Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 9 de 9
Filtrar
1.
Cell ; 166(5): 1215-1230.e20, 2016 Aug 25.
Artículo en Inglés | MEDLINE | ID: mdl-27523608

RESUMEN

Methionine-1 (M1)-linked ubiquitin chains regulate the activity of NF-κB, immune homeostasis, and responses to infection. The importance of negative regulators of M1-linked chains in vivo remains poorly understood. Here, we show that the M1-specific deubiquitinase OTULIN is essential for preventing TNF-associated systemic inflammation in humans and mice. A homozygous hypomorphic mutation in human OTULIN causes a potentially fatal autoinflammatory condition termed OTULIN-related autoinflammatory syndrome (ORAS). Four independent OTULIN mouse models reveal that OTULIN deficiency in immune cells results in cell-type-specific effects, ranging from over-production of inflammatory cytokines and autoimmunity due to accumulation of M1-linked polyubiquitin and spontaneous NF-κB activation in myeloid cells to downregulation of M1-polyubiquitin signaling by degradation of LUBAC in B and T cells. Remarkably, treatment with anti-TNF neutralizing antibodies ameliorates inflammation in ORAS patients and rescues mouse phenotypes. Hence, OTULIN is critical for restraining life-threatening spontaneous inflammation and maintaining immune homeostasis.


Asunto(s)
Enfermedades Autoinmunes/genética , Autoinmunidad/genética , Enzimas Desubicuitinizantes/metabolismo , Endopeptidasas/metabolismo , Inflamación/genética , Animales , Anticuerpos Neutralizantes/uso terapéutico , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/terapia , Linfocitos B/inmunología , Citocinas/metabolismo , Enzimas Desubicuitinizantes/genética , Modelos Animales de Enfermedad , Endopeptidasas/genética , Mutación de Línea Germinal , Humanos , Inflamación/inmunología , Inflamación/terapia , Infliximab/uso terapéutico , Metionina/metabolismo , Ratones , Ratones Mutantes , Células Mieloides/inmunología , Poliubiquitina/metabolismo , Eliminación de Secuencia , Síndrome , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores
2.
Mol Cell ; 54(3): 335-48, 2014 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-24726323

RESUMEN

The linear ubiquitin (Ub) chain assembly complex (LUBAC) generates Met1-linked "linear" Ub chains that regulate the activation of the nuclear factor κB (NFκB) transcription factor and other processes. We recently discovered OTULIN as a deubiquitinase that specifically cleaves Met1-linked polyUb. Now, we show that OTULIN binds via a conserved PUB-interacting motif (PIM) to the PUB domain of the LUBAC component HOIP. Crystal structures and nuclear magnetic resonance experiments reveal the molecular basis for the high-affinity interaction and explain why OTULIN binds the HOIP PUB domain specifically. Analysis of LUBAC-induced NFκB signaling suggests that OTULIN needs to be present on LUBAC in order to restrict Met1-polyUb signaling. Moreover, LUBAC-OTULIN complex formation is regulated by OTULIN phosphorylation in the PIM. Phosphorylation of OTULIN prevents HOIP binding, whereas unphosphorylated OTULIN is part of the endogenous LUBAC complex. Our work exemplifies how coordination of ubiquitin assembly and disassembly activities in protein complexes regulates individual Ub linkage types.


Asunto(s)
Endopeptidasas/química , Ubiquitina-Proteína Ligasas/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Endopeptidasas/metabolismo , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Ubiquitina-Proteína Ligasas/metabolismo
3.
Br J Cancer ; 125(12): 1666-1676, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34663950

RESUMEN

BACKGROUND: The absence of the putative DNA/RNA helicase Schlafen11 (SLFN11) is thought to cause resistance to DNA-damaging agents (DDAs) and PARP inhibitors. METHODS: We developed and validated a clinically applicable SLFN11 immunohistochemistry assay and retrospectively correlated SLFN11 tumour levels to patient outcome to the standard of care therapies and olaparib maintenance. RESULTS: High SLFN11 associated with improved prognosis to the first-line treatment with DDAs platinum-plus-etoposide in SCLC patients, but was not strongly linked to paclitaxel-platinum response in ovarian cancer patients. Multivariate analysis of patients with relapsed platinum-sensitive ovarian cancer from the randomised, placebo-controlled Phase II olaparib maintenance Study19 showed SLFN11 tumour levels associated with sensitivity to olaparib. Study19 patients with high SLFN11 had a lower progression-free survival (PFS) hazard ratio compared to patients with low SLFN11, although both groups had the benefit of olaparib over placebo. Whilst caveated by small sample size, this trend was maintained for PFS, but not overall survival, when adjusting for BRCA status across the olaparib and placebo treatment groups, a key driver of PARP inhibitor sensitivity. CONCLUSION: We provide clinical evidence supporting the role of SLFN11 as a DDA therapy selection biomarker in SCLC and highlight the need for further clinical investigation into SLFN11 as a PARP inhibitor predictive biomarker.


