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

Liu T, Kishton RJ, Macintyre AN, Gerriets VA, Xiang H, Liu X, Abel ED, Rizzieri D, Locasale JW, Rathmell JC - Cell Death Dis (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27710, USA [2] Department of Immunology, Duke Cancer Institute and Duke Molecular Physiology Institute, Duke University, Durham, NC 27710, USA.

ABSTRACT
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.

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In vitro Glut1 deletion induces pro-apoptotic Bim expression and sensitizes B-ALL to cell death stimulus. (a and b) WT Cre-ER and Glut1fl/fl 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. (c) WT Cre-ER and Glut1fl/fl 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. *P<0.05, **P<0.005, ***P<0.001, ****P<0.0001
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fig5: In vitro Glut1 deletion induces pro-apoptotic Bim expression and sensitizes B-ALL to cell death stimulus. (a and b) WT Cre-ER and Glut1fl/fl 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. (c) WT Cre-ER and Glut1fl/fl 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. *P<0.05, **P<0.005, ***P<0.001, ****P<0.0001

Mentions: Glucose metabolism is closely linked to cell survival, and Glut1 deletion may have also impaired cell accumulation in vitro through increased apoptosis. Consistent with this notion, Glut1 deficiency specifically induced expression of the pro-apoptotic protein Bim and only modestly impacted expression of other Bcl-2 family proteins, including pro-apoptotic protein Bax, Bak, Bid and anti-apoptotic protein Mcl-1 and Bcl-xL (Figure 5a and Supplementary Figure 5A). Bim can be induced in response to ER stress and the unfolded protein response,24 but only very modest markers of these pathways were detected relative to those induced by the glycosylation inhibitor, tunicamycin (Supplementary Figure 5B). Thus, ER stress may contribute to Bim induction, but this response was not strongly induced.


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

Liu T, Kishton RJ, Macintyre AN, Gerriets VA, Xiang H, Liu X, Abel ED, Rizzieri D, Locasale JW, Rathmell JC - Cell Death Dis (2014)

In vitro Glut1 deletion induces pro-apoptotic Bim expression and sensitizes B-ALL to cell death stimulus. (a and b) WT Cre-ER and Glut1fl/fl 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. (c) WT Cre-ER and Glut1fl/fl 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. *P<0.05, **P<0.005, ***P<0.001, ****P<0.0001
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fig5: In vitro Glut1 deletion induces pro-apoptotic Bim expression and sensitizes B-ALL to cell death stimulus. (a and b) WT Cre-ER and Glut1fl/fl 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. (c) WT Cre-ER and Glut1fl/fl 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. *P<0.05, **P<0.005, ***P<0.001, ****P<0.0001
Mentions: Glucose metabolism is closely linked to cell survival, and Glut1 deletion may have also impaired cell accumulation in vitro through increased apoptosis. Consistent with this notion, Glut1 deficiency specifically induced expression of the pro-apoptotic protein Bim and only modestly impacted expression of other Bcl-2 family proteins, including pro-apoptotic protein Bax, Bak, Bid and anti-apoptotic protein Mcl-1 and Bcl-xL (Figure 5a and Supplementary Figure 5A). Bim can be induced in response to ER stress and the unfolded protein response,24 but only very modest markers of these pathways were detected relative to those induced by the glycosylation inhibitor, tunicamycin (Supplementary Figure 5B). Thus, ER stress may contribute to Bim induction, but this response was not strongly induced.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27710, USA [2] Department of Immunology, Duke Cancer Institute and Duke Molecular Physiology Institute, Duke University, Durham, NC 27710, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus