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• W2617361678 abstract "Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common hereditary renal disease with no currently available targeted therapies. Based on the established connection between β-catenin signaling and renal ciliopathies, and on data from our and other laboratories showing striking similarities of this disease and cancer, we evaluated the use of an orally bioavailable small molecule, KPT-9274 (a dual inhibitor of the protein kinase PAK4 and nicotinamide phosphoribosyl transferase), for treatment of ADPKD. Treatment of PKD-derived cells with this compound not only reduces PAK4 steady-state protein levels and regulates β-catenin signaling, but also inhibits nicotinamide phosphoribosyl transferase, the rate-limiting enzyme in a key NAD salvage pathway. KPT-9274 can attenuate cellular proliferation and induce apoptosis associated with a decrease in active (phosphorylated) PAK4 and β-catenin in several Pkd1-null murine cell lines, with a less pronounced effect on the corresponding phenotypically normal cells. Additionally, KPT-9274 shows inhibition of cystogenesis in an ex vivo model of cyclic AMP-induced cystogenesis as well as in the early stage Pkd1flox/flox:Pkhd1-Cre mouse model, the latter showing confirmation of specific anti-proliferative, apoptotic, and on-target effects. NAD biosynthetic attenuation by KPT-9274, while critical for highly proliferative cancer cells, does not appear to be important in the slower growing cystic epithelial cells during cystogenesis. KPT-9274 was not toxic in our ADPKD animal model or in other cancer models. Thus, this small molecule inhibitor could be evaluated in a clinical trial as a viable therapy of ADPKD. Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common hereditary renal disease with no currently available targeted therapies. Based on the established connection between β-catenin signaling and renal ciliopathies, and on data from our and other laboratories showing striking similarities of this disease and cancer, we evaluated the use of an orally bioavailable small molecule, KPT-9274 (a dual inhibitor of the protein kinase PAK4 and nicotinamide phosphoribosyl transferase), for treatment of ADPKD. Treatment of PKD-derived cells with this compound not only reduces PAK4 steady-state protein levels and regulates β-catenin signaling, but also inhibits nicotinamide phosphoribosyl transferase, the rate-limiting enzyme in a key NAD salvage pathway. KPT-9274 can attenuate cellular proliferation and induce apoptosis associated with a decrease in active (phosphorylated) PAK4 and β-catenin in several Pkd1-null murine cell lines, with a less pronounced effect on the corresponding phenotypically normal cells. Additionally, KPT-9274 shows inhibition of cystogenesis in an ex vivo model of cyclic AMP-induced cystogenesis as well as in the early stage Pkd1flox/flox:Pkhd1-Cre mouse model, the latter showing confirmation of specific anti-proliferative, apoptotic, and on-target effects. NAD biosynthetic attenuation by KPT-9274, while critical for highly proliferative cancer cells, does not appear to be important in the slower growing cystic epithelial cells during cystogenesis. KPT-9274 was not toxic in our ADPKD animal model or in other cancer models. Thus, this small molecule inhibitor could be evaluated in a clinical trial as a viable therapy of ADPKD. Autosomal dominant polycystic kidney disease (ADPKD) is a common hereditary renal disease associated with the formation of fluid-filled cysts in the kidney as well as in other epithelial organs including the liver and pancreas.1Gabow P.A. Autosomal dominant polycystic kidney disease.N Engl J Med. 1993; 329: 332-342Crossref PubMed Scopus (851) Google Scholar With a prevalence of 1 in 500 to 1000 individuals, it is the most prevalent monogenic disorder in humans and a common cause of end-stage renal disease.2Torres V.E. Harris P.C. Pirson Y. Autosomal dominant polycystic kidney disease.Lancet. 2007; 369: 1287-1301Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar, 3Gabow P.A. Johnson A.M. Kaehny W.D. et al.Factors affecting the progression of renal disease in autosomal-dominant polycystic kidney disease.Kidney Int. 1992; 41: 1311-1319Abstract Full Text PDF PubMed Scopus (439) Google Scholar While there is a great deal known about the signaling defects underlying this disease, an enormous effort to evaluate targetable pathways, and new discoveries frequently added, there are still no ADPKD-specific therapies currently available. Efforts in our4Hwang V.J. Kim J. Rand A. et al.The cpk model of recessive PKD shows glutamine dependence associated with the production of the oncometabolite 2-hydroxyglutarate.Am J Physiol Renal Physiol. 2015; 309: F492-F498Crossref PubMed Scopus (26) Google Scholar and other5Rowe I. Chiaravalli M. Mannella V. et al.Defective glucose metabolism in polycystic kidney disease identifies a new therapeutic strategy.Nat Med. 2013; 19: 488-493Crossref PubMed Scopus (309) Google Scholar, 6Riwanto M. Kapoor S. Rodriguez D. et al.Inhibition of aerobic glycolysis attenuates disease progression in polycystic kidney disease.PLoS One. 2016; 11: e0146654Crossref Scopus (66) Google Scholar laboratories have focused on metabolic reprogramming in PKD, which in many cases shows a striking similarity to malignancy. Through conventional principles that drive oncologic research, studies on cyst formation in PKD can lead to repurposing of effective cancer drugs for use in these patients. Building on studies in cancer,7Sampath D. Zabka T.S. Misner D.L. et al.Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) as a therapeutic strategy in cancer.Pharmacol Ther. 2015; 151: 16-31Crossref PubMed Scopus (171) Google Scholar, 8Dart A.E. Wells C.M. P21-activated kinase 4—not just one of the PAK.Eur J Cell Biol. 2013; 92: 129-138Crossref PubMed Scopus (73) Google Scholar, 9Abu Aboud O. Chen C.H. Senapedis W. et al.Dual and specific inhibition of NAMPT and PAK4 by KPT-9274 decreases kidney cancer growth.Mol Cancer Ther. 2016; 15: 2119-2129Crossref PubMed Scopus (94) Google Scholar we have identified the p21 activated kinase (PAK)/WNT/β-catenin signaling as a potential target for PKD. Overexpression of PAKs influence a broad range of cellular activities with involvement in several oncogenic signaling pathways, including cell proliferation and mitogenesis,9Abu Aboud O. Chen C.H. Senapedis W. et al.Dual and specific inhibition of NAMPT and PAK4 by KPT-9274 decreases kidney cancer growth.Mol Cancer Ther. 2016; 15: 2119-2129Crossref PubMed Scopus (94) Google Scholar hence their evaluation as potential targets for dysregulated proliferative diseases including renal cystic disease. PAK4, the first of the group II PAKs to be cloned and characterized, is involved with regulating such cyst-promoting events as actin-mediated cell morphology, embryonic development, and cell cycle regulation.10Nekrasova T. Minden A. PAK4 is required for regulation of the cell-cycle regulatory protein p21, and for control of cell-cycle progression.J Cell Biochem. 2011; 112: 1795-1806Crossref PubMed Scopus (39) Google Scholar, 11Abo A. Qu J. Cammarano M.S. et al.PAK4, a novel effector for Cdc42Hs, is implicated in the reorganization of the actin cytoskeleton and in the formation of filopodia.EMBO J. 1998; 17: 6527-6540Crossref PubMed Scopus (316) Google Scholar, 12Qu J. Li X. Novitch B.G. et al.PAK4 kinase is essential for embryonic viability and for proper neuronal development.Mol Cell Biol. 2003; 23: 7122-7133Crossref PubMed Scopus (125) Google Scholar In addition, deletion of PAK4 reduces tumor formation through inhibition of cell growth13Liu Y. Xiao H. Tian Y. et al.The pak4 protein kinase plays a key role in cell survival and tumorigenesis in athymic mice.Mol Cancer Res. 2008; 6: 1215-1224Crossref PubMed Scopus (115) Google Scholar and would thus be expected to have similar effects on the dysregulated renal tubular epithelial cell proliferation seen in renal cystic disease. In this report, we investigate the utility of the dual PAK4/nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, KPT-9274 (and by inference analogs such as KPT-7523, KPT-8752, and KPT-9331),14Senapedis W. Crochiere M. Baloglu E. Landesman Y. Therapeutic potential of targeting PAK signaling.Anticancer Agents Med Chem. 2015; 16: 75-88Crossref Scopus (26) Google Scholar, 15Jiang YY, Lin DC, Mayakonda A, et al. Targeting super-enhancer-associated oncogenes in oesophageal squamous cell carcinoma [e-pub ahead of print]. Gut. http://dx.doi.org/10.1136/gutjnl-2016-311818.Google Scholar to reduce PAK4 steady-state protein levels in PKD. We show here that KPT-9274 treatment, primarily through PAK4 inhibition, causes reduction of cell proliferation and induction of apoptosis in PKD. In addition, administration of KPT-9274 reduces cystogenesis both ex vivo and in vivo with the expected on-target effects. Based on our data presented in this report, the minimal toxicity of oral administration of KPT-9274 given either for short or long periods in other animal models9Abu Aboud O. Chen C.H. Senapedis W. et al.Dual and specific inhibition of NAMPT and PAK4 by KPT-9274 decreases kidney cancer growth.Mol Cancer Ther. 2016; 15: 2119-2129Crossref PubMed Scopus (94) Google Scholar, 16Fulciniti M. Martinez-Lopez J. Senapedis W. et al.Functional role and therapeutic targeting of p21-associated kinase 4 in multiple myeloma.Blood. 2017; 129: 2233-2245Crossref PubMed Scopus (33) Google Scholar, 17Rane C SW Baloglu E. Landesman Y. et al.A novel orally bioavailable compound KPT-9274 inhibits PAK4, and blocks triple negative breast cancer tumor growth.Sci Rep. 2017; 7: 42555Crossref PubMed Scopus (48) Google Scholar, 18Aboukameel A. Muqbil I. Senapedis W. et al.Novel p21-activated kinase 4 (PAK4) allosteric modulators overcome drug resistance and stemness in pancreatic ductal adenocarcinoma.Mol Cancer Ther. 2017; 16: 76-87Crossref PubMed Scopus (65) Google Scholar and the fact that KPT-9274 has entered human phase-1 clinical trials in advanced solid malignancies (NCT02702492), there is strong evidence that this small molecule could be directly evaluated in a clinical trial of ADPKD patients. KPT-9274 is an orally bioavailable small molecule (Figure 1a) that shows dual inhibition of PAK4 and NAMPT.9Abu Aboud O. Chen C.H. Senapedis W. et al.Dual and specific inhibition of NAMPT and PAK4 by KPT-9274 decreases kidney cancer growth.Mol Cancer Ther. 2016; 15: 2119-2129Crossref PubMed Scopus (94) Google Scholar, 15Jiang YY, Lin DC, Mayakonda A, et al. Targeting super-enhancer-associated oncogenes in oesophageal squamous cell carcinoma [e-pub ahead of print]. Gut. http://dx.doi.org/10.1136/gutjnl-2016-311818.Google Scholar, 17Rane C SW Baloglu E. Landesman Y. et al.A novel orally bioavailable compound KPT-9274 inhibits PAK4, and blocks triple negative breast cancer tumor growth.Sci Rep. 2017; 7: 42555Crossref PubMed Scopus (48) Google Scholar, 18Aboukameel A. Muqbil I. Senapedis W. et al.Novel p21-activated kinase 4 (PAK4) allosteric modulators overcome drug resistance and stemness in pancreatic ductal adenocarcinoma.Mol Cancer Ther. 2017; 16: 76-87Crossref PubMed Scopus (65) Google Scholar When injected into rats, the compound and its acetylated metabolite were found by liquid chromatography–mass spectrometry analysis to be present in plasma and bile but were undetectable in urine (unpublished observations). Specificity of inhibition of PAK4 by KPT-9274 was previously demonstrated using a clustered regularly interspaced short palindromic repeat PAK4 knockout cell line9Abu Aboud O. Chen C.H. Senapedis W. et al.Dual and specific inhibition of NAMPT and PAK4 by KPT-9274 decreases kidney cancer growth.Mol Cancer Ther. 2016; 15: 2119-2129Crossref PubMed Scopus (94) Google Scholar, 16Fulciniti M. Martinez-Lopez J. Senapedis W. et al.Functional role and therapeutic targeting of p21-associated kinase 4 in multiple myeloma.Blood. 2017; 129: 2233-2245Crossref PubMed Scopus (33) Google Scholar and is demonstrated here by a dose-dependent reduction in phosphorylated PAK4 and total PAK4 in lysates of both postnatal Pkd1-null (PN24) and Pkd1-heterozygous (PH2; control) cells as well as Pkd1-null versus wild-type (WT) control mouse embryonic kidney (MEK) cells, with a more substantial decrease in Pkd1-null MEK null cells as compared to MEK-WT (Figure 1b). While likely less important in PKD signaling (vide infra), KPT-9274 also shows inhibition of NAMPT (rate-limiting enzyme necessary for nicotinamide adenine dinucleotide [NAD] biosynthesis) as evidenced by decreased total NAD + reduced NAD (NADH) levels in both PH2 and PN24 cells with no significant difference between them (Figure 1c). After incubation with KPT-9274 for 72 hours, the viability of PN24 cells was attenuated more markedly than PH2 cells, with a 50% inhibitory concentration of 2.68 μmol/l for PN24 cells (Figure 2a); a similar qualitative result was seen with MEK-null compared with MEK-WT cells, with a 50% inhibitory concentration of 0.469 μmol/l for MEK-null cells. To determine whether KPT-9274 attenuates tubular epithelial cell growth and apoptosis, properties associated with progression of ADPKD,3Gabow P.A. Johnson A.M. Kaehny W.D. et al.Factors affecting the progression of renal disease in autosomal-dominant polycystic kidney disease.Kidney Int. 1992; 41: 1311-1319Abstract Full Text PDF PubMed Scopus (439) Google Scholar, 19Ali S.M. Wong V.Y. Kikly K. et al.Apoptosis in polycystic kidney disease: involvement of caspases.Am J Physiol Regul Integr Comp Physiol. 2000; 278: R763-R769PubMed Google Scholar we demonstrated a dose-dependent decrease in cell proliferation as assessed by methylene blue staining in both sets of cell lines (Figure 2b), and an increase in apoptosis as determined by annexin V flow cytometry (Figure 2c) with both proliferation and apoptosis of both PDK-null cell lines being significantly affected by a lower dose of KPT-9274 compared with their respective normal cell line. To further evaluate the mechanism of the attenuated cell number seen with KPT-9274 incubation, we quantified the number of cell division events using live-cell imaging. Consistent with the methylene blue data, KPT-9274 was more effective in suppressing cell division events in PN24 cells when compared with PH2 cells by about 2-fold (p < 0.001) (Figure 2d, Supplementary Movies S1 to S4). Thus, KPT-9274 attenuates cell proliferation and decreases cell viability with a predilection for homozygous null ADPKD cells (PN24 and MEK null) as compared to their heterozygous or homozygous normal control cells (PH2 and MEK-WT). Dysregulation of the β-catenin-dependent WNT signaling pathway leads to cystic disease,20Kim E. Arnould T. Sellin L.K. et al.The polycystic kidney disease 1 gene product modulates Wnt signaling.J Biol Chem. 1999; 274: 4947-4953Crossref PubMed Scopus (239) Google Scholar, 21Saadi-Kheddouci S. Berrebi D. Romagnolo B. et al.Early development of polycystic kidney disease in transgenic mice expressing an activated mutant of the beta-catenin gene.Oncogene. 