RESUMEN
INTRODUCTION: The activation of the phosphoinositide 3-kinase (PI3K)/AKT signalling pathway is one the most frequent genetic events in breast cancer, consequently the development of PI3K inhibitors has attracted much attention. Here we evaluate the effect of PI3K inhibition on global gene expression in breast cancer cells. METHODS: We used a range of methodologies that include in silico compound analysis, in vitro kinase assays, cell invasion assays, proliferation assays, genome-wide transcription studies (Agilent Technologies full genome arrays), gene set enrichment analysis, quantitative real-time PCR, immunoblotting in addition to chromatin immunoprecipitation. RESULTS: We defined the physico-chemical and the biological properties of ETP-45658, a novel potent PI3K inhibitor. We demonstrated that ETP-45658 potently inhibited cell proliferation within a broad range of human cancer cells, most potently suppressing the growth of breast cancer cells via inhibiting cell cycle. We show that this response is Forkhead box O (FOXO) protein dependent and p53 independent. Our genome-wide microarray analysis revealed that the cell cycle was the most affected biological process after exposure to ETP-45658 (or our control PI3K inhibitor PI-103), that despite the multiple transcription factors that are regulated by the PI3K/AKT signalling cascade, only the binding sites for FOXO transcription factors were significantly enriched and only a subset of all FOXO-dependent genes were induced. This disparity in gene transcription was not due to differential FOXO promoter recruitment. CONCLUSIONS: The constitutive activation of PI3Ks and thus the exclusion of FOXO transcription factors from the nucleus is a key feature of breast cancer. Our results presented here highlight that PI3K inhibition activates specific FOXO-dependent genes that mediate cell cycle arrest in breast cancer cells.
Asunto(s)
Adenocarcinoma , Neoplasias de la Mama , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Factores de Transcripción Forkhead/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Pirazoles/farmacología , Pirimidinas/farmacología , Línea Celular Tumoral , Simulación por Computador , Femenino , Factores de Transcripción Forkhead/metabolismo , Células HCT116 , Humanos , Técnicas In Vitro , Células MCF-7 , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Zalypsis(®) is a marine compound in phase II clinical trials for multiple myeloma, cervical and endometrial cancer, and Ewing's sarcoma. However, the determinants of the response to Zalypsis are not well known. The identification of biomarkers for Zalypsis activity would also contribute to broaden the spectrum of tumors by selecting those patients more likely to respond to this therapy. METHODS: Using in vitro drug sensitivity data coupled with a set of molecular data from a panel of sarcoma cell lines, we developed molecular signatures that predict sensitivity to Zalypsis. We verified these results in culture and in vivo xenograft studies. RESULTS: Zalypsis resistance was dependent on the expression levels of PDGFRα or constitutive phosphorylation of c-Kit, indicating that the activation of tyrosine kinase receptors (TKRs) may determine resistance to Zalypsis. To validate our observation, we measured the levels of total and active (phosphorylated) forms of the RTKs PDGFRα/ß, c-Kit, and EGFR in a new panel of diverse solid tumor cell lines and found that the IC50 to the drug correlated with RTK activation in this new panel. We further tested our predictions about Zalypsis determinants for response in vivo in xenograft models. All cells lines expressing low levels of RTK signaling were sensitive to Zalypsis in vivo, whereas all cell lines except two with high levels of RTK signaling were resistant to the drug. CONCLUSIONS: RTK activation might provide important signals to overcome the cytotoxicity of Zalypsis and should be taken into consideration in current and future clinical trials.
Asunto(s)
Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Sarcoma/tratamiento farmacológico , Sarcoma/genética , Biomarcadores Farmacológicos , Línea Celular Tumoral , Resistencia a Antineoplásicos , Receptores ErbB/biosíntesis , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Proto-Oncogénicas c-kit/biosíntesis , ARN Mensajero/biosíntesis , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Sarcoma/patología , Tetrahidroisoquinolinas/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Inhibitors of PI3K signaling are of great therapeutic interest in oncology. The phosphoinositide-3-kinase signaling pathway is activated in a variety of solid and non-solid tumors. We have identified an imidazopyrazine derivative, ETP-46321, as a potent inhibitor of PI3Kα [Formula: see text]. The compound was 6 times less potent towards PI3Kδ and more than 200 and 60 times less potent at inhibiting PI3Kß and PI3Kγ and did not significantly inhibit the related phosphoinositide-3-kinase-related protein kinase family kinases mTOR or DNA PK (IC(50)'s > 5 µM), or an additional 287 protein kinases that were screened. ETP-46321 inhibited PI3K signaling in treated tumor cell lines, induced cell cycle arrest and inhibited VEGF-dependent sprouting of HUVEC cells. The compound was anti-proliferative and synergized with both cytotoxic and targeted therapeutics. The compound induced a reduction in the phosphorylation of Akt in U87 MG xenografts after a single treatment. The growth of colon and lung cancinoma HT-29 and A549 xenografts was delayed by once a day treatment with ETP-46321. The compound synergized with Doxotaxel in a model of ovarian cancer.
Asunto(s)
Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Imidazoles/uso terapéutico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Pirazinas/uso terapéutico , Animales , Antineoplásicos/sangre , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/sangre , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Imidazoles/sangre , Imidazoles/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Fosfatidilinositol 3-Quinasas/metabolismo , Pirazinas/sangre , Pirazinas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PIM kinases are a family of serine/threonine kinases composed of three different isoforms (PIM1, PIM 2 and PIM 3) that are highly homologous. Their expression is mediated by the JAK/STAT signalling pathway, providing survival and cell cycle transition signals. PIM kinases are heavily targeted for anticancer drug discovery. However, very little is known about the relative contribution of the different isoforms to the tumourigenesis process in vivo, and how their individual inhibition might affect tumour growth. Taking advantage of genetically modified mice, we explored whether the inhibition of specific isoforms is required to prevent sarcomas induced by 3-methylcholanthrene carcinogenic treatment. We found that absence of Pim2 and Pim3 greatly reduced sarcoma growth to a similar extent to the absence of all three isoforms. This model of sarcoma generally produces bone invasion by the tumour cells. Lack of Pim2 and Pim3 reduced tumour-induced bone invasion by 70%, which is comparable with the reduction of tumour-induced bone invasion in the absence of all three isoforms. Similar results were obtained in mouse embryonic fibroblasts (MEFs) derived from these knockout (KO) mice, where double Pim2/3 KO MEFs already showed reduced proliferation and were resistant to oncogenic transformation by the RAS oncogene. Our data also suggest an important role of Gsk3ß phosphorylation in the process of tumourigenesis.
Asunto(s)
Huesos/patología , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Sarcoma Experimental/patología , Animales , Carcinógenos/toxicidad , Línea Celular , Proliferación Celular , Células Cultivadas , Metilcolantreno/toxicidad , Ratones , Ratones Noqueados , Invasividad Neoplásica , Sarcoma Experimental/inducido químicamente , Sarcoma Experimental/enzimologíaRESUMEN
Phosphoinositide-3-kinases (PI3K) are a family of lipid kinases mediating numerous cell processes such as proliferation, migration and differentiation. PI3K is an important target for cancer therapeutics due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein, we describe the rapid identification of ETP-46992, within 2-aminocarbonyl imidazo [1,2-a] pyrazine series, with suitable pharmacokinetic (PK) properties that allows the establishment of mechanism of action and efficacy in vivo studies. ETP-46992 showed tumor growth inhibition in a GEMM mouse tumor model driven by a K-Ras(G12V) oncogenic mutation and in tumor xenograft models with PI3K pathway deregulated (BT474).
Asunto(s)
Imidazoles/química , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/química , Pirazinas/química , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Administración Oral , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Citocromos/metabolismo , Modelos Animales de Enfermedad , Semivida , Humanos , Imidazoles/síntesis química , Imidazoles/farmacocinética , Ratones , Ratones Endogámicos BALB C , Microsomas Hepáticos/metabolismo , Neoplasias/tratamiento farmacológico , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirazinas/síntesis química , Pirazinas/farmacocinética , Serina-Treonina Quinasas TOR/metabolismo , Trasplante HeterólogoRESUMEN
Phosphoinositide-3-kinase (PI3K) is an important target for cancer therapeutics due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein, we describe the optimization of imidazo [1,2-a] pyrazines, which allow us to identify compound 14 (ETP-46321), with potent biochemical and cellular activity and good pharmacokinetic properties (PK) after oral dosing. ETP-46321 PK/PD studies showed time dependent downregulation of AKT(Ser473) phosphorylation, which correlates with compound levels in tumor tissue and demonstrating to be efficacious in a GEMM mouse tumor model driven by a K-Ras(G12V) oncogenic mutation. Treatment with ETP-46321 resulted in significant tumor growth inhibition.
Asunto(s)
Imidazoles/farmacología , Isoenzimas/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Pirazinas/farmacología , Administración Oral , Disponibilidad Biológica , Humanos , Imidazoles/administración & dosificación , Imidazoles/farmacocinética , Tomografía de Emisión de Positrones , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacocinética , Pirazinas/administración & dosificación , Pirazinas/farmacocinética , Tomografía Computarizada por Rayos XRESUMEN
Activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is one the most frequent genetic events in human cancer. A cell-based imaging assay that monitored the translocation of the Akt effector protein, Forkhead box O (FOXO), from the cytoplasm to the nucleus was employed to screen a collection of 33,992 small molecules. The positive compounds were used to screen kinases known to be involved in FOXO translocation. Pyrazolopyrimidine derivatives were found to be potent FOXO relocators as well as biochemical inhibitors of PI3Kalpha. A combination of virtual screening and molecular modeling led to the development of a structure-activity relationship, which indicated the preferred substituents on the pyrazolopyrimidine scaffold. This leads to the synthesis of ETP-45658, which is a potent and selective inhibitor of phosphoinositide 3-kinases and demonstrates mechanism of action in tumor cell lines and in vivo in treated mice.
Asunto(s)
Núcleo Celular/metabolismo , Inhibidores Enzimáticos/metabolismo , Factores de Transcripción Forkhead/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Pirazoles/metabolismo , Pirimidinas/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Animales , Línea Celular , Cromonas/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Femenino , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Furanos/metabolismo , Humanos , Ratones , Ratones Transgénicos , Estructura Molecular , Morfolinas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirazoles/química , Pirazoles/farmacología , Piridinas/metabolismo , Pirimidinas/química , Pirimidinas/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiologíaRESUMEN
PIM kinase family (PIM-1, PIM-2 and PIM-3) is an appealing target for the discovery and development of selective inhibitors, useful in various disease conditions in which these proteins are highly expressed, such as cancer. The significant effort put, in the recent years, towards the development of small molecules exhibiting inhibitory activity against this protein family has ended up with several molecules entering clinical trials. As part of our ongoing exploration for potential drug candidates that exhibit affinity towards this protein family, we have generated a novel chemical series of triazolo[4,3-b]pyridazine based tricycles by applying a scaffold hopping strategy over our previously reported potent pan-PIM inhibitor ETP-47453 (compound 2). The structure-activity relationship studies presented herein demonstrate a rather selective PIM-1/PIM-3 biochemical profile for this novel series of tricycles, although pan-PIM and PIM-1 inhibitors have also been identified. Selected examples show significant inhibition of the phosphorylation of BAD protein in a cell-based assay. Moreover, optimized and highly selective compounds, such as 42, did not show significant hERG inhibition at 20⯵M concentration, and proved its antiproliferative activity and utility in combination with particular antitumoral agents in several tumor cell lines.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Piridazinas/farmacología , Quinolinas/farmacología , Triazoles/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Piridazinas/síntesis química , Piridazinas/química , Quinolinas/síntesis química , Quinolinas/química , Relación Estructura-Actividad , Triazoles/síntesis química , Triazoles/químicaRESUMEN
