Supplementary Figure 4 from Inhibition of the Ras-Net (Elk-3) Pathway by a Novel Pyrazole that Affects Microtubules

Supplementary Methods and Materials, Figure Legends 1-5, Tables 1-6 from Inhibition of the Ras-Net (Elk-3) Pathway by a Novel Pyrazole that Affects Microtubules

Mesenchymal stem cells (MSCs) are promising candidates for the development of cell-based drug delivery systems for autoimmune inflammatory diseases, such as multiple sclerosis (MS). Here, we investigated the effect of Ro-31-8425, an ATP-competitive kinase inhibitor, on the therapeutic properties of MSCs. Upon a simple pretreatment procedure, MSCs spontaneously took up and then gradually released significant amounts of Ro-31-8425. Ro-31-8425 (free or released by MSCs) suppressed the proliferation of CD4+ T cells in vitro following polyclonal and antigen-specific stimulation. Systemic administration of Ro-31-8425-loaded MSCs ameliorated the clinical course of experimental autoimmune encephalomyelitis (EAE), a murine model of MS, displaying a stronger suppressive effect on EAE than control MSCs or free Ro-31-8425. Ro-31-8425-MSC administration resulted in sustained levels of Ro-31-8425 in the serum of EAE mice, modulating immune cell trafficking and the autoimmune response during EAE. Collectively, these results identify MSC-based drug delivery as a potential therapeutic strategy for the treatment of autoimmune diseases. KEY MESSAGES: MSCs can spontaneously take up the ATP-competitive kinase inhibitor Ro-31-8425. Ro-31-8425-loaded MSCs gradually release Ro-31-8425 and exhibit sustained suppression of T cells. Ro-31-8425-loaded MSCs have more sustained serum levels of Ro-31-8425 than free Ro-31-8425. Ro-31-8425-loaded MSCs are more effective than MSCs and free Ro-31-8425 for EAE therapy.

From the moment of birth, each human being builds a microbe-host symbiosis which is key for the preservation of its health and well-being. This personal symbiotic coexistence is the result of progressive enrichments in microorganism diversity through external supplies. This diversity is nowadays massively overthrown by drastic changes related to clinical practice in birth management, environmental exposure, nutrition and healthcare behaviors. The last two generations have been the frame of massive modifications in life and food habits, with people being more and more sedentary, overfed and permeated with drugs and pollutants. We are now able to measure the impact of these changes on the gut microbiota diversity. Concomitantly, these modifications of lifestyle were associated with a dramatic increase in incidence of immune-mediated diseases including metabolic, allergic and inflammatory diseases and most likely neurodegenerative and psychiatric disorders. Microbiota is becoming a hot topic in the scientific community and in the mainstream media. The number of scientific publications increased by up to a factor three over the last five years, with gastrointestinal and metabolic diseases being the most productive areas. In the intellectual property landscape, the patent families on microbiota have more than doubled in the meantime. In parallel, funding either from National Institutes (e.g. from NIH which funds research mainly in the field of allergies, infections, cancer and cardiovascular diseases, from the White House which launched the national microbiome initiative) or by pharmaceutical companies follow the same trend, showing a boost and a strong support in the research field on microbiota. All major health players are investing in microbiome research as shown by the number of deals signed and by funding during 2015. The Giens round table addressed how the medicine of tomorrow, considering human beings as a human-microbe symbiotic supraorganism, could leverage microbiome knowledge and tools. The rationale for our working group has been structured around four domains of innovation that could derive from ongoing efforts in deciphering the interactions between human cells and intestinal microbiome as a central component of human health, namely: (1) development of stratification and monitoring tools; (2) identification of new target and drug discovery, as a part of our supra-genome; (4) exploitation of microbiota as a therapeutic target that can be modulated; (4) and finally as a source of live biotherapeutics and adjuvants. These four streams will exemplify how microbiota has changed the way we consider a wide range of chronic and incurable diseases and the consequences of long-lasting dysbiosis. In-depth microbiota analysis is opening one of the broadest fields of investigation for improving human and animal health and will be a source of major therapeutic innovations for tackling today's medical unmet needs. We thus propose a range of recommendations for basic researchers, care givers as well as for health authorities to gain reliability in microbiome analysis and accelerate discovery processes and their translation into applications for the benefits of the people. Finally, les Ateliers de Giens round table on microbiota benefited from the richness of the French ecosystem. France represents a center of excellence in the microbiota research field, with French institutions as Institut national de la recherche agronomique (INRA [Metagenopolis, Micalis]), Centre national de la recherché scientifique (CNRS), Unité de recherche sur les maladies infectieuses et tropicales émergentes (URMITE), Institut of Cardiometabolism and Nutrition (ICAN), Institut des maladies métaboliques et cardiovasculaires (I2MC), Institut national de la santé et de la recherche médicale (Inserm), Pasteur Institute and Gustave-Roussy being top-players for the number of publications.

À partir de la naissance, chaque être humain établit une symbiose avec son microbiote qui joue un rôle clef dans le maintien de sa santé et de son bien-être. Cette co-existence symbiotique individuelle est le résultat d’une succession d’enrichissements de la diversité des microorganismes commensaux par des apports extérieurs. Cette diversité se trouve néanmoins menacée depuis peu par des changements drastiques dans nos habitudes de vie comme la prise en charge des naissances, l’environnement extérieur, l’alimentation et les pratiques médicales. Les deux dernières générations ont été le cadre de modifications importantes des modes de vies et d’alimentation, avec une augmentation de la sédentarité, une suralimentation et une exposition importante aux médicaments et aux polluants. Nous sommes maintenant en mesure d’évaluer l’impact de ces changements sur la diversité du microbiote intestinal. Parallèlement à ces modifications du mode de vie, on a pu observer une explosion de l’incidence de maladies liées à un dysfonctionnement du système immunitaire comme les maladies métaboliques, les allergies et les maladies inflammatoires et plus étonnamment des maladies neurodégénératives et des désordres psychiatriques. Le microbiote est devenu un sujet d’actualité dans la communauté scientifique ainsi que dans les medias grand public. Le nombre de publications scientifiques a augmenté d’un facteur trois sur les cinq dernières années, les domaines des maladies gastro-intestinales et liées aux dysfonctionnements métaboliques étant les plus étudiés. D’un point de vue de la propriété intellectuelle, les familles de brevets traitant du microbiote ont plus que doublé sur la même période. En parallèle, les financements provenant soit des organisations étatiques (par exemple du National Institute of Health [NIH] qui finance les recherches essentiellement dans le domaine des allergies, des anti-infectieux, du cancer et des maladies cardio-vasculaires, ou de la Maison Blanche qui a lancé l’initiative nationale sur le microbiome) soit des entreprises pharmaceutiques ont suivi la même tendance et reflètent une poussée des investissements et un soutien important de ce domaine de la recherche sur le microbiote. Tous les acteurs majeurs de la santé investissent actuellement dans la recherche sur le microbiome ce qui se traduit par le grand nombre de contrats et de financements signés en 2015. La table ronde des Ateliers de Giens s’est intéressée à envisager comment la médecine de demain, en considérant l’homme comme un supra-organisme symbiotique homme–microbes, pourra bénéficier du savoir acquis sur les fonctions du microbiote et sur le microbiome en tant qu’outil thérapeutique. La réflexion de notre groupe de travail a été structurée autour de quatre domaines d’innovation qui pourraient bénéficier des efforts réalisés actuellement afin de déchiffrer les interactions entre cellules humaines et le microbiome intestinal, en tant qu’élément central de la santé humaine, c’est-à-dire : 1) le développement d’outils de stratification et de monitorage ; 2) l’identification de cibles originales et de nouvelles drogues à partir du microbiome en tant que partie de notre supra-génome ; 3) l’exploitation du microbiote en tant que cible thérapeutique qui peut être modulable ; et enfin, 4) comme source de biothérapeutiques vivants ou d’adjuvants. Ces quatre chapitres vont permettre de montrer comment le microbiote a modifié notre façon de considérer un grand nombre de maladies chroniques et incurables comme les conséquences d’une dysbiose établie et durable. L’analyse en profondeur du microbiote est le point de départ d’un des plus vastes chantiers de recherches qui permettra l’amélioration de la santé humaine et animale et qui deviendra la source d’innovations médicales pour couvrir les gaps thérapeutiques d’aujourd’hui. Nous proposons donc une série de recommandations que nous adressons aux chercheurs académiques, aux cliniciens ainsi qu’aux autorités de santé, afin que les analyses du microbiote gagnent en fiabilité et solidité. Ceci permettra d’accélérer le processus de découvertes et ainsi que leurs applications afin que tous puissent en bénéficier.

