The JAK2V617F oncogene requires expression of inducible phosphofructokinase/fructose-bisphosphatase 3 for cell growth and increased metabolic activity - PubMed (original) (raw)

The JAK2V617F oncogene requires expression of inducible phosphofructokinase/fructose-bisphosphatase 3 for cell growth and increased metabolic activity

M M Reddy et al. Leukemia. 2012 Mar.

Abstract

Myeloproliferative neoplasms are characterized by overproduction of myeloid lineage cells with frequent acquisition of oncogenic JAK2V617F kinase mutations. The molecular mechanisms that regulate energy requirements in these diseases are poorly understood. Transformed cells tend to rely on fermentation instead of more efficient oxidative phosphorylation for energy production. Our data in JAK2V617F-transformed cells show that growth and metabolic activity were strictly dependent on the presence of glucose. Uptake of glucose and cell surface expression of the glucose transporter Glut1 required the oncogenic tyrosine kinase. Importantly, JAK2V617F as well as active STAT5 increased the expression of the inducible rate-limiting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), which controls glycolytic flux through 6-phosphofructo-1-kinase. PFKFB3 was required for JAK2V617F-dependent lactate production, oxidative metabolic activity and glucose uptake. Targeted knockdown of PFKFB3 also limited cell growth under normoxic and hypoxic conditions and blocked in vivo tumor formation in mice. Overall, these data suggest that inducible PFKFB3 is required for increased growth, metabolic activity and is regulated through active JAK2 and STAT5. Novel therapies that specifically block PFKFB3 activity or expression would, therefore, be expected to inhibit JAK2/STAT5-dependent malignancies and related cancers.

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Figures

Figure 1

Figure 1. Glucose metabolism is stimulated in JAK2V617F expressing cells

A and B, changes in 2-NBD-glucose uptake were measured by flow cytometry in KU812, HEL and Molm13 cells in response to inhibitors of their respective oncogenic tyrosine kinases (A) and in BaF3 cells compared to cells containing BCR-ABL, JAK2V617F or FLT3-ITD (B) (n=3). Typical experiments are shown in the left panel. C, Growth of HEL cells was determined by trypan blue exclusion (left panel) and changes in lactate production were measured (right panel) in response to control medium, Jak inhibitor (1 µM) or medium lacking (△) glutamine (Q) and/or glucose, as indicated (n=3). *, indicates that significant differences (p<0.05) were observed between control and treated cells (A, C) or parental BaF3 cells and their transformed counterparts containing oncogenic tyrosine kinases (B).

Figure 2

Figure 2. JAK2V617F is associated with increased expression of Glut1 and PFKFB3

A, cell surface expression of Glut1 was determined by flow cytometry. BaF3 cells were compared to cells expressing JAK2V617F or HEL and BaF3.EpoR.JAK2V617F cells were left untreated or treated with Jak inhibitor (1µM, 18 h), as indicated (typical experiment shown). B, expression of PFKFB3, MYC, HIF1α, HIF2α and β-actin was determined by immunoblotting in HEL cells maintained under normoxic (20% O2) and hypoxic (0.1% O2) conditions. C, expression of PFKFB3 and β-actin was determined by immunoblotting in parental BaF3 and cells containing BCR-ABL, JAK2V617F or FLT3-ITD. D, mRNA expression of PFKFB3 was compared between healthy donors (HD) and polycythemia vera patient (PV) specimens. Changes were calculated relative to the average expression of PFKFB3 in healthy donors.

Figure 3

Figure 3. Activation of the STAT5 pathway is sufficient for increased metabolism

Untreated BaF3 cells with doxycycline inducible constitutively active STAT5 (−) were used and compared to cells treated (+) with 1 µg/mL doxycycline (DOX). Expression of active STAT5, PFKFB3 and β-actin was determined by immunoblotting (A). Changes in glucose uptake (B) and lactate production (C) were determined in response to expression of active STAT5 (n=3). *, indicates that significant differences (p<0.05) were observed between active STAT5-expressing and control cells.

Figure 4

Figure 4. PFKFB3 is associated with increased metabolism

A, HEL cells were treated for 18 hours with 3PO and lactate production was measured. B and C, HEL cells containing scrambled shRNA (Scr) or two different PFKFB3-targeting constructs C and D were used, as indicated. B, expression of PFKFB3 and β-actin was determined by immunoblotting. C, relative changes in lactate production (left panel), levels of intracellular ROS (middle panel) and 2-NBD-glucose uptake (right panel) were measured in response to PFKFB3 knockdown (n=3). *, indicates that significant differences (p<0.05) were observed between control and treated cells.

Figure 5

Figure 5. PFKFB3 is required for increased growth

A, HEL cells were treated for 48h with different concentrations of 3PO and cell growth was determined. B, HEL cells containing scrambled shRNA or PFKFB3-targeting constructs C and D were used to measure changes in growth of cells maintained under normoxic (20% O2) or hypoxic (0.1% O2) conditions (n=3). *, indicates that significant differences (p<0.05) were observed between control and treated cells. C, in vivo tumor formation of HEL cells with targeted knockdown of PFKFB3 (■, △) or scrambled shRNA (●) was determined in SCID/Beige mice (n=8). The volumes of subcutaneous tumors were measured and compared 18 days after initial injection. **, indicates that significant differences (p<0.0001) were observed between scrambled and PFKFB3-targeting constructs C and D (11, 14 and 18 days after tumor cell injection).

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