The JAK2V617F Oncogene, Associated with Myeloproliferative Neoplasms, Requires Expression of Inducible Phosphofructokinase/Fructose Bisphosphatase 3 for Cell Growth and Increased Metabolic Activity

Abstract 793

Myeloproliferative neoplasms (MPNs) and myeloid leukemias, characterized by overproduction of myeloid lineage cells, are frequently associated with transforming oncogenic kinases, including JAK2V617F, BCR-ABL, or FLT3-ITD. The mechanisms that regulate altered energy metabolism in these diseases are poorly understood but cancer cells tend to produce energy through increased glycolysis instead of oxidative phosphorylation, even under normoxic conditions (Warburg effect). Our data in JAK2V617F-transformed HEL cells show that glucose uptake, the first step in glucose metabolism, is reduced in response to a JAK2 inhibitor (−22.29%, p<0.05, n=3). Further, introduction of JAK2V617F into murine BaF3 cells resulted in increased glucose uptake (103.72%, p<0.05, n=3), compared to parental BaF3 cells. Exposure of cells transformed by BCR/ABL or FLT3-ITD with appropriate kinase inhibitors similarly resulted in a 30 to 40% decrease in glucose uptake, and introduction of either BCR-ABL or FLT3-ITD into BaF3 cells resulted in substantial increases in glucose uptake (BCR-ABL, +122.74%, and FLT3-ITD, +142.77%; p<0.05, n=3). Consistent with an increase in glucose uptake, we also found elevated cell surface expression of the glucose transporter Glut1 in BaF3.JAK2V617F cells. Importantly, cell growth and metabolic activity were strictly dependent on the presence of glucose in the culture medium. Also, treatment with the hexokinase inhibitor, 2-deoxyglucose, led to reduced cell growth, further supporting the notion that JAK2V617F transformed cells rely on glucose for their metabolic functions. JAK2V617F increased the expression of at least two rate-limiting enzymes in the glycolytic pathway, including hexokinase 2 (HK2) as well as 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and in particular, a JAK2 inhibitor substantially decreased the expression of PFKFB3 in HEL cells. To determine the significance of altered PFKFB3 expression, we targeted PFKFB3 in HEL cells using a lentiviral-based shRNA approach. We found that PFKFB3 knockdown reduced cell growth by 46.3 to 46.8% in HEL cells (p<0.05, n=3) compared to control shRNA. The impact on cell growth was similar under normoxic (20% O₂) compared to hypoxic (0.1% O₂) conditions (−58.2% to −45.5%; p<0.05, n=3), further underlining the importance of this pathway for cell growth. We also observed a reduction in oxidative metabolic activity (−32.26% to −34.14%, p<0.05, n=3) and glucose uptake (−28.58% to −22.5%, p<0.05, n=3) in response to PFKFB3 knockdown. Finally, in order to understand the role of the JAK2V617F target STAT5 in the regulation of increased PFKFB3 expression, we used BaF3 cells with a doxycycline inducible form of active STAT5. These cells, upon induction of active STAT5, showed increased growth and metabolic activity as well as elevated expression of PFKFB3, compared to controls. It is not known whether PFKFB3 is a direct transcriptional target of STAT5. Overall, these data suggest that inducible PFKFB3 is required for increased growth, metabolic activity and is regulated through the JAK2V617F/STAT5 pathway, hinting at novel targets for drug development. Small molecule drugs that target PFKFB3 would be expected to specifically inhibit this pathway and to have activity in diseases dependent on JAK2V617F or active STAT5 in related malignancies.