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1.
Cell ; 175(3): 766-779.e17, 2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30340042

RESUMO

The super elongation complex (SEC) is required for robust and productive transcription through release of RNA polymerase II (Pol II) with its P-TEFb module and promoting transcriptional processivity with its ELL2 subunit. Malfunction of SEC contributes to multiple human diseases including cancer. Here, we identify peptidomimetic lead compounds, KL-1 and its structural homolog KL-2, which disrupt the interaction between the SEC scaffolding protein AFF4 and P-TEFb, resulting in impaired release of Pol II from promoter-proximal pause sites and a reduced average rate of processive transcription elongation. SEC is required for induction of heat-shock genes and treating cells with KL-1 and KL-2 attenuates the heat-shock response from Drosophila to human. SEC inhibition downregulates MYC and MYC-dependent transcriptional programs in mammalian cells and delays tumor progression in a mouse xenograft model of MYC-driven cancer, indicating that small-molecule disruptors of SEC could be used for targeted therapy of MYC-induced cancer.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Fator B de Elongação Transcricional Positiva/metabolismo , Proteínas Repressoras/metabolismo , Elongação da Transcrição Genética/efeitos dos fármacos , Fatores de Elongação da Transcrição/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Drosophila , Feminino , Células HCT116 , Células HEK293 , Resposta ao Choque Térmico , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Polimerase II/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
2.
Anal Chem ; 91(6): 3790-3794, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30835099

RESUMO

Mass spectrometry (MS) has many advantages as a quantitative detection technology for applications within drug discovery. However, current methods of liquid sample introduction to a detector are slow and limit the use of mass spectrometry for kinetic and high-throughput applications. We present the development of an acoustic mist ionization (AMI) interface capable of contactless nanoliter-scale "infusion" of up to three individual samples per second into the mass detector. Installing simple plate handling automation allowed us to reach a throughput of 100 000 samples per day on a single mass spectrometer. We applied AMI-MS to identify inhibitors of a human histone deacetylase from AstraZeneca's collection of 2 million small molecules and measured their half-maximal inhibitory concentration. The speed, sensitivity, simplicity, robustness, and consumption of nanoliter volumes of sample suggest that this technology will have a major impact across many areas of basic and applied research.


Assuntos
Acústica , Inibidores de Histona Desacetilases/análise , Espectrometria de Massas/instrumentação , Inibidores de Histona Desacetilases/química , Humanos
3.
Cancer Immunol Immunother ; 66(11): 1411-1424, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28634816

RESUMO

T-cell receptor (TCR)-pMHC affinity has been generally accepted to be the most important factor dictating antigen recognition in gene-modified T-cells. As such, there is great interest in optimizing TCR-based immunotherapies by enhancing TCR affinity to augment the therapeutic benefit of TCR gene-modified T-cells in cancer patients. However, recent clinical trials using affinity-enhanced TCRs in adoptive cell transfer (ACT) have observed unintended and serious adverse events, including death, attributed to unpredicted off-tumor or off-target cross-reactivity. It is critical to re-evaluate the importance of other biophysical, structural, or cellular factors that drive the reactivity of TCR gene-modified T-cells. Using a model for altered antigen recognition, we determined how TCR-pMHC affinity influenced the reactivity of hepatitis C virus (HCV) TCR gene-modified T-cells against a panel of naturally occurring HCV peptides and HCV-expressing tumor targets. The impact of other factors, such as TCR-pMHC stabilization and signaling contributions by the CD8 co-receptor, as well as antigen and TCR density were also evaluated. We found that changes in TCR-pMHC affinity did not always predict or dictate IFNγ release or degranulation by TCR gene-modified T-cells, suggesting that less emphasis might need to be placed on TCR-pMHC affinity as a means of predicting or augmenting the therapeutic potential of TCR gene-modified T-cells used in ACT. A more complete understanding of antigen recognition by gene-modified T-cells and a more rational approach to improve the design and implementation of novel TCR-based immunotherapies is necessary to enhance efficacy and maximize safety in patients.


Assuntos
Transferência Adotiva/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Linfócitos T/transplante , Animais , Ligação Competitiva/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Técnicas de Cocultura , Citometria de Fluxo , Células HEK293 , Células Hep G2 , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Células Jurkat , Camundongos , Peptídeos/genética , Peptídeos/imunologia , Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/metabolismo
4.
Cancer Immunol Immunother ; 65(3): 293-304, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26842125

RESUMO

The success in recent clinical trials using T cell receptor (TCR)-genetically engineered T cells to treat melanoma has encouraged the use of this approach toward other malignancies and viral infections. Although hepatitis C virus (HCV) infection is being treated with a new set of successful direct anti-viral agents, potential for virologic breakthrough or relapse by immune escape variants remains. Additionally, many HCV+ patients have HCV-associated disease, including hepatocellular carcinoma (HCC), which does not respond to these novel drugs. Further exploration of other approaches to address HCV infection and its associated disease are highly warranted. Here, we demonstrate the therapeutic potential of PBL-derived T cells genetically engineered with a high-affinity, HLA-A2-restricted, HCV NS3:1406-1415-reactive TCR. HCV1406 TCR-transduced T cells can recognize naturally processed antigen and elicit CD8-independent recognition of both peptide-loaded targets and HCV+ human HCC cell lines. Furthermore, these cells can mediate regression of established HCV+ HCC in vivo. Our results suggest that HCV TCR-engineered antigen-reactive T cells may be a plausible immunotherapy option to treat HCV-associated malignancies, such as HCC.


Assuntos
Carcinoma Hepatocelular/terapia , Genes Codificadores dos Receptores de Linfócitos T/fisiologia , Hepatite C/complicações , Neoplasias Hepáticas/terapia , Linfócitos T/imunologia , Animais , Carcinoma Hepatocelular/etiologia , Linhagem Celular Tumoral , Engenharia Genética , Antígeno HLA-A2/imunologia , Humanos , Imunoterapia , Neoplasias Hepáticas/etiologia , Camundongos , Proteínas não Estruturais Virais/genética
5.
Mol Ecol ; 25(16): 3801-16, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27286564

RESUMO

Baker's Law predicts uniparental reproduction will facilitate colonization success in novel habitats. While evidence supports this prediction among colonizing plants and animals, few studies have investigated shifts in reproductive mode in haplo-diplontic species in which both prolonged haploid and diploid stages separate meiosis and fertilization in time and space. Due to this separation, asexual reproduction can yield the dominance of one of the ploidy stages in colonizing populations. We tested for shifts in ploidy and reproductive mode across native and introduced populations of the red seaweed Gracilaria vermiculophylla. Native populations in the northwest Pacific Ocean were nearly always attached by holdfasts to hard substrata and, as is characteristic of the genus, haploid-diploid ratios were slightly diploid-biased. In contrast, along North American and European coastlines, introduced populations nearly always floated atop soft-sediment mudflats and were overwhelmingly dominated by diploid thalli without holdfasts. Introduced populations exhibited population genetic signals consistent with extensive vegetative fragmentation, while native populations did not. Thus, the ecological shift from attached to unattached thalli, ostensibly necessitated by the invasion of soft-sediment habitats, correlated with shifts from sexual to asexual reproduction and slight to strong diploid bias. We extend Baker's Law by predicting other colonizing haplo-diplontic species will show similar increases in asexuality that correlate with the dominance of one ploidy stage. Labile mating systems likely facilitate colonization success and subsequent range expansion, but for haplo-diplontic species, the long-term eco-evolutionary impacts will depend on which ploidy stage is lost and the degree to which asexual reproduction is canalized.


Assuntos
Diploide , Ecossistema , Genética Populacional , Gracilaria/genética , Haploidia , Evolução Biológica , Oceano Pacífico
6.
Sci Adv ; 5(7): eaax2887, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31281901

RESUMO

Using biochemical characterization of fusion proteins associated with endometrial stromal sarcoma, we identified JAZF1 as a new subunit of the NuA4 acetyltransferase complex and CXORF67 as a subunit of the Polycomb Repressive Complex 2 (PRC2). Since CXORF67's interaction with PRC2 leads to decreased PRC2-dependent H3K27me2/3 deposition, we propose a new name for this gene: CATACOMB (catalytic antagonist of Polycomb; official gene name: EZHIP ). We map CATACOMB's inhibitory function to a short highly conserved region and identify a single methionine residue essential for diminution of H3K27me2/3 levels. Remarkably, the amino acid sequence surrounding this critical methionine resembles the oncogenic histone H3 Lys27-to-methionine (H3K27M) mutation found in high-grade pediatric gliomas. As CATACOMB expression is regulated through DNA methylation/demethylation, we propose CATACOMB as the potential interlocutor between DNA methylation and PRC2 activity. We raise the possibility that similar regulatory mechanisms could exist for other methyltransferase complexes such as Trithorax/COMPASS.


Assuntos
Glioma/metabolismo , Histonas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Oncogênicas/biossíntese , Complexo Repressor Polycomb 2/metabolismo , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Metilação de DNA , DNA de Neoplasias , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Glioma/patologia , Células HCT116 , Histonas/genética , Humanos , Metilação , Proteínas de Neoplasias/genética , Proteínas Oncogênicas/genética , Complexo Repressor Polycomb 2/genética
7.
Mol Ther Oncolytics ; 5: 105-115, 2017 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-28573185

RESUMO

T cell receptor (TCR)-gene-modified T cells for adoptive cell transfer can mediate objective clinical responses in melanoma and other malignancies. When introducing a second TCR, mispairing between the endogenous and introduced α and ß TCR chains limits expression of the introduced TCR, which can result in impaired efficacy or off-target reactivity and autoimmunity. One approach to promote proper TCR chain pairing involves modifications of the introduced TCR genes: introducing a disulfide bridge, substituting murine for human constant regions, codon optimization, TCR chain leucine zipper fusions, and a single-chain TCR. We have introduced these modifications into our hepatitis C virus (HCV) reactive TCR and utilize a marker gene, CD34t, which allows us to directly compare transduction efficiency with TCR expression and T cell function. Our results reveal that of the TCRs tested, T cells expressing the murine Cß2 TCR or leucine zipper TCR have the highest levels of expression and the highest percentage of lytic and interferon-γ (IFN-γ)-producing T cells. Our studies give us a better understanding of how TCR modifications impact TCR expression and T cell function that may allow for optimization of TCR-modified T cells for adoptive cell transfer to treat patients with malignancies.

8.
Ecol Evol ; 7(12): 4432-4447, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28649353

RESUMO

The identification of native sources and vectors of introduced species informs their ecological and evolutionary history and may guide policies that seek to prevent future introductions. Population genetics provides a powerful set of tools to identify origins and vectors. However, these tools can mislead when the native range is poorly sampled or few molecular markers are used. Here, we traced the introduction of the Asian seaweed Gracilaria vermiculophylla (Rhodophyta) into estuaries in coastal western North America, the eastern United States, Europe, and northwestern Africa by genotyping more than 2,500 thalli from 37 native and 53 non-native sites at mitochondrial cox1 and 10 nuclear microsatellite loci. Overall, greater than 90% of introduced thalli had a genetic signature similar to thalli sampled from the coastline of northeastern Japan, strongly indicating this region served as the principal source of the invasion. Notably, northeastern Japan exported the vast majority of the oyster Crassostrea gigas during the 20th century. The preponderance of evidence suggests G. vermiculophylla may have been inadvertently introduced with C. gigas shipments and that northeastern Japan is a common source region for estuarine invaders. Each invaded coastline reflected a complex mix of direct introductions from Japan and secondary introductions from other invaded coastlines. The spread of G. vermiculophylla along each coastline was likely facilitated by aquaculture, fishing, and boating activities. Our ability to document a source region was enabled by a robust sampling of locations and loci that previous studies lacked and strong phylogeographic structure along native coastlines.

9.
J Leukoc Biol ; 100(3): 545-57, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-26921345

RESUMO

A major obstacle hindering the development of effective immunity against viral infections, their associated disease, and certain cancers is their inherent genomic instability. Accumulation of mutations can alter processing and presentation of antigens recognized by antibodies and T cells that can lead to immune escape variants. Use of an agent that can intrinsically combat rapidly mutating viral or cancer-associated antigens would be quite advantageous in developing effective immunity against such disease. We propose that T cells harboring cross-reactive TCRs could serve as a therapeutic agent in these instances. With the use of hepatitis C virus, known for its genomic instability as a model for mutated antigen recognition, we demonstrate cross-reactivity against immunogenic and mutagenic nonstructural protein 3:1406-1415 and nonstructural protein 3:1073-1081 epitopes in PBL-derived, TCR-gene-modified T cells. These single TCR-engineered T cells can CD8-independently recognize naturally occurring and epidemiologically relevant mutant variants. TCR-peptide MHC modeling data allow us to rationalize how TCR structural properties accommodate recognition of certain mutated epitopes and how these substitutions impact the requirement of CD8 affinity enhancement for recognition. A better understanding of such TCRs' promiscuous behavior may allow for exploitation of these properties to develop novel, adoptive T cell-based therapies for viral infections and cancers exhibiting similar genomic instability.


Assuntos
Epitopos de Linfócito T/imunologia , Instabilidade Genômica , Hepacivirus/imunologia , Hepatite C/prevenção & controle , Antígenos de Histocompatibilidade Classe I/imunologia , Imunoterapia , Receptores de Antígenos de Linfócitos T/imunologia , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Reações Cruzadas , Hepacivirus/genética , Hepatite C/etiologia , Humanos
10.
Clin Cancer Res ; 10(5): 1780-8, 2004 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-15014032

RESUMO

PURPOSE: The majority of cases of Cushing's disease are due to the presence of a corticotroph microadenoma. Less frequently no adenoma is found and histology shows either corticotroph hyperplasia, or apparently normal pituitary. In this study we have used molecular pathology to determine whether the tissue labeled histologically as "normal" is indeed abnormal. EXPERIMENTAL DESIGN: Tissue from 31 corticotroph adenomas and 16 nonadenomatous pituitaries were subject to methylation-sensitive PCR to determine the methylation status of the p16 gene CpG island. The proportion of methylated versus unmethylated CpG island was determined using combined bisulphite restriction analysis. Methylation status was correlated with immunohistochemical detection of p16. RESULTS: Seventeen of 31 adenomas (54.8%), 4 of 6 cases of corticotroph hyperplasia, and 7 of 10 apparently normal pituitaries showed p16 methylation. Ten of 14 (71%; P = 0.01) adenomas and 2 of 3 cases of corticotroph hyperplasia, which were methylated, failed to express p16 protein. However, only 2 of 7 apparently normal pituitaries that were methylated failed to express p16 protein. Quantitative analysis of methylation using combined bisulphite restriction analysis showed only unmethylated CpG islands in postmortem normal pituitaries; however, in adenomas 80-90% of the cells within a specimen were methylated. The reverse was true for corticotroph hyperplasia and apparently normal pituitaries where only 10-20% of the cells were methylated. Thus, the decreased proportion of cells that were methylated, particularly in those cases of apparently normal pituitary, is the most likely explanation for the lack of association between this change and loss of cognate protein in these cases. CONCLUSIONS: To our knowledge this is the first report that describes an intrinsic molecular change, namely methylation of the p16 gene CpG island, common to all three histological patterns associated with Cushing's disease. Thus, the use of molecular pathology reveals abnormalities undetected by routine pathological investigation. In cases of "apparently" normal pituitaries it is not possible to determine whether the change is associated with adenoma cells "scattered" throughout the gland, albeit few in number, or with the ancestor-clonal origin of these tumor cells.


Assuntos
Síndrome de Cushing/genética , Metilação de DNA , DNA de Neoplasias/genética , Genes p16 , Hipófise/patologia , Neoplasias Hipofisárias/genética , Adenoma/genética , Adenoma/patologia , Sequência de Bases , Síndrome de Cushing/patologia , Inibidor p16 de Quinase Dependente de Ciclina/análise , Inibidor p16 de Quinase Dependente de Ciclina/genética , Primers do DNA , Fosfatos de Dinucleosídeos/genética , Humanos , Hiperplasia , Imuno-Histoquímica , Neoplasias Hipofisárias/patologia , Reação em Cadeia da Polimerase , Valores de Referência
11.
Curr Opin Chem Biol ; 26: 104-10, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25909818

RESUMO

The pharmaceutical industry has historically relied on high throughput screening as a cornerstone to identify chemical equity for drug discovery projects. However, with pharmaceutical companies moving through a phase of diminished returns and alternative hit identification strategies proving successful, it is more important than ever to understand how this approach can be used more effectively to increase the delivery of next generation therapeutics from high throughput screening libraries. There is a wide literature that describes HTS and fragment based screening approaches which offer clear direction on the process for these two distinct activities. However, few people have considered how best to identify medium to low molecular weight compounds from large diversity screening sets and increase downstream success.


Assuntos
Biblioteca Gênica , Ensaios de Triagem em Larga Escala/economia , Peptidomiméticos/síntese química , Bibliotecas de Moléculas Pequenas/síntese química , Desenho de Fármacos , Descoberta de Drogas/economia , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Peptidomiméticos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade
12.
J Am Soc Nephrol ; 11(5): 912-916, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10770969

RESUMO

Echocardiographic abnormalities are the rule in patients starting dialysis therapy and are associated with the development of cardiac failure and death. It is unknown, however, whether regression of these abnormalities is associated with an improvement in prognosis. As part of a prospective cohort study with mean follow-up of 41 mo, 227 patients had echocardiography at inception and after 1 yr of dialysis therapy. Improvements in left ventricular (LV) mass index, volume index, and fractional shortening were seen in 48, 48, and 46%, respectively. Ninety patients had developed cardiac failure by 1 yr of dialysis therapy. Twenty-six percent of the remaining 137 patients subsequently developed new-onset cardiac failure. The mean changes in LV mass index were 17 g/m(2) in those who subsequently developed cardiac failure compared with 0 g/m(2) among those who did not (P = 0.05). The corresponding values were -8 versus 0% for fractional shortening (P < 0.0001). The associations between serial change in both LV mass index and fractional shortening and subsequent cardiac failure persisted after adjusting for baseline age, diabetes, ischemic heart disease, and the corresponding baseline echocardiographic parameter. Regression of LV abnormalities is associated with an improved cardiac outcome in dialysis patients. Serial echocardiography adds prognostic information to one performed at baseline.


Assuntos
Ecocardiografia , Insuficiência Cardíaca/diagnóstico por imagem , Falência Renal Crônica/complicações , Análise de Variância , Distribuição de Qui-Quadrado , Feminino , Seguimentos , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Hipertrofia Ventricular Esquerda/diagnóstico por imagem , Hipertrofia Ventricular Esquerda/patologia , Falência Renal Crônica/sangue , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Estudos Prospectivos , Diálise Renal/efeitos adversos , Fatores de Risco , Análise de Sobrevida , Resultado do Tratamento , Disfunção Ventricular Esquerda/diagnóstico por imagem , Disfunção Ventricular Esquerda/fisiopatologia
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