RESUMEN
Test compounds used on in vitro model systems are conventionally delivered to cell culture wells as fixed concentration bolus doses; however, this poorly replicates the pharmacokinetic (PK) concentration changes seen in vivo and reduces the predictive value of the data. Herein, proof-of-concept experiments were performed using a novel microfluidic device, the Microformulator, which allows in vivo like PK profiles to be applied to cells cultured in microtiter plates and facilitates the investigation of the impact of PK on biological responses. We demonstrate the utility of the device in its ability to reproduce in vivo PK profiles of different oncology compounds over multiweek experiments, both as monotherapy and drug combinations, comparing the effects on tumour cell efficacy in vitro with efficacy seen in in vivo xenograft models. In the first example, an ERK1/2 inhibitor was tested using fixed bolus dosing and Microformulator-replicated PK profiles, in 2 cell lines with different in vivo sensitivities. The Microformulator-replicated PK profiles were able to discriminate between cell line sensitivities, unlike the conventional fixed bolus dosing. In a second study, murine in vivo PK profiles of multiple Poly(ADP-Ribose) Polymerase 1/2 (PARP) and DNA-dependent protein kinase (DNA-PK) inhibitor combinations were replicated in a FaDu cell line resulting in a reduction in cell growth in vitro with similar rank ordering to the in vivo xenograft model. Additional PK/efficacy insight into theoretical changes to drug exposure profiles was gained by using the Microformulator to expose FaDu cells to the DNA-PK inhibitor for different target coverage levels and periods of time. We demonstrate that the Microformulator enables incorporating PK exposures into cellular assays to improve in vitro-in vivo translation understanding for early therapeutic insight.
Asunto(s)
Técnicas de Cultivo de Célula , Microfluídica , Animales , ADN , Humanos , Ratones , Modelos BiológicosRESUMEN
The function of the tumor suppressor RE1 silencing transcription factor (REST) is lost in colon and small cell lung cancers and is known to induce anchorage-independent growth in human mammary epithelial cells. However, nothing is currently known about the role of this tumor suppressor in breast cancer. Here, we test the hypothesis that loss of REST function plays a role in breast cancer. To assay breast tumors for REST function, we developed a 24-gene signature composed of direct targets of the transcriptional repressor. Using the 24- gene signature, we identified a previously undefined RESTless breast tumor subtype. Using gene set enrichment analysis, we confirmed the aberrant expression of REST target genes in the REST-less tumors, including neuronal gene targets of REST that are normally not expressed outside the nervous system. Examination of REST mRNA identified a truncated splice variant of REST present in the REST-less tumor population, but not other tumors. Histological analysis of 182 outcome-associated breast tumor tissues also identified a subpopulation of tumors that lack full-length, functional REST and over-express the neuroendocrine marker and REST target gene Chromogranin A. Importantly, patients whose tumors were found to be REST-less using either the 24-gene signature or histology had significantly poorer prognosis and were more than twice as likely to undergo disease recurrence within the first 3 years after diagnosis. We show here that REST function is lost in breast cancer, at least in part via an alternative splicing mechanism. Patients with REST-less breast cancer undergo significantly more early disease recurrence than those with fully functional REST, regardless of estrogen receptor or HER2 status. Importantly, REST status may serve as a predictor of poor prognosis, helping to untangle the heterogeneity inherent in disease course and response to treatment. Additionally, the alternative splicing observed in REST-less breast cancer is an attractive therapeutic target.
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Empalme Alternativo , Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Represoras/genética , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/terapia , Línea Celular , Perfilación de la Expresión Génica , Humanos , Pronóstico , ARN Mensajero/genética , Resultado del TratamientoRESUMEN
BACKGROUND: Glioblastomas are the most common central nervous system neoplasia in adults, with 9,000 cases in the US annually. Glioblastoma multiformae, the most aggressive glioma subtype, has an 18% one-year survival rate, and 3% two year survival rate. Recent work has highlighted the role of the transcription factor RE1 Silencing Transcription Factor, REST in glioblastoma but how REST function correlates with disease outcome has not been described. METHOD: Using a bioinformatic approach and mining of publicly available microarray datasets, we describe an aggressive subtype of gliomas defined by a gene signature derived from REST. Using this REST gene signature we predict that REST function is enhanced in advanced glioblastoma. We compare disease outcomes between tumors based on REST status and treatment regimen, and describe downstream targets of REST that may contribute to the decreased benefits observed with high dose chemotherapy in REM tumors. RESULTS: We present human data showing that patients with "REST Enhanced Malignancies" (REM) tumors present with a shorter disease free survival compared to non-REM gliomas. Importantly, REM tumors are refractory to multiple rounds of chemotherapy and patients fail to respond to this line of treatment. CONCLUSIONS: This report is the first to describe a REST gene signature that predicts response to multiple rounds of chemotherapy, the mainline therapy for this disease. The REST gene signature may have important clinical implications for the treatment of glioblastoma.
