PlGF blockade does not inhibit angiogenesis during primary tumor growth.

It has been recently reported that treatment with an anti-placenta growth factor (PlGF) antibody inhibits metastasis and primary tumor growth. Here we show that, although anti-PlGF treatment inhibited wound healing, extravasation of B16F10 cells, and growth of a tumor engineered to overexpress the PlGF receptor (VEGFR-1), neutralization of PlGF using four novel blocking antibodies had no significant effect on tumor angiogenesis in 15 models. Also, genetic ablation of the tyrosine kinase domain of VEGFR-1 in the host did not result in growth inhibition of the anti-VEGF-A sensitive or resistant tumors tested. Furthermore, combination of anti-PlGF with anti-VEGF-A antibodies did not result in greater antitumor efficacy than anti-VEGF-A monotherapy. In conclusion, our data argue against an important role of PlGF during primary tumor growth in most models and suggest that clinical evaluation of anti-PlGF antibodies may be challenging.

Mathematical modeling of the microtubule detyrosination/tyrosination cycle for cell-based drug screening design.

Microtubules and their post-translational modifications are involved in major cellular processes. In severe diseases such as neurodegenerative disorders, tyrosinated tubulin and tyrosinated microtubules are in lower concentration. We present here a mechanistic mathematical model of the microtubule tyrosination cycle combining computational modeling and high-content image analyses to understand the key kinetic parameters governing the tyrosination status in different cellular models. That mathematical model is parameterized, firstly, for neuronal cells using kinetic values taken from the literature, and, secondly, for proliferative cells, by a change of two parameter values obtained, and shown minimal, by a continuous optimization procedure based on temporal logic constraints to formalize experimental high-content imaging data. In both cases, the mathematical models explain the inability to increase the tyrosination status by activating the Tubulin Tyrosine Ligase enzyme. The tyrosinated tubulin is indeed the product of a chain of two reactions in the cycle: the detyrosinated microtubule depolymerization followed by its tyrosination. The tyrosination status at equilibrium is thus limited by both reaction rates and activating the tyrosination reaction alone is not effective. Our computational model also predicts the effect of inhibiting the Tubulin Carboxy Peptidase enzyme which we have experimentally validated in MEF cellular model. Furthermore, the model predicts that the activation of two particular kinetic parameters, the tyrosination and detyrosinated microtubule depolymerization rate constants, in synergy, should suffice to enable an increase of the tyrosination status in living cells.

An integrative analysis of colon cancer identifies an essential function for PRPF6 in tumor growth.

The spliceosome machinery is composed of multimeric protein complexes that generate a diverse repertoire of mRNA through coordinated splicing of heteronuclear RNAs. While somatic mutations in spliceosome components have been discovered in several cancer types, the molecular bases and consequences of spliceosome aberrations in cancer are poorly understood. Here we report for the first time that PRPF6, a member of the tri-snRNP (small ribonucleoprotein) spliceosome complex, drives cancer proliferation by preferential splicing of genes associated with growth regulation. Inhibition of PRPF6 and other tri-snRNP complex proteins, but not other snRNP spliceosome complexes, selectively abrogated growth in cancer cells with high tri-snRNP levels. High-resolution transcriptome analyses revealed that reduced PRPF6 alters the constitutive and alternative splicing of a discrete number of genes, including an oncogenic isoform of the ZAK kinase. These findings implicate an essential role for PRPF6 in cancer via splicing of distinct growth-related gene products.

A putative new melatonin binding site in sheep brain, MTx: preliminary observations and characteristics.

