RESUMEN
PURPOSE: Alpelisib plus fulvestrant demonstrated a significant progression-free survival benefit versus fulvestrant in patients with PIK3CA-mutated HR+ /HER2- advanced breast cancer (ABC) (SOLAR-1). Hyperglycemia, an on-target adverse effect of PI3Kα inhibition, can lead to dose modifications, potentially impacting alpelisib efficacy. We report data from preclinical models and two clinical trials (SOLAR-1 and BYLieve) on Sodium glucose cotransporter 2 inhibitor (SGLT2i) use to improve PI3Kα inhibitor-associated hyperglycemia. METHODS: Healthy Brown Norway (BN), mild diabetic Zucker diabetic fatty (ZDF), and Rat1-myr-p110α/HBRX3077 tumor-bearing nude rats treated with alpelisib were analyzed for glucose and insulin control with metformin and dapagliflozin (SGLT2i) and alpelisib efficacy. Hyperglycemia adverse events (AEs) were compared between patients receiving SGLT2i with alpelisib (n = 19) and a propensity score-matched cohort not receiving SGLT2i (n = 74) in both trials. RESULTS: Dapagliflozin and metformin in BN and ZDF rats treated with alpelisib normalized blood glucose and reduced insulin levels. No signs of ketosis or drug-drug interaction were observed when metformin and dapagliflozin was administered with alpelisib. Alpelisib antitumor efficacy was maintained when used with dapagliflozin in tumor-bearing rats. Compared with a matched set of patients without SGLT2i, patients receiving SGLT2i had 4.9 and 6.4 times lower rates of grade ≥ 3 hyperglycemia AEs and hyperglycemia AEs resulting in alpelisib dose adjustments, interruptions, or withdrawals, respectively, and a relative reduction in risk of experiencing these AEs (70.6% and 35.7%). CONCLUSION: These data suggest adding an SGLT2i can effectively manage hyperglycemia, resulting in fewer alpelisib dose modifications and discontinuations in patients with PIK3CA-mutated HR+ /HER2- ABC (SOLAR-1: NCT02437318; BYLieve: NCT03056755).
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BACKGROUND: Cell replacement therapy (CRT) for Huntington disease (HD) requires a source of striatal (STR) progenitors capable of restoring the function lost due to STR degeneration. Authentic STR progenitors can be collected from the fetal putative striatum, or whole ganglionic eminence (WGE), but these tissues remain impractical for widespread clinical application, and alternative donor sources are required. Here we begin exploring the possibility that induced pluripotent stem cells (iPSC) derived from WGE may retain an epigenetic memory of their tissue of origin, which could enhance their ability to differentiate into STR cells. RESULTS: We generate four iPSC lines from human WGE (hWGE) and establish that they have a capacity similar to human embryonic stem cells with regard to their ability to differentiate toward an STR phenotype, as measured by expression and demethylation of key STR genes, while maintaining an overall different methylome. Finally, we demonstrate that these STR-differentiated hWGE iPSCs share characteristics with hWGE (i.e., authentic STR tissues) both in vitro and following transplantation into an HD model. Overall, iPSCs derived from human WGE show promise as a donor source for CRT for HD.
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Tratamiento Basado en Trasplante de Células y Tejidos , Cuerpo Estriado , Enfermedad de Huntington , Células Madre Pluripotentes Inducidas , Diferenciación Celular , Cuerpo Estriado/citología , Humanos , Enfermedad de Huntington/terapiaRESUMEN
Background CLR457 is an orally bioavailable pan-phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) inhibitor. Methods CLR457 anti-tumor activity and pharmacokinetics (PK) were characterized by in vitro biochemical assays and in vivo tumor xenografts. A first-in-human study was conducted to determine the maximum tolerated dose (MTD), safety, PK, and efficacy of CLR457. Successive cohorts of patients with advanced solid tumors with PI3K pathway activation received increasing CLR457 doses according to a Bayesian escalation model based on the rate of dose limiting toxicity (DLT) in the first 28-day cycle. Results CLR457 inhibited p110α, p110ß, p110δ and p110γ isoforms with an IC50 of 89 ± 29 nM, 56 ± 35 nM, 39 ± 10 nM and 230 ± 31 nM, respectively. CLR457 exhibited dose-dependent antitumor activity and interfered with glucose homeostasis in PI3K-mutant tumor xenografts. 31 patients received doses ranging from 5 to 100 mg. DLTs included grade 3 hyperglycemia and rash (3). In the 100 mg cohort (n = 11), 3 (27.3%) patients had DLTs and all patients (100%) experienced ≥ grade 3 toxicity with rash (45.5%) as the most common event. The MTD was not determined. For the entire study population, stomatitis (45.2%), diarrhea (38.7%), rash (35.5%) were the most common any grade toxicities-51.6% patients experienced ≥ Grade 3 toxicity. CLR457 was rapidly absorbed with limited accumulation and linear PK. PK modeling indicated that pharmacologically active concentrations were achieved at the highest dose tested (100 mg), though no objective responses were observed. Conclusion CLR457 clinical development was terminated due to poor tolerability and limited antitumor activity. These results emphasize the difficulty of achieving a wide therapeutic index when targeting all class I PI3K-isoforms.