Asunto(s)
Daño del ADN/genética , Proteínas Nucleares/metabolismo , Animales , Femenino , Humanos , Masculino , Ratones , Ratones Desnudos , Estudios Retrospectivos , Resultado del Tratamiento
4.
Mol Cancer Ther ; 22(10): 1154-1165, 2023 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-37486983

RESUMEN

AZD5153, a reversible, bivalent inhibitor of the bromodomain and extraterminal family protein BRD4, has preclinical activity in multiple tumors. This first-in-human, phase I study investigated AZD5153 alone or with olaparib in patients with relapsed/refractory solid tumors or lymphoma. Adults with relapsed tumors intolerant of, or refractory to, prior therapies received escalating doses of oral AZD5153 once daily or twice daily continuously (21-day cycles), or AZD5153 once daily/twice daily continuously or intermittently plus olaparib 300 mg twice daily, until disease progression or unacceptable toxicity. Between June 30, 2017 and April 19, 2021, 34 patients received monotherapy and 15 received combination therapy. Dose-limiting toxicities were thrombocytopenia/platelet count decreased (n = 4/n = 2) and diarrhea (n = 1). The recommended phase II doses (RP2D) were AZD5153 30 mg once daily or 15 mg twice daily (monotherapy) and 10 mg once daily (intermittent schedule) with olaparib. With AZD5153 monotherapy, common treatment-emergent adverse events (TEAE) included fatigue (38.2%), thrombocytopenia, and diarrhea (each 32.4%); common grade ≥ 3 TEAEs were thrombocytopenia (14.7%) and anemia (8.8%). With the combination, common TEAEs included nausea (66.7%) and fatigue (53.3%); the most common grade ≥ 3 TEAE was thrombocytopenia (26.7%). AZD5153 had dose-dependent pharmacokinetics, with minimal accumulation, and demonstrated dose-dependent modulation of peripheral biomarkers, including upregulation of HEXIM1. One patient with metastatic pancreatic cancer receiving combination treatment had a partial response lasting 4.2 months. These results show AZD5153 was tolerable as monotherapy and in combination at the RP2Ds; common toxicities were fatigue, hematologic AEs, and gastrointestinal AEs. Strong evidence of peripheral target engagement was observed.


Asunto(s)
Antineoplásicos , Linfoma , Neoplasias , Trombocitopenia , Adulto , Humanos , Antineoplásicos/farmacología , Antineoplásicos/toxicidad , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidad , Proteínas de Ciclo Celular , Diarrea/inducido químicamente , Fatiga/inducido químicamente , Fatiga/tratamiento farmacológico , Linfoma/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Proteínas Nucleares , Proteínas de Unión al ARN , Trombocitopenia/inducido químicamente , Factores de Transcripción
5.
J Vis Exp ; (169)2021 03 20.
Artículo en Inglés | MEDLINE | ID: mdl-33818572

RESUMEN

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of treatment, and help guide dose and schedule setting of therapeutics. From a single blood draw, peripheral blood mononuclear cells can be isolated and processed to analyze and quantify protein markers, and plasma samples can be used for the analysis of circulating tumor DNA, cytokines, and plasma metabolomics. Longitudinal samples from a treatment provide information on the evolution of a given protein marker, the mutational status and immunological landscape of the patient. This can only be achieved if the processing of the peripheral blood is carried out effectively in clinical sites and samples are properly preserved from the bedside to bench. Here, we present an optimized general-purpose protocol that can be implemented at clinical sites for obtaining PBMC pellets and plasma samples in multi-center clinical trials, that will enable clinical professionals in hospital laboratories to successfully provide high quality samples, regardless of their level of technical expertise. Alternative protocol variations are also presented that are optimized for more specific downstream analytical methods. We apply this protocol for studying protein biomarkers against DNA damage response (DDR) on X-ray irradiated blood to demonstrate the suitability of the approach in oncology settings where DDR drugs and/or radiotherapy have been practiced as well as in preclinical stages where mechanistic hypothesis testing is required.


Asunto(s)
Biomarcadores/sangre , Leucocitos Mononucleares/inmunología , Plasma/inmunología , Humanos
6.
Cancer Med ; 10(9): 3045-3058, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33811746

RESUMEN

BACKGROUND: In high grade serous ovarian cancer (HGSOC), there is a spectrum of sensitivity to first line platinum-based chemotherapy. This study molecularly characterizes HGSOC patients from two distinct groups of chemotherapy responders (good vs. poor). METHODS: Following primary debulking surgery and intravenous carboplatin/paclitaxel, women with stage III-IV HGSOC were grouped by response. Patients in the good response (GR) and poor response (PR) groups respectively had a progression-free intervals (PFI) of ≥12 and ≤6 months. Analysis of surgical specimens interrogated genomic and immunologic features using whole exome sequencing. RNA-sequencing detected gene expression outliers and inference of immune infiltrate, with validation by targeted NanoString arrays. PD-L1 expression was scored by immunohistochemistry (IHC). RESULTS: A total of 39 patient samples were analyzed (GR = 20; PR = 19). Median PFI for GR and PR patient cohorts was 32 and 3 months, respectively. GR tumors were enriched for loss-of-function BRCA2 mutations and had a significantly higher nonsynonymous mutation rate compared to PR tumors (p = 0.001). Samples from the PR cohort were characterized by mutations in MGA and RAD51B and trended towards a greater rate of amplification of PIK3CA, MECOM, and ATR in comparison to GR tumors. Gene expression analysis by NanoString correlated increased PARP4 with PR and increased PD-L1 and EMSY with GR. There was greater tumor immune cell infiltration and higher immune cell PD-L1 protein expression in the GR group. CONCLUSIONS: Our research demonstrates that tumors from HGSOC patients responding poorly to first line chemotherapy have a distinct molecular profile characterized by actionable drug targets including PARP4.


