Multiplexed cellular profiling identifies an organoselenium compound as an inhibitor of CRM1-mediated nuclear export.

Chromosomal region maintenance 1 (CRM1 also known as Xpo1 and exportin-1) is the receptor for the nuclear export controlling the intracellular localization and function of many cellular and viral proteins that play a crucial role in viral infections and cancer. The inhibition of CRM1 has emerged as a promising therapeutic approach to interfere with the lifecycle of many viruses, for the treatment of cancer, and to overcome therapy resistance. Recently, selinexor has been approved as the first CRM1 inhibitor for the treatment of multiple myeloma, providing proof of concept for this therapeutic option with a new mode of action. However, selinexor is associated with dose-limiting toxicity and hence, the discovery of alternative small molecule leads that could be developed as less toxic anticancer and antiviral therapeutics will have a significant impact in the clinic. Here, we report a CRM1 inhibitor discovery platform. The development of this platform includes reporter cell lines that monitor CRM1 activity by using red fluorescent protein or green fluorescent protein-labeled HIV-1 Rev protein with a strong heterologous nuclear export signal. Simultaneously, the intracellular localization of other proteins, to be interrogated for their capacity to undergo CRM1-mediated export, can be followed by co-culturing stable cell lines expressing fluorescent fusion proteins. We used this platform to interrogate the mode of nuclear export of several proteins, including PDK1, p110α, STAT5A, FOXO1, 3, 4 and TRIB2, and to screen a compound collection. We show that while p110α partially relies on CRM1-dependent nuclear export, TRIB2 is exported from the nucleus in a CRM1-independent manner. Compound screening revealed the striking activity of an organoselenium compound on the CRM1 nuclear export receptor.

FOXO1 represses PPARα-Mediated induction of FGF21 gene expression.

Fibroblast growth factor 21 (FGF21) has emerged as a metabolic regulator that exerts potent anti-diabetic and lipid-lowering effects in animal models of obesity and type 2 diabetes, showing a protective role in fatty liver disease and hepatocellular carcinoma progression. Hepatic expression of FGF21 is regulated by PPARα and is induced by fasting. Ablation of FoxO1 in liver has been shown to increase FGF21 expression in hyperglycemia. To better understand the role of FOXO1 in the regulation of FGF21 expression we have modified HepG2 human hepatoma cells to overexpress FoxO1 and PPARα. Here we show that FoxO1 represses PPARα-mediated FGF21 induction, and that the repression acts on the FGF21 gene promoter without affecting other PPARα target genes. Additionally, we demonstrate that FoxO1 physically interacts with PPARα and that FoxO1/3/4 depletion in skeletal muscle contributes to increased Fgf21 tissue levels. Taken together, these data indicate that FOXO1 is a PPARα-interacting protein that antagonizes PPARα activity on the FGF21 promoter. Because other PPARα target genes remained unaffected, these results suggest a highly specific mechanism implicated in FGF21 regulation. We conclude that FGF21 can be specifically modulated by FOXO1 in a PPARα-dependent manner.

Genetic analysis of global faba bean diversity, agronomic traits and selection signatures.

KEY MESSAGE: We identified marker-trait associations for key faba bean agronomic traits and genomic signatures of selection within a global germplasm collection. Faba bean (Vicia faba L.) is a high-protein grain legume crop with great potential for sustainable protein production. However, little is known about the genetics underlying trait diversity. In this study, we used 21,345 high-quality SNP markers to genetically characterize 2678 faba bean genotypes. We performed genome-wide association studies of key agronomic traits using a seven-parent-MAGIC population and detected 238 significant marker-trait associations linked to 12 traits of agronomic importance. Sixty-five of these were stable across multiple environments. Using a non-redundant diversity panel of 685 accessions from 52 countries, we identified three subpopulations differentiated by geographical origin and 33 genomic regions subjected to strong diversifying selection between subpopulations. We found that SNP markers associated with the differentiation of northern and southern accessions explained a significant proportion of agronomic trait variance in the seven-parent-MAGIC population, suggesting that some of these traits were targets of selection during breeding. Our findings point to genomic regions associated with important agronomic traits and selection, facilitating faba bean genomics-based breeding.

Modulating undruggable targets to overcome cancer therapy resistance.

Many cancer patients frequently fail to respond to anti-cancer treatment due to therapy resistance which is the major obstacle towards curative cancer treatment. Therefore, identification of the molecular mechanisms underlying resistance is of paramount clinical and economic importance. The advent of targeted therapies based on a molecular understanding of cancer could serve as a model for strategies to overcome drug resistance. Accordingly, the identification and validation of proteins critically involved in resistance mechanisms represent a path towards innovative therapeutic strategies to improve the clinical outcome of cancer patients. In this review, we discuss emerging targets, small molecule therapeutics and drug delivery strategies to overcome therapy resistance. We focus on rational treatment strategies based on transcription factors, pseudokinases, nuclear export receptors and immunogenic cell death strategy. Historically, unliganded transcription factors and pseudokinases were considered undruggable while blocking the nuclear export e.g., through inhibition of the nuclear export receptor CRM1 was predicted as highly toxic. Recent success inhibiting Gli-1, HIF-1α, HIF-2α and reactivating the tumor suppressor transcription factors p53 and FOXO illustrates the feasibility and power of this targeting approach. Similarly, progress has been made in modulating the activity of pseudokinase proteins implicated in therapy resistance including members of the Tribbles protein family. On the other hand, the recent clinical approval of Selinexor, a specific inhibitor of CRM-1, a protein that mediates the transport of cargos with leucine-rich nuclear export signals and known to be a driver of drug resistance, represents the proof-of-concept for inhibiting the nuclear export as a feasible strategy to overcome therapy resistance. The ever-growing capacity to target resistance mechanisms with judiciously selected small molecules, some of which are being formulated within smart nanoparticles, will pave the way towards the improvement of the clinical outcome and realize the full potential of targeted therapies and immunotherapies.

mTORC2 Is the Major Second Layer Kinase Negatively Regulating FOXO3 Activity.

Forkhead box O (FOXO) proteins are transcription factors involved in cancer and aging and their pharmacological manipulation could be beneficial for the treatment of cancer and healthy aging. FOXO proteins are mainly regulated by post-translational modifications including phosphorylation, acetylation and ubiquitination. As these modifications are reversible, activation and inactivation of FOXO factors is attainable through pharmacological treatment. One major regulatory input of FOXO signaling is mediated by protein kinases. Here, we use specific inhibitors against different kinases including PI3K, mTOR, MEK and ALK, and other receptor tyrosine kinases (RTKs) to determine their effect on FOXO3 activity. While we show that inhibition of PI3K efficiently drives FOXO3 into the cell nucleus, the dual PI3K/mTOR inhibitors dactolisib and PI-103 induce nuclear FOXO translocation more potently than the PI3Kδ inhibitor idelalisib. Furthermore, specific inhibition of mTOR kinase activity affecting both mTORC1 and mTORC2 potently induced nuclear translocation of FOXO3, while rapamycin, which specifically inhibits the mTORC1, failed to affect FOXO3. Interestingly, inhibition of the MAPK pathway had no effect on the localization of FOXO3 and upstream RTK inhibition only weakly induced nuclear FOXO3. We also measured the effect of the test compounds on the phosphorylation status of AKT, FOXO3 and ERK, on FOXO-dependent transcriptional activity and on the subcellular localization of other FOXO isoforms. We conclude that mTORC2 is the most important second layer kinase negatively regulating FOXO activity.

Amino acid digestibility and metabolisable energy of spring and winter faba beans grown on two sites and effects of dehulling in caecectomised laying hens.

BACKGROUND: The variation in amino acid (AA) digestibility and metabolisable energy (MEN ) in four spring and four winter faba bean genotypes differing in vicine/convicine (V/C) concentrations grown on two sites was investigated in caecectomised LSL-Classic laying hens. Effects of dehulling one faba bean genotype were also examined. Diets containing one out of 17 faba bean variants each and a basal diet were fed to ten caecectomised laying hens in a row-column design to achieve five replicates per diet. RESULTS: Ranges and levels of digestibility of the hulled variants differed widely among AA with the lowest and highest range determined for Arg (90-93%) and Cys (-12-65%), respectively. MEN ranged between 10.3 and 12.3 MJ kg-1 dry matter. Lower MEN and digestibility of Cys, Glx, Phe, Pro, Tyr, and Val (P < 0.050) was determined for the winter genotypes grown in Nimtitz compared to the other variants. Digestibility of Ser was lower for the spring than for the winter genotypes (P < 0.050). Negative correlations with AA digestibility were determined for phytate, but not for tannin and V/C concentrations (P < 0.050). Negative correlations between tannin fractions and MEN were weak (P = 0.082-0.099). Dehulling increased MEN by 1.8 MJ kg-1 dry matter and raised the digestibility of Pro, His, and Glx (P < 0.050). CONCLUSIONS: The results indicated that the digestible AA and MEN supply of laying hens was increased by using low phytate faba beans while breeding for low V/C genotypes did not affect AA digestibility or MEN . Dehulling increased MEN and the digestibility of some AA. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Paternal outcrossing success differs among faba bean genotypes and impacts breeding of synthetic cultivars.

KEY MESSAGE: Faba bean genotypes showed significant and marked genetic differences in their success as pollen donors to cross-fertilized seeds. The findings may improve exploitation of heterosis in synthetic cultivars. In partially allogamous crops such as faba bean (Vicia faba L.), increasing the share of heterosis in a synthetic cultivar can improve yield and yield stability. The share of heterosis in such synthetic cultivars is increased by higher degrees of cross-fertilization. This trait is defined as percentage of cross-fertilized seeds among all seeds and is a crucial parameter in breeders' yield predictions. Current approaches use degree of cross-fertilization to predict inbreeding and share of heterosis, they even consider genotype-specific degrees; yet, all genotypes are assumed to contribute equally to the cross-fertilized seeds. Here, we expect faba bean genotypes to differ in their success rates as pollen donors, i.e. in paternal outcrossing success. To quantify the variation of both, the degree of cross-fertilization and the paternal outcrossing success, we assessed these parameters in inbred lines and F1 hybrids, grown in four polycrosses composed of eight genotypes each. We identified the paternal genotype of 500 to 800 seeds per genotype and polycross using SNP markers. In both traits, we found marked and significant variation among inbred lines and among F1 hybrids, as well as between inbred lines and F1. Based on our findings, we discuss how differential paternal outcrossing success influences the amount of inbreeding in synthetic cultivars. Our findings offer the potential for a better management and exploitation of heterotic yield increase in faba bean.

CRISPR/Cas9-mediated genome editing: From basic research to translational medicine.

The recent development of the CRISPR/Cas9 system as an efficient and accessible programmable genome-editing tool has revolutionized basic science research. CRISPR/Cas9 system-based technologies have armed researchers with new powerful tools to unveil the impact of genetics on disease development by enabling the creation of precise cellular and animal models of human diseases. The therapeutic potential of these technologies is tremendous, particularly in gene therapy, in which a patient-specific mutation is genetically corrected in order to treat human diseases that are untreatable with conventional therapies. However, the translation of CRISPR/Cas9 into the clinics will be challenging, since we still need to improve the efficiency, specificity and delivery of this technology. In this review, we focus on several in vitro, in vivo and ex vivo applications of the CRISPR/Cas9 system in human disease-focused research, explore the potential of this technology in translational medicine and discuss some of the major challenges for its future use in patients.

FOXO transcription factors at the interface of metabolism and cancer.

Diabetes refers to a group of metabolic diseases characterized by impaired insulin signalling and high blood glucose. A growing body of epidemiological evidence links diabetes to several types of cancer but the underlying molecular mechanisms are poorly understood. The signalling cascade connecting insulin and FOXO proteins provides a compelling example for a conserved pathway at the interface between insulin signalling and cancer. FOXOs are transcription factors that orchestrate programs of gene expression known to control a variety of processes in response to cellular stress. Genes regulated by this family of proteins are involved in the regulation of cellular energy production, oxidative stress resistance and cell viability and proliferation. Accordingly, FOXO factors have been shown to play an important role in the suppression of tumour growth and in the regulation of metabolic homeostasis. There is emerging evidence that deregulation of FOXO factors might account for the association between insulin resistance-related metabolic disorders and cancer.

A SNP-based consensus genetic map for synteny-based trait targeting in faba bean (Vicia faba L.).

Faba bean (Vicia faba L.) is a globally important nitrogen-fixing legume, which is widely grown in a diverse range of environments. In this work, we mine and validate a set of 845 SNPs from the aligned transcriptomes of two contrasting inbred lines. Each V. faba SNP is assigned by BLAST analysis to a single Medicago orthologue. This set of syntenically anchored polymorphisms were then validated as individual KASP assays, classified according to their informativeness and performance on a panel of 37 inbred lines, and the best performing 757 markers used to genotype six mapping populations. The six resulting linkage maps were merged into a single consensus map on which 687 SNPs were placed on six linkage groups, each presumed to correspond to one of the six V. faba chromosomes. This sequence-based consensus map was used to explore synteny with the most closely related crop species, lentil and the most closely related fully sequenced genome, Medicago. Large tracts of uninterrupted colinearity were found between faba bean and Medicago, making it relatively straightforward to predict gene content and order in mapped genetic interval. As a demonstration of this, we mapped a flower colour gene to a 2-cM interval of Vf chromosome 2 which was highly colinear with Mt3. The obvious candidate gene from 78 gene models in the collinear Medicago chromosome segment was the previously characterized MtWD40-1 gene controlling anthocyanin production in Medicago and resequencing of the Vf orthologue showed a putative causative deletion of the entire 5' end of the gene.

Moving to the core: spatiotemporal analysis of Forkhead box O (FOXO) and nuclear factor-κB (NF-κB) nuclear translocation.

Nuclear translocation of proteins is an essential aspect of normal cell function, and defects in this process have been detected in many disease-associated conditions. The detection and quantification of nuclear translocation was significantly boosted by the association of robotized microscopy with automated image analysis, a technology designated as high-content screening. Image-based high-content screening and analysis provides the means to systematically observe cellular translocation events in time and space in response to chemical or genetic perturbation at large scale. This approach yields powerful insights into the regulation of complex signaling networks independently of preconceived notions of mechanistic relationships. In this review, we briefly overview the different mechanisms involved in nucleocytoplasmic protein trafficking. In addition, we discuss high-content approaches used to interrogate the mechanistic and spatiotemporal dynamics of cellular signaling events using Forkhead box O (FOXO) proteins and the nuclear factor-κB (NF-κB) as important and clinically relevant examples.

TRIB2 as a biomarker for diagnosis and progression of melanoma.

Malignant melanoma is the most deadly form of skin cancer. There is a critical need to identify the patients that could be successfully treated by surgery alone and those that require adjuvant treatment. In this study, we demonstrate that the expression of tribbles2 (TRIB2) strongly correlates with both the presence and progression of melanocyte-derived malignancies. We examined the expression of TRIB2 in addition to 12 previously described melanoma biomarkers across three independent full genome microarray studies. TRIB2 expression was consistently and significantly increased in benign nevi and melanoma, and was highest in samples from patients with metastatic melanoma. The expression profiles for the 12 biomarkers were poorly conserved throughout these studies with only TYR, S100B and SPP1 showing consistently elevated expression in metastatic melanoma versus normal skin. Strikingly we confirmed these findings in 20 freshly obtained primary melanoma tissue samples from metastatic lesions where the expression of these biomarkers were evaluated revealing that TRIB2 expression correlated with disease stage and clinical prognosis. Our results suggest that TRIB2 is a meaningful biomarker reflecting diagnosis and progression of melanoma, as well as predicting clinical response to chemotherapy.

