RESUMO
Two recent studies examining the clinical and economic value of next-generation sequencing (NGS)-based diagnostic testing (multi-gene panel examining ≥ 30 genes) for non-small-cell lung cancer therapy compared with single gene ALK, EGFR testing to select therapy demonstrated statistically insignificant improvement in population-level overall survival and only a moderate incremental cost-effectiveness ratio associated with the NGS testing approach. The data, however, revealed a key practice gap: many patients with actionable mutations did not receive targeted therapies. This gap is attributed, in part, to limitations in the availability and interpretation of NGS results, sample processing constraints, limited access to targeted therapies, and lagging awareness of the rapidly evolving field of personalized medicine, all of which result in "clinical inertia," (ie, suboptimal use of targeted therapy against an actionable driver alteration identified by NGS testing). Additional analysis estimated that cost-effectiveness would improve significantly if a higher percentage of patients received testing and if all patients who were eligible for targeted therapies received them. Strategies to address implementation barriers will help to realize the full value of NGS testing in cancer care.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Medicina de PrecisãoRESUMO
Despite being one of the most common cancers, treatment options for prostate cancer are limited. Novel approaches for advanced disease are needed. We evaluated the relative rate of use of clinical-grade next generation sequencing (NGS) in prostate cancer, as well as genomic alterations identified and their potential actionability. Of 4864 patients from multiple institutions for whom NGS was ordered by physicians, only 67 (1.4%) had prostate cancer, representing 1/10 the ordering rate for lung cancer. Prostate cancers harbored 148 unique alterations affecting 63 distinct genes. No two patients had an identical molecular portfolio. The median number of characterized genomic alterations per patient was 3 (range, 1 to 9). Fifty-six of 67 patients (84%) had ≥ 1 potentially actionable alteration. TMPRSS2 fusions affected 28.4% of patients. Genomic aberrations were most frequently detected in TP53 (55.2% of patients), PTEN (29.9%), MYC (17.9%), PIK3CA (13.4%), APC (9.0%), BRCA2 (9.0%), CCND1 (9.0%), and RB1 genes (9.0%). The PI3K (52.2% of patients), WNT (13.5%), DNA repair (17.9%), cell cycle (19.4%), and MAPK (14.9%) machinery were commonly impacted. A minority of patients harbored BRAF, NTRK, ERBB2, or mismatch repair gene abnormalities, which are highly druggable in some cancers. Only ~ 10% of prostate cancer trials (clinicaltrials.gov, year 2017) applied a (non-hormone) biomarker before intervention. In conclusion, though use of clinical-grade NGS is relatively low and only a minority of trials deploy DNA-based biomarkers, many prostate cancer-associated molecular alterations may be pharmacologically tractable with genomcially targeted therapy or, in the case of mismatch repair anomalies, with checkpoint inhibitor immunotherapy.
Assuntos
Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias da Próstata/genética , Humanos , Masculino , Neoplasias da Próstata/patologia , RiscoRESUMO
Purpose: Aberrations in genetic sequences encoding the tyrosine kinase receptor RET lead to oncogenic signaling that is targetable with anti-RET multikinase inhibitors. Understanding the comprehensive genomic landscape of RET aberrations across multiple cancers may facilitate clinical trial development targeting RETExperimental Design: We interrogated the molecular portfolio of 4,871 patients with diverse malignancies for the presence of RET aberrations using Clinical Laboratory Improvement Amendments-certified targeted next-generation sequencing of 182 or 236 gene panels.Results: Among diverse cancers, RET aberrations were identified in 88 cases [1.8% (88/4, 871)], with mutations being the most common alteration [38.6% (34/88)], followed by fusions [30.7% (27/88), including a novel SQSTM1-RET] and amplifications [25% (22/88)]. Most patients had coexisting aberrations in addition to RET anomalies [81.8% (72/88)], with the most common being in TP53-associated genes [59.1% (52/88)], cell cycle-associated genes [39.8% (35/88)], the PI3K signaling pathway [30.7% (27/88)], MAPK effectors [22.7% (20/88)], or other tyrosine kinase families [21.6% (19/88)]. RET fusions were mutually exclusive with MAPK signaling pathway alterations. All 72 patients harboring coaberrations had distinct genomic portfolios, and most [98.6% (71/72)] had potentially targetable coaberrations with either an FDA-approved or an investigational agent. Two cases with lung (KIF5B-RET) and medullary thyroid carcinoma (RET M918T) that responded to a vandetanib (multikinase RET inhibitor)-containing regimen are shown.Conclusions:RET aberrations were seen in 1.8% of diverse cancers, with most cases harboring actionable, albeit distinct, coexisting alterations. The current report suggests that optimal targeting of patients with RET anomalies will require customized combination strategies. Clin Cancer Res; 23(8); 1988-97. ©2016 AACR.
Assuntos
Proteínas de Ligação a DNA/genética , Neoplasias/genética , Proteínas Nucleares/genética , Aberrações Cromossômicas , Análise Mutacional de DNA , Sequenciamento de Nucleotídeos em Larga Escala , HumanosRESUMO
Understanding the genomic landscape of malignant mesothelioma may identify novel molecular drivers of this ultra-rare disease, which can lead to an expanded roster of targeted therapies and clinical trial options for patients with mesothelioma. We examined the molecular profiles of 42 patients with malignant mesothelioma (including pleural, peritoneal, and pericardial) that were referred by clinicians to be tested in a Clinical Laboratory Improvement Amendments (CLIA) laboratory using next-generation sequencing (NGS; 182 or 236 genes). Among 42 patients, there were 116 alterations, with 92 being distinct. The number of genomic alterations per patient ranged from 1 to 5 (median = 3). No two patients had identical molecular portfolios. The most common aberrations were in BAP1 (BRCA1-associated protein 1; 47.6% [20/42]), NF2 (38.1% [16/42]), and CDKN2A/B (loss) (35.7% [15/42]). BAP1 alterations and CDKN2A/B loss were associated with pleural mesothelioma (OR 3.4, P = 0.059 [BAP1] [trend]; OR 5.8, P = 0.01 [CDKN2A/B]). All 42 patients had a molecular abnormality that was potentially actionable (median = three actionable alterations per patient; range, 1 to 5), and, in 40 patients (95.2%), a drug approved by the FDA was applicable. In conclusion, each individual with malignant mesothelioma harbored a unique set of genomic aberrations, suggesting that NGS-based profiling of patients will be needed if patients are to be optimally matched to cognate treatments. All 42 patients had at least one alteration that was, in theory, pharmacologically tractable. Mol Cancer Ther; 15(10); 2498-507. ©2016 AACR.
Assuntos
Variação Genética , Genômica , Neoplasias Pulmonares/genética , Mesotelioma/genética , Biomarcadores Tumorais , Análise por Conglomerados , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Genômica/métodos , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Mesotelioma/diagnóstico , Mesotelioma/tratamento farmacológico , Mesotelioma/mortalidade , Mesotelioma Maligno , Terapia de Alvo Molecular , Mutação , Estadiamento de NeoplasiasRESUMO
Alterations in the cyclin-dependent kinase (CDK)-retinoblastoma (RB) machinery disrupt cell-cycle regulation and are being targeted in drug development. To understand the cancer types impacted by this pathway, we analyzed frequency of abnormalities in key cell-cycle genes across 4,864 tumors using next-generation sequencing (182 or 236 genes; Clinical Laboratory Improvement Amendments laboratory). Aberrations in the cell-cycle pathway were identified in 39% of cancers, making this pathway one of the most commonly altered in cancer. The frequency of aberrations was as follows: CDKN2A/B (20.1% of all patients), RB1 (7.6%), CCND1 (6.1%), CCNE1 (3.6%), CDK4 (3.2%), CCND3 (1.8%), CCND2 (1.7%), and CDK6 (1.7%). Rates and types of aberrant cell-cycle pathway genes differed between cancer types and within histologies. Analysis of coexisting and mutually exclusive genetic aberrations showed that CCND1, CCND2, and CCND3 aberrations were all positively associated with CDK6 aberrations [OR and P values, multivariate analysis: CCND1 and CDK6 (OR = 3.5; P < 0.0001), CCND2 and CDK6 (OR = 4.3; P = 0.003), CCND3 and CDK6 (OR = 3.6; P = 0.007)]. In contrast, RB1 alterations were negatively associated with multiple gene anomalies in the cell-cycle pathway, including CCND1 (OR = 0.25; P = 0.003), CKD4 (OR = 0.10; P = 0.001), and CDKN2A/B (OR = 0.21; P < 0.0001). In conclusion, aberrations in the cell-cycle pathway were very common in diverse cancers (39% of 4,864 neoplasms). The frequencies and types of alterations differed between and within tumor types and will be informative for drug development strategies. Mol Cancer Ther; 15(7); 1682-90. ©2016 AACR.
Assuntos
Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias/genética , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Biologia Computacional/métodos , Análise Mutacional de DNA , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genômica/métodos , Humanos , Masculino , Terapia de Alvo Molecular , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/metabolismoRESUMO
Merkel cell carcinoma is an ultra-rare cutaneous neuroendocrine cancer for which approved treatment options are lacking. To better understand potential actionability, the genomic landscape of Merkel cell cancers was assessed. The molecular aberrations in 17 patients with Merkel cell carcinoma were, on physician request, tested in a Clinical Laboratory Improvement Amendments (CLIA) laboratory (Foundation Medicine, Cambridge, MA) using next-generation sequencing (182 or 236 genes) and analyzed by N-of-One, Inc. (Lexington, MA). There were 30 genes harboring aberrations and 60 distinct molecular alterations identified in this patient population. The most common abnormalities involved the TP53 gene (12/17 [71% of patients]) and the cell cycle pathway (CDKN2A/B, CDKN2C or RB1) (12/17 [71%]). Abnormalities also were observed in the PI3K/AKT/mTOR pathway (AKT2, FBXW7, NF1, PIK3CA, PIK3R1, PTEN or RICTOR) (9/17 [53%]) and DNA repair genes (ATM, BAP1, BRCA1/2, CHEK2, FANCA or MLH1) (5/17 [29%]). Possible cognate targeted therapies, including FDA-approved drugs, could be identified in most of the patients (16/17 [94%]). In summary, Merkel cell carcinomas were characterized by multiple distinct aberrations that were unique in the majority of analyzed cases. Most patients had theoretically actionable alterations. These results provide a framework for investigating tailored combinations of matched therapies in Merkel cell carcinoma patients.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Célula de Merkel/genética , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Terapia de Alvo Molecular , Neoplasias Cutâneas/genética , Carcinoma de Célula de Merkel/tratamento farmacológico , Humanos , Medicina de Precisão , Neoplasias Cutâneas/tratamento farmacológicoRESUMO
PURPOSE: Molecular profiling may have prognostic and predictive value, and is increasingly used in the clinical setting. There are more than a dozen fibroblast growth factor receptor (FGFR) inhibitors in development. Optimal therapeutic application of FGFR inhibitors requires knowledge of the rates and types of FGFR aberrations in a variety of cancer types. EXPERIMENTAL DESIGN: We analyzed frequencies of FGFR aberrations in 4,853 solid tumors that were, on physician request, tested in a Clinical Laboratory Improvement Amendments (CLIA) laboratory (Foundation Medicine) using next-generation sequencing (182 or 236 genes), and analyzed by N-of-One. RESULTS: FGFR aberrations were found in 7.1% of cancers, with the majority being gene amplification (66% of the aberrations), followed by mutations (26%) and rearrangements (8%). FGFR1 (mostly amplification) was affected in 3.5% of 4,853 patients; FGFR2 in 1.5%; FGFR3 in 2.0%; and FGFR4 in 0.5%. Almost every type of malignancy examined showed some patients with FGFR aberrations, but the cancers most commonly affected were urothelial (32% FGFR-aberrant); breast (18%); endometrial (â¼13%), squamous lung cancers (â¼13%), and ovarian cancer (â¼9%). Among 35 unique FGFR mutations seen in this dataset, all but two are found in COSMIC. Seventeen of the 35 are known to be activating, and 11 are transforming. CONCLUSIONS: FGFR aberrations are common in a wide variety of cancers, with the majority being gene amplifications or activating mutations. These data suggest that FGFR inhibition could be an important therapeutic option across multiple tumor types.
Assuntos
Perfilação da Expressão Gênica , Neoplasias/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Feminino , Amplificação de Genes , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mutação , Neoplasias/diagnóstico , Neoplasias/patologia , Translocação GenéticaRESUMO
Effective treatment options for advanced salivary gland tumors are lacking. To better understand these tumors, we report their genomic landscape. We studied the molