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1.
Proc Natl Acad Sci U S A ; 108(4): 1513-8, 2011 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-21187386

RESUMEN

Massively parallel DNA sequencing technologies are revolutionizing genomics by making it possible to generate billions of relatively short (~100-base) sequence reads at very low cost. Whereas such data can be readily used for a wide range of biomedical applications, it has proven difficult to use them to generate high-quality de novo genome assemblies of large, repeat-rich vertebrate genomes. To date, the genome assemblies generated from such data have fallen far short of those obtained with the older (but much more expensive) capillary-based sequencing approach. Here, we report the development of an algorithm for genome assembly, ALLPATHS-LG, and its application to massively parallel DNA sequence data from the human and mouse genomes, generated on the Illumina platform. The resulting draft genome assemblies have good accuracy, short-range contiguity, long-range connectivity, and coverage of the genome. In particular, the base accuracy is high (≥99.95%) and the scaffold sizes (N50 size = 11.5 Mb for human and 7.2 Mb for mouse) approach those obtained with capillary-based sequencing. The combination of improved sequencing technology and improved computational methods should now make it possible to increase dramatically the de novo sequencing of large genomes. The ALLPATHS-LG program is available at http://www.broadinstitute.org/science/programs/genome-biology/crd.


Asunto(s)
Algoritmos , Genómica/métodos , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Animales , Genoma/genética , Humanos , Internet , Ratones , Reproducibilidad de los Resultados
2.
Am J Cancer Res ; 12(6): 2733-2743, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35812049

RESUMEN

Hepatocellular carcinoma (HCC) is an aggressive liver malignancy that is difficult to treat with no approved biomarker based targeted therapies. FGF19-FGFR4 signaling blockade has been recently identified as a promising avenue for treatment of a subset of HCC patients. Using HCC relevant xenograft and PDX models, we show that Lenvatinib, an approved multi-kinase inhibitor, strongly enhanced the efficacy of FGFR4 inhibitor H3B-6527. This enhanced combination effect is not due to enhanced FGFR4 inhibition and it is likely due to cell non-autonomous VEGFR activity of Lenvatinib. This cell non-autonomous mode of action was further supported by strong in vivo combination efficacy with the mouse specific VEGFR2 antibody, DC101, which cannot cell-autonomously inhibit pathways in human xenografts. Mechanistic studies showed that the combination resulted in enhanced efficacy through increased anti-angiogenic and anti-tumorigenic activities. Overall, our results indicate that this combination can be a highly effective treatment option for FGF19 driven HCC patients, and provide preclinical validation of a combination that can be readily tested in the clinical setting.

3.
PLoS Pathog ; 4(11): e1000197, 2008 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-18989460

RESUMEN

Phylogenetic analyses have provided strong evidence that amino acid changes in spike (S) protein of animal and human SARS coronaviruses (SARS-CoVs) during and between two zoonotic transfers (2002/03 and 2003/04) are the result of positive selection. While several studies support that some amino acid changes between animal and human viruses are the result of inter-species adaptation, the role of neutralizing antibodies (nAbs) in driving SARS-CoV evolution, particularly during intra-species transmission, is unknown. A detailed examination of SARS-CoV infected animal and human convalescent sera could provide evidence of nAb pressure which, if found, may lead to strategies to effectively block virus evolution pathways by broadening the activity of nAbs. Here we show, by focusing on a dominant neutralization epitope, that contemporaneous- and cross-strain nAb responses against SARS-CoV spike protein exist during natural infection. In vitro immune pressure on this epitope using 2002/03 strain-specific nAb 80R recapitulated a dominant escape mutation that was present in all 2003/04 animal and human viruses. Strategies to block this nAb escape/naturally occurring evolution pathway by generating broad nAbs (BnAbs) with activity against 80R escape mutants and both 2002/03 and 2003/04 strains were explored. Structure-based amino acid changes in an activation-induced cytidine deaminase (AID) "hot spot" in a light chain CDR (complementarity determining region) alone, introduced through shuffling of naturally occurring non-immune human VL chain repertoire or by targeted mutagenesis, were successful in generating these BnAbs. These results demonstrate that nAb-mediated immune pressure is likely a driving force for positive selection during intra-species transmission of SARS-CoV. Somatic hypermutation (SHM) of a single VL CDR can markedly broaden the activity of a strain-specific nAb. The strategies investigated in this study, in particular the use of structural information in combination of chain-shuffling as well as hot-spot CDR mutagenesis, can be exploited to broaden neutralization activity, to improve anti-viral nAb therapies, and directly manipulate virus evolution.


Asunto(s)
Anticuerpos Antivirales/genética , Evolución Biológica , Selección Genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Animales , Regiones Determinantes de Complementariedad/genética , Citidina Desaminasa/genética , Epítopos , Humanos , Sueros Inmunes/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología
4.
Nat Commun ; 10(1): 137, 2019 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-30635584

RESUMEN

Dysregulation of RNA splicing by spliceosome mutations or in cancer genes is increasingly recognized as a hallmark of cancer. Small molecule splicing modulators have been introduced into clinical trials to treat solid tumors or leukemia bearing recurrent spliceosome mutations. Nevertheless, further investigation of the molecular mechanisms that may enlighten therapeutic strategies for splicing modulators is highly desired. Here, using unbiased functional approaches, we report that the sensitivity to splicing modulation of the anti-apoptotic BCL2 family genes is a key mechanism underlying preferential cytotoxicity induced by the SF3b-targeting splicing modulator E7107. While BCL2A1, BCL2L2 and MCL1 are prone to splicing perturbation, BCL2L1 exhibits resistance to E7107-induced splicing modulation. Consequently, E7107 selectively induces apoptosis in BCL2A1-dependent melanoma cells and MCL1-dependent NSCLC cells. Furthermore, combination of BCLxL (BCL2L1-encoded) inhibitors and E7107 remarkably enhances cytotoxicity in cancer cells. These findings inform mechanism-based approaches to the future clinical development of splicing modulators in cancer treatment.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Melanoma/tratamiento farmacológico , Antígenos de Histocompatibilidad Menor/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Empalme del ARN/efectos de los fármacos , Proteína bcl-X/genética , Células A549 , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Doxiciclina/farmacología , Sinergismo Farmacológico , Compuestos Epoxi/farmacología , Femenino , Humanos , Neoplasias Pulmonares/genética , Macrólidos/farmacología , Melanoma/genética , Ratones , Ratones Desnudos , Interferencia de ARN , Empalme del ARN/genética , ARN Interferente Pequeño/genética , Empalmosomas/efectos de los fármacos , Empalmosomas/genética , Secuenciación del Exoma , Ensayos Antitumor por Modelo de Xenoinjerto
5.
Nat Commun ; 8: 15522, 2017 05 25.
Artículo en Inglés | MEDLINE | ID: mdl-28541300

RESUMEN

Pladienolide, herboxidiene and spliceostatin have been identified as splicing modulators that target SF3B1 in the SF3b subcomplex. Here we report that PHF5A, another component of this subcomplex, is also targeted by these compounds. Mutations in PHF5A-Y36, SF3B1-K1071, SF3B1-R1074 and SF3B1-V1078 confer resistance to these modulators, suggesting a common interaction site. RNA-seq analysis reveals that PHF5A-Y36C has minimal effect on basal splicing but inhibits the global action of splicing modulators. Moreover, PHF5A-Y36C alters splicing modulator-induced intron-retention/exon-skipping profile, which correlates with the differential GC content between adjacent introns and exons. We determine the crystal structure of human PHF5A demonstrating that Y36 is located on a highly conserved surface. Analysis of the cryo-EM spliceosome Bact complex shows that the resistance mutations cluster in a pocket surrounding the branch point adenosine, suggesting a competitive mode of action. Collectively, we propose that PHF5A-SF3B1 forms a central node for binding to these splicing modulators.