In mammals, MT1 and MT2 melatonin receptors are high affinity G protein-coupled receptors and are thought to be involved in the integration of the melatonin signaling throughout the brain and periphery. In the present study, we describe a new melatonin binding site, named MTx, with a peculiar pharmacological profile. This site had a low affinity for 2-[125I]-melatonin in saturation assays in hypothalamus and retina (pKD = 9.13 {plus minus} 0.05, Bmax = 1.12 {plus minus} 0.11 fmol/mg protein and pKD = 8.81 {plus minus} 0.50, Bmax = 7.65 {plus minus} 2.64 fmol/mg protein, respectively) and a very high affinity, in competition assays, for melatonin (pKi = 13.08 {plus minus} 0.18), and other endogenous compounds. Using autoradiography, we showed a preferential localization of the MTx in periventricular areas of the sheep brain, with a density 3 to 8 times higher than those observed for ovine MT1 In addition, using a set of well-characterized ligands, we showed that this site did not correspond to any of the following receptors: MT1, MT2, MT3 , D1, D2, noradrenergic, nor 5-HT2 Based on its affinity for melatonin, MTx did not seem to be implicated in the integration of cerebral melatonin concentration variations since they were saturating for MTx. Nevertheless, it remained of prime importance because of its periventricular distribution, in close contact with the CSF, and its peculiar pharmacological profile responding to both melatoninergic and serotoninergic compounds. Significance Statement Herein a putative new melatonin binding site is described in sheep brain parts in close contact with the 3rd ventricle. The characteristics of the pharmacological profile of this site is different from anything previously reported in the literature. The present work forms the basis of future full pharmacological characterization.

TRAF2 Sets a threshold for extrinsic apoptosis by tagging caspase-8 with a ubiquitin shutoff timer.

Apoptotic caspase activation mechanisms are well defined, yet inactivation modes remain unclear. The death receptors (DRs), DR4, DR5, and Fas, transduce cell-extrinsic apoptotic signals by recruiting caspase-8 into a death-inducing signaling complex (DISC). At the DISC, Cullin3-dependent polyubiquitination on the small catalytic subunit of caspase-8 augments stimulation. Here we report that tumor necrosis factor receptor-associated factor 2 (TRAF2) interacts with caspase-8 at the DISC, downstream of Cullin3. TRAF2 directly mediates RING-dependent, K48-linked polyubiquitination on the large catalytic domain of caspase-8. This modification destines activated caspase-8 molecules to rapid proteasomal degradation upon autoprocessing and cytoplasmic translocation. TRAF2 depletion lowers the signal threshold for DR-mediated apoptosis, altering cell life versus death decisions in vitro and in vivo. Thus, TRAF2 sets a critical barrier for cell-extrinsic apoptosis commitment by tagging activated caspase-8 with a K48-ubiquitin shutoff timer. These results may have important implications for caspase regulation mechanisms.

Kinome Screen Identifies PFKFB3 and Glucose Metabolism as Important Regulators of the Insulin/Insulin-like Growth Factor (IGF)-1 Signaling Pathway.

The insulin/insulin-like growth factor (IGF)-1 signaling pathway (ISP) plays a fundamental role in long term health in a range of organisms. Protein kinases including Akt and ERK are intimately involved in the ISP. To identify other kinases that may participate in this pathway or intersect with it in a regulatory manner, we performed a whole kinome (779 kinases) siRNA screen for positive or negative regulators of the ISP, using GLUT4 translocation to the cell surface as an output for pathway activity. We identified PFKFB3, a positive regulator of glycolysis that is highly expressed in cancer cells and adipocytes, as a positive ISP regulator. Pharmacological inhibition of PFKFB3 suppressed insulin-stimulated glucose uptake, GLUT4 translocation, and Akt signaling in 3T3-L1 adipocytes. In contrast, overexpression of PFKFB3 in HEK293 cells potentiated insulin-dependent phosphorylation of Akt and Akt substrates. Furthermore, pharmacological modulation of glycolysis in 3T3-L1 adipocytes affected Akt phosphorylation. These data add to an emerging body of evidence that metabolism plays a central role in regulating numerous biological processes including the ISP. Our findings have important implications for diseases such as type 2 diabetes and cancer that are characterized by marked disruption of both metabolism and growth factor signaling.

High-throughput drug profiling with voltage- and calcium-sensitive fluorescent probes in human iPSC-derived cardiomyocytes.

Cardiomyocytes derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) are increasingly used for in vitro assays and represent an interesting opportunity to increase the data throughput for drug development. In this work, we describe a 96-well recording of synchronous electrical activities from spontaneously beating hiPSC-derived cardiomyocyte monolayers. The signal was obtained with a fast-imaging plate reader using a submillisecond-responding membrane potential recording assay, FluoVolt, based on a newly derived voltage-sensitive fluorescent dye. In our conditions, the toxicity of the dye was moderate and compatible with episodic recordings for >3 h. We show that the waveforms recorded from a whole well or from a single cell-sized zone are equivalent and make available critical functional parameters that are usually accessible only with gold standard techniques like intracellular microelectrode recording. This approach allows accurate identification of the electrophysiological effects of reference drugs on the different phases of the cardiac action potential as follows: fast depolarization (lidocaine), early repolarization (nifedipine, Bay K8644, and veratridine), late repolarization (dofetilide), and diastolic slow depolarization (ivabradine). Furthermore, the data generated with the FluoVolt dye can be pertinently complemented with a calcium-sensitive dye for deeper characterization of the pharmacological responses. In a semiautomated plate reader, the two probes used simultaneously in 96-well plates provide an easy and powerful multiparametric assay to rapidly and precisely evaluate the cardiotropic profile of compounds for drug discovery or cardiac safety.

Application of a new wall-less plate technology to complex multistep cell-based investigations using suspension cells.

Despite significant progresses, cell-based assays still have major limitations part to because of their plate format. Here, we present a wall-less plate technology based on unique liquid dynamics named DropArray that takes advantage of hydrophobic and hydrophilic surface properties. Liquid velocities within the DropArray plate were quantified through fluid dynamics simulation and complete retention of suspension cells experimentally demonstrated within the range of simulated shear stresses. Subsequently, we compared the DropArray technology with conventional microtiter plates in a cell-based protein-binding assay. Although the wall-less plate produced similar results with adherent cells, the advantage of the DropArray technology was absolutely clear when semiadherent or suspension cells were used in this multistep experimental procedure. The technology also was evaluated for the cell viability assay and generated similar results to conventional plate format while enabling significant reduction in toxic reagent use. Finally, we developed a DropArray cell-based assay to evaluate a bispecific antibody designed to engage cytotoxic T cells and trigger tumor cell killing. This assay enables for the first time the visualization and quantification of the specific killing events and represents a very powerful tool to further investigate functional aspects of the cancer immunotherapy.

Antibodies against the gH/gL/UL128/UL130/UL131 complex comprise the majority of the anti-cytomegalovirus (anti-CMV) neutralizing antibody response in CMV hyperimmune globulin.

Anti-cytomegalovirus (anti-CMV) hyperimmune globulin (HIG) has demonstrated efficacy in preventing CMV disease in solid-organ transplant patients as well as congenital disease when administered to pregnant women. To identify the neutralizing component of cytomegalovirus hyperimmune globulin (CMV-HIG), we performed serial depletions of CMV-HIG on cell-surface-expressed CMV antigens as well as purified antigens. Using this approach, we demonstrate that the major neutralizing antibody response is directed at the gH/gL/UL128/UL130/UL131 complex, suggesting little role for anti-gB antibodies in CMV-HIG neutralization.

Ultrasensitive detection of cellular protein interactions using bioluminescence resonance energy transfer quantum dot-based nanoprobes.

Sensitive detection of protein interactions is a critical step toward understanding complex cellular processes. As an alternative to fluorescence-based detection, Renilla reniformis luciferase conjugated to quantum dots results in self-illuminating bioluminescence resonance energy transfer quantum dot (BRET-Qdot) nanoprobes that emit red to near-infrared bioluminescence light. Here, we report the development of an ultrasensitive technology based on BRET-Qdot conjugates modified with streptavidin ([BRET-Qdot]-SA) to detect cell-surface protein interactions. Transfected COS7 cells expressing human cell-surface proteins were interrogated with a human Fc tagged protein of interest. Specific protein interactions were detected using a biotinylated anti-human Fc region specific antibody followed by incubation with [BRET-Qdot]-SA. The luciferase substrate coelenterazine activated bioluminescence light emission was detected with an ultra-fast and -sensitive imager. Protein interactions barely detectable by the fluorescence-based approach were readily quantified using this technology. The results demonstrate the successful application and the flexibility of the BRET-Qdot-based imaging technology to the ultrasensitive investigation of cell-surface proteins and protein-protein interactions.

[From high content screening to target deconvolution: New insights for phenotypic approaches]. / Du criblage à haut contenu à la déconvolution de cibles - Nouvelle donne pour les approches phénotypiques.

The advent of the molecular biology and the completion of the human genome sequencing prompted the pharmaceutical industry to progressively implement target-centric drug discovery strategies. However, concerns regarding the research and development productivity during the last ten years, combined with technological developments in high-content screening, automation, image analysis and artificial intelligence triggered a renewed interest for the phenotypic drug discovery approaches. Target-centric and phenotypic approaches are more and more considered complementary, hence, positioning the target deconvolution on the critical path. This review analyzes the evolution of the target-centric and phenotypic approaches, focusing more specifically on the high-content screening and the target deconvolution technologies currently available.

TITLE: Du criblage à haut contenu à la déconvolution de cibles - Nouvelle donne pour les approches phénotypiques. ABSTRACT: L'avènement de la biologie moléculaire et l'achèvement du séquençage du génome humain ont conduit l'industrie pharmaceutique à progressivement implémenter des approches dites cible-centriques pour identifier les candidats médicaments. Cependant, la faible productivité de la recherche et du développement en ce début de millénaire, combinée aux évolutions technologiques dans des domaines tels que l'ingénierie cellulaire, le criblage à haut contenu, la robotique, l'analyse d'images et l'intelligence artificielle, ont nourri un fort regain d'intérêt pour les approches phénotypiques. De plus en plus fréquemment, les approches cible-centriques et phénotypiques sont considérées de façon complémentaire, positionnant ainsi les techniques de déconvolution1 de cible sur le chemin critique de la découverte et du développement de médicaments. Cette revue analyse l'évolution des approches cible-centriques versus phénotypiques, en se focalisant plus particulièrement sur le criblage à haut contenu et les différentes techniques de déconvolution de cible aujourd'hui disponibles.

Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake.

The insulin-like growth factor (IGF) system consists of two ligands (IGF-I and IGF-II), which both signal through IGF-I receptor (IGF-IR) to stimulate proliferation and inhibit apoptosis, with activity contributing to malignant growth of many types of human cancers. We have developed a humanized, affinity-matured anti-human IGF-IR monoclonal antibody (h10H5), which binds with high affinity and specificity to the extracellular domain. h10H5 inhibits IGF-IR-mediated signaling by blocking IGF-I and IGF-II binding and by inducing cell surface receptor down-regulation via internalization and degradation, with the extracellular and intracellular domains of IGF-IR being differentially affected by the proteasomal and lysosomal inhibitors. In vitro, h10H5 exhibits antiproliferative effects on cancer cell lines. In vivo, h10H5 shows single-agent antitumor efficacy in human SK-N-AS neuroblastoma and SW527 breast cancer xenograft models and even greater efficacy in combination with the chemotherapeutic agent docetaxel or an anti-vascular endothelial growth factor antibody. Antitumor activity of h10H5 is associated with decreased AKT activation and glucose uptake and a 316-gene transcription profile with significant changes involving DNA metabolic and cell cycle machineries. These data support the clinical testing of h10H5 as a biotherapeutic for IGF-IR-dependent human tumors and furthermore illustrate a new method of monitoring its activity noninvasively in vivo via 2-fluoro-2-deoxy-d-glucose-positron emission tomography imaging.

High CD21 expression inhibits internalization of anti-CD19 antibodies and cytotoxicity of an anti-CD19-drug conjugate.

