLC cell's abilities in migration, clone formation, invasion, and proliferation. TRIM38 regulated the AMPK/NF-κB/NLRP3 pathway to suppress the NSCLC's progression and development.

Roflumilast ameliorates ovariectomy-induced depressive-like behavior in rats via activation of AMPK/mTOR/ULK1-dependent autophagy pathway

AimsRoflumilast, a well-known phosphodiesterase-4 (PDE-4) inhibitor, possess an anti-inflammatory activity with approved indications in chronic obstructive pulmonary disease. This study aimed to evaluate the neuroprotective role of roflumilast in ovariectomy (OVX)-induced depressive-like behavior in female rats and to shed light on a potential autophagy enhancing effect. Main methodsRats were randomly divided into four groups: sham, OVX, OVX + roflumilast (1 mg/kg, p.o), and OVX + roflumilast + chloroquine (CQ) (50 mg/kg, i.p). Drugs were administered for 4 weeks starting 2 weeks after OVX. Key findingsRoflumilast improved the depressive-like behaviors observed in OVX rats as evidenced by decreasing both forced swimming and open field immobility times while, increasing % sucrose preference and number of open field crossed squares. Histopathological analysis provides further evidence of roflumilast's beneficial effects, demonstrating that roflumilast ameliorated the neuronal damage caused by OVX. Roflumilast antidepressant potential was mediated via restoring hippocampal cAMP and BDNF levels as well as down-regulating PDE4 expression. Moreover, roflumilast revealed anti-inflammatory and anti-apoptotic effects via hindering TNF-α level and diminishing Bax/Bcl2 ratio. Roflumilast restored the autophagic function via up-regulation of p-AMPK, p-ULK1, Beclin-1 and LC3II/I expression, along with downregulation of P62 level and p-mTOR protein expression. The autophagy inhibitor CQ was used to demonstrate the suggested pathway. SignificanceThe present study revealed that roflumilast showed an anti-depressant activity in OVX female rats via turning on AMPK/mTOR/ULK1-dependent autophagy pathway; and neurotrophic, anti-inflammatory, and anti-apoptotic activities. Roflumilast could offer a more secure alternative to hormone replacement therapy for postmenopausal depression treatment.

AMPK/mTOR/ULK1 Axis-Mediated Pathway Participates in Apoptosis and Autophagy Induction by Oridonin in Colon Cancer DLD-1 Cells.

BackgroundOridonin has been demonstrated to exert strong antitumor activities in various types of human cancers. Our previous study established that oridonin induced the apoptosis of and exerted an inhibitory effect on colon cancer cells in vitro and in vivo. However, the mechanisms behind the antitumor effects of oridonin on colorectal cancer are not clearly known. This study explored whether autophagy was involved in antitumorigenesis effects caused by the usage of oridonin in colon cancer and examined whether the AMPK/mTOR/ULK1 signaling pathway was involved in this process.MethodsCell viability was determined using CCK-8 assay. The distribution of cell apoptosis was evaluated using flow cytometry. RT-PCR and Western blotting analysis were conducted to identify the key target genes and proteins involved in the AMPK/mTOR cascade. AMPK siRNA was used to disturb AMPK expression. A DLD-1 cell orthotopic transplantation tumor model was established to explore the anti-cancer effects in vivo.ResultsOridonin exhibited a suppressive effect on DLD-1 cells in a concentration- and time-dependent manner. Additionally, in a dose-dependent manner, oridonin induced cell apoptosis via inducing the protein expression levels of cleaved caspase-3, cleaved PARP and stimulated autophagy by increasing protein expression levels of Becin1, LC3-II, decreasing protein expression levels of LC3-I, p62, which were respectively attenuated and elevated by autophagy inhibitor 3-MA. Furthermore, oridonin upregulated the expression level of p-AMPK and downregulated the expression levels of p-mTOR, p-ULK1 in the DLD-1 cells in a dose-dependent manner. Moreover, knockdown of AMPK by a specific siRNA reversed the expression levels of proteins involved in the AMPK/mTOR pathway, autophagy and apoptosis. In addition, outcomes from the in vivo experiments also showed that oridonin treatment significantly repressed tumorigenic growth of DLD-1 cells without any side effects, which was accompanied by the upregulation of p-AMPK, LC3-II, active caspase-3 protein expression levels and the downregulation of p-mTOR and p-ULK1 protein expression levels.ConclusionThis study demonstrated that oridonin induced apoptosis and autophagy of colon cancer DLD-1 cells via regulating the AMPK/mTOR/ULK1 pathway, which indicated that oridonin may be used as a novel therapeutic intervention for patients with colorectal cancer.

