PFKFB3 Knockdown Research Articles

PFKFB3-mediated endothelial glycolysis promotes pulmonary hypertension

Increased glycolysis in the lung vasculature has been connected to the development of pulmonary hypertension (PH). We therefore investigated whether glycolytic regulator 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (PFKFB3)-mediated endothelial glycolysis plays a critical role in the development of PH. Heterozygous global deficiency of Pfkfb3 protected mice from developing hypoxia-induced PH, and administration of the PFKFB3 inhibitor 3PO almost completely prevented PH in rats treated with Sugen 5416/hypoxia, indicating a causative role of PFKFB3 in the development of PH. Immunostaining of lung sections and Western blot with isolated lung endothelial cells showed a dramatic increase in PFKFB3 expression and activity in pulmonary endothelial cells of rodents and humans with PH. We generated mice that were constitutively or inducibly deficient in endothelial Pfkfb3 and found that these mice were incapable of developing PH or showed slowed PH progression. Compared with control mice, endothelial Pfkfb3-knockout mice exhibited less severity of vascular smooth muscle cell proliferation, endothelial inflammation, and leukocyte recruitment in the lungs. In the absence of PFKFB3, lung endothelial cells from rodents and humans with PH produced lower levels of growth factors (such as PDGFB and FGF2) and proinflammatory factors (such as CXCL12 and IL1β). This is mechanistically linked to decreased levels of HIF2A in lung ECs following PFKFB3 knockdown. Taken together, these results suggest that targeting PFKFB3 is a promising strategy for the treatment of PH.

Ablation of endothelial Pfkfb3 protects mice from acute lung injury in LPS-induced endotoxemia

Acute lung injury (ALI) is one of the leading causes of death in sepsis. Endothelial inflammation and dysfunction play a prominent role in development of ALI. Glycolysis is the predominant bioenergetic pathway for endothelial cells (ECs). However, the role of EC glycolysis in ALI of sepsis remains unclear. Here we show that both the expression and activity of PFKFB3, a key glycolytic activator, were markedly increased in lipopolysaccharide (LPS)-treated human pulmonary arterial ECs (HPAECs) in vitro and in lung ECs of mice challenged with LPS in vivo. PFKFB3 knockdown significantly reduced LPS-enhanced glycolysis in HPAECs. Compared with LPS-challenged wild-type mice, endothelial-specific Pfkfb3 knockout (Pfkfb3ΔVEC) mice exhibited reduced endothelium permeability, lower pulmonary edema, and higher survival rate. This was accompanied by decreased expression of intracellular adhesion molecule-1 (Icam-1) and vascular cell adhesion molecule 1 (Vcam-1), as well as decreased neutrophil and macrophage infiltration to the lung. Consistently, PFKFB3 silencing or PFKFB3 inhibition in HPAECs and human pulmonary microvascular ECs (HPMVECs) significantly downregulated LPS-induced expression of ICAM-1 and VCAM-1, and monocyte adhesion to human pulmonary ECs. In contrast, adenovirus-mediated PFKFB3 overexpression upregulated ICAM-1 and VCAM-1 expression in HPAECs. Mechanistically, PFKFB3 silencing suppressed LPS-induced nuclear translocation of nuclear factor κB (NF-κB)-p65, and NF-κB inhibitors abrogated PFKFB3-induced expression of ICAM-1 and VCAM-1. Finally, administration of the PFKFB3 inhibitor 3PO also reduced the inflammatory response of vascular endothelium and protected mice from LPS-induced ALI. Overall, these findings suggest that targeting PFKFB3-mediated EC glycolysis is an efficient therapeutic strategy for ALI in sepsis.

The Glycolytic Enzyme PFKFB3 Controls TNF-α-Induced Endothelial Proinflammatory Responses.