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Biomarcadores de Tumor/metabolismo , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Compuestos Orgánicos/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Administración Oral , Adulto , Anciano , Anciano de 80 o más Años , Animales , Apoptosis , Proliferación Celular , Relación Dosis-Respuesta a Droga , Femenino , Estudios de Seguimiento , Humanos , Masculino , Dosis Máxima Tolerada , Ratones , Ratones Desnudos , Persona de Mediana Edad , Neoplasias/metabolismo , Neoplasias/patología , Pronóstico , Ratas Desnudas , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Although numerous protocols have been developed for differentiation of neurons from a variety of pluripotent stem cells, most have concentrated on being able to specify effectively appropriate neuronal subtypes and few have been designed to enhance or accelerate functional maturity. Of those that have, most employ time courses of functional maturation that are rather protracted, and none have fully characterized all aspects of neuronal function, from spontaneous action potential generation through to postsynaptic receptor maturation. Here, we describe a simple protocol that employs the sequential addition of just two supplemented media that have been formulated to separate the two key phases of neural differentiation, the neurogenesis and synaptogenesis, each characterized by different signaling requirements. Employing these media, this new protocol synchronized neurogenesis and enhanced the rate of maturation of pluripotent stem cell-derived neural precursors. Neurons differentiated using this protocol exhibited large cell capacitance with relatively hyperpolarized resting membrane potentials; moreover, they exhibited augmented: 1) spontaneous electrical activity; 2) regenerative induced action potential train activity; 3) Na(+) current availability, and 4) synaptic currents. This was accomplished by rapid and uniform development of a mature, inhibitory GABAAreceptor phenotype that was demonstrated by Ca(2+) imaging and the ability of GABAAreceptor blockers to evoke seizurogenic network activity in multielectrode array recordings. Furthermore, since this protocol can exploit expanded and frozen prepatterned neural progenitors to deliver mature neurons within 21 days, it is both scalable and transferable to high-throughput platforms for the use in functional screens.
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Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/fisiología , Medios de Cultivo/química , Células Madre Pluripotentes Inducidas/citología , Células-Madre Neurales/citología , Western Blotting , Ciclo Celular/fisiología , Línea Celular , Técnicas de Cocultivo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Células Madre Pluripotentes Inducidas/metabolismo , Microscopía Electrónica de Rastreo , Células-Madre Neurales/metabolismo , Neurogénesis/fisiología , Técnicas de Placa-Clamp , Receptores de GABA-A/metabolismoRESUMEN
Neurons differentiated from pluripotent stem cells using established neural culture conditions often exhibit functional deficits. Recently, we have developed enhanced media which both synchronize the neurogenesis of pluripotent stem cell-derived neural progenitors and accelerate their functional maturation; together these media are termed SynaptoJuice. This pair of media are pro-synaptogenic and generate authentic, mature synaptic networks of connected forebrain neurons from a variety of induced pluripotent and embryonic stem cell lines. Such enhanced rate and extent of synchronized maturation of pluripotent stem cell-derived neural progenitor cells generates neurons which are characterized by a relatively hyperpolarized resting membrane potential, higher spontaneous and induced action potential activity, enhanced synaptic activity, more complete development of a mature inhibitory GABAA receptor phenotype and faster production of electrical network activity when compared to standard differentiation media. This entire process - from pre-patterned neural progenitor to active neuron - takes 3 weeks or less, making it an ideal platform for drug discovery and disease modelling in the fields of human neurodegenerative and neuropsychiatric disorders, such as Huntington's disease, Parkinson's disease, Alzheimer's disease and Schizophrenia.
