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1
Sun, Chen-min, Da-bo Xiong, Yang Yan, Jiang Geng, Min Liu, and Xu-dong Yao. "Genetic Alteration in Phosphofructokinase Family Promotes Growth of Muscle-Invasive Bladder Cancer." International Journal of Biological Markers 31, no. 3 (July 2016): 286–93. http://dx.doi.org/10.5301/jbm.5000189.
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Aims Metabolic alterations in cancer, including bladder cancer, have been addressed in recent years. We aimed to study the role of phosphofructokinase (PFK) in muscle-invasive bladder cancer (MIBC). Method By in silico analysis of the bladder cancer data from the Cancer Genome Atlas (TCGA) database using the cBioPortal platform, we studied genetic alteration of genes within the PFK family (PFKL, PFKM, PFKP, PFKFB1, PFKFB2, PFKFB3, and PFKFB4). In vitro studies were carried out using the PFK inhibitor 2,5-anhydro-D-glucitol-6-phosphate. Results Genetic alterations of PFK family genes were observed in ~44% of MIBC cases in TCGA. The main alterations were amplification and upregulation. Patients with altered PFK gene status were more likely to have a history of noninvasive bladder cancer. Altered PFK status was not associated with survival or disease relapse. Use of the PFK inhibitor significantly decreased the level of glycolysis and inhibited the growth and invasion of bladder cancer cells. Conclusions PFKs were critical genes in charge of glycolysis and were upregulated in bladder cancer. Targeting this pathway could inhibit cell growth in bladder cancer.
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Kotowski, Krzysztof, Jakub Rosik, Filip Machaj, Stanisław Supplitt, Daniel Wiczew, Karolina Jabłońska, Emilia Wiechec, Saeid Ghavami, and Piotr Dzięgiel. "Role of PFKFB3 and PFKFB4 in Cancer: Genetic Basis, Impact on Disease Development/Progression, and Potential as Therapeutic Targets." Cancers 13, no. 4 (February 22, 2021): 909. http://dx.doi.org/10.3390/cancers13040909.
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Glycolysis is a crucial metabolic process in rapidly proliferating cells such as cancer cells. Phosphofructokinase-1 (PFK-1) is a key rate-limiting enzyme of glycolysis. Its efficiency is allosterically regulated by numerous substances occurring in the cytoplasm. However, the most potent regulator of PFK-1 is fructose-2,6-bisphosphate (F-2,6-BP), the level of which is strongly associated with 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase activity (PFK-2/FBPase-2, PFKFB). PFK-2/FBPase-2 is a bifunctional enzyme responsible for F-2,6-BP synthesis and degradation. Four isozymes of PFKFB (PFKFB1, PFKFB2, PFKFB3, and PFKFB4) have been identified. Alterations in the levels of all PFK-2/FBPase-2 isozymes have been reported in different diseases. However, most recent studies have focused on an increased expression of PFKFB3 and PFKFB4 in cancer tissues and their role in carcinogenesis. In this review, we summarize our current knowledge on all PFKFB genes and protein structures, and emphasize important differences between the isoenzymes, which likely affect their kinase/phosphatase activities. The main focus is on the latest reports in this field of cancer research, and in particular the impact of PFKFB3 and PFKFB4 on tumor progression, metastasis, angiogenesis, and autophagy. We also present the most recent achievements in the development of new drugs targeting these isozymes. Finally, we discuss potential combination therapies using PFKFB3 inhibitors, which may represent important future cancer treatment options.
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Regueira, Mariana, Silvana Lucía Artagaveytia, María Noel Galardo, Eliana Herminia Pellizzari, Selva Beatriz Cigorraga, Silvina Beatriz Meroni, and María Fernanda Riera. "Novel molecular mechanisms involved in hormonal regulation of lactate production in Sertoli cells." REPRODUCTION 150, no. 4 (October 2015): 311–21. http://dx.doi.org/10.1530/rep-15-0093.
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The aim of the study was to analyze molecular mechanisms involved in FSH and basic fibroblast growth factor (bFGF) regulation of lactate production in rat Sertoli cells. The regulation of the availability of pyruvate, which is converted to lactate, could be a mechanism utilized by hormones to ensure lactate supply to germ cells. On one hand, the regulation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB) expression could result in increased glycolysis, while an increase in pyruvate availability may also result from a lower conversion to acetyl-CoA by negative regulation of pyruvate dehydrogenase complex (PDC) activity by phosphorylation. Sertoli cell cultures obtained from 20-day-old rats were used. Stimulation of the cultures with FSH or bFGF showed that FSH increases Pfkfb1 and Pfkfb3 expression while bFGF increases Pfkfb1 mRNA levels. Additionally, we observed that FSH-stimulated lactate production was inhibited in the presence of a PFKFB3 inhibitor, revealing the physiological relevance of this mechanism. As for the regulation of PDC, analysis of pyruvate dehydrogenase kinase (Pdk) expression showed that FSH increases Pdk3 and decreases Pdk4 mRNA levels while bFGF increases the expression of all Pdks. In addition, we showed that bFGF increases phosphorylated PDC levels and that bFGF-stimulated lactate production is partially inhibited in the presence of a PDK inhibitor. Altogether, these results add new information regarding novel molecular mechanisms involved in hormonal regulation of lactate production in Sertoli cells. Considering that lactate is essential for the production of energy in spermatocytes and spermatids, these mechanisms might be relevant in maintaining spermatogenesis and male fertility.
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Minchenko, Oleksandr H., Iryna L. Opentanova, Tsutomu Ogura, Dmytro O. Minchenko, Sergiy V. Komisarenko, Jaime Caro, and Hiroyasu Esumi. "Expression and hypoxia-responsiveness of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 in mammary gland malignant cell lines." Acta Biochimica Polonica 52, no. 4 (July 11, 2005): 881–88. http://dx.doi.org/10.18388/abp.2005_3402.
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Recently, we have shown that PFKFB4 gene which encodes the testis isoenzyme of PFKFB is also expressed in the prostate and hepatoma cancer cell lines. Here we have studied expression and hypoxic regulation of the testis isoenzyme of PFKFB4 in several malignant cell lines from a female organ--the mammary gland. Our studies clearly demonstrated that PFKFB4 mRNA is also expressed in mammary gland malignant cells (MCF-7 and T47D cell lines) in normoxic conditions and that hypoxia strongly induces it expression. To better understand the mechanism of hypoxic regulation of PFKFB4 gene expression, we used dimethyloxalylglycine, a specific inhibitor of HIF-1alpha hydroxylase enzymes, which strongly increases HIF-1alpha levels and mimics the effect of hypoxia. It was observed that PFKFB4 expression in the MCF7 and T47D cell lines was highly responsive to dimethyloxalylglycine, suggesting that the hypoxia responsiveness of PFKFB4 gene in these cell lines is regulated by HIF-1 proteins. Moreover, desferrioxamine and cobalt chloride, which mimic the effect of hypoxia by chelating or substituting for iron, had a similar stimulatory effect on the expression of PFKFB mRNA. In other mammary gland malignant cell lines (BT549, MDA-MB-468, and SKBR-3) hypoxia and hypoxia mimics also induced PFKFB4 mRNA, but to variable degrees. The hypoxic induction of PFKFB4 mRNA was equivalent to the expression of PFKFB3, Glut1, and VEGF, which are known HIF-1-dependent genes. Hypoxia and dimethyloxalylglycine increased the PFKFB4 protein levels in all cell lines studied except MDA-MB-468. Through site-specific mutagenesis in the 5'-flanking region of PFKFB4 gene the hypoxia response could be limited. Thus, this study provides evidence that PFKFB4 gene is also expressed in mammary gland cancer cells and strongly responds to hypoxia via an HIF-1alpha dependent mechanism. Moreover, the PFKFB4 and PFKFB3 gene expression in mammary gland cancer cells has also a significant role in the Warburg effect which is found in all malignant cells.
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Emini Veseli, Besa, Pieter Van Wielendaele, Mirela Delibegovic, Wim Martinet, and Guido R. Y. De Meyer. "The PFKFB3 Inhibitor AZ67 Inhibits Angiogenesis Independently of Glycolysis Inhibition." International Journal of Molecular Sciences 22, no. 11 (May 31, 2021): 5970. http://dx.doi.org/10.3390/ijms22115970.
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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.
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Truong, Thu Ha, Elizabeth A. Benner, Kyla M. Hagen, Nuri A. Temiz, Carlos Perez Kerkvliet, Ying Wang, Emilio Cortes-Sanchez, et al. "Steroid Receptor Co-Activators Regulate Metabolic Kinases to Drive Therapy Resistant ER+ Breast Cancer." Journal of the Endocrine Society 5, Supplement_1 (May 1, 2021): A1031—A1032. http://dx.doi.org/10.1210/jendso/bvab048.2111.
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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.
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Zhou, Z., L. G. Plug, E. S. M. de Jonge-Muller, A. Abou Elmagd, A. E. van der Meulen-de Jong, M. C. Barnhoorn, and L. J. A. C. Hawinkels. "P028 Inhibition of stromal glycolysis by targeting PFKFB3 decreases experimental colitis." Journal of Crohn's and Colitis 16, Supplement_1 (January 1, 2022): i150. http://dx.doi.org/10.1093/ecco-jcc/jjab232.157.
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Abstract Background Studies in fibrotic diseases revealed that glycolysis is the preferred energy source for fibroblasts. 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) has the highest kinase activity to shunt glucose toward glycolysis. Therefore inhibition of PFKFB3 has been proposed as a potential target for several cancers and inflammatory diseases. However, the metabolic status of fibroblasts in patients with inflammatory bowel disease (IBD) and the role of PFKFB3 are currently unknown. Methods Single-sample gene set enrichment analysis (ssGSEA) of GSE16879 was performed to evaluate metabolic changes in IBD. Seahorse real-time cell metabolic analysis was performed to explore the metabolic activity of fibroblasts. Next the expression of PFKFB3 in primary patient derived intestinal fibroblast was determined by quantitative PCR (qPCR) and western blot under normal and inflammatory conditions. Proliferation and migration of fibroblasts were measured using colony formation and wound healing assays. In order to evaluate the effect of the inhibition of PFKFB3 in vivo, PFK15, a specific inhibitor of PFKFB3, was intraperitoneal injected in mice with dextran sodium sulfate (DSS)-induced colitis and in the T-cell transfer model for colitis. Next to clinical parameters, the abundance of α-smooth muscle actin (α-SMA) expressing fibroblasts, immune cells (CD45) and endothelial cells (CD105) was determined by immunohistochemistry. Results The ssGSEA analysis revealed glycolysis was significantly higher in IBD patients, compared to healthy controls. Consistently, the expression of PFKFB3 was also elevated in a cohort of inflamed intestinal tissues from IBD patients compared to non-inflamed sites from the same patient or healthy controls. On the cellular levels, this analysis showed that PFKB3 expression was higher in IBD-derived stromal cells compared to healthy or non-inflamed stromal cells. In vitro PFKFB3 expression in fibroblasts was increased after the stimulation with pro-inflammatory cytokines like TNF-α and a mix of cytokines often upregulated in IBD patients: interleukin (IL)-17A, oncostatin M (OSM) and IL-1β. As for the metabolic changes, inflamed fibroblasts had a higher extracellular acidification rate and a lower oxygen consumption rate, which could be reverted by inhibition of PFKFB3 using PFK15. Furthermore, PFK15 suppressed the proliferation and migration of fibroblasts. The in vivo experiments showed that PFK15 reduced the severity of the colitis, accompanied by a reduction of the total amount of immune cells (CD45), activated fibroblasts (α-SMA) and angiogenesis (CD105). Conclusion Increased PFKFB3 expression seems to contribute the inflammation and the pathological function of fibroblasts in IBD.
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Yu, Hongbin, Chuang Dai, Wei Zhu, Yude Jin, and Chunhui Wang. "PFKFB3 Increases IL-1β and TNF-α in Intestinal Epithelial Cells to Promote Tumorigenesis in Colitis-Associated Colorectal Cancer." Journal of Oncology 2022 (August 16, 2022): 1–8. http://dx.doi.org/10.1155/2022/6367437.
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Colorectal cancer (CRC) is significantly correlated with inflammatory bowel disease, which usually manifests as chronic relapsing-remitting colitis. Phosphofructo-2-kinase/fructose-2,6-biophosphatase 3 (PFKFB3) can catalyze to produce fructose-2,6-bisphosphate and function as an oncogene. In this study, we revealed the function of PFKFB3 in colitis-associated CRC (CAC) and the potential mechanism. RT-qPCR and Western blot were utilized to detect the level of PFKFB3 expression. Increased PFKFB3 expression was observed in the mouse CAC model and CAC patient samples. We identified that overexpression of PFKFB3 in intestinal epithelial cells (IECs) could increase the proliferation, migration, and invasion of CRC cells by the coculture system. Mechanistically, overexpression of PFKFB3 induced phospho-p65 and promoted the expression of IL-1β and tumor necrosis factor alpha (TNF-α) in the development of colitis and CAC. In addition, PFK158, the PFKFB3 inhibitor, could reduce the CRC cell viability, migration, and invasion caused by PFKFB3 overexpression. In conclusion, overexpression of PFKFB3 promoted tumorigenesis in CAC by inducing phospho-p65 and expression of IL-1β and TNF-α. Our study suggested that PFKFB3 acted as a potential treatment target for CAC.
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Grewal, Jaspreet, Jamaal Ricthie, Numan Al Rayyan, Sucheta Telang, Kavitha Yaddanapudi, and Jason Chesney. "6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) is necessary for human melanoma MDSC differentiation and function." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 74.8. http://dx.doi.org/10.4049/jimmunol.196.supp.74.8.
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Abstract Myeloid derived suppressor cells (MDSCs) inhibit the expansion of tumor antigen-specific effector CD8+ T cells via arginase, transforming growth factor – β (TGF – β) and indoleamine 2,3-dioxygenase (IDO). Recently, MDSCs were found to over-express hypoxia inducible factor 1 alpha (HIF-1α) which is required for their differentiation. An essential transcriptional target of HIF-1α is 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) which synthesizes fructose 2,6-bisphosphate, an allosteric stimulator of glycolysis and of proliferation via stimulation of cyclin dependent kinase-1 (CDK1). We hypothesized that MDSCs might over-express PFKFB3 which in turn might be required for their function as T cell suppressors. We demonstrate that monocytic MDSCs (M-MDSCs) induced by co-culture with A375 melanoma cells express increased PFKFB3 and that exposure to the PFKFB3 inhibitor, PFK-158, blocks the suppressive function of these M-MDSCs on T cell activation. Furthermore, we analyzed three advanced cancer patients for circulating MDSCs before and after PFK-158 administration as part of a multi-center phase 1 clinical trial. And, we found that the MDSCs were markedly reduced in each patient. Taken together, these data indicate that selective inhibition of PFKFB3 may be a novel approach to target MDSCs and combinations of PFKFB3 inhibitors with immunotherapies may be a rational strategy to promote durable immune-mediated remissions in cancer patients.
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Reddy, Mamatha M., Margret Fernandes, James D. Griffin, and Martin Sattler. "The JAK2V617F Oncogene, Associated with Myeloproliferative Neoplasms, Requires Expression of Inducible Phosphofructokinase/Fructose Bisphosphatase 3 for Cell Growth and Increased Metabolic Activity." Blood 116, no. 21 (November 19, 2010): 793. http://dx.doi.org/10.1182/blood.v116.21.793.793.
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Abstract 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. Disclosures: No relevant conflicts of interest to declare.
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Cao, Yapeng, Xiaoyu Zhang, Lina Wang, Qiuhua Yang, Qian Ma, Jiean Xu, Jingjing Wang, et al. "PFKFB3-mediated endothelial glycolysis promotes pulmonary hypertension." Proceedings of the National Academy of Sciences 116, no. 27 (June 18, 2019): 13394–403. http://dx.doi.org/10.1073/pnas.1821401116.
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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.
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Jones, Brandon C., Surojeet Sengupta, Catherine M. Sevigny, Lu Jin, Paula R. Pohlmann, Ayesha Shajahan-Haq, and Robert Clarke. "Abstract P4-02-10: Pfkfb3 inhibition significantly decreases endocrine-resistant breast cancer growth and induces necroptotic cell death." Cancer Research 82, no. 4_Supplement (February 15, 2022): P4–02–10—P4–02–10. http://dx.doi.org/10.1158/1538-7445.sabcs21-p4-02-10.
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Abstract Estrogen receptor-positive breast cancers constitute the majority of newly diagnosed breast cancers in patients. While commonly treated with various approved endocrine therapy strategies, these cancers can unfortunately also develop resistance to such therapies, indicating a need to explore other avenues of treatment in endocrine-resistant breast cancer. Rapidly dividing cancer cells are known to have an increased energy demand and altered reprogramming of glucose metabolism, leading to a subsequent heavy reliance on glycolysis. A critical rate-limiting step in the glycolysis pathway, the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate, is greatly influenced by an allosteric activator produced by the enzyme PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3), thus making PFKFB3 an essential regulator of glycolytic flux. Furthermore, PFKFB3 has been shown to be overexpressed in many human cancers, including breast cancer, as well as be upregulated by estrogen. Through the following work, we show that endocrine-resistant BC cell growth is strongly reduced by PFKFB3 targeting using different commercial PFKFB3 inhibitors, which can be further exacerbated by combination treatment with well-established endocrine therapies like fulvestrant in some cell lines. Moreover, we relatedly show that PFKFB3 inhibition reduced tumor size in mouse xenografts of endocrine-resistant BC cells. Endocrine-resistant cells also presented higher basal glucose uptake and greater reduction of glucose uptake in response to PFKFB3 inhibition than endocrine-sensitive cells, indicating that endocrine-resistant breast cancer cells may have a more significant reliance on glycolysis and PFKFB3 activity for their metabolic needs than sensitive cells. Additionally, PFKFB3 inhibition produced an increase in phosphorylation of the necroptotic markers RIPK1 (receptor-interacting kinase 1), and MLKL (Mixed Lineage Kinase Domain Like Pseudokinase), and pre-treatment with the necroptosis inhibitor necrostatin-1 was able to partially rescue cell death caused by PFKFB3 targeting, suggesting that necroptosis is a primary method of cell death induced by PFKFB3 inhibition. In summation, this study showcases the glycolytic enzyme PFKFB3 as an encouraging therapeutic target in endocrine-resistant breast cancer, and warrants further review as a monotherapy or in combination with current endocrine therapies. Citation Format: Brandon C Jones, Surojeet Sengupta, Catherine M Sevigny, Lu Jin, Paula R Pohlmann, Ayesha Shajahan-Haq, Robert Clarke. Pfkfb3 inhibition significantly decreases endocrine-resistant breast cancer growth and induces necroptotic cell death [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-10.
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Zhang, Yongsheng, Yukun Liu, Zhenxing Xie, Qinxin Liu, Yangfan Zhuang, Weiming Xie, Xiang Wang, et al. "Inhibition of PFKFB Preserves Intestinal Barrier Function in Sepsis by Inhibiting NLRP3/GSDMD." Oxidative Medicine and Cellular Longevity 2022 (December 23, 2022): 1–13. http://dx.doi.org/10.1155/2022/8704016.
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Intestinal barrier dysfunction is associated with the occurrence and development of sepsis. Further, aerobic glycolysis plays an essential role in inflammation and cell death. This study is aimed at investigating the protective effect and mechanism of PFKFB3 inhibition on intestinal barrier dysfunction in sepsis mice. Sepsis mouse models were established by cecal ligation and puncture (CLP) in wild-type mice and Gsdmd-/- mice. The results showed that the expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) in the small intestines was significantly upregulated in sepsis. 3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), the specific inhibitor of PFKFB3, and Gsdmd gene knockout significantly inhibited the inflammatory response and cell death caused by sepsis, thus alleviating intestinal damage and barrier dysfunction. 3PO was also shown to significantly inhibit oxidative stress and NLRP3/caspase-1/GSDMD-dependent cell pyroptosis in the small intestines. The in vitro studies revealed that 3PO reduced NLRP3/caspase-1/GSDMD-dependent cell pyroptosis by inhibiting ROS. Taken together, our results suggest that PFKFB3 is involved in inflammation, oxidative stress, and pyroptosis during sepsis and enhances intestinal damage, which may provide important clues about the potential targets to be exploited in this highly lethal disease.
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Zhu, Yu, Jianyong Li, Chun Qiao, Yi Shan, Sixuan Qian, Yaoyu Chen, Ming Hong, et al. "PFKFB3 Is a Crucial Target in the Treatment of Tyrosine Kinase Inhibitor Resistant Chronic Myelogenous Leukemia." Blood 128, no. 22 (December 2, 2016): 3936. http://dx.doi.org/10.1182/blood.v128.22.3936.3936.
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Abstract Resistance to the BCR-ABL tyrosine kinase inhibitor (TKI) imatinib mesylate (IM) remains challenge for the treatment of chronic myeloid leukemia (CML). IM resistance often results from unknown mechanisms with wild type BCR-ABL that have no effects on TKIs binding to ABL kinase domain. The basis of such BCR-ABL-independent IM resistance remains to be elucidated. To gain insight into BCR-ABL-independent IM resistance mechanisms, we performed an initial bioinformatics screen on over represented CML genes, followed by a quantitative PCR screen of genes that were elevated in TKIs resistant CML samples. We identified a total of 33 candidate genes that were highly expressed in TKIs resistant patients. Among these genes, 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3 (PFKFB3) controlling the limiting step of glycolysis, was found to strongly associated with TKIs resistance. RNA interference of the expression of PFKFB3 and pharmacological inhibition of its kinase activity markedly increased the sensitivity of TKIs resistant CML cells to TKIs. Furthermore, pharmacological inhibition of PFKFB3 prevented CML cells growth and significantly improved the survival of both allograft and xenograft CML mice. ChIP-seq data analysis combined with subsequent knockdown experiment demonstrated that the Ets transcription factor PU.1 regulates the elevated expression of PFKFB3 in TKIs resistant CML cells. Collectively, our results identify a therapeutically targetable mechanism of BCR-ABL-independent TKIs resistant CML. Disclosures No relevant conflicts of interest to declare.
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De Oliveira, Tiago, Tina Goldhardt, Marcus Edelmann, Torben Rogge, Karsten Rauch, Nikola Dobrinov Kyuchukov, Kerstin Menck, et al. "Effects of the Novel PFKFB3 Inhibitor KAN0438757 on Colorectal Cancer Cells and Its Systemic Toxicity Evaluation In Vivo." Cancers 13, no. 5 (February 28, 2021): 1011. http://dx.doi.org/10.3390/cancers13051011.
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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.
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Warrier, Govind, Lilibeth Lanceta, Yoannis Imbert-Fernandez, and Jason Alan Chesney. "Inhibition of glucose metabolism through treatment of BRAF mutated metastatic melanoma with vemurafenib." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e21005-e21005. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e21005.
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e21005 Background: Increased glucose metabolism is a hallmark of neoplastic cells that allows self-promotion of growth and survival. The enzyme 6-phosphofructo-2-kinase (PFKFB3) is an integral controller of glycolysis by promoting the synthesis of fructose 2,6-bisphosphonate (F2,6BP) which activates 6-phoshofructo-1-kinase (PFK-1), a rate-limiting enzyme and essential control point in the glycolytic pathway. Additionally, mitogen-activated protein kinase (MAPK) is a key signaling pathway in a number of cancers with mutations of the BRAF component, described most commonly in melanoma, resulting in constitutive activation of the MAPK pathway. We aim to demonstrate that vemurafenib, a BRAF inhibitor, has antiglycolytic activity in sensitive melanoma cell lines which may help guide development of future therapies with specific attention to PFKFB3 as a potential enzymatic target to decrease glycolytic flux thereby inhibiting tumor growth and survival. Methods: Vemurafenib sensitive and resistant variants of two separate human metastatic melanoma cell lines (451Lu and WM983) were treated with 3 mM Vemurafenib for 24 and 48 hours. Additionally, cells from aforementioned lines were probed for PFKFB3 after 24 hours of treatment with vemurafenib. Glycolysis was measured by incubating cells in complete media containing 1 mCi [5-3H]glucose for 60 minutes. [3H]H2O produced by glycolysis through enolase was measured. Results: A decrease in PFKFB3 protein expression was found in vemurafenib sensitive cells compared to controls but not in resistant cells after 24h treatment with 3 mM vemurafenib in both 451Lu and WM983 metastatic melanoma cell lines (n = 2). Treatment with vemurafenib led to decrease in glycolysis compared to untreated controls in both vemurafenib sensitive metastatic melanoma cell lines but not in resistant cell lines (n = 5). Additionally, there was a significant reduction in glycolysis in vemurafenib resistant WM983 at 48 hours compared to resistant untreated control. Conclusions: BRAF mutated metastatic melanoma cells showed decrease in PFKFB3 protein expression and decreased glycolysis after treatment with BRAF inhibitor vemurafenib. Future studies will focus on assessing metastatic melanoma cell viability and glycolytic activity after treatment with combination BRAF inhibition and PFKFB3 specific inhibition.
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Zhang, Yang, Weifang Liu, Yanqi Zhong, Qi Li, Mengying Wu, Liu Yang, Xiaoxia Liu, and Li Zou. "Metformin Corrects Glucose Metabolism Reprogramming and NLRP3 Inflammasome-Induced Pyroptosis via Inhibiting the TLR4/NF-κB/PFKFB3 Signaling in Trophoblasts: Implication for a Potential Therapy of Preeclampsia." Oxidative Medicine and Cellular Longevity 2021 (November 11, 2021): 1–22. http://dx.doi.org/10.1155/2021/1806344.
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NOD-like receptor family, pyrin domain-containing protein 3 (NLRP3) inflammasome-mediated pyroptosis is a crucial event in the preeclamptic pathogenesis, tightly linked with the uteroplacental TLR4/NF-κB signaling. Trophoblastic glycometabolism reprogramming has now been noticed in the preeclampsia pathogenesis, plausibly modulated by the TLR4/NF-κB signaling as well. Intriguingly, cellular pyroptosis and metabolic phenotypes may be inextricably linked and interacted. Metformin (MET), a widely accepted NF-κB signaling inhibitor, may have therapeutic potential in preeclampsia while the underlying mechanisms remain unclear. Herein, we investigated the role of MET on trophoblastic pyroptosis and its relevant metabolism reprogramming. The safety of pharmacologic MET concentration to trophoblasts was verified at first, which had no adverse effects on trophoblastic viability. Pharmacological MET concentration suppressed NLRP3 inflammasome-induced pyroptosis partly through inhibiting the TLR4/NF-κB signaling in preeclamptic trophoblast models induced via low-dose lipopolysaccharide. Besides, MET corrected the glycometabolic reprogramming and oxidative stress partly via suppressing the TLR4/NF-κB signaling and blocking transcription factor NF-κB1 binding on the promoter PFKFB3, a potent glycolytic accelerator. Furthermore, PFKFB3 can also enhance the NF-κB signaling, reduce NLRP3 ubiquitination, and aggravate pyroptosis. However, MET suppressed pyroptosis partly via inhibiting PFKFB3 as well. These results provided that the TLR4/NF-κB/PFKFB3 pathway may be a novel link between metabolism reprogramming and NLRP3 inflammasome-induced pyroptosis in trophoblasts. Further, MET alleviates the NLRP3 inflammasome-induced pyroptosis, which partly relies on the regulation of TLR4/NF-κB/PFKFB3-dependent glycometabolism reprogramming and redox disorders. Hence, our results provide novel insights into the pathogenesis of preeclampsia and propose MET as a potential therapy.
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Zhu, Yu, Luo Lu, Chun Qiao, Yi Shan, Huapeng Li, Sixuan Qian, Ming Hong, et al. "Targeting PFKFB3 sensitizes chronic myelogenous leukemia cells to tyrosine kinase inhibitor." Oncogene 37, no. 21 (March 7, 2018): 2837–49. http://dx.doi.org/10.1038/s41388-018-0157-8.
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Parker, Gretchen E., Bartholomew A. Pederson, Mariko Obayashi, Jill M. Schroeder, Robert A. Harris, and Peter J. Roach. "Gene expression profiling of mice with genetically modified muscle glycogen content." Biochemical Journal 395, no. 1 (March 15, 2006): 137–45. http://dx.doi.org/10.1042/bj20051456.
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Glycogen, a branched polymer of glucose, forms an energy re-serve in numerous organisms. In mammals, the two largest glyco-gen stores are in skeletal muscle and liver, which express tissue-specific glycogen synthase isoforms. MGSKO mice, in which mGys1 (mouse glycogen synthase) is disrupted, are devoid of muscle glycogen [Pederson, Chen, Schroeder, Shou, DePaoli-Roach and Roach (2004) Mol. Cell. Biol. 24, 7179–7187]. The GSL30 mouse line hyper-accumulates glycogen in muscle [Manchester, Skurat, Roach, Hauschka and Lawrence (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 10707–10711]. We performed a microarray analysis of mRNA from the anterior tibialis, medial gastrocnemius and liver of MGSKO mice, and from the gastroc-nemius of GSL30 mice. In MGSKO mice, transcripts of 79 genes varied in their expression in the same direction in both the anterior tibialis and gastrocnemius. These included several genes encoding proteins proximally involved in glycogen metabolism. The Ppp1r1a [protein phosphatase 1 regulatory (inhibitor) sub-unit 1A] gene underwent the greatest amount of downregulation. In muscle, the downregulation of Pfkfb1 and Pfkfb3, encoding isoforms of 6-phosphofructo-2-kinase/fructose-2,6-bisphospha-tase, is consistent with decreased glycolysis. Pathways for branched-chain amino acid, and ketone body utilization appear to be downregulated, as is the capacity to form the gluconeogenic precursors alanine, lactate and glutamine. Expression changes among several members of the Wnt signalling pathway were identified, suggesting an as yet unexplained role in glycogen meta-bolism. In liver, the upregulation of Pfkfb1 and Pfkfb3 expression is consistent with increased glycolysis, perhaps as an adaptation to altered muscle metabolism. By comparing changes in muscle expression between MGSKO and GSL30 mice, we found a subset of 44 genes, the expression of which varied as a function of muscle glycogen content. These genes are candidates for regulation by glycogen levels. Particularly interesting is the observation that 11 of these genes encode cardiac or slow-twitch isoforms of muscle contractile proteins, and are upregulated in muscle that has a greater oxidative capacity in MGSKO mice.
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Benner, Elizabeth, Camila Dos Santos, Kyla Hagen, Carol Lange, Julie Ostrander, Nuri Temiz, Marygrace Trousdell, and Thu Truong. "OR16-2 Steroid Receptor Co-Activator Complexes Regulate Metabolic PFKFB Enzymes to Drive Therapy Resistant ER+ Breast Cancer." Journal of the Endocrine Society 6, Supplement_1 (November 1, 2022): A879—A880. http://dx.doi.org/10.1210/jendso/bvac150.1821.
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Abstract Recurrence of metastatic breast cancer stemming from acquired endocrine and chemotherapy resistance remains a health burden for women with luminal (ER+) breast cancer. ER+ tumor cells can remain viable but quiescent for years to decades. Contributing factors to metastatic spread include the survival and expansion of breast cancer stem cells (CSCs). Breast CSCs are poorly proliferative and frequently exist as a minority population in recurrent tumors. Our objective is to define novel signaling pathways that govern therapy resistance in ER+ breast cancer. We previously showed 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. PELP1 interacts with PFKFB proteins, and inhibition of PFKFB3 and PFKFB4 kinase activity blocks PELP1-induced tumorspheres and protein-protein interactions with SRC-3. PFKFB inhibitors in combination with ER targeted therapies blocked proliferation and tumorsphere formation in multiple models of advanced breast cancer, including paclitaxel (TaxR) and tamoxifen (TamR) resistant models and ER+ patient-derived organoids (PDxO). Chemotherapy and endocrine therapy resistant models contained increased levels of cytoplasmic PELP1, PELP1/SRC-3 complexes, and phenocopied PELP1/SRC-3/PFKFB biology and CSC behavior. To better understand CSC-mediated pathways in the context of therapy resistance, we performed RNA-seq on 3D-cultured TaxR tumorspheres to enrich for CSCs. Pathway analysis revealed that TaxR tumorspheres upregulate stem (Sox2-Oct4-Nanog), HIF, and progesterone mediated pathways. Progesterone receptor (PR) is a known contributor to CSC populations, and we observed increased PR mRNA and protein expression in TaxR cells grown as tumorspheres. Additionally, in vivo emergence of circulating tumor cell (CTC) populations was observed in ER+ TaxR mammary intraductal (MIND) xenografts. Together, our data suggest that PELP1, SRC-3, and PFKFBs cooperate to drive ER+ tumor cells that include CSCs and CTCs. Our findings are applicable to therapy resistant models, indicating that SR co-activator complexes are a key mediator of resistance in ER+ breast cancer and may cooperate with PR to promote therapy resistant CSCs. 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. Presentation: Sunday, June 12, 2022 11:15 a.m. - 11:30 a.m.
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Okabe, Seiichi, Yuko Tanaka, Mitsuru Moriyama, and Akihiko Gotoh. "Targeting Glycolysis in Multiple Myeloma: Novel Strategies in the Treatment of Proteasome Inhibitor Resistant in Hypoxic Conditions." Blood 134, Supplement_1 (November 13, 2019): 4344. http://dx.doi.org/10.1182/blood-2019-129818.
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Introduction: Multiple myeloma (MM) is one of the hematological malignancy and characterized by the clonal expansion of plasma cells in the bone marrow. The treatment of MM patients has been dramatically changed by new agents such as proteasome inhibitors and immunomodulatory drugs, however, many patients will relapse even if new agents provide therapeutic advantages. Therefore, a new strategy is still needed to increase MM patient survival. Hypoxia is an important component of the bone marrow microenvironment. Hypoxia may increase myeloma cell survival. Because cells shift primarily to a glycolytic mode for generation of energy in hypoxic conditions, glycolytic activities can be targeted therapeutically in MM patients. The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) is responsible for maintaining the cellular levels of fructose-2,6-bisphosphate which is a regulator of glycolysis. Materials and Methods: In this study, we investigated whether PFKFB was involved in myeloma cells in hypoxia condition. We also investigated whether PFKFB inhibitors could suppress myeloma cells and enhance the sensitivity of myeloma cells to proteasome inhibition. Results: We first investigated the expression of PFKBP in the myeloma cell lines in hypoxia condition. PFKFB family contains four tissue-specific isoenzymes encoded by four different genes. We found expression of PFKBP3 and PFKBP4 were increased in hypoxia condition. We found gene expression of PFKBP3 and PFKBP4 were involved in myeloma cell lines and myeloma patient samples in hypoxia condition from the public microarray datasets (GSE80140 and GSE80545). In hypoxia condition, expression of hypoxia-inducible factor 1α (HIF1α) was increased and phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was activated in myeloma cell lines. Expression of PFKBP3 and PFKBP4 were inhibited by HIF1α inhibitor and p38 MAPK inhibitor treatment. In the hypoxia condition, activity of proteasome inhibitors were reduced compared to normoxia condition. We next investigated whether PFKBP3 inhibitor, PFK158 and PFKBP4 inhibitor, 5MPN could inhibit the proliferation of myeloma cells. We found PFK158 and 5MPN treatment inhibited the growth of myeloma cells in a dose dependent manner in hypoxia condition. Combined treatment of myeloma cells with carfilzomib and PFK158 or 5MPN caused more cytotoxicity than each drug alone. Caspase 3/7 activity and cellular cytotoxicity was also increased. We found proteasomal activity was also reduced by carfilzomib and PFK158 or 5MPN treatment. Adenosine triphosphate (ATP) is the most important source of energy for intracellular reactions. Intracellular ATP levels drastically decreased after carfilzomib and PFK158 or 5MPN treatment. Because mitochondria generate ATP and participate in signal transduction and cellular pathology and cell death. The quantitative analysis of JC-1 stained cells changed mitochondrial membrane potential in cell death, which were induced by carfilzomib and PFK158 or 5MPN on myeloma cells. In the hypoxia condition and inhibitor treatment, glycolytic activities (e.g. glucose and lactate) were changed in myeloma cells. Conclusion: The PFKBP3 and PFKBP4 are enhanced in hypoxia condition and involved in proteasome inhibitor sensitivity. Our data also suggested that administration of PFKBP3 and PFKBP4 inhibitors may be a powerful strategy against myeloma cells and enhance cytotoxic effects of proteasome inhibitors in hypoxia condition. Disclosures No relevant conflicts of interest to declare.
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Gómez, Marta, Anna Manzano, Agnes Figueras, Francesc Viñals, Francesc Ventura, Jose Luis Rosa, Ramon Bartrons, and Àurea Navarro-Sabaté. "Sertoli-secreted FGF-2 induces PFKFB4 isozyme expression in mouse spermatogenic cells by activation of the MEK/ERK/CREB pathway." American Journal of Physiology-Endocrinology and Metabolism 303, no. 6 (September 15, 2012): E695—E707. http://dx.doi.org/10.1152/ajpendo.00381.2011.
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Sertoli cells play a central role in the control and maintenance of spermatogenesis by secreting growth factors, in response to hormonal stimulation, that participate in the paracrine regulation of this process. In this study, we investigated how the hormonal regulation of spermatogenesis modulates 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) isozyme expression in two mouse spermatogenic cell lines, GC-1 spg and GC-2 spd (ts). For this purpose, TM4 Sertoli cells were used to obtain conditioned medium that was treated or not with dihydrotestosterone for 2 days [dihydrotestosterone conditioned medium (TCM) and basal conditioned medium (BCM), respectively]. We observed an increase in the expression of PFKFB4 along with a decrease in PFKFB3 in spermatogenic cell lines treated with TCM. These effects were inhibited by the antiandrogen drug flutamide and by heat-inactivated TCM, indicating the protein nature of the TCM mediator and its dependence on Sertoli cell stimulation by dihydrotestosterone. In addition, adult rat testes treated with the GnRH antagonist Degarelix exhibited a reduction in the expression of PFKFB4 in germ cells. Addition of exogenous FGF-2 mimicked the changes in the Pfkfb gene expression, whereas neutralizing antibodies against FGF-2 abolished them. Interestingly, similar effects on Pfkfb gene expression were observed using different MAPK inhibitors (U-0126, PD-98059, and H-89). Luciferase analysis of Pfkfb4 promoter constructs demonstrated that a putative CRE-binding sequence located at −1,463 relative to the transcription start site is required to control Pfkfb4 gene expression after TCM treatment. Pulldown assays showed the binding of the CREB transcription factor to this site. Altogether, these results show how the paracrine regulation orchestrated by Sertoli cells in response to testosterone controls glycolysis in germ cells.
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Simula, Luca, Marco Alifano, and Philippe Icard. "How Phosphofructokinase-1 Promotes PI3K and YAP/TAZ in Cancer: Therapeutic Perspectives." Cancers 14, no. 10 (May 18, 2022): 2478. http://dx.doi.org/10.3390/cancers14102478.
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PI3K/AKT is one of the most frequently altered signaling pathways in human cancers, supporting the activation of many proteins sustaining cell metabolism, proliferation, and aggressiveness. Another important pathway frequently altered in cancer cells is the one regulating the YAP/TAZ transcriptional coactivators, which promote the expression of genes sustaining aerobic glycolysis (such as WNT, MYC, HIF-1), EMT, and drug resistance. Of note, the PI3K/AKT pathway can also regulate the YAP/TAZ one. Unfortunately, although PI3K and YAP inhibitors are currently tested in highly resistant cancers (both solid and hematologic ones), several resistance mechanisms may arise. Resistance mechanisms to PI3K inhibitors may involve the stimulation of alternative pathways (such as RAS, HER, IGFR/AKT), the inactivation of PTEN (the physiologic inhibitor of PI3K), and the expression of anti-apoptotic Bcl-xL and MCL1 proteins. Therefore, it is important to improve current therapeutic strategies to overcome these limitations. Here, we want to highlight how the glycolytic enzyme PFK1 (and its product F-1,6-BP) promotes the activation of both PI3K/AKT and YAP/TAZ pathways by several direct and indirect mechanisms. In turn, PI3K/AKT and YAP/TAZ can promote PFK1 activity and F-1,6-BP production in a positive feedback loop, thus sustaining the Warburg effect and drug resistance. Thus, we propose that the inhibition of PFK1 (and of its key activator PFK2/PFKFB3) could potentiate the sensitivity to PI3K and YAP inhibitors currently tested. Awaiting the development of non-toxic inhibitors of these enzymes, we propose to test the administration of citrate at a high dosage, because citrate is a physiologic inhibitor of both PFK1 and PFK2/PFKFB3. Consistently, in various cultured cancer cells (including melanoma, sarcoma, hematologic, and epithelial cancer cells), this “citrate strategy” efficiently inhibits the IGFR1/AKT pathway, promotes PTEN activity, reduces Bcl-xL and MCL1 expression, and increases sensitivity to standard chemotherapy. It also inhibits the development of sarcoma, pancreatic, mammary HER+ and lung RAS-driven tumors in mice without apparent toxicities.
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Zlacká, Jana, Miroslav Murár, Gabriela Addová, Roman Moravčík, Andrej Boháč, and Michal Zeman. "Synthesis of Glycolysis Inhibitor PFK15 and Its Synergistic Action with an Approved Multikinase Antiangiogenic Drug on Human Endothelial Cell Migration and Proliferation." International Journal of Molecular Sciences 23, no. 22 (November 18, 2022): 14295. http://dx.doi.org/10.3390/ijms232214295.
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Activated endothelial, immune, and cancer cells prefer glycolysis to obtain energy for their proliferation and migration. Therefore, the blocking of glycolysis can be a promising strategy against cancer and autoimmune disease progression. Inactivation of the glycolytic enzyme PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase) suppresses glycolysis level and contributes to decreased proliferation and migration of cancer (tumorigenesis) and endothelial (angiogenesis) cells. Recently, several glycolysis inhibitors have been developed, among them (E)-1-(pyridin-4-yl)-3-(quinolin-2-yl)prop-2-en-1-one (PFK15) that is considered as one of the most promising. It is known that PFK15 decreases glucose uptake into the endothelial cells and efficiently blocks pathological angiogenesis. However, no study has described sufficiently PFK15 synthesis enabling its general availability. In this paper we provide all necessary details for PFK15 preparation and its advanced characterization. On the other hand, there are known tyrosine kinase inhibitors (e.g., sunitinib), that affect additional molecular targets and efficiently block angiogenesis. From a biological point of view, we have studied and proved the synergistic inhibitory effect by simultaneous administration of glycolysis inhibitor PFK15 and multikinase inhibitor sunitinib on the proliferation and migration of HUVEC. Our results suggest that suppressing the glycolytic activity of endothelial cells in combination with growth factor receptor blocking can be a promising antiangiogenic treatment.
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Redman, Rebecca Ann, Paula Raffin Pohlmann, Michael R. Kurman, Gilles Tapolsky, and Jason Chesney. "A phase I, dose-escalation, multicenter study of ACT-PFK-158, 2HCl in patients with advanced solid malignancies explores a first-in-human inhibitor of glycolysis." Journal of Clinical Oncology 33, no. 3_suppl (January 20, 2015): TPS494. http://dx.doi.org/10.1200/jco.2015.33.3_suppl.tps494.
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TPS494 Background: In human cancers, loss of PTEN, stabilization of hypoxia inducible factor-1α, and activation of Ras and AKT converge to increase the activity of a regulator of glycolysis, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). This enzyme synthesizes fructose-2,6-bisphosphate (F2,6BP), which is an activator of 6-phosphofructo-1-kinase, a crucial step of glycolysis that is tightly controlled by multiple metabolic feedback mechanisms and dictates the rate of glycolytic flow. The vast majority of pancreatic ductal adenocarcinomas and approximately 50% of colon adenocarcinomas harbor activating mutations in Ras and these tumors have been reported to be highly glycolytic. PFK158 is a potent small molecule inhibitor of PFKFB3 that is selectively cytotoxic to Ras-transformed epithelial cells and displays broad anti-tumor activity causing ~80% growth inhibition in several mouse models of human-derived tumors and syngeneic murine models of colon cancer. Importantly, IND-enabling safety and toxicity studies have demonstrated that PFK158 is well tolerated in rats and dogs with an expected good therapeutic index, lending support for a phase 1 trial that is now underway. Methods: The primary objective of the study is to describe the dose limiting toxicity and to determine either the maximum tolerated dose or biological effective dose of PFK-158 in a “3+3” cohort-based dose escalation design that follows a modified Fibonacci scheme. Multiple secondary endpoints have been incorporated to assess the effects of PFK-158 on peripheral blood mononuclear cell F2,6BP activity and on glucose uptake using FDG-PET imaging. This trial is currently enrolling at two US sites; Cohort 1 has been completed without dose-limiting toxicity and Cohort 2 is enrolling with two subjects under treatment as of September 2014. In conclusion, PFK158 is the first-in-man and first-in-class PFKFB3 inhibitor to be examined in a phase I trial and may have significant clinical utility either as a monotherapy or when combined with other targeted agents. Clinical trial information: NCT02044861.
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Telang, Sucheta, Julie O’Neal, Gilles Tapolsky, Brian Clem, Alan Kerr, Yoannis Imbert-Ferndandez, and Jason Chesney. "Discovery of a PFKFB3 inhibitor for phase I trial testing that synergizes with the B-Raf inhibitor vemurafenib." Cancer & Metabolism 2, Suppl 1 (2014): P14. http://dx.doi.org/10.1186/2049-3002-2-s1-p14.
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Guan, Shuyuan, Lingbin Sun, Xihua Wang, Xirui Huang, and Tao Luo. "Isoschaftoside Inhibits Lipopolysaccharide-Induced Inflammation in Microglia through Regulation of HIF-1α-Mediated Metabolic Reprogramming." Evidence-Based Complementary and Alternative Medicine 2022 (November 23, 2022): 1–8. http://dx.doi.org/10.1155/2022/5227335.
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Isoschaftoside is a C-glycosyl flavonoid extracted from the root exudates of Desmodium uncinatum and Abrus cantoniensis. Previous studies suggested that C-glycosyl flavonoid has neuroprotective effects with the property of reducing oxidative stress and inflammatory markers. Microglia are key cellular mediators of neuroinflammation in the central nervous system. The aim of this study was to investigate the effect of isoschaftoside on lipopolysaccharide-induced activation of BV-2 microglial cells. The BV-2 cells were exposed to 10 ng/ml lipopolysaccharide and isoschaftoside (0–1000 μM). Isoschaftoside effectively inhibited lipopolysaccharide-induced nitric oxide production and proinflammatory cytokines including iNOS, TNF-α, IL-1β, and COX2 expression. Isoschaftoside also significantly reduced lipopolysaccharide-induced HIF-1α, HK2, and PFKFB3 protein expression. Induction of HIF-1α accumulation by CoCl2 was inhibited by isoschaftoside, while the HIF-1α specific inhibitor Kc7f2 mitigated the metabolic reprogramming and anti-inflammatory effect of isoschaftoside. Furthermore, isoschaftoside attenuated lipopolysaccharide-induced phosphorylation of ERK1/2 and mTOR. These results suggest that isoschaftoside can suppress inflammatory responses in lipopolysaccharide-activated microglia, and the mechanism was partly due to inhibition of the HIF-1α-mediated metabolic reprogramming pathway.
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Mondal, Susmita, Debarshi Roy, Sayantani Sarkar Bhattacharya, Ling Jin, Deokbeom Jung, Song Zhang, Eleftheria Kalogera, et al. "Therapeutic targeting of PFKFB3 with a novel glycolytic inhibitor PFK158 promotes lipophagy and chemosensitivity in gynecologic cancers." International Journal of Cancer 144, no. 1 (October 30, 2018): 178–89. http://dx.doi.org/10.1002/ijc.31868.
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Erlandsson, M., K. M. Andersson, N. Oparina, S. Töyrä Silfverswärd, and M. I. Bokarewa. "OP0315 EFFECTOR CD4 T CELLS REQUIRE SURVIVIN FOR REGULATION OF GLUCOSE METABOLISM AND IFNg PRODUCTION." Annals of the Rheumatic Diseases 80, Suppl 1 (May 19, 2021): 192.2–193. http://dx.doi.org/10.1136/annrheumdis-2021-eular.2295.
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Background:Interferon-gamma (IFNg) producing effector T cells play the leading role in triggering and perpetuation of inflammation in rheumatoid arthritis. Inflammation leads to metabolic reprogramming of T cells and high energy consumption supporting proliferation and IFNg production. Being a part of chromosomal passenger complex, oncoprotein survivin is essential for cell proliferation. It has also been identified as a marker of severe therapy-resistant rheumatoid arthritis. Thus, we aimed to explore the association between survivin and IFNg producing phenotype of CD4 T cells.Objectives:We study if survivin mediates the glucose dependent mechanism of IFNg production in CD4 T cells.Methods:CD4 cells were sorted from the peripheral blood of RA patients and healthy controls, activated with aCD3, cultured in presence of survivin inhibitor YM155 and subjected to RNA sequencing (Illumina, Life Science). IFNg levels in supernatants were measured by ELISA. To study glucose uptake in presence of YM155, CD4 cells were treated with IFNg+aCD3 overnight followed by 2NBD-glucose challenge for 30 min. Uptake of fluorescent 2NBD-glucose probe was measured by flow cytometry. Statistical analysis of RNAseq was performed in R-studio using the Bioconductor package DESeq2.Results:Comparison of the whole-genome transcription profile of CD4 cells different by levels of BIRC5, coding for survivin, demonstrated that the BIRC5hi group expressed significantly higher levels of IFNg (mRNA, p=10-26 and protein, p=10-4). Also, BIRC5hi CD4 cells had higher expression of glucose transporter GLUT1 (SLC2A1, p=0.0064) and of glycolytic enzymes glucose-6-phosphate dehydrogenase (G6PD, p=10-6), pyruvate kinase (PFKP, p=10-6), and lactate dehydrogenase (LDHA, p=10-14). On the contrary, expression of the key regulator of glycolysis 6-phosphofructo-2-kinase (PFKFB3) was significantly lower in the BIRC5hi group (p=4.4x10-5). Notably, expression of glycolytic enzymes G6PD and PFKFB3 correlated strongly to IFNg (r=0.880 and -0.698, respectively), TBX21 (r=0.811 and -0.698) and perforin (r=0.781 and -0.698). To demonstrate functional relevance of the connection between BIRC5 and glucose metabolism, survivin was inhibited in CD4 cell cultures. Survivin inhibition resulted in significant increase of PFKFB3 (p=7x10-6) and LDHA (p=0.0089), leading to inhibition of phosphoglycerate mutase PGAM1 and ATF citrate lyase ACLY (p=0.021 and p=0.0074, respectively), which dignify the restoration of aerobic glycolysis. Importantly, inhibition of survivin decreased 2NBD-glucose uptake by CD4 cells (p=0.031) and reduced expression of GLUT1 (p=0.034). These changes in glucose metabolism were followed by decreased IFNg production in supernatants (p=0.037).Conclusion:The study demonstrates a strong connection between IFNg production and glucose metabolism in CD4 cells. Survivin emerges as an important regulator of glycolysis acting through expression of glycolytic enzymes and glucose transport.Disclosure of Interests:None declared.
