RESUMEN
Most breast cancer deaths are caused by estrogen receptor-αpositive (ER+) disease. Preclinical progress is hampered by a shortage of therapy-naïve ER+ tumor models that recapitulate metastatic progression and clinically relevant therapy resistance. Human prolactin (hPRL) is a risk factor for primary and metastatic ER+ breast cancer. Because mouse prolactin fails to activate hPRL receptors, we developed a prolactin-humanized Nod-SCID-IL2Rγ (NSG) mouse (NSG-Pro) with physiological hPRL levels. Here, we show that NSG-Pro mice facilitate establishment of therapy-naïve, estrogen-dependent PDX tumors that progress to lethal metastatic disease. Preclinical trials provide first-in-mouse efficacy of pharmacological hPRL suppression on residual ER+ human breast cancer metastases and document divergent biology and drug responsiveness of tumors grown in NSG-Pro versus NSG mice. Oncogenomic analyses of PDX lines in NSG-Pro mice revealed clinically relevant therapy-resistance mechanisms and unexpected, potently actionable vulnerabilities such as DNA-repair aberrations. The NSG-Pro mouse unlocks previously inaccessible precision medicine approaches for ER+ breast cancers.
Asunto(s)
Laparoscopía/métodos , Leiomioma/patología , Leiomioma/cirugía , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/cirugía , Adulto , Enfermedades Asintomáticas , Biopsia con Aguja , Cistectomía/métodos , Cistostomía/métodos , Estudios de Seguimiento , Humanos , Inmunohistoquímica , Hallazgos Incidentales , Leiomioma/diagnóstico , Masculino , Invasividad Neoplásica/patología , Estadificación de Neoplasias , Medición de Riesgo , Robótica , Tomografía Computarizada por Rayos X/métodos , Resultado del Tratamiento , Neoplasias de la Vejiga Urinaria/diagnósticoRESUMEN
Human epidermal growth factor receptor 2 (HER2) is a well-studied therapeutic target as well as a biomarker of breast cancer. HER2-targeting affibody (ZHER2:342) is a novel small scaffold protein with an extreme high affinity against HER2 screened by phage display. However, the small molecular weight of ZHER2:342 has limited its pharmaceutical application. Human serum albumin (HSA) and ZHER2:342 fusion protein may not only extend the serum half-life of ZHER2:342 but also preserve the biological function of HSA to bind and transport fatty acids, which can be used to deliver fatty acid-modified therapeutics to HER2-positive cancer cells. Two HSA and ZHER2:342 fusion proteins, one with a single ZHER2:342 domain fused to the C terminus of HSA (rHSA-ZHER2) and another with two tandem copies of ZHER2:342 fused to the C terminus of HSA (rHSA-(ZHER2)2), have been constructed, expressed, and purified. Both fusion proteins possessed the HER2 and fatty acid (FA) binding abilities demonstrated by in vitro assays. Interestingly, rHSA-(ZHER2)2, not rHSA-ZHER2, was able to inhibit the proliferation of SK-BR-3 cells at a relatively low concentration, and the increase of HER2 and ERK1/2 phosphorylation followed by rHSA-(ZHER2)2 treatment has been observed. HSA fusion proteins are easy and economical to express, purify, and formulate. As expected, HSA fusion proteins and fusion protein-bound fatty acid-modified FITC could be efficiently taken up by cells. These results proved the feasibility of using HSA fusion proteins as therapeutic agents as well as carriers for targeted drug delivery.