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Biomarcadores de Tumor/genética , Glioblastoma/clasificación , Glioblastoma/genética , Glioblastoma/fisiopatología , Proteínas Represoras/genética , Análisis por Conglomerados , Biología Computacional , Dosificación de Gen , Perfilación de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Análisis por MicromatricesRESUMEN
Phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) modulates a plethora of cytoskeletal interactions that control the dynamics of actin assembly and, ultimately, cell migration. We show that the type Igamma phosphatidylinositol phosphate kinase 661 (PIPKIgamma661), an enzyme that generates PI4,5P(2), is required for growth factor but not G protein-coupled receptor-stimulated directional migration. By generating PI4,5P(2) and regulating talin assembly, PIPKIgamma661 modulates nascent adhesion formation at the leading edge to facilitate cell migration. The epidermal growth factor (EGF) receptor directly phosphorylates PIPKIgamma661 at tyrosine 634, and this event is required for EGF-induced migration. This phosphorylation regulates the interaction between PIPKIgamma661 and phospholipase Cgamma1 (PLCgamma1, an enzyme previously shown to be involved in the regulation of EGF-stimulated migration). Our results suggest that phosphorylation events regulating specific PIPKIgamma661 interactions are required for growth factor-induced migration. These interactions in turn define the spatial and temporal generation of PI4,5P(2) and derived messengers required for directional migration.
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Movimiento Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Línea Celular Tumoral , Células HeLa , Humanos , Hidrólisis , Modelos Biológicos , Mutación , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipasa C gamma/metabolismo , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Recombinantes/metabolismo , Talina/metabolismo , Tirosina/metabolismoRESUMEN
Drug-induced neurotoxicity is a leading cause of safety-related attrition for therapeutics in clinical trials, often driven by poor predictivity of preclinical in vitro and in vivo models of neurotoxicity. Over a dozen different iPSC-derived 3D spheroids have been described in recent years, but their ability to predict neurotoxicity in patients has not been evaluated nor compared with the predictive power of nonclinical species. To assess the predictive capabilities of human iPSC-derived neural spheroids (microBrains), we used 84 structurally diverse pharmaceuticals with robust clinical and pre-clinical datasets with varying degrees of seizurogenic and neurodegenerative liability. Drug-induced changes in neural viability and phenotypic calcium bursts were assessed using 7 endpoints based on calcium oscillation profiles and cel-lular ATP levels. These endpoints, normalized by therapeutic exposure, were used to build logistic regression models to establish endpoint cutoffs and evaluate probability for clinical neurotoxicity. The neurotoxicity score calculated from the logistic regression model could distinguish neurotoxic from non-neurotoxic clinical molecules with a specificity as high as 93.33% and a sensitivity of 53.49%, demonstrating a very low false positive rate for the prediction of seizures, convulsions, and neurodegeneration. In contrast, nonclinical species showed a higher sensitivity (75%) but much lower specificity (30.4%). The neural spheroids demonstrated higher likelihood ratio positive and inverse likelihood ratio neg-ative values compared with nonclinical safety studies. This assay has the potential to be used as a predictive assay to detect neurotoxicity in early drug discovery, aiding in the early identification of compounds that eventually may fail due to neurotoxicity.