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Zhu, Wei, Liang Ye, Jianzhao Zhang, Pengfei Yu, Hongbo Wang, Zuguang Ye, and Jingwei Tian. "PFK15, a Small Molecule Inhibitor of PFKFB3, Induces Cell Cycle Arrest, Apoptosis and Inhibits Invasion in Gastric Cancer." PLOS ONE 11, no. 9 (September 26, 2016): e0163768. http://dx.doi.org/10.1371/journal.pone.0163768.
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Zhang, Y., A. Nguyen, A. Rudin, and C. Maglio. "POS0425 METABOLIC ALTERATIONS IN ACTIVATED FIBROBLAST-LIKE SYNOVIOCYTES FROM NON-INFLAMED SUBJECTS - MIMICKING EARLY STAGE OF RHEUMATOID ARTHRITIS." Annals of the Rheumatic Diseases 81, Suppl 1 (May 23, 2022): 467.1–467. http://dx.doi.org/10.1136/annrheumdis-2022-eular.2806.
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BackgroundProliferative cells, such as inflamed cells, depend on altered metabolic pathways to support their active proliferation. Synovial samples from patients with rheumatoid arthritis (RA) show reprogramming of different metabolic pathways such as glucose and glutamine metabolism [1]. However, it is unknown which metabolic pathways are altered in the early phases of RA pathogenesis, when non-inflamed fibroblast-like synoviocytes (FLS) are activated to a pro-inflammatory state. Our group has created an in-vitro cell model using FLS from subjects without inflammatory arthritis (non-inflamed FLS) after activation with adiponectin and tumour necrosis factor (TNF) to mimic the early stage of RA [2].ObjectivesWe aim to determine if the stimulation of non-inflamed FLS upregulates the expression of key-enzymes involved in glucose and glutamine metabolism and how the inhibition of those enzymes affects FLS activation.MethodsFLS (passage 6-8) were isolated from synovial tissues of patients without inflammatory arthritis or osteoarthritis who underwent diagnostic arthroscopy due to a previous injury. FLS were cultured in DMEM medium (high glucose and GlutaMAX) containing 10% FBS and stimulated by known FLS-activators, i.e. TNF, interleukin 1 beta (IL-1 β), or adiponectin. For the inhibition experiments, cells were pre-treated with 25 mM 2-DG or 300 nM CB-839 for 4 hours before stimulation. Expressions of the enzymes were measured by western blot in whole-cell lysates and IL-6 was measured using ELISA in cell culture supernatants at 24 hours after stimulation. Cell proliferation was determined using MTT assay after 48 hours of stimulation.ResultsFirst, we compared the expression of hexokinase 2 (HK2), glutaminase C (GAC), and PFKFB3 in non-inflamed FLS before and after activation with adiponectin, TNF, and IL-1β. Expression of HK2 and GAC were upregulated in adiponectin- and TNF-activated FLS compared to unstimulated FLS. PFKFB3 was not affected by any of the stimuli. IL-1β did not affect the expression of the analysed enzymes (Figure 1A-C). As IL-1β did not affect the expression of metabolic enzymes, we continued stimulation only with adiponectin and TNF. TNF but not adiponectin significantly enhanced the proliferation of FLS without inhibition (Figure 1D). However, FLS proliferation was significantly reduced by pre-treatment with 2-DG, a glycolysis inhibitor, in unstimulated as well as TNF- and adiponectin-stimulated cells. CB-839, a glutaminase inhibitor, did not affect the proliferation of FLS (Figure 1D). Both TNF and adiponectin significantly upregulated the production of IL-6 in FLS. Pre-treatment with 2-DG significantly reduced the production of IL-6. CB-839 pre-treatment significantly reduced the production of IL-6 only in unstimulated FLS (Figure 1E).Figure 1.Metabolic alterations in non-inflamed FLS. Expression of hexokinase 2 (HK2; A), glutaminase C (GAC; B) and PFKFB3 (C) in non-inflamed FLS measured by western blot. Proliferation assay (MTT assay; D) and production of IL-6 (ELISA; E) was performed using cells treated with HK2 inhibitor 2-DG and glutaminase inhibitor CB-839. Statistical significance was determined as by one sample t test (A-C; n=7) or paired t test (D-E; n=4-6).ConclusionOur results show that the expression of key-enzymes regulating metabolic pathways can be enhanced by adiponectin and TNF in non-inflamed FLS. Moreover, we also show that inhibition of specific metabolic pathways can affect FLS activation differently depending on the cytokine stimulation. These results provide a deeper understanding of metabolic reprogramming in FLS in early RA.References[1]Bustamante, M.F., et al., Fibroblast-like synoviocyte metabolism in the pathogenesis of rheumatoid arthritis. Arthritis Res Ther, 2017. 19(1): p. 110.[2]Zhang, Y., et al., Recombinant Adiponectin Induces the Production of Pro-Inflammatory Chemokines and Cytokines in Circulating Mononuclear Cells and Fibroblast-Like Synoviocytes From Non-Inflamed Subjects. Front Immunol, 2020. 11: p. 569883.Disclosure of InterestsNone declared.
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Chang, Simone, Evan Meiman, and Sucheta Telang. "DDRE-34. TARGETING RESISTANCE IN MEDULLOBLASTOMA." Neuro-Oncology Advances 3, Supplement_1 (March 1, 2021): i14. http://dx.doi.org/10.1093/noajnl/vdab024.056.
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Abstract Medulloblastoma is the most commonly diagnosed pediatric brain tumor. Although therapeutic advances have improved survival from this cancer, they result in devastating sequelae and, additionally, have proven inadequate in metastatic disease and recurrence where survival remains <5%. Effective therapies are urgently needed to improve outcomes in medulloblastoma. Medulloblastoma development is driven by dysregulation of normal cerebellar proliferation. Mutations in the sonic hedgehog (Shh) pathway are found in ~30% of these tumors and responsible for their aggressive growth. The poor outcomes in Shh-driven medulloblastoma have prompted the evaluation of Shh-targeting agents in their treatment – with limited success likely attributable in part to the upregulation of alternate survival pathways (e.g. Ras/MAPK and HIF-1α). These alternate mechanisms stimulate glycolysis, in part by increasing the activity of the 6-phosphofructo-2-kinase/fructose-2,6 bisphosphatases (PFKFB1-4) to produce fructose-2,6-bisphosphate (F26BP), a potent activator of the rate-limiting glycolytic enzyme, 6-phosphofructo-1-kinase. In recent studies, we have determined that the PFKFB4 enzyme is highly expressed in patient-derived Shh medulloblastomas. We have found that hypoxia, through HIF-1α, strongly induced PFKFB4 expression in Shh-driven medulloblastoma cells and that silencing PFKFB4 suppressed F26BP, glycolysis and proliferation in normoxia and, more markedly, in hypoxia, indicating that PFKFB4 may be required for growth under hypoxia. We found that simultaneously silencing PFKFB4 and Shh pathway effectors significantly reduced cell survival and that co-targeting PFKFB4 (with a novel inhibitor) and Shh effectors synergistically decreased cell viability. In order to simulate Shh antagonist resistance, we have now subjected Shh medulloblastoma cells to prolonged Shh inhibitor exposure and found that these cells exhibit increased proliferation, glycolysis and PFKFB4. Studies are underway to delineate their metabolic alterations. Taken together, our data indicate that targeting PFKFB4 may be a valid therapeutic option in aggressive, treatment-resistant medulloblastoma and strongly support the further examination of PFKFB4 inhibitors in these tumors.
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Cao, Xiankun, Xin Wang, Kewei Rong, Kexin Liu, Xiao Yang, Tangjun Zhou, Pu Zhang, et al. "Specific PFKFB3 Inhibitor Memorably Ameliorates Intervertebral Disc Degeneration via Inhibiting NF-κB and MAPK Signaling Pathway and Reprogramming of Energy Metabolism of Nucleus Pulposus Cells." Oxidative Medicine and Cellular Longevity 2022 (September 21, 2022): 1–17. http://dx.doi.org/10.1155/2022/7548145.
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Intervertebral disc (IVD) degeneration (IVDD) is a characteristic of the dominating pathological processes of nucleus pulposus (NP) cell senescence, abnormal synthesis and irregular distribution of extracellular matrix (ECM), and tumor necrosis factor-α (TNF-α) induced inflammation. Nowadays, IVD acid environment variation which accelerates the pathological processes mentioned above arouses researchers’ attention. KAN0438757 (KAN) is an effective inhibitor of selective metabolic kinase phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3) that has both energy metabolism reprogramming and anti-inflammatory effects. Therefore, a potential therapeutic benefit of KAN lies in its ability to inhibit the development of IVDD. This study examined in vitro KAN toxicity in NP primary cells (NPPs). Moreover, KAN influenced tumor necrosis factor-α (TNF-α) induced ECM anabolism and catabolism; the inflammatory signaling pathway activation and the energy metabolism phenotype were also examined in NPPs. Furthermore, KAN’s therapeutic effect was investigated in vivo using the rat tail disc puncture model. Phenotypically speaking, the KAN treatment partially rescued the ECM degradation and glycolysis energy metabolism phenotypes of NPPs induced by TNF-α. In terms of mechanism, KAN inhibited the activation of MAPK and NF-κB inflammatory signaling pathways induced by TNF-α and reprogramed the energy metabolism. For the therapeutic aspect, the rat tail disc puncture model demonstrated that KAN has a significant ameliorated effect on the progression of IVDD. To sum up, our research successfully authenticated the potential therapeutic effect of KAN on IVDD and declaimed its mechanisms of both novel energy metabolism reprogramming and conventional anti-inflammation effect.
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Yan, Siyuan, Nan Zhou, Deru Zhang, Kaile Zhang, Wenao Zheng, Yonghua Bao, and Wancai Yang. "PFKFB3 Inhibition Attenuates Oxaliplatin-Induced Autophagy and Enhances Its Cytotoxicity in Colon Cancer Cells." International Journal of Molecular Sciences 20, no. 21 (October 30, 2019): 5415. http://dx.doi.org/10.3390/ijms20215415.
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6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3), a glycolytic enzyme highly expressed in cancer cells, has been reported to participate in regulating metabolism, angiogenesis, and autophagy. Although anti-cancer drug oxaliplatin (Oxa) effectively inhibits cell proliferation and induces apoptosis, the growing resistance and side-effects make it urgent to improve the therapeutic strategy of Oxa. Although Oxa induces the autophagy process, the role of PFKFB3 in this process remains unknown. In addition, whether PFKFB3 affects the cytotoxicity of Oxa has not been investigated. Here, we show that Oxa-inhibited cell proliferation and migration concomitant with the induction of apoptosis and autophagy in SW480 cells. Both inhibition of autophagy by small molecule inhibitors and siRNA modification decreased the cell viability loss and apoptosis induced by Oxa. Utilizing quantitative PCR and immunoblotting, we observed that Oxa increased PFKFB3 expression in a time- and dose-dependent manner. Meanwhile, suppression of PFKFB3 attenuated both the basal and Oxa-induced autophagy, by monitoring the autophagic flux and phosphorylated-Ulk1, which play essential roles in autophagy initiation. Moreover, PFKFB3 inhibition further inhibited the cell proliferation/migration, and cell viability decreased by Oxa. Collectively, the presented data demonstrated that PFKFB3 inhibition attenuated Oxa-induced autophagy and enhanced its cytotoxicity in colorectal cancer cells.
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Ährlund-Richter, Lars, Katarina Färnegårdh, Elisee Wiita, Mattias Jönsson, Carina Norström, Jessica Martinsson, Kenth Hallberg, Rune Ringom, and Hakan Mellstedt. "Metabolic intervention targeting 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) using a structure-based design." Journal of Clinical Oncology 30, no. 15_suppl (May 20, 2012): e13518-e13518. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.e13518.
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e13518 Background: By producing fructose-2,6-bisphosphate, PFKFB3 functions as an activator of anaerobic glycolysis. PFKFB3 is both over expressed and over activated in many of the types of human cancer. Specific inhibition of the PFKFB3 enzyme results in a reduction in metabolism and cell growth in oxygen-deficient cancer environments. Methods: High-throughput screening. Medicinal Chemistry. Structure-Based Drug Design, X-ray Crystallography. NMR. Isothermal Calorimetry. Dynamic Light Scatttering. ADME. Results: A high-throughput screening of 50.000 selected compounds, by means of a biochemical assay, generated 105 hits including both ATP-and non-ATP competitive hits as identified by NMR binding experiments. The latter type was prioritized and two hits with a similar “ring-linker-ring structure” were selected for further expansions. Interestingly, although structurally similar, the two hits were found by means of X-ray crystallography to exhibit different binding modes within the fructose pocket. Based on their respective binding mode, two chemical series were developed displaying different ADME properties and PFKFB isoenzyme selectivity. Calorimetry verified a reversible strong enthalpy driven, direct binding for both chemical series. A third chemical series was developed towards yet another unoccupied binding pocket within the fructose-site, yielding a 5-fold increase in potency. Strong interactions within the new pocket were confirmed using X-ray crystallography. Our PFKFB3 inhibitors were shown to reduce tumor cell growth in vitro and to exhibit combinatory effects with Cisplatin. Conclusions: We have targeted the fructose-binding pocket of PFKFB3, developed compounds with nM binding potency and have gained a detailed understanding of SAR via structural information. The structure-based analysis has provided a good understanding of the molecular interactions, which is important for further biological/clinical positioning: e.g., combination with chemotherapy, optimization of PK properties and proof of principle in vivo.
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Lyu, Zhong-Shi, Wei-Li Yao, Qi Wen, Hong-Yan Zhao, Fei-Fei Tang, Yu Wang, Lan-Ping Xu, et al. "Glycolysis Restoration Attenuates Damaged Bone Marrow Endothelial Cells." Blood 134, Supplement_1 (November 13, 2019): 2491. http://dx.doi.org/10.1182/blood-2019-122794.
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Background: Bone marrow(BM) endothelial cells(ECs), a key component of BM microenvironment, is essential for the physiology and regeneration of hematopoietic stem cells (HSCs). The damage of ECs is recognized by us and other researchers as a mainstay in the pathophysiology of a serious of life-threatening complications after chemoradiotherapy and myeloablative hematopoietic cell transplantation(HSCT), including poor graft function (PGF) (2013BBMT, 2015BMT, 2016Blood, 2019Blood Advances). Despite numerous researches focused on the BM ECs contributing to HSC regeneration following myelotoxicity, the mechanisms underlying the injured BM ECs itself remain to be elucidated. Under physiological conditions, energy metabolism plays an instrumental role in maintaining EC function, and markedly perturbed of EC metabolism is linked to many pathologies, like cancer and diabetes. However, little is known about the metabolism state and its role in impaired BM ECs. Aims: The current study was performed to investigate the metabolism status in BM ECs after chemotherapy-induced injury. Moreover, we evaluated the metabolic state and its role in BM ECs of PGF patients post-allotransplant. Finally, we evaluated the therapeutical potential of anti-metabolic drugs to the dysfunctional BM ECs derived from PGF patients. Methods: Two EC injury models in vitro were established with the cultivated human BM ECs treated by 5-Fluorouracil (5-FU) and hydrogen peroxide. The findings from the above models were further validated by a prospective case-control study enrolled 15 patients with PGF, 30 matched patients with good graft function (GGF) and 15 healthy donors (HD). To determine the metabolic status of BM ECs, the expression of metabolism regulating genes was analyzed by qRT-PCR (mRNA level) and flow cytometry (protein level). Glucose metabolism levels were measured by glucose consumption and lactate production assays. To evaluate the functions of BM ECs, apoptosis, migration and tube formation assays were performed. To investigate the effect of anti-metabolic drugs to injured BM ECs, the glycolysis inhibitor 3PO and PPARd agonist GW501516 were administrated to the cultivated BM ECs treated by 5-FU , hydrogen peroxide or derived from PGF. Results: We demonstrated that the glycolysis in BM ECs could be induced by the treatment with either 5-FU or hydrogen peroxide in vitro, consistent with the dysfunction(impaired migration, angiogenesis, and higher level of apoptosis) of BM ECs, which could be attenuated by glycolysis restoration. Mechanistically, we revealed that the aberrant glycolysis and dysfunction of BM ECs could be triggered by PPARd knockdown in vitro, while the PPARd were down-regulated by either 5-FU or hydrogen peroxide treatment in vitro, Furthermore, PPARd agonist GW501516 treatment attenuated the perturbed function and number of injured BM ECs treated by either 5-FU or hydrogen peroxide. Subsequently, the prospective case-control study demonstrated elevated expressions of the glycolytic activator PFKFB3 and decreased PPARd were observed in BM ECs of PGF patients, when compared with those of GGF patients and HD, indicating that BM ECs of PGF patients have a hyper-glycolytic metabolism. Moreover, either glycolysis (PFKFB3) inhibitor 3PO or PPARd agonist GW501516 treatment reduced the aberrant glycolysis and improved the number and function of BM ECs derived from patients with PGF in vitro, revealing the critical role of defective glycolysis in the impaired BM ECs of PGF. Summary / Conclusions: These findings reveal that hyper-glycolysis mediated by PPARd inhibition is involved in the dysfunction of BM ECs after injury. Defective glycolysis may contribute to the pathobiology of BM ECs of PGF patients, which could be attenuated by glycolysis inhibitor 3PO or PPARd agonist GW501516 in vitro. Our findings might merit further consideration of targeting BM ECs glycolysis or PPARd as a promising therapeutic approach for PGF patients post-allotransplant in the future. Disclosures No relevant conflicts of interest to declare.
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Wen, Qi, Song Yang, Zhong-Shi Lyu, Wei-Li Yao, Yu-Hong Chen, Ting-Ting Han, Yu Wang, et al. "Regulation of the Elevated T Cell Glycolysis May Alleviate Acute Graft-Versus-Host Disease Post-Allotransplant." Blood 134, Supplement_1 (November 13, 2019): 600. http://dx.doi.org/10.1182/blood-2019-122795.
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Background: Acute graft-versus-host disease(aGVHD) remains a major complication following allogeneic hematopoietic stem cell transplantation(allo-HSCT). The pathogenesis of aGVHD is commonly considered to be caused by exaggerated and undesirable immune responses. Metabolism not only provide energy and substrates for T cell growth and survival, but also instruct effector functions, differentiation, and gene expression of T cells. In this regard, the metabolic profile of T cells was reported to play a critical role in the occurrence and development of many immunological disorders such as systemic lupus erythematosus and rheumatoid arthritis. Murine studies found that alloreactive T cells use aerobic glycolysis as the predominant metabolic process to meet activation and proliferation demand after allo-HSCT. However, the metabolic profile of T cells and the approach for regulating T cell metabolism in aGVHD patients remains to be elucidated. Aims: To determine the metabolic state in T cells of patients with aGVHD. Moreover, to investigate the effect of the novel approach targeting the abnormal metabolism in T cells of aGVHD patients, which may provide a potential therapeutic target for aGVHD patients after allo-HSCT. Methods: In this prospective case-control study, a total of 25 patients with aGVHD and 25 matched patients without aGVHD(non-aGVHD) after allo-HSCT were enrolled. T cell subsets were analyzed in aGVHD and non-aGVHD patients by flow cytometry. Th1, Th2, Th17, and Treg cells were identified as CD4+IFN-γ+, CD4+IL-4+, CD4+IL17A+, and CD4+CD25+Foxp3+, respectively. Tc1 and Tc2 cells were identified as CD8+IFN-γ+ and CD8+IL-4+, respectively. In order to determine the metabolic state in T cells of patients with aGVHD and non-aGVHD, the metabolic profile was determined using a Seahorse XF96 Analyzer. The glucose consumption and lactate production rates were detected by glucose assay kit and lactate assay kit. The mitochondrial mass, the mitochondrial membrane potential, the protein expressions for the lipid metabolism enzyme CTP1a and the glycolytic activator PFKFB3 were measured by flow cytometry. To further understand the metabolic state of T cells in aGVHD and non-aGVHD patients and investigate its mechanism, RNA sequencing (RNA-Seq) was performed to analyze the gene expression profiles of T cells. Subsequently, to explore the potential way of targeting the abnormal metabolism in T cells, the glycolysis inhibitor 3-PO was administrated to T cells from aGVHD patients. Results: When compared with T cells in non-aGVHD patients, T cells in aGVHD patients were polarized towards pro-inflammatory T cells, characterized by an elevated proportion of Tc1, Th1 and Th17. Furthermore, T cells isolated from aGVHD patients exhibited higher extracellular acidification rate, as well as the increased glucose consumption rate and lactate production rate compared to those in non-aGVHD patients. Moreover, elevated expression of PFKFB3 was observed in T cells, especially in naïve T cells of aGVHD patients, but oxygen consumption rate, CPT1A, mitochondrial mass or membrane potential showed no significant differences in T cells between aGVHD and non-aGVHD patients. These results implied higher glycolytic activity of T cells in aGVHD patients when compared with those in non-aGVHD patients. Consistent with the increased glycolytic activity observed in T cells from aGVHD patients, the mRNA levels of genes involved in the glycolytic pathway were substantially elevated in T cells of aGVHD patients compared to those in non-aGVHD patients. Importantly, in vitro treatment with glycolysis inhibitor 3-PO improved the activity of T cells derived from aGVHD patients through down-regulating glycolytic activity of T cells. Summary/Conclusion: The current study demonstrated that T cells in aGVHD patients preferentially depend on glycolysis to meet activation and proliferation demands. Furthermore, the activity of T cells from aGVHD patients could be ameliorated by glycolysis inhibitor 3-PO in vitro. Although further validation is required, T cell glycolysis promises to be a novel therapeutic target for aGVHD patients after allo-HSCT. Disclosures No relevant conflicts of interest to declare.
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Ikeda, Sho, Fumito Abe, Matsuda Yuka, Akihiro Kitadate, Takahiro Kobayashi, Naoto Takahashi, and Hiroyuki Tagawa. "Hexokinase-2 Regulates Hypoxia-Inducible Autophagy, Leading to Enhance Anti-Apoptotic Capability of Refractory Multiple Myeloma." Blood 134, Supplement_1 (November 13, 2019): 1787. http://dx.doi.org/10.1182/blood-2019-123360.
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(background) The drug resistance of multiple myeloma (MM) cells is thought to be induced by various factors of the bone marrow microenvironment. Of these factors, hypoxic stress may be associated with drug resistance in various hematologic malignancies, including MM. Hypoxic stress lead MM cells to induce distinct gene expressions. It has been reported that oncogenic transcription factors such as IRF4 and Myc are suppressed under hypoxia. Instead, accumulation of another transcription factor, HIF-1α upregulates anti-apoptotic proteins, increases glycolysis, and enhances neovascularization leading MM cells to represent anti-apoptotic phenotype. Autophagy is an intracellular process that encapsulates cytoplasmic components, which are directed to the lysosome for degradation. Autophagy and proteasomal degradation prevent apoptosis caused by endoplasmic reticulum (ER) stress. Although proteasome inhibitor such as bortezomib, is a key drug for MM, it may induce treatment resistance. This might be because autophagy is induced in hypoxic microenvironment. Autophagy associated molecules might be therapeutic target in MM cells adapted to hypoxia. (Aim and methods) To clarify the association of hypoxia inducible genes and autophagy, and to obtain rational basis for a new therapeutic strategy against MM, we performed following experiments in vitro using myeloma cell lines (MM.1S, KMS-12-PE, KMS-11, and H929) and primary samples (n=6) that were subjected to hypoxia (1% O2). (Results) First, we examined volcano plot analysis on our cDNA microarray data (GSE80545) of patient samples incubated in normoxia or hypoxia for 48 hours. 546 probes were significantly elevated in hypoxia (fold change > 2.0, p < 0.05). Gene ontology analysis revealed that "Glycolytic Process" contained 13 genes such as PFKFB4, ENO2, ALDOC, PFKFB3, HK2, PFKP, GPI, PGK1, LDHA, ALDOA, ENO1, PKM, and GAPDH. We focused on hexokinase-2 (HK2) because it has been reported that HK2 activates autophagy under stress conditions. Western blot analysis for patient samples revealed that HK2 expression was remarkably upregulated under hypoxia. Apoptosis assay showed that viable cells of HK2 knockdowned cell lines were significantly lower than that of control cells under hypoxia, but not under normoxia. Also, in hypoxia, we found that number of 3-bromopyruvate (3-BrPA, a HK2 inhibitor) subjected viable cells were significantly lower than that of normoxia. This suggested that HK2 contributes to anti-apoptotic phenotype of MM cells under hypoxia. Next, we examined the role of HK2 in autophagy under hypoxia. Because degradation of p62 and increase of LC3-II/LC3-I ratio is considered to be useful for autophagy detection, we examined these factors by Western blot analysis. We found that hypoxic stress decreased expression of p62 and increased the ratio of LC3-II/LC3-I in myeloma cell lines, indicating that hypoxia activates autophagy. However, under hypoxia, these changes were canceled by HK2 knockdown. We confirmed that the number of autophagosome were significantly decreased in HK2-knockdowned myeloma cells by electron microscopy analysis. These data suggested that HK2 is required for hypoxia-inducible autophagy in MM. Finally, we examined the effect of combined inhibition of HK2 and proteasome. In hypoxia, apoptosis by bortezomib was significantly increased in HK2-knockdowned myeloma cells when compared with control. Moreover, we found that the combination of 3-BrPA and bortezomib increased apoptotic cells in both normoxia and hypoxia. These results suggested that HK2-inhibition can induce apoptosis against MM cells with enhancement of sensitivity to proteasome inhibitors. (Conclusion) These results suggest that hypoxia induced HK2 promotes autophagy and inhibits apoptosis. Thus, the combination of proteasome inhibitors and HK2 inhibition may bring about a deep response against treatment resistant MM. Disclosures Ikeda: Nippon Shinyaku Research Grant: Research Funding. Takahashi:Bristol-Myers Squibb: Speakers Bureau; Eisai Pharmaceuticals: Research Funding; Pfizer: Research Funding, Speakers Bureau; Otsuka Pharmaceutical: Research Funding, Speakers Bureau; Kyowa Hakko Kirin: Research Funding; Chug Pharmaceuticals: Research Funding; Ono Pharmaceutical: Research Funding; Novartis Pharmaceuticals: Research Funding, Speakers Bureau; Astellas Pharma: Research Funding; Asahi Kasei Pharma: Research Funding.
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Petrasca, Andreea, James J. Phelan, Sharon Ansboro, Douglas J. Veale, Ursula Fearon, and Jean M. Fletcher. "Targeting bioenergetics prevents CD4 T cell–mediated activation of synovial fibroblasts in rheumatoid arthritis." Rheumatology 59, no. 10 (February 11, 2020): 2816–28. http://dx.doi.org/10.1093/rheumatology/kez682.
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Abstract Objectives We investigated the reciprocal relationship linking fibroblast-like synoviocytes (FLS) and T lymphocytes in the inflamed RA synovium and subsequently targeted cellular metabolic pathways in FLS to identify key molecular players in joint inflammation. Methods RA FLS were cultured with CD4 T cells or T cell conditioned medium (CD4CM); proliferation, expression of adhesion molecules and intracellular cytokines were examined by flow cytometry. FLS invasiveness and secreted cytokines were measured by transwell matrigel invasion chambers and ELISA, while metabolic profiles were determined by extracellular Seahorse flux analysis. Gene expression was quantified by real-time quantitative RT-PCR. Results Our results showed mutual activation between CD4 T cells and FLS, which resulted in increased proliferation and expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 by both CD4 T cells and FLS. Furthermore, interaction between CD4 T cells and FLS resulted in an increased frequency of TNF-α+, IFN-γ+ and IL-17A+ CD4 T cells and augmented TNF-α, IFN-γ, IL-17A, IL-6, IL-8 and VEGF secretion. Moreover, CD4CM promoted invasiveness and boosted glycolysis in FLS while downregulating oxidative phosphorylation, effects paralleled by increased glucose transporters GLUT1 and GLUT3; key glycolytic enzymes GSK3A, HK2, LDHA and PFKFB3; angiogenic factor VEGF and MMP-3 and MMP-9. Importantly, these effects were reversed by the glycolytic inhibitor 2-DG and AMP analogue 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Conclusion This study demonstrates that CD4 T cells elicit an aggressive phenotype in FLS, which subsequently upregulate glycolysis to meet the increased metabolic demand. Accordingly, 2-DG and AICAR prevent this activation, suggesting that glycolytic manipulation could have clinical implications for RA treatment.
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Lu, Yan, Lei Zhang, Ran Zhu, Huijuan Zhou, Huaying Fan, and Qiang Wang. "PFKFB3, a key glucose metabolic enzyme regulated by pathogen recognition receptor TLR4 in liver cells." Therapeutic Advances in Endocrinology and Metabolism 11 (January 2020): 204201882092347. http://dx.doi.org/10.1177/2042018820923474.
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Aims: Toll-like receptor 4 (TLR4) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB3) are involved in the progress of inflammation and glucose metabolism. Here, we aimed to assess the relationship between TLR4 and PFKFB3 in liver cells. Methods: We detected the expression of TLR4 and PFKFB3 in both normal liver cell lines and liver cancer cell lines. Then, a small interfering RNA (siRNA) was used to knock down the expression of TLR4 and analyze the expression of PFKFB3 in the HL-7702 cell line. Further, following stimulation of the HL-7702 cell line with free fatty acids (FFA) or insulin, we observed the expression of TLR4 and PFKFB3, respectively. Results: Knocking down siRNA-mediated TLR4 significantly reduced PFKFB3 expression at the mRNA and protein level. Furthermore, activating TLR4 with FFA dramatically increased PFKFB3 expression. Insulin increased the expression of TLR4 and PFKFB3, which could be inhibited by TLR siRNA. Conclusion: These findings suggest that PFKFB3 expression is regulated via the TLR4–PFKFB3 axis, which might be a bridge linking fat and glucose metabolism.
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Khodova, Kristina, Mikhail Pyatnitskiy, Tatiana Eremeeva, and Peter Fedichev. "Application of bioinformatics approach to identify indications for PFKFB3-mediated glycolysis inhibition as potential anticancer treatment." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): e23158-e23158. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.e23158.
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e23158 Background: Glycolysis is one of the mechanisms which enables cancer cells to adapt to intermittent oxygen deprivation and obtain the best available metabolic strategy in the lack of resources. Therefore, inhibition of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) enzyme, one of the glycolysis pathway regulators, may result in decreased tumor metabolism, as well as suppressed cell growth. Additionally it has been shown that PFKFB3 is involved in angiogenesis regulation, which contributes to tumor growth, and in immune cells activation, which mediates anti-tumor immune response. Methods: We performed the analysis of available data on the role of PFKFB3 gene expression in various cancer types, i.e. patients’ stratification to identify tumors in which PFKFB3 inhibition improved survival. We exploited TCGA expression data and applied cluster analysis and Cox-regression analysis. The objective was to detect the relation between the increased level of PFKFB3 gene expression and patients’ survival. We also compared the clusters we identified with TCGA metadata to define whether any of them fall down into well-established tumor subtypes. Results: As a result we selected four clusters among solid tumor types, in which increased expression of the target was related to significant decrease in overall survival rate. These include breast cancer, renal cell carcinoma, cervical carcinoma, and leiomyosarcoma. We managed to cross-validate the obtained results on independent datasets. The results show potential indications for PFKFB3 inhibitors. Using our proprietary molecular modelling tool, we designed a series of novel small molecule PFKFB3 inhibitors to target the fructose binding pocket rather than occupying the ATP-pocket which leads to the highly selective inhibition and strong reduction of potential off-target binding. We proved target coverage by performing in vitro biochemical assays. Conclusions: Our current data provide strong rationale for further preclinical and clinical development of PFKFB3 inhibitors as novel anticancer treatment with the potential to overcome resistance to chemo- or radiotherapy.
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Gao, Liansheng, Chun Wang, Bing Qin, Tao Li, Weilin Xu, Cameron Lenahan, Guangyu Ying, et al. "6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase Suppresses Neuronal Apoptosis by Increasing Glycolysis and “cyclin-dependent kinase 1-Mediated Phosphorylation of p27 After Traumatic Spinal Cord Injury in Rats." Cell Transplantation 29 (January 1, 2020): 096368972095022. http://dx.doi.org/10.1177/0963689720950226.
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Apoptosis is a vital pathological factor that accounts for the poor prognosis of traumatic spinal cord injury (t-SCI). The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) is a critical regulator for energy metabolism and proven to have antiapoptotic effects. This study aimed to investigate the neuroprotective role of PFKFB3 in t-SCI. A compressive clip was introduced to establish the t-SCI model. Herein, we identified that PFKFB3 was extensively distributed in neurons, and PFKFB3 levels significantly increased and peaked 24 h after t-SCI. Additionally, knockdown of PFKFB3 inhibited glycolysis, accompanied by aggravated neuronal apoptosis and white matter injury, while pharmacological activation of PFKFB3 with meclizine significantly enhanced glycolysis, attenuated t-SCI-induced spinal cord injury, and alleviated neurological impairment. The PFKFB3 agonist, meclizine, activated cyclin-dependent kinase 1 (CDK1) and promoted the phosphorylation of p27, ultimately suppressing neuronal apoptosis. However, the neuroprotective effects of meclizine against t-SCI were abolished by the CDK1 antagonist, RO3306. In summary, our data demonstrated that PFKFB3 contributes robust neuroprotection against t-SCI by enhancing glycolysis and modulating CDK1-related antiapoptotic signals. Moreover, targeting PFKFB3 may be a novel and promising therapeutic strategy for t-SCI.
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Lypova, Nadiia, Susan M. Dougherty, Lilibeth Lanceta, Jason Chesney, and Yoannis Imbert-Fernandez. "PFKFB3 Inhibition Impairs Erlotinib-Induced Autophagy in NSCLCs." Cells 10, no. 7 (July 3, 2021): 1679. http://dx.doi.org/10.3390/cells10071679.
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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.
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St-Gallay, Stephen A., Neil Bennett, Susan E. Critchlow, Nicola Curtis, Gareth Davies, Judit Debreczeni, Nicola Evans, et al. "A High-Throughput Screening Triage Workflow to Authenticate a Novel Series of PFKFB3 Inhibitors." SLAS DISCOVERY: Advancing the Science of Drug Discovery 23, no. 1 (September 25, 2017): 11–22. http://dx.doi.org/10.1177/2472555217732289.
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A high-throughput screen (HTS) of human 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) resulted in several series of compounds with the potential for further optimization. Informatics was used to identify active chemotypes with lead-like profiles and remove compounds that commonly occurred as actives in other HTS screens. The activities were confirmed with IC50 measurements from two orthogonal assay technologies, and further analysis of the Hill slopes and comparison of the ratio of IC50 values at 10 times the enzyme concentration were used to identify artifact compounds. Several series of compounds were rejected as they had both high slopes and poor ratios. A small number of compounds representing the different leading series were assessed using isothermal titration calorimetry, and the X-ray crystal structure of the complex with PFKFB3 was solved. The orthogonal assay technology and isothermal calorimetry were demonstrated to be unreliable in identifying false-positive compounds in this case. Presented here is the discovery of the dihydropyrrolopyrimidinone series of compounds as active and novel inhibitors of PFKFB3, shown by X-ray crystallography to bind to the adenosine triphosphate site. The crystal structures of this series also reveal it is possible to flip the binding mode of the compounds, and the alternative orientation can be driven by a sigma-hole interaction between an aromatic chlorine atom and a backbone carbonyl oxygen. These novel inhibitors will enable studies to explore the role of PFKFB3 in driving the glycolytic phenotype of tumors.
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Novellasdemunt, Laura, Laurent Bultot, Anna Manzano, Francesc Ventura, Jose Luis Rosa, Didier Vertommen, Mark H. Rider, Àurea Navarro-Sabate, and Ramon Bartrons. "PFKFB3 activation in cancer cells by the p38/MK2 pathway in response to stress stimuli." Biochemical Journal 452, no. 3 (May 31, 2013): 531–43. http://dx.doi.org/10.1042/bj20121886.
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PFK-2/FBPase-2 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) catalyses the synthesis and degradation of Fru-2,6-P2 (fructose 2,6-bisphosphate), a key modulator of glycolysis and gluconeogenesis. The PFKFB3 gene is involved in cell proliferation owing to its role in carbohydrate metabolism. In the present study we analysed the mechanism of regulation of PFKFB3 as an immediate early gene controlled by stress stimuli that activates the p38/MK2 [MAPK (mitogen-activated protein kinase)-activated protein kinase 2] pathway. We report that exposure of HeLa and T98G cells to different stress stimuli (NaCl, H2O2, UV radiation and anisomycin) leads to a rapid increase (15–30 min) in PFKFB3 mRNA levels. The use of specific inhibitors in combination with MK2-deficient cells implicate control by the protein kinase MK2. Transient transfection of HeLa cells with deleted gene promoter constructs allowed us to identify an SRE (serum-response element) to which SRF (serum-response factor) binds and thus transactivates PFKFB3 gene transcription. Direct binding of phospho-SRF to the SRE sequence (−918 nt) was confirmed by ChIP (chromatin immunoprecipiation) assays. Moreover, PFKFB3 isoenzyme phosphorylation at Ser461 by MK2 increases PFK-2 activity. Taken together, the results of the present study suggest a multimodal mechanism of stress stimuli affecting PFKFB3 transcriptional regulation and kinase activation by protein phosphorylation, resulting in an increase in Fru-2,6-P2 concentration and stimulation of glycolysis in cancer cells.
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Lane, Daniel Scot, Priyanka Talukdar, Beth Fallert Junecko, and Joshua T. Mattila. "Inhibiting glycolysis by targeting the enzyme PFKFB3 restricts macrophage anti-mycobacterial activity and neutrophil phagocytosis of Mycobacterium tuberculosis." Journal of Immunology 208, no. 1_Supplement (May 1, 2022): 110.03. http://dx.doi.org/10.4049/jimmunol.208.supp.110.03.
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Abstract Mycobacterium tuberculosis (Mtb) causes tuberculosis (TB), a leading cause of infectious disease-related mortality around the world. Myeloid cells are important in TB as Mtb host cells and anti-Mtb effector cells but the factors that differentiate these roles are not clearly defined. Immunometabolism is a driver of immune function and may be an important determinant of this interaction and represents a potential target for host-directed therapies against TB. To better understand how immunometabolism relates to TB, we incubated macrophages and neutrophils from Mtb-infected macaques with a panel of metabolic inhibitors and measured how this affected cellular responses to Mtb infection. Specifically, we targeted glycolysis, oxidative phosphorylation, and fatty acid oxidation and measured how this affected macrophage and neutrophil phagocytosis, macrophage control over Mtb viability and replication, and neutrophil production of extracellular traps. We found that targeting glycolysis by inhibiting PFKFB3, a rate-limiting enzyme in glycolysis, reduced macrophage anti-Mtb activity and neutrophil phagocytosis, while inhibition with the glucose analog 2-DG did not. In contrast, incubating cell-free mycobacteria with PFKFB3 inhibitors severely inhibited their growth, suggesting that these bacteria use different metabolic pathways in cells and culture to survive. Inhibiting oxidative phosphorylation with metformin or fatty acid oxidation with etomoxir did not change how macrophages or neutrophils responded to Mtb or affect mycobacterial growth in culture. These results indicate a role for immunometabolism in myeloid responses to Mtb and may inform efforts for development of metabolism-targeted therapies for TB. Supported by grants from NIH (R01AI134183)
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Grewal, Jaspreet Singh, Numan Al-Rayyan, Jamaal Ritchie, Paxton Schowe, Cam Falkner, Sucheta Telang, Kavitha Yaddanapudi, and Jason Alan Chesney. "Targeting the glucose metabolism of monocytic myeloid-derived suppressor cells to stimulate cancer immunity." Journal of Clinical Oncology 35, no. 7_suppl (March 1, 2017): 126. http://dx.doi.org/10.1200/jco.2017.35.7_suppl.126.
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126 Background: Myeloid derived suppressor cells (MDSCs) inhibit the expansion of tumor antigen-specific effector CD8+ T cells via different mechanisms including increased expression of arginase, transforming growth factor – β (TGF – β) and indoleamine 2,3-dioxygenase (IDO). Recently, MDSCs were found to over-express hypoxia inducible factor 1 alpha (HIF-1α) which is required for their differentiation. An essential transcriptional target of HIF-1α is 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) which synthesizes fructose 2,6-bisphosphate, an allosteric stimulator of glycolysis and of proliferation via stimulation of cyclin dependent kinase-1 (CDK1). We hypothesized that MDSCs might over-express PFKFB3 which in turn might be required for their function as T cell suppressors. Methods: We used monocytic MDSCs (M-MDSCs) induced by co-culture with A375 melanoma cells, M-MDSCs from metastatic melanoma patients, murine bone marrow MDSCs and splenic M-MDSCs from B16 F10 tumor bearing mice for our studies. T cell suppression assays were performed to analyze M-MDSC suppression and reversal following PFKFB3 blockade. Results: We found that M-MDSCs have increased PFKFB3 expression. We also found that PFK-158 administration in B16 (wild-type) melanoma-bearing mice results in a marked reduction in MDSCs and a simultaneous increase in CD8+ T cell infiltration in the tumors. We analyzed three advanced cancer patients for circulating MDSCs before and after PFK-158 administration as part of a multi-center phase 1 clinical trial. And, we found that the MDSCs were markedly reduced in each patient. In addition, we have generated data for MDSC suppressive activity following in vitro treatment with PFK-158 showing reversal of suppressive activity. Conclusions: Taken together, these data indicate that selective inhibition of PFKFB3 may be a novel approach to target MDSCs and combinations of PFKFB3 inhibitors with immunotherapies may be a rational strategy to promote durable immune-mediated remissions in cancer patients.
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Erlandsson, M., E. Malmhäll-Bah, K. M. Andersson, S. Töyrä Silfverswärd, R. Pullerits, and M. I. Bokarewa. "POS0702 ANTI-RHEUMATIC TREATMENT ALTERS PFKFB3 EXPRESSION, KEY OF GLYCOLYSIS, IN CD14+ MONOCYTES OF RA PATIENTS, WHICH CONTRIBUTES TO DISSIMILARITIES OF THE IFNg-SIGNATURE." Annals of the Rheumatic Diseases 81, Suppl 1 (May 23, 2022): 631.1–631. http://dx.doi.org/10.1136/annrheumdis-2022-eular.4562.
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BackgroundInterferon-gamma (IFNαnterferon-gamma (IFNRheumatology clinic, Gothenburg, Swedenh, Göteborg, Swedenmonocytes of RA patients, which contributes to dissimilarities of the IFNg-signature Peter C. Taylor Consultant of: AbbVie, Biogen, Eli Lilly and Company, Fresenius, Galapagos, mulation is energy consuming and often demands metabolic adaptation of a cell by switching glucose metabolism from the tricarboxylic acid cycle (TCA) to the pentose phosphate pathway (PPP) alternative of glycolysis that dramatically increases glucose intake. Fructose-2,6-biphosphatase 3 (coded by PFKFB3) has been identified as the rate-limiting regulator of glucose metabolism suppressed in leukocytes of RA patients (2).ObjectivesTo study effect of anti-rheumatic treatment on the cellular energy metabolism of CD14+ mononuclear cells and its connection to the phenotype and IFNαmononuclear.MethodsCD14+ cells were sorted from the peripheral blood of the randomly recruited 60 RA patients (mean age 59.6y, DD 13.8y). The cells were LPS activated for 2h and submitted to RNA sequencing (RNAseq, IluminaNextseq). The patients were divided by actual DMARD treatment into those who had no DMARDs (n=8), methotrexate (MTX) only (n=15), biologics (MTX+aTNF n=12, MTX+RTX n=4) and JAK-inhibitors (JAKi, n=24). Differentially expressed gene (DEG) analysis between the groups was performed in R-studio, Bioconductor package, DESeq2. Reported IFN signatures (1) were combined in a set of 51 IFN signature genes (ISG) and analyzed in relation to the transcriptomic profile behind the cellular energy metabolism and DMARD treatment.ResultsMTX and JAKi but not biologics make a significant and opposing contribution of the transcriptomic of energy metabolism in CD14+ cells. MTX-treated patients had significantly higher levels of the rate-limiting enzyme of glycolysis PFKFB3 and PGAM1 compared to those with no DMARDS, which normalized aerobic glycolysis by increasing expression of the pyruvate dehydrogenase complex proteins PDHA1, PHDX and PDK3 linking glycolysis with TCA and decreasing PPP enzymes PGLS, RPIA and TKT. In contrast, PFKFB3 was suppressed in patients treated with JAK-inhibitors compared to those treated with MTX (cor.p=1.32e-8), which significantly activated glycolysis downstream of PFKFB3 and shunting metabolism to PPP inducing expression of G6PD (cor.p=5.0e-92) and PGLS (cor.p=3.1e-46) and increased the major glucose transporter SLC2A1 (cor.p=1.11e-24).These differences in glucose metabolism were linked to divergent phenotype of CD14+ cells being short-lived CD14intCD11chi cells and IL6 producing in MTX-treated patients and long-lived mature CD14hiCD11bhiCX3CR1hi cells and IFNαcells and IFN producing in MTX-treated pa+ cells of JAKi-treated patients expressed low levels of STAT1 and ISGs compared to MTX-treated patients.ConclusionDMARD treatment har divergent effect on glycolysis of CD14+ cells, acting through PFKFB3. This has significant impact on the phenotype of CD14+ cells and inflammatory ability.References[1]Lamot L. Et.al. Clin Exp Rheumatol 2019, 37, 1077.[2]Yang Z. et al, Science Translational Medicine 2016, 8, 331, 331ra38.Disclosure of InterestsNone declared
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Bobarykina, Anastasiya Y., Dmytro O. Minchenko, Iryna L. Opentanova, Michel Moenner, Jaime Caro, Hiroyasu Esumi, and Oleksandr H. Minchenko. "Hypoxic regulation of PFKFB-3 and PFKFB-4 gene expression in gastric and pancreatic cancer cell lines and expression of PFKFB genes in gastric cancers." Acta Biochimica Polonica 53, no. 4 (December 4, 2006): 789–99. http://dx.doi.org/10.18388/abp.2006_3308.
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Previously we have shown that hypoxia strongly induces the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 and -4 (PFKFB-3 and PFKFB-4) genes in several cancer cell lines via a HIF-dependent mechanism. In this paper we studied the expression and hypoxic regulation of PFKFB-4 and PFKFB-3 mRNA as well as its correlation with HIF-1alpha, HIF-2alpha, VEGF and Glut1 mRNA expression in the pancreatic cancer cell line Panc1 and two gastric cancer cell lines MKN45 and NUGC3. This study clearly demonstrated that PFKFB-3 and PFKFB-4 mRNA are expresses in MKN45, NUGC3 and Panc1 cancers cells and that both genes are responsive to hypoxia in vitro. However, their basal level of expression and hypoxia responsiveness vary in the different cells studied. Particularly, PFKFB-3 mRNA is highly expressed in MKN45 and NUGC3 cancer cells, with the highest response to hypoxia in the NUGC3 cell line. The PFKFB-4 mRNA has a variable low basal level of expression in both gastric and pancreatic cancer cell lines. However, the highest hypoxia response of PFKFB-4 mRNA is found in the pancreatic cancer cell line Panc1. The basal level of PFKFB-4 protein expression is the highest in NUGC3 gastric cancer cell line and lowest in Panc1 cells, with the highest response to hypoxia in the pancreatic cancer cell line. Further studies showed that PFKFB-3 and PFKFB-4 gene expression was highly responsive to the hypoxia mimic dimethyloxalylglycine, a specific inhibitor of HIF-alpha hydroxylase enzymes, suggesting that the hypoxia responsiveness of PFKFB-3 and PFKFB-4 genes in these cell lines is regulated by the HIF transcription complex. The expression of VEGF and Glut1, which are known HIF-dependent genes, is also strongly induced under hypoxic conditions in gastric and pancreatic cancer cell lines. The levels of HIF-1alpha protein are increased in both gastric and pancreatic cancer cell lines under hypoxic conditions. However, the basal level of HIF-1alpha as well as HIF-2alpha mRNA expression and their hypoxia responsiveness are different in the MKN45 and NUGC3 cancer cells. Thus, the expression of HIF-1alpha mRNA is decreased in both gastric cancer cell lines treated by hypoxia or dimethyloxalylglycine, but HIF-2alpha mRNA expression is not changed significantly in NUGC3 and slightly increased in MKN45 cells. Expression of PFKFB-4 and PFKFB-3 was also studied in gastric cancers and corresponding nonmalignant tissue counterparts from the same patients on both the mRNA and protein levels. The expression of PFKFB-3 and PFKFB-4 mRNA as well as PFKFB-1 and PFKFB-2 mRNA was observed in normal human gastric tissue and was increased in malignant gastric tumors. The basal level of PFKFB-4 protein expression in gastric cancers was much higher as compared to the PFKFB-3 isoenzyme. In conclusion, this study provides evidence that PFKFB-4 and PFKFB-3 genes are also expressed in gastric and pancreatic cancer cells, they strongly respond to hypoxia via a HIF-1alpha dependent mechanism and, together with the expression of PFKFB-1 and PFKFB-2 genes, possibly have a significant role in the Warburg effect which is found in malignant cells.
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Boyd, Scott, Joanna L. Brookfield, Susan E. Critchlow, Iain A. Cumming, Nicola J. Curtis, Judit Debreczeni, Sébastien L. Degorce, et al. "Structure-Based Design of Potent and Selective Inhibitors of the Metabolic Kinase PFKFB3." Journal of Medicinal Chemistry 58, no. 8 (April 13, 2015): 3611–25. http://dx.doi.org/10.1021/acs.jmedchem.5b00352.
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