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1.
Oncologist ; 25(10): 833-e1438, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32311798

RESUMO

LESSONS LEARNED: Despite strong preclinical rationale, combined cobimetinib-mediated MEK inhibition and GDC-0994-mediated ERK inhibition was not tolerable on two 28-day dosing schedules in which GDC-0994 was given for 21 days continuously and cobimetinib administered over 21 days either continuously or intermittently. Adverse events were as expected for mitogen-activated protein kinase pathway inhibition, but overlapping and cumulative toxicities could not be managed on either dosing schedule. Pharmacokinetic parameters of cobimetinib and GDC-0994 given in combination were similar to those previously observed in monotherapy studies, so that there was no evidence of drug-drug interaction. Cycle 1 metabolic responses were observed by 18F-fluorodeoxyglucose-positron emission tomography but were not predictive of outcome measured by RECIST 1.1. BACKGROUND: Simultaneous targeting of multiple nodes in the mitogen-activated protein kinase (MAPK) pathway offers the prospect of enhanced activity in RAS-RAF-mutant tumors. This phase Ib trial evaluated the combination of cobimetinib (MEK inhibitor) and GDC-0994 (ERK inhibitor) in patients with locally advanced or metastatic solid tumors. METHODS: Cobimetinib and GDC-0994 were administered orally on two separate dosing schedules. Arm A consisted of concurrent cobimetinib and GDC-0994 once daily for 21 days of a 28-day cycle; Arm B consisted of intermittent dosing of cobimetinib on a 28-day cycle concurrent with GDC-0994 daily for 21 days of a 28-day cycle. RESULTS: In total, 24 patients were enrolled. For Arm A, owing to cumulative grade 1-2 toxicity, the dose of cobimetinib was decreased. For Arm B, dose increases of GDC-0994 and cobimetinib were intolerable with grade 3 dose-limiting toxicities of myocardial infarction and rash. Pharmacokinetic data did not show evidence of a drug-drug interaction. Overall, seven patients had a best overall response of stable disease (SD) and one patient with pancreatic adenocarcinoma had an unconfirmed partial response. CONCLUSION: The safety profile of MEK and ERK inhibition demonstrated classic MAPK inhibitor-related adverse events (AEs). However, overlapping AEs and cumulative toxicity could not be adequately managed on either dosing schedule, restricting the ability to further develop this combination.


Assuntos
Adenocarcinoma , Neoplasias , Neoplasias Pancreáticas , Azetidinas , Humanos , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno , Neoplasias/tratamento farmacológico , Piperidinas , Inibidores de Proteínas Quinases/efeitos adversos
2.
Clin Cancer Res ; 26(6): 1229-1236, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31848189

RESUMO

PURPOSE: ERK1/2 signaling can be dysregulated in cancer. GDC-0994 is an oral inhibitor of ERK1/2. A first-in-human, phase I dose escalation study of GDC-0994 was conducted in patients with locally advanced or metastatic solid tumors. PATIENTS AND METHODS: GDC-0994 was administered once daily on a 21-day on/7-day off schedule to evaluate safety, pharmacokinetics, and preliminary signs of efficacy. Patients with pancreatic adenocarcinoma and BRAF-mutant colorectal cancer were enrolled in the expansion stage. RESULTS: Forty-seven patients were enrolled in six successive cohorts (50-800 mg). A single DLT of grade 3 rash occurred at 600 mg. The most common drug-related adverse events (AE) were diarrhea, rash, nausea, fatigue, and vomiting. Pharmacokinetic data showed dose-proportional increases in exposure, with a mean half-life of 23 hours, supportive of once daily dosing. In evaluable paired biopsies, MAPK pathway inhibition ranged from 19% to 51%. Partial metabolic responses by FDG-PET were observed in 11 of 20 patients across dose levels in multiple tumor types. Overall, 15 of 45 (33%) patients had a best overall response of stable disease and 2 patients with BRAF-mutant colorectal cancer had a confirmed partial response. CONCLUSIONS: GDC-0994 had an acceptable safety profile and pharmacodynamic effects were observed by FDG-PET and in serial tumor biopsies. Single-agent activity was observed in 2 patients with BRAF-mutant colorectal cancer.


