RESUMEN
The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation. Loss of G6b-B results in severe macrothrombocytopenia, myelofibrosis and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into how G6b-B is regulated and contribute to our understanding of the interaction of megakaryocytes and platelets with glycans.
Asunto(s)
Plaquetas/fisiología , Heparitina Sulfato/metabolismo , Megacariocitos/fisiología , Receptores Inmunológicos/metabolismo , Animales , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica , Multimerización de Proteína , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/genética , Transducción de SeñalRESUMEN
UNLABELLED: FGFR1 and FGFR2 are amplified in many tumor types, yet what determines response to FGFR inhibition in amplified cancers is unknown. In a translational clinical trial, we show that gastric cancers with high-level clonal FGFR2 amplification have a high response rate to the selective FGFR inhibitor AZD4547, whereas cancers with subclonal or low-level amplification did not respond. Using cell lines and patient-derived xenograft models, we show that high-level FGFR2 amplification initiates a distinct oncogene addiction phenotype, characterized by FGFR2-mediated transactivation of alternative receptor kinases, bringing PI3K/mTOR signaling under FGFR control. Signaling in low-level FGFR1-amplified cancers is more restricted to MAPK signaling, limiting sensitivity to FGFR inhibition. Finally, we show that circulating tumor DNA screening can identify high-level clonally amplified cancers. Our data provide a mechanistic understanding of the distinct pattern of oncogene addiction seen in highly amplified cancers and demonstrate the importance of clonality in predicting response to targeted therapy. SIGNIFICANCE: Robust single-agent response to FGFR inhibition is seen only in high-level FGFR-amplified cancers, with copy-number level dictating response to FGFR inhibition in vitro, in vivo, and in the clinic. High-level amplification of FGFR2 is relatively rare in gastric and breast cancers, and we show that screening for amplification in circulating tumor DNA may present a viable strategy to screen patients. Cancer Discov; 6(8); 838-51. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 803.
Asunto(s)
Antineoplásicos/farmacología , Benzamidas/farmacología , Evolución Clonal/genética , Amplificación de Genes , Piperazinas/farmacología , Pirazoles/farmacología , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptores de Factores de Crecimiento de Fibroblastos/genética , Animales , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Ratones , Terapia Molecular Dirigida , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Tomografía de Emisión de Positrones , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Taquicininas/metabolismo , Tomografía Computarizada por Rayos X , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
FGFR1 amplification occurs in ~20% of sqNSCLC and trials with FGFR inhibitors have selected FGFR1 amplified patients by FISH. Lung cancer cell lines were profiled for sensitivity to AZD4547, a potent, selective inhibitor of FGFRs 1-3. Sensitivity to FGFR inhibition was associated with but not wholly predicted by increased FGFR1 gene copy number. Additional biomarker assays evaluating expression of FGFRs and correlation between amplification and expression in clinical tissues are therefore warranted. We validated nanoString for mRNA expression analysis of 194 genes, including FGFRs, from clinical tumour tissue. In a panel of sqNSCLC tumours 14.4% (13/90) were FGFR1 amplified by FISH. Although mean FGFR1 expression was significantly higher in amplified samples, there was significant overlap in the range of expression levels between the amplified and non-amplified cohorts with several non-amplified samples expressing FGFR1 to levels equivalent to amplified samples. Statistical analysis revealed increased expression of FGFR1 neighboring genes on the 8p12 amplicon (BAG4, LSM1 and WHSC1L1) in FGFR1 amplified tumours, suggesting a broad rather than focal amplicon and raises the potential for codependencies. High resolution aCGH analysis of pre-clinical and clinical samples supported the presence of a broad and heterogeneous amplicon around the FGFR1 locus. In conclusion, the range of FGFR1 expression levels in both FGFR1 amplified and non-amplified NSCLC tissues, together with the breadth and intra-patient heterogeneity of the 8p amplicon highlights the need for gene expression analysis of clinical samples to inform the understanding of determinants of response to FGFR inhibitors. In this respect the nanoString platform provides an attractive option for RNA analysis of FFPE clinical samples.
Asunto(s)
Benzamidas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Terapia Molecular Dirigida/métodos , Piperazinas/farmacología , Pirazoles/farmacología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Línea Celular Tumoral/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cromosomas Humanos Par 8 , Hibridación Genómica Comparativa , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Neoplasias Pulmonares/tratamiento farmacológico , Reproducibilidad de los ResultadosRESUMEN
Analysis of clinical trial specimens such as formalin-fixed paraffin-embedded (FFPE) tissue for molecular mechanisms of disease progression or drug response is often challenging and limited to a few markers at a time. This has led to the increasing importance of highly multiplexed assays that enable profiling of many biomarkers within a single assay. Methods for gene expression analysis have undergone major advances in biomedical research, but obtaining a robust dataset from low-quality RNA samples, such as those isolated from FFPE tissue, remains a challenge. Here, we provide a detailed evaluation of the NanoString Technologies nCounter platform, which provides a direct digital readout of up to 800 mRNA targets simultaneously. We tested this system by examining a broad set of human clinical tissues for a range of technical variables, including sensitivity and limit of detection to varying RNA quantity and quality, reagent performance over time, variability between instruments, the impact of the number of fields of view sampled, and differences between probe sequence locations and overlapping genes across CodeSets. This study demonstrates that Nanostring offers several key advantages, including sensitivity, reproducibility, technical robustness, and utility for clinical application.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Nanotecnología/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Perfilación de la Expresión Génica/normas , Humanos , Nanotecnología/normas , Análisis de Secuencia por Matrices de Oligonucleótidos/normas , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Deregulation of the cell cycle has long been recognized as an essential driver of tumorigenesis, and agents that selectively target key cell cycle components continue to hold promise as potential therapeutics. We have developed AZD5438, a 4-(1-isopropyl-2-methylimidazol-5-yl)-2-(4-methylsulphonylanilino) pyrimidine, as a potent inhibitor of cyclin-dependent kinase (cdk) 1, 2, and 9 (IC(50), 16, 6, and 20 nmol/L, respectively). In vitro, AZD5438 showed significant antiproliferative activity in human tumor cell lines (IC(50) range, 0.2-1.7 micromol/L), causing inhibition of the phosphorylation of cdk substrates pRb, nucleolin, protein phosphatase 1a, and RNA polymerase II COOH-terminal domain and blocking cell cycling at G(2)-M, S, and G(1) phases. In vivo, when orally administered at either 50 mg/kg twice daily or 75 mg/kg once daily, AZD5438 inhibited human tumor xenograft growth (maximum percentage tumor growth inhibition, range, 38-153; P < 0.05). In vivo, AZD5438 reduced the proportion of actively cycling cells. Further pharmacodynamic analysis of AZD5438-treated SW620 xenografts showed that efficacious doses of AZD5438 (>40% tumor growth inhibition) maintained suppression of biomarkers, such as phospho-pRbSer(249)/Thr(252), for up to 16 hours following a single oral dose. A comparison of different schedules indicated that chronic daily oral dosing provided optimal cover to ensure antitumor efficacy. These data indicate that broad cdk inhibition may provide an effective method to impair the dysregulated cell cycle that drives tumorigenesis and AZD5438 has the pharmacologic profile that provides an ideal probe to test this premise.
Asunto(s)
Antineoplásicos/farmacología , Proteína Quinasa CDC2/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Imidazoles/farmacología , Neoplasias/tratamiento farmacológico , Pirimidinas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Western Blotting , Proteína Quinasa CDC2/metabolismo , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 9 Dependiente de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Femenino , Humanos , Ratones , Ratones Desnudos , Neoplasias/metabolismo , Neoplasias/patología , Ratas , Ratas Desnudas , Células Tumorales CultivadasRESUMEN
The development of a novel series of imidazole pyrimidine amides as cyclin-dependent kinase (CDK) inhibitors is described. Optimisation of inhibitory potency against multiple CDK's (1, 2 and 9) resulted in imidazole pyrimidine amides with potent in vitro anti-proliferative effects against a range of cancer cell lines. Excellent physiochemical properties and large margins against inhibition of CYP isoforms and the hERG ion channel were achieved by modification of lipophilicity and amine basicity. A candidate with disease model activity in human cancer cell line xenografts and with suitable physiochemical and pharmacokinetic profiles for intravenous (i.v.) dosing was selected for further development as AZD5597.
Asunto(s)
Amidas/química , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/química , Imidazoles/síntesis química , Inhibidores de Proteínas Quinasas/síntesis química , Pirimidinas/química , Línea Celular Tumoral , Química Física/métodos , Cristalografía por Rayos X , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/farmacología , Diseño de Fármacos , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/metabolismo , Humanos , Imidazoles/farmacología , Infusiones Intravenosas , Modelos Químicos , Conformación Molecular , Trasplante de Neoplasias , Isoformas de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/síntesis química , Pirimidinas/farmacologíaRESUMEN
The development of a novel series of imidazole pyrimidine amides as cyclin-dependent kinase (CDK) inhibitors is described. The series was found to have much improved CDK2 inhibition and potent in vitro anti-proliferative effects against cancer cell lines. Control of overall lipophilicity was important to achieve good in vitro potency along with acceptable physiochemical properties and margins against inhibition of both CYP isoforms and the hERG potassium ion channel. A compound with an attractive overall balance of properties was profiled in vivo and possessed suitable physiochemical and pharmacokinetic profiles for oral dosing.
Asunto(s)
Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/síntesis química , Imidazoles/química , Pirimidinas/química , Administración Oral , Animales , Línea Celular Tumoral , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Concentración 50 Inhibidora , Masculino , Ratones , Ratones Desnudos , Ratas , Ratas WistarRESUMEN
Because the majority of cancers exhibit direct or indirect deregulation of cyclin-dependent kinase (CDK) function, members of the CDK family are attractive targets for the development of anticancer agents. As part of an ongoing program, novel imidazopyridines were identified and developed as potent and selective CDK inhibitors. Here, we describe data on the in vitro biological activities of one of these compounds, AZ703. The selectivity profile of AZ703 was investigated in kinase assays against a range of CDK enzymes as well as a panel of protein kinases in vitro. IC50s were assessed against different tumor cell lines in vitro. The mechanism of action of AZ703 was determined by observing changes in phosphorylation of CDK substrates and cell cycle effects on tumor and normal cells. In vitro studies revealed that AZ703 is a selective inhibitor of CDK1 and CDK2 and displays a mode of action consistent with the induction of G1-, S-, and G2-M-phase arrest. AZ703 also showed potent antiproliferative activity across a wide range of tumor cell lines in vitro. Moreover, AZ703 induced reversible blockade of normal cells while causing tumor cells to undergo apoptosis. We have identified AZ703 as a novel selective imidazo[1,2-a]pyridine CDK inhibitor that shows promising antitumor properties in vitro.