CD19 and CD21 (CR2) are co-receptors found on B-cells and various B-cell lymphomas, including non-Hodgkin lymphoma. To evaluate their suitability as targets for therapy of such lymphomas using internalization-dependent antibody-drug conjugates [such as antibody-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, (N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine) (MCC-DM1) conjugates, which require lysosomal degradation of the antibody moiety for efficacy], we examined uptake of antibodies to CD19 and CD21 in a panel of B-cell lines. Anti-CD21 antibodies were not sufficiently internalized even in the highest CD21-expressing Raji cells, resulting in lack of efficacy with anti-CD21-MCC-DM1 conjugates. Anti-CD19 antibody uptake was variable, and was unexpectedly negatively correlated with CD21 expression. Thus, high CD21-expressing Raji, ARH77 and primary B-cells only very slowly internalized anti-CD19 antibodies, while CD21-negative or low expressing cells, including Ramos and Daudi, rapidly internalized these antibodies in clathrin-coated vesicles followed by lysosomal delivery. Anti-CD19-MCC-DM1 caused greater cytotoxicity in the faster anti-CD19-internalizing cell lines, implying that the rate of lysosomal delivery and subsequent drug release is important. Furthermore, transfection of Ramos cells with CD21 impeded anti-CD19 uptake and decreased anti-CD19-MCC-DM1 efficacy, suggesting that CD21-negative tumours should respond better to such anti-CD19 conjugates. This may have possible clinical implications, as anti-CD21 immunohistochemistry revealed only approximately 30% of 54 diffuse large B-cell lymphoma patients lack CD21 expression.

Anti-CD22-MCC-DM1 and MC-MMAF conjugates: impact of assay format on pharmacokinetic parameters determination.

CD22 represents a promising target for antibody-drug conjugate therapy in the context of B cell malignancies since it rapidly internalizes, importing specifically bound antibodies with it. To determine the pharmacokinetic parameters of anti-CD22-MCC-DM1 and MC-MMAF conjugates, various approaches to quantifying total and conjugated antibody were investigated. Although the total antibody assay formats gave similar results for both conjugates, the mouse pharmacokinetic profile for the anti-CD22-MCC-DM1 and MC-MMAF appeared significantly different depending on the conjugated antibody assay format. Since these differences significantly impacted the PK parameters determination, we investigated the effect of the drug/antibody ratio on the total and conjugated antibody quantification using multiple assay formats. Our investigations revealed the limitations of some assay formats to quantify anti-CD22-MCC-DM1 and MC-MMAF with different drug load and in the context of a heterogeneous ADC population highlight the need to carefully plan the assay strategy for the total and conjugated antibody quantification in order to accurately determine the ADC PK parameters.

Filling the drug discovery gap: is high-content screening the missing link?

In recent years, questions about the sustainability of the current drug discovery process have triggered a revival of interest in phenotypic drug discovery approaches. This trend has clearly been amplified by the emergence of multiple cell-based assay technologies enabling a higher degree of translatability between in vitro conditions and physio-pathological situations, including induced pluripotent stem cells, three-dimensional models, co-culture and organ-on-a-chip systems, complemented by advances in gene editing technologies. Progress in High-Content Screening technology has also contributed to the recent excitement for phenotypic drug discovery approaches, bringing image-capture and processing, and data-analysis, to a level of content and throughput fully compatible with large scale drug discovery efforts. Nevertheless, implementation of HCS in discovery projects must be carefully considered, to ensure optimal performance and the generation of relevant data to enable the discovery of first-in-class medicines.

SUV420H2 is an epigenetic regulator of epithelial/mesenchymal states in pancreatic cancer.

Epithelial-to-mesenchymal transition is implicated in metastasis, where carcinoma cells lose sessile epithelial traits and acquire mesenchymal migratory potential. The mesenchymal state is also associated with cancer stem cells and resistance to chemotherapy. It might therefore be therapeutically beneficial to promote epithelial identity in cancer. Because large-scale cell identity shifts are often orchestrated on an epigenetic level, we screened for candidate epigenetic factors and identified the histone methyltransferase SUV420H2 (KMT5C) as favoring the mesenchymal identity in pancreatic cancer cell lines. Through its repressive mark H4K20me3, SUV420H2 silences several key drivers of the epithelial state. Its knockdown elicited mesenchymal-to-epithelial transition on a molecular and functional level, and cells displayed decreased stemness and increased drug sensitivity. An analysis of human pancreatic cancer biopsies was concordant with these findings, because high levels of SUV420H2 correlated with a loss of epithelial characteristics in progressively invasive cancer. Together, these data indicate that SUV420H2 is an upstream epigenetic regulator of epithelial/mesenchymal state control.

Gene expression profiling during hibernation in the European hamster.

Hibernation is an exceptional physiological response to a hostile environment, characterized by a seasonal period of torpor cycles involving dramatic reductions of body temperature and metabolism, and arousal back to normothermia. As the mechanisms regulating hibernation are still poorly understood, here we analysed the expression of genes involved in energy homeostasis, torpor regulation, and daily or seasonal timing using digital droplet PCR in various central and peripheral tissues sampled at different stages of torpor/arousal cycles in the European hamster. During torpor, the hypothalamus exhibited strongly down-regulated gene expression, suggesting that hypothalamic functions were reduced during this period of low metabolic activity. During both torpor and arousal, many structures (notably the brown adipose tissue) exhibited altered expression of deiodinases, potentially leading to reduced tissular triiodothyronine availability. During the arousal phase, all analysed tissues showed increased expression of the core clock genes Per1 and Per2. Overall, our data indicated that the hypothalamus and brown adipose tissue were the tissues most affected during the torpor/arousal cycle, and that clock genes may play critical roles in resetting the body's clocks at the beginning of the active period.

Repression of Stress-Induced LINE-1 Expression Protects Cancer Cell Subpopulations from Lethal Drug Exposure.

Maintenance of phenotypic heterogeneity within cell populations is an evolutionarily conserved mechanism that underlies population survival upon stressful exposures. We show that the genomes of a cancer cell subpopulation that survives treatment with otherwise lethal drugs, the drug-tolerant persisters (DTPs), exhibit a repressed chromatin state characterized by increased methylation of histone H3 lysines 9 and 27 (H3K9 and H3K27). We also show that survival of DTPs is, in part, maintained by regulators of H3K9me3-mediated heterochromatin formation and that the observed increase in H3K9me3 in DTPs is most prominent over long interspersed repeat element 1 (LINE-1). Disruption of the repressive chromatin over LINE-1 elements in DTPs results in DTP ablation, which is partially rescued by reducing LINE-1 expression or function.

Nucleic acid capture assay, a new method for direct quantitation of nucleic acids.

Technologies allowing direct detection of specific RNA/DNA sequences occasionally serve as an alternative to amplification methods for gene expression studies. In these direct methods the hybridization of probes takes place in complex mixtures, thus specificity and sensitivity still limit the use of current technologies. To address these challenges, we developed a new technique called the nucleic acid capture assay, involving a direct multi-capture system. This approach combines a 3'-ethylene glycol scaffolding with the incorporation of 2'-methoxy deoxyribonucleotides in the capture sequences. In our design, all nucleotides other than those complementary to the target mRNA have been replaced by an inert linker, resulting in significant reductions in non-specific binding. We also provide a versatile method to detect the presence of captured targets by using specific labeled probes with alkaline phosphatase-conjugated anti-label antibodies. This direct, flexible and reliable technique for gene expression analysis is well suited for high-throughput screening and has potential for DNA microarray applications.

Proline Starvation Induces Unresolved ER Stress and Hinders mTORC1-Dependent Tumorigenesis.

The role of essential amino acids in metabolic reprogramming of cancer cells is now well established, whereas the role of non-essential amino acids (NEAAs) in malignancy remains less clear. Here, we have identified an important role for the NEAA proline in the tumorigenic potential of a subset of cancer cells. By profiling a large panel of cancer cell lines, we observed that proline consumption and expression of proline biosynthesis enzymes were well correlated with clonogenic and tumorigenic potential. Moreover, proline starvation or inhibition of proline biosynthesis enzymes impaired clonogenic/tumorigenic potential. Cancer cells exhibiting dependency on exogenous proline displayed hyperactivation of the mTORC1-mediated 4EBP1 signaling axis, as well as unresolved ER stress. Exogenous proline alleviated ER stress and promoted cellular homeostasis and clonogenicity. Increased dependence on proline may therefore define a specific vulnerability in some cancers that can be exploited by proline depletion.