Asunto(s)
Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/inmunología , Neoplasias Ováricas/genética , Neoplasias Ováricas/inmunología , Transcriptoma/genética , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Proteínas de la Ataxia Telangiectasia Mutada/genética , Antígeno B7-H1/metabolismo , Carboplatino/administración & dosificación , Fosfatidilinositol 3-Quinasa Clase I/genética , Cistadenocarcinoma Seroso/tratamiento farmacológico , Cistadenocarcinoma Seroso/patología , Procedimientos Quirúrgicos de Citorreducción , Femenino , Amplificación de Genes , Perfilación de la Expresión Génica , Genes BRCA1 , Genes BRCA2 , Genes p53 , Humanos , Proteína del Locus del Complejo MDS1 y EV11/genética , Persona de Mediana Edad , Mutación , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Paclitaxel/administración & dosificación , Supervivencia sin Progresión , Proteínas Represoras/metabolismo , Estudios Retrospectivos , Factores de Tiempo , Resultado del Tratamiento , Secuenciación del Exoma
7.
Cancers (Basel) ; 13(15)2021 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-34359745

RESUMEN

The ATM serine/threonine kinase (HGNC: ATM) is involved in initiation of repair of DNA double-stranded breaks, and ATM inhibitors are currently being tested as anti-cancer agents in clinical trials, where pharmacodynamic (PD) assays are crucial to help guide dose and scheduling and support mechanism of action studies. To identify and quantify PD biomarkers of ATM inhibition, we developed and analytically validated a 51-plex assay (DDR-2) quantifying protein expression and DNA damage-responsive phosphorylation. The median lower limit of quantification was 1.28 fmol, the linear range was over 3 orders of magnitude, the median inter-assay variability was 11% CV, and 86% of peptides were stable for storage prior to analysis. Use of the assay was demonstrated to quantify signaling following ionizing radiation-induced DNA damage in both immortalized lymphoblast cell lines and primary human peripheral blood mononuclear cells, identifying PD biomarkers for ATM inhibition to support preclinical and clinical studies.

8.
Methods Mol Biol ; 2102: 441-457, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31989571

RESUMEN

A fully optimized staining method for detecting sister chromatid exchanges in cultured cells is presented. The method gives reproducibly robust quantitative results. Sister chromatid exchange is a classic toxicology assay for genotoxicity and for detecting alterations to the biochemistry underlying cellular homologous recombination. Growth of cells in the presence of 5'-bromo-deoxyuridine for two rounds of DNA replication followed by collecting metaphase spreads on glass slides, treatment with the UV-sensitive dye Hoechst 33258, long-wave UV light exposure, and Giemsa staining gives a permanent record of the exchanges.


Asunto(s)
Metafase , Pruebas de Mutagenicidad/métodos , Intercambio de Cromátides Hermanas , Colorantes Azulados , Bioensayo/métodos , Bisbenzimidazol , Bromodesoxiuridina/metabolismo , Células Cultivadas , Cromátides/efectos de los fármacos , Cromátides/metabolismo , Cromátides/efectos de la radiación , Cromosomas/efectos de los fármacos , Cromosomas/metabolismo , Cromosomas/efectos de la radiación , Recombinación Homóloga/efectos de los fármacos , Recombinación Homóloga/efectos de la radiación , Humanos , Metafase/efectos de los fármacos , Metafase/efectos de la radiación , Flujo de Trabajo
9.
Methods Mol Biol ; 2102: 459-482, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31989572

RESUMEN

A newly developed method for quantitatively detecting genomic restructuring in cultured human cell lines as the result of recombination is presented: the "gene cluster instability" (GCI) assay. The assay is physiological in that it detects spontaneous restructuring without the need for exogenous recombination-initiating treatments such as DNA damage. As an assay for genotoxicity, the GCI assay is complementary to well-established sister chromatid exchange (SCE) methods. Analysis of the U-2 OS osteosarcoma cell line is presented as an illustration of the method.


Asunto(s)
Pruebas de Mutagenicidad/métodos , Recombinación Genética , Southern Blotting , Línea Celular Tumoral , ADN/efectos de los fármacos , ADN/genética , ADN/aislamiento & purificación , Enzimas de Restricción del ADN , Electroforesis en Gel de Agar , Inestabilidad Genómica , Humanos , Familia de Multigenes , Flujo de Trabajo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA