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1.
Cell ; 137(3): 391-5, 2009 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-19410533

RESUMEN

Recent findings of gene fusions in carcinomas recapitulate the discovery of chromosomal abnormalities in leukemias and sarcomas decades ago. A recurring feature of carcinoma gene fusions, in contrast to those in hematopoietic and mesenchymal malignancies, is that they result in aberrant cell signaling. This may reflect differences in the differentiation programs of these tissues.


Asunto(s)
Aberraciones Cromosómicas , Neoplasias/genética , Fusión de Oncogenes , Proteínas de Fusión Oncogénica/genética , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Femenino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Neoplasias/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología
2.
Proc Natl Acad Sci U S A ; 116(7): 2545-2550, 2019 02 12.
Artículo en Inglés | MEDLINE | ID: mdl-30683716

RESUMEN

The RAS gene family is frequently mutated in human cancers, and the quest for compounds that bind to mutant RAS remains a major goal, as it also does for inhibitors of protein-protein interactions. We have refined crystallization conditions for KRAS169Q61H-yielding crystals suitable for soaking with compounds and exploited this to assess new RAS-binding compounds selected by screening a protein-protein interaction-focused compound library using surface plasmon resonance. Two compounds, referred to as PPIN-1 and PPIN-2, with related structures from 30 initial RAS binders showed binding to a pocket where compounds had been previously developed, including RAS effector protein-protein interaction inhibitors selected using an intracellular antibody fragment (called Abd compounds). Unlike the Abd series of RAS binders, PPIN-1 and PPIN-2 compounds were not competed by the inhibitory anti-RAS intracellular antibody fragment and did not show any RAS-effector inhibition properties. By fusing the common, anchoring part from the two new compounds with the inhibitory substituents of the Abd series, we have created a set of compounds that inhibit RAS-effector interactions with increased potency. These fused compounds add to the growing catalog of RAS protein-protein inhibitors and show that building a chemical series by crossing over two chemical series is a strategy to create RAS-binding small molecules.


Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Proteína Oncogénica p21(ras)/antagonistas & inhibidores , Cristalografía por Rayos X , Desarrollo de Medicamentos , Estructura Molecular , Proteína Oncogénica p21(ras)/metabolismo , Unión Proteica , Resonancia por Plasmón de Superficie
3.
Proc Natl Acad Sci U S A ; 113(13): 3603-8, 2016 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-26979953

RESUMEN

The cell surface proteome of tumors mediates the interface between the transformed cells and the general microenvironment, including interactions with stromal cells in the tumor niche and immune cells such as T cells. In addition, the cell surface proteome of individual cancers defines biomarkers for that tumor type and potential proteins that can be the target of antibody-mediated therapy. We have used next-generation deep RNA sequencing (RNA-seq) coupled to an in-house database of genes encoding cell surface proteins (herein referred to as the surfaceome) as a tool to define a cell surface proteome of Ewing sarcoma compared with progenitor mesenchymal stem cells. This subtractive RNA-seq analysis revealed a specific surfaceome of Ewing and showed unexpectedly that the leucine-rich repeat and Ig domain protein 1 (LINGO1) is expressed in over 90% of Ewing sarcoma tumors, but not expressed in any other somatic tissue apart from the brain. We found that the LINGO1 protein acts as a gateway protein internalizing into the tumor cells when engaged by antibody and can carry antibody conjugated with drugs to kill Ewing sarcoma cells. Therefore, LINGO1 is a new, unique, and specific biomarker and drug target for the treatment of Ewing sarcoma.


Asunto(s)
Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/inmunología , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Proteínas de la Membrana/inmunología , Células Madre Mesenquimatosas/metabolismo , Proteínas del Tejido Nervioso/inmunología , Proteoma/genética , Proteoma/metabolismo , ARN Neoplásico/genética , Sarcoma de Ewing/terapia , Análisis de Secuencia de ARN
4.
Blood ; 124(25): 3738-47, 2014 Dec 11.
Artículo en Inglés | MEDLINE | ID: mdl-25301704

RESUMEN

T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subtype of acute lymphoblastic leukemia (ALL) with gradually improved survival through introduction of intensified chemotherapy. However, therapy-resistant or refractory T-ALL remains a major clinical challenge. Here, we evaluated B-cell lymphoma (BCL)-2 inhibition by the BH3 mimetic ABT-199 as a new therapeutic strategy in human T-ALL. The T-ALL cell line LOUCY, which shows a transcriptional program related to immature T-ALL, exhibited high in vitro and in vivo sensitivity for ABT-199 in correspondence with high levels of BCL-2. In addition, ABT-199 showed synergistic therapeutic effects with different chemotherapeutic agents including doxorubicin, l-asparaginase, and dexamethasone. Furthermore, in vitro analysis of primary patient samples indicated that some immature, TLX3- or HOXA-positive primary T-ALLs are highly sensitive to BCL-2 inhibition, whereas TAL1 driven tumors mostly showed poor ABT-199 responses. Because BCL-2 shows high expression in early T-cell precursors and gradually decreases during normal T-cell differentiation, differences in ABT-199 sensitivity could partially be mediated by distinct stages of differentiation arrest between different molecular genetic subtypes of human T-ALL. In conclusion, our study highlights BCL-2 as an attractive molecular target in specific subtypes of human T-ALL that could be exploited by ABT-199.


Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonamidas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Western Blotting , Compuestos Bicíclicos Heterocíclicos con Puentes/administración & dosificación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Niño , Sinergismo Farmacológico , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Concentración 50 Inhibidora , Células Jurkat , Ratones Endogámicos NOD , Ratones SCID , Análisis de Secuencia por Matrices de Oligonucleótidos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfonamidas/administración & dosificación , Análisis de Supervivencia , Células Tumorales Cultivadas
5.
Biochim Biophys Acta ; 1844(11): 1970-1976, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24881582

RESUMEN

Many proteins of interest in basic biology, translational research studies and for clinical targeting in diseases reside inside the cell and function by interacting with other macromolecules. Protein complexes control basic processes such as development and cell division but also abnormal cell growth when mutations occur such as found in cancer. Interfering with protein-protein interactions is an important aspiration in both basic and disease biology but small molecule inhibitors have been difficult and expensive to isolate. Recently, we have adapted molecular biology techniques to develop a simple set of protocols for isolation of high affinity antibody fragments (in the form of single VH domains) that function within the reducing environment of higher organism cells and can bind to their target molecules. The method called Intracellular Antibody Capture (IAC) has been used to develop inhibitory anti-RAS and anti-LMO2 single domains that have been used for target validation of these antigens in pre-clinical cancer models and illustrate the efficacy of the IAC approach to generation of drug surrogates. Future use of inhibitory VH antibody fragments as drugs in their own right (we term these macrodrugs to distinguish them from small molecule drugs) requires their delivery to target cells in vivo but they can also be templates for small molecule drug development that emulate the binding sites of the antibody fragments. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

6.
Blood ; 122(12): 2039-46, 2013 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-23908472

RESUMEN

Mixed Lineage Leukemia (MLL1) translocations encode fusion proteins retaining the N terminus of MLL1, which interacts with the tumor suppressor, menin. This interaction is essential for leukemogenesis and thus is a promising drug target. However, wild-type MLL1 plays a critical role in sustaining hematopoietic stem cells (HSCs); therefore, disruption of an essential MLL1 cofactor would be expected to obliterate normal hematopoiesis. Here we show that rather than working together as a complex, menin and MLL1 regulate distinct pathways during normal hematopoiesis, particularly in HSCs and B cells. We demonstrate the lack of genetic interaction between menin and MLL1 in steady-state or regenerative hematopoiesis and in B-cell differentiation despite the fact that MLL1 is critical for these processes. In B cells, menin- or MLL1-regulated genes can be classified into 3 categories: (1) a relatively small group of coregulated genes including previously described targets Hoxa9 and Meis1 but also Mecom and Eya1, and much larger groups of (2) exclusively menin-regulated and (3) exclusively MLL1-regulated genes. Our results highlight the large degree of independence of these 2 proteins and demonstrate that menin is not a requisite cofactor for MLL1 during normal hematopoiesis. Furthermore, our data support the development of menin-MLL1-disrupting drugs as safe and selective leukemia targeting agents.


Asunto(s)
Linfocitos B/metabolismo , Células Madre Hematopoyéticas/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Animales , Linfocitos B/citología , Diferenciación Celular/fisiología , Epistasis Genética , Femenino , Regulación de la Expresión Génica , Hematopoyesis/genética , Células Madre Hematopoyéticas/citología , Linfopoyesis/genética , Ratones , Ratones Transgénicos , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas Proto-Oncogénicas/genética
7.
Blood ; 122(12): 2093-103, 2013 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-23926305

RESUMEN

Lmo2 is an oncogenic transcription factor that is frequently overexpressed in T-cell acute lymphoblastic leukemia (T-ALL), including early T-cell precursor ALL (ETP-ALL) cases with poor prognosis. Lmo2 must be recruited to DNA by binding to the hematopoietic basic helix-loop-helix factors Scl/Tal1 or Lyl1. However, it is unknown which of these factors can mediate the leukemic activity of Lmo2. To address this, we have generated Lmo2-transgenic mice lacking either Scl or Lyl1 in the thymus. We show that although Scl is dispensable for Lmo2-driven leukemia, Lyl1 is critical for all oncogenic functions of Lmo2, including upregulation of a stem cell-like gene signature, aberrant self-renewal of thymocytes, and subsequent generation of T-cell leukemia. Lyl1 expression is restricted to preleukemic and leukemic stem cell populations in this model, providing a molecular explanation for the stage-specific expression of the Lmo2-induced gene expression program. Moreover, LMO2 and LYL1 are coexpressed in ETP-ALL patient samples, and LYL1 is required for growth of ETP-ALL cell lines. Thus, the LMO2-LYL1 interaction is a promising therapeutic target for inhibiting self-renewing cancer stem cells in T-ALL, including poor-prognosis ETP-ALL cases.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas con Dominio LIM/genética , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/genética , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Análisis por Conglomerados , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Humanos , Proteínas con Dominio LIM/metabolismo , Ratones , Ratones Transgénicos , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/mortalidad , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteína 1 de la Leucemia Linfocítica T Aguda , Linfocitos T/citología , Linfocitos T/metabolismo , Timocitos/metabolismo , Timocitos/patología
8.
J Biol Chem ; 286(5): 3707-16, 2011 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-20980262

RESUMEN

Interfering intracellular antibodies are valuable for biological studies as drug surrogates and as potential macromolecular drugs per se. Their application is still limited because of the difficulty of acquisition of functional intracellular antibodies. We describe the use of the new intracellular antibody capture procedure (IAC(3)) to facilitate direct isolation of functional single domain antibody fragments using four independent target molecules (LMO2, TP53, CRAF1, and Hoxa9) from a set of diverse libraries. Initially, these have variability in only one of the three antigen-binding CDR regions of VH or VL and first round single domains are affinity matured by iterative randomization of the two other CDRs and reselection. We highlight the approach using a single domain binding to LMO2 protein. Our results show that interfering with LMO2 protein function demonstrates a role specifically in erythroid differentiation, confirm a necessary and sufficient function for LMO2 as a cancer therapy target in T-cell neoplasia and allowed for the first time production of soluble recombinant LMO2 protein by co-expression with intracellular domain antibodies. Co-crystallization of LMO2 and the anti-LMO2 VH protein was successful. These results demonstrate that this third generation IAC(3) offers a robust toolbox for various biomedical applications and consolidates functional features of the LMO2 protein complex, which includes the importance of Lmo2-Ldb1 protein interaction.


Asunto(s)
Proteínas de Unión al ADN/inmunología , Región Variable de Inmunoglobulina/farmacología , Metaloproteínas/inmunología , Proteínas Adaptadoras Transductoras de Señales , Animales , Anticuerpos , Antineoplásicos/inmunología , Diferenciación Celular/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Células Eritroides , Cadenas Pesadas de Inmunoglobulina , Región Variable de Inmunoglobulina/uso terapéutico , Proteínas con Dominio LIM , Metaloproteínas/metabolismo , Metaloproteínas/fisiología , Ratones , Biblioteca de Péptidos , Unión Proteica/efectos de los fármacos
9.
Pharm Res ; 29(4): 1098-109, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22183511

