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Glucose transporter 1-mediated glucose uptake is limiting for B-cell acute lymphoblastic leukemia anabolic metabolism and resistance to apoptosis

Glucose transporter 1-mediated glucose uptake is limiting for B-cell acute lymphoblastic leukemia... Citation: Cell Death and Disease (2014) 5, e1470; doi:10.1038/cddis.2014.431 OPEN & 2014 Macmillan Publishers Limited All rights reserved 2041-4889/14 www.nature.com/cddis Glucose transporter 1-mediated glucose uptake is limiting for B-cell acute lymphoblastic leukemia anabolic metabolism and resistance to apoptosis This article has been corrected since Online Publication and a corrigendum has also been published 1,2 1,2 1,2 1,2 1,2 3 4 5 3 ,1,2 T Liu , RJ Kishton , AN Macintyre , VA Gerriets , H Xiang , X Liu , ED Abel , D Rizzieri , JW Locasale and JC Rathmell* The metabolic profiles of cancer cells have long been acknowledged to be altered and to provide new therapeutic opportunities. In particular, a wide range of both solid and liquid tumors use aerobic glycolysis to supply energy and support cell growth. This metabolic program leads to high rates of glucose consumption through glycolysis with secretion of lactate even in the presence of oxygen. Identifying the limiting events in aerobic glycolysis and the response of cancer cells to metabolic inhibition is now essential to exploit this potential metabolic dependency. Here, we examine the role of glucose uptake and the glucose transporter Glut1 in the metabolism and metabolic stress response of BCR-Abl+ B-cell acute lymphoblastic leukemia cells (B-ALL). B-ALL cells were highly glycolytic and primary human B-ALL samples were dependent on glycolysis. We show B-ALL cells express multiple glucose transporters and conditional genetic deletion of Glut1 led to a partial loss of glucose uptake. This reduced glucose transport capacity, however, was sufficient to metabolically reprogram B-ALL cells to decrease anabolic and increase catabolic flux. Cell proliferation decreased and a limited degree of apoptosis was also observed. Importantly, Glut1-deficient B-ALL cells failed to accumulate in vivo and leukemic progression was suppressed by Glut1 deletion. Similarly, pharmacologic inhibition of aerobic glycolysis with moderate doses of 2-deoxyglucose (2-DG) slowed B-ALL cell proliferation, but extensive apoptosis only occurred at high doses. Nevertheless, 2-DG induced the pro-apoptotic protein Bim and sensitized B-ALL cells to the tyrosine kinase inhibitor Dasatinib in vivo. Together, these data show that despite expression of multiple glucose transporters, B-ALL cells are reliant on Glut1 to maintain aerobic glycolysis and anabolic metabolism. Further, partial inhibition of glucose metabolism is sufficient to sensitize cancer cells to specifically targeted therapies, suggesting inhibition of aerobic glycolysis as a plausible adjuvant approach for B-ALL therapies. Cell Death and Disease (2014) 5, e1470; doi:10.1038/cddis.2014.431; published online 16 October 2014 Many cancer cells have elevated rates of glycolysis and acute lymphoblastic leukemia cells (B-ALL) and is associated lactate production even in the presence of oxygen. This with poor prognosis. The metabolic program of B-ALL cells is program, termed aerobic glycolysis, occurs in a wide range of undefined, although diffuse large B-cell lymphoma (DLBCL) both solid and liquid tumors and is driven by oncogenic signals can either be highly glycolytic or use oxidative phosphorylation 1 3 and microenvironmental pressures. Aerobic glycolysis is and mitochondrial metabolism. It has been suggested that proposed to allow metabolism in low oxygen tensions and to BCR-Abl signaling is associated with elevated glucose provide biosynthetic intermediates for cell growth. Indeed, metabolism, as BCR-Abl can promote glucose uptake and aerobic glycolysis readily supports both generation of ATP and trafficking of glucose transporter Glut1 to the cell surface. biosynthesis of lipids, nucleic acids, and amino acids. Given Conversely, inhibition of BCR-Abl in leukemic cells sup- 4–7 the high rates of glucose consumption and aerobic glycolysis presses glucose uptake and glycolysis. This regulation of in most cancers, targeting glucose metabolism has become of glucose metabolism may be critical for survival of BCR-Abl significant interest as an approach to eliminate cancer cells. B-ALL, as enforced expression of Glut1 protected B-ALL cells It is now important to establish mechanisms of aerobic from imatinib-induced apoptosis. These data show that BCR- glycolysis and the response of cancer cells to metabolic Abl promotes glucose uptake and aerobic glycolysis, and inhibition. BCR-Abl-transformed cells may rely on this pathway. The t(9;22) chromosomal translocation that generates the Targeting glucose metabolism can have efficacy against a oncogenic kinase BCR-Abl occurs in ~ 25% of adult B-cell variety of cancers. Mechanistic understanding of cancer cell 1 2 Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27710, USA; Department of Immunology, Duke Cancer Institute and Duke Molecular 3 4 Physiology Institute, Duke University, Durham, NC 27710, USA; Division of Nutritional Sciences, Cornell University, Ithaca, NY 14850, USA; Fraternal Order of Eagles Diabetes Research Center, Division of Endocrinology and Metabolism, Department of Medicine, Carver College of Medicine University of Iowa, Iowa City, IA 52242, USA and Division of Cell Therapy, Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA *Corresponding author: JC Rathmell, Department of Pharmacology and Cancer Biology, Duke University, DUMC 3813, Durham, NC 27710, USA. Tel: +1 1919 681 1084; Fax: +1 919 668 6044; E-mail: jeff.rathmell@duke.edu Abbreviations: B-ALL, B-cell acute lymphoblastic leukemia; 2-DG, 2-deoxyglucose; DLBCL, diffuse large B cell lymphoma; ECAR, extracellular acidification rate; OCR, oxygen consumption rate; 4-OHT, 4-hydroxytamoxifen; BrDU, BromodeoxyUridine Received 19.7.14; revised 28.8.14; accepted 01.9.14; Edited by C Munoz-Pinedo Glut1 is limiting in B-ALL anabolic metabolism T Liu et al metabolic requirements or response to inhibition using complete media with or without the addition of 10 mM 2-DG to strongly inhibit glycolysis. Cell survival was measured over pharmacologic approaches, however, has been limited. time and normal B cells were found only modestly affected by It has been shown using the glycolytic inhibitor 2-deoxyglucose glycolytic inhibition (Figure 1b). In contrast, primary B-ALL (2-DG) or glucose deprivation culture conditions that inhibition cells treated with 2-DG rapidly underwent cell death and were of glucose metabolism impacts cancer cell growth and viability highly dependent on glycolysis regardless of the BCR-Abl status. through several different mechanisms, including cell cycle arrest or cell death by activating AMPK pathway and B-ALL cells express multiple Glut family members and inactivating mTOR signaling. Reduced glucose metabolism deletion of Glut1 partially inhibits glucose uptake. Glu- has also been found to impact the stability and synthesis of cose uptake is the first essential step in glucose metabolism Bcl-2 family proteins. Glucose deprivation induces expression 19–21 7,11–15 and can be limiting in lymphocyte proliferation. Measure- of pro-apoptotic molecules, including Bim and can ment of Glut family members in BCR-Abl+ murine B-ALL induce apoptosis in cells transformed with oncogenic K-Ras found expression of Glut1, Glut3, Glut6, Glut8, and Glut9, through the unfolded protein response pathway. whereas the remaining Glut family members were undetected Here we examine the mechanism and role of glucose (Figure 1c). Similarly, human B-ALL cell lines expressed a uptake in B-ALL metabolism and leukemia progression by variable array of Glut family members, including Glut1, Glut3, genetically targeting glucose transport. The Glut family of Glut4, Glut5, Glut6, Glut7, Glut8, and Glut9 (Supplementary hexose transporters consists of 14 members and B-ALL Figure 1B). Of these transporters, Glut1 was previously cells expressed multiple family members. Conditional deletion shown important for proliferation and antibody secretion of of Glut1, however, demonstrated that B-ALL cells are reliant normal B cells and appeared broadly abundant in B-ALL. on this specific glucose transporter to sustain anabolic To genetically target B-ALL glucose uptake, therefore, we metabolism, proliferation, and resistance to cell death. fl/fl generated primary BCR-Abl B-ALL cells from Glut1 /Ubi-Cre- Consistent with our data showing a key role for glucose T2 ER mice to enable inducible deletion of glucose transporter uptake, we found that pharmacologic inhibition of glycolysis Glut1 upon treatment with 4-hydroxytamoxifen (4-OHT). sensitized B-ALL cells to caspase activation and apoptosis to fl/fl T2 Treatment of Glut1 /Ubi-Cre-ER B-ALL with 4-OHT led reduce leukemia burden in vivo. Glut1 and glucose uptake to efficient deletion of Glut1 with minimal compensation from have a key role, therefore, to maintain BCR-Abl B-ALL cell other Glut transporters, and 4-OHT had no effect on control growth and resistance to cell death. B-ALL cells (Figures 1c and d, Supplementary Figure 2). Deletion of Glut1 reduced glucose uptake by approximately Results half, showing Glut1 was limiting for maximal but does not mediate all glucose uptake (Figure 1e). To test whether this B-ALL cells utilize glucose through aerobic glycolysis. partial impairment of glucose transport impacted BCR-Abl Oncogenic signals can increase glucose uptake and drive oncogenic activity, we examined phosphorylation of BCR-Abl aerobic glycolysis. However, usage of glucose in B-ALL and immediate downstream targets and found BCR-Abl and and the response of B-ALL cells to inhibition of aerobic its substrate CRKL remained phosphorylated (Figure 1f). glycolysis are poorly understood. To examine glucose Glut1, therefore, contributes significantly to B-ALL glucose metabolism activity in B-ALL, real-time extracellular flux was uptake, and reduction of glucose metabolism does not alter measured in six human B-ALL cell lines, including BCR-Abl+ BCR-Abl activity. and BCR-Abl− cell lines, and peripheral B cells from three healthy donors in the absence of nutrients, after addition of Glut1 deficiency suppresses anabolic metabolism. Given glutamine, then upon addition of glucose. Extracellular the remaining capacity for glucose uptake in Glut1-deficient acidification rate (ECAR), reflective of lactate production, cells, the metabolic impact of Glut1 deletion was unclear. was similar in each cell type in the absence of nutrients To better understand the metabolic role of Glut1, the (Figure 1a). Glutamine, which is used as another important steady-state levels of 410 metabolites were measured metabolic fuel for many types of cancer cells in addition to through non-targeted LC/MS in B-ALL cells, with or without glucose, did not increase ECAR in any of the B-ALL cell expression of Glut1. Principal component analyses of the lines or normal B cells. The addition of glucose following metabolome of each population showed a modest difference T2 fl/fl T2 glutamine, however, led to a sharp increase in ECAR in all between control CreER B-ALL cells and Glut1 CreER T2 the B-ALL cells (Figure 1a). A modest increase in ECAR cells. However, control CreER B-ALL were largely was also observed in normal B cells. Importantly, under no unaffected by 4-OHT treatment, whereas Glut1 deletion led fl/fl condition did the oxygen consumption rate (OCR) change to a distinct metabolic profile in 4-OHT-treated Glut1 T2 with glutamine or glucose addition (Supplementary Figure CreER B-ALL cells (Figure 2a and Supplementary 1a), and we did not observe a clear difference of metabolic Table 1). Only five metabolites were significantly altered T2 program between BCR-Abl+ and BCR-Abl − cell lines. after 4-OHT treatment of control CreER B-ALL cells, B-ALL cells thus selectively metabolize glucose through whereas 43 metabolites were specifically changed in aerobic glycolysis with minimal contribution to oxidative response to Glut1 deletion (Figure 2b and Supplementary metabolism. Table 1). Affected metabolites represented a wide