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1.
Nature ; 519(7544): 425-30, 2015 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-25799996

RESUMO

Cell migration is a stepwise process that coordinates multiple molecular machineries. Using in vitro angiogenesis screens with short interfering RNA and chemical inhibitors, we define here a MAP4K4-moesin-talin-ß1-integrin molecular pathway that promotes efficient plasma membrane retraction during endothelial cell migration. Loss of MAP4K4 decreased membrane dynamics, slowed endothelial cell migration, and impaired angiogenesis in vitro and in vivo. In migrating endothelial cells, MAP4K4 phosphorylates moesin in retracting membranes at sites of focal adhesion disassembly. Epistasis analyses indicated that moesin functions downstream of MAP4K4 to inactivate integrin by competing with talin for binding to ß1-integrin intracellular domain. Consequently, loss of moesin (encoded by the MSN gene) or MAP4K4 reduced adhesion disassembly rate in endothelial cells. Additionally, α5ß1-integrin blockade reversed the membrane retraction defects associated with loss of Map4k4 in vitro and in vivo. Our study uncovers a novel aspect of endothelial cell migration. Finally, loss of MAP4K4 function suppressed pathological angiogenesis in disease models, identifying MAP4K4 as a potential therapeutic target.


Assuntos
Movimento Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Integrinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Motivos de Aminoácidos , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Forma Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Epistasia Genética , Adesões Focais/metabolismo , Humanos , Integrina alfa1/efeitos dos fármacos , Integrina alfa1/metabolismo , Integrina beta1/química , Integrina beta1/efeitos dos fármacos , Integrina beta1/metabolismo , Integrinas/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Neovascularização Patológica , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Talina/química , Talina/metabolismo
2.
Blood ; 122(22): 3678-90, 2013 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-23886837

RESUMO

Establishment and stabilization of endothelial tubes with patent lumens is vital during vertebrate development. Ras-interacting protein 1 (RASIP1) has been described as an essential regulator of de novo lumenogenesis through modulation of endothelial cell (EC) adhesion to the extracellular matrix (ECM). Here, we show that in mouse and zebrafish embryos, Rasip1-deficient vessels transition from an angioblast cord to a hollow tube, permit circulation of primitive erythrocytes, but ultimately collapse, leading to hemorrhage and embryonic lethality. Knockdown of RASIP1 does not alter EC-ECM adhesion, but causes cell-cell detachment and increases permeability of EC monolayers in vitro. We also found that endogenous RASIP1 in ECs binds Ras-related protein 1 (RAP1), but not Ras homolog gene family member A or cell division control protein 42 homolog. Using an exchange protein directly activated by cyclic adenosine monophosphate 1 (EPAC1)-RAP1-dependent model of nascent junction formation, we demonstrate that a fraction of the RASIP1 protein pool localizes to cell-cell contacts. Loss of RASIP1 phenocopies loss of RAP1 or EPAC1 in ECs by altering junctional actin organization, localization of the actin-bundling protein nonmuscle myosin heavy chain IIB, and junction remodeling. Our data show that RASIP1 regulates the integrity of newly formed blood vessels as an effector of EPAC1-RAP1 signaling.


Assuntos
Proteínas de Transporte/fisiologia , Endotélio Vascular/embriologia , Endotélio Vascular/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Junções Intercelulares/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Neovascularização Fisiológica , Gravidez , Interferência de RNA , Transdução de Sinais , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologia
3.
Mol Cancer Ther ; 21(6): 986-998, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35642431

RESUMO

In the past year, four antibody-drug conjugates (ADC) were approved, nearly doubling the marketed ADCs in oncology. Among other attributes, successful ADCs optimize targeting antibody, conjugation chemistry, and payload mechanism of action. Here, we describe the development of ABBV-011, a novel SEZ6-targeted, calicheamicin-based ADC for the treatment of small cell lung cancer (SCLC). We engineered a calicheamicin conjugate that lacks the acid-labile hydrazine linker that leads to systemic release of a toxic catabolite. We then screened a patient-derived xenograft library to identify SCLC as a tumor type with enhanced sensitivity to calicheamicin ADCs. Using RNA sequencing (RNA-seq) data from primary and xenograft SCLC samples, we identified seizure-related homolog 6 (SEZ6) as a surface-expressed SCLC target with broad expression in SCLC and minimal normal tissue expression by both RNA-seq and IHC. We developed an antibody targeting SEZ6 that is rapidly internalized upon receptor binding and, when conjugated to the calicheamicin linker drug, drives potent tumor regression in vitro and in vivo. These preclinical data suggest that ABBV-011 may provide a novel treatment for patients with SCLC and a rationale for ongoing phase I studies (NCT03639194).


