RESUMEN
BACKGROUND: Most patients with KIT-mutant gastrointestinal stromal tumours (GISTs) benefit from imatinib, but treatment resistance results from outgrowth of heterogeneous subclones with KIT secondary mutations. Once resistance emerges, targeting KIT with tyrosine kinase inhibitors (TKIs) sunitinib and regorafenib provides clinical benefit, albeit of limited duration. METHODS: We systematically explored GIST resistance mechanisms to KIT-inhibitor TKIs that are either approved or under investigation in clinical trials: the studies draw upon GIST models and clinical trial correlative science. We subsequently modelled in vitro a rapid TKI alternation approach against subclonal heterogeneity. RESULTS: Each of the KIT-inhibitor TKIs targets effectively only a subset of KIT secondary mutations in GIST. Regorafenib and sunitinib have complementary activity in that regorafenib primarily inhibits imatinib-resistance mutations in the activation loop, whereas sunitinib inhibits imatinib-resistance mutations in the ATP-binding pocket. We find that rapid alternation of sunitinib and regorafenib suppresses growth of polyclonal imatinib-resistant GIST more effectively than either agent as monotherapy. CONCLUSIONS: Our data highlight that heterogeneity of KIT secondary mutations is the main mechanism of tumour progression to KIT inhibitors in imatinib-resistant GIST patients. Therapeutic combinations of TKIs with complementary activity against resistant mutations may be useful to suppress growth of polyclonal imatinib-resistance in GIST.
Asunto(s)
Neoplasias Gastrointestinales/tratamiento farmacológico , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Animales , Células CHO , Ensayos Clínicos Fase II como Asunto , Cricetulus , Resistencia a Antineoplásicos , Femenino , Neoplasias Gastrointestinales/enzimología , Neoplasias Gastrointestinales/genética , Tumores del Estroma Gastrointestinal/enzimología , Tumores del Estroma Gastrointestinal/genética , Humanos , Mesilato de Imatinib/farmacología , Ratones , Ratones Desnudos , Compuestos de Fenilurea/farmacología , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Piridinas/farmacología , Sunitinib/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The additional information of this manuscript originally stated that the authors declare no competing interests. This statement was incorrect, and should instead have stated the following:M.C.H. has the following competing interests to declare: Equity interest at Molecular MD; Consulting at Molecular MD, Blueprint Medicines, Deciphera Pharmaceuticals; Expert Testimony at Novartis; Licensed patent with royalty payments at Novartis. The remaining authors have no competing interests to declare.The authors apologise for any convenience this may have caused.
RESUMEN
Mammalian target of rapamycin (mTOR) is a kinase that plays a key role in a wide array of cellular processes and exists in two distinct functional complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Although mTORC2 is primarily activated by growth factors, mTORC1 is regulated by numerous extracellular and intracellular signals such as nutrients, growth factors, and cellular redox. Previous study has shown that cysteine oxidants sufficiently activate mTORC1 activity under amino acid-depleted conditions and that a reducing agent effectively suppresses amino acid-induced mTORC1 activity, thereby raising the possibility that redox-sensitive mechanisms underlie amino acid-dependent mTORC1 regulation. However, the molecular mechanism by which redox regulates mTORC1 activity is not well understood. In this study, we show that the redox-sensitive regulation of mTORC1 occurs via Rheb but not the Rag small GTPase. Enhancing cellular redox potential with cysteine oxidants significantly increases Rheb GTP levels. Importantly, modulation of the cellular redox potential with a cysteine oxidant or reducing agent failed to alter mTORC1 activity in TSC1(-/-) or TSC2(-/-) mouse embryonic fibroblast cells. Furthermore, a cysteine oxidant has little effect on mTOR localization but sufficiently activates mTORC1 activity in both p18(-/-) and control mouse embryonic fibroblast cells, suggesting that the redox-sensitive regulation of mTORC1 occurs independent of the Ragulator·Rag complex. Taken together, our results suggest that the TSC complex plays an important role in redox-sensitive mTORC1 regulation and argues for the activation of mTORC1 in places other than the lysosome upon inhibition of the TSC complex.