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Calcio/metabolismo , Diferenciación Celular/fisiología , Neuronas/metabolismo , Neuronas/fisiología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/fisiología , Receptores de GABA-A/metabolismo , Animales , Humanos , Neurogénesis/fisiologíaRESUMEN
In vitro metabolic identification studies with a PI3K-α inhibitor lead molecule 1 identified a single predominant site of oxidative metabolism to be occurring within a tert.butyl moiety. Modification of the tert.butyl group within the lead molecule 1, to the corresponding d9-tert.butyl analogue 2, led to an increase in both the in vitro and in vivo metabolic stability. This increase in metabolic stability resulted in a 2-fold increase in the oral bioavailability measured in the rat, and a 3-fold increase in potency in a chronic in vivo study in the mouse, for 2 when compared to 1.
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Deuterio/metabolismo , Inhibidores Enzimáticos/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Amidas/química , Animales , Disponibilidad Biológica , Fosfatidilinositol 3-Quinasa Clase I , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacocinética , Cinética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Prolina/química , Ratas , Tiazoles/química , Urea/químicaRESUMEN
Taking the pyrrolopyrimidine derived IGF-1R inhibitor NVP-AEW541 as the starting point, the benzyl ether back-pocket binding moiety was replaced with a series of 2-cyclic ether methyl ethers leading to the identification of novel achiral [2.2.1]-bicyclic ether methyl ether containing analogues with improved IGF-1R activities and kinase selectivities. Further exploration of the series, including a fluorine scan of the 5-phenyl substituent, and optimisation of the sugar-pocket binding moiety identified compound 33 containing (S)-2-tetrahydrofuran methyl ether 6-fluorophenyl ether back-pocket, and cis-N-Ac-Pip sugar-pocket binding groups. Compound 33 showed improved selectivity and pharmacokinetics compared to NVP-AEW541, and produced comparable in vivo efficacy to linsitinib in inhibiting the growth of an IGF-1R dependent tumour xenograft model in the mouse.
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Antineoplásicos/farmacología , Imidazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirazinas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Receptor IGF Tipo 1/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Imidazoles/síntesis química , Imidazoles/química , Ratones , Ratones Desnudos , Estructura Molecular , Células 3T3 NIH , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Pirazinas/síntesis química , Pirazinas/química , Pirimidinas/síntesis química , Pirimidinas/química , Pirroles/síntesis química , Pirroles/química , Receptor IGF Tipo 1/metabolismo , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A cyclisation within a 4',5-bisthiazole (S)-proline-amide-urea series of selective PI3Kα inhibitors led to a novel 4,5-dihydrobenzo[1,2-d:3,4-d]bisthiazole tricyclic sub-series. The synthesis and optimisation of this 4,5-dihydrobenzo[1,2-d:3,4-d]bisthiazole sub-series and the expansion to a related tricyclic 4,5-dihydrothiazolo[4,5-h]quinazoline sub-series are described. From this work analogues including 11, 12, 19 and 23 were identified as potent and selective PI3Kα inhibitor in vivo tool compounds.
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Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/química , Quinazolinas/farmacología , Tiazoles/química , Tiazoles/farmacología , Animales , Células CACO-2 , Fosfatidilinositol 3-Quinasa Clase I , Femenino , Humanos , Ratones Desnudos , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinazolinas/farmacocinética , Quinazolinas/uso terapéutico , Relación Estructura-Actividad , Tiazoles/farmacocinética , Tiazoles/uso terapéuticoRESUMEN
Activating mutations in GNAQ/GNA11 occur in over 90% of uveal melanomas (UMs), the most lethal melanoma subtype; however, targeting these oncogenes has proven challenging and inhibiting their downstream effectors show limited clinical efficacy. Here, we performed genome-scale CRISPR screens along with computational analyses of cancer dependency and gene expression datasets to identify the inositol-metabolizing phosphatase INPP5A as a selective dependency in GNAQ/11-mutant UM cells in vitro and in vivo. Mutant cells intrinsically produce high levels of the second messenger inositol 1,4,5 trisphosphate (IP3) that accumulate upon suppression of INPP5A, resulting in hyperactivation of IP3-receptor signaling, increased cytosolic calcium and p53-dependent apoptosis. Finally, we show that GNAQ/11-mutant UM cells and patients' tumors exhibit elevated levels of IP4, a biomarker of enhanced IP3 production; these high levels are abolished by GNAQ/11 inhibition and correlate with sensitivity to INPP5A depletion. Our findings uncover INPP5A as a synthetic lethal vulnerability and a potential therapeutic target for GNAQ/11-mutant-driven cancers.
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Melanoma , Humanos , Melanoma/tratamiento farmacológico , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/uso terapéutico , Mutación , Transducción de Señal , Inositol Polifosfato 5-Fosfatasas/genéticaRESUMEN
Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinson's disease (PD), but the underlying pathophysiological mechanisms and the normal function of this large multidomain protein remain speculative. To address the role of this protein in vivo, we generated three different LRRK2 mutant mouse lines. Mice completely lacking the LRRK2 protein (knock-out, KO) showed an early-onset (age 6 weeks) marked increase in number and size of secondary lysosomes in kidney proximal tubule cells and lamellar bodies in lung type II cells. Mice expressing a LRRK2 kinase-dead (KD) mutant from the endogenous locus displayed similar early-onset pathophysiological changes in kidney but not lung. KD mutants had dramatically reduced full-length LRRK2 protein levels in the kidney and this genetic effect was mimicked pharmacologically in wild-type mice treated with a LRRK2-selective kinase inhibitor. Knock-in (KI) mice expressing the G2019S PD-associated mutation that increases LRRK2 kinase activity showed none of the LRRK2 protein level and histopathological changes observed in KD and KO mice. The autophagy marker LC3 remained unchanged but kidney mTOR and TCS2 protein levels decreased in KD and increased in KO and KI mice. Unexpectedly, KO and KI mice suffered from diastolic hypertension opposed to normal blood pressure in KD mice. Our findings demonstrate a role for LRRK2 in kidney and lung physiology and further show that LRRK2 kinase function affects LRRK2 protein steady-state levels thereby altering putative scaffold/GTPase activity. These novel aspects of peripheral LRRK2 biology critically impact ongoing attempts to develop LRRK2 selective kinase inhibitors as therapeutics for PD.
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Homeostasis , Riñón/enzimología , Pulmón/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/patología , Células Epiteliales Alveolares/ultraestructura , Animales , Presión Sanguínea/efectos de los fármacos , Dopamina/metabolismo , Agonistas de Dopamina/farmacología , Antagonistas de Dopamina/farmacología , Estabilidad de Enzimas/efectos de los fármacos , Homeostasis/efectos de los fármacos , Riñón/patología , Riñón/fisiopatología , Riñón/ultraestructura , Túbulos Renales Proximales/enzimología , Túbulos Renales Proximales/patología , Túbulos Renales Proximales/fisiopatología , Túbulos Renales Proximales/ultraestructura , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Pulmón/efectos de los fármacos , Pulmón/patología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/ultraestructura , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Mutantes , Actividad Motora , Transducción de Señal/efectos de los fármacosRESUMEN
Anti-tumor efficacy of targeted therapies is variable across patients and cancer types. Even in patients with initial deep response, tumors are typically not eradicated and eventually relapse. To address these challenges, we present a systematic screen for targets that limit the anti-tumor efficacy of EGFR and ALK inhibitors in non-small cell lung cancer and BRAF/MEK inhibitors in colorectal cancer. Our approach includes genome-wide CRISPR screens with or without drugs targeting the oncogenic driver ("anchor therapy"), and large-scale pairwise combination screens of anchor therapies with 351 other drugs. Interestingly, targeting of a small number of genes, including MCL1, BCL2L1, and YAP1, sensitizes multiple cell lines to the respective anchor therapy. Data from drug combination screens with EGF816 and ceritinib indicate that dasatinib and agents disrupting microtubules act synergistically across many cell lines. Finally, we show that a higher-order-combination screen with 26 selected drugs in two resistant EGFR-mutant lung cancer cell lines identified active triplet combinations.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Proteínas Proto-Oncogénicas B-raf/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Recurrencia Local de Neoplasia/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptores ErbB/genética , Proteínas Tirosina Quinasas Receptoras/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Mutación , Línea Celular TumoralRESUMEN
NVP-BEZ235 is a dual PI3K/mTOR inhibitor currently in phase I clinical trials. We profiled this compound against a panel of breast tumor cell lines to identify the patient populations that would benefit from such treatment. In this setting, NVP-BEZ235 selectively induced cell death in cell lines presenting either HER2 amplification and/or PIK3CA mutation, but not in cell lines with PTEN loss of function or KRAS mutations, for which resistance could be attributed, in part to ERK pathway activity. An in depth analysis of death markers revealed that the cell death observed upon NVP-BEZ235 treatment could be recapitulated with other PI3K inhibitors and that this event is linked to active PARP cleavage indicative of an apoptotic process. Moreover, the effect seemed to be partly independent of the caspase-9 executioner and mitochondrial activated caspases, suggesting an alternate route for apoptosis induction by PI3K inhibitors. Overall, this study will provide guidance for patient stratification for forthcoming breast cancer phase II trials for NVP-BEZ235.
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Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Genes erbB-2 , Imidazoles/farmacología , Péptidos y Proteínas de Señalización Intracelular/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Quinolinas/farmacología , Apoptosis/genética , Apoptosis/fisiología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Caspasa 9/metabolismo , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/fisiología , Inhibidores Enzimáticos/farmacología , Femenino , Amplificación de Genes , Genes erbB-2/efectos de los fármacos , Humanos , Mutación , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TORRESUMEN
Balanced pan-class I phosphoinositide 3-kinase inhibition as an approach to cancer treatment offers the prospect of treating a broad range of tumor types and/or a way to achieve greater efficacy with a single inhibitor. Taking buparlisib as the starting point, the balanced pan-class I PI3K inhibitor 40 (NVP-CLR457) was identified with what was considered to be a best-in-class profile. Key to the optimization to achieve this profile was eliminating a microtubule stabilizing off-target activity, balancing the pan-class I PI3K inhibition profile, minimizing CNS penetration, and developing an amorphous solid dispersion formulation. A rationale for the poor tolerability profile of 40 in a clinical study is discussed.
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Antineoplásicos , Fosfatidilinositol 3-Quinasas , Aminopiridinas/farmacología , Antineoplásicos/farmacología , Línea Celular Tumoral , Compuestos Orgánicos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Covalent inhibitors of KRASG12C have shown antitumor activity against advanced/metastatic KRASG12C-mutated cancers, though resistance emerges and additional strategies are needed to improve outcomes. JDQ443 is a structurally unique covalent inhibitor of GDP-bound KRASG12C that forms novel interactions with the switch II pocket. JDQ443 potently inhibits KRASG12C-driven cellular signaling and demonstrates selective antiproliferative activity in KRASG12C-mutated cell lines, including those with G12C/H95 double mutations. In vivo, JDQ443 induces AUC exposure-driven antitumor efficacy in KRASG12C-mutated cell-derived (CDX) and patient-derived (PDX) tumor xenografts. In PDX models, single-agent JDQ443 activity is enhanced by combination with inhibitors of SHP2, MEK, or CDK4/6. Notably, the benefit of JDQ443 plus the SHP2 inhibitor TNO155 is maintained at reduced doses of either agent in CDX models, consistent with mechanistic synergy. JDQ443 is in clinical development as monotherapy and in combination with TNO155, with both strategies showing antitumor activity in patients with KRASG12C-mutated tumors. SIGNIFICANCE: JDQ443 is a structurally novel covalent KRASG12C inhibitor with a unique binding mode that demonstrates potent and selective antitumor activity in cell lines and in vivo models. In preclinical models and patients with KRASG12C-mutated malignancies, JDQ443 shows potent antitumor activity as monotherapy and in combination with the SHP2 inhibitor TNO155. This article is highlighted in the In This Issue feature, p. 1397.