RESUMEN

PURPOSE: Bcr-Abl, the causative agent of chronic myelogenous leukemia (CML), localizes in the cytoplasm where its oncogenic signaling leads to proliferation of cells. If forced into the nucleus Bcr-Abl causes apoptosis. To achieve nuclear translocation, binding domains for capture of Bcr-Abl were generated and attached to proteins with signals destined for the nucleus. These resulting proteins would be capable of binding and translocating endogenous Bcr-Abl to the nucleus. METHODS: Bcr-Abl was targeted at 3 distinct domains for capture: by construction of high affinity intracellular antibody domains (iDabs) to regions of Bcr-Abl known to promote cytoplasmic retention, via its coiled coil domain (CC), and through a naturally occurring protein-protein interaction domain (RIN1). These binding domains were then tested for their ability to escort Bcr-Abl into the nucleus using a "protein switch" or attachment of 4 nuclear localization signals (NLSs). RESULTS: Although RIN1, ABI7-iDab, and CCmut3 constructs all produced similar colocalization with Bcr-Abl, only 4NLS-CCmut3 produced efficient nuclear translocation of Bcr-Abl. CONCLUSIONS: We demonstrate that a small binding domain can be used to control the subcellular localization of Bcr-Abl, which may have implications for CML therapy. Our ultimate future goal is to change the location of critical proteins to alter their function.


Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Fusión bcr-abl/metabolismo , Animales , Apoptosis/fisiología , Sitios de Unión , Células COS , Procesos de Crecimiento Celular/fisiología , Células Cultivadas , Chlorocebus aethiops , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Ratones , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Transporte de Proteínas , Transducción de Señal/fisiología
10.
Cancer Cell ; 3(5): 449-58, 2003 May.
Artículo en Inglés | MEDLINE | ID: mdl-12781363

RESUMEN

The etiology of human tumors often involves chromosomal translocations. Models that emulate translocations are essential to understanding the determinants of frank malignancy, those dictating the restriction of translocations to specific lineages, and as a basis for development of rational therapeutic methods. We demonstrate that developmentally regulated Cre-loxP-mediated interchromosomal recombination between the Mll gene, whose human counterpart is involved in a spectrum of leukemias, and the Enl gene creates reciprocal chromosomal translocations that cause myeloid tumors. There is a rapid onset and high penetrance of leukemogenesis in these translocator mice, and high proportions of cells carrying chromosomal translocations can be found in bone marrow as early as 12 days after birth. This de novo strategy is a direct recapitulation of naturally occurring human cancer-associated translocations.


Asunto(s)
Cromosomas/ultraestructura , Proteínas de Unión al ADN/genética , Técnicas Genéticas , Neoplasias/genética , Proteínas de Fusión Oncogénica/genética , Proto-Oncogenes , Factores de Transcripción , Translocación Genética , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Modelos Animales de Enfermedad , Citometría de Flujo , Genotipo , N-Metiltransferasa de Histona-Lisina , Humanos , Hibridación Fluorescente in Situ , Leucocitos/metabolismo , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Proteína de la Leucemia Mieloide-Linfoide , Fenotipo , Recombinación Genética , Factores de Tiempo
11.
Bioessays ; 32(7): 589-98, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-20544739

RESUMEN

Since the realisation that the antigen-binding regions of antibodies, the variable (V) regions, can be uncoupled from the rest of the molecule to create fragments that recognise and abrogate particular protein functions in cells, the use of antibody fragments inside cells has become an important tool in bioscience. Diverse libraries of antibody fragments plus in vivo screening can be used to isolate single chain variable fragments comprising VH and VL segments or single V-region domains. Some of these are interfering antibody fragments that compete with protein-protein interactions, providing lead molecules for drug interactions that until now have been considered difficult or undruggable. It may be possible to deliver or express antibody fragments in target cells as macrodrugs per se. In future incarnations of intracellular antibodies, however, the structural information of the interaction interface of target and antibody fragment should facilitate development of binding site mimics as small drug-like molecules. This is a new dawn for intracellular antibody fragments both as macrodrugs and as precursors of drugs to treat human diseases and should finally lead to the removal of the epithet of the 'undruggable' protein-protein interactions.


Asunto(s)
Anticuerpos/uso terapéutico , Espacio Intracelular/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Ingeniería de Proteínas/métodos , Anticuerpos/aislamiento & purificación , Evaluación Preclínica de Medicamentos , Humanos , Fragmentos de Péptidos/aislamiento & purificación , Unión Proteica
12.
Nucleic Acids Res ; 38(6): e86, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20008102

RESUMEN

Protein-protein interactions (PPIs) are ubiquitous in Biology, and thus offer an enormous potential for the discovery of novel therapeutics. Although protein interfaces are large and lack defining physiochemical traits, is well established that only a small portion of interface residues, the so-called hot spot residues, contribute the most to the binding energy of the protein complex. Moreover, recent successes in development of novel drugs aimed at disrupting PPIs rely on targeting such residues. Experimental methods for describing critical residues are lengthy and costly; therefore, there is a need for computational tools that can complement experimental efforts. Here, we describe a new computational approach to predict hot spot residues in protein interfaces. The method, called Presaging Critical Residues in Protein interfaces (PCRPi), depends on the integration of diverse metrics into a unique probabilistic measure by using Bayesian Networks. We have benchmarked our method using a large set of experimentally verified hot spot residues and on a blind prediction on the protein complex formed by HRAS protein and a single domain antibody. Under both scenarios, PCRPi delivered consistent and accurate predictions. Finally, PCRPi is able to handle cases where some of the input data is either missing or not reliable (e.g. evolutionary information).


Asunto(s)
Mapeo de Interacción de Proteínas/métodos , Aminoácidos/química , Teorema de Bayes , Biología Computacional , Región Variable de Inmunoglobulina/química , Modelos Moleculares , Conformación Proteica , Proteínas/química , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/inmunología , Programas Informáticos
13.
Sci Rep ; 12(1): 7226, 2022 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-35508511

RESUMEN

The transcription factor complex, consisting of LMO2, TAL1 or LYL1, and GATA2, plays an important role in capillary sprouting by regulating VEGFR2, DLL4, and angiopoietin 2 in tip cells. Overexpression of the basic helix-loop-helix transcription factor LYL1 in transgenic mice results in shortened tails. This phenotype is associated with vessel hyperbranching and a relative paucity of straight vessels due to DLL4 downregulation in tip cells by forming aberrant complex consisting of LMO2 and LYL1. Knockdown of LMO2 or TAL1 inhibits capillary sprouting in spheroid-based angiogenesis assays, which is associated with decreased angiopoietin 2 secretion. In the same assay using mixed TAL1- and LYL1-expressing endothelial cells, TAL1 was found to be primarily located in tip cells, while LYL1-expressing cells tended to occupy the stalk position in sprouts by upregulating VEGFR1 than TAL1. Thus, the interaction between LMO2 and TAL1 in tip cells plays a key role in angiogenic switch of sprouting angiogenesis.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Angiopoyetina 2 , Proteínas con Dominio LIM/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Células Endoteliales , Proteínas con Dominio LIM/genética , Ratones , Proteínas de Neoplasias/genética , Neovascularización Fisiológica/genética , Proteína 1 de la Leucemia Linfocítica T Aguda/genética
14.
Sci Adv ; 8(45): eabm3548, 2022 11 11.
Artículo en Inglés | MEDLINE | ID: mdl-36351009

RESUMEN

Metastasis is responsible for most breast cancer-related deaths; however, identifying the cellular determinants of metastasis has remained challenging. Here, we identified a minority population of immature THY1+/VEGFA+ tumor epithelial cells in human breast tumor biopsies that display angiogenic features and are marked by the expression of the oncogene, LMO2. Higher abundance of LMO2+ basal cells correlated with tumor endothelial content and predicted poor distant recurrence-free survival in patients. Using MMTV-PyMT/Lmo2CreERT2 mice, we demonstrated that Lmo2 lineage-traced cells integrate into the vasculature and have a higher propensity to metastasize. LMO2 knockdown in human breast tumors reduced lung metastasis by impairing intravasation, leading to a reduced frequency of circulating tumor cells. Mechanistically, we find that LMO2 binds to STAT3 and is required for STAT3 activation by tumor necrosis factor-α and interleukin-6. Collectively, our study identifies a population of metastasis-initiating cells with angiogenic features and establishes the LMO2-STAT3 signaling axis as a therapeutic target in breast cancer metastasis.


Asunto(s)
Neoplasias de la Mama , Neoplasias Pulmonares , Humanos , Ratones , Animales , Femenino , Neoplasias de la Mama/patología , Neoplasias Pulmonares/metabolismo , Transducción de Señal , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/metabolismo
15.
Am J Respir Crit Care Med ; 182(1): 83-91, 2010 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-20299530

RESUMEN

RATIONALE: Amplification of distal 3q is the most common genomic aberration in squamous lung cancer (SQC). SQC develops in a multistage progression from normal bronchial epithelium through dysplasia to invasive disease. Identifying the key driver events in the early pathogenesis of SQC will facilitate the search for predictive molecular biomarkers and the identification of novel molecular targets for chemoprevention and therapeutic strategies. For technical reasons, previous attempts to analyze 3q amplification in preinvasive lesions have focused on small numbers of predetermined candidate loci rather than an unbiased survey of copy-number variation. OBJECTIVES: To perform a detailed analysis of the 3q amplicon in bronchial dysplasia of different histological grades. METHODS: We use molecular copy-number counting (MCC) to analyze the structure of chromosome 3 in 19 preinvasive bronchial biopsy specimens from 15 patients and sequential biopsy specimens from 3 individuals. MEASUREMENTS AND MAIN RESULTS: We demonstrate that no low-grade lesions, but all high-grade lesions, have 3q amplification. None of seven low-grade lesions progressed clinically, whereas 8 of 10 patients with high-grade disease progressed to cancer. We identify a minimum commonly amplified region on chromosome 3 consisting of 17 genes, including 2 known oncogenes, SOX2 and PIK3CA. We confirm that both genes are amplified in all high-grade dysplastic lesions tested. We further demonstrate, in three individuals, that the clinical progression of high-grade preinvasive disease is associated with incremental amplification of SOX2, suggesting this promotes malignant progression. CONCLUSIONS: These findings demonstrate progressive 3q amplification in the evolution of preinvasive SQC and implicate SOX2 as a key target of this dynamic process.


Asunto(s)
Cromosomas Humanos Par 3/genética , Amplificación de Genes/fisiología , Neoplasias de Células Escamosas/genética , Lesiones Precancerosas/genética , Factores de Transcripción SOXB1/genética , Anciano , Neoplasias de los Bronquios/genética , Neoplasias de los Bronquios/patología , Neoplasias de los Bronquios/fisiopatología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Invasividad Neoplásica , Neoplasias de Células Escamosas/fisiopatología , Lesiones Precancerosas/clasificación , Lesiones Precancerosas/patología
16.
Nucleic Acids Res ; 37(5): e41, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19208637

RESUMEN

Antibodies are now indispensable tools for all areas of cell biology and biotechnology as well as for diagnosis and therapy. Antigen-specific single immunoglobulin variable domains that bind to native antigens can be isolated and manipulated using yeast intracellular antibody capture technology but converting these to whole monoclonal antibody requires that complementary variable domains (VH or VL) bind to the same antigenic site. We describe a simple approach (CatcherAb) for specific isolation of such complementary single domains allowing the constitution of functional Fv, forming the basis of antigen-specific whole immunoglobulin and thus antibody production. We illustrate this approach by developing high-affinity Fv from single variable domains binding to RAS and LMO2 oncogenic proteins.