Asunto(s)
Proteínas de Unión al GTP Monoméricas/metabolismo , Neuropéptidos/metabolismo , Proteínas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisosomas/genética , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Noqueados , Proteínas de Unión al GTP Monoméricas/genética , Complejos Multiproteicos , Neuropéptidos/genética , Oxidación-Reducción , Transporte de Proteínas/fisiología , Proteínas/genética , Proteína Homóloga de Ras Enriquecida en el Cerebro , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genéticaRESUMEN
The arenavirus genome encodes for a Z-protein, which contains a RING domain that coordinates two zinc ions, and has been identified as having several functional roles at various stages of the virus life cycle. Z-protein binds to multiple host proteins and has been directly implicated in the promotion of viral budding, repression of mRNA translation, and apoptosis of infected cells. Using homology models of the Z-protein from Lassa strain arenavirus, replica exchange molecular dynamics (MD) was used to refine the structures, which were then subsequently clustered. Population-weighted ensembles of low-energy cluster representatives were predicted based upon optimal agreement of the chemical shifts computed with the SPARTA program with the experimental NMR chemical shifts. A member of the refined ensemble was identified to be a potential binder of budding factor Tsg101 based on its correspondence to the structure of the HIV-1 Gag late domain when bound to Tsg101. Members of these ensembles were docked against the crystal structure of human eIF4E translation initiation factor. Two plausible binding modes emerged based upon their agreement with experimental observation, favorable interaction energies and stability during MD trajectories. Mutations to Z are proposed that would either inhibit both binding mechanisms or selectively inhibit only one mode. The C-terminal domain conformation of the most populated member of the representative ensemble shielded protein-binding recognition motifs for Tsg101 and eIF4E and represents the most populated state free in solution. We propose that C-terminal flexibility is key for mediating the different functional states of the Z-protein.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Virus Lassa/metabolismo , Dominios RING Finger , Proteínas Virales/química , Proteínas Virales/metabolismo , Zinc/química , Proteínas Portadoras/genética , Biología Computacional , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Bases de Datos de Proteínas , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Factor 4E Eucariótico de Iniciación/química , Factor 4E Eucariótico de Iniciación/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Proteínas Virales/genética , Zinc/metabolismoRESUMEN
Hundreds of genetically characterized cell lines are available for the discovery of genotype-specific cancer vulnerabilities. However, screening large numbers of compounds against large numbers of cell lines is currently impractical, and such experiments are often difficult to control. Here we report a method called PRISM that allows pooled screening of mixtures of cancer cell lines by labeling each cell line with 24-nucleotide barcodes. PRISM revealed the expected patterns of cell killing seen in conventional (unpooled) assays. In a screen of 102 cell lines across 8,400 compounds, PRISM led to the identification of BRD-7880 as a potent and highly specific inhibitor of aurora kinases B and C. Cell line pools also efficiently formed tumors as xenografts, and PRISM recapitulated the expected pattern of erlotinib sensitivity in vivo.
Asunto(s)
Código de Barras del ADN Taxonómico/métodos , Resistencia a Antineoplásicos/genética , Técnicas de Genotipaje/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias/clasificación , Neoplasias/genética , Animales , Línea Celular Tumoral , Humanos , RatonesRESUMEN
mTOR, an evolutionarily conserved Ser/Thr protein kinase, belongs to the PI3K-related kinase family, which also includes DNA-PKcs, ATM, and ATR. Although other PI3K-related kinase family members have been shown to secure genomic integrity by sensing DNA damage and related stresses, mTOR is known to function as a nutrient and growth factor sensor. mTOR is the catalytic subunit of two distinct multiprotein complexes known as mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). In response to growth factor and nutrient availability, these complexes regulate a variety of cellular processes, such as cell growth, proliferation, and survival by modulating downstream effectors, such as S6K1, 4EBP1, and AKT. Therefore, evaluation of mTOR activity has been a clear readout in order to monitor the physiological status of cells in response to environmental cues. Here, we present the current techniques for the assessment of mTOR kinase activity in different experimental settings.