PFKFB3 Inhibitor Research Articles

Effect of inflammation-mediated endothelial metabolic shift on endothelial barrier function

Abstract Background The integrity of the endothelial cell barrier of the microvasculature is compromised by inflammation. The increased vascular permeability leads to tissue injury and organ dysfunction. In recent years, considerable advances have been made in the understanding of signalling mechanisms regulating the endothelial barrier integrity. The role of endothelial metabolism as a modulator of endothelial barrier integrity is not yet well-studied. The aim of the present study was to investigate the effect of inflammation on endothelial metabolism and its role in the maintenance of endothelial barrier integrity. Methods The study was carried out on cultured human umbilical vein endothelial cells and rat coronary microvascular endothelial cells. Inflammatory condition was simulated by treating cells with low concentrations (1 ng/mL) of TNFα for 24h. Endothelial barrier function was analysed by measuring the flux of albumen through endothelial monolayers cultured on filter membranes. Gene expression was analysed by qPCR-based assays. The capacity of endothelial cells for maximal ATP synthesis rate was investigated by the real-time live-cell imaging using FRET-based ATP-biosensor (live cell FRET). Total cellular ATP concentration was measured using luminescence-based commercial kit (ATPLite, PerkinElmer). Mitochondrial mass was analysed by the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA). The cellular glucose uptake was measured by fluorescent microscopy using a fluorescent analogue of glucose (2-NBDG). Results Treatment of human endothelial cells with TNFα resulted in significant suppression of mitochondrial and upregulation of glycolytic ATP synthesis rate, suggesting a metabolic switch. This was accompanied by a reduction in mitochondrial content (mtDNA/nDNA), reduction in total cellular ATP levels, an enhanced expression of glycolytic enzymes 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and phosphofructokinase 1 (PFK1), and enhanced glucose uptake by endothelial cells (n=5; p<0.05 for all parameters tested). Moreover, TNFα caused a 3-fold increase in endothelial permeability. Pharmacological inhibition of glycolysis either by partial replacement of glucose with 2-deoxy glucose (2DG) or an inhibition of PFKFB3 resulted in further worsening (a 5-fold increase in permeability) of TNFα-induced endothelial barrier failure. On the other hand pharmacological activation of AMPK, a potent inducer of mitochondrial biogenesis, could attenuate TNFα-induced but not 2DG-induced endothelial hyperpermeability. Conclusion The study demonstrates that TNFα induces metabolic switch towards glycolysis in endothelial cells. Moreover, the data suggest that upregulation of glycolysis may serve as an endogenous metabolic adaptation to the TNFα-induced suppression of mitochondrial ATP synthesis, which protects endothelial barrier integrity. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Justus-Liebig University GiessenDZHK (German Centre for Cardiovascular Research), partner site Rhein-Main, Bad Nauheim, Germany

Deficiency of Myeloid Pfkfb3 Protects Mice From Lung Edema and Cardiac Dysfunction in LPS-Induced Endotoxemia.

Sepsis, a pathology resulting from excessive inflammatory response that leads to multiple organ failure, is a major cause of mortality in intensive care units. Macrophages play an important role in the pathophysiology of sepsis. Accumulating evidence has suggested an upregulated rate of aerobic glycolysis as a key common feature of activated proinflammatory macrophages. Here, we identified a crucial role of myeloid 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (Pfkfb3), a glycolytic activator in lipopolysaccharide (LPS)-induced endotoxemia in mice. Pfkfb3 expression is substantially increased in bone marrow derived macrophages (BMDMs) treated with LPS in vitro and in lung macrophages of mice challenged with LPS in vivo. Myeloid-specific knockout of Pfkfb3 in mice protects against LPS-induced lung edema, cardiac dysfunction and hypotension, which were associated with decreased expression of interleukin 1 beta (Il1b), interleukin 6 (Il6) and nitric oxide synthase 2 (Nos2), as well as reduced infiltration of neutrophils and macrophages in lung tissue. Pfkfb3 ablation in cultured macrophages attenuated LPS-induced glycolytic flux, resulting in a decrease in proinflammatory gene expression. Mechanistically, Pfkfb3 ablation or inhibition with a Pfkfb3 inhibitor AZ26 suppresses LPS-induced proinflammatory gene expression via the NF-κB signaling pathway. In summary, our study reveals the critical role of Pfkfb3 in LPS-induced sepsis via reprogramming macrophage metabolism and regulating proinflammatory gene expression. Therefore, PFKFB3 is a potential target for the prevention and treatment of inflammatory diseases such as sepsis.

Abstract P275: Deletion Of Endothelial Leptin Receptor Elevates Blood Pressure And Impairs Endothelial-dependent Relaxation Via Upregulation Of Endothelial Glycolytic Enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3)

The adipokine leptin plays a crucial role in blood pressure (BP) regulation notably by exerting pressor effects centrally via sympatho-activation and depressor effects via direct activation of its receptor (LepR) peripherally resulting in nitric oxide (NO)-mediated vasodilation. However, the predominant effects and cell type responsible for leptin-mediated NO production is not clearly understood. Herein, we examined the effect of selective deletion of LepR in endothelial cells (LepR EC-/- , KO) on BP and vascular function. BP recording via radiotelemetry in male KO and WT (LepR EC+/+ ) mice revealed significant increases in diastolic and mean arterial pressure in KO mice (DBP, WT: 90.2±2.1 vs. KO: 100.1±3.6; MAP, WT: 105.7±2.1 vs. KO: 113.7±2.6 mmHg, n=5, p<0.05). There was no difference in Systolic blood pressure or heart rate between KO and WT. Leptin infusion (0.9mg/kg/day,7 days) elicits a significant increase in BP of WT but not KO mice (DBP, WT: 89.2± 2.6 vs WT+Leptin 95.7±3.3; MAP, WT: 104 ±2.8 vs WT+Leptin: 110 ±2.7, n=5, p<0.05). We quantified sympathetic contribution to BP elevation by measuring BP response to glanglionic blockade (Hexamethonium). At baseline, KO mice exhibited a lower BP response than WT supporting a reduced neurogenic control of BP regulation in KO mice. Vascular contribution to high BP was investigated using wire myography in thoracic aorta. LepR deficiency impaired endothelial-dependent relaxation (EDR) to acetylcholine (n=7, p<0.05). L-NAME completely abolished EDR in KO and WT indicating that EC LepR deficiency reduced NO bioavailability. Recent evidence presents PFKFB3-mediated EC glycolysis as a new regulator of endothelial homeostasis. We found that aortic EC from KO exhibited increased PFKFB3 mRNA expression (p=0.065) and PFKFB3 inhibition restored EDR in KO. Remarkably, overexpression of PFKFB3 increased EC glycolysis in vitro and impaired EDR in WT aortic rings ex vivo . Collectively, our data suggest that impaired endothelial leptin receptor signaling induces a PFKFB3-dependent hyper-glycolytic phenotype resulting in NO deficiency and endothelial dysfunction that predisposes to higher BP regardless the reduced sympatho-activation which might prevent the increase in BP induced by exogenous leptin.

Endothelial cell PHD2-HIF1α-PFKFB3 contributes to right ventricle vascular adaptation in pulmonary hypertension.

Humans and animals with pulmonary hypertension (PH) show right ventricular (RV) capillary growth, which positively correlates with overall RV hypertrophy. However, molecular drivers of RV vascular augmentation in PH are unknown. Prolyl hydroxylase (PHD2) is a regulator of hypoxia-inducible factors (HIFs), which transcriptionally activates several proangiogenic genes, including the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). We hypothesized that a signaling axis of PHD2-HIF1α-PFKFB3 contributes to adaptive coupling between the RV vasculature and tissue volume to maintain appropriate vascular density in PH. We used design-based stereology to analyze endothelial cell (EC) proliferation and the absolute length of the vascular network in the RV free wall, relative to the tissue volume in mice challenged with hypoxic PH. We observed increased RV EC proliferation starting after 6 h of hypoxia challenge. Using parabiotic mice, we found no evidence for a contribution of circulating EC precursors to the RV vascular network. Mice with transgenic deletion or pharmacological inhibition of PHD2, HIF1α, or PFKFB3 all had evidence of impaired RV vascular adaptation following hypoxia PH challenge. PHD2-HIF1α-PFKFB3 contributes to structural coupling between the RV vascular length and tissue volume in hypoxic mice, consistent with homeostatic mechanisms that maintain appropriate vascular density. Activating this pathway could help augment the RV vasculature and preserve RV substrate delivery in PH, as an approach to promote RV function.

Simultaneous inhibition of PFKFB3 and GLS1 selectively kills KRAS-transformed pancreatic cells

Activating mutations of the oncogenic KRAS in pancreatic ductal adenocarcinoma (PDAC) are associated with an aberrant metabolic phenotype that may be therapeutically exploited. Increased glutamine utilization via glutaminase-1 (GLS1) is one such feature of the activated KRAS signaling that is essential to cell survival and proliferation; however, metabolic plasticity of PDAC cells allow them to adapt to GLS1 inhibition via various mechanisms including activation of glycolysis, suggesting a requirement for combinatorial anti-metabolic approaches to combat PDAC. We investigated whether targeting the glycolytic regulator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) in combination with GLS1 can selectively prevent the growth of KRAS-transformed cells. We show that KRAS-transformation of pancreatic duct cells robustly sensitizes them to the dual targeting of GLS1 and PFKFB3. We also report that this sensitivity is preserved in the PDAC cell line PANC-1 which harbors an activating KRAS mutation. We then demonstrate that GLS1 inhibition reduced fructose-2,6-bisphosphate levels, the product of PFKFB3, whereas PFKFB3 inhibition increased glutamine consumption, and these effects were augmented by the co-inhibition of GLS1 and PFKFB3, suggesting a reciprocal regulation between PFKFB3 and GLS1. In conclusion, this study identifies a novel mutant KRAS-induced metabolic vulnerability that may be targeted via combinatorial inhibition of GLS1 and PFKFB3 to suppress PDAC cell growth.

PFKFB3 Inhibition Sensitizes DNA Crosslinking Chemotherapies by Suppressing Fanconi Anemia Repair.

Simple SummaryDNA-damaging chemotherapeutics, such as platinum drugs, are cornerstones in cancer treatment. The efficacy of such treatment is intimately linked to the DNA repair capacity of the cancer cells, as DNA damage above a tolerable threshold culminates in cell death. Cancer cells often have deregulated DNA repair mechanisms, making them initially more sensitive to DNA-damaging chemotherapies. Unfortunately, over time, cancer cells often develop resistance to such treatments by rewiring their DNA damage response pathways. Here, we identify that targeting the recognized anti-cancer target 6-phosphofructo-2-kinase/fructose-2,6,-bisphophatase 3 (PFKFB3), commonly overexpressed in cancer, with the small molecule inhibitor KAN0438757, selectively sensitizes cancer cells to platinum drugs, including treatment-resistant cancer cells, while sparing normal cells. Mechanistically, PFKFB3 promotes tolerance to and the repair of platinum-induced DNA interstrand crosslinks (ICLs) through modulation of the Fanconi anemia (FA) DNA repair pathway. Thus targeting PFKFB3 opens up therapeutic possibilities to improve the efficacy of ICL-inducing cancer treatments.Replicative repair of interstrand crosslinks (ICL) generated by platinum chemotherapeutics is orchestrated by the Fanconi anemia (FA) repair pathway to ensure resolution of stalled replication forks and the maintenance of genomic integrity. Here, we identify novel regulation of FA repair by the cancer-associated glycolytic enzyme PFKFB3 that has functional consequences for replication-associated ICL repair and cancer cell survival. Inhibition of PFKFB3 displays a cancer-specific synergy with platinum compounds in blocking cell viability and restores sensitivity in treatment-resistant models. Notably, the synergies are associated with DNA-damage-induced chromatin association of PFKFB3 upon cancer transformation, which further increases upon platinum resistance. FA pathway activation triggers the PFKFB3 assembly into nuclear foci in an ATR- and FANCM-dependent manner. Blocking PFKFB3 activity disrupts the assembly of key FA repair factors and consequently prevents fork restart. This results in an incapacity to replicate cells to progress through S-phase, an accumulation of DNA damage in replicating cells, and fork collapse. We further validate PFKFB3-dependent regulation of FA repair in ex vivo cultures from cancer patients. Collectively, targeting PFKFB3 opens up therapeutic possibilities to improve the efficacy of ICL-inducing cancer treatments.

PFKFB3 Inhibition Impairs Erlotinib-Induced Autophagy in NSCLCs.

Tyrosine kinase inhibitors (TKIs) targeting the kinase domain of the epidermal growth factor receptor (EGFR), such as erlotinib, have dramatically improved clinical outcomes of patients with EGFR-driven non-small cell lung carcinomas (NSCLCs). However, intrinsic or acquired resistance remains a clinical barrier to the success of FDA-approved EGFR TKIs. Multiple mechanisms of resistance have been identified, including the activation of prosurvival autophagy. We have previously shown that the expression and activity of PFKFB3—a known driver of glycolysis—is associated with resistance to erlotinib and that PFKFB3 inhibition improves the response of NSCLC cells to erlotinib. This study focuses on investigating the role of PFKFB3 in regulating erlotinib-driven autophagy to escape resistance to erlotinib. We evaluated the consequence of pharmacological inhibition of PFKFB3 on erlotinib-driven autophagy in NSCLC cells with different mutation statuses. Here, we identify PFKFB3 as a mediator of erlotinib-induced autophagy in NSCLCs. We demonstrate that PFKFB3 inhibition sensitizes NCSLCs to erlotinib via impairing autophagy flux. In summary, our studies uncovered a novel crosstalk between PFKFB3 and EGFR that regulates erlotinib-induced autophagy, thus contributing to erlotinib sensitivity in NSCLCs.

Abstract 3085: Steroid receptor co-activators regulate metabolic kinases to drive therapy resistant ER+ breast cancer

Abstract Recurrence of metastatic breast cancer stemming from acquired endocrine and chemotherapy resistance remains a health burden for women with luminal (ER+) breast cancer. Disseminated ER+ tumor cells can remain viable but quiescent for years to decades. Contributing factors to metastatic spread include the maintenance and expansion of breast cancer stem cells (CSCs). Breast CSCs are poorly proliferative and frequently exist as a minority population in therapy resistant tumors. Our objective is to define novel signaling pathways that govern therapy resistance in ER+ breast cancer. In this study, we show that cytoplasmic complexes composed of steroid receptor (SR) co-activators, PELP1 and SRC-3, modulate breast CSC expansion through upregulation of the HIF-activated metabolic target genes PFKFB3 and PFKFB4. Seahorse metabolic assays demonstrated that cytoplasmic PELP1 influences cellular metabolism by increasing both glycolysis and mitochondrial respiration. PELP1 interacts with PFKFB3 and PFKFB4 proteins, and inhibition of PFKFB3 and PFKFB4 kinase activity blocks PELP1-induced tumorspheres and protein-protein interactions with SRC-3. PFKFB4 knockdown inhibited in vivo emergence of circulating tumor cell (CTC) populations in ER+ mammary intraductal (MIND) xenografts. Application of PFKFB inhibitors in combination with ER targeted therapies blocked tumorsphere formation in multiple models of advanced breast cancer, including tamoxifen (TamR) and paclitaxel (TaxR) resistant models and ER+ patient-derived organoids (PDxO). Together, our data suggest that PELP1, SRC-3, and PFKFBs cooperate to drive ER+ tumor cells that include CSCs and CTCs. Identifying non-ER pharmacological targets offers a useful approach to blocking metastatic escape from standard of care ER/estrogen (E2)-targeted strategies to overcome endocrine and chemotherapy resistance in ER+ breast cancer. Citation Format: Thu Ha Truong, Elizabeth Benner, Kyla M. Hagen, Nuri A. Temiz, Carlos Perez Kerkvliet, Ying Wang, Emilio Cortes-Sanchez, Chieh-Hsiang Yang, Thomas Pengo, Katrin P. Guillen, Bryan E. Welm, Sucheta Telang, Carol A. Lange, Julie H. Ostrander. Steroid receptor co-activators regulate metabolic kinases to drive therapy resistant ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3085.

Abstract 1052: 6-phosphofructo-2-kinase enhances cytotoxicity of the EGFR inhibitor erlotinib via regulation of cell cycle in non-small lung cancer cell lines

Abstract Lung tumor development and aggressiveness is largely driven by a multitude of oncogenes including the epidermal growth factor receptor (EGFR). Targeted EGFR inhibition with small-molecule tyrosine kinase inhibitors (EGFR-TKIs) such as erlotinib have initially improved progression-free survival in patients with EGFR-driven NSCLCs but failed to provide an overall survival benefit due to the development of resistance. Clinically, responses to EGFR-TKIs are evaluated by positron emission tomography using 2[18F]- fluoro-2-deoxy-glucose (18FDG-PET) as tumors consume significantly more glucose compared to adjacent normal tissue in vivo. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3), a known driver of glycolytic flux, plays a key role in the metabolic shift that occurs during tumor progression. The product of PFKFB3, F2,6BP, regulates glucose metabolism by activating PFK1, which in turn facilitates glycolysis. Increased glycolysis fulfills the elevated tumor demands for energetic and biosynthetic precursors needed for survival and proliferation - the functions that confer resistance to targeted therapies. Here, we interrogated the hypothesis that PFKFB3 inhibition increases the efficacy of EGFR-TKIs in NSCLCs with different EGFR mutation status in vitro. We treated multiple NSCLCs models (wild type EGFR: H522, H1437; and mutated EGFR: PC9, HCC827) with erlotinib and observed a significant inhibition of cell proliferation in vitro. Erlotinib treatment resulted in G1 arrest, decreased expression and phosphorylation of cyclin dependent kinases (CDKs), Rb and accumulation of p27 in all NSCLCs. Notably, PFKFB3 silencing in TKI-treated cells dramatically decreased cell viability, indicating that PFKFB3 expression is required to maintain cell viability of G1 arrested cells. Further, co-treatment with the PFKFB3 inhibitor PFK-158 increased cell death in all NSCLCs. We found that inhibition of PFKFB3 reduced TKI-induced G1 arrest and altered cell cycle progression, switching the effect of erlotinib from cytostatic to cytotoxic. Importantly, dual treatment caused only minor changes in glycolytic flux when compared to the effects of PFK-158 or erlotinib alone suggesting that glucose metabolism did not contribute to the observed effect. Taken together, our findings suggest that a non-metabolic function of PFKFB3 limits the usefulness of EGFR-TKI in vitro and that targeting of PFKFB3 can be a promising approach to improve clinical efficacy of FDA-approved EGFR-TKIs in lung cancer patients. Citation Format: Nadiia Lypova, Lilibeth Lanceta, Jason Chesney, Yoannis Imbert-Fernandez. 6-phosphofructo-2-kinase enhances cytotoxicity of the EGFR inhibitor erlotinib via regulation of cell cycle in non-small lung cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1052.

Abstract 2519: Targeting glucose metabolism in HER2+breast cancer

Abstract Primary and metastatic breast cancers are driven by oncogenic mutations to rely on glucose metabolism to support their rapid proliferation. Of these genetic changes, the increased activity of the human epidermal growth factor receptor 2 (HER2/ErbB2) is observed in a third of all breast tumors. HER2 has been shown to activate downstream effectors (e.g. Ras, HIF-1α) that stimulate glycolysis - in part by activation of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase family of enzymes (PFKFB1-4). The PFKFBs produce fructose-2,6-bisphosphate (F26BP) which activates a key rate-limiting glycolytic enzyme, 6-phosphofructo-1-kinase (PFK1). In preliminary studies, we found that the PFKFB4 family member was highly expressed in a series of patient-derived HER2+ breast tumors relative to normal breast with markedly higher expression in their matched metastatic lymph nodes. We next determined that silencing PFKFB4 markedly reduced F26BP, glycolysis and cell survival in HER2+ breast cancer cells and inhibited tumor growth in vivo. Through virtual screening, we have discovered a first-in-class small molecule inhibitor of PFKFB4, termed 5MPN, that selectively inhibits PFKFB4 activity with a resultant decrease in F26BP, glycolysis and cancer cell growth. 5MPN also caused a significant decrease in the invasion and migration of HER2+ breast cancer cells in vitro, importantly at concentrations well below levels that affect cell viability and marked suppression in the growth of established tumors, without systemic toxicity. In additional studies, we have found that the related PFKFB3 isoform is also expressed in HER+ breast tumors. Our previous work found that the PFKFB4 enzyme localizes to the cytoplasm (where PFK1 and the glycolytic pathway function) indicating a possible dominant role for PFKFB4 in glycolytic regulation. PFKFB3 however largely localized to the nucleus where it serves an important function in proliferation via activation of cyclin-dependent kinases. Based on the different roles played by these enzymes in cancer cells, we next evaluated the effects of simultaneous administration of 5MPN ± a clinical candidate PFKFB3 inhibitor on survival and proliferation in HER2+ breast cancer and found that the combination synergistically and significantly decreased viability and growth in several examined HER2+ breast cancer cell lines. Our data indicate that targeting PFKFB4 may be a viable therapeutic option against breast cancer and strongly support the further exploration of co-targeting PFKFB4 and PFKFB3 as a potential therapeutic strategy for the successful treatment of HER2+ breast cancer. Citation Format: Simone Chang, Jason Chesney, John O. Trent, Sucheta Telang. Targeting glucose metabolism in HER2+breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2519.

PFKFB3 Promotes Liver Fibrosis by Regulating Aerobic Glycolysis of Hepatic Stellate Cells

Background: Hepatic stellate cells (HSCs) are the key effector cells in the occurrence and development of liver fibrosis, while aerobic glycolysis is one of the important metabolic characteristics of HSC activation. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) is a homodimeric bifunctional enzyme, which is a rate-limiting enzyme in glycolysis. This metabolite is important for the dynamic regulation of glycolytic flux. However, little is known about the role of PFKFB3 in liver fibrosis. Objectives: In this study, we aimed to explore the effects of PFKFB3 on aerobic glycolysis in the process of HSC trans-differentiation and liver fibrosis. Methods: Immunohistochemical (IHC) staining and immunofluorescence assays were used to examine PFKFB3 expression in mice fibrotic liver tissue. The determination of extracellular acidification rate was used to examine changes in aerobic glycolytic flux, lactate production levels, and glucose consumption levels in HSCs upon TGF-β1 stimulation. Western blot analysis of the expression of PFKFB3, α-SMA protein, and type I collagen was done. Liver histopathology was also examined. Besides, glycolytic inhibition by pharmacologic approaches was used to demonstrate the critical role of glycolysis in liver fibrosis. Results: The PFKFB3 protein expression was increased in mouse fibrotic liver tissue. In addition, immunofluorescence revealed the colocalization of PFKFB3 and alpha-smooth muscle actin (α-SMA) protein. In vitro experiments showed that PFKFB3 could promote glycolysis flux, lactic acid production, and glucose consumption of hepatic stellate cells. The PFKFB3 inhibitor was used in a mouse model of liver fibrosis, and the inhibition of PFKFB3 reduced the degree of liver inflammation and liver fibrosis. Conclusions: PFKFB3 can promote HSC aerobic glycolysis, which, in turn, promotes HSC activation and liver fibrosis.

PELP1/SRC-3-dependent regulation of metabolic PFKFB kinases drives therapy resistant ER+ breast cancer.

Recurrence of metastatic breast cancer stemming from acquired endocrine and chemotherapy resistance remains a health burden for women with luminal (ER+) breast cancer. Disseminated ER+ tumor cells can remain viable but quiescent for years to decades. Contributing factors to metastatic spread include the maintenance and expansion of breast cancer stem cells (CSCs). Breast CSCs frequently exist as a minority population in therapy resistant tumors. In this study, we show that cytoplasmic complexes composed of steroid receptor (SR) co-activators, PELP1 and SRC-3, modulate breast CSC expansion through upregulation of the HIF-activated metabolic target genes PFKFB3 and PFKFB4. Seahorse metabolic assays demonstrated that cytoplasmic PELP1 influences cellular metabolism by increasing both glycolysis and mitochondrial respiration. PELP1 interacts with PFKFB3 and PFKFB4 proteins, and inhibition of PFKFB3 and PFKFB4 kinase activity blocks PELP1-induced tumorspheres and protein-protein interactions with SRC-3. PFKFB4 knockdown inhibited in vivo emergence of circulating tumor cell (CTC) populations in mammary intraductal (MIND) models. Application of PFKFB inhibitors in combination with ER targeted therapies blocked tumorsphere formation in multiple models of advanced breast cancer including tamoxifen (TamR) and paclitaxel (TaxR) resistant models, murine tumor cells, and ER+ patient-derived organoids (PDxO). Together, our data suggest that PELP1, SRC-3, and PFKFBs cooperate to drive ER+ tumor cell populations that include CSCs and CTCs. Identifying non-ER pharmacological targets offers a useful approach to blocking metastatic escape from standard of care ER/estrogen (E2)-targeted strategies to overcome endocrine and chemotherapy resistance.

The PFKFB3 Inhibitor AZ67 Inhibits Angiogenesis Independently of Glycolysis Inhibition.

Angiogenesis is the process of new blood vessel formation. In this complex orchestrated growth, many factors are included. Lately, focus has shifted to endothelial cell metabolism, particularly to the PFKFB3 protein, a key regulatory enzyme of the glycolytic pathway. A variety of inhibitors of this important target have been studied, and a plethora of biological effects related to the process of angiogenesis have been reported. However, recent studies have disputed their mechanism of action, questioning whether all the effects are indeed due to PFKFB3 inhibition. Remarkably, the most well-studied inhibitor, 3PO, does not bind to PFKFB3, raising questions about this target. In our study, we aimed to elucidate the effects of PFKFB3 inhibition in angiogenesis by using the small molecule AZ67. We used isothermal titration calorimetry and confirmed binding to PFKFB3. In vitro, AZ67 did not decrease lactate production in endothelial cells (ECs), nor ATP levels, but exhibited good inhibitory efficacy in the tube-formation assay. Surprisingly, this was independent of EC migratory and proliferative abilities, as this was not diminished upon treatment. Strikingly however, even the lowest dose of AZ67 demonstrated significant inhibition of angiogenesis in vivo. To our knowledge, this is the first study to demonstrate that the process of angiogenesis can be disrupted by targeting PFKFB3 independently of glycolysis inhibition.

Steroid Receptor Co-Activators Regulate Metabolic Kinases to Drive Therapy Resistant ER+ Breast Cancer

Recurrence of metastatic breast cancer stemming from acquired endocrine and chemotherapy resistance remains a health burden for women with luminal (ER+) breast cancer. Disseminated ER+ tumor cells can remain viable but quiescent for years to decades. Contributing factors to metastatic spread include the maintenance and expansion of breast cancer stem cells (CSCs). Breast CSCs are poorly proliferative and frequently exist as a minority population in therapy resistant tumors. Our objective is to define novel signaling pathways that govern therapy resistance in ER+ breast cancer. In this study, we show that cytoplasmic complexes composed of steroid receptor (SR) co-activators, PELP1 and SRC-3, modulate breast CSC expansion through upregulation of the HIF-activated metabolic target genes PFKFB3 and PFKFB4. Seahorse metabolic assays demonstrated that cytoplasmic PELP1 influences cellular metabolism by increasing both glycolysis and mitochondrial respiration. PELP1 interacts with PFKFB3 and PFKFB4 proteins, and inhibition of PFKFB3 and PFKFB4 kinase activity blocks PELP1-induced tumorspheres and protein-protein interactions with SRC-3. PFKFB4 knockdown inhibited in vivo emergence of circulating tumor cell (CTC) populations in ER+ mammary intraductal (MIND) xenografts. Application of PFKFB inhibitors in combination with ER targeted therapies blocked tumorsphere formation in multiple models of advanced breast cancer, including tamoxifen (TamR) and paclitaxel (TaxR) resistant models and ER+ patient-derived organoids (PDxO). Together, our data suggest that PELP1, SRC-3, and PFKFBs cooperate to drive ER+ tumor cells that include CSCs and CTCs. Identifying non-ER pharmacological targets offers a useful approach to blocking metastatic escape from standard of care ER/estrogen (E2)-targeted strategies to overcome endocrine and chemotherapy resistance in ER+ breast cancer.

Targeting Glucose Metabolism in HER2 + Breast Cancer

The rapid growth and progression of breast tumors is driven by multiple oncogenic mutations. Of these genetic alterations, one of the most important is the increased activity of the human epidermal growth factor receptor 2 (HER2) which is present in a third of breast cancers and associated with aggressive disease and poor survival. Although new agents have improved outcomes in HER2+ breast cancer, many patients continue to progress, develop metastases and die of their disease. Novel and effective targeted therapies are urgently required to improve survival from this devastating cancer. HER2+ cancers consistently exhibit a high reliance on glycolysis to fuel their rapid growth and metastasis. The high glycolytic flux in these tumors is driven by HER2-mediated effectors including the PI3K/AKT and Ras/MAPK pathways and HIF-1α which increase glucose metabolism in part by increasing the activity of a family of enzymes termed the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4). The PFKFBs produce fructose-2,6-bisphosphate (F26BP) which regulates glycolysis by activating a key rate-limiting glycolytic enzyme, 6-phosphofructo-1-kinase (PFK1). In recent studies, we have found that the PFKFB4 family member is highly expressed in a series of HER2+ patient-derived breast tumors relative to normal breast tissue with markedly higher expression in their matched metastatic lymph nodes. We have determined that silencing PFKFB4 markedly reduced F26BP, glycolysis and cell survival in HER2+ breast cancer cells in vitro. We have now developeda novel and potent small molecule PFKFB4 inhibitor that selectively inhibits recombinant PFKFB4 activity, decreases glycolysis and proliferation in HER2+ breast cancer cells and limits their migration and invasion at concentrations that do not affect their viability and suppresses xenograft tumor growth in mice without systemic toxicity. During our studies, we observed that inhibition of a frequently co-expressed PFKFB family member, PFKFB3, increased PFKFB4 expression, indicating that PFKFB4 may compensate for decreased PFKFB3 expression. We now have also found that the simultaneous administration of PFKFB4 and PFKFB3 inhibitors synergistically and significantly decreases proliferation in HER2+ breast cancer cells. These observations carry additional relevance since a novel PFKFB3 inhibitor has recently completed Phase I clinical trial evaluation and compensation by PFKFB4 may limit the efficacy of this and other PFKFB3 inhibitors. Taken together, our current data indicate that targeting PFKFB4 may prove to be a successful treatment option against HER2+ breast tumors and strongly support the further exploration of co-targeting PFKFB4 and PFKFB3 as a potential therapeutic strategy to improve outcomes in this cancer.

Perivascular adipose tissue (PVAT)‐derived leptin improves aortic endothelial function via attenuating endothelial glycolysis in a mouse model of lipodystrophy

Endothelial dysfunction, characterized by impaired endothelial-dependent relaxation (EDR), is a hallmark and first step in the chain of events leading to cardiovascular disorders. Several vascular pathologies have been associated with aberrant increases in endothelial glycolysis and 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) expression/activity, however, whether endothelial glycolysis contributes to EDR regulation remains unknown. Additionally, endothelial dysfunction is present in conditions associated with severe alterations in body fat content including obesity (excess) and lipodystrophy (absence of adipose tissue). However, the exact link between endothelial function and adipose tissue, particularly the one surrounding blood vessels (perivascular adipose tissue, PVAT) remains ill-defined. As leptin, the adipokine highly involved in the regulation of cardiovascular function, has also been shown to modulate cellular bioenergetics in immune and cancer cells, we hypothesized that leptin derived from PVAT controls EDR via endothelial leptin receptor activation and attenuation of PFKFB3-mediated glycolysis. To determine the role of PFKFB3 in endothelial function, we overexpressed PFKFB3 in mouse endothelial cells via adenovirus transduction (Ad-PFKFB3), and reported impaired aorta EDR. Additionally, using seahorse analyzer and glycolysis stress test we found that human aortic endothelial cells (HAEC) transduced with Ad-PFKFB3 showed a significant increase in basal glycolysis (p<0.05) which was blunted by exposure to leptin (10µg/ml, 2h). To determine the role of PVAT-derived leptin in EDR, we used a mouse model of lipodystrophy, with total deficiency in adipose tissue and leptin (Bscl2-/-) characterized by vascular and metabolic dysfunctions as evidenced by impaired EDR and glucose intolerance. We also found that aortas from Bscl2-/- mice exhibited a marked increase in basal and maximum glycolysis measured using seahorse analyzer and glycolysis stress test. This is in line with the PCR data showing increased expression of PFKFB3 mRNA compared to WT mice. Interestingly, inhibition of PFKFB3 using 3 PO or PFK158 (20uM) restored EDR in Bscl2-/- mice. Additionally, we transplanted 25 mg PVAT from WT mice to the abdominal aorta of Bscl2-/-. After 4 weeks, PVAT significantly reduced aortic expression of PFKFB3 and corrected systemic glycemia as well as EDR in Bscl2-/- vs sham (P<0.05). However, leptin receptor blockade (Allo-Aca, 0.05 mg/kg/day) blunted PVAT-mediated restoration of EDR in PVAT-transplanted Bscl2-/- mice. In conclusion, these data demonstrate that PVAT releases a vasodilatory factor, potentially, leptin that acts via activation of endothelial leptin receptor to improve endothelial function via a mechanism that involves regulation of PFKFB3-mediated endothelial glycolysis.

Inhibition of Glycolysis Suppresses Cell Proliferation and Tumor Progression In Vivo: Perspectives for Chronotherapy.

A high rate of glycolysis is considered a hallmark of tumor progression and is caused by overexpression of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Therefore, we analyzed the possibility of inhibiting tumor and endothelial cell metabolism through the inhibition of PFKFB3 by a small molecule, (E)-1-(pyridin-4-yl)-3-(quinolin-2-yl)prop-2-en-1-one (PFK15), as a promising therapy. The effects of PFK15 on cell proliferation and apoptosis were analyzed on human umbilical vein endothelial cells (HUVEC) and the human colorectal adenocarcinoma cell line DLD1 through cytotoxicity and proliferation assays, flow cytometry, and western blotting. The results showed that PFK15 inhibited the proliferation of both cell types and induced apoptosis with decreasing the Bcl-2/Bax ratio. On the basis of the results obtained from in vitro experiments, we performed a study on immunodeficient mice implanted with DLD1 cells. We found a reduced tumor mass after morning PFK15 treatment but not after evening treatment, suggesting circadian control of underlying processes. The reduction in tumor size was related to decreased expression of Ki-67, a marker of cell proliferation. We conclude that inhibition of glycolysis can represent a promising therapeutic strategy for cancer treatment and its efficiency is circadian dependent.

Effects of the Novel PFKFB3 Inhibitor KAN0438757 on Colorectal Cancer Cells and Its Systemic Toxicity Evaluation In Vivo.

Simple SummaryGlycolysis is one of the hallmarks of cancer. Therefore, the development of novel therapeutical strategies for colorectal cancer targeting glycolysis may improve treatment responses. PFKFB3 expression has been directly associated with enhanced glycolysis, not only in cancer cells but also within the tumor environment. The aim of this study was to evaluate PFKFB3 expression and its correlation with outcome in rectal and colon tumors and to assess the effects of the newly developed PFKFB3 inhibitor KAN0438757 on colorectal cancer cells and intestinal patient-derived organoids. Our results showed that KAN0438757 efficiently targets PFKFB3 expression and was able to affect cancer cell motility, invasion and survival. Additionally, a tumor specific cytotoxic-effect was observed in patient-derived organoids. In vivo, KAN0438757 showed to be well tolerated by mice without systemic toxicity. Our work re-enforces the concept that targeting of glycolysis may be a promising therapeutical approach for colorectal cancer.Background: Despite substantial progress made in the last decades in colorectal cancer (CRC) research, new treatment approaches are still needed to improve patients’ long-term survival. To date, the promising strategy to target tumor angiogenesis metabolically together with a sensitization of CRC to chemo- and/or radiotherapy by PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3) inhibition has never been tested. Therefore, initial evaluation and validation of newly developed compounds such as KAN0438757 and their effects on CRC cells are crucial steps preceding to in vivo preclinical studies, which in turn may consolidate new therapeutic targets. Materials and Methods: The efficiency of KAN0438757 to block PFKFB3 expression and translation in human CRC cells was evaluated by immunoblotting and real-time PCR. Functional in vitro assays assessed the effects of KAN0438757 on cell viability, proliferation, survival, adhesion, migration and invasion. Additionally, we evaluated the effects of KAN0438757 on matched patient-derived normal and tumor organoids and its systemic toxicity in vivo in C57BL6/N mice. Results: High PFKFB3 expression is correlated with a worse survival in CRC patients. KAN0438757 reduces PFKFB3 protein expression without affecting its transcriptional regulation. Additionally, a concentration-dependent anti-proliferative effect was observed. The migration and invasion capacity of cancer cells were significantly reduced, independent of the anti-proliferative effect. When treating colonic patient-derived organoids with KAN0438757 an impressive effect on tumor organoids growth was apparent, surprisingly sparing normal colonic organoids. No high-grade toxicity was observed in vivo. Conclusion: The PFKFB3 inhibitor KAN0438757 significantly reduced CRC cell migration, invasion and survival. Moreover, on patient-derived cancer organoids KAN0438757 showed significant effects on growth, without being overly toxic in normal colon organoids and healthy mice. Our findings strongly encourage further translational studies to evaluate KAN0438757 in CRC therapy.

PFKFB3 regulates lipopolysaccharide-induced excessive inflammation and cellular dysfunction in HTR-8/Svneo cells: Implications for the role of PFKFB3 in preeclampsia

IntroductionPreeclampsia is characterized by overactive inflammation at the uteroplacental interface, leading to trophoblasts dysfunction. 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) is a crucial glycolytic regulator which has recently been found to participate in the pathological inflammatory states. This study aimed to investigate the role of PFKFB3 in the inflammation-induced damage in trophoblasts, and elucidate the underlying mechanisms. MethodsImmunohistochemistry, qRT-PCR, and Western blot analysis (WB) were used to detect the expression of PFKFB3 in preeclamptic and normal placentas. Lipopolysaccharide (LPS)-induced HTR8/SVneo cells were established as the in vitro model to simulate the overactive inflammation at the uteroplacental interface of PE, which were subsequently transfected with PFKFB3 siRNA. The expression of PFKFB3, NF-κB-p-p65, phosphorylation states of NF-κB-p65, ICAM-1, Bcl-2, BAX, and MMP2 were detected by WB. qRT-PCR was used to detect the expression of TNF-α and IL-1β. The ICAM-1 expression was also reflected by monocyte adhesion assay. Reactive Oxygen Species (ROS) levels were detected by DCFH-DA (2,7-Dichlorodi-hydrofluorescein diacetate). Apoptosis was detected using Annexin V-FITC staining. Migration and invasion were measured by wound-healing and transwell assays. ResultsPFKFB3 was up-regulated in the preeclamptic placenta. In LPS-treated HTR-8/Svneo cells, the inhibition of PFKFB3 blocked the NF-κB signal pathway, thereby downregulating the expression of proinflammatory cytokines and adhesion molecules, meanwhile, PFKFB3 knockdown significantly alleviated monocyte adhesion, oxidative stress, apoptosis, and reinstated migration and invasive capacity. DiscussionPFKFB3 controls the LPS-induced inflammation via the NF-κB pathway and impacts trophoblasts function such as adhesion, oxidative stress, apoptosis, migration, and invasion, thereby potentially participating in the preeclamptic etiopathogenesis.

Mice with a specific deficiency of Pfkfb3 in myeloid cells are protected from hypoxia-induced pulmonary hypertension.

Macrophage infiltration into the lungs is a characteristic of pulmonary hypertension (PH). Glycolysis is the main metabolic pathway for macrophage activation. However, the effect of macrophage glycolysis on the development of PH remains unknown. We investigated the effect of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKBF3), a critical enzyme of macrophage glycolysis, on PH development. Lung tissues from PH patients were examined by immunostaining with macrophage markers. PH was induced in Wistar rats with SU5416/hypoxia and in mice with hypoxia. Lungs and macrophages were isolated for analysis by RT-PCR, western blot, flow cytometry, and immunostaining. Expression of glycolytic molecules was increased in circulating peripheral blood mononuclear cells (PBMCs) and lung macrophages of PH patients. These results were also found in lung macrophages of SU5416/hypoxia (Su/Hx)-induced PH rats and hypoxia-induced PH mice. PH was ameliorated in myeloid-specific Pfkfb3-deficient mice (Pfkfb3ΔMϕ ) or mice treated with the PFKFB3 inhibitor 3PO, compared with their controls. Alveolar macrophages of PH Pfkfb3ΔMϕ mice produced lower levels of growth factors and pro-inflammatory cytokines than those of control mice. Circulating myeloid cells and lung myeloid cells were much fewer in PH Pfkfb3ΔMϕ mice than controls. Mechanistically, overexpression of Hif1a or Hif2a in bone marrow-derived macrophages (BMDMs) cultured with bone marrow of Pfkfb3ΔMϕ mice restored the decreased expression of pro-inflammatory cytokines and growth factors. Myeloid Pfkfb3 deficiency protects mice from PH, thereby suggesting that myeloid PFKFB3 is one of the important targets in the therapeutic effect of PFKFB3 inhibition in PH treatment.