array of To test whether primary B-ALL cells depended on main- pathways (Figure 2c) with particular reductions in anabolic tenance of high glycolysis, BCR-Abl+ and BCR-Abl− B-ALL pathways, including pyrimidine and purine metabolism, patient samples and two normal donor B cells were cultured in glycolysis, and the amino and nucleotide sugar metabolism Cell Death and Disease Glut1 is limiting in B-ALL anabolic metabolism T Liu et al No Fuel Healthy 1 BCR-Abl + B-ALL Glutamine Healthy 2 BCR-Abl - B-ALL Glutamine + Glucose BV173 Tom-1 Nalm Nalm KOPN Sup 1 2 3 16 19 8 B15 0 10203040 50 Hours BCR-Abl+ BCR-Abl- BCR-Abl+ Healthy B cells ns ns ns ns 0.9 *** 0.8 Glut1 0.7 fl/fl Glut1 Vehicle 0.6 fl/fl Glut1 4-OHT 0.5 0.4 Actin 0.3 4-OHT - - + + 0.2 fl/fl 0.1 Glut1 CreER WT CreER Glut1 Glut3 Glut6 Glut8 Glut9 pBCR-Abl (Tyr 177) ns ** 35000 BCR-Abl BCR pCRKL (Tyr 207) CRKL 4-OHT - - + + Actin fl/fl Glut1 CreER WT CreER 4-OHT - - + + Normal B cell fl/fl Glut1 WT CreER CreER Figure 1 B-ALL cells require glycolysis and express multiple glucose transporters, but Glut1 is limiting for maximal glucose uptake. (a) ECAR was measured in human B-ALL cell lines and primary healthy-donor peripheral blood B cells at baseline and followed by sequential injection of 2 mM glutamine and 25 mM glucose. (b) Viability of human primary B-ALL cells and normal B cells from two donors each over time upon treatment of 2-DG (10 mM). Values were normalized at each time point to the vehicle control for that sample. fl/fl The BCR-Abl status of primary B-ALL is indicated. (c–e) Control and Glut1 CreER B-ALL cells were treated with vehicle or 4-OHT for 96 h followed by 48 h culture without 4-OHT prior to analyses. (c) Glucose transporter family member mRNA expression was measured by qrtPCR. Shown are expression of detected glucose transporters relative to expression of beta-actin. Other Glut family members were not detected. (d) Glut1 protein was measured by immunoblot and (e) glucose uptake was measured with radiolabeled glucose. (f) BCR-Abl activity was analyzed by immunoblot. Means of three or more replicates and S.D. are shown ***P≤ 0.001, **P≤ 0.005. NS, not significant that support glycosylation. Metabolic levels were lowered for increased (Figure 2d). Surprisingly, pyruvate and lactate, glycolytic intermediates, a key pentose phosphate pathway the majority of TCA cycle intermediates, and free intracellular intermediate, and multiple nucleotides and intermediates amino acids were unaffected by Glut1 deletion (Figure 2d, in nucleotide synthesis (Figure 2d, Supplementary Figure 3 Supplementary Figure 3 and Supplementary Table 1). and Supplementary Table 1). In contrast, catabolic pathways Pyruvate and lactate can be derived from non-glucose including glycerophospholipid metabolism and acyl-carni- sources, such as glutamine, and this may account for tines, which reflect fatty acid and lipid oxidation, were maintenance of these metabolites even with reduced glucose Cell Death and Disease mRNA expression relative ECAR (mpH/Min/Million cells) Glucose uptake (CPM) to Actin % Viable (Normalized to vehicle control) ∗ ∗ ∗ ∗ ∗∗ ∗ ns ns ns ns ns Glut1 is limiting in B-ALL anabolic metabolism T Liu et al Glycolysis Hexose phosphate WT CreER Vehicle WT CreER 4-OHT Fructose 1,6-biphosphate fl/fl Glut1 CreER Vehicle fl/fl Glut1 CreER 4-OHT Dihydroxyacetone phosphate Biphosphoglycerate D-Glycerate 3-phosphate/ D-Glycerate 2-phosphate Phosphenolpyruvate PC 1 (45.1%) Pyruvate 6 Lactate fl/fl Glut1 CreER +/-4OHT 0 0.5 1 1.5 Pentose Phosphate Pathway 2-deoxyribose phosphate N=410 D-gluconate -6 -4 -2 0 2 4 6 6 Sedoheptulose 7-phosphate WT CreER +/-4OHT 0 0.5 1 1.5 2 Nucleotides ATP UTP N=410 KL(" CTP -6 -4 -2 0 2 4 6 0 0.5 1 1.5 Log (fold change+/-4OHT) TCA Cycle Malate Pyrimidine N-Carbamoyl-L-aspartate, (S)- Fumarate metabolism dihydroorotatedCTP, CTP, UTP, Succinate Deoxyuridine, Glycerophospho- CDP-ethanolamine, choline, CDPcholine, N- -Ketoglutarate lipid metabolism methylethanolaminium phosphate, Dihydroxyacetone phosphate, Aconitate Glycolysis or Hexose phosphate, Phosphoenolpyruvate, Citrate/Isocitrate Gluconeogenesis Dihydroxyacetone phosphate, Fructose 1,6- biphosphate, D-Glycerate 3-phosphate/D- 0 0.5 1 1.5 2 Glycerate 2-phosphate Lipid Oxidation (Acyl-Carnitines) Acyl Carnitines Heptadecanoyl-, Linoleyl-, tetradecenoyl-, L-palmitoyl-carnitine Linoleyl carnitine Purine dGMP/AMP/3-AMP, ATP metabolism Heptadecanoyl carnitine Pentose Sedoheptulose 7-phosphate, phosphate Tetradecenoyl carnitine pathway Stearoyl carnitine Amino sugar and GDP-alpha-D-mannose, UDP-N-acetyl-D- nucleotide sugar galactosamine/UDP-N-acetyl-alpha-D- Palmitoyl carnitine metabolism glucosamine 0 0.5 1 1.5 2 2.5 3 Glycine, serine Choline, O-Phospho-L-serine and threonine Fold change to vehicle control metabolism fl/fl fl/fl Glut1 Vehicle Glut1 4-OHT Decreased >1.5 fold, p<0.05 Increased >1.5 fold, p<0.05 fl/fl Figure 2 In vitro Glut1 deletion leads to metabolic reprogramming of B-ALL cells. (a–c) Steady-state metabolite levels in wild-type (WT) Cre-ER and Glut1 CreER B-ALL cells treated with vehicle or 4-OHT were determined using LC/MS. (a) Principal component, (b) volcano plots of metabolites changed 41.5-fold, Po0.05, and (c)key metabolites categorized by KEGG metabolic pathways are shown in order of significance. (d) Relative levels of specific metabolites in key pathways are shown following Glut1 deletion. Data represent mean and S.D. for triplicate samples. *Po0.05. NS, not significant metabolism. These data suggest that Glut1-dependent To further investigate glucose contribution to downstream glucose mainly supports biosynthetic pathways in B-ALL metabolic pathways and how Glut1 deficiency alters these cells, and reduced glucose uptake led to metabolic repro- pathway activities, glucose fate was traced and metabolic flux gramming to favor catabolism. analysis was performed using C-labeled glucose. B-ALL Cell Death and Disease -Log(p value) PC2 (20.1%) Glut1 is limiting in B-ALL anabolic metabolism T Liu et al cells were cultured in vehicle or 4-OHT for 4 days to delete cells efficiently converted 13C-glucose to uniformly labeled Glut1 and then labeled with 13C-glucose for 24 h prior to C phosphoenolpyruvate, dihydroxyacetone phosphate, and LC/MS mass spectrometry. Despite partial maintenance of ribose phosphate through glycolysis and the pentose glucose uptake, flux to anabolic pathways was sharply phosphate pathway, respectively (Figure 3a, Supplementary curtailed following Glut1 deletion. Control Glut1-expressing Figure 4 and Supplementary Table 2). Glut1-deficient cells, Vehicle 4-OHT Phosphoenolpyruvate ns ∗∗∗ ∗∗ 0[13C] 1[13C] 2[13C] 3[13C] Ribose-phosphate ∗∗ ∗ ns ∗ ns ∗∗∗ ∗ 0[13C] 1[13C] 2[13C] 3[13C] 4[13C] 5[13C] Lactate ns ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ 40000000 40 0[13C] 1[13C] 2[13C] 3[13C] Malate ∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ 0 0 0[13C] 1[13C] 2[13C] 3[13C] 4[13C] Pentose Phosphate Pathway Palmitate Oxidation ∗∗∗ ns ∗∗∗∗ ns 700 400 - + - + 4OHT - + - + 4OHT fl/fl fl/fl Glut1 CreER WT CreER Glut1 CreER WT CreER Figure 3 Glut1 deletion suppresses glucose contribution to anabolic pathways and increases catabolic metabolism. (a) 13C-glucose tracing contribution of glucose to indicated metabolite pools. Graphs on the left indicate the total quantity of each metabolite and the graph on the right indicates the relative distribution of C carbons in each fl/fl metabolite. (b and c) WT Cre-ER and Glut1 Cre-ER B-ALL cells were treated with vehicle or 4-OHT for 96 h followed by 48 h of culture without 4-OHT and (b) pentose 14 14 phosphate pathway flux was measured by oxidation of 1-C -glucose and (c) lipid oxidation was measured by oxidation of C palmitate. Means and S.D. of triplicate measurements are shown. *Po0.05, **Po0.005, ***Po0.001, ****Po0.0001. NS, not significant Cell Death and Disease Total AUC Total AUC Total AUC Total AUC CPM/Million cells Percentage Percentage Percentage Percentage CPM/Million cells Glut1 is limiting in B-ALL anabolic metabolism T Liu et al however, produced very little total levels of these metabolites following Glut1 deletion (Figure 3b). Conversely, Glut1 relative to control cells and that which was generated deletion led to a sharp increase in palmitate oxidation contained a significantly lower fraction of 13C-glucose- (Figure 3c). Together, these data show that B-ALL cells are derived carbon (Figures 2d and 3a, Supplementary Figure 4 highly glycolytic and primarily use glucose to support and Supplementary Table 2). Pyruvate and lactate were biosynthetic reactions and pathways, such as the pentose present in similar levels, and control cells generated these phosphate pathway. metabolites through both C-labeled glucose and unlabeled sources, whereas the majority of these metabolites were derived from non-glucose sources in Glut1-deficient cells. Thus alternative sources, such as glutamine, contribute Metabolic reprogramming suppresses B-ALL prolifera- tion. The sharp decrease in flux toward biosynthetic meta- significantly to pyruvate and lactate in control cells and these bolic pathways and increased catabolism following Glut1 pathways become increasingly dominant after Glut1 deletion. deletion suggested that Glut1 deficiency may impede B-ALL Surprisingly, glucose did not contribute significantly to the TCA cell growth and proliferation. Indeed, 4-OHT treatment led to cycle in B-ALL regardless of Glut1 expression, as malate, a sharp reduction in cell accumulation rates over time citrate, succinate, and alpha-ketoglutarate were unlabeled in (Figure 4a). This was at least partially due to reduced both control and Glut1-deficient cells (Figure 3a, proliferation, as BromodeoxyUridine (BrDU) incorporation in Supplementary Figure 4 and Supplementary Table 2). Thus, fl/fl T2 Glut1 CreER B-ALL cells was significantly decreased, glucose was not the main fuel resource for oxidative T2 whereas control CreER cells were unaffected by 4-OHT metabolism in B-ALL, nor was it redirected toward oxidative (Figures 4a and b). Cell cycle profiling of control and 4-OHT- metabolism in Glut1-deficient B-ALL cells. Rather, other treated B-ALL cells, however, showed that Glut1 deletion did metabolic fuels sustained the TCA cycle. Radiolabeled tracer assays were next conducted in pentose not lead to a clear cell cycle arrest or accumulation of cells in phosphate and lipid oxidation pathways to independently a specific phase of the cell cycle (Figure 4c). Rather Glut1 confirm these findings. Consistent with previous results, deficiency appeared to slow cell proliferation than activate a pentose phosphate pathway activity was significantly reduced specific cell cycle arrest checkpoint. fl/fl WT CreER Glut1 CreER Vehicle Vehicle 6 6 4-OHT 4-OHT 4 4 3 3 2 2 0 20 40 60 80 100 0 20 40 60 80 100 Hours Hours fl/fl Glut1 CreER ns Vehicle 4OHT WT CreER - + - + 4OHT fl/fl Glut1 CreER WT CreER DNA content fl/fl Figure 4 In vitro Glut1 suppresses B-ALL accumulation through decreased proliferation. (a) WT Cre-ER and Glut1 Cre-ER B-ALL cells were treated with vehicle or 4-OHT for 96 h followed by 48 h of culture without 4-OHT, and cell numbers were counted over time. (b and c) After 4 days treatment with vehicle or 4-OHT followed by 2 days culture without 4-OHT, cells were cultured with BrDU for 1.5 additional hours and (b) BrDU incorporation was measured by intracellular flow cytometry. (c) DNA content was determined flow cytometrically by propidium iodide staining to indicate cell cycle status. Means and S.D. are shown for triplicate samples in representative experiments repeated three or more times. ****Po0.0001. NS, not significant Cell Death and Disease BrDU Positive Cells (%) Cell Density (Million cells/ml) Cell Percentage Glut1 is limiting in B-ALL anabolic metabolism T Liu et al Glut1 deletion sensitizes B-ALL cells to apoptosis with accumulation in vitro through increased apoptosis. Consis- targeted agents. Glucose metabolism is closely linked to tent with this notion, Glut1 deficiency specifically induced cell survival, and Glut1 deletion may have also impaired cell expression of the pro-apoptotic protein Bim and only fl/fl a b Glut1 CreER WT CreER 100 100 Bim 80 80 60 60 Actin 40 40 Vehicle 4OHT -+ -+ 