Key Transport Proteins Research Articles

Evolution of Plant Na+-P-Type ATPases: From Saline Environments to Land Colonization.

Soil salinity is one of the major factors obstructing the growth and development of agricultural crops. Eukaryotes have two main transport systems involved in active Na+ removal: cation/H+ antiporters and Na+-P-type ATPases. Key transport proteins, Na+/K+-P-ATPases, are widely distributed among the different taxa families of pumps which are responsible for keeping cytosolic Na+ concentrations below toxic levels. Na+/K+-P-ATPases are considered to be absent in flowering plants. The data presented here are a complete inventory of P-type Na+/K+-P-ATPases in the major branches of the plant kingdom. We also attempt to elucidate the evolution of these important membrane pumps in plants in comparison with other organisms. We were able to observe the gradual replacement of the Na+-binding site to the Ca2+-binding site, starting with cyanobacteria and moving to modern land plants. Our results show that the α-subunit likely evolved from one common ancestor to bacteria, fungi, plants, and mammals, whereas the β-subunit did not evolve in green algae. In conclusion, our results strongly suggest the significant differences in the domain architecture and subunit composition of plant Na+/K+-P-ATPases depending on plant taxa and the salinity of the environment. The obtained data clarified and broadened the current views on the evolution of Na+/K+-P-ATPases. The results of this work would be helpful for further research on P-type ATPase functionality and physiological roles.

Human placental uptake of glutamine and glutamate is reduced in fetal growth restriction

Fetal growth restriction (FGR) is a significant risk factor for stillbirth, neonatal complications and adulthood morbidity. Compared with those of appropriate weight for gestational age (AGA), FGR babies have smaller placentas with reduced activity of amino acid transporter systems A and L, thought to contribute to poor fetal growth. The amino acids glutamine and glutamate are essential for normal placental function and fetal development; whether transport of these is altered in FGR is unknown. We hypothesised that FGR is associated with reduced placental glutamine and glutamate transporter activity and expression, and propose the mammalian target of rapamycin (mTOR) signaling pathway as a candidate mechanism. FGR infants [individualised birth weight ratio (IBR) < 5th centile] had lighter placentas, reduced initial rate uptake of 14C-glutamine and 14C-glutamate (per mg placental protein) but higher expression of key transporter proteins (glutamine: LAT1, LAT2, SNAT5, glutamate: EAAT1) versus AGA [IBR 20th–80th]. In further experiments, in vitro exposure to rapamycin inhibited placental glutamine and glutamate uptake (24 h, uncomplicated pregnancies) indicating a role of mTOR in regulating placental transport of these amino acids. These data support our hypothesis and suggest that abnormal glutamine and glutamate transporter activity is part of the spectrum of placental dysfunction in FGR.

Photoaffinity Labeling and Quantitative Chemical Proteomics Identify Liver X Receptor β as the Functional Target of Enhancers of Astrocytic Apolipoprotein E

Apolipoprotein E (apoE) is a 34 kDa key lipid transport protein in both the brain and the periphery. In the brain, it is produced predominantly by astrocytes and has been shown to play an important role in promoting neurite outgrowth and synaptogenesis, clearing amyloid β42, promoting cerebrovascular integrity and in neuroimmune modulation. Given the critical role of apoE in the brain, it is important to understand its regulation in the CNS. To that end, utilizing a phenotypic screen, we identified novel chemical matter that increased astrocytic apoE secretion in vitro. We designed a clickable photoaffinity probe and carried out quantitative chemical proteomics to identify liver x receptor β (LXRβ) as the target. Binding of the ligand stabilized LXRβ, as shown by CETSA. Our findings demonstrated that the lead chemical matter bound directly to LXRβ, and highlight the power of chemical proteomics to identify the target of a phenotypic screening hit.

How Reciprocal Interactions Between the Tumor Microenvironment and Ion Transport Proteins Drive Cancer Progression.