Assuntos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Piridonas/farmacocinética , Piridonas/uso terapêutico , Pirimidinas/farmacocinética , Pirimidinas/uso terapêutico , Adulto , Idoso , Relação Dose-Resposta a Droga , Fadiga/induzido quimicamente , Feminino , Humanos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Náusea/induzido quimicamente , Neoplasias/induzido quimicamente , Neoplasias/patologia , Segurança do Paciente , Distribuição Tecidual , Vômito/induzido quimicamente
3.
J Cell Sci ; 127(Pt 7): 1454-63, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24481816

RESUMO

Coatomer (COPI)-coated vesicles mediate membrane trafficking in the early secretory pathway. There are at least three subclasses of COPI coats and two classes of Arf GTPases that couple COPI coat proteins to membranes. Whether mechanisms exist to link specific Arfs to specific COPI subcomplexes is unknown. We now demonstrate that Scy1-like protein 1 (Scyl1), a member of the Scy1-like family of catalytically inactive protein kinases, oligomerizes through centrally located HEAT repeats and uses a C-terminal RKXX-COO(-) motif to interact directly with the appendage domain of coatomer subunit γ-2 (also known as COPG2 or γ2-COP). Through a distinct site, Scyl1 interacts selectively with class II Arfs, notably Arf4, thus linking class II Arfs to γ2-bearing COPI subcomplexes. Therefore, Scyl1 functions as a scaffold for key components of COPI coats, and disruption of the scaffolding function of Scyl1 causes tubulation of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and the cis-Golgi, similar to that observed following the loss of Arf and Arf-guanine-nucleotide-exchange factor (GEF) function. Our data reveal that Scyl1 is a key organizer of a subset of the COPI machinery.


Assuntos
Complexo I de Proteína do Envoltório/metabolismo , Proteína Coatomer/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ligação a DNA , Complexo de Golgi/metabolismo , Células HEK293 , Células HeLa , Humanos , Dados de Sequência Molecular , Transporte Proteico
4.
PLoS Biol ; 11(10): e1001670, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24130457

RESUMO

AP-2 is the core-organizing element in clathrin-mediated endocytosis. During the formation of clathrin-coated vesicles, clathrin and endocytic accessory proteins interact with AP-2 in a temporally and spatially controlled manner, yet it remains elusive as to how these interactions are regulated. Here, we demonstrate that the endocytic protein NECAP 1, which binds to the α-ear of AP-2 through a C-terminal WxxF motif, uses an N-terminal PH-like domain to compete with clathrin for access to the AP-2 ß2-linker, revealing a means to allow AP-2-mediated coordination of accessory protein recruitment and clathrin polymerization at sites of vesicle formation. Knockdown and functional rescue studies demonstrate that through these interactions, NECAP 1 and AP-2 cooperate to increase the probability of clathrin-coated vesicle formation and to control the number, size, and cargo content of the vesicles. Together, our data demonstrate that NECAP 1 modulates the AP-2 interactome and reveal a new layer of organizational control within the endocytic machinery.


Assuntos
Complexo 2 de Proteínas Adaptadoras/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Clatrina/metabolismo , Endocitose , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Células COS , Chlorocebus aethiops , Sequência Conservada , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteínas de Membrana/química , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Vesículas Sinápticas/metabolismo
5.
J Biol Chem ; 286(16): 13791-800, 2011 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-21330364

RESUMO

The DENN domain is a common, evolutionarily ancient, and conserved protein module, yet it has gone largely unstudied; until recently, little was known regarding its functional roles. New studies reveal that various DENN domains interact directly with members of the Rab family of small GTPases and that DENN domains function enzymatically as Rab-specific guanine nucleotide exchange factors. Thus, DENN domain proteins appear to be generalized regulators of Rab function. Study of these proteins will provide new insights into Rab-mediated membrane trafficking pathways.


Assuntos
Proteínas rab de Ligação ao GTP/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Endocitose , Genoma Humano , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de Sinais
6.
J Biol Chem ; 284(2): 774-83, 2009 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-18990695

RESUMO

Entry of the bacterial pathogen Listeria monocytogenes into host epithelial cells is critical for infection and virulence. One major pathway for Listeria entry involves binding of the bacterial protein Internalin B to the host receptor tyrosine kinase Met (hepatocyte growth factor receptor). Activation of Met and downstream signaling cascades is critical for Listeria entry. Internalin B is composed of several structural domains including an N-terminal leucine-rich repeat that is sufficient for binding Met and stimulating downstream signal transduction. Internalin B is monomeric, whereas the leucine-rich repeat is dimeric when expressed as an isolated fragment. The different quaternary states of Internalin B and the leucine-rich repeat suggest that these two Met ligands might cause distinct biological effects. Here we demonstrate that Internalin B and the leucine-rich repeat fragment exhibit agonist properties that differentially influence Met down-regulation in lysosomes. Specifically, Met stability is increased in response to the leucine-rich repeat fragment compared with Internalin B. Interestingly, Internalin B and the leucine-rich repeat stimulate equivalent rates of clathrin-mediated Met internalization. However, the leucine-rich repeat is defective in promoting lysosomal down-regulation of Met and instead enhances receptor recycling to the cell surface. In addition, the leucine-rich repeat causes prolonged Met activation (phosphorylation) and increased cell motility compared with Internalin B. Taken together, our findings indicate that individual domains of Internalin B differentially regulate Met trafficking. The ability of the leucine-rich repeat fragment to promote Met recycling could account for the increased cell motility induced by this ligand.


Assuntos
Proteínas de Bactérias/metabolismo , Leucina/metabolismo , Listeria monocytogenes/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Apoptose , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Linhagem Celular , Clatrina/metabolismo , Cães , Endocitose , Ativação Enzimática/efeitos dos fármacos , Humanos , Listeria monocytogenes/genética , Proteínas de Membrana/genética , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-met/deficiência , Proteínas Proto-Oncogênicas c-met/genética
7.
Cell Microbiol ; 9(10): 2497-516, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17848169

RESUMO

The intracellular bacterial pathogen Listeria monocytogenes causes food-borne illnesses leading to gastroenteritis, meningitis or abortion. Listeria induces its internalization into some mammalian cells through binding of the bacterial surface protein InlB to its host receptor, the Met Receptor Tyrosine Kinase. InlB-induced activation of Met stimulates host signal transduction pathways that culminate in cell surface changes driving pathogen engulfment. One mammalian protein with the potential to couple Met to downstream signalling is the adaptor CrkII. CrkII contains an unusual carboxyl-terminal SH3 domain (SH3C) that promotes entry of Listeria. However, binding partners or downstream effectors of SH3C remain unknown. Here, we use RNA interference and overexpression studies to demonstrate that SH3C affects bacterial uptake, at least in part, through stimulation of host phosphatidylinositide (PI) 3-kinase. Experiments with latex beads coated with InlB protein indicated that one potential role of SH3C and PI 3 kinase is to promote changes in the F-actin cytoskeleton necessary for particle engulfment. Taken together, our results indicate that the CrkII SH3C domain engages a cellular ligand that regulates PI 3 kinase activity and host cell surface rearrangements.


Assuntos
Listeria monocytogenes/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-crk/fisiologia , Domínios de Homologia de src , Actinas/fisiologia , Proteínas de Bactérias/metabolismo , Membrana Celular/fisiologia , Cromonas/farmacologia , Endocitose , Ativação Enzimática , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Proteínas de Membrana/metabolismo , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Transdução de Sinais
8.
Traffic ; 6(6): 459-73, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15882443

RESUMO

Increased hepatocyte growth factor receptor (HGFR) signaling correlates closely with neoplastic invasion and metastatic potential of many human cancers. Hepatocyte growth factor receptor signaling is initiated by binding the physiological ligand HGF or the internalin B (InlB) protein of Listeria monocytogenes. Subsequent degradation of endocytosed HGFR terminates receptor signaling. Previously reported discrepancies in InlB and HGF-induced HGFR signaling could reflect differences in receptor internalization and degradation in response to these distinct ligands. We report that soluble InlB and HGF are mechanistically equivalent in triggering clathrin-dependent endocytosis and lysosomal degradation of HGFR. After internalization, InlB and HGF colocalize with Rab5, EEA1 and the transferrin receptor in classical early endosomes. Hepatocyte growth factor receptor internalization was prevented by overexpression of dominant negative mutants of dynamin 1 and epidermal growth factor phosphorylation substrate 15, but not caveolin 1, the GTPase Arf6 or the cholesterol-chelating drug Nystatin. Thus, HGFR internalization is principally clathrin-mediated and is not regulated by clathrin- independent pathways. Phosphatidylinositol 3-kinase signaling and HGF-regulated tyrosine kinase substrate were not required for ligand-triggered internalization of HGFR but were essential for subsequent lysosomal degradation. Thus, soluble InlB and HGF induce HGFR endocytosis and degradation by indistinguishable mechanisms, suggesting that InlB may be exploited to regulate pathogenic HGFR signaling.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Fator de Crescimento de Hepatócito/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Biotina/química , Biotinilação , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Colesterol/metabolismo , Clatrina/metabolismo , Dinamina I/metabolismo , Endocitose , Endossomos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Genes Dominantes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Ligantes , Listeria/metabolismo , Lisossomos/metabolismo , Microdomínios da Membrana/metabolismo , Microscopia Confocal , Nistatina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Plasmídeos/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/química , Transdução de Sinais , Fatores de Tempo , Transfecção
9.
Cell Microbiol ; 7(3): 443-57, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15679846

RESUMO

The bacterial surface protein InlB mediates internalization of Listeria monocytogenes into mammalian cells through interaction with the host receptor tyrosine kinase, Met. InlB/Met interaction results in activation of the host phosphoinositide (PI) 3-kinase p85-p110, an event required for bacterial entry. p85-p110 activation coincides with tyrosine phosphorylation of the host adaptor Gab1, and formation of complexes between Gab1 and the p85 regulatory subunit of PI 3-kinase. When phosphorylated in response to agonists, Gab1 is known to recruit several Src-homology 2 (SH2) domain-containing proteins including p85, the tyrosine phosphatase Shp2 and the adaptor CrkII. Here, we demonstrate that Gab1.p85 and Gab1.CrkII complexes promote entry of Listeria. Overexpression of wild-type Gab1 stimulated entry, whereas Gab1 alleles unable to recruit all SH2 proteins known to bind wild-type Gab1 inhibited internalization. Further analysis with Gab1 alleles defective in binding individual effectors suggested that recruitment of p85 and CrkII are critical for entry. Consistent with this data, overexpression of wild-type CrkII stimulated bacterial uptake. Experiments with mutant CrkII alleles indicated that both the first and second SH3 domains of this adaptor participate in entry, with the second domain playing the most critical role. Taken together, these findings demonstrate novel roles for Gab1 and CrkII in Listeria internalization.


Assuntos
Proteínas de Bactérias/fisiologia , Listeria monocytogenes/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Chlorocebus aethiops , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Listeria monocytogenes/patogenicidade , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-crk , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Transdução de Sinais , Células Vero , Domínios de Homologia de src
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