Asunto(s)
Adenosina/química , Empalme Alternativo , Proteínas Portadoras/química , Fosfoproteínas/química , Factores de Empalme de ARN/química , Proliferación Celular , Supervivencia Celular , Microscopía por Crioelectrón , Cristalografía por Rayos X , Compuestos Epoxi/química , Exones , Alcoholes Grasos/química , Células HCT116 , Humanos , Intrones , Macrólidos/química , Espectrometría de Masas , Mutagénesis Sitio-Dirigida , Mutación , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/química , Fosfoproteínas/metabolismo , Unión Proteica , Conformación Proteica , Piranos/química , Interferencia de ARN , Factores de Empalme de ARN/metabolismo , Proteínas de Unión al ARN , Proteínas Recombinantes/química , Análisis de Secuencia de ARN , Compuestos de Espiro/química , Empalmosomas/metabolismo , Transactivadores
6.
Cell Rep ; 13(5): 1033-45, 2015 Nov 03.
Artículo en Inglés | MEDLINE | ID: mdl-26565915

RESUMEN

Recurrent mutations in the spliceosome are observed in several human cancers, but their functional and therapeutic significance remains elusive. SF3B1, the most frequently mutated component of the spliceosome in cancer, is involved in the recognition of the branch point sequence (BPS) during selection of the 3' splice site (ss) in RNA splicing. Here, we report that common and tumor-specific splicing aberrations are induced by SF3B1 mutations and establish aberrant 3' ss selection as the most frequent splicing defect. Strikingly, mutant SF3B1 utilizes a BPS that differs from that used by wild-type SF3B1 and requires the canonical 3' ss to enable aberrant splicing during the second step. Approximately 50% of the aberrantly spliced mRNAs are subjected to nonsense-mediated decay resulting in downregulation of gene and protein expression. These findings ascribe functional significance to the consequences of SF3B1 mutations in cancer.


Asunto(s)
Empalme Alternativo , Mutación , Neoplasias/genética , Fosfoproteínas/genética , Ribonucleoproteína Nuclear Pequeña U2/genética , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Células HEK293 , Humanos , Datos de Secuencia Molecular , Tasa de Mutación , Degradación de ARNm Mediada por Codón sin Sentido , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Factores de Empalme de ARN , Ribonucleoproteína Nuclear Pequeña U2/química , Ribonucleoproteína Nuclear Pequeña U2/metabolismo
7.
Nat Genet ; 45(3): 299-303, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23396133

RESUMEN

Although genetic lesions responsible for some mendelian disorders can be rapidly discovered through massively parallel sequencing of whole genomes or exomes, not all diseases readily yield to such efforts. We describe the illustrative case of the simple mendelian disorder medullary cystic kidney disease type 1 (MCKD1), mapped more than a decade ago to a 2-Mb region on chromosome 1. Ultimately, only by cloning, capillary sequencing and de novo assembly did we find that each of six families with MCKD1 harbors an equivalent but apparently independently arising mutation in sequence markedly under-represented in massively parallel sequencing data: the insertion of a single cytosine in one copy (but a different copy in each family) of the repeat unit comprising the extremely long (∼1.5-5 kb), GC-rich (>80%) coding variable-number tandem repeat (VNTR) sequence in the MUC1 gene encoding mucin 1. These results provide a cautionary tale about the challenges in identifying the genes responsible for mendelian, let alone more complex, disorders through massively parallel sequencing.


Asunto(s)
Repeticiones de Minisatélite/genética , Mucina-1/genética , Mutación , Riñón Poliquístico Autosómico Dominante , Citosina/metabolismo , Femenino , Ligamiento Genético , Haplotipos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Mucina-1/metabolismo , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/patología
8.
Genome Biol ; 12(2): R18, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21338519

RESUMEN

Despite the ever-increasing output of Illumina sequencing data, loci with extreme base compositions are often under-represented or absent. To evaluate sources of base-composition bias, we traced genomic sequences ranging from 6% to 90% GC through the process by quantitative PCR. We identified PCR during library preparation as a principal source of bias and optimized the conditions. Our improved protocol significantly reduces amplification bias and minimizes the previously severe effects of PCR instrument and temperature ramp rate.


Asunto(s)
Artefactos , Composición de Base/genética , Genoma Humano , Genómica/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Escherichia coli/genética , Sitios Genéticos , Biblioteca Genómica , Humanos , Desnaturalización de Ácido Nucleico , Plasmodium falciparum/genética , Análisis de Secuencia de ADN , Temperatura
9.
Nat Struct Mol Biol ; 16(3): 265-73, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19234466

RESUMEN

Influenza virus remains a serious health threat, owing to its ability to evade immune surveillance through rapid genetic drift and reassortment. Here we used a human non-immune antibody phage-display library and the H5 hemagglutinin ectodomain to select ten neutralizing antibodies (nAbs) that were effective against all group 1 influenza viruses tested, including H5N1 'bird flu' and the H1N1 'Spanish flu'. The crystal structure of one such nAb bound to H5 shows that it blocks infection by inserting its heavy chain into a conserved pocket in the stem region, thus preventing membrane fusion. Nine of the nAbs employ the germline gene VH1-69, and all seem to use the same neutralizing mechanism. Our data further suggest that this region is recalcitrant to neutralization escape and that nAb-based immunotherapy is a promising strategy for broad-spectrum protection against seasonal and pandemic influenza viruses.


Asunto(s)
Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Internalización del Virus/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/uso terapéutico , Cristalografía por Rayos X , Células HeLa , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Datos de Secuencia Molecular , Pruebas de Neutralización , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Biblioteca de Péptidos , Unión Proteica , Estructura Cuaternaria de Proteína , Alineación de Secuencia , Análisis de Supervivencia
10.
PLoS One ; 3(9): e3181, 2008 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-18784843

RESUMEN

BACKGROUND: Isolation of human antibodies using current display technologies can be limited by constraints on protein expression, folding and post-translational modifications. Here we describe a discovery platform that utilizes self-inactivating (SIN) lentiviral vectors for the surface display of high-affinity single-chain variable region (scFv) antibody fragments on human cells and lentivirus particles. METHODOLOGY/PRINCIPAL FINDINGS: Bivalent scFvFc human antibodies were fused in frame with different transmembrane (TM) anchoring moieties to allow efficient high-level expression on human cells and the optimal TM was identified. The addition of an eight amino acid HIV-1 gp41 envelope incorporation motif further increased scFvFc expression on human cells and incorporation into lentiviral particles. Both antibody-displaying human cells and virus particles bound antigen specifically. Sulfation of CDR tyrosine residues, a property recently shown to broaden antibody binding affinity and antigen recognition was also demonstrated. High level scFvFc expression and stable integration was achieved in human cells following transduction with IRES containing bicistronic SIN lentivectors encoding ZsGreen when scFvFc fusion proteins were expressed from the first cassette. Up to 10(6)-fold enrichment of antibody expressing cells was achieved with one round of antigen coupled magnetic bead pre-selection followed by FACS sorting. Finally, the scFvFc displaying human cells could be used directly in functional biological screens with remarkable sensitivity. CONCLUSIONS/SIGNIFICANCE: This antibody display platform will complement existing technologies by virtue of providing properties unique to lentiviruses and antibody expression in human cells, which, in turn, may aid the discovery of novel therapeutic human mAbs.


Asunto(s)
Anticuerpos/química , Membrana Celular/virología , Regulación Viral de la Expresión Génica , Lentivirus/genética , Biblioteca de Péptidos , Virión/metabolismo , Anticuerpos Monoclonales/química , Línea Celular , Separación Celular , Citometría de Flujo , Técnicas Genéticas , Humanos , Fragmentos de Inmunoglobulinas/genética , Región Variable de Inmunoglobulina/genética , Glicoproteínas de Membrana/química , Glicoproteína de la Espiga del Coronavirus , Proteínas del Envoltorio Viral/química
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