Solid tumors comprise two major components: the cancer cells and the tumor stroma. The stroma is a mixture of cellular and acellular components including fibroblasts, mesenchymal and cancer stem cells, endothelial cells, immune cells, extracellular matrix, and tumor interstitial fluid. The insufficient tumor perfusion and the highly proliferative state and dysregulated metabolism of the cancer cells collectively create a physicochemical microenvironment characterized by altered nutrient concentrations and varying degrees of hypoxia and acidosis. Furthermore, both cancer and stromal cells secrete numerous growth factors, cytokines, and extracellular matrix proteins which further shape the tumor microenvironment (TME), favoring cancer progression.Transport proteins expressed by cancer and stromal cells localize at the interface between the cells and the TME and are in a reciprocal relationship with it, as both sensors and modulators of TME properties. It has been amply demonstrated how acid-base and nutrient transporters of cancer cells enable their growth, presumably by contributing both to the extracellular acidosis and the exchange of metabolic substrates and waste products between cells and TME. However, the TME also impacts other transport proteins important for cancer progression, such as multidrug resistance proteins. In this review, we summarize current knowledge of the cellular and acellular components of solid tumors and their interrelationship with key ion transport proteins. We focus in particular on acid-base transport proteins with known or proposed roles in cancer development, and we discuss their relevance for novel therapeutic strategies.

K+ and the renin-angiotensin-aldosterone system: new insights into their role in blood pressure control and hypertension treatment.

Approximately 25% of the adult population is diagnosed with hypertension and it is therefore one of the biggest challenges for the health sector. The renin-angiotensin-aldosterone system (RAAS) adjusts effective circulating volume and ultimately blood pressure (BP). Accordingly, antihypertensive drugs targeting the RAAS have been a major focus in modern medical treatment. Low and high dietary K+ intakes are associated with increased or decreased BP and risk of cardiac failure, respectively, suggesting that dietary K+ augmentation has the potential to supplement or replace conventional anti-hypertensive drugs. Animal studies have indicated that the beneficial effects of high dietary K+ may be linked to a dominant regulatory role of plasma K+ on key renal transport proteins controlled by the RAAS. However, only a limited number of studies have investigated whether the reported mechanisms in animal models apply to humans. Furthermore, hypertension is often treated with so-called 'K+ sparing' drugs, thus complicating co-treatment with K+ supplementation. In this review, we revisit old concepts of RAAS effects in the kidney, relate them to effects of dietary K+ manipulation, and finally consider the clinical potential of treating hypertension with K+ supplementation alone or in combination with RAAS inhibitors. Collectively, a wealth of data suggest that increased dietary K+ intake may have beneficial effects on BP in the general population, but underlying medical conditions or current treatment regimens need to be carefully considered before implementing K+ supplementation in patients.

Targeting Apolipoprotein E for Alzheimer's Disease: An Industry Perspective.

Apolipoprotein E (apoE), a key lipid transport protein in the brain, is predominantly produced by astrocytes. Astrocytes are the most numerous cell type in the brain and are the main support network for neurons. They play a critical role in the synthesis and delivery of cholesterol in the brain. Humans have three common apoE isoforms, apoE2, apoE3 and apoE4, that show a strong genotype effect on the risk and age of onset for sporadic and late onset forms of Alzheimer’s disease (AD). Carriers of an ε4 allele have an increased risk of developing AD, while those with an ε2 allele are protected. Investigations into the contribution of apoE to the development of AD has yielded conflicting results and there is still much speculation about the role of this protein in disease. Here, we review the opposing hypotheses currently described in the literature and the approaches that have been considered for targeting apoE as a novel therapeutic strategy for AD. Additionally, we provide our perspective on the rationale for targeting apoE and the challenges that arise with respect to “drug-ability” of this target.

Guard cells in fern stomata are connected by plasmodesmata, but control cytosolic Ca2+ levels autonomously.

Recent studies have revealed that some responses of fern stomata to environmental signals differ from those of their relatives in seed plants. However, it is unknown whether the biophysical properties of guard cells differ fundamentally between species of both clades. Intracellular micro-electrodes and the fluorescent Ca2+ reporter FURA2 were used to study voltage-dependent cation channels and Ca2+ signals in guard cells of the ferns Polypodium vulgare and Asplenium scolopendrium. Voltage clamp experiments with fern guard cells revealed similar properties of voltage-dependent K+ channels as found in seed plants. However, fluorescent dyes moved within the fern stomata, from one guard cell to the other, which does not occur in most seed plants. Despite the presence of plasmodesmata, which interconnect fern guard cells, Ca2+ signals could be elicited in each of the cells individually. Based on the common properties of voltage-dependent channels in ferns and seed plants, it is likely that these key transport proteins are conserved in vascular plants. However, the symplastic connections between fern guard cells in mature stomata indicate that the biophysical mechanisms that control stomatal movements differ between ferns and seed plants.

Regulation of Muscle Glycogen Metabolism during Exercise: Implications for Endurance Performance and Training Adaptations

Since the introduction of the muscle biopsy technique in the late 1960s, our understanding of the regulation of muscle glycogen storage and metabolism has advanced considerably. Muscle glycogenolysis and rates of carbohydrate (CHO) oxidation are affected by factors such as exercise intensity, duration, training status and substrate availability. Such changes to the global exercise stimulus exert regulatory effects on key enzymes and transport proteins via both hormonal control and local allosteric regulation. Given the well-documented effects of high CHO availability on promoting exercise performance, elite endurance athletes are typically advised to ensure high CHO availability before, during and after high-intensity training sessions or competition. Nonetheless, in recognition that the glycogen granule is more than a simple fuel store, it is now also accepted that glycogen is a potent regulator of the molecular cell signaling pathways that regulate the oxidative phenotype. Accordingly, the concept of deliberately training with low CHO availability has now gained increased popularity amongst athletic circles. In this review, we present an overview of the regulatory control of CHO metabolism during exercise (with a specific emphasis on muscle glycogen utilization) in order to discuss the effects of both high and low CHO availability on modulating exercise performance and training adaptations, respectively.

Transgenic corn decreased total and key storage and lipid transport protein levels in honey bee hemolymph while seed treatment with imidacloprid reduced lipophorin levels

Pollen substitutes are currently widely used in apiculture because of a lack of natural forage and an increasing need for bees for pollination services. A major concern in the confection of bee diets is whether specific ingredients, such as GMO grain, or contaminants such as pesticides that are applied to crops, negatively impact bee health. We measured hemolymph protein levels and the lifespan of honey bees fed on diets containing non-hybrid (IAC-Airan), hybrid (AG 7088) or transgenic (AG 8088YG) maize, the seeds of which had been treated with fipronil, imidacloprid or left untreated. Corn from these plantings was harvested, dried and finely ground, and then used for the preparation of corn-based diets. Groups of 100 newly emerged bees were placed in cages, where the diets were offered ad libitum. Honey bee lifespan was similar for all the corn diets, though shorter than for bees fed beebread and longer than for bees fed only honey. Total protein in the hemolymph of bees fed with transgenic maize was 15% lower than in bees fed on non-hybrid maize; lipophorin and vitellogenin, were more reduced, by over 30%. Irrespective of corn variety, imidacloprid resulted in over 25% lower levels of lipophorin compared with the control. Lower storage and transport protein levels would compromise the ability of the bees to rear brood and develop an adequate immune response to pathogen challenge. Consequently, transgenic maize, as well as any maize produced from seed treated with imidacloprid should not be included as ingredients in honey bee diets.

New insights in integrated response mechanism of Lactobacillus plantarum under excessive manganese stress

As a natural inhabitant of gastrointestinal tract, Lactobacillus plantarum is well known for its health-promoting effects, especially in heavy metal-removal function. The response mechanism integrated physiology and proteomics of Lactobacillus plantarum under excessive manganese stress was systematically performed. More extracellular polymeric substance was secreted, whereas cells preferred to aggregate for resisting manganese stress. The stability of the membrane was maintained by up-regulating the proportion of unsaturated fatty acids, especially cyclopropane fatty acids. The contents of intracellular Asp and Arg, closely related with energy metabolism, decreased under excessive manganese stress. Comparative 2-DE proteomic analysis identified 52 proteins that significantly differed under different levels of manganese stress. The differentially expressed proteins, involved in the carbohydrate, amino acid, stress response and nucleotide metabolisms, were categorized as the crucial components during resisting manganese stress. Moreover, MntH 4 and MntH 5 were identified as the functional elements in regulation of manganese transportation model. The differential expression of six key transporter proteins under different manganese stresses indicated that the MntH 1, 2 and 3 was negatively regulated by MntR, while the regulation of MntH 4 and 5 presented the contrary model. Taken altogether, the study provides new insights into the response mechanism of probiotics to similar environmental stress.

Relaxation and the Role of Calcium in Isolated Contracting Myocardium From Patients With Hypertensive Heart Disease and Heart Failure With Preserved Ejection Fraction.

Relaxation characteristics and Ca2+ homeostasis have not been studied in isolated myocardium from patients with hypertensive heart disease (HHD) and heart failure with preserved ejection fraction (HFpEF). Prolonged myocardial relaxation is believed to play an important role in the pathophysiology of these conditions. In this study, we evaluated relaxation parameters, myocardial calcium (Ca2+), and sodium (Na+) handling, as well as ion transporter expression and tested the effect of Na+-influx inhibitors on relaxation in isolated myocardium from patients with HHD and HFpEF. Relaxation characteristics were studied in myocardial strip preparations under physiological conditions at stimulation rates of 60 and 180 per minute. Intracellular Ca2+ and Na+ were simultaneously assessed using Fura-2 and AsanteNATRIUMGreen-2, whereas elemental analysis was used to measure total myocardial concentrations of Ca, Na, and other elements. Quantitative polymerase chain reaction was used to measure expression levels of key ion transport proteins. The lusitropic effect of Na+-influx inhibitors ranolazine, furosemide, and amiloride was evaluated. Myocardial left ventricular biopsies were obtained from 36 control patients, 29 HHD and 19 HHD+HFpEF. When compared with control patients, half maximal relaxation time (RT50) at 60 per minute was prolonged by 13% in HHD and by 18% in HHD+HFpEF (both P<0.05). Elevated resting Ca2+ levels and a tachycardia-induced increase in diastolic Ca2+ were associated with incomplete relaxation and an increase in diastolic tension in HHD and HHD+HFpEF. Na+ levels were not increased, and expression levels of Ca2+- or Na+-handling proteins were not altered. Na+-influx inhibitors did not improve relaxation or prevent incomplete relaxation at high stimulation rates. Contraction and relaxation are prolonged in isolated myocardium from patients with HHD and HHD+HFpEF. This leads to incomplete relaxation at higher rates. Elevated calcium levels in HFpEF are neither a result of an impaired Na+ gradient nor expression changes in key ion transporters and regulatory proteins.

EFFECT OF INCREASING APOLIPOPROTEIN E ON INFLAMMATION IN THE BRAIN

Apolipoprotein E (apoE) is a key lipid transport protein in brain and plasma. Humans have three apoE isoforms; namely, apoE2, apoE3 and apoE4. Of the three isoforms, apoE4 is strongly associated with late-onset and sporadic forms of Alzheimer's disease (AD). In addition to its role in neuronal repair, synaptogenesis, and clearance of toxic Aß fragments from brain, apoE has been suggested to play a critical role in inflammation in the brain. Using pan LXR agonists as a tool compound for increasing brain and astrocytic apoE levels, we sought to investigate the effect of increasing astrocytic apoE on brain inflammation induced by lipopolysaccharide (LPS) both in vitro and in vivo. Using primary mouse microglia and astrocytes and C57/bl6 wild type mice as our model system, we evaluated the effect of increasing apoE protein levels using T0901317 (pan LXR agonist) on LPS induced inflammation. We used RT-qPCR to evaluate the mRNA levels of apoE and apoE lipidating genes and MSD assays to evaluate cytokine protein levels in cell media and brain homogenates. TO901317 treatment led to an increase in brain apoE levels in vivo in wild type mice and in vitro, in both primary murine astrocytes and microglia. This increase in apoE was accompanied by an increase in apoE lipidating genes (Abca1 and Abcg1). Treatment with LPS led to a significant inflammatory response as seen by an increase in pro-inflammatory cytokines, TNFα and IL6. However, TO901317 treatment led to a significant reduction in the levels of the pro-inflammatory cytokines both in vitro (astrocytes and microglia) and in vivo in the brain. Our results indicate that increasing brain and gial apoE using a pan LXR agonist attenuates LPS induced inflammation. Our data suggests that apoE could play an important role in promoting anti- inflammatory effects in the brain. Given the strong association of apoE with AD, increasing lipidated apoE in the brain could have multi-prong benefits including anti-inflammatory effects.

CRM1 Inhibitors for Antiviral Therapy.

Infectious diseases are a major global concern and despite major advancements in medical research, still cause significant morbidity and mortality. Progress in antiviral therapy is particularly hindered by appearance of mutants capable of overcoming the effects of drugs targeting viral components. Alternatively, development of drugs targeting host proteins essential for completion of viral lifecycle holds potential as a viable strategy for antiviral therapy. Nucleocytoplasmic trafficking pathways in particular are involved in several pathological conditions including cancer and viral infections, where hijacking or alteration of function of key transporter proteins, such as Chromosome Region Maintenance1 (CRM1) is observed. Overexpression of CRM1-mediated nuclear export is evident in several solid and hematological malignancies. Interestingly, CRM1-mediated nuclear export of viral components is crucial in various stages of the viral lifecycle and assembly. This review summarizes the role of CRM1 in cancer and selected viruses. Leptomycin B (LMB) is the prototypical inhibitor of CRM1 potent against various cancer cell lines overexpressing CRM1 and in limiting viral infections at nanomolar concentrations in vitro. However, the irreversible shutdown of nuclear export results in high cytotoxicity and limited efficacy in vivo. This has prompted search for synthetic and natural CRM1 inhibitors that can potentially be developed as broadly active antivirals, some of which are summarized in this review.

Living with a leaky skin: upregulation of ion transport proteins during sloughing.

Amphibian skin is a multifunctional organ providing protection from the external environment and facilitating the physiological exchange of gases, water and salts with the environment. In order to maintain these functions, the outer layer of skin is regularly replaced in a process called sloughing. During sloughing, the outermost layer of the skin is removed in its entirety, which has the potential to interfere with skin permeability and ion transport, disrupting homeostasis. In this study, we measured, in vivo, the effects of sloughing on the cutaneous efflux of ions in toads Rhinella marina kept in freshwater conditions. We also measured transepithelial potential, cutaneous resistance, active ion transport and the distribution, abundance and gene expression of the key ion transport proteins sodium-potassium ATPase (NKA) and epithelial sodium channel (ENaC) during sloughing. We hypothesised that the increase in transepithelial efflux of ions during sloughing is a consequence of increased permeability and/or a reduction in the abundance or expression of cutaneous ion transport proteins, resulting in disruption of internal ion homeostasis. There was a significant increase in sodium and chloride efflux during sloughing in R. marina However, although in vitro skin resistance decreased after sloughing, active sodium transport increased commensurate with an increase in NKA and ENaC protein abundance in the skin. These changes in skin function associated with sloughing did not affect the maintenance of internal electrolyte homeostasis. These results suggest that during sloughing, amphibians actively maintain internal homeostasis by increasing cutaneous rates of ion uptake.

The Second-Generation Exportin-1 Inhibitor KPT-8602 Demonstrates Potent Activity against Acute Lymphoblastic Leukemia.

Purpose: Human exportin-1 (XPO1) is the key nuclear-cytoplasmic transport protein that exports different cargo proteins out of the nucleus. Inducing nuclear accumulation of these proteins by inhibiting XPO1 causes cancer cell death. First clinical validation of pharmacological inhibition of XPO1 was obtained with the Selective Inhibitor of Nuclear Export (SINE) compound selinexor (KPT-330) demonstrating activity in phase-II/IIb clinical trials when dosed 1 to 3 times weekly. The second-generation SINE compound KPT-8602 shows improved tolerability and can be dosed daily. Here, we investigate and validate the drug-target interaction of KPT-8602 and explore its activity against acute lymphoblastic leukemia (ALL).Experimental Design: We examined the effect of KPT-8602 on XPO1 function and XPO1-cargo as well as on a panel of leukemia cell lines. Mutant XPO1 leukemia cells were designed to validate KPT-8602's drug-target interaction. In vivo, anti-ALL activity was measured in a mouse ALL model and patient-derived ALL xenograft models.Results: KPT-8602 induced caspase-dependent apoptosis in a panel of leukemic cell lines in vitro Using CRISPR/Cas9 genome editing, we demonstrated the specificity of KPT-8602 for cysteine 528 in the cargo-binding groove of XPO1 and validated the drug target interaction. In vivo, KPT-8602 showed potent anti-leukemia activity in a mouse ALL model as well as in patient-derived T- and B-ALL xenograft models without affecting normal hematopoiesis.Conclusions: KPT-8602 is highly specific for XPO1 inhibition and demonstrates potent anti-leukemic activity supporting clinical application of the second-generation SINE compound for the treatment of ALL. Clin Cancer Res; 23(10); 2528-41. ©2016 AACR.

Common variation in the sodium/glucose cotransporter 2 gene SLC5A2 does neither affect fasting nor glucose-suppressed plasma glucagon concentrations.

AimInhibition of sodium/glucose cotransporter 2 (SGLT2), the key transport protein in renal glucose reabsorption, promotes glucose excretion and represents a new concept in the therapy of type-2 diabetes. In addition, SGLT2 inhibition elevates circulating glucagon concentrations and enhances hepatic glucose production. Since SGLT2 is expressed in human pancreatic α-cells and regulates glucagon release, we tested whether common variants of the SGLT2 gene SLC5A2 associate with altered plasma glucagon concentrations in the fasting state and upon glucose challenge.MethodsA study population of 375 healthy subjects at increased risk for type-2 diabetes, phenotyped by a 5-point oral glucose tolerance test (OGTT) and genotyped for recently described SLC5A2 tagging single nucleotide polymorphisms (SNPs), was selected for plasma glucagon measurements.ResultsAfter adjustment for gender, age, body mass index, and insulin sensitivity, the four tagging SNPs (rs9924771, rs3116150, rs3813008, rs9934336), tested separately or as genetic score, were neither significantly nor nominally associated with plasma glucagon concentrations at any time during the OGTT, with the inverse AUC of glucagon or the glucagon fold-change during the OGTT (p ≥ 0.2, all). Testing for genotype-related differences in the time course of the glucagon response using MANOVA did also not reveal any significant or nominal associations (p ≥ 0.5, all).ConclusionWe could not obtain statistically significant evidence for a role of common SLC5A2 variants in the regulation of glucagon release in the fasting state or upon glucose challenge. Moreover, the reported nominal effects of individual SLC5A2 variants on fasting and post-challenge glucose levels may probably not be mediated by altered glucagon release.

Oestrogen upregulates the expression levels and functional activities of duodenal mucosal CFTR and SLC26A6.

What is the central question of this study? Duodenal ulcer is a common disease. A sex-based difference in the incidence of duodenal ulcer has long been observed clinically, but the cause is unclear. What is the main finding and its importance? Duodenal mucosal bicarbonate secretion is the most important protective factor in duodenal mucosa against acid-induced damage. The cystic fibrosis transmembrane conductance regulator (CFTR) and the solute-linked carrier 26 gene family A6 (SLC26A6) are two key bicarbonate transport proteins that mediate duodenal mucosal bicarbonate secretion. We demonstrate that endogenous oestrogen upregulates the expression levels and functional activities of duodenal mucosal CFTR and SLC26A6, which contributes to the sex difference in the prevalence of duodenal ulcer. The incidence of duodenal ulcer is markedly lower in women than men, but the cause of the sex difference is not clear. The cystic fibrosis transmembrane conductance regulator (CFTR) and the solute-linked carrier 26 gene family A6 (SLC26A6) are two key bicarbonate transport proteins that mediate duodenal mucosal bicarbonate secretion, which is an important protective factor against acid-induced duodenal injury. The aim of this study was to investigate the effect of oestrogen on the expressions and functional activities of CFTR and SLC26A6 in duodenal mucosa. We found that the expression levels of duodenal CFTR and SLC26A6 were markedly higher in young (20- to 30-year-old) women than in young men and old (60- to 70-year-old) women and men. The expression levels of CFTR and SLC26A6 in young women were markedly higher in preovulatory phases than in premenstrual phases, which was consistent with the changes of serum estradiol concentrations. Further results showed that duodenal CFTR and SLC26A6 expression levels in female mice were markedly decreased after ovariectomy, and supplementation with estradiol reversed the changes in CFTR and SLC26A6. 17β-Estradiol increased CFTR and SLC26A6 expression levels of human duodenocytes in experiments in vitro. Functional experiments showed that basal and forskolin- and prostaglandin E2 -stimulated duodenal bicarbonate secretion in ovariectomized mice was markedly decreased and, likewise, supplementation with 17β-estradiol reversed the changes. In conclusion, endogenous oestrogen upregulates the expressions and functional activities of CFTR and SLC26A6 in duodenal mucosa, which could contribute to protection of the duodenum and explain the sex difference in the prevalence of duodenal ulcer.

Abstract LB-210: KPT-8602 is a second-generation XPO1 inhibitor with improved in vivo tolerability that demonstrates potent acute lymphoblastic leukemia activity

Abstract Human exportin-1 (XPO1) is the key nuclear-cytoplasmic transport protein that exports a wide variety of different cargo proteins including tumor suppressors out of the cell's nucleus. Inhibition of XPO1 function consequently restores nuclear localization of these proteins and is a promising therapeutic strategy for cancer. Selective inhibitor of nuclear export (SINE) compounds are inhibitors of the XPO1-mediated nuclear export with potent anti-cancer activity. The oral clinical candidate SINE selinexor (KPT-330) is currently in Phase-II/IIb clinical trials and demonstrated remission in patients as a single agent or in combination therapy in trials for pre-treated, relapsed and refractory hematological and solid tumor malignancies. The drug is generally well tolerated when dosed every other day 1-3 times a week. However, XPO1 inhibitors with improved tolerability allowing more frequent dosing can be expected to have a substantial clinical benefit. Here we present the anti-leukemic activity of a second-generation XPO1 inhibitor KPT-8602 with improved tolerability allowing for a daily dosing regimen. First, its anti-XPO1 activity was assessed; KPT-8602 potently inhibited the XPO1-mediated nuclear protein export at nanomolar concentrations and it blocked the interaction of XPO1 with cargo protein. KPT-8602 also induced potent cytotoxicity on a panel of T-ALL and B-ALL cell lines. Cytotoxicity correlated with the induction of caspase-dependent apoptosis and the nuclear accumulation of p53 as well as the subsequent induction of p53 response. To further investigate the mechanism of action of KPT-8602 we applied CRISPR/Cas9 to introduce a Cys528Ser mutation in the XPO1 gene of four different leukemia cell lines. Mutant cells were over 100 times resistant to KPT-8602. In addition, drug-target interaction was confirmed by pull-down of wild-type XPO1 protein out of cells using biotinylated KPT-8602 while it was unable to pull down mutated XPO1C528S out of mutant cells. These results illustrate the highly specific interaction of the drug for its target and prove that the anti-leukemic activity of KPT-8602 is caused by inhibition of XPO1. To examine the anti-leukemic activity of KPT-8062 in vivo, mice engrafted with patient-derived T-cell acute lymphoblastic leukemia were treated with KPT-8602 or placebo. Mice were daily treated by oral gavage for 3 weeks. Treatment with KPT-8602 led to a significant reduction of leukemia cell numbers in blood as measured by weekly blood counts, without affecting normal erythropoiesis. Animals treated with KPT-8602 had prolonged survival compared to placebo treated animals. KPT-8602 also showed potent anti-leukemia activity in a mouse T-ALL leukemia model. In conclusion, KPT-8062 is a second-generation XPO1 inhibitor with high specificity for its target and with potent anti-ALL activity. It displays better tolerability as compared to the first-generation SINE selinexor allowing it for daily dosing resulting in effective anti-ALL activity in in vivo PDX models warranting further evaluation of this new drug in patients. As such, in January 2016 a phase I/II study with KPT-8602 has been initiated. Citation Format: Dirk Daelemans, Jasper Edgar Neggers, Jolien De Bie, Maarten Jacquemyn, Astrid D’Hoore, Els Vanstreels, Erkan Baloglu, Yosef Landesman, Sharon Shacham, William Senapedis, Antonis Dagklis, Thomas Vercruysse, Jan Cools. KPT-8602 is a second-generation XPO1 inhibitor with improved in vivo tolerability that demonstrates potent acute lymphoblastic leukemia activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-210.

Transferrin Receptor Levels are increased by Interleukin 1β in a Human Kidney Proximal Tubular Cell line

BackgroundIron is required for several essential cellular processes such as DNA synthesis and energy metabolism, but when present in excess can cause cellular injury. Although kidney iron levels and urinary excretion of iron and iron transport proteins such as transferrin are increased in patients with kidney disease, it remains unclear how iron is handled by the kidneys during inflammation. We hypothesize that inflammation increases renal tubular iron uptake mechanisms, which in turn may promote cellular injury.Methods and ResultsTo understand how iron is handled by proximal tubule cells during inflammation, we turned to HK‐2 cells, a human proximal tubular cell line, and investigated the levels and distribution of transferrin receptor and divalent metal transporter 1 (DMT‐1) in response to interleukin 1β, a pro‐inflammatory cytokine. Transferrin receptor allows the uptake of iron after binding and internalization of iron‐loaded transferrin while DMT‐1 carries divalent metals like iron, zinc and copper inside the cells. Our data with HK‐2 cells indicated that transferrin receptor levels doubled upon treatment with 0.1 ng/mL interleukin 1β for 24 hours (P&lt;0.005 by one‐way ANOVA, followed by Dunnett's test). Immunofluorescent staining of HK‐2 cells showed no apparent change in transferrin receptor distribution. In contrast, DMT‐1 levels in HK‐2 cells remained unchanged upon treatment with interleukin 1β for 24 hours at concentrations as high as 50 ng/mL. Increased intracellular levels of iron may lead to mitochondrial oxidative stress, which in turn may impact ATP production. After treating HK‐2 cells with 1 ng/mL interleukin 1β for 24 hours, intracellular ATP levels were measured and indicated no significant difference with untreated cells.ConclusionsThese data suggest that under inflammatory conditions, the uptake mechanism for the key iron transport protein transferrin is increased in the proximal tubule. This could have important implications in the setting of glomerular injury, which permits greater amounts of transferrin to be filtered, resulting in increased exposure of proximal tubular cells to transferrin and thus iron uptake.

The choroid plexus epithelium displays conventional cell polarization

The choroid plexus epithelium (CPE) has been studied extensively as a model cell in e.g. high rate fluid secretion because of the direct access to the luminal surface. The high‐rate secretion is orchestrated by an unusual plasma membrane distribution of key transport proteins in the CPE cells. Par example, the ubiquitously expressed Na, K‐ATPase accumulates in the luminal membrane domain of CPE cells. The same has been observed for other normally basolateral proteins such as NKCC1 and NHE1, but not for all basolateral proteins (e.g. AE2). The observation led to the hypotheses that the CPE cells exhibit an inverse cell polarity, despite a conventional localization of structural polarity elements in epithelial cells: basal membrane facing the interstitium, tight junctions close to the luminal surface and luminal specializations such as microvilli and cilia. In this study, we investigated the expression and subcellular distribution of basic cell polarity markers in the CPE cells. Immunohistochemical analysis revealed a basolateral domain accumulation of the membrane lipid PtdIns(3,4,5)P3, Scrib, E‐cadherin, and partitioning‐defective protein (Par) 1. Luminal membrane domain accumulation was observed for the atypical protein kinase aPKC, the plasma membrane target snare syntaxin‐3, whereas Par3 was localized at the tight junction area. These markers represent the molecular complexes known to determine luminal and basolateral identity and were, thus, all located as expected for mature and normally polarized epithelial cells. We conclude that these central polarity proteins are distributed normally in the choroid plexus cells, and that cell polarity as such does not seem to be involved in the unusual localizations of key transport proteins in these cells.Support or Funding InformationThe work was supported by the Danish Medical Research Council and Aarhus University Ideas programme.