Tribbles breaking bad: TRIB2 suppresses FOXO and acts as an oncogenic protein in melanoma.

TRIB2 (tribbles homolog 2) encodes one of three members of the tribbles family in mammals. These members share a Trb (tribbles) domain, which is homologous to protein serine-threonine kinases, but lack the active site lysine. The tribbles proteins interact and modulate the activity of signal transduction pathways in a number of physiological and pathological processes. TRIB2 has been identified as an oncogene that inactivates the transcription factor CCAAT/enhancer-binding protein α (C/EBPα) and causes acute myelogenous leukaemia (AML). Recent research provided compelling evidence that TRIB2 can also act as oncogenic driver in solid tumours, such as lung and liver cancer. In particular, our recent work demonstrated that TRIB2 is dramatically overexpressed in malignant melanomas compared with normal skin and promotes the malignant phenotype of melanoma cells via the down-regulation of FOXO (forkhead box protein O) tumour suppressor activity in vitro and in vivo. TRIB2 was found to be expressed in normal skin, but its expression consistently increased in benign nevi, melanoma and was highest in samples from patients with malignant melanoma. The observation that TRIB2 strongly correlates with the progression of melanocyte-derived malignancies suggests TRIB2 as a meaningful biomarker to both diagnose and stage melanoma. In addition, interfering with TRIB2 activity might be a therapeutic strategy for the treatment of several different tumour types.

Methylseleninic acid promotes antitumour effects via nuclear FOXO3a translocation through Akt inhibition.

Selenium supplement has been shown in clinical trials to reduce the risk of different cancers including lung carcinoma. Previous studies reported that the antiproliferative and pro-apoptotic activities of methylseleninic acid (MSA) in cancer cells could be mediated by inhibition of the PI3K pathway. A better understanding of the downstream cellular targets of MSA will provide information on its mechanism of action and will help to optimize its use in combination therapies with PI3K inhibitors. For this study, the effects of MSA on viability, cell cycle, metabolism, apoptosis, protein and mRNA expression, and reactive oxygen species production were analysed in A549 cells. FOXO3a subcellular localization was examined in A549 cells and in stably transfected human osteosarcoma U2foxRELOC cells. Our results demonstrate that MSA induces FOXO3a nuclear translocation in A549 cells and in U2OS cells that stably express GFP-FOXO3a. Interestingly, sodium selenite, another selenium compound, did not induce any significant effects on FOXO3a translocation despite inducing apoptosis. Single strand break of DNA, disruption of tumour cell metabolic adaptations, decrease in ROS production, and cell cycle arrest in G1 accompanied by induction of apoptosis are late events occurring after 24h of MSA treatment in A549 cells. Our findings suggest that FOXO3a is a relevant mediator of the antiproliferative effects of MSA. This new evidence on the mechanistic action of MSA can open new avenues in exploiting its antitumour properties and in the optimal design of novel combination therapies. We present MSA as a promising chemotherapeutic agent with synergistic antiproliferative effects with cisplatin.

A novel phosphatidylinositol 3-kinase (PI3K) inhibitor directs a potent FOXO-dependent, p53-independent cell cycle arrest phenotype characterized by the differential induction of a subset of FOXO-regulated genes.