20 4-OHT fl/fl Glut1 WT CreER 0 0 CreER 050 100 150 0 50 100 150 Hours c Dasatinib Dasatinib fl/fl Glut1 CreER WT CreER 100 100 Vehicle 50nM Dasatinib 4OHT 20 4OHT 50nM Dasatinib 0 50 100 150 0 50 100 150 Hours d Post Glut1 deletion and prior to 48 hours after Dasatinib addition Dasatinib addition *** ** *** **** * 60 **** **** Annexin V+/PI- Annexin V+/PI- 50 * Annexin V+/PI+ Annexin V+/PI+ 4-OHT - - - - ++++ - - - - ++++ 4-OHT - - ++ -- ++ Q-vd - +- + - +- + Dasatinib - - + + -- ++ - - + + -- ++ Q-vd - + - + - ++ - - + - + - ++ - fl/fl Glut1 WT CreER fl/fl CreER WT CreER Glut1 CreER fl/fl Figure 5 In vitro Glut1 deletion induces pro-apoptotic Bim expression and sensitizes B-ALL to cell death stimulus. (a and b) WT Cre-ER and Glut1 Cre-ER B-ALL cells treated with vehicle or 4-OHT for 96 h followed by 48 h of culture without 4-OHT and (a) examined by immunoblot on day 6 or (b) analyzed by flow cytometry for survival over time. fl/fl (c) WT Cre-ER and Glut1 Cre-ER B-ALL cells were cultured with vehicle or 4-OHT for 96 h, washed, then cultured an additional 48 h alone or with addition of Dasatinib (50 nM), and cell viability was determined over time by flow cytometry. (d) Apoptosis in Glut1-deleted cells with or without Dasatinib treatment was assessed by annexin V/PI staining. Cells were treated with vehicle or 4-OHT for 96 h and apoptosis was assessed by annexin V/PI staining (left panel). After 96 h of culture with vehicle or 4-OHT, cells were washed and cultured for an additional 48 h alone or with Dasatinib (50 nM). Cell apoptosis was assessed at the end of the 48 h (right panel). Ten μM pan caspases inhibitor Q-vd-oph was added in some cell cultures as indicated. Gray bar, annexin V+/PI− cell percentage. Black bar, annexin V+/PI+ cell percentage. Means and S.D. are shown for triplicate samples from representative experiments repeated three or more times. *Po0.05, **Po0.005, ***Po0.001, ****Po0.0001 Cell Death and Disease % Annexin V Positive % Viable % Viable Glut1 is limiting in B-ALL anabolic metabolism T Liu et al modestly impacted expression of other Bcl-2 family proteins, cells were dependent on continued glycolysis and a low dose including pro-apoptotic protein Bax, Bak, Bid and anti- of 2-DG prevented cell accumulation (Figure 7a). A higher apoptotic protein Mcl-1 and Bcl-xL (Figure 5a and dose of 2-DG to further inhibit glycolysis was required to Supplementary Figure 5A). Bim can be induced in response induce B-ALL cell death, similar to the observation in human to ER stress and the unfolded protein response, but only primary B-ALL cells (Figure 7b). This dose of 2-DG treatment very modest markers of these pathways were detected induced Bim expression (Figure 7c), which was essential to − /− relative to those induced by the glycosylation inhibitor, induce apoptosis, as Bim B-ALL cells resisted cell death tunicamycin (Supplementary Figure 5B). Thus, ER stress even with high doses of 2-DG (Figure 7d). may contribute to Bim induction, but this response was not The potential of 2-DG to provide an adjuvant metabolic strongly induced. stress to specifically sensitize cancer cells to Dasatinib was Glut1 deletion led to reduced B-ALL cell viability over time next tested in vivo. Wild-type B-ALL cells were adoptively (Figure 5b). The majority of cells, however, remained viable transferred into recipient animals and allowed 2 days to even without Glut1. However, increased expression of Bim in engraft. To provoke metabolic stress and prime B-ALL cells for Glut1-deficient cells suggested that sensitivity of the surviving targeted therapy, animals were treated with vehicle or 2-DG for cells to apoptosis was increased. Indeed, treatment of BCR- 4 days alone, followed by three additional days with or without Abl B-ALL with a low dose of the tyrosine kinase inhibitor, addition of Dasatinib. B-ALL cell percentages and numbers Dasatinib, only mildly impacted control B-ALL cells, but B-ALL were then determined 11 days after transfer (Figure 7e). 2-DG cell death was markedly increased in Glut1-deleted cells and Dasatinib alone each reduced B-ALL cell burden. (Figure 5c). Cell death appeared to occur in part through Importantly, combined treatment of 2-DG and Dasatinib apoptosis, as Annexin V+ early-stage apoptotic cells were together led to significantly further depletion of B-ALL cells. detected (Figure 5d) and caspase activity increased Likewise, 2-DG treatment also increased efficacy of Dasatinib (Supplementary Figure 6). Importantly, caspase inhibition to induce cell death in human BCR-Abl+ B-ALL cell lines with Q-VD partially protected cells from death (Figure 5d). (Supplementary Figure 7). Thus, partial non-cytotoxic phar- macologic inhibition of glucose metabolism slows tumor Glut1 deletion suppresses B-ALL progression growth and provides an enhanced response to specifically in vivo. Despite sharply curtailed growth and proliferation targeted therapy. in vitro, how B-ALL progressed in vivo without Glut1 remained unclear. Presence of in vivo nutrients and stromal cell support Discussion may allow B-ALL cells to persist and proliferate even without UbiCreERT2 Glut1 and with reduced glucose uptake. Control Elevated rates of glucose uptake and glycolysis can have fl/fl UbiCreERT2 and Glut1 B-ALL cells were, therefore, trans- significant roles in cancer cell survival and progression by ferred into immunocompromised hosts that were treated with supporting cellular energetics and providing biosynthetic T2 25 vehicle or tamoxifen to activate CreER , and in vivo and substrates. It is now important to establish limiting compo- B-ALL growth was assessed with or without Glut1 expression nents of aerobic glycolysis and how cancer cells respond to (Figure 6a). B-ALL cells were monitored by IRES-driven GFP metabolic inhibition. Here we show that B-ALL cells are highly expression from the BCR-Abl expressing retroviral vector. dependent on glucose and that glucose uptake through Glut1 Two days after cell transfer, recipients were treated with is essential for BCR-Abl B-ALL cells to maintain anabolic tamoxifen to delete Glut1 in transferred B-ALL cells. Animals metabolism to support proliferation. Glut1-deficient B-ALL were then analyzed for B-ALL cell number after an additional cells both proliferated at a lower rate and had moderate levels week. Glut1 was efficiently deleted in vivo, and Glut1 protein of apoptosis. Importantly, B-ALL cells required Glut1 to progress in vivo, demonstrating a clear role for this specific levels were sharply reduced in B-ALL cells purified from glucose transporter in B-ALL metabolism. Similar to Glut1 splenocytes of tamoxifen-treated recipient animals UbiCreERT2 fl/fl UbiCreERT2 deletion, pharmacologic inhibition of aerobic glycolysis with 2- (Figure 6b). Both control and Glut1 DG also suppressed proliferation and led to increased B-ALL cells were present in high levels in both spleen and expression of Bim to sensitize to the tyrosine kinase inhibitor bone marrow of vehicle-treated mice (Figures 6c and d). Dasatinib in vivo. Partial metabolic stress thus impaired cell Importantly, B-ALL cells did not accumulate in vivo and proliferation and sensitized BCR-Abl-driven B-ALL cells to disease did not rapidly progress after Glut1 deletion apoptosis with specifically targeted therapy in vivo. (Figure 6c) and numbers of Glut1-deficient B-ALL cells were Similar to the glycolytic form of DLBCL, we show here that significantly reduced in spleen and bone marrow (Figure 6d), both BCR-Abl+ and BCR-Abl− human B-ALL cell lines are relative to vehicle-treated or control B-ALL. These data indicate that Glut1 deletion suppresses B-ALL progression, highly glycolytic and metabolize glucose through aerobic and B-ALL cells are dependent on Glut1 even in physiological glycolysis. Importantly, in vitro, B-ALL cells were sensitive to conditions. inhibition of glycolysis with reduced proliferation followed by apoptosis. In vitro culture conditions, however, do not faithfully Pharmacological inhibition of glycolysis impairs prolif- mimic in vivo nutrient conditions. Presence of alternate eration and leads to Bim-dependent cell death to nutrients, stromal cells and growth factors in a physiological sensitize to targeted therapy. Given the dependence of setting may substantially modulate sensitivity of cells to B-ALL cells on Glut1, the cellular response to pharmacolo- therapy. Primary B-ALL cells with conditional deletion of Glut1 gical disruption of glucose metabolism was next examined by allows for a direct test of glucose metabolism contribution to treating BCR-Abl B-ALL with 2-DG. BCR-Abl+ murine B-ALL B-ALL progression in vivo. Glut1 deletion did not fully prevent Cell Death and Disease Glut1 is limiting in B-ALL anabolic metabolism T Liu et al Day 0: B-ALL cell transfer Glut1 Day 3-6: Injection of corn oil control or tamoxifen Actin Tumor burden Tamoxifen -- - + + + analysis Vehicle Tamoxifen fl/fl WT CreER Glut1 CreER Spleen Bone Marrow Spleen Bone Marrow Veh: 35% Veh: 50% Veh: 37% Veh: 69% Tam: 43% Tam: 11% Tam: 4.6% Tam: 73% GFP WT CreER ns 80 ns ns 60 0 0 0 Vehicle Tamoxifen Vehicle Tamoxifen Vehicle Tamoxifen fl/fl Glut1 CreER **** **** **** 6 60 2 20 0 0 Vehicle Tamoxifen Vehicle Tamoxifen Vehicle Tamoxifen fl/fl Figure 6 In vivo Glut1 deletion decreases leukemic tumor burden. (a) Schematic diagram showing tamoxifen treatment to induce Glut1 deletion in vivo.(b) Glut1 CreER B-ALL were transferred and hosts treated with vehicle or tamoxifen on day 3, and purified B-ALL cells were analyzed by immunoblot from individual mice.(c) Flow cytometry from fl/fl representative spleen and bone marrow on day 10 of recipient animals that received WT Cre-ER or Glut1 Cre-ER and were treated with vehicle or tamoxifen. (d) Percentages and numbers of GFP+ transferred B-ALL cells on day 10 in individual mice treated as indicated with vehicle or tamoxifen. Means and S.D. from n= 5 mice/group for WT CreER fl/fl group and n= 10 mice/group for Glut1 CreER group are shown. ****Po0.0001. NS, not significant glucose uptake, but instead glucose transport was reduced to provide potentially different capacities to adapt and compen- approximately half in Glut1-deficient cells. The remaining sate for metabolic inhibition. Our data suggest, however, that glucose uptake was likely mediated through other glucose Glut1 or Glut3 may have dominant roles. 26–28 transporters expressed by B-ALL cells. In particular, Despite the incomplete inhibition of glucose uptake, Glut1 Gluts 3 and 6 may increase activity to support this glucose deletion led to striking shifts in metabolic pathways that transport. Human B-ALL cells expressed additional trans- demonstrate glucose transport as a limiting component of porters, including Glut4 and Glut5 (Supplementary Figure B-ALL cell metabolism. Glut1-deficient cells had significantly S1B). The array and expression levels of these different decreased flux through biosynthetic pathways, including the transporters, however, differed across individual cell lines to pentose phosphate pathway, nucleotide and phospho-lipid Cell Death and Disease BM GFP+ cell number BM GFP+ cell number 6 6 Cell Percentage (x10 ) (x10 ) Splenic GFP+ (%) Splenic GFP+ (%) Splenic GFP+ cell number Splenic GFP+ cell (x10 ) number (x10 ) Glut1 is limiting in B-ALL anabolic metabolism T Liu et al 3.5 Vehicle 1mM 2DG 2.5 1.5 Vehicle 1mM 2DG 0.5 5mM 2DG 0 10 20 30 40 50 010 20 30 40 50 Hours Hours Bim Actin WT Vehicle WT 5mM 2DG 5mM 20 -/- Vehicle Bim Vehicle 2DG -/- 0h Bim 5mM 2DG 24h 010 20 30 40 50 Hours **** **** 15 80 ** * **** 2-DG - - + + - - + + Dasatinib - + - + - + - + Figure 7 Pharmacological inhibition of glycolysis impairs B-ALL proliferation and sensitizes to apoptosis in vitro and in vivo.