Palbociclib induces activation of AMPK and inhibits hepatocellular carcinoma in a CDK4/6-independent manner.

Palbociclib, a CDK4/6 inhibitor, has recently been approved for hormone receptor‐positive breast cancer patients. The effects of palbociclib as a treatment for other malignancies, including hepatocellular carcinoma (HCC), are of great clinical interest and are under active investigation. Here, we report the effects and a novel mechanism of action of palbociclib in HCC. We found that palbociclib induced both autophagy and apoptosis in HCC cells through a mechanism involving 5′ AMP‐activated protein kinase (AMPK) activation and protein phosphatase 5 (PP5) inhibition. Blockade of AMPK signals or ectopic expression of PP5 counteracted the effect of palbociclib, confirming the involvement of the PP5/AMPK axis in palbociclib‐mediated HCC cell death. However, CDK4/6 inhibition by lentivirus‐mediated shRNA expression did not reproduce the effect of palbociclib‐treated cells, suggesting that the anti‐HCC effect of palbociclib is independent of CDK4/6. Moreover, two other CDK4/6 inhibitors (ribociclib and abemaciclib) had minimal effects on HCC cell viability and the PP5/AMPK axis. Palbociclib also demonstrated significant tumor‐suppressive activity in a HCC xenograft model, which was associated with upregulation of pAMPK and PP5 inhibition. Finally, we analyzed 153 HCC clinical samples and found that PP5 expression was highly tumor specific and was associated with poor clinical features. Taken together, we conclude that palbociclib exerted antitumor activity against HCC through the PP5/AMPK axis independent of CDK4/6. Our findings provide a novel mechanistic basis for palbociclib and reveal the therapeutic potential of targeting PP5/AMPK signaling with a PP5 inhibitor for the treatment of hepatocellular carcinoma.

Dutasteride and Metformin Reduce the Growth of LNCaP Cells and Alter the SREBP-1 Pathway

Prostate cancer (PCa) is the second most prevalent cancer in men after lung cancer. Prostate cancer develop- ment and progression is associated with the dysregulation of a number of molecular pathways; hence, therapeutic strate- gies targeting such pathways bring great promise. Recently we have shown that metformin, the anti-diabetic drug, can in- hibit tumor progression when combined with dutasteride, a 5-alpha-reductase inhibitor (5ARI). Interestingly, both met- formin and dutasteride have been reported to alter the Sterol Regulatory Element Binding Proteins (SREBP) Fatty Acid Synthase (FASN) pathway. The SREBP pathway is involved with lipid and energy homeostasis. In our present study, we investigated if dutasteride in combination with metformin can reduce the proliferation of LNCaP PCa cells, and whether this is mediated through the SREBP-1/FASN pathway. Human PCa cells were treated with either dutasteride (0-100 µM) or metformin (0-50 mM) alone or in combination. The treated cells were then incubated for up to 24 hours, and prolifera- tion assessed using the MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazo- lium) assay. Western blot analysis was performed on cell lysates to assess alterations in key signaling molecules including cleaved SREBP-1, FASN, AR, PSA, pAMPK and apoptotic markers. Results revealed that there was a significant (p<0.05) decrease in cellular proliferation of LNCaP cells treated with a combination of metformin and dutasteride, this effect being greater than either treatment alone. Treating LNCaP cells with both metformin and dutasteride reduced the expression of FASN, cleaved SREBP-1 and pro-caspase-3 with expression of cleaved PARP; suggesting a possible inter- action between FASN and apoptosis. All treatments resulted in reductions in AR, PSA and an upregulation of pAMPK, with the highest expression seen in combination treatment. We report for the first time that metformin and dutasteride in combination can reduce the proliferation of androgen-sensitive cell line through the activation of p-AMPK, and SREBP- 1/FASN pathway and highlight the importance of targeting the SREBP-1 pathway, for improving future therapeutic strate- gies for prostate cancer.