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Inhibidores Enzimáticos , Indazoles , Neoplasias , Proteínas Proto-Oncogénicas p21(ras) , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Indazoles/química , Indazoles/farmacología , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/genética , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismoRESUMEN
The G protein-coupled receptor GPR4 is activated by acidic pH and recent evidence indicates that it is expressed in endothelial cells. In agreement with these reports, we observe a high correlation of GPR4 mRNA expression with endothelial marker genes, and we confirm expression and acidic pH dependent function of GPR4 in primary human vascular endothelial cells. GPR4-deficient mice were generated; these are viable and fertile and show no gross abnormalities. However, these animals show a significantly reduced angiogenic response to VEGF (vascular endothelial growth factor), but not to bFGF (basic fibroblast growth factor), in a growth factor implant model. Accordingly, in two different orthotopic models, tumor growth is strongly reduced in mice lacking GPR4. Histological analysis of tumors indicates reduced tumor cell proliferation as well as altered vessel morphology, length and density. Moreover, GPR4 deficiency results in reduced VEGFR2 (VEGF Receptor 2) levels in endothelial cells, accounting, at least in part, for the observed phenotype. Our data suggest that endothelial cells sense local tissue acidosis via GPR4 and that this signal is required to generate a full angiogenic response to VEGF.
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Células Endoteliales/metabolismo , Neoplasias/fisiopatología , Neovascularización Patológica/metabolismo , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/metabolismo , Animales , Western Blotting , Proliferación Celular , Biología Computacional , Cartilla de ADN/genética , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neoplasias/metabolismo , ARN Interferente Pequeño/genética , Receptores Acoplados a Proteínas G/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The objective of this study was to evaluate the use of the generation of 3D models and 3D prints of complex cases for physicians at the example of an intricate left ventricular outflow tract obstruction (LVOTO). LVOTO is a known complication of mitral valve surgery. A 38-year-old female patient with increasing dyspnoea after mitral valve replacement was referred to our centre. Echocardiography showed a strut of the bioprosthetic heart valve protruding into the left ventricular outflow tract. However, the diagnosis of a LVOTO was difficult based on echocardiography alone. Therefore, we fabricated a physical model of the left ventricular outflow tract, the mitral valve, the aortic valve and the left ventricle. With this physical model in hand, we were able to visualize the LVOTO and to discuss potential therapeutic options. Moreover, we were able to plan the subsequent redo surgery in detail using the model. This case shows the benefit of 3D printing technologies for surgeons and patients, not only for analysis, but also during the decision-making and pre-operative planning process.
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Procedimientos Quirúrgicos Cardíacos , Insuficiencia de la Válvula Mitral , Impresión Tridimensional , Adulto , Femenino , Humanos , Válvula Mitral/diagnóstico por imagen , Válvula Mitral/cirugía , Obstrucción del Flujo Ventricular Externo/diagnóstico por imagen , Obstrucción del Flujo Ventricular Externo/etiología , Obstrucción del Flujo Ventricular Externo/cirugíaRESUMEN
EphB4 and its cognitive ligand ephrinB2 play an important role in embryonic vessel development and vascular remodeling. In addition, several reports suggest that this receptor ligand pair is also involved in pathologic vessel formation in adults including tumor angiogenesis. Eph/ephrin signaling is a complex phenomena characterized by receptor forward signaling through the tyrosine kinase of the receptor and ephrin reverse signaling through various protein-protein interaction domains and phosphorylation motifs of the ephrin ligands. Therefore, interfering with EphR/ephrin signaling by the means of targeted gene ablation, soluble receptors, dominant negative mutants or antisense molecules often does not allow to discriminate between inhibition of Eph/ephrin forward and reverse signaling. We developed a specific small molecular weight kinase inhibitor of the EphB4 kinase, NVP-BHG712, which inhibits EphB4 kinase activity in the low nanomolar range in cellular assays showed high selectivity for targeting the EphB4 kinase when profiled against other kinases in biochemical as well as in cell based assays. Furthermore, NVP-BHG712 shows excellent pharmacokinetic properties and potently inhibits EphB4 autophosphorylation in tissues after oral administration. In vivo, NVP-BHG712 inhibits VEGF driven vessel formation, while it has only little effects on VEGF receptor (VEGFR) activity in vitro or in cellular assays. The data shown here suggest a close cross talk between the VEGFR and EphR signaling during vessel formation. In addition to its established function in vascular remodeling and endothelial arterio-venous differentiation, EphB4 forward signaling appears to be an important mediator of VEGF induced angiogenesis since inhibition of EphB4 forward signaling is sufficient to inhibit VEGF induced angiogenesis.