Aplidin (plitidepsin) is a novel anticancer drug isolated from the marine tunicate Aplidium albicans. Aplidin shows potent antitumor activity in preclinical models against a wide variety of human tumors. Aplidin is currently in phase II clinical trials in a variety of solid tumors and hematologic malignancies. Moreover, clinical studies of Aplidin in combination with other agents are ongoing because it generally lacks cross-resistance with other known cytotoxic drugs. The mode of action of Aplidin in tumor cells is only partially understood. Aplidin induces an early oxidative stress response, which results in a rapid and sustained activation of the epidermal growth factor receptor, the nonreceptor protein tyrosine kinase Src, and the serine threonine kinases c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinase. Here, we show that sensitivity to Aplidin correlates inversely with the levels of expression of the cyclin-dependent kinase inhibitor p27(kip1) (p27) in a panel of low passaged human sarcoma cell lines. Aplidin induces p27 through an oxidation-dependent mechanism and the reduction of p27 levels by specific short hairpin RNA increases Aplidin sensitivity. We confirmed these results in p27 null mouse embryonic fibroblasts corroborating the specificity of the p27 role in Aplidin response because p21(waf1) null mouse embryonic fibroblasts do not show this increased sensitivity. We propose a mechanism of action of Aplidin involving p27 and support the analysis of p27 in the response to Aplidin in currently ongoing clinical trials to establish the levels of this protein as response predictor.
Asunto(s)
Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Depsipéptidos/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/deficiencia , Ensayos de Selección de Medicamentos Antitumorales , Flavonoides/farmacología , Humanos , Ratones , Péptidos Cíclicos , Piperidinas/farmacología , Vinblastina/farmacologíaRESUMEN
Here we report the development and validation of a complete solution to manage and analyze the data produced by image-based phenotypic screening campaigns of small-molecule libraries. In one step initial crude images are analyzed for multiple cytological features, statistical analysis is performed and molecules that produce the desired phenotypic profile are identified. A naïve Bayes classifier, integrating chemical and phenotypic spaces, is built and utilized during the process to assess those images initially classified as "fuzzy"-an automated iterative feedback tuning. Simultaneously, all this information is directly annotated in a relational database containing the chemical data. This novel fully automated method was validated by conducting a re-analysis of results from a high-content screening campaign involving 33 992 molecules used to identify inhibitors of the PI3K/Akt signaling pathway. Ninety-two percent of confirmed hits identified by the conventional multistep analysis method were identified using this integrated one-step system as well as 40 new hits, 14.9% of the total, originally false negatives. Ninety-six percent of true negatives were properly recognized too. A web-based access to the database, with customizable data retrieval and visualization tools, facilitates the posterior analysis of annotated cytological features which allows identification of additional phenotypic profiles; thus, further analysis of original crude images is not required.
Asunto(s)
Evaluación Preclínica de Medicamentos/estadística & datos numéricos , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Línea Celular , Sistemas de Administración de Bases de Datos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Biología de SistemasRESUMEN
Yondelis (Trabectedin, ET-743) is a marine anticancer agent currently in Phase II/III development in patients with advanced pretreated soft tissue sarcoma. In the present study, we generated a panel of low passaged tumor cell lines from samples explanted from chemonaive sarcoma patients with different tumor types. We assessed in vitro sensitivity/resistance to Trabectedin and doxorubicin in a panel of sarcoma cell lines and examined the correlation between molecular alterations in DNA repair genes and sensitivity to Trabectedin. We treated cell lines with Trabectedin and doxorubicin in both 96-h and clonogenic assays. In both assays, well-defined groups of resistant and sensitive cell lines were observed. Resistance to Trabectedin did not correlate with resistance to doxorubicin, indicating that the two drugs may have different mechanisms of resistance. p53 mutations and deletions correlated with extreme sensitivity (IC50 < 1 nM) to Trabectedin (P < 0.01). In a pair of isogenic cell lines differing only in the presence or absence of wild-type p53, the absence of p53 rendered cells threefold more sensitive to Trabectedin.