2001; 20: 5972-5981Crossref PubMed Scopus (204) Google Scholar, 22Qian C.N. Knol J. Igarashi P. et al.Cystic renal neoplasia following conditional inactivation of apc in mouse renal tubular epithelium.J Biol Chem. 2005; 280: 3938-3945Crossref PubMed Scopus (114) Google Scholar possibly through disruption of the ciliary signaling machinery.23Simons M. Gloy J. Ganner A. et al.Inversin, the gene product mutated in nephronophthisis type II, functions as a molecular switch between Wnt signaling pathways.Nat Genet. 2005; 37: 537-543Crossref PubMed Scopus (598) Google Scholar Cilia are microtubule-based sensory organelles that play a critical role in kidney homeostasis, and it is well established that defects in the assembly or function of cilia cause PKD.24Pan J. Seeger-Nukpezah T. Golemis E.A. The role of the cilium in normal and abnormal cell cycles: emphasis on renal cystic pathologies.Cell Mol Life Sci. 2013; 70: 1849-1874Crossref PubMed Scopus (60) Google Scholar However, the mechanism and significance of this interaction is currently unknown. As PAK4 is known to phosphorylate β-catenin (serine 675)25Li Y. Shao Y. Tong Y. et al.Nucleo-cytoplasmic shuttling of PAK4 modulates β-catenin intracellular translocation and signaling.Biochim Biophys Acta. 2012; 1823: 465-475Crossref PubMed Scopus (106) Google Scholar leading to activation of the WNT/β-catenin signaling pathway in both cancer25Li Y. Shao Y. Tong Y. et al.Nucleo-cytoplasmic shuttling of PAK4 modulates β-catenin intracellular translocation and signaling.Biochim Biophys Acta. 2012; 1823: 465-475Crossref PubMed Scopus (106) Google Scholar and cystic disease,26Wuebken A. Schmidt-Ott K.M. WNT/beta-catenin signaling in polycystic kidney disease.Kidney Int. 2011; 80: 135-138Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar we analyzed the expression levels of β-catenin in cell lysates. We noted a decrease in expression of phosphorylated β-catenin levels in the presence of KPT-9274 in all 4 cell lines and a decrease in total β-catenin expression in both homozygous null ADPKD lines (PN24, MEK null) and the heterozygous ADPKD line (PH2) (Figure 3a).Figure 3KPT-9274 treatment disrupts β-catenin levels but does not affect ciliary length and number. (a) Pkd1-heterozygous (PH2), postnatal Pkd1-null (PN24) cells, and mouse embryonic kidney (MEK)–wild-type (WT) and MEK-null cells were grown to 50% confluence, treated with KPT-9274 for 24 hours at the concentrations indicated, and subjected to immunoblotting with total and phosphorylated (serine 675) β-catenin (p-β-catenin) antibodies. Experiments shown are representative of at least 3 repeats. ImageJ quantification of protein expression corrected for β-actin is above the blots. Data are means ± SEM (n = 3–6). ∗P < 0.05 as compared to no drug. (b) Inner medullary collecting duct cells were grown to confluence and serum-starved for 72 hours to induce ciliogenesis in the presence of 10 μmol/l (μM) KPT-9274. Cells were fixed and immunostained with antibodies against acetylated tubulin to mark cilia (green), ZO-1 to highlight cell-cell junctions (red), and DNA (4′,6-diamidino-2-phenylindole, blue). Bar = 10 μm. (c) Quantification of ciliogenesis and ciliary length in KPT-9274-treated inner medullary collecting duct cells. Data are means ± SD; n = 300 cells for each sample, from 3 independent experiments. (d) Analysis of ciliogenesis in KPT-9274–treated mice. Kidney sections from WT (Pkd+/+), untreated Pkd1flox/flox:Pkhd1-Cre (vehicle), and KPT-9274–treated Pkd1flox/flox:Pkhd1-Cre (KPT-9274) mice were immunostained with antibodies against acetylated tubulin to mark cilia, and the percentage of ciliated cells were determined. There were 286 cells from 59 tubules scored (WT), 321 cells from 52 tubules (Pkd1flox/flox vehicle), and 427 cells from 67 tubules (Pkd1flox/flox KPT-9274). Data are means ± SD. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next explored the influence of KPT-9274 on ciliogenesis in murine inner medullary collecting duct-3 (mIMCD-3) cells. In contrast to PH2/PN24 and MEK-WT/MEK null cells, mIMCD-3 cells are a widely studied and reliable model of ciliogenesis because they can be consistently induced to form cilia that are representative of those in the normal kidney.27Sang L. Miller J.J. Corbit K.C. et al.Mapping the NPHP-JBTS-MKS protein network reveals ciliopathy disease genes and pathways.Cell. 2011; 145: 513-528Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar, 28Mahjoub M.R. Stearns T. Supernumerary centrosomes nucleate extra cilia and compromise primary cilium signaling.Curr Biol. 2012; 22: 1628-1634Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar Analysis of quiescent mIMCD-3 cells incubated with KPT-9274 showed no change in the fraction of cells that assembled cilia (Figure 3b), nor in the average length of cilia per cell (Figure 3c). Moreover, quantification of ciliogenesis in kidney sections from mice treated with KPT-9274 (described in more detail in the next section) showed no difference in the percentage of cells that assembled cilia in vivo (Figure 3d). Thus, while the WNT signaling cascade is attenuated by KPT-9274, higher concentrations of this compound do not have adverse effects on ciliogenesis and are therefore less likely to be toxic to normal renal and nonrenal ciliated cells. Metanephric organ cultures have been used to determine whether embryonic kidney tubules can be stimulated to form cysts and as a way to rapidly assess whether various drugs can inhibit or slow cystic progression.29Avner E.D. Epithelial polarity and differentiation in polycystic kidney disease.J Cell Sci Suppl. 1993; 17: 217-222Crossref PubMed Google Scholar, 30Magenheimer B.S. St John P.L. Isom K.S. et al.Early embryonic renal tubules of wild-type and polycystic kidney disease kidneys respond to cAMP stimulation with cystic fibrosis transmembrane conductance regulator/Na(+),K(+),2Cl(-) Co-transporter-dependent cystic dilation.J Am Soc Nephrol. 2006; 17: 3424-3437Crossref PubMed Scopus (105) Google Scholar, 31Anders C. Ashton N. Ranjzad P. Dilworth M.R. Woolf A.S. Ex vivo modeling of chemical synergy in prenatal kidney cystogenesis.PLoS One. 2013; 8: e57797Crossref PubMed Scopus (7) Google Scholar Under basal culture conditions, Pkd-1 WT kidneys from embryonic day 13.5 mice grow in size and continue ureteric bud branching and tubule formation over a 4- to 5-day period. Treatment of these kidneys with cyclic adenosine monophosphatase (cAMP) analogues (or the cAMP agonist forskolin) can induce the formation of dilated tubules within 1 hour, which enlarge over several days and result in dramatically expanded cyst-like structures of proximal tubule and collecting duct origin. Using this organ culture model, we evaluated direct effects of KPT-9274 on cyst growth in the kidney. Treatment of kidneys isolated from embryonic mice at embryonic day 13.5 and cultured ex vivo for up to 4 days with KPT-9274, in the presence of exogenous cAMP, resulted in significant decreases in the fraction of cystic area in a dose-dependent manner (Figure 4). In light of its inhibition of cancer signaling pathways, many of which we and others have shown to be relevant to ADPKD, we next asked whether KPT-9274 would have a salutary effect on cyst growth in a well-established early stage ADPKD mouse model.16Fulciniti M. Martinez-Lopez J. Senapedis W. et al.Functional role and therapeutic targeting of p21-associated kinase 4 in multiple myeloma.Blood. 