Assuntos
Antineoplásicos , Imunoconjugados , Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Antineoplásicos/farmacologia , Calicheamicinas , Ensaios Clínicos Fase I como Assunto , Humanos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética
4.
Transl Oncol ; 14(1): 100883, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33074129

RESUMO

Rovalpituzumab tesirine (Rova-T) offers a targeted therapy for ~85% of SCLC patients whose tumors express DLL3, but clinical dosing is limited due to off-target toxicities. We hypothesized that a sub-efficacious dose of Rova-T combined with anti-PD1, which alone shows a clinical benefit to ~15% of SCLC patients, might elicit a novel mechanism of action and extend clinical utility. Using a pre-clinical murine SCLC tumor model that expresses Dll3 and has an intact murine immune system, we found that sub-efficacious doses of Rova-T with anti-PD1 resulted in enhanced anti-tumor activity, compared to either monotherapy. Multiplex immunohistochemistry (IHC) showed CD4 and CD8 T-cells primarily in normal tissue immediately adjacent to the tumor. Combination treatment, but not anti-PD1 alone, increased Ki67+/CD8 T-cells and Granzyme B+/CD8 in tumors by flow cytometry and IHC. Antibody depletion of T-cell populations showed CD8+ T-cells are required for in vivo anti-tumor efficacy. Whole transcriptome analysis as well as flow cytometry and IHC showed that Rova-T activates dendritic cells and increases Ccl5, Il-12, and Icam more than anti-PD1 alone. Increased tumor expression of PDL1 and MHC1 following Rova-T treatment also supports combination with anti-PD1. Mice previously treated with Rova-T + anti-PD1 withstood tumor re-challenge, demonstrating sustained anti-tumor immunity. Collectively our pre-clinical data support clinical combination of sub-efficacious Rova-T with anti-PD1 to extend the benefit of immune checkpoint inhibitors to more SCLC patients.

5.
ACS Med Chem Lett ; 6(8): 913-8, 2015 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-26288693

RESUMO

Diverse biological roles for mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) have necessitated the identification of potent inhibitors in order to study its function in various disease contexts. In particular, compounds that can be used to carry out such studies in vivo would be critical for elucidating the potential for therapeutic intervention. A structure-based design effort coupled with property-guided optimization directed at minimizing the ability of the inhibitors to cross into the CNS led to an advanced compound 13 (GNE-495) that showed excellent potency and good PK and was used to demonstrate in vivo efficacy in a retinal angiogenesis model recapitulating effects that were observed in the inducible Map4k4 knockout mice.

6.
J Med Chem ; 57(8): 3484-93, 2014 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-24673130

RESUMO

Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is a serine/threonine kinase implicated in the regulation of many biological processes. A fragment-based lead discovery approach was used to generate potent and selective MAP4K4 inhibitors. The fragment hit pursued in this article had excellent ligand efficiency (LE), an important attribute for subsequent successful optimization into drug-like lead compounds. The optimization efforts eventually led us to focus on the pyridopyrimidine series, from which 6-(2-fluoropyridin-4-yl)pyrido[3,2-d]pyrimidin-4-amine (29) was identified. This compound had low nanomolar potency, excellent kinase selectivity, and good in vivo exposure, and demonstrated in vivo pharmacodynamic effects in a human tumor xenograft model.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/síntese química , Animais , Descoberta de Drogas , Feminino , Peptídeos e Proteínas de Sinalização Intracelular/química , Camundongos , Modelos Moleculares , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/química , Pirimidinas/farmacologia , Relação Estrutura-Atividade
7.
Neoplasia ; 15(7): 694-711, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23814482