Asunto(s)
Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Albúmina Sérica/metabolismo , Western Blotting , Línea Celular Tumoral , Ácidos Grasos/química , Humanos , Inmunoprecipitación , Células MCF-7 , Fosforilación , Unión Proteica , Receptor ErbB-2/química , Proteínas Recombinantes de Fusión/química , Albúmina Sérica/químicaRESUMEN
Bypassing tyrosine kinases responsible for Stat5a/b phosphorylation would be advantageous for therapy development for Stat5a/b-regulated cancers. Here, we sought to identify small molecule inhibitors of Stat5a/b for lead optimization and therapy development for prostate cancer and Bcr-Abl-driven leukemias. In silico screening of chemical structure databases combined with medicinal chemistry was used for identification of a panel of small molecule inhibitors to block SH2 domain-mediated docking of Stat5a/b to the receptor-kinase complex and subsequent phosphorylation and dimerization. We tested the efficacy of the lead compound IST5-002 in experimental models and patient samples of two known Stat5a/b-driven cancers, prostate cancer and chronic myeloid leukemia (CML). The lead compound inhibitor of Stat5-002 (IST5-002) prevented both Jak2 and Bcr-Abl-mediated phosphorylation and dimerization of Stat5a/b, and selectively inhibited transcriptional activity of Stat5a (IC50 = 1.5µmol/L) and Stat5b (IC50 = 3.5 µmol/L). IST5-002 suppressed nuclear translocation of Stat5a/b, binding to DNA and Stat5a/b target gene expression. IST5-002 induced extensive apoptosis of prostate cancer cells, impaired growth of prostate cancer xenograft tumors, and induced cell death in patient-derived prostate cancers when tested ex vivo in explant organ cultures. Importantly, IST5-002 induced robust apoptotic death not only of imatinib-sensitive but also of imatinib-resistant CML cell lines and primary CML cells from patients. IST5-002 provides a lead structure for further chemical modifications for clinical development for Stat5a/b-driven solid tumors and hematologic malignancies.
Asunto(s)
Antineoplásicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Neoplasias de la Próstata/metabolismo , Relación Estructura-Actividad Cuantitativa , Factor de Transcripción STAT5/química , Proteínas Supresoras de Tumor/química , Animales , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Análisis por Conglomerados , Bases de Datos Factuales , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Expresión Génica , Perfilación de la Expresión Génica , Genes Reporteros , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Masculino , Ratones , Modelos Moleculares , Conformación Molecular , Fosforilación , Neoplasias de la Próstata/tratamiento farmacológico , Multimerización de Proteína , Factor de Transcripción STAT5/antagonistas & inhibidores , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas , Técnicas de Cultivo de Tejidos , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Bacterial lipopolysaccharide (LPS) is a key mediator in the vascular leak syndromes associated with Gram-negative bacterial infections. LPS opens the paracellular pathway in pulmonary vascular endothelia through protein tyrosine phosphorylation. We now have identified the protein-tyrosine kinases (PTKs) and their substrates required for LPS-induced protein tyrosine phosphorylation and opening of the paracellular pathway in human lung microvascular endothelial cells (HMVEC-Ls). LPS disrupted barrier integrity in a dose- and time-dependent manner, and prior broad spectrum PTK inhibition was protective. LPS increased tyrosine phosphorylation of zonula adherens proteins, VE-cadherin, gamma-catenin, and p120(ctn). Two SRC family PTK (SFK)-selective inhibitors, PP2 and SU6656, blocked LPS-induced increments in tyrosine phosphorylation of VE-cadherin and p120(ctn) and paracellular permeability. In HMVEC-Ls, c-SRC, YES, FYN, and LYN were expressed at both mRNA and protein levels. Selective small interfering RNA-induced knockdown of c-SRC, FYN, or YES diminished LPS-induced SRC Tyr(416) phosphorylation, tyrosine phosphorylation of VE-cadherin and p120(ctn), and barrier disruption, whereas knockdown of LYN did not. For VE-cadherin phosphorylation, knockdown of either c-SRC or FYN provided total protection, whereas YES knockdown was only partially protective. For p120(ctn) phosphorylation, knockdown of FYN, c-SRC, or YES each provided comparable but partial protection. Toll-like receptor 4 (TLR4) was expressed both on the surface and intracellular compartment of HMVEC-Ls. Prior knockdown of TLR4 blocked both LPS-induced SFK activation and barrier disruption. These data indicate that LPS recognition by TLR4 activates the SFKs, c-SRC, FYN, and YES, which, in turn, contribute to tyrosine phosphorylation of zonula adherens proteins to open the endothelial paracellular pathway.
Asunto(s)
Uniones Adherentes/metabolismo , Células Endoteliales/metabolismo , Pulmón/metabolismo , Fosfotirosina/metabolismo , Transducción de Señal , Receptor Toll-Like 4/metabolismo , Familia-src Quinasas/metabolismo , Animales , Bovinos , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Activación Enzimática , Humanos , Lipopolisacáridos/farmacología , Pulmón/irrigación sanguínea , Pulmón/efectos de los fármacos , Microcirculación/efectos de los fármacos , ARN Interferente Pequeño/genética , Albúmina Sérica Bovina/metabolismo , Transducción de Señal/efectos de los fármacos , Especificidad por Sustrato , Receptor Toll-Like 4/genética , Familia-src Quinasas/genéticaRESUMEN
Compound selection based on chemical similarity has been used to validate active "parent" compounds identified via database searching as viable lead compounds and to obtain initial structure-activity relationships for those leads. Twelve parent compounds that have inhibitory activity against the SH2 domain of the p56 T-cell tyrosine kinase (Lck) are the focus of this study. Lck is involved in the T-cell mediated immune response, and inhibitors of Lck protein-protein interactions could potentially be used to develop novel immunosuppressants. Similarity searches for each parent compound were performed using 2D structural fingerprints on a database containing 1,300,000 commercially available compounds. The inhibitory activity of the selected compounds was assessed using enzyme immunoassay (EIA). In general, the most active parent compounds yield the most high activity similar compounds; however, in two cases low activity parent compounds (i.e. inhibitory activity < 25% at 100 microM) yielded multiple similar compounds with activities > 60%. Such compounds may, therefore, be considered as viable lead compounds for optimization. Structure-activity relationships were explored by examining both ligand structures and their computed bound conformations to the protein. Functional groups common to the active compounds as well as key amino acid residues that form hydrogen bonds with the active compounds were identified. This information will act as the basis for the rational optimization of the lead compounds.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/química , Dominios Homologos src/efectos de los fármacos , Simulación por Computador , Evaluación Preclínica de Medicamentos , Enlace de Hidrógeno , Immunoblotting , Técnicas para Inmunoenzimas , Prueba de Cultivo Mixto de Linfocitos , Modelos Químicos , Modelos Moleculares , Conformación Proteica , Relación Estructura-ActividadRESUMEN
The protein p56 lymphoid T cell tyrosine kinase (Lck) is predominantly expressed in T lymphocytes where it plays a critical role in T-cell-mediated immune response. Lck participates in phosphotyrosine-dependent protein-protein interactions through its modular binding unit, the Src homology-2 (SH2) domain. Accordingly, virtual screening methods combined with experimental assays were used to identify small molecular weight nonpeptidic compounds that block Lck SH2 domain-dependent interactions. Virtual screening included scoring normalization procedures and postdocking structural clustering that is shown to facilitate the selection of active compounds. By targeting the well-defined hydrophobic binding pocket known to impart specificity on Lck-protein interactions (i.e., pY + 3 site), inhibitors of the Lck SH2 domain were discovered that omit the phosphotyrosine (pY) or related moieties. The 34 out of 196 computationally selected compounds were shown to inhibit Lck SH2 domain association with phosphorylated immunoreceptor tyrosine based activation motifs peptide. Twenty-four of the active compounds were further tested for their ability to modulate biological function. Thirteen of these compounds showed inhibitory activity in mixed lymphocyte culture assay. Fluorescence titration experiments on four of these active compounds further verified their binding to the SH2 domain. Because of their simple chemical structures, these small organic compounds have the potential to act as lead compounds for the development of novel immunosuppressant drugs.