2017; 129: 2233-2245Crossref PubMed Scopus (33) Google Scholar Sixteen Pkd1flox/flox:Pkhd1-Cre mice were treated i.p. daily with 60 mg/kg of KPT-9274 or vehicle control for 15 days and then, 24 hours after the last dose of KPT-9274, the mice were killed and the kidneys were harvested. Phosphorylated and total β-catenin were markedly elevated in Pkd1flox/flox:Pkhd1-Cre mice as compared to Pkd1+/+:Pkhd1-Cre mice (Figure 5a), suggesting basal and constitutive activation of this pathway in PKD. To show target specificity of KPT-9274, we performed a separate experiment with Pkd1+/+:Pkhd1-Cre mice treated daily with i.p. KPT-9274 (100 mg/kg) or vehicle and harvested the kidneys only 16 hours after the last dose. It can be seen that total-PAK4 and to a lesser extent total-β-catenin were decreased in the kidneys from treated animals (Figure 5b), which is consistent with target engagement of KPT-9274.17Rane C SW Baloglu E. Landesman Y. et al.A novel orally bioavailable compound KPT-9274 inhibits PAK4, and blocks triple negative breast cancer tumor growth.Sci Rep. 2017; 7: 42555Crossref PubMed Scopus (48) Google Scholar In the Pkd1flox/flox:Pkhd1-Cre mice, KPT-9274 treatment resulted in significantly decreased cyst growth, kidney size, and blood urea nitrogen when compared with vehicle-treated control mice (Figure 5c–f). Consistent with this data, proliferating cell nuclear antigen staining demonstrated less cell proliferation in KPT-9274–treated mice when compared with vehicle-treated control mice (Figure 5f) and more apoptotic cell death in KPT-9274–treated mice was demonstrated by more terminal deoxynucleotidyltransferase–mediated deoxyuridine triphosphate nick end-labeling (TUNEL)-positive epithelial cells lining cysts (Figure 5g). Thus, KPT-9274 effectively inhibits cyst growth by decreasing cell proliferation in Pkd1flox/flox:Pkhd1-Cre mice, suggesting an attractive therapeutic approach for treatment of at least early stage ADPKD. KPT-9274 treatment did not affect mouse body weight at the conclusion of the experiment (vehicle control: 7.28 ± 0.66; KPT-9274 treated: 6.6 ± 1.0; P = 0.13), suggesting a lack of general toxicity.Figure 5KPT-9274 treatment delays cyst growth in Pkd1flox/flox:Pkhd1-Cre mice. (a) Kidneys from Pkd1flox/flox:Pkhd1-Cre mouse kidneys and Pkd1+/+:Pkhd1-Cre mice were treated for 15 days with KPT-9274 (i.p.; 60 mg/kg), and the kidneys were harvested 24 hours after the last dose of KPT-9274, immunoblotted, and probed with antibodies to total and phosphorylated β-catenin and β-actin as a loading control. (b) Kidneys from Pkd1+/+:Pkhd1-Cre mice were treated for 7 days with KPT-9274 (i.p.; 100 mg/kg) or vehicle. The mice were killed 16 hours after the last dose of KPT-9274, and lysates were subject to immunoblotting for total-PAK4, total-β-catenin, and β-actin as loading control. *P < 0.05 when compared with vehicle. (c) Hematoxylin and eosin staining of Pkd1flox/flox:Pkhd1-Cre mouse kidneys treated with vehicle or KPT-9274. Bar = 2 mm. (d) Cystic index was quantified from sagittal sections of whole kidneys. Whole kidney images were acquired and total kidney area, cystic area, and noncystic area were measured by ImageJ. Cystic index = (total cystic area ÷ total kidney area) × 100 and is expressed as a percentage. Data are means ± SD (n = 8). (e) Kidney