RESUMO

The phosphatidylinositol 3-kinase (PI3K) pathway is a central mediator of vascular endothelial growth factor (VEGF)-driven angiogenesis. The discovery of small molecule inhibitors that selectively target PI3K or PI3K and mammalian target of rapamycin (mTOR) provides an opportunity to pharmacologically determine the contribution of these key signaling nodes in VEGF-A-driven tumor angiogenesis in vivo. This study used an array of micro-vascular imaging techniques to monitor the antivascular effects of selective class I PI3K, mTOR, or dual PI3K/mTOR inhibitors in colorectal and prostate cancer xenograft models. Micro-computed tomography (micro-CT) angiography, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), vessel size index (VSI) MRI, and DCE ultrasound (DCE-U/S) were employed to quantitatively evaluate the vascular (structural and physiological) response to these inhibitors. GDC-0980, a dual PI3K/mTOR inhibitor, was found to reduce micro-CT angiography vascular density, while VSI MRI demonstrated a significant reduction in vessel density and an increase in mean vessel size, consistent with a loss of small functional vessels and a substantial antivascular response. DCE-MRI showed that GDC-0980 produces a strong functional response by decreasing the vascular permeability/perfusion-related parameter, K (trans). Interestingly, comparable antivascular effects were observed for both GDC-980 and GNE-490 (a selective class I PI3K inhibitor). In addition, mTOR-selective inhibitors did not affect vascular density, suggesting that PI3K inhibition is sufficient to generate structural changes, characteristic of a robust antivascular response. This study supports the use of noninvasive microvascular imaging techniques (DCE-MRI, VSI MRI, DCE-U/S) as pharmacodynamic assays to quantitatively measure the activity of PI3K and dual PI3K/mTOR inhibitors in vivo.


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Inibidores Enzimáticos , Neoplasias/diagnóstico , Neovascularização Patológica/diagnóstico , Angiografia/métodos , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Xenoenxertos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Imageamento por Ressonância Magnética/métodos , Camundongos , Imagem Multimodal , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Pirimidinas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Carga Tumoral/efeitos dos fármacos , Ultrassonografia/métodos , Microtomografia por Raio-X/métodos
8.
Mol Cell Biol ; 31(2): 342-50, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20974808

RESUMO

Cells in endothelial cell monolayers maintain a tight barrier between blood and tissue, but it is not well understood how endothelial cells move within monolayers, pass each other, migrate when stimulated with growth factor, and also retain monolayer integrity. Here, we develop a quantitative steering model based on functional classes of genes identified previously in a small interfering RNA (siRNA) screen to explain how cells locally coordinate their movement to maintain monolayer integrity and collectively migrate in response to growth factor. In the model, cells autonomously migrate within the monolayer and turn in response to mechanical cues resulting from adhesive, drag, repulsive, and directed steering interactions with neighboring cells. We show that lateral-drag steering explains the local coordination of cell movement and the maintenance of monolayer integrity by allowing closure of small lesions. We further demonstrate that directional steering of cells at monolayer boundaries, combined with adhesive steering of cells behind, can explain growth factor-triggered collective migration into open space. Together, this model provides a mechanistic explanation for the observed genetic modularity and a conceptual framework for how cells can dynamically maintain sheet integrity and undergo collective directed migration.


Assuntos
Movimento Celular/fisiologia , Células Endoteliais/fisiologia , Modelos Biológicos , Adesão Celular/fisiologia , Células Cultivadas , Células Endoteliais/citologia , Humanos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
9.
Genes Dev ; 22(23): 3268-81, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19056882

RESUMO

Growth factor-induced migration of endothelial cell monolayers enables embryonic development, wound healing, and angiogenesis. Although collective migration is widespread and therapeutically relevant, the underlying mechanism by which cell monolayers respond to growth factor, sense directional signals, induce motility, and coordinate individual cell movements is only partially understood. Here we used RNAi to identify 100 regulatory proteins that enhance or suppress endothelial sheet migration into cell-free space. We measured multiple live-cell migration parameters for all siRNA perturbations and found that each targeted protein primarily regulates one of four functional outputs: cell motility, directed migration, cell-cell coordination, or cell density. We demonstrate that cell motility regulators drive random, growth factor-independent motility in the presence or absence of open space. In contrast, directed migration regulators selectively transduce growth factor signals to direct cells along the monolayer boundary toward open space. Lastly, we found that regulators of cell-cell coordination are growth factor-independent and reorient randomly migrating cells inside the sheet when boundary cells begin to migrate. Thus, cells transition from random to collective migration through a modular control system, whereby growth factor signals convert boundary cells into pioneers, while cells inside the monolayer reorient and follow pioneers through growth factor-independent migration and cell-cell coordination.


Assuntos
Movimento Celular/fisiologia , Células Endoteliais/fisiologia , Fatores de Crescimento de Fibroblastos/fisiologia , Comunicação Celular , Proliferação de Células , Células Cultivadas , Humanos , Neovascularização Fisiológica , RNA Interferente Pequeno/farmacologia
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