(a–c) WT B-ALL cells were cultured in low (1 mM) or high (5 mM) dose of 2-DG and (a) cell number was counted and (b) cell viability was analyzed by flow cytometry over time. (c) Cell lysates were analyzed by immunoblot. − /− (d) WTor Bim B-ALL cells were treated with 5 mM 2-DG and cell viability was analyzed by flow cytometry over time. (e) WT B-ALL cells were adoptively transferred into host animals that were treated starting on day 2 with vehicle alone or with 500 mg/kg per day of 2-DG alone for 4 days or with additional 10 mg/kg per day of Dasatinib for 3 days. GFP+ B-ALL cell percentages and numbers were determined by flow cytometry. Means and S.D. are shown of (a, b and d) triplicate and (e) n= 5 mice/group. ****Po0.0001, ***Po0.001, **Po0.005, *Po0.05 synthetic pathways, but had elevated lipid metabolism inter- Similar to 2-DG treatment, Glut1 deletion also induced mediates and oxidation rates. Surprisingly, the remaining expression of pro-apoptotic protein Bim and decreased B-ALL glucose uptake and glycolytic flux in Glut1-deficient B-ALL viability partially through apoptosis, although non-apoptotic was not redirected to oxidative metabolism and the TCA cycle cell death may have occurred as well. Bim appears to be the for maximum ATP generation. Rather, even Glut1-deficient primary Bcl-2 family protein mediating apoptotic events, as B-ALL cells remained dependent on non-glucose sources for expression levels of other Bcl-2 family proteins were only TCA intermediates. It is possible that a BCR-Abl signal may modestly altered by Glut1 deletion (Supplementary Figure S5). suppress pyruvate oxidation even under nutrient-limiting Glut1 deletion did not fully eliminate all B-ALL cells, possibly conditions. As a consequence, B-ALL cell growth and because the level of metabolic stress from Glut1 deletion was proliferation were sharply curtailed by Glut1 deletion. Cells insufficient to kill all the cells. Nevertheless, Glut1 deficiency did not arrest in a specific cell cycle stage, but rather cell sensitized the remaining B-ALL cells to cell death stimulus. A proliferation appeared slowed in each phase in a balanced sub-lethal dose of BCR-Abl inhibitor, Dasatinib, rapidly fashion. It may be that a more severe form of metabolic induced apoptosis in Glut1-deleted cells. It is unclear what inhibition is essential to induce cell cycle checkpoints and pathway was directly responsible for Bim induction. It has arrest, such as p21 induction that can be induced through been reported that decreased protein glycosylation upon 8,29 AMPK activation of p53, but we observed B-ALL cells to glucose deprivation or 2-DG can lead to endoplasmic reticulum 11,30 instead undergo apoptosis in these conditions. stress, which may promote Bim induction through the Cell Death and Disease GFP+ cell number in Cell Density bone marrow (x10 ) (Million cell/ml) GFP+ cell number in spleen % Viable % Viable (x10^6) Glut1 is limiting in B-ALL anabolic metabolism T Liu et al 24,31 transcription factor CHOP. However, Glut1 deficiency only Diego, CA, USA). Use of the human subject-derived samples was approved by the Duke University Institutional Review Board. imposed mild, if any, ER stress to B-ALL cells. The role of this mild ER stress on Bim induction remains unclear. Primary murine BCR-Abl+ B-ALL. Bone marrow cells from C57B6/J Interactions of B-ALL cells with stromal cells and the − /− T2 (Jackson Labs, Bar Harbor, ME, USA), Bim (Jackson Labs), Ubi-Cre-ER availability of alternate nutrients may support metabolic fl/fl T2 transgenic (Jackson Labs), and Glut1 mice crossed to Ubi-Cre-ER mice were flexibility to allow cancer progression in vivo even with limited cultured in IL-7 (10 ng/ml) (eBioscience) in IMDM supplemented with 20% fetal glucose uptake. In vivo deletion of Glut1 from B-ALL cells, bovine serum and infected with MSCV-BCR-Abl-IRES-GFP retrovirus (gift of D Fruman, UC Irvine) with polybrene (4 μg/ml) (Millipore, Billerica, MA, USA). however, markedly reduced leukemic tumor burden in Infected cells were cultured in methylcellulose medium containing IL-7 (Stem Cell recipient animals and prevented disease progression. The Technologies) and individual colonies were isolated on day 7 and transferred into significant reduction of tumor burden may be due to the complete IMDM media with 20% fetal bovine serum, but no IL-7. After 7 days, viable combinational effects of suppressed cell proliferation and GFP+ colonies were selected for expansion. The Institutional Animal Care and Use impaired cell viability. These data demonstrate that glucose Committee of Duke University approved all animal protocols. uptake and Glut1 are limiting components in the support of aerobic glycolysis in BCR-Abl+ B-ALL cells in vivo. Similarly, Immunoblot and flow cytometry. Cell lysates for immunoblots were 4,7,12 prepared as described. Primary antibodies used were rabbit anti-Bim (BD Glut1 deficiency suppressed breast cancer progression. Pharmingen, San Jose, CA, USA), rabbit anti-Glut1 (Abcam, Cambridge, MA, USA), Other glycolytic enzymes have also been shown to have rabbit anti-phospho-BCR (Cell Signaling, Danvers, MA, USA), rabbit anti-BCR (Cell key-limiting roles in cancer metabolism, including pyruvate Signaling), rabbit anti-phospho-CRKL (Cell signaling), rabbit anti-CRKL (Santa 33 34 kinase M2, phospho-fructokinase, and lactate dehydro- Cruz, Dallas, TX, USA), rabbit anti-Bcl-xL (Cell signaling), rabbit anti-Mcl-1 genase A. Our data support Glut1 as an additional potential (Biolegend, San Diego, CA, USA), rabbit anti-Bax (Cell signaling), rabbit anti-Bak restriction point in cancer metabolism. (Cell signaling), rabbit anti-Bid (mouse specific, Cell signaling), rabbit anti-PDI (Cell signaling), rabbit anti-IRE1α (Cell signaling), rabbit anti-Bip (Cell Signaling), mouse These data collectively suggest Glut1 may provide a anti-CHOP (Cell signaling), mouse anti-actin (Sigma-Aldrich) and were detected therapeutic target to reduce glucose uptake and treat cancers with anti-rabbit horseradish peroxidase-labeled antibody (Promega, Madison, WI, that use aerobic glycolysis. Although Glut1 is essential for USA) and fluorescent-labeled anti-mouse antibody (LiCor, Lincoln, NE, USA). Blots 36,37 glucose uptake in some tissues, incomplete inhibition of were visualized using Supersignal West Pico Chemiluminescent Substrate (Thermo Glut1 may be feasible and provide benefits. Indeed, several Scientific, Waltham, MA, USA) or the Odyssey infrared imaging system (LiCor). groups have now described inhibitors of Glut1 with partial Antibodies for cytometry include anti-human CD34, CD19, CD10 and CD20 38–41 (eBioscience), annexin V (Invitrogen, Grand Island, NY, USA) and anti-BrDU activity that can suppress tumor growth. The HIV (Invitrogen). Cell viability was measured by flow cytometry for propidium iodide protease inhibitor, ritonavir, also shows non-selective partial 12,13 42 (PI; Invitrogen) exclusion as described. Apoptotic cell populations were assessed inhibition of both Glut1 and Glut4 with low toxicity. Although it by annexin V/PI staining. Cells were incubated with Alexa Fluor 488 annexin V remains unclear to what extent Glut1 may provide a direct (Invitrogen) for 15 min in dark and then 2 μg/ml PI was added. Cell cycle profile and pharmacologic target, our data indicate that it is not essential DNA content were measured in cells fixed in ethanol and stained with PI. BrDU to fully suppress glucose uptake to prevent cancer cell incorporation was measured by culture of cells with 10 uM BrDU (Sigma-Aldrich) for 1.5 h followed by ethanol fixation, denaturation with 2 M HCl for 20 min and staining proliferation and disease progression. with anti-BrdU. Flow cytometry data were collected on MACSQuant (Miltenyi, Together, we show B-ALL is a highly glycolytic cancer Bergisch Gladbach, Germany) or FACScan (Becton Dickinson, San Jose, CA, USA) dependent on Glut1 as a limiting component of glucose and flow cytometers and analyzed using FlowJo software (Treestar, Ashland, OR, USA). anabolic metabolism. Glycolytic inhibition triggers a graded response of reduced cell proliferation followed by increased Quantitative real-time-PCR. Total mRNA was extracted (RNeasy mini kit; sensitivity to apoptosis both in vitro and in vivo. Impairment of Qiagen, Valencia, CA, USA) and 1 μg of RNA was reverse transcribed (iScript; Bio- glucose uptake through Glut1 deletion sufficiently suppressed Rad, Hercules, CA, USA) to perform SYBR Green (Bio-Rad) semi-quantitative real- time-PCR for Glut1. Relative expression levels were calculated using the ΔCt/ΔCt biosynthetic reactions and shifted B-ALL metabolic state to method, with expression normalized to 18S or actin RNA. Primers for mouse glucose catabolism. This metabolic reprogramming impeded B-ALL transporters and mouse actin: Glut1, 5ʹ-AGCCCTGCTACAGTGTAT-3ʹ,5ʹ-AGGTCT proliferation and prevented disease progression in vivo. The CGGGTCACATC-3ʹ; Glut3, 5ʹ-TAAACCAGCTGGGCATCGTTGTTG-3ʹ,5ʹ-AATGAT glucose dependence observed in B-ALL is likely also present GGTTAAGCCAAGGAGCCC-3ʹ; Glut6, 5ʹ-TTGGTGCTGTGAGGCT-3ʹ,5ʹ-TGGC in other types of cancer, and partial blockade of aerobic ACAAACTGGACGTA-3ʹ;Glut8, 5ʹ-ACATCTCGGAAATCGCCT-3ʹ,5ʹ-ACACAGCCC glycolysis may provide an adjuvant approach to augment the AGCACG-3ʹ;Glut9, 5ʹ-TGCTTCCTCGTCTTCGCCACAATA-3ʹ,5ʹ-CTCTTGGCAAA TGCCTGGCTGATT-3ʹ;actin,5ʹ-CCTTCCTTCTTGGGTATGGA-3ʹ,5ʹ-TGGTACCAC efficacy of targeted agents with minimal additional toxicity. CAGACAGCACT-3ʹ. Primers for human glucose transporters and 18S: Glut1, 5ʹ-CACTCCTGTTACTTACCTAA-3ʹ,5ʹ-CACTTACTTCTGTCTCACT-3ʹ;Glut3,5ʹ-GACC CAGAGATGCTGTAATGGT-3ʹ,5ʹ-GGGGTGACCTTCTGTGTCCC-3ʹ;Glut4, 5ʹ-CTTC Materials and Methods Human primary B-ALL and B-cell culture. Human B-ALL cell lines CAACAGATAGGCTCCG-3ʹ,5ʹ-CCCCAATGTTGTACCCAAAC-3ʹ; Glut5, 5ʹ-GCAA (BV-173, TOM-1, Nalm-16, Nalm-19, and KOPN-8 from DSMZ (Braunschweig, CAGGATCAGAGCATGA-3ʹ,5ʹ-CCATACTGGAAGGATGACCC-3ʹ; Glut6, 5ʹ-GTCCA Germany); Sup-B15 from ATCC (Manassas, VA, USA)) were cultured in complete TCTTCGACAGCACCG-3ʹ,5ʹ-GCAAACATGATGGCCGCTGA-3ʹ; Glut7, 5ʹ-CACC RPMI 1640 (Mediatech, Manassas, VA, USA) or complete IMDM (Gibco, Grand GTCTCCATGTTTCCTC-3ʹ,5ʹ-TGTTGTTGATCAGCAGGGTC-3ʹ; Glut8, 5ʹ-TCCT Island, NY, USA) with 10 or 20% fetal bovine serum (Gemini Bioproducts, West GGTTCGGGGCTGTC-3ʹ,5ʹ-GAGCACAGCAAGAGGCTCAG-3ʹ; Glut9, 5ʹ-GAGTAT Sacramento, CA, USA). De-identified primary human B-ALL samples were cultured CGTGGGCATTCTGG-3ʹ,5ʹ-AGTTGGAGAGCCAGTTGACG-3ʹ; 18S, 5ʹ-GTAAC in Hybridoma SFM media (Gibco) supplemented with cytokines (refer to CCGTTGAACCCCATT-3ʹ,5ʹ-CCATCCAATCGGTAGTAGCG-3ʹ. Supplementary Materials for list of cytokines). Human B cells from peripheral blood (Gulf Coast Regional Blood Center, Houston, TX, USA) were purified by Metabolomic profiling. Metabolomic analyses were performed as described negative selection (Stem Cell Technologies, Vancouver, BC, Canada) and cultured before using la iquid chromatography Q Exactive Mass Spectrometer (LC-QE-MS) 22 fl/fl in the same media as B-ALL cells. Some samples were treated with 2-DG (Sigma- (Thermo Scientific). In non-targeted metabolomics analyses, Glut1 CreER and Aldrich, St. Louis, MO, USA) at indicated doses. Samples were monitored by flow WT CreER cells were treated with vehicle or 0.4 μM 4-OHT for 96 h followed by an cytometry using anti-human CD34, CD19, CD10 and CD20 (eBioscience, San additional 48 h in complete IMDM media containing 1 mM sodium pyruvate. For Cell Death and Disease Glut1 is limiting in B-ALL anabolic metabolism T Liu et al 13C-glucose flux studies, cells were cultured as described above for 5 days culture, 8. 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Kraus M, Malenke E, Gogel J, Muller H, Ruckrich T, Overkleeft H et al. Ritonavir induces Licence. The images or other third party material in this article are endoplasmic reticulum stress and sensitizes sarcoma cells toward bortezomib-induced apoptosis. Mol Cancer Ther 2008; 7: 1940–1948. included in the article’s Creative Commons licence, unless indicated 40. Tuccinardi T, Granchi C, Iegre J, Paterni I, Bertini S, Macchia M et al. Oxime-based inhibitors otherwise in the credit line; if the material is not included under the of glucose transporter 1 displaying antiproliferative effects in cancer cells. Bioorg Med Chem Creative Commons licence, users will need to obtain permission from Lett 2013; 23: 6923–6927. the licence holder to reproduce the material. To view a copy of this 41. Wood TE, Dalili S, Simpson CD, Hurren R, Mao X, Saiz FS et al. A novel inhibitor of glucose uptake sensitizes cells to FAS-induced cell death. 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Glucose transporter 1-mediated glucose uptake is limiting for B-cell acute lymphoblastic leukemia anabolic metabolism and resistance to apoptosis

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Springer Journals
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Copyright © 2014 by The Author(s)
Subject
Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Immunology; Cell Culture; Antibodies
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2041-4889
DOI
10.1038/cddis.2014.431
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Abstract

Citation: Cell Death and Disease (2014) 5, e1470; doi:10.1038/cddis.2014.431 OPEN & 2014 Macmillan Publishers Limited All rights reserved 2041-4889/14 www.nature.com/cddis Glucose transporter 1-mediated glucose uptake is limiting for B-cell acute lymphoblastic leukemia anabolic metabolism and resistance to apoptosis This article has been corrected since Online Publication and a corrigendum has also been published 1,2 1,2 1,2 1,2 1,2 3 4 5 3 ,1,2 T Liu , RJ Kishton , AN Macintyre , VA Gerriets , H Xiang , X Liu , ED Abel , D Rizzieri , JW Locasale and JC Rathmell* The metabolic profiles of cancer cells have long been acknowledged to be altered and to provide new therapeutic opportunities. In particular, a wide range of both solid and liquid tumors use aerobic glycolysis to supply energy and support cell growth. This metabolic program leads to high rates of glucose consumption through glycolysis with secretion of lactate even in the presence of oxygen. Identifying the limiting events in aerobic glycolysis and the response of cancer cells to metabolic inhibition is now essential to exploit this potential metabolic dependency. Here, we examine the role of glucose uptake and the glucose transporter Glut1 in the metabolism and metabolic stress response of BCR-Abl+ B-cell acute lymphoblastic leukemia cells (B-ALL). B-ALL cells were highly glycolytic and primary human B-ALL samples were dependent on glycolysis. We show B-ALL cells express multiple glucose transporters and conditional genetic deletion of Glut1 led to a partial loss of glucose uptake. This reduced glucose transport capacity, however, was sufficient to metabolically reprogram B-ALL cells to decrease anabolic and increase catabolic flux. Cell proliferation decreased and a limited degree of apoptosis was also observed. Importantly, Glut1-deficient B-ALL cells failed to accumulate in vivo and leukemic progression was suppressed by Glut1 deletion. Similarly, pharmacologic inhibition of aerobic glycolysis with moderate doses of 2-deoxyglucose (2-DG) slowed B-ALL cell proliferation, but extensive apoptosis only occurred at high doses. Nevertheless, 2-DG induced the pro-apoptotic protein Bim and sensitized B-ALL cells to the tyrosine kinase inhibitor Dasatinib in vivo. Together, these data show that despite expression of multiple glucose transporters, B-ALL cells are reliant on Glut1 to maintain aerobic glycolysis and anabolic metabolism. Further, partial inhibition of glucose metabolism is sufficient to sensitize cancer cells to specifically targeted therapies, suggesting inhibition of aerobic glycolysis as a plausible adjuvant approach for B-ALL therapies. Cell Death and Disease (2014) 5, e1470; doi:10.1038/cddis.2014.431; published online 16 October 2014 Many cancer cells have elevated rates of glycolysis and acute lymphoblastic leukemia cells (B-ALL) and is associated lactate production even in the presence of oxygen. This with poor prognosis. The metabolic program of B-ALL cells is program, termed aerobic glycolysis, occurs in a wide range of undefined, although diffuse large B-cell lymphoma (DLBCL) both solid and liquid tumors and is driven by oncogenic signals can either be highly glycolytic or use oxidative phosphorylation 1 3 and microenvironmental pressures. Aerobic glycolysis is and mitochondrial metabolism. It has been suggested that proposed to allow metabolism in low oxygen tensions and to BCR-Abl signaling is associated with elevated glucose provide biosynthetic intermediates for cell growth. Indeed, metabolism, as BCR-Abl can promote glucose uptake and aerobic glycolysis readily supports both generation of ATP and trafficking of glucose transporter Glut1 to the cell surface. biosynthesis of lipids, nucleic acids, and amino acids. Given Conversely, inhibition of BCR-Abl in leukemic cells sup- 4–7 the high rates of glucose consumption and aerobic glycolysis presses glucose uptake and glycolysis. This regulation of in most cancers, targeting glucose metabolism has become of glucose metabolism may be critical for survival of BCR-Abl significant interest as an approach to eliminate cancer cells. B-ALL, as enforced expression of Glut1 protected B-ALL cells It is now important to establish mechanisms of aerobic from imatinib-induced apoptosis. These data show that BCR- glycolysis and the response of cancer cells to metabolic Abl promotes glucose uptake and aerobic glycolysis, and inhibition. BCR-Abl-transformed cells may rely on this pathway. The t(9;22) chromosomal translocation that generates the Targeting glucose metabolism can have efficacy against a oncogenic kinase BCR-Abl occurs in ~ 25% of adult B-cell variety of cancers. Mechanistic understanding of cancer cell 1 2 Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27710, USA; Department of Immunology, Duke Cancer Institute and Duke Molecular 3 4 Physiology Institute, Duke University, Durham, NC 27710, USA; Division of Nutritional Sciences, Cornell University, Ithaca, NY 14850, USA; Fraternal Order of Eagles Diabetes Research Center, Division of Endocrinology and Metabolism, Department of Medicine, Carver College of Medicine University of Iowa, Iowa City, IA 52242, USA and Division of Cell Therapy, Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA *Corresponding author: JC Rathmell, Department of Pharmacology and Cancer Biology, Duke University, DUMC 3813, Durham, NC 27710, USA. Tel: +1 1919 681 1084; Fax: +1 919 668 6044; E-mail: jeff.rathmell@duke.edu Abbreviations: B-ALL, B-cell acute lymphoblastic leukemia; 2-DG, 2-deoxyglucose; DLBCL, diffuse large B cell lymphoma; ECAR, extracellular acidification rate; OCR, oxygen consumption rate; 4-OHT, 4-hydroxytamoxifen; BrDU, BromodeoxyUridine Received 19.7.14; revised 28.8.14; accepted 01.9.14; Edited by C Munoz-Pinedo Glut1 is limiting in B-ALL anabolic metabolism T Liu et al metabolic requirements or response to inhibition using complete media with or without the addition of 10 mM 2-DG to strongly inhibit glycolysis. Cell survival was measured over pharmacologic approaches, however, has been limited. time and normal B cells were found only modestly affected by It has been shown using the glycolytic inhibitor 2-deoxyglucose glycolytic inhibition (Figure 1b). In contrast, primary B-ALL (2-DG) or glucose deprivation culture conditions that inhibition cells treated with 2-DG rapidly underwent cell death and were of glucose metabolism impacts cancer cell growth and viability highly dependent on glycolysis regardless of the BCR-Abl status. through several different mechanisms, including cell cycle arrest or cell death by activating AMPK pathway and B-ALL cells express multiple Glut family members and inactivating mTOR signaling. Reduced glucose metabolism deletion of Glut1 partially inhibits glucose uptake. Glu- has also been found to impact the stability and synthesis of cose uptake is the first essential step in glucose metabolism Bcl-2 family proteins. Glucose deprivation induces expression 19–21 7,11–15 and can be limiting in lymphocyte proliferation. Measure- of pro-apoptotic molecules, including Bim and can ment of Glut family members in BCR-Abl+ murine B-ALL induce apoptosis in cells transformed with oncogenic K-Ras found expression of Glut1, Glut3, Glut6, Glut8, and Glut9, through the unfolded protein response pathway. whereas the remaining Glut family members were undetected Here we examine the mechanism and role of glucose (Figure 1c). Similarly, human B-ALL cell lines expressed a uptake in B-ALL metabolism and leukemia progression by variable array of Glut family members, including Glut1, Glut3, genetically targeting glucose transport. The Glut family of Glut4, Glut5, Glut6, Glut7, Glut8, and Glut9 (Supplementary hexose transporters consists of 14 members and B-ALL Figure 1B). Of these transporters, Glut1 was previously cells expressed multiple family members. Conditional deletion shown important for proliferation and antibody secretion of of Glut1, however, demonstrated that B-ALL cells are reliant normal B cells and appeared broadly abundant in B-ALL. on this specific glucose transporter to sustain anabolic To genetically target B-ALL glucose uptake, therefore, we metabolism, proliferation, and resistance to cell death. fl/fl generated primary BCR-Abl B-ALL cells from Glut1 /Ubi-Cre- Consistent with our data showing a key role for glucose T2 ER mice to enable inducible deletion of glucose transporter uptake, we found that pharmacologic inhibition of glycolysis Glut1 upon treatment with 4-hydroxytamoxifen (4-OHT). sensitized B-ALL cells to caspase activation and apoptosis to fl/fl T2 Treatment of Glut1 /Ubi-Cre-ER B-ALL with 4-OHT led reduce leukemia burden in vivo. Glut1 and glucose uptake to efficient deletion of Glut1 with minimal compensation from have a key role, therefore, to maintain BCR-Abl B-ALL cell other Glut transporters, and 4-OHT had no effect on control growth and resistance to cell death. B-ALL cells (Figures 1c and d, Supplementary Figure 2). Deletion of Glut1 reduced glucose uptake by approximately Results half, showing Glut1 was limiting for maximal but does not mediate all glucose uptake (Figure 1e). To test whether this B-ALL cells utilize glucose through aerobic glycolysis. partial impairment of glucose transport impacted BCR-Abl Oncogenic signals can increase glucose uptake and drive oncogenic activity, we examined phosphorylation of BCR-Abl aerobic glycolysis. However, usage of glucose in B-ALL and immediate downstream targets and found BCR-Abl and and the response of B-ALL cells to inhibition of aerobic its substrate CRKL remained phosphorylated (Figure 1f). glycolysis are poorly understood. To examine glucose Glut1, therefore, contributes significantly to B-ALL glucose metabolism activity in B-ALL, real-time extracellular flux was uptake, and reduction of glucose metabolism does not alter measured in six human B-ALL cell lines, including BCR-Abl+ BCR-Abl activity. and BCR-Abl− cell lines, and peripheral B cells from three healthy donors in the absence of nutrients, after addition of Glut1 deficiency suppresses anabolic metabolism. Given glutamine, then upon addition of glucose. Extracellular the remaining capacity for glucose uptake in Glut1-deficient acidification rate (ECAR), reflective of lactate production, cells, the metabolic impact of Glut1 deletion was unclear. was similar in each cell type in the absence of nutrients To better understand the metabolic role of Glut1, the (Figure 1a). Glutamine, which is used as another important steady-state levels of 410 metabolites were measured metabolic fuel for many types of cancer cells in addition to through non-targeted LC/MS in B-ALL cells, with or without glucose, did not increase ECAR in any of the B-ALL cell expression of Glut1. Principal component analyses of the lines or normal B cells. The addition of glucose following metabolome of each population showed a modest difference T2 fl/fl T2 glutamine, however, led to a sharp increase in ECAR in all between control CreER B-ALL cells and Glut1 CreER T2 the B-ALL cells (Figure 1a). A modest increase in ECAR cells. However, control CreER B-ALL were largely was also observed in normal B cells. Importantly, under no unaffected by 4-OHT treatment, whereas Glut1 deletion led fl/fl condition did the oxygen consumption rate (OCR) change to a distinct metabolic profile in 4-OHT-treated Glut1 T2 with glutamine or glucose addition (Supplementary Figure CreER B-ALL cells (Figure 2a and Supplementary 1a), and we did not observe a clear difference of metabolic Table 1). Only five metabolites were significantly altered T2 program between BCR-Abl+ and BCR-Abl − cell lines. after 4-OHT treatment of control CreER B-ALL cells, B-ALL cells thus selectively metabolize glucose through whereas 43 metabolites were specifically changed in aerobic glycolysis with minimal contribution to oxidative response to Glut1 deletion (Figure 2b and Supplementary metabolism. Table 1). Affected metabolites represented a wide array of To test whether primary B-ALL cells depended on main- pathways (Figure 2c) with particular reductions in anabolic tenance of high glycolysis, BCR-Abl+ and BCR-Abl− B-ALL pathways, including pyrimidine and purine metabolism, patient samples and two normal donor B cells were cultured in glycolysis, and the amino and nucleotide sugar metabolism Cell Death and Disease Glut1 is limiting in B-ALL anabolic metabolism T Liu et al No Fuel Healthy 1 BCR-Abl + B-ALL Glutamine Healthy 2 BCR-Abl - B-ALL Glutamine + Glucose BV173 Tom-1 Nalm Nalm KOPN Sup 1 2 3 16 19 8 B15 0 10203040 50 Hours BCR-Abl+ BCR-Abl- BCR-Abl+ Healthy B cells ns ns ns ns 0.9 *** 0.8 Glut1 0.7 fl/fl Glut1 Vehicle 0.6 fl/fl Glut1 4-OHT 0.5 0.4 Actin 0.3 4-OHT - - + + 0.2 fl/fl 0.1 Glut1 CreER WT CreER Glut1 Glut3 Glut6 Glut8 Glut9 pBCR-Abl (Tyr 177) ns ** 35000 BCR-Abl BCR pCRKL (Tyr 207) CRKL 4-OHT - - + + Actin fl/fl Glut1 CreER WT CreER 4-OHT - - + + Normal B cell fl/fl Glut1 WT CreER CreER Figure 1 B-ALL cells require glycolysis and express multiple glucose transporters, but Glut1 is limiting for maximal glucose uptake. (a) ECAR was measured in human B-ALL cell lines and primary healthy-donor peripheral blood B cells at baseline and followed by sequential injection of 2 mM glutamine and 25 mM glucose. (b) Viability of human primary B-ALL cells and normal B cells from two donors each over time upon treatment of 2-DG (10 mM). Values were normalized at each time point to the vehicle control for that sample. fl/fl The BCR-Abl status of primary B-ALL is indicated. (c–e) Control and Glut1 CreER B-ALL cells were treated with vehicle or 4-OHT for 96 h followed by 48 h culture without 4-OHT prior to analyses. (c) Glucose transporter family member mRNA expression was measured by qrtPCR. Shown are expression of detected glucose transporters relative to expression of beta-actin. Other Glut family members were not detected. (d) Glut1 protein was measured by immunoblot and (e) glucose uptake was measured with radiolabeled glucose. (f) BCR-Abl activity was analyzed by immunoblot. Means of three or more replicates and S.D. are shown ***P≤ 0.001, **P≤ 0.005. NS, not significant that support glycosylation. Metabolic levels were lowered for increased (Figure 2d). Surprisingly, pyruvate and lactate, glycolytic intermediates, a key pentose phosphate pathway the majority of TCA cycle intermediates, and free intracellular intermediate, and multiple nucleotides and intermediates amino acids were unaffected by Glut1 deletion (Figure 2d, in nucleotide synthesis (Figure 2d, Supplementary Figure 3 Supplementary Figure 3 and Supplementary Table 1). and Supplementary Table 1). In contrast, catabolic pathways Pyruvate and lactate can be derived from non-glucose including glycerophospholipid metabolism and acyl-carni- sources, such as glutamine, and this may account for tines, which reflect fatty acid and lipid oxidation, were maintenance of these metabolites even with reduced glucose Cell Death and Disease mRNA expression relative ECAR (mpH/Min/Million cells) Glucose uptake (CPM) to Actin % Viable (Normalized to vehicle control) ∗ ∗ ∗ ∗ ∗∗ ∗ ns ns ns ns ns Glut1 is limiting in B-ALL anabolic metabolism T Liu et al Glycolysis Hexose phosphate WT CreER Vehicle WT CreER 4-OHT Fructose 1,6-biphosphate fl/fl Glut1 CreER Vehicle fl/fl Glut1 CreER 4-OHT Dihydroxyacetone phosphate Biphosphoglycerate D-Glycerate 3-phosphate/ D-Glycerate 2-phosphate Phosphenolpyruvate PC 1 (45.1%) Pyruvate 6 Lactate fl/fl Glut1 CreER +/-4OHT 0 0.5 1 1.5 Pentose Phosphate Pathway 2-deoxyribose phosphate N=410 D-gluconate -6 -4 -2 0 2 4 6 6 Sedoheptulose 7-phosphate WT CreER +/-4OHT 0 0.5 1 1.5 2 Nucleotides ATP UTP N=410 KL(" CTP -6 -4 -2 0 2 4 6 0 0.5 1 1.5 Log (fold change+/-4OHT) TCA Cycle Malate Pyrimidine N-Carbamoyl-L-aspartate, (S)- Fumarate metabolism dihydroorotatedCTP, CTP, UTP, Succinate Deoxyuridine, Glycerophospho- CDP-ethanolamine, choline, CDPcholine, N- -Ketoglutarate lipid metabolism methylethanolaminium phosphate, Dihydroxyacetone phosphate, Aconitate Glycolysis or Hexose phosphate, Phosphoenolpyruvate, Citrate/Isocitrate Gluconeogenesis Dihydroxyacetone phosphate, Fructose 1,6- biphosphate, D-Glycerate 3-phosphate/D- 0 0.5 1 1.5 2 Glycerate 2-phosphate Lipid Oxidation (Acyl-Carnitines) Acyl Carnitines Heptadecanoyl-, Linoleyl-, tetradecenoyl-, L-palmitoyl-carnitine Linoleyl carnitine Purine dGMP/AMP/3-AMP, ATP metabolism Heptadecanoyl carnitine Pentose Sedoheptulose 7-phosphate, phosphate Tetradecenoyl carnitine pathway Stearoyl carnitine Amino sugar and GDP-alpha-D-mannose, UDP-N-acetyl-D- nucleotide sugar galactosamine/UDP-N-acetyl-alpha-D- Palmitoyl carnitine metabolism glucosamine 0 0.5 1 1.5 2 2.5 3 Glycine, serine Choline, O-Phospho-L-serine and threonine Fold change to vehicle control metabolism fl/fl fl/fl Glut1 Vehicle Glut1 4-OHT Decreased >1.5 fold, p<0.05 Increased >1.5 fold, p<0.05 fl/fl Figure 2 In vitro Glut1 deletion leads to metabolic reprogramming of B-ALL cells. (a–c) Steady-state metabolite levels in wild-type (WT) Cre-ER and Glut1 CreER B-ALL cells treated with vehicle or 4-OHT were determined using LC/MS. (a) Principal component, (b) volcano plots of metabolites changed 41.5-fold, Po0.05, and (c)key metabolites categorized by KEGG metabolic pathways are shown in order of significance. (d) Relative levels of specific metabolites in key pathways are shown following Glut1 deletion. Data represent mean and S.D. for triplicate samples. *Po0.05. NS, not significant metabolism. These data suggest that Glut1-dependent To further investigate glucose contribution to downstream glucose mainly supports biosynthetic pathways in B-ALL metabolic pathways and how Glut1 deficiency alters these cells, and reduced glucose uptake led to metabolic repro- pathway activities, glucose fate was traced and metabolic flux gramming to favor catabolism. analysis was performed using C-labeled glucose. B-ALL Cell Death and Disease -Log(p value) PC2 (20.1%) Glut1 is limiting in B-ALL anabolic metabolism T Liu et al cells were cultured in vehicle or 4-OHT for 4 days to delete cells efficiently converted 13C-glucose to uniformly labeled Glut1 and then labeled with 13C-glucose for 24 h prior to C phosphoenolpyruvate, dihydroxyacetone phosphate, and LC/MS mass spectrometry. Despite partial maintenance of ribose phosphate through glycolysis and the pentose glucose uptake, flux to anabolic pathways was sharply phosphate pathway, respectively (Figure 3a, Supplementary curtailed following Glut1 deletion. Control Glut1-expressing Figure 4 and Supplementary Table 2). Glut1-deficient cells, Vehicle 4-OHT Phosphoenolpyruvate ns ∗∗∗ ∗∗ 0[13C] 1[13C] 2[13C] 3[13C] Ribose-phosphate ∗∗ ∗ ns ∗ ns ∗∗∗ ∗ 0[13C] 1[13C] 2[13C] 3[13C] 4[13C] 5[13C] Lactate ns ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ 40000000 40 0[13C] 1[13C] 2[13C] 3[13C] Malate ∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ 0 0 0[13C] 1[13C] 2[13C] 3[13C] 4[13C] Pentose Phosphate Pathway Palmitate Oxidation ∗∗∗ ns ∗∗∗∗ ns 700 400 - + - + 4OHT - + - + 4OHT fl/fl fl/fl Glut1 CreER WT CreER Glut1 CreER WT CreER Figure 3 Glut1 deletion suppresses glucose contribution to anabolic pathways and increases catabolic metabolism. (a) 13C-glucose tracing contribution of glucose to indicated metabolite pools. Graphs on the left indicate the total quantity of each metabolite and the graph on the right indicates the relative distribution of C carbons in each fl/fl metabolite. (b and c) WT Cre-ER and Glut1 Cre-ER B-ALL cells were treated with vehicle or 4-OHT for 96 h followed by 48 h of culture without 4-OHT and (b) pentose 14 14 phosphate pathway flux was measured by oxidation of 1-C -glucose and (c) lipid oxidation was measured by oxidation of C palmitate. Means and S.D. of triplicate measurements are shown. *Po0.05, **Po0.005, ***Po0.001, ****Po0.0001. NS, not significant Cell Death and Disease Total AUC Total AUC Total AUC Total AUC CPM/Million cells Percentage Percentage Percentage Percentage CPM/Million cells Glut1 is limiting in B-ALL anabolic metabolism T Liu et al however, produced very little total levels of these metabolites following Glut1 deletion (Figure 3b). Conversely, Glut1 relative to control cells and that which was generated deletion led to a sharp increase in palmitate oxidation contained a significantly lower fraction of 13C-glucose- (Figure 3c). Together, these data show that B-ALL cells are derived carbon (Figures 2d and 3a, Supplementary Figure 4 highly glycolytic and primarily use glucose to support and Supplementary Table 2). Pyruvate and lactate were biosynthetic reactions and pathways, such as the pentose present in similar levels, and control cells generated these phosphate pathway. metabolites through both C-labeled glucose and unlabeled sources, whereas the majority of these metabolites were derived from non-glucose sources in Glut1-deficient cells. Thus alternative sources, such as glutamine, contribute Metabolic reprogramming suppresses B-ALL prolifera- tion. The sharp decrease in flux toward biosynthetic meta- significantly to pyruvate and lactate in control cells and these bolic pathways and increased catabolism following Glut1 pathways become increasingly dominant after Glut1 deletion. deletion suggested that Glut1 deficiency may impede B-ALL Surprisingly, glucose did not contribute significantly to the TCA cell growth and proliferation. Indeed, 4-OHT treatment led to cycle in B-ALL regardless of Glut1 expression, as malate, a sharp reduction in cell accumulation rates over time citrate, succinate, and alpha-ketoglutarate were unlabeled in (Figure 4a). This was at least partially due to reduced both control and Glut1-deficient cells (Figure 3a, proliferation, as BromodeoxyUridine (BrDU) incorporation in Supplementary Figure 4 and Supplementary Table 2). Thus, fl/fl T2 Glut1 CreER B-ALL cells was significantly decreased, glucose was not the main fuel resource for oxidative T2 whereas control CreER cells were unaffected by 4-OHT metabolism in B-ALL, nor was it redirected toward oxidative (Figures 4a and b). Cell cycle profiling of control and 4-OHT- metabolism in Glut1-deficient B-ALL cells. Rather, other treated B-ALL cells, however, showed that Glut1 deletion did metabolic fuels sustained the TCA cycle. Radiolabeled tracer assays were next conducted in pentose not lead to a clear cell cycle arrest or accumulation of cells in phosphate and lipid oxidation pathways to independently a specific phase of the cell cycle (Figure 4c). Rather Glut1 confirm these findings. Consistent with previous results, deficiency appeared to slow cell proliferation than activate a pentose phosphate pathway activity was significantly reduced specific cell cycle arrest checkpoint. fl/fl WT CreER Glut1 CreER Vehicle Vehicle 6 6 4-OHT 4-OHT 4 4 3 3 2 2 0 20 40 60 80 100 0 20 40 60 80 100 Hours Hours fl/fl Glut1 CreER ns Vehicle 4OHT WT CreER - + - + 4OHT fl/fl Glut1 CreER WT CreER DNA content fl/fl Figure 4 In vitro Glut1 suppresses B-ALL accumulation through decreased proliferation. (a) WT Cre-ER and Glut1 Cre-ER B-ALL cells were treated with vehicle or 4-OHT for 96 h followed by 48 h of culture without 4-OHT, and cell numbers were counted over time. (b and c) After 4 days treatment with vehicle or 4-OHT followed by 2 days culture without 4-OHT, cells were cultured with BrDU for 1.5 additional hours and (b) BrDU incorporation was measured by intracellular flow cytometry. (c) DNA content was determined flow cytometrically by propidium iodide staining to indicate cell cycle status. Means and S.D. are shown for triplicate samples in representative experiments repeated three or more times. ****Po0.0001. NS, not significant Cell Death and Disease BrDU Positive Cells (%) Cell Density (Million cells/ml) Cell Percentage Glut1 is limiting in B-ALL anabolic metabolism T Liu et al Glut1 deletion sensitizes B-ALL cells to apoptosis with accumulation in vitro through increased apoptosis. Consis- targeted agents. Glucose metabolism is closely linked to tent with this notion, Glut1 deficiency specifically induced cell survival, and Glut1 deletion may have also impaired cell expression of the pro-apoptotic protein Bim and only fl/fl a b Glut1 CreER WT CreER 100 100 Bim 80 80 60 60 Actin 40 40 Vehicle 4OHT -+ -+ 20 4-OHT fl/fl Glut1 WT CreER 0 0 CreER 050 100 150 0 50 100 150 Hours c Dasatinib Dasatinib fl/fl Glut1 CreER WT CreER 100 100 Vehicle 50nM Dasatinib 4OHT 20 4OHT 50nM Dasatinib 0 50 100 150 0 50 100 150 Hours d Post Glut1 deletion and prior to 48 hours after Dasatinib addition Dasatinib addition *** ** *** **** * 60 **** **** Annexin V+/PI- Annexin V+/PI- 50 * Annexin V+/PI+ Annexin V+/PI+ 4-OHT - - - - ++++ - - - - ++++ 4-OHT - - ++ -- ++ Q-vd - +- + - +- + Dasatinib - - + + -- ++ - - + + -- ++ Q-vd - + - + - ++ - - + - + - ++ - fl/fl Glut1 WT CreER fl/fl CreER WT CreER Glut1 CreER fl/fl Figure 5 In vitro Glut1 deletion induces pro-apoptotic Bim expression and sensitizes B-ALL to cell death stimulus. (a and b) WT Cre-ER and Glut1 Cre-ER B-ALL cells treated with vehicle or 4-OHT for 96 h followed by 48 h of culture without 4-OHT and (a) examined by immunoblot on day 6 or (b) analyzed by flow cytometry for survival over time. fl/fl (c) WT Cre-ER and Glut1 Cre-ER B-ALL cells were cultured with vehicle or 4-OHT for 96 h, washed, then cultured an additional 48 h alone or with addition of Dasatinib (50 nM), and cell viability was determined over time by flow cytometry. (d) Apoptosis in Glut1-deleted cells with or without Dasatinib treatment was assessed by annexin V/PI staining. Cells were treated with vehicle or 4-OHT for 96 h and apoptosis was assessed by annexin V/PI staining (left panel). After 96 h of culture with vehicle or 4-OHT, cells were washed and cultured for an additional 48 h alone or with Dasatinib (50 nM). Cell apoptosis was assessed at the end of the 48 h (right panel). Ten μM pan caspases inhibitor Q-vd-oph was added in some cell cultures as indicated. Gray bar, annexin V+/PI− cell percentage. Black bar, annexin V+/PI+ cell percentage. Means and S.D. are shown for triplicate samples from representative experiments repeated three or more times. *Po0.05, **Po0.005, ***Po0.001, ****Po0.0001 Cell Death and Disease % Annexin V Positive % Viable % Viable Glut1 is limiting in B-ALL anabolic metabolism T Liu et al modestly impacted expression of other Bcl-2 family proteins, cells were dependent on continued glycolysis and a low dose including pro-apoptotic protein Bax, Bak, Bid and anti- of 2-DG prevented cell accumulation (Figure 7a). A higher apoptotic protein Mcl-1 and Bcl-xL (Figure 5a and dose of 2-DG to further inhibit glycolysis was required to Supplementary Figure 5A). Bim can be induced in response induce B-ALL cell death, similar to the observation in human to ER stress and the unfolded protein response, but only primary B-ALL cells (Figure 7b). This dose of 2-DG treatment very modest markers of these pathways were detected induced Bim expression (Figure 7c), which was essential to − /− relative to those induced by the glycosylation inhibitor, induce apoptosis, as Bim B-ALL cells resisted cell death tunicamycin (Supplementary Figure 5B). Thus, ER stress even with high doses of 2-DG (Figure 7d). may contribute to Bim induction, but this response was not The potential of 2-DG to provide an adjuvant metabolic strongly induced. stress to specifically sensitize cancer cells to Dasatinib was Glut1 deletion led to reduced B-ALL cell viability over time next tested in vivo. Wild-type B-ALL cells were adoptively (Figure 5b). The majority of cells, however, remained viable transferred into recipient animals and allowed 2 days to even without Glut1. However, increased expression of Bim in engraft. To provoke metabolic stress and prime B-ALL cells for Glut1-deficient cells suggested that sensitivity of the surviving targeted therapy, animals were treated with vehicle or 2-DG for cells to apoptosis was increased. Indeed, treatment of BCR- 4 days alone, followed by three additional days with or without Abl B-ALL with a low dose of the tyrosine kinase inhibitor, addition of Dasatinib. B-ALL cell percentages and numbers Dasatinib, only mildly impacted control B-ALL cells, but B-ALL were then determined 11 days after transfer (Figure 7e). 2-DG cell death was markedly increased in Glut1-deleted cells and Dasatinib alone each reduced B-ALL cell burden. (Figure 5c). Cell death appeared to occur in part through Importantly, combined treatment of 2-DG and Dasatinib apoptosis, as Annexin V+ early-stage apoptotic cells were together led to significantly further depletion of B-ALL cells. detected (Figure 5d) and caspase activity increased Likewise, 2-DG treatment also increased efficacy of Dasatinib (Supplementary Figure 6). Importantly, caspase inhibition to induce cell death in human BCR-Abl+ B-ALL cell lines with Q-VD partially protected cells from death (Figure 5d). (Supplementary Figure 7). Thus, partial non-cytotoxic phar- macologic inhibition of glucose metabolism slows tumor Glut1 deletion suppresses B-ALL progression growth and provides an enhanced response to specifically in vivo. Despite sharply curtailed growth and proliferation targeted therapy. in vitro, how B-ALL progressed in vivo without Glut1 remained unclear. Presence of in vivo nutrients and stromal cell support Discussion may allow B-ALL cells to persist and proliferate even without UbiCreERT2 Glut1 and with reduced glucose uptake. Control Elevated rates of glucose uptake and glycolysis can have fl/fl UbiCreERT2 and Glut1 B-ALL cells were, therefore, trans- significant roles in cancer cell survival and progression by ferred into immunocompromised hosts that were treated with supporting cellular energetics and providing biosynthetic T2 25 vehicle or tamoxifen to activate CreER , and in vivo and substrates. It is now important to establish limiting compo- B-ALL growth was assessed with or without Glut1 expression nents of aerobic glycolysis and how cancer cells respond to (Figure 6a). B-ALL cells were monitored by IRES-driven GFP metabolic inhibition. Here we show that B-ALL cells are highly expression from the BCR-Abl expressing retroviral vector. dependent on glucose and that glucose uptake through Glut1 Two days after cell transfer, recipients were treated with is essential for BCR-Abl B-ALL cells to maintain anabolic tamoxifen to delete Glut1 in transferred B-ALL cells. Animals metabolism to support proliferation. Glut1-deficient B-ALL were then analyzed for B-ALL cell number after an additional cells both proliferated at a lower rate and had moderate levels week. Glut1 was efficiently deleted in vivo, and Glut1 protein of apoptosis. Importantly, B-ALL cells required Glut1 to progress in vivo, demonstrating a clear role for this specific levels were sharply reduced in B-ALL cells purified from glucose transporter in B-ALL metabolism. Similar to Glut1 splenocytes of tamoxifen-treated recipient animals UbiCreERT2 fl/fl UbiCreERT2 deletion, pharmacologic inhibition of aerobic glycolysis with 2- (Figure 6b). Both control and Glut1 DG also suppressed proliferation and led to increased B-ALL cells were present in high levels in both spleen and expression of Bim to sensitize to the tyrosine kinase inhibitor bone marrow of vehicle-treated mice (Figures 6c and d). Dasatinib in vivo. Partial metabolic stress thus impaired cell Importantly, B-ALL cells did not accumulate in vivo and proliferation and sensitized BCR-Abl-driven B-ALL cells to disease did not rapidly progress after Glut1 deletion apoptosis with specifically targeted therapy in vivo. (Figure 6c) and numbers of Glut1-deficient B-ALL cells were Similar to the glycolytic form of DLBCL, we show here that significantly reduced in spleen and bone marrow (Figure 6d), both BCR-Abl+ and BCR-Abl− human B-ALL cell lines are relative to vehicle-treated or control B-ALL. These data indicate that Glut1 deletion suppresses B-ALL progression, highly glycolytic and metabolize glucose through aerobic and B-ALL cells are dependent on Glut1 even in physiological glycolysis. Importantly, in vitro, B-ALL cells were sensitive to conditions. inhibition of glycolysis with reduced proliferation followed by apoptosis. In vitro culture conditions, however, do not faithfully Pharmacological inhibition of glycolysis impairs prolif- mimic in vivo nutrient conditions. Presence of alternate eration and leads to Bim-dependent cell death to nutrients, stromal cells and growth factors in a physiological sensitize to targeted therapy. Given the dependence of setting may substantially modulate sensitivity of cells to B-ALL cells on Glut1, the cellular response to pharmacolo- therapy. Primary B-ALL cells with conditional deletion of Glut1 gical disruption of glucose metabolism was next examined by allows for a direct test of glucose metabolism contribution to treating BCR-Abl B-ALL with 2-DG. BCR-Abl+ murine B-ALL B-ALL progression in vivo. Glut1 deletion did not fully prevent Cell Death and Disease Glut1 is limiting in B-ALL anabolic metabolism T Liu et al Day 0: B-ALL cell transfer Glut1 Day 3-6: Injection of corn oil control or tamoxifen Actin Tumor burden Tamoxifen -- - + + + analysis Vehicle Tamoxifen fl/fl WT CreER Glut1 CreER Spleen Bone Marrow Spleen Bone Marrow Veh: 35% Veh: 50% Veh: 37% Veh: 69% Tam: 43% Tam: 11% Tam: 4.6% Tam: 73% GFP WT CreER ns 80 ns ns 60 0 0 0 Vehicle Tamoxifen Vehicle Tamoxifen Vehicle Tamoxifen fl/fl Glut1 CreER **** **** **** 6 60 2 20 0 0 Vehicle Tamoxifen Vehicle Tamoxifen Vehicle Tamoxifen fl/fl Figure 6 In vivo Glut1 deletion decreases leukemic tumor burden. (a) Schematic diagram showing tamoxifen treatment to induce Glut1 deletion in vivo.(b) Glut1 CreER B-ALL were transferred and hosts treated with vehicle or tamoxifen on day 3, and purified B-ALL cells were analyzed by immunoblot from individual mice.(c) Flow cytometry from fl/fl representative spleen and bone marrow on day 10 of recipient animals that received WT Cre-ER or Glut1 Cre-ER and were treated with vehicle or tamoxifen. (d) Percentages and numbers of GFP+ transferred B-ALL cells on day 10 in individual mice treated as indicated with vehicle or tamoxifen. Means and S.D. from n= 5 mice/group for WT CreER fl/fl group and n= 10 mice/group for Glut1 CreER group are shown. ****Po0.0001. NS, not significant glucose uptake, but instead glucose transport was reduced to provide potentially different capacities to adapt and compen- approximately half in Glut1-deficient cells. The remaining sate for metabolic inhibition. Our data suggest, however, that glucose uptake was likely mediated through other glucose Glut1 or Glut3 may have dominant roles. 26–28 transporters expressed by B-ALL cells. In particular, Despite the incomplete inhibition of glucose uptake, Glut1 Gluts 3 and 6 may increase activity to support this glucose deletion led to striking shifts in metabolic pathways that transport. Human B-ALL cells expressed additional trans- demonstrate glucose transport as a limiting component of porters, including Glut4 and Glut5 (Supplementary Figure B-ALL cell metabolism. Glut1-deficient cells had significantly S1B). The array and expression levels of these different decreased flux through biosynthetic pathways, including the transporters, however, differed across individual cell lines to pentose phosphate pathway, nucleotide and phospho-lipid Cell Death and Disease BM GFP+ cell number BM GFP+ cell number 6 6 Cell Percentage (x10 ) (x10 ) Splenic GFP+ (%) Splenic GFP+ (%) Splenic GFP+ cell number Splenic GFP+ cell (x10 ) number (x10 ) Glut1 is limiting in B-ALL anabolic metabolism T Liu et al 3.5 Vehicle 1mM 2DG 2.5 1.5 Vehicle 1mM 2DG 0.5 5mM 2DG 0 10 20 30 40 50 010 20 30 40 50 Hours Hours Bim Actin WT Vehicle WT 5mM 2DG 5mM 20 -/- Vehicle Bim Vehicle 2DG -/- 0h Bim 5mM 2DG 24h 010 20 30 40 50 Hours **** **** 15 80 ** * **** 2-DG - - + + - - + + Dasatinib - + - + - + - + Figure 7 Pharmacological inhibition of glycolysis impairs B-ALL proliferation and sensitizes to apoptosis in vitro and in vivo.