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Células Madre Pluripotentes Inducidas , Síndromes de Neurotoxicidad , Humanos , Síndromes de Neurotoxicidad/etiología , Convulsiones/inducido químicamente , Señalización del Calcio , Preparaciones FarmacéuticasRESUMEN
Complex in vitro models (CIVM) offer the potential to improve pharmaceutical clinical drug attrition due to safety and/ or efficacy concerns. For this technology to have an impact, the establishment of robust characterization and qualification plans constructed around specific contexts of use (COU) is required. This article covers the output from a workshop between the Food and Drug Administration (FDA) and Innovation and Quality Microphysiological Systems (IQ MPS) Affiliate. The intent of the workshop was to understand how CIVM technologies are currently being applied by pharmaceutical companies during drug development and are being tested at the FDA through various case studies in order to identify hurdles (real or perceived) to the adoption of microphysiological systems (MPS) technologies, and to address evaluation/qualification pathways for these technologies. Output from the workshop includes the alignment on a working definition of MPS, a detailed description of the eleven CIVM case studies presented at the workshop, in-depth analysis, and key take aways from breakout sessions on ADME (absorption, distribution, metabolism, and excretion), pharmacology, and safety that covered topics such as qualification and performance criteria, species differences and concordance, and how industry can overcome barriers to regulatory submission of CIVM data. In conclusion, IQ MPS Affiliate and FDA scientists were able to build a general consensus on the need for animal CIVMs for preclinical species to better determine species concordance. Furthermore, there was acceptance that CIVM technologies for use in ADME, pharmacology and safety assessment will require qualification, which will vary depending on the specific COU.
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Alternativas a las Pruebas en Animales , Dispositivos Laboratorio en un Chip , Animales , Evaluación Preclínica de Medicamentos , Industria Farmacéutica , Preparaciones Farmacéuticas/metabolismo , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Cell migration requires the coordination of many biochemical events, including cell-matrix contact turnover and cytoskeletal restructuring. Recent advances further implicate phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P(2)] in the control of these events. Many proteins that are crucial to the assembly of the migration machinery are regulated by PtdIns(4,5)P(2). Coordinated synthesis of PtdIns(4,5)P(2) at these sites is dependent on the precise targeting of the type I phosphatidylinositol phosphate kinases (PIPKs). Two PIPKI isoforms target to, and generate, PtdIns(4,5)P(2) at membrane ruffles and focal adhesions during cell migration. Here, we discuss our current understanding of PtdIns(4,5)P(2) in the regulation of cell responses to migratory stimuli and how the migrating cell controls PtdIns(4,5)P(2) availability.
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Movimiento Celular/fisiología , Fosfatidilinositol 4,5-Difosfato/fisiología , Animales , Extensiones de la Superficie Celular/fisiología , Adhesiones Focales/fisiología , Modelos Biológicos , Fosfatidilinositol 4,5-Difosfato/biosíntesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Transducción de Señal , Distribución TisularRESUMEN
An important mechanism of chemical toxicity is the induction of oxidative stress through the production of excess reactive oxygen species (ROS). In this study, we show that the level of drug-induced ROS production between NRK52E and HepG2 cells is significantly different for several marketed drugs and a number of Takeda's internal proprietary compounds. Nifedipine, a calcium channel blocker and the initial focus of the study, was demonstrated to promote in vitro ROS production and a decrease in cell viability in NRK52E cells but not HepG2 cells. ROS production after nifedipine treatment was inhibited by a NOX inhibitor (GKT136901) but not the mitochondrial NADH dehydrogenase inhibitor, rotenone, suggesting that nifedipine decreases NRK52E cell viability primarily through a NOX-mediated pathway. To understand the breadth of NOX-mediated ROS production, 12 commercially available compounds that are structurally and/or pharmacologically related to nifedipine as well as 172 internal Takeda candidate drugs, were also evaluated against these two cell types. Over 15 % of compounds not cytotoxic to HepG2 cells (below 50 µM) were cytotoxic to NRK52E cells. Our results suggest that a combination of cell viability data from both NRK52E and HepG2 cells was superior for the prediction of in vivo toxicity findings when compared to use of only one cell line. Further, the NRK52E cell viability assay is a good predictor of NOX-mediated ROS production and can be used as a follow up assay following a negative HepG2 response to aid in the selection of suitable compounds for in vivo toxicity studies.
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Células Epiteliales/efectos de los fármacos , Riñón/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Bioensayo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Drogas en Investigación/toxicidad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Células Hep G2 , Humanos , Concentración 50 Inhibidora , Riñón/metabolismo , Riñón/patología , NADPH Oxidasa 4/genética , Nifedipino/toxicidadRESUMEN
Drug induced kidney injury (DIKI) is a common reason for compound attrition in drug development pipelines with proximal tubule epithelial cells (PTECs) most commonly associated with DIKI. Here, we investigated freshly isolated human (hPTECs) as an in vitro model for assessing renal tubular toxicity. The freshly isolated hPTECs were first characterized to confirm gene expression of important renal transporters involved in drug handling which was further corroborated by confirming the functional activity of organic cation transporter 2 and organic anion transporter 1 by using transporter specific inhibitors. Additionally, functionality of megalin/cubilin endocytic receptors was also confirmed. A training set of 36 compounds was used to test the ability of the model to classify them using six different endpoints which included three biomarkers (Kidney Injury Molecule-1, Neutrophil gelatinase-associated lipocalin, and Clusterin) and three non-specific injury endpoints (ATP depletion, LDH leakage, and barrier permeability via transepithelial electrical resistance) in a dose-dependent manner across two independent kidney donors. In general, biomarkers showed higher predictivity than non-specific endpoints, with Clusterin showing the highest predictivity (Sensitivity/Specificity - 65.0/93.8 %). By using the thresholds generated from the training set, nine candidate internal Takeda compounds were screened where PTEC toxicity was identified as one of the findings in preclinical animal studies. The model correctly classified four of six true positives and two of three true negatives, showing validation of the in vitro model for detection of tubular toxicants. This work thus shows the potential application of freshly isolated primary hPTECs using translational biomarkers in assessment of tubular toxicity within the drug discovery pipeline.
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Síndrome de Fanconi/inducido químicamente , Síndrome de Fanconi/patología , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/patología , Cultivo Primario de Células/métodos , Biomarcadores/análisis , Determinación de Punto Final , Síndrome de Fanconi/genética , Expresión Génica/genética , Humanos , Factor 1 de Transcripción de Unión a Octámeros/genética , Transportador 2 de Cátion Orgánico/genética , Reproducibilidad de los ResultadosRESUMEN
Drug-induced gastrointestinal toxicities (GITs) rank among the most common clinical side effects. Preclinical efforts to reduce incidence are limited by inadequate predictivity of in vitro assays. Recent breakthroughs in in vitro culture methods support intestinal stem cell maintenance and continual differentiation into the epithelial cell types resident in the intestine. These diverse cells self-assemble into microtissues with in vivo-like architecture. Here, we evaluate human GI microtissues grown in transwell plates that allow apical and/or basolateral drug treatment and 96-well throughput. Evaluation of assay utility focused on predictivity for diarrhea because this adverse effect correlates with intestinal barrier dysfunction which can be measured in GI microtissues using transepithelial electrical resistance (TEER). A validation set of widely prescribed drugs was assembled and tested for effects on TEER. When the resulting TEER inhibition potencies were adjusted for clinical exposure, a threshold was identified that distinguished drugs that induced clinical diarrhea from those that lack this liability. Microtissue TEER assay predictivity was further challenged with a smaller set of drugs whose clinical development was limited by diarrhea that was unexpected based on 1-month animal studies. Microtissue TEER accurately predicted diarrhea for each of these drugs. The label-free nature of TEER enabled repeated quantitation with sufficient precision to develop a mathematical model describing the temporal dynamics of barrier damage and recovery. This human 3D GI microtissue is the first in vitro assay with validated predictivity for diarrhea-inducing drugs. It should provide a platform for lead optimization and offers potential for dose schedule exploration.
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Diarrea/inducido químicamente , Evaluación de Medicamentos/métodos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Células Epiteliales/fisiología , Células Epiteliales/ultraestructura , Células CACO-2 , Diferenciación Celular , Impedancia Eléctrica , Humanos , Preparaciones Farmacéuticas , Cultivo Primario de CélulasRESUMEN
The epithelial to mesenchymal transition (EMT) is the breakdown of epithelial cell morphology that gives way to a more mobile, mesenchymal phenotype. Although this process is fundamental to the development of multicellular organisms, it is also a key occurrence in many diseases, including cancers of epithelial origin E-cadherin is a central component of adherens junctions (AJs), which act as structural and signaling hubs in epithelial cells that oppose EMT. The loss of E-cadherin from the plasma membrane is an early indication of EMT and a marker of poor prognosis in many cancers making the trafficking of E-cadherin an area of great interest. Recent work from the authors' laboratory has established the role of type I gamma phosphatidylinositol 4-phosphate 5-kinase (PIPKI gamma) in the trafficking of E-cadherin by studying the surface accessibility of E-cadherin in endocytosis and recycling assays. Additionally, immunofluorescence data demonstrated that cells lacking PIPKI gamma lost E-cadherin at the plasma membrane. The biochemical and microscopic techniques used to investigate the trafficking of E-cadherin are presented herein.