Asunto(s)
Anticuerpos Monoclonales/química , Región Variable de Inmunoglobulina/aislamiento & purificación , Ingeniería de Proteínas/métodos , Proteínas/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Células CHO , Cricetinae , Cricetulus , Biblioteca de Genes , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Pesadas de Inmunoglobulina/aislamiento & purificación , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/aislamiento & purificación , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Ratones , Células 3T3 NIH , Proteína Oncogénica p21(ras)/inmunología , Proteínas Oncogénicas/inmunología , Estructura Terciaria de Proteína
17.
Sci Adv ; 7(15)2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33837087

RESUMEN

Intracellular antibodies are tools that can be used directly for target validation by interfering with properties like protein-protein interactions. An alternative use of intracellular antibodies in drug discovery is developing small-molecule surrogates using antibody-derived (Abd) technology. We previously used this strategy with an in vitro competitive surface plasmon resonance method that relied on high-affinity antibody fragments to obtain RAS-binding compounds. We now describe a novel implementation of the Abd method with a cell-based intracellular antibody-guided screening method that we have applied to the chromosomal translocation protein LMO2. We have identified a chemical series of anti-LMO2 Abd compounds that bind at the same LMO2 location as the inhibitory anti-LMO2 intracellular antibody combining site. Intracellular antibodies could therefore be used in cell-based screens to identify chemical surrogates of their binding sites and potentially be applied to any challenging proteins, such as transcription factors that have been considered undruggable.


Asunto(s)
Anticuerpos , Translocación Genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sitios de Unión , Sitios de Unión de Anticuerpos , Humanos , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas/genética
18.
Antiviral Res ; 194: 105147, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34375715

RESUMEN

The SARS-CoV-2 receptor angiotensin converting enzyme 2 (ACE2) was previously engineered into a high affinity tetravalent format (ACE2-Fc-TD) that is a potential decoy protein in SARS-CoV-2 infection.We report that this protein shows greatly enhanced binding to SARS-CoV-2 spike proteins of the SARS-CoV-2 variants of concern B.1.1.7 (alpha variant, originally isolated in the United Kingdom) and B.1.351 (beta variant, originally isolated in South Africa) with picomolar compared with nanomolar Kd values. In addition, ACE2-Fc-TD displays greater neutralization of SARS-CoV-2 pseudotype viruses compared to a dimeric ACE2-Fc, with enhanced activity on variant B.1.351. This tetrameric decoy protein would be a valuable addition to SARS-CoV-2 therapeutic approaches, especially where vaccination cannot be used but also should there be any future coronavirus pandemics.


Asunto(s)
Enzima Convertidora de Angiotensina 2/farmacología , Antivirales/metabolismo , COVID-19/prevención & control , SARS-CoV-2/efectos de los fármacos , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/metabolismo , Antivirales/química , COVID-19/enzimología , COVID-19/virología , Línea Celular , Humanos , Cinética , Mutación , Unión Proteica , Dominios Proteicos , SARS-CoV-2/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/metabolismo , Tratamiento Farmacológico de COVID-19
19.
Bio Protoc ; 10(13): e3666, 2020 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-33659336

RESUMEN

In drug development programmes, multiple assays are needed for the determination of protein-compound interactions and evaluation of potential use in assays with protein-protein interactions. In this protocol we describe the waterLOGSY NMR method for confirming protein-ligand binding events.

20.
Commun Biol ; 3(1): 342, 2020 07 03.
Artículo en Inglés | MEDLINE | ID: mdl-32620833

RESUMEN

Protein-based affinity reagents (like antibodies or alternative binding scaffolds) offer wide-ranging applications for basic research and therapeutic approaches. However, whereas small chemical molecules efficiently reach intracellular targets, the delivery of macromolecules into the cytosol of cells remains a major challenge; thus cytosolic applications of protein-based reagents are rather limited. Some pathogenic bacteria have evolved a conserved type III secretion system (T3SS) which allows the delivery of effector proteins into eukaryotic cells. Here, we enhance the T3SS of an avirulent strain of Salmonella typhimurium to reproducibly deliver multiple classes of recombinant proteins into eukaryotic cells. The efficacy of the system is probed with both DARPins and monobodies to functionally inhibit the paradigmatic and largely undruggable RAS signaling pathway. Thus, we develop a bacterial secretion system for potent cytosolic delivery of therapeutic macromolecules.


Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Citosol/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/metabolismo , Sistemas de Secreción Tipo III/metabolismo , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Células HCT116 , Células HeLa , Humanos , Proteínas de Neoplasias/genética , Proteínas Recombinantes/genética , Salmonella typhimurium/genética , Salmonella typhimurium/crecimiento & desarrollo , Sistemas de Secreción Tipo III/genética
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