Previous reports demonstrate that metformin, an anti-diabetic drug, can decrease the risk of cancer and inhibit cancer cell growth. However, its mechanism in cancer cells is still unknown. Metformin significantly blocks cell cycle and inhibits cell proliferation and colony formation of leukemic cells. However, the apoptotic response to metformin varies. Furthermore, daily treatment with metformin induces apoptosis and reduces tumor growth in vivo. While metformin induces early and transient activation of AMPK, inhibition of AMPKα1/2 does not abrogate anti-proliferative or pro-apoptotic effects of metformin. Metformin decreases electron transport chain complex I activity, oxygen consumption and mitochondrial ATP synthesis, while stimulating glycolysis for ATP and lactate production, pentose phosphate pathway for purine biosynthesis, fatty acid metabolism, as well as anaplerotic and mitochondrial gene expression. Importantly, leukemic cells with high basal AKT phosphorylation, glucose consumption or glycolysis exhibit a markedly reduced induction of the Pasteur effect in response to metformin and are resistant to metformin-induced apoptosis. Accordingly, glucose starvation or treatment with deoxyglucose or an AKT inhibitor induces sensitivity to metformin. Overall, metformin elicits reprogramming of intermediary metabolism leading to inhibition of cell proliferation in all leukemic cells and apoptosis only in leukemic cells responding to metformin with AKT phosphorylation and a strong Pasteur effect.

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by GAA triplet expansions or point mutations in the FXN gene on chromosome 9q13. The gene product called frataxin, a mitochondrial protein that is severely reduced in FRDA patients, leads to mitochondrial iron accumulation, FeS cluster deficiency and oxidative damage. The tissue specificity of this mitochondrial disease is complex and poorly understood. While frataxin is ubiquitously expressed, the cellular phenotype is most severe in neurons and cardiomyocytes. Here, we conducted comprehensive proteomic, metabolic and functional studies to determine whether subclinical abnormalities exist in mitochondria of blood cells from FRDA patients. Frataxin protein levels were significantly decreased in platelets and peripheral blood mononuclear cells from FRDA patients. Furthermore, the most significant differences associated with frataxin deficiency in FRDA blood cell mitochondria were the decrease of two mitochondrial heat shock proteins. We did not observe profound changes in frataxin-targeted mitochondrial proteins or mitochondrial functions or an increase of apoptosis in peripheral blood cells, suggesting that functional defects in these mitochondria are not readily apparent under resting conditions in these cells.

4351 Metformin is a biguanide compound widely used for the treatment of type 2 diabetes. Several epidemiological studies have shown that metformin may reduce the risk of cancer in these patients and recent works in cancer suggest that this drug class may have anti-neoplastic activity. Metformin is known to have at least two mechanisms of action, which may be interrelated, inhibition of electron transport chain complex I and modulation of intracellular signal transduction pathways especially AMP Kinase. We hypothesized that acute myeloid leukemia (AML) cells may be sensitive to this agent and have studied its effects on cell survival and cellular metabolism in several AML cell lines. Metformin consistently and markedly decreased oxygen consumption of six leukemic cell lines in a concentration-dependent manner. However, only MOLM14 cells showed significant apoptosis when treated with metformin alone or in combination with a conventional chemotherapeutic agent (cytosine arabinoside). In addition, only MOLM14 cells exhibit a significant increase of the extracellular lactate level (Pasteur effect) in response to metformin-induced inhibition of the mitochondrial electron transport chain complex I. By contrast, U937 cells, another AML cell line are insensitive to metformin with a marked decrease of the Pasteur effect, suggesting that intrinsic metabolic differences may contribute to the cytotoxic effect of metformin in vitro. Interestingly, we first observed highest glucose consumption and glutathione content as well as differentially expressed genes encoding several enzymes that catalyze glycolytic and anapleurotic reactions in metformin-insensitive U937 cells compared to metformin-sensitive MOLM14 cells. Accordingly, treatment of U937 cells with an inhibitor of glycolysis sensitized U937 cells to metformin while their treatment with an inhibitor of the glutathione synthesis did not abrogate their insensitivity. Finally, treatment of insensitive HL60 cells with activators of mitochondrial oxygen consumption and cell differentiation sensitized these cells to metformin. Taken together, these findings suggest that a high glycolytic flux for production of ATP and biosynthetic precursors coupled to significant routing to the pentose phosphate pathway for NADPH for biosynthesis and GSH regeneration are key components which counterbalance the metformin-induced cytotoxic stress in U937 cells. Furthermore, based on these results, we can hypothesize that AML cell lines, and perhaps primary AML patient samples undergo a reprogramming of diverse metabolic pathways, which might be exploited by targeted therapies. Experiments on metabolic and signaling pathways as well as in vivo studies are in progress to better characterize alterations in different metabolic pathways which mediate the cytotoxic response of metformin in both AML cell lines and primary patient specimens, and thereby impact the therapeutic potential of metformin in vivo. Disclosures: Carroll: Cephalon Oncology: Consultancy; Sanofi Aventis Corporation: Research Funding; Agios Pharmaceuticals: Research Funding; Tetralogic Pharmaceuticals: Research Funding.

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2601 An emerging hallmark of cancer cells is the reprogramming of intermediary and energy metabolism these cells undergo. Several epidemiological studies have shown that metformin, widely used to treat patients with type 2 diabetes, may reduce their risk of cancer. Despite several reports of anti-neoplastic activity of metformin, the mechanisms responsible for this activity have not been fully elucidated in cancer or leukemic cells. We hypothesized that metformin elicits a metabolic reprogramming driven by alterations in mitochondrial function and signaling, which induces apoptosis in leukemic cells, and that metabolic flexibility determines the variation(s) of the cytotoxic response to metformin among different leukemic cell lines. We first demonstrated that metformin markedly decreased oxygen consumption of six leukemic cell lines in a concentration-dependent manner. We also observed that the cytotoxic effect of metformin varies between cell lines reflecting their energetic capacity to compensate for the mitochondrial inhibition induced by metformin (eg. to induce the Pasteur effect). Importantly, metformin-insensitive leukemic cells did not exhibit a Pasteur effect in response to metformin. All leukemic cells exhibited high basal conversion of glucose to lactate (eg. aerobic glycolysis) and specific expression of key metabolic genes as compared to normal mononuclear cells. Despite dependence on glucose catabolism, metformin sensitivity was associated with relative resistance to glucose starvation. Metformin effects in drug-resistant cells were potentiated by the addition of a glycolytic inhibitor, but not by inhibitors of the pentose phosphate pathway or glutaminolysis. Leukemic cells with broad metabolic capacities to utilize other energetic substrates in response to diverse nutrient starvation showed insensitivity to metformin. Metformin induced a significant decrease in metabolites of the upper segment of glycolysis and the oxidative branch of the pentose phosphate pathway as well as a clear increase of PRPP and IMP biosynthesis. Energy charge, the nucleotide phosphate pool and lactate/glucose ratio remained stable after metformin treatment. Furthermore, our results showed that basal glucose uptake/consumption and the activity of the lower segment of the glycolytic pathway are key determinants of a cytotoxic response to metformin. In addition, high glutathione, malate, IMP and orotate content were observed in metformin-insensitive leukemic cells. Moreover, the cytotoxic effect of metformin was independent of AMPK/LKB1 status of the leukemic cells while p53 expression abrogated this effect. The presence of wild-type p53 appears to partially protect tumor cells from glucose starvation and metformin cytotoxicity and prevents the induction of the Pasteur effect. Finally, we demonstrated that metformin increased the cytotoxicity of chemotherapy agent, cytarabine, on all leukemic cell lines in vitro and significantly reduced leukemic colony-forming units (CFU-L) from six primary AML patient samples in a concentration-dependent manner. Additional experiments on metabolic and signaling pathways as well as in vivo studies are in progress to better understand the cytotoxic response of metformin in both AML cell lines and primary AML patient specimens that impact the therapeutic potential of metformin in vivo. Disclosures: Carroll: Agios Pharmaceuticals: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Sanofi Aventis Corporation: Research Funding; Glaxo Smith Kline, Inc.: Research Funding.