Endothelial cells play an important role in health and a variety of diseases. Recent evidences show that endothelial cells rely on glycolysis rather than on oxidative phosphorylation to generate energy to support cellular functions such as angiogenesis. However, the effect of endothelial glycolysis on vascular inflammation remains little known. Here, we investigate the role of key glycolytic enzyme PFKFB3 in tumor necrosis factor-α (TNF-α)-induced endothelial proinflammatory responses. siRNAs were used to knockdown the expression of PFKFB3. In some experiments, PFKFB3 inhibitors were also used. TNF-α at 20ng/ml was added to confluent endothelial cells for different time period of stimulation. PFKFB3 expression was examined by RT-PCR and western blotting. Cytokine antibody panel membranes were employed to detect different cytokines/chemokines in culture supernatant of endothelial cells. The determination of monocyte adhesion to endothelial cells after TNF-α treatment was conducted using THP-1 cells. The monocyte attraction was performed using Transwell filters. For further mechanisms, NF-κB-p65 localization was examined by immunofluorescence. Expression of total IκB, phospho-IκB, phospho-NF-κB-p65, and Ikkβ was detected by western blotting. DNA-binding activity of NF-κB was assessed using electrophoretic mobility shift assay. We found that TNF-α increased endothelial PFKFB3 expression. Knockdown of PFKFB3 almost blocked all TNF-α-induced release of the proinflammatory cytokines/chemokines (MCP-1, IL-8, CXCL1, GMCSF, RANTES, TNF-α) and ICAM-1. PFKFB3 knockdown also significantly inhibited TNF-α-induced monocyte adhesion and transmigration. Furthermore, inhibition of PFKFB3 inhibited TNF-α-induced Ikkβ phosphorylation, IκBα phosphorylation and degradation, NF-κB-p65 phosphorylation, nuclear translocation, and DNA-binding activity. Thus, our results demonstrate that glycolytic enzyme PFKFB3 plays a critical role in TNF-α-induced endothelial inflammation.

Abstract 668: Myeloid Pfkfb3 Deficiency Protect Mice From Hypoxia Induced Pulmonary Hypertension

Background: Pulmonary hypertension is a progressive disease and perivascular inflammation is found in all types of pulmonary hypertension patients. Macrophage are innate inflammatory cells and the number of macrophages are significantly increased in the lungs of PAH patients. Glycolysis is the main pathway to generate energy and intermediate products for macrophage activation. However, the effect of macrophage glycolysis on the development of pulmonary hypertension remains unknown. As PFKFB3 is a critical regulator in glycolysis, we investigated the effect of macrophage glycolysis on pulmonary hypertension using myeloid PFKFB3 specific deficiency mice and hypoxia induced pulmonary hypertension model. Results: In mouse and rat hypoxia induced pulmonary hypertension models, we found that PFKFB3 was upregulated in macrophages. PFKFB3 myeloid specific depletion protected mice from the formation of pulmonary hypertension. Knockdown of PFKFB3 in macrophages suppressed the expression of growth factors and inflammatory cytokines in whole lung tissue. Also, we found that PFKFB3 knockdown decreased the numbers of perivascular macrophages and interstitial macrophages using immunostaining and FACS. The number of alveolar macrophages showed no significant difference, while the expression of growth factors and inflammatory cytokines were decreased in alveolar macrophage when PFKFB3 was depleted. Conclusions: PFKFB3 depletion in macrophage decreases the number of perivascular macrophages and interstitial macrophages in the lung, and suppresses the expression of growth factors and inflammatory cytokines in alveolar macrophage, thus protecting mice from the formation of pulmonary hypertension.

PFKFB3 blockade inhibits hepatocellular carcinoma growth by impairing DNA repair through AKT

Overexpression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a key molecule of glucose metabolism in cytoplasm, has been found in various tumors. Emerging evidence has suggested that PFKFB3 is also located in the nucleus and apparent in regulatory functions other than glycolysis. In this study, we found that PFKFB3 expression is associated with hepatocellular carcinoma (HCC) growth and located mainly in the nucleus of tumor cells. PFKFB3 overexpression was associated with large tumor size (p = 0.04) and poor survival of patients with HCC (p = 0.027). Knockdown of PFKFB3 inhibited HCC growth, not only by reducing glucose consumption but also by damaging the DNA repair function, leading to G2/M phase arrest and apoptosis. In animal studies, overexpression of PFKFB3 is associated with increased tumor growth. Mechanistically, PFKFB3 silencing decreased AKT phosphorylation and reduced the expression of ERCC1, which is an important DNA repair protein. Moreover, PFK15, a selective PFKFB3 inhibitor, significantly inhibited tumor growth in a xenograft model of human HCC. PFKFB3 is a potential novel target in the treatment of HCC.