(a–c) WT B-ALL cells were cultured in low (1 mM) or high (5 mM) dose of 2-DG and (a) cell number was counted and (b) cell viability was analyzed by flow cytometry over time. (c) Cell lysates were analyzed by immunoblot. − /− (d) WTor Bim B-ALL cells were treated with 5 mM 2-DG and cell viability was analyzed by flow cytometry over time. (e) WT B-ALL cells were adoptively transferred into host animals that were treated starting on day 2 with vehicle alone or with 500 mg/kg per day of 2-DG alone for 4 days or with additional 10 mg/kg per day of Dasatinib for 3 days. GFP+ B-ALL cell percentages and numbers were determined by flow cytometry. Means and S.D. are shown of (a, b and d) triplicate and (e) n= 5 mice/group. ****Po0.0001, ***Po0.001, **Po0.005, *Po0.05 synthetic pathways, but had elevated lipid metabolism inter- Similar to 2-DG treatment, Glut1 deletion also induced mediates and oxidation rates. Surprisingly, the remaining expression of pro-apoptotic protein Bim and decreased B-ALL glucose uptake and glycolytic flux in Glut1-deficient B-ALL viability partially through apoptosis, although non-apoptotic was not redirected to oxidative metabolism and the TCA cycle cell death may have occurred as well. Bim appears to be the for maximum ATP generation. Rather, even Glut1-deficient primary Bcl-2 family protein mediating apoptotic events, as B-ALL cells remained dependent on non-glucose sources for expression levels of other Bcl-2 family proteins were only TCA intermediates. It is possible that a BCR-Abl signal may modestly altered by Glut1 deletion (Supplementary Figure S5). suppress pyruvate oxidation even under nutrient-limiting Glut1 deletion did not fully eliminate all B-ALL cells, possibly conditions. As a consequence, B-ALL cell growth and because the level of metabolic stress from Glut1 deletion was proliferation were sharply curtailed by Glut1 deletion. Cells insufficient to kill all the cells. Nevertheless, Glut1 deficiency did not arrest in a specific cell cycle stage, but rather cell sensitized the remaining B-ALL cells to cell death stimulus. A proliferation appeared slowed in each phase in a balanced sub-lethal dose of BCR-Abl inhibitor, Dasatinib, rapidly fashion. It may be that a more severe form of metabolic induced apoptosis in Glut1-deleted cells. It is unclear what inhibition is essential to induce cell cycle checkpoints and pathway was directly responsible for Bim induction. It has arrest, such as p21 induction that can be induced through been reported that decreased protein glycosylation upon 8,29 AMPK activation of p53, but we observed B-ALL cells to glucose deprivation or 2-DG can lead to endoplasmic reticulum 11,30 instead undergo apoptosis in these conditions. stress, which may promote Bim induction through the Cell Death and Disease GFP+ cell number in Cell Density bone marrow (x10 ) (Million cell/ml) GFP+ cell number in spleen % Viable % Viable (x10^6) Glut1 is limiting in B-ALL anabolic metabolism T Liu et al 24,31 transcription factor CHOP. However, Glut1 deficiency only Diego, CA, USA). Use of the human subject-derived samples was approved by the Duke University Institutional Review Board. imposed mild, if any, ER stress to B-ALL cells. The role of this mild ER stress on Bim induction remains unclear. Primary murine BCR-Abl+ B-ALL. Bone marrow cells from C57B6/J Interactions of B-ALL cells with stromal cells and the − /− T2 (Jackson Labs, Bar Harbor, ME, USA), Bim (Jackson Labs), Ubi-Cre-ER availability of alternate nutrients may support metabolic fl/fl T2 transgenic (Jackson Labs), and Glut1 mice crossed to Ubi-Cre-ER mice were flexibility to allow cancer progression in vivo even with limited cultured in IL-7 (10 ng/ml) (eBioscience) in IMDM supplemented with 20% fetal glucose uptake. In vivo deletion of Glut1 from B-ALL cells, bovine serum and infected with MSCV-BCR-Abl-IRES-GFP retrovirus (gift of D Fruman, UC Irvine) with polybrene (4 μg/ml) (Millipore, Billerica, MA, USA). however, markedly reduced leukemic tumor burden in Infected cells were cultured in methylcellulose medium containing IL-7 (Stem Cell recipient animals and prevented disease progression. The Technologies) and individual colonies were isolated on day 7 and transferred into significant reduction of tumor burden may be due to the complete IMDM media with 20% fetal bovine serum, but no IL-7. After 7 days, viable combinational effects of suppressed cell proliferation and GFP+ colonies were selected for expansion. The Institutional Animal Care and Use impaired cell viability. These data demonstrate that glucose Committee of Duke University approved all animal protocols. uptake and Glut1 are limiting components in the support of aerobic glycolysis in BCR-Abl+ B-ALL cells in vivo. Similarly, Immunoblot and flow cytometry. Cell lysates for immunoblots were 4,7,12 prepared as described. Primary antibodies used were rabbit anti-Bim (BD Glut1 deficiency suppressed breast cancer progression. Pharmingen, San Jose, CA, USA), rabbit anti-Glut1 (Abcam, Cambridge, MA, USA), Other glycolytic enzymes have also been shown to have rabbit anti-phospho-BCR (Cell Signaling, Danvers, MA, USA), rabbit anti-BCR (Cell key-limiting roles in cancer metabolism, including pyruvate Signaling), rabbit anti-phospho-CRKL (Cell signaling), rabbit anti-CRKL (Santa 33 34 kinase M2, phospho-fructokinase, and lactate dehydro- Cruz, Dallas, TX, USA), rabbit anti-Bcl-xL (Cell signaling), rabbit anti-Mcl-1 genase A. Our data support Glut1 as an additional potential (Biolegend, San Diego, CA, USA), rabbit anti-Bax (Cell signaling), rabbit anti-Bak restriction point in cancer metabolism. (Cell signaling), rabbit anti-Bid (mouse specific, Cell signaling), rabbit anti-PDI (Cell signaling), rabbit anti-IRE1α (Cell signaling), rabbit anti-Bip (Cell Signaling), mouse These data collectively suggest Glut1 may provide a anti-CHOP (Cell signaling), mouse anti-actin (Sigma-Aldrich) and were detected therapeutic target to reduce glucose uptake and treat cancers with anti-rabbit horseradish peroxidase-labeled antibody (Promega, Madison, WI, that use aerobic glycolysis. Although Glut1 is essential for USA) and fluorescent-labeled anti-mouse antibody (LiCor, Lincoln, NE, USA). Blots 36,37 glucose uptake in some tissues, incomplete inhibition of were visualized using Supersignal West Pico Chemiluminescent Substrate (Thermo Glut1 may be feasible and provide benefits. Indeed, several Scientific, Waltham, MA, USA) or the Odyssey infrared imaging system (LiCor). groups have now described inhibitors of Glut1 with partial Antibodies for cytometry include anti-human CD34, CD19, CD10 and CD20 38–41 (eBioscience), annexin V (Invitrogen, Grand Island, NY, USA) and anti-BrDU activity that can suppress tumor growth. The HIV (Invitrogen). Cell viability was measured by flow cytometry for propidium iodide protease inhibitor, ritonavir, also shows non-selective partial 12,13 42 (PI; Invitrogen) exclusion as described. Apoptotic cell populations were assessed inhibition of both Glut1 and Glut4 with low toxicity. Although it by annexin V/PI staining. Cells were incubated with Alexa Fluor 488 annexin V remains unclear to what extent Glut1 may provide a direct (Invitrogen) for 15 min in dark and then 2 μg/ml PI was added. Cell cycle profile and pharmacologic target, our data indicate that it is not essential DNA content were measured in cells fixed in ethanol and stained with PI. BrDU to fully suppress glucose uptake to prevent cancer cell incorporation was measured by culture of cells with 10 uM BrDU (Sigma-Aldrich) for 1.5 h followed by ethanol fixation, denaturation with 2 M HCl for 20 min and staining proliferation and disease progression. with anti-BrdU. Flow cytometry data were collected on MACSQuant (Miltenyi, Together, we show B-ALL is a highly glycolytic cancer Bergisch Gladbach, Germany) or FACScan (Becton Dickinson, San Jose, CA, USA) dependent on Glut1 as a limiting component of glucose and flow cytometers and analyzed using FlowJo software (Treestar, Ashland, OR, USA). anabolic metabolism. Glycolytic inhibition triggers a graded response of reduced cell proliferation followed by increased Quantitative real-time-PCR. Total mRNA was extracted (RNeasy mini kit; sensitivity to apoptosis both in vitro and in vivo. Impairment of Qiagen, Valencia, CA, USA) and 1 μg of RNA was reverse transcribed (iScript; Bio- glucose uptake through Glut1 deletion sufficiently suppressed Rad, Hercules, CA, USA) to perform SYBR Green (Bio-Rad) semi-quantitative real- time-PCR for Glut1. Relative expression levels were calculated using the ΔCt/ΔCt biosynthetic reactions and shifted B-ALL metabolic state to method, with expression normalized to 18S or actin RNA. Primers for mouse glucose catabolism. This metabolic reprogramming impeded B-ALL transporters and mouse actin: Glut1, 5ʹ-AGCCCTGCTACAGTGTAT-3ʹ,5ʹ-AGGTCT proliferation and prevented disease progression in vivo. The CGGGTCACATC-3ʹ; Glut3, 5ʹ-TAAACCAGCTGGGCATCGTTGTTG-3ʹ,5ʹ-AATGAT glucose dependence observed in B-ALL is likely also present GGTTAAGCCAAGGAGCCC-3ʹ; Glut6, 5ʹ-TTGGTGCTGTGAGGCT-3ʹ,5ʹ-TGGC in other types of cancer, and partial blockade of aerobic ACAAACTGGACGTA-3ʹ;Glut8, 5ʹ-ACATCTCGGAAATCGCCT-3ʹ,5ʹ-ACACAGCCC glycolysis may provide an adjuvant approach to augment the AGCACG-3ʹ;Glut9, 5ʹ-TGCTTCCTCGTCTTCGCCACAATA-3ʹ,5ʹ-CTCTTGGCAAA TGCCTGGCTGATT-3ʹ;actin,5ʹ-CCTTCCTTCTTGGGTATGGA-3ʹ,5ʹ-TGGTACCAC efficacy of targeted agents with minimal additional toxicity. CAGACAGCACT-3ʹ. Primers for human glucose transporters and 18S: Glut1, 5ʹ-CACTCCTGTTACTTACCTAA-3ʹ,5ʹ-CACTTACTTCTGTCTCACT-3ʹ;Glut3,5ʹ-GACC CAGAGATGCTGTAATGGT-3ʹ,5ʹ-GGGGTGACCTTCTGTGTCCC-3ʹ;Glut4, 5ʹ-CTTC Materials and Methods Human primary B-ALL and B-cell culture. Human B-ALL cell lines CAACAGATAGGCTCCG-3ʹ,5ʹ-CCCCAATGTTGTACCCAAAC-3ʹ; Glut5, 5ʹ-GCAA (BV-173, TOM-1, Nalm-16, Nalm-19, and KOPN-8 from DSMZ (Braunschweig, CAGGATCAGAGCATGA-3ʹ,5ʹ-CCATACTGGAAGGATGACCC-3ʹ; Glut6, 5ʹ-GTCCA Germany); Sup-B15 from ATCC (Manassas, VA, USA)) were cultured in complete TCTTCGACAGCACCG-3ʹ,5ʹ-GCAAACATGATGGCCGCTGA-3ʹ; Glut7, 5ʹ-CACC RPMI 1640 (Mediatech, Manassas, VA, USA) or complete IMDM (Gibco, Grand GTCTCCATGTTTCCTC-3ʹ,5ʹ-TGTTGTTGATCAGCAGGGTC-3ʹ; Glut8, 5ʹ-TCCT Island, NY, USA) with 10 or 20% fetal bovine serum (Gemini Bioproducts, West GGTTCGGGGCTGTC-3ʹ,5ʹ-GAGCACAGCAAGAGGCTCAG-3ʹ; Glut9, 5ʹ-GAGTAT Sacramento, CA, USA). De-identified primary human B-ALL samples were cultured CGTGGGCATTCTGG-3ʹ,5ʹ-AGTTGGAGAGCCAGTTGACG-3ʹ; 18S, 5ʹ-GTAAC in Hybridoma SFM media (Gibco) supplemented with cytokines (refer to CCGTTGAACCCCATT-3ʹ,5ʹ-CCATCCAATCGGTAGTAGCG-3ʹ. Supplementary Materials for list of cytokines). Human B cells from peripheral blood (Gulf Coast Regional Blood Center, Houston, TX, USA) were purified by Metabolomic profiling. Metabolomic analyses were performed as described negative selection (Stem Cell Technologies, Vancouver, BC, Canada) and cultured before using la iquid chromatography Q Exactive Mass Spectrometer (LC-QE-MS) 22 fl/fl in the same media as B-ALL cells. Some samples were treated with 2-DG (Sigma- (Thermo Scientific). In non-targeted metabolomics analyses, Glut1 CreER and Aldrich, St. Louis, MO, USA) at indicated doses. Samples were monitored by flow WT CreER cells were treated with vehicle or 0.4 μM 4-OHT for 96 h followed by an cytometry using anti-human CD34, CD19, CD10 and CD20 (eBioscience, San additional 48 h in complete IMDM media containing 1 mM sodium pyruvate. For Cell Death and Disease Glut1 is limiting in B-ALL anabolic metabolism T Liu et al 13C-glucose flux studies, cells were cultured as described above for 5 days culture, 8. 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Kraus M, Malenke E, Gogel J, Muller H, Ruckrich T, Overkleeft H et al. Ritonavir induces Licence. The images or other third party material in this article are endoplasmic reticulum stress and sensitizes sarcoma cells toward bortezomib-induced apoptosis. Mol Cancer Ther 2008; 7: 1940–1948. included in the article’s Creative Commons licence, unless indicated 40. Tuccinardi T, Granchi C, Iegre J, Paterni I, Bertini S, Macchia M et al. Oxime-based inhibitors otherwise in the credit line; if the material is not included under the of glucose transporter 1 displaying antiproliferative effects in cancer cells. Bioorg Med Chem Creative Commons licence, users will need to obtain permission from Lett 2013; 23: 6923–6927. the licence holder to reproduce the material. To view a copy of this 41. Wood TE, Dalili S, Simpson CD, Hurren R, Mao X, Saiz FS et al. A novel inhibitor of glucose uptake sensitizes cells to FAS-induced cell death. 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