Abstract PD03-02: Evidence for the anti-cancer action of metformin mediated via tumor AMPK, Akt and Ki67, in a preoperative window of opportunity trial

Abstract Background: Metformin reduces the overall risk of cancer incidence by 31%, improves cancer-related mortality and enhances response to neoadjuvant chemotherapy in type 2-diabetics receiving metformin. There is evidence that metformin works through activation of Adenosine Monophosphate Protein Kinase (AMPK), an energy-sensing kinase that maintains cellular energy homeostasis. In a pre-operative window-of-opportunity randomized trial we have demonstrated that metformin significantly reduces proliferation and blunts the insulin response in primary operable breast cancer. This study further analysed cancer tissue from the trial to dissect the molecular mechanisms involved. Methodology: Non-diabetic women with operable invasive breast cancer were randomised to receive pre-operative metformin or no drug. Forty seven patients had core biopsy at diagnosis then were randomized to metformin (metformin 500mg o.d. for 1 week increased to 1g b.d for a further week continued to surgery) or no drug, and 2 weeks later had core biopsy at surgery. Insulin receptor, phospho-AMPK (pAMPK), phospho-Akt (pAkt) and Ki67 immunohistochemistry was performed on formalin-fixed paraffin-embedded cores and scored blinded to treatment. Paired t-test was used for analysis. Results: Significant up-regulation of pAMPK (p = 0.04) and down-regulation of pAkt (p = 0.04) in metformin treated patients was demonstrated compared to the control group. No change in insulin receptor expression was identified but, as previously reported, there was a fall in ki67. Changes were independent of Body Mass Index. Seven patients (7/24) receiving metformin withdrew because of gastro-intestinal upset and were excluded from the immunohistochemical analyses. Conclusion: These findings suggest that metformin works in vivo in breast cancer patients via up-regulation of tumor pAMPK and down-regulation of pAkt and proliferation. Since down-regulation of pAMPK is a feature of breast cancer, this suggests mechanistic evidence for the therapeutic effect of metformin. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD03-02.

The BCR-ABL Fusion Protein Sensitizes Acute Lymphoblastic Leukemia to Apoptotic Death Induced by the Dual Glycolytic and Glycosylation Inhibitor 2-Deoxy-D-Glucose.

Abstract 2439Tumor metabolism has emerged as a hallmark of cancer by which the oncogenic profile of cancer cells pairs energy availability with cell growth and survival. BCR-ABL positive acute lymphoblastic leukemia (BCR-ABL+ ALL) is a highly resistant phenotype, and the BCR-ABL fusion protein has been correlated with alterations in glucose metabolism. The glucose analogue 2-deoxy-D-glucose (2DG) has been found to be an effective antitumor agent in both animal models and human clinical trials for solid tumors based on the dependency of hypoxic tumor cells on anaerobic glycolysis to generate ATP. 2DG inhibits two key glycolytic enzymes, hexokinase (HK) and phosphoglucose isomerase (PGI), and interfers with N-linked glycosylation by its incorporation in place of mannose into lipid-linked oligosaccharide (LLO) chains which leads to premature termination of LLO synthesis. Unlike hypoxic solid tumors, we recently demonstrated that 2DG induces cell death in ALL under normoxia preferentially by inhibiting N-linked glycosylation resulting in ER stress/UPR-mediated apoptosis, although inhibition of glycolysis also contributed to cell death in a phenotype specific-manner (Mol Cancer Res 10:969, 2012). Among ALL subtypes, BCR-ABL+ ALL t(9;22) cell lines SupB15 and TOM1 exhibited the highest sensitivity to 2-DG suggesting that BCR-ABL expression may be linked to sensitivity to 2DG. To investigate the role of BCR-ABL expression in this process, we constructed NALM6 (Bp-ALL) stable cell lines expressing BCR-ABL p190 fusion, and assessed their level of sensitivity to 2DG under normoxia. Expression of the BCR-ABL p190 fusion in NALM6 cells significantly increased 2DG-induced apoptosis compared to mock transfected NALM6 cells, indicating that BCR-ABL expression results in higher sensitivity to 2DG in this experimental system. Western blot analysis of BCR-ABL+ ALL (SUPB15 and TOM1) and Bp-ALL (NALM6) cell lines demonstrated that BCR-ABL expression induced alterations in the PI3K/Akt/p70S6K, GSK-3β/Mcl-1, p-STAT3 (Ser727), p-PKCδ (Thr505), and GRP78/p-eIF2α (Ser51) signaling pathways. Treatment with 2DG resulted in upregulation of p-AMPK (Thr172) and p-GSK-3β (Ser9), and downregulation of p-mTOR (Ser2448), p-p70S6K (Thr389), p-STAT3 (Ser727) and Mcl-1 in both BCR-ABL+ ALL (SUPB15 and TOM1) and Bp-ALL cells (NALM6). As we have previously described, 2DG induces ER stress and the expression of the unfolded protein response (UPR) markers (Mol Cancer Res 10:969, 2012). In BCR-ABL+ ALL cells, 2DG led to upregulation of IRE1α, GRP78 and CHOP (a marker of UPR induced cell death) and resulted in apoptotic cell death (>60%), which correlated with lower level of Mcl-1 expression. To further investigate the role of Mcl-1 on 2DG’s sensitivity we used pharmacological inhibitors and shRNA. We found that Mcl-1 down-regulation sensitized Bp-ALL cells to 2DG (36% ± 4% cell death; p<0.001 for NALM6/shMcl-1 vs. NALM6/shScramble treated with 2DG 0.5mM). We then examined the role of GSK-3β/Mcl-1 signaling in K562, a highly 2DG resistant CML cell line expressing BCR-ABL p210 fusion. We found that K562 cells expressed high levels of Mcl-1. Inhibition of PI3K/Akt/GSK-3β pathway using LY294002 sensitized K562 cells to 2DG, suggesting that downregulation of Mcl-1 by GSK-3β activation leads to 2DG sensitivity. Finally, we co-targeted BCR-ABL+ ALL cells with TKIs (Imatinib) plus the glycolytic/glycosylation inhibitor 2DG and found significant increase in cell death for the combination compared to single drug (62.6% ± 0.8% cell death; p<0.001 for 2DG 4mM + Imatinib 0.5μM vs. single drug). Further, no cross-resistance was detected between 2DG and Imatinib in TKI-resistant BCR-ABL+ ALL cells. Taken together, our data demonstrate that BCR-ABL p190 expression sensitizes Bp-ALL cells to 2DG by altering the GSK-3β/Mcl-1 signaling pathway and inducing ER stress/UPR mediated cell death. This study supports further consideration of strategies combining inhibitors of glucose metabolism such as 2DG with BCR-ABL TKIs for future clinical translation in BCR-ABL+ ALL. Disclosures:No relevant conflicts of interest to declare.

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

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 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 AICAR's 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&amp;lt;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&amp;lt;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&amp;lt;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