Targeting PFKFB3 sensitizes chronic myelogenous leukemia cells to tyrosine kinase inhibitor.

Resistance to the BCR-ABL tyrosine kinase inhibitor (TKI) remains a challenge for curing the disease in chronic myeloid leukemia (CML) patients as leukemia cells may survive through BCR-ABL kinase activity-independent signal pathways. To gain insight into BCR-ABL kinase activity-independent mechanisms, we performed an initial bioinformatics screen and followed by a quantitative PCR screen of genes that were elevated in CML samples. A total of 33 candidate genes were identified to be highly expressed in TKIs resistant patients. Among those genes, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), controlling the limiting step of glycolysis, was found to be strongly associated with TKIs resistance. PFKFB3 knockdown or pharmacological inhibition of its kinase activity markedly enhanced the sensitivity of CML cells to TKIs. Furthermore, pharmacological inhibition of PFKFB3 inhibited CML cells growth and significantly prolonged the survival of both allograft and xenograft CML mice. ChIP-seq data analysis combined with subsequent knockdown experiment showed that the Ets transcription factor PU.1 regulated the elevated expression of PFKFB3 in TKIs-resistant CML cells. Therefore, our results showed that targeting PFKFB3 sensitizes CML cells to TKIs and PFKFB3 may be a potential BCR-ABL kinase activity-independent mechanism in CML.

TGF-β1 targets Smad, p38 MAPK, and PI3K/Akt signaling pathways to induce PFKFB3 gene expression and glycolysis in glioblastoma cells.

In human cancers, transforming growth factor-β1 (TGF-β1) plays a dual role by acting as both a tumor suppressor and a promoter of tumor metastasis. Although TGF-β1 contributes to the metabolic reprogramming of cancer cells and tumor-associated stromal cells, little is known of the molecular mechanisms connecting this cytokine with enhanced glycolysis. PFKFB3 is a homodymeric bifunctional enzyme, belonging to the family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases, that controls the conversion of fructose-6-phosphate (Fru-6-P) to fructose-2,6-bisphosphate (Fru-2,6-P2 ). This metabolite is important for the dynamic regulation of glycolytic flux by allosterically activating phosphofructokinase-1, a rate-limiting enzyme in glycolysis. The PFKFB3 gene is involved in cell proliferation via its role in carbohydrate metabolism. Here, we studied the mechanisms connecting TGF-β1, glucose metabolism, and PFKFB3 in glioblastoma cell lines. We demonstrate that TGF-β1 upregulates PFKFB3 mRNA and protein expression resulting in an increase in fructose 2,6-bisphosphate concentration, glucose uptake, glycolytic flux and lactate production. Moreover, these increases in PFKFB3 mRNA and protein expression and Fru-2,6-P2 concentration were reduced when the Smad3, p38 mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase (PI3K)/Akt signaling pathways were inhibited. We demonstrate that inhibition of PFKFB3 activity with 3PO or siRNA-mediated knockdown of PFKFB3 significantly eliminated the capacity of the T98G cells to form colonies by TGF-β1, one of the hallmarks of transformation. Taken together, these results show that TGF-β1 induces PFKFB3 expression through activation of the p38 MAPK and PI3K/Akt signaling pathways that complement and converge with early activation of Smad signaling. This suggests that PFKFB3 induction by TGF-β1 can be one of the main mechanisms mediating the reprogramming of glioma cells.

PFKFB3 was overexpressed in gastric cancer patients and promoted the proliferation and migration of gastric cancer cells.

Gastric cancer is one of the most common cancers worldwide, and the prognosis is still very poor due to the lack of specific and sensitive biomarkers. Aerobic glycolysis is one of the critical hallmarks of gastric cancer cells, and several glycolytic enzymes are highly expressed in gastric cancer patients. However, the expression and clinical significances of phosphofructokinase-2/fructose-2,6-bisphosphatase3 (PFKFB3, one of the glycolytic enzymes) in a large sample of gastric cancer patients remain unclear. The expression of PFKFB3 was detected in 134 gastric cancer patients by qRT-PCR, immunohistochemistry, and western blot analyses. The correlation between PFKFB3 expression and clinicopathological factors was analyzed by χ 2 test. In addition, we also analyzed whether the knockdown of PFKFB3 by siRNAs could inhibit the ability of gastric cancer cells (MGC-803 and AGS) to proliferate and migrate by MTT analysis and transwell analyses. PFKFB3 was highly expressed in 81.3% (109/134) of gastric cancer patients. The overexpression of PFKFB3 was associated with lymph node metastasis (P = 0.045) and TNM stage (P = 0.033). Knockdown of PFKFB3 by siRNAs significantly inhibited the proliferation and migration abilities of gastric cancer cells. Our data suggest that PFKFB3 might be a potential biomarker for gastric cancer and anti-neoplastic targeting gene.

Role of Akt/PKB and PFKFB isoenzymes in the control of glycolysis, cell proliferation and protein synthesis in mitogen-stimulated thymocytes

Proliferating cells depend on glycolysis mainly to supply precursors for macromolecular synthesis. Fructose 2,6-bisphosphate (Fru-2,6-P2) is the most potent positive allosteric effector of 6-phosphofructo-1-kinase (PFK-1), and hence of glycolysis. Mitogen stimulation of rat thymocytes with concanavalin A (ConA) led to time-dependent increases in lactate accumulation (6-fold), Fru-2,6-P2 content (4-fold), 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase isoenzyme 3 and 4 (PFKFB3 and PFKFB4) protein levels (~2-fold and ~15-fold, respectively) and rates of cell proliferation (~40-fold) and protein synthesis (10-fold) after 68h of incubation compared with resting cells. After 54h of ConA stimulation, PFKFB3 mRNA levels were 45-fold higher than those of PFKFB4 mRNA. Although PFKFB3 could be phosphorylated at Ser461 by protein kinase B (PKB) in vitro leading to PFK-2 activation, PFKFB3 Ser461 phosphorylation was barely detectable in resting cells and only increased slightly in ConA-stimulated cells. On the other hand, PFKFB3 and PFKFB4 mRNA levels were decreased (90% and 70%, respectively) by exposure of ConA-stimulated cells to low doses of PKB inhibitor (MK-2206), suggesting control of expression of the two PFKFB isoenzymes by PKB. Incubation of thymocytes with ConA resulted in increased expression and phosphorylation of the translation factors eukaryotic initiation factor-4E-binding protein-1 (4E-BP1) and ribosomal protein S6 (rpS6). Treatment of ConA-stimulated thymocytes with PFK-2 inhibitor (3PO) or MK-2206 led to significant decreases in Fru-2,6-P2 content, medium lactate accumulation and rates of cell proliferation and protein synthesis. These data were confirmed by using siRNA knockdown of PFKFB3, PFKFB4 and PKB α/β in the more easily transfectable Jurkat E6-1 cell line. The findings suggest that increased PFKFB3 and PFKFB4 expression, but not increased PFKFB3 Ser461 phosphorylation, plays a role in increasing glycolysis in mitogen-stimulated thymocytes and implicate PKB in the upregulation of PFKFB3 and PFKFB4. The results also support a role for Fru-2,6-P2 in coupling glycolysis to cell proliferation and protein synthesis in this model.

PFKFB3-mediated glycolysis is involved in reactive astrocyte proliferation after oxygen-glucose deprivation/reperfusion and is regulated by Cdh1

Reactive astrocyte proliferation is involved in many central degenerative diseases. The enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3), an allosteric activator of 6-phosphofructo-1-kinase (PFK1), controls glycolytic flux. Furthermore, APC/C-Cdh1 plays a crucial role in brain metabolism by regulating PFKFB3 expression. Previous studies have defined the roles of PFKFB3-mediated glycolysis in pathological angiogenesis, cell autophagy, and amyloid plaque deposition in proliferating cells. However, the role of PFKFB3 in reactive astrocyte proliferation after cerebral ischemia is unknown. In this study, we cultured rat primary cortical astrocytes and established an oxygen-glucose deprivation/reperfusion (OGD/R) model to mimic cerebral ischemia in vivo. Astrocyte proliferation was measured by western blotting for proliferating cell nuclear antigen (PCNA) and by EdU incorporation. We found that OGD/R up-regulated PFKFB3 and PFK1 expression, which was accompanied by reactive astrocyte proliferation. Knockdown of PFKFB3 by siRNA transfection significantly inhibited reactive astrocyte proliferation and lactate release, an indicator of glycolysis. We found that PFKFB3 and PFK1 expression were down-regulated and lactate release was decreased when OGD/R-induced astrocyte proliferation was inhibited by a Cdh1-expressing lentivirus. Thus, reactive astrocyte proliferation can be effectively suppressed by down-regulation of PFKFB3 through control of glycolytic flux, which is downstream of APC/C-Cdh1.

Abstract 719: PFKFB3 Mediated Glycolysis in Vascular Smooth Muscle Cells Is Essential for Arterial Neointima Formation

Background: Mature vascular smooth muscle cells (VSMCs) exhibit limited proliferation and migration ability. However, in response to stresses, VSMCs undergo phenotypic modulation characterized by increased proliferation and motility. This phenotypic modulation is associated with several vascular diseases, including atherosclerosis, arterial neointima formation. Elevated glycolysis has been described in proliferative VSMCs. The isoform 3 of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (PFKFB3) is a regulatory factor for VSMC glycolysis. PFKFB3 catalyze the synthesis of fructose 2, 6-bishosphate (F2, 6 BP). The latter is a powerful activator of 6-phosphofructo-1-kinase, the rate-limiting enzyme in glycolytic flux. It is unclear whether PFKFB3 mediated glycolysis play a role in VSMC phenotypic modulation andarterial neointima formation. Methods and results: PDGF treatment increased PFKFB3 expression in human aortic smooth muscle cells (HASMCs). This was accompanied with enhanced proliferation and migration of HASMCs. Knockdown of PFKFB3 with adenovirus carrying PFKFB3 short hairpin RNA suppressed PDGF-induced HASMC proliferation and migration. Examination of intracellular signaling indicated PI3K/Akt participated into PFKFB3-mediated glycolysis, and further cooperated with PFKFB3 to modulate phenotype of HASMCs. In a model of the left common carotid artery ligation, both PFKFB3 heterozygotes and mice with deficiency of PFKFB3 in VSMCs showed the decreased arterial neointima compared with controls mice. Conclusions: PFKFB3 mediates glycolysis and cooperates with PI3K/AKT, thereafter promoting proliferation and migration of VSMCs and leading to the formation of an increased arterial neointima. Thus, PFKFB3 is a promising therapeutic target for the treatment of atherosclerosis, arterial neointima and other peripheral arterial diseases.

Laminar shear stress inhibits endothelial cell metabolism via KLF2-mediated repression of PFKFB3.

Cellular metabolism was recently shown to regulate endothelial cell phenotype profoundly. Whether the atheroprotective biomechanical stimulus elicited by laminar shear stress modulates endothelial cell metabolism is not known. Here, we show that laminar flow exposure reduced glucose uptake and mitochondrial content in endothelium. Shear stress-mediated reduction of endothelial metabolism was reversed by silencing the flow-sensitive transcription factor Krüppel-like factor 2 (KLF2). Endothelial-specific deletion of KLF2 in mice induced glucose uptake in endothelial cells of perfused hearts. KLF2 overexpression recapitulates the inhibitory effects on endothelial glycolysis elicited by laminar flow, as measured by Seahorse flux analysis and glucose uptake measurements. RNA sequencing showed that shear stress reduced the expression of key glycolytic enzymes, such as 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3 (PFKFB3), phosphofructokinase-1, and hexokinase 2 in a KLF2-dependent manner. Moreover, KLF2 represses PFKFB3 promoter activity. PFKFB3 knockdown reduced glycolysis, and overexpression increased glycolysis and partially reversed the KLF2-mediated reduction in glycolysis. Furthermore, PFKFB3 overexpression reversed KLF2-mediated reduction in angiogenic sprouting and network formation. Our data demonstrate that shear stress-mediated repression of endothelial cell metabolism via KLF2 and PFKFB3 controls endothelial cell phenotype.

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

Myeloproliferative neoplasms (MPNs) 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.

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

AbstractAbstract 793Myeloproliferative 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. Disclosures:No relevant conflicts of interest to declare.