weight/body weight (KW/BW) ratio was quantified as total kidney weight (n = 2 kidneys per mouse) divided by total body weight. Data are means ± SD (n = 8). (f) Blood urea nitrogen (BUN) was quantified by a Quantichrom urea assay kit. Data are means ± SD (n = 8). (g) Proliferating cell nuclear antigen (PCNA) staining and (h) terminal deoxynucleotidyltransferase–mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay of Pkd1flox/flox:Pkhd1-Cre mouse kidneys treated with vehicle or KPT-9274. Data are means ± SD (n = 8). Bars = 50 μm for both (g) and (h). p-β-catenin, phosphorylated β-catenin. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) By virtue of its inhibition of NAMPT, KPT-9274 attenuates NAD regeneration through a NAD salvage pathway.9Abu Aboud O. Chen C.H. Senapedis W. et al.Dual and specific inhibition of NAMPT and PAK4 by KPT-9274 decreases kidney cancer growth.Mol Cancer Ther. 2016; 15: 2119-2129Crossref PubMed Scopus (94) Google Scholar In normal cells, there exists 2 salvage pathways for NAD regeneration, but in some cancers,7Sampath D. Zabka T.S. Misner D.L. et al.Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) as a therapeutic strategy in cancer.Pharmacol Ther. 2015; 151: 16-31Crossref PubMed Scopus (171) Google Scholar including renal cell carcinoma (RCC),9Abu Aboud O. Chen C.H. Senapedis W. et al.Dual and specific inhibition of NAMPT and PAK4 by KPT-9274 decreases kidney cancer growth.Mol Cancer Ther. 2016; 15: 2119-2129Crossref PubMed Scopus (94) Google Scholar, 32Wettersten H.I. Hakimi A.A. Morin D. et al.Grade-dependent metabolic reprogramming in kidney cancer revealed by combined proteomics and metabolomics analysis.Cancer Res. 2015; 75: 2541-2552Crossref PubMed Scopus (187) Google Scholar the NAD synthetic pathway operates primarily through NAMPT. In RCC, hypermethylation of the nicotinate phosphoribosyltransferase domain containing-1 (NAPRT1) promoter results in down-regulation of NAPRT1 mRNA and protein, causing rapidly proliferative cancer cells to be highly dependent on NAMPT activity33Kirkland J.B. Niacin status, NAD distribution and ADP-ribose metabolism.Curr Pharm Des. 2009; 15: 3-11Crossref PubMed Scopus (60) Google Scholar for NAD regeneration and cell survival.34Watson M. Roulston A. Belec L. et al.The small molecule GMX1778 is a potent inhibitor of NAD+ biosynthesis: strategy for enhanced therapy in nicotinic acid phosphoribosyltransferase 1-deficient tumors.Mol Cell Biol. 2009; 29: 5872-5888Crossref PubMed Scopus (183) Google Scholar To evaluate this requirement in PKD, we show (as expected) that KPT-9274 decreased total NAD+NADH in both PN24 and PH2 cells by a similar magnitude (see Figure 1c), which argues against any differential requirement in PKD. The lack of change in baseline NAMPT or NAPRT1 levels in these cells support this hypothesis (Figure 6a). Furthermore, unlike in the cell lines, the Pkd1flox/flox:Pkhd1-Cre model tissues (Figure 6b) as well as human PKD tissues (Figure 6c) showed the opposite changes in NAMPT as are seen in cancer,9Abu Aboud O. Chen C.H. Senapedis W. et al.Dual and specific inhibition of NAMPT and PAK4 by KPT-9274 decreases kidney cancer growth.Mol Cancer Ther. 2016; 15: 2119-2129Crossref PubMed Scopus (94) Google Scholar notably lower levels of NAMPT in ADPKD (which regulates the salvage pathway of NAD generation attenuated by KPT-9274). This may explain why KPT-9274 did not significantly" @default.
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• W2617361678 title "Anticystogenic activity of a small molecule PAK4 inhibitor may be a novel treatment for autosomal dominant polycystic kidney disease" @default.
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