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Cadherinas/metabolismo , Membrana Celular/metabolismo , Biología Molecular/métodos , Animales , Bioensayo , Biotina/metabolismo , Western Blotting , Línea Celular , Perros , Endocitosis , Técnica del Anticuerpo Fluorescente , Fosfotransferasas (Aceptor de Grupo Alcohol)/deficiencia , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transporte de Proteínas , Coloración y EtiquetadoRESUMEN
Despite six decades of clinical experience with the polymyxin class of antibiotics, their dose-limiting nephrotoxicity remains difficult to predict due to a paucity of sensitive biomarkers. Here, we evaluate the performance of standard of care and next-generation biomarkers of renal injury in the detection and monitoring of polymyxin-induced acute kidney injury in male Han Wistar rats using colistin (polymyxin E) and a polymyxin B (PMB) derivative with reduced nephrotoxicity, PMB nonapeptide (PMBN). This study provides the first histopathological and biomarker analysis of PMBN, an important test of the hypothesis that fatty acid modifications and charge reductions in polymyxins can reduce their nephrotoxicity. The results indicate that alterations in a panel of urinary kidney injury biomarkers can be used to monitor histopathological injury, with Kim-1 and α-GST emerging as the most sensitive biomarkers outperforming clinical standards of care, serum or plasma creatinine and blood urea nitrogen. To enable the prediction of polymyxin-induced nephrotoxicity, an in vitro cytotoxicity assay was employed using human proximal tubule epithelial cells (HK-2). Cytotoxicity data in these HK-2 cells correlated with the renal toxicity detected via safety biomarker data and histopathological evaluation, suggesting that in vitro and in vivo methods can be incorporated within a screening cascade to prioritize polymyxin class analogs with more favorable renal toxicity profiles.
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Antibacterianos/toxicidad , Colistina/toxicidad , Enfermedades Renales/orina , Polimixina B/análogos & derivados , Animales , Antibacterianos/administración & dosificación , Antibacterianos/farmacocinética , Biomarcadores/orina , Línea Celular , Supervivencia Celular/efectos de los fármacos , Colistina/administración & dosificación , Colistina/farmacocinética , Interpretación Estadística de Datos , Relación Dosis-Respuesta a Droga , Diagnóstico Precoz , Enfermedades Renales/inducido químicamente , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Masculino , Polimixina B/administración & dosificación , Polimixina B/farmacocinética , Polimixina B/toxicidad , Pronóstico , Ratas , Ratas WistarRESUMEN
The transcription factor RE1 silencing transcription factor (REST) is lost in approximately 20% of breast cancers. Although it is known that these RESTless tumors are highly aggressive and include all tumor subtypes, the underlying tumorigenic mechanisms remain unknown. In this study, we show that loss of REST results in upregulation of LIN28A, a known promoter of tumor development, in breast cancer cell lines and human breast tumors. We found that LIN28A was a direct transcriptional target of REST in cancer cells and that loss of REST resulted in increased LIN28A expression and enhanced tumor growth both in vitro and in vivo, effects that were dependent on heightened LIN28A expression. Tumors lacking REST expression were locally invasive, consistent with the increased lymph node involvement observed in human RESTless tumors. Clinically, human RESTless breast tumors also displayed significantly enhanced LIN28A expression when compared with non-RESTless tumors. Our findings therefore show a critical role for the REST-LIN28A axis in tumor aggression and suggest a causative relationship between REST loss and tumorigenicity in vivo.
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Neoplasias de la Mama/patología , División Celular/fisiología , Proteínas de Unión al ADN/biosíntesis , Proteínas Represoras/metabolismo , Animales , Western Blotting , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/fisiología , Femenino , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Proteínas de Unión al ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Represoras/genéticaRESUMEN
The phosphatidylinositol phosphate (PIP) kinases are a unique family of enzymes that generate an assortment of lipid messengers, including the pivotal second messenger phosphatidylinositol 4,5-bisphosphate (PI4,5P2). While members of the PIP kinase family function by catalyzing a similar phosphorylation reaction, the specificity loop of each PIP kinase subfamily determines substrate preference and partially influences distinct subcellular targeting. Specific protein-protein interactions that are unique to particular isoforms or splice variants play a key role in targeting PIP kinases to appropriate subcellular compartments to facilitate the localized generation of PI4,5P2 proximal to effectors, a mechanism key for the function of PI4,5P2 as a second messenger. This review documents the discovery of the PIP kinases and their signaling products, and summarizes our current understanding of the mechanisms underlying the localized generation of PI4,5P2 by PIP kinases for the regulation of cellular events including actin cytoskeleton dynamics, vesicular trafficking, cell migration, and an assortment of nuclear events.