INTRODUCTION: The activation of the phosphoinositide 3-kinase (PI3K)/AKT signalling pathway is one the most frequent genetic events in breast cancer, consequently the development of PI3K inhibitors has attracted much attention. Here we evaluate the effect of PI3K inhibition on global gene expression in breast cancer cells. METHODS: We used a range of methodologies that include in silico compound analysis, in vitro kinase assays, cell invasion assays, proliferation assays, genome-wide transcription studies (Agilent Technologies full genome arrays), gene set enrichment analysis, quantitative real-time PCR, immunoblotting in addition to chromatin immunoprecipitation. RESULTS: We defined the physico-chemical and the biological properties of ETP-45658, a novel potent PI3K inhibitor. We demonstrated that ETP-45658 potently inhibited cell proliferation within a broad range of human cancer cells, most potently suppressing the growth of breast cancer cells via inhibiting cell cycle. We show that this response is Forkhead box O (FOXO) protein dependent and p53 independent. Our genome-wide microarray analysis revealed that the cell cycle was the most affected biological process after exposure to ETP-45658 (or our control PI3K inhibitor PI-103), that despite the multiple transcription factors that are regulated by the PI3K/AKT signalling cascade, only the binding sites for FOXO transcription factors were significantly enriched and only a subset of all FOXO-dependent genes were induced. This disparity in gene transcription was not due to differential FOXO promoter recruitment. CONCLUSIONS: The constitutive activation of PI3Ks and thus the exclusion of FOXO transcription factors from the nucleus is a key feature of breast cancer. Our results presented here highlight that PI3K inhibition activates specific FOXO-dependent genes that mediate cell cycle arrest in breast cancer cells.

Protein localization in disease and therapy.

The eukaryotic cell is organized into membrane-covered compartments that are characterized by specific sets of proteins and biochemically distinct cellular processes. The appropriate subcellular localization of proteins is crucial because it provides the physiological context for their function. In this Commentary, we give a brief overview of the different mechanisms that are involved in protein trafficking and describe how aberrant localization of proteins contributes to the pathogenesis of many human diseases, such as metabolic, cardiovascular and neurodegenerative diseases, as well as cancer. Accordingly, modifying the disease-related subcellular mislocalization of proteins might be an attractive means of therapeutic intervention. In particular, cellular processes that link protein folding and cell signaling, as well as nuclear import and export, to the subcellular localization of proteins have been proposed as targets for therapeutic intervention. We discuss the concepts involved in the therapeutic restoration of disrupted physiological protein localization and therapeutic mislocalization as a strategy to inactivate disease-causing proteins.

Biological characterization of ETP-46321 a selective and efficacious inhibitor of phosphoinositide-3-kinases.

Inhibitors of PI3K signaling are of great therapeutic interest in oncology. The phosphoinositide-3-kinase signaling pathway is activated in a variety of solid and non-solid tumors. We have identified an imidazopyrazine derivative, ETP-46321, as a potent inhibitor of PI3Kα [Formula: see text]. The compound was 6 times less potent towards PI3Kδ and more than 200 and 60 times less potent at inhibiting PI3Kß and PI3Kγ and did not significantly inhibit the related phosphoinositide-3-kinase-related protein kinase family kinases mTOR or DNA PK (IC(50)'s > 5 µM), or an additional 287 protein kinases that were screened. ETP-46321 inhibited PI3K signaling in treated tumor cell lines, induced cell cycle arrest and inhibited VEGF-dependent sprouting of HUVEC cells. The compound was anti-proliferative and synergized with both cytotoxic and targeted therapeutics. The compound induced a reduction in the phosphorylation of Akt in U87 MG xenografts after a single treatment. The growth of colon and lung cancinoma HT-29 and A549 xenografts was delayed by once a day treatment with ETP-46321. The compound synergized with Doxotaxel in a model of ovarian cancer.

Pseudokinases, Tribbles Proteins and Cancer.

The human kinome comprises 518 protein kinases, of which approximately 10% lack one or more of the conserved amino acids necessary for catalytic activity [...].