Poor homing of systemically infused cells to disease sites may limit the success of exogenous cell-based therapy. In this study, we screened 9,000 signal-transduction modulators to identify hits that increase mesenchymal stromal cell (MSC) surface expression of homing ligands that bind to intercellular adhesion molecule 1 (ICAM-1), such as CD11a. Pretreatment of MSCs with Ro-31-8425, an identified hit from this screen, increased MSC firm adhesion to an ICAM-1-coated substrate in vitro and enabled targeted delivery of systemically administered MSCs to inflamed sites in vivo in a CD11a- (and other ICAM-1-binding domains)-dependent manner. This resulted in a heightened anti-inflammatory response. This represents a new strategy for engineering cell homing to enhance therapeutic efficacy and validates CD11a and ICAM-1 as potential targets. Altogether, this multi-step screening process may significantly improve clinical outcomes of cell-based therapies.

Reprogramming of somatic cells into induced pluripotent stem cells is achieved by the expression of defined transcription factors. In the last few years, reprogramming strategies on the basis of doxycycline-inducible lentiviruses in mouse cells became highly powerful for screening purposes when the expression of a GFP gene, driven by the reactivation of endogenous stem cell specific promoters, was used as a reprogramming reporter signal. However, similar reporter systems in human cells have not been generated. Here, we describe the derivation of drug-inducible human fibroblast-like cell lines that express different subsets of reprogramming factors containing a GFP gene under the expression of the endogenous OCT4 promoter. These cell lines can be used to screen functional substitutes for reprogramming factors or modifiers of reprogramming efficiency. As a proof of principle of this system, we performed a screening of a library of pluripotent-enriched microRNAs and identified hsa-miR-519a as a novel inducer of reprogramming efficiency.

Objective— To characterize the role of a vascular-expressed class 3 semaphorin (semaphorin 3G [Sema3G]). Methods and Results— Semaphorins have been identified as axon guidance molecules. Yet, they have more recently also been characterized as attractive and repulsive regulators of angiogenesis. Through a transcriptomic screen, we identified Sema3G as a molecule of angiogenic endothelial cells. Sema3G-deficient mice are viable and exhibit no overt vascular phenotype. Yet, LacZ expression in the Sema3G locus revealed intense arterial vascular staining in the angiogenic vasculature, starting at E9.5, which was detectable throughout adolescence and downregulated in adult vasculature. Sema3G is expressed as a full-length 100-kDa secreted molecule that is processed by furin proteases to yield 95- and a 65-kDa Sema domain–containing subunits. Full-length Sema3G binds to NP2, whereas processed Sema3G binds to NP1 and NP2. Expression profiling and cellular experiments identified autocrine effects of Sema3G on endothelial cells and paracrine effects on smooth muscle cells. Conclusion— Although the mouse knockout phenotype suggests compensatory mechanisms, the experiments identify Sema3G as a primarily endothelial cell–expressed class 3 semaphorin that controls endothelial and smooth muscle cell functions in autocrine and paracrine manners, respectively.

Abstract Net (Elk-3/SAP-2/Erp) is a transcription factor that is phosphorylated and activated by the Ras–extracellular signal-regulated kinase (Erk) signaling pathway and is involved in wound healing, angiogenesis, and tumor growth. In a cell-based screen for small molecule inhibitors of Ras activation of Net transcriptional activity, we identified a novel pyrazole, XRP44X. XRP44X inhibits fibroblast growth factor 2 (FGF-2)–induced Net phosphorylation by the Ras-Erk signaling upstream from Ras. It also binds to the colchicine-binding site of tubulin, depolymerizes microtubules, stimulates cell membrane blebbing, and affects the morphology of the actin skeleton. Interestingly, Combretastin-A4, which produces similar effects on the cytoskeleton, also inhibits FGF-2 Ras-Net signaling. This differs from other classes of agents that target microtubules, which have either little effect (vincristine) or no effect (docetaxel and nocodazole) on the Ras-Net pathway. XRP44X inhibits various cellular properties, including cell growth, cell cycle progression, and aortal sprouting, similar to other molecules that bind to the tubulin colchicine site. XRP44X has the potentially interesting property of connecting two important pathways involved in cell transformation and may thereby represent an interesting class of molecules that could be developed for cancer treatment. [Cancer Res 2008;68(5):1275–83]

Angiogenesis is fundamental to physiological and pathological processes. Despite intensive efforts, little is known about the intracellular circuits that regulate angiogenesis. The transcription factor Net is activated by phosphorylation induced by Ras, an indirect regulator of angiogenesis. Net is expressed at sites of vasculogenesis and angiogenesis during early mouse development, suggesting that it could have a role in blood vessel formation. We show here that down-regulation of Net inhibits angiogenesis and vascular endothelial growth factor (VEGF) expression in vivo, ex vivo, and in vitro. Ras-activated phosphorylated Net (P-Net) stimulates the mouse VEGF promoter through the -80 to -53 region that principally binds Sp1. P-Net and VEGF are coexpressed in angiogenic processes in wild-type mouse tissues and in human tumors. We conclude that Net is a regulator of angiogenesis that can switch to an activator following induction by pro-angiogenic molecules.

We have previously isolated the hpttg proto-oncogene, which is expressed in normal tissues containing proliferating cells and in several kinds of tumors. In fact, expression of hPTTG correlates with cell proliferation in a cell cycle-dependent manner. Recently it was reported that PTTG is a vertebrate analog of the yeast securins Pds1 and Cut2, which are involved in sister chromatid separation. Here we show that hPTTG binds to Ku, the regulatory subunit of the DNA-dependent protein kinase (DNA-PK). hPTTG and Ku associate both in vitro and in vivo and the DNA-PK catalytic subunit phosphorylates hPTTG in vitro. Furthermore, DNA double-strand breaks prevent hPTTG-Ku association and disrupt the hPTTG-Ku complexes, indicating that genome damaging events, which result in the induction of pathways that activate DNA repair mechanisms and halt cell cycle progression, might inhibit hPTTG-Ku interaction in vivo. We propose that hPTTG might connect DNA damage-response pathways with sister chromatid separation, delaying the onset of mitosis while DNA repair occurs.

The MAPK pathway is required for T-cell activation; however, its role in modulating T-cell function following human immunodeficiency virus type 1 (HIV-1) infection is poorly understood. In this report, we investigated whether Grb3-3, an isoform of the Grb2 (growth factor receptor-bound protein-2) adaptor molecule that is associated with the MAPK pathway, could be involved. We found that Grb3-3, but not its isoform Grb2, is markedly up-regulated in CD4(+) peripheral blood mononuclear cells derived from either in vitro HIV-1-infected cultures or HIV-1-infected human subjects. Analysis of HIV-1 gene products indicated that Tat and Nef, both of which have been implicated in modulating T-cell function, can independently induce expression of Grb3-3. By using NFAT/AP-1, AP-1, or NFAT reporter assays, we found that Grb3-3 can potentiate NFAT (but not AP-1) promoter activity in Jurkat T-cells upon engagement of the T-cell receptor and CD28 co-receptor. In addition, potentiation of NFAT by Grb3-3 is substantially suppressed by MEKK1, a kinase that may play an important role in retaining NFAT in the cytoplasm, and by cyclosporin A. Finally, we also found that Grb3-3 potentiates HIV-1 long terminal (LTR) repeat promoter activity following T-cell receptor stimulation, an effect that can be largely suppressed by cyclosporin A. Taken together, this study indicates that Grb3-3 is a cellular factor that can be up-regulated by HIV-1. In addition, Grb3-3 can also function as a positive factor for T-cell activation and, in doing so, may aid in establishing an intracellular environment that can optimally support HIV-1 replication.

The mechanism(s) by which HIV-1 infection contributes to depletion of CD4(+) T cell is not well understood. In this report, we investigated whether a recently identified isoform of growth factor receptor bound protein (Grb2), named Grb3-3, a signaling molecule that is associated with the MAP kinase pathway and with apoptosis could be involved. We find that Grb3-3 is markedly up-regulated following HIV-1 infection of CD4(+) peripheral blood mononuclear cells undergoing apoptosis. Although IL-2 deprived CD4(+) cells also undergo apoptosis to a similar extent, Grb3-3 upregulation is not detected under these experimental conditions. Transient overexpression of Grb3-3 in Jurkat T-cells also causes apoptosis. Upon staurosporine stimulation, Grb3-3 predisposes Sup-T1 cell to apoptosis. Finally, analysis of the HIV-1 genes responsible for Grb3-3 expression demonstrates that Tat and Nef can independently induces its expression, suggesting these two earliest viral gene products of HIV-1 may share some common pathway(s) in up-regulating Grb3-3 expression.

We recently isolated a cDNA for hpttg, the human homolog of rat pituitary tumor transforming gene. Now we have analysed the expression of hpttg as a function of cell proliferation. hPTTG protein level is up-regulated in rapidly proliferating cells, is down-regulated in response to serum starvation or cell confluence, and is regulated in a cell cycle-dependent manner, peaking in mitosis. In addition, we show that hPTTG is phosphorylated during mitosis. Immunodepletion and in vitro phosphorylation experiments, together with the use of a specific inhibitor, indicate that Cdc2 is the kinase that phosphorylates hPTTG. These results suggest that hpttg is induced by, and may have a role in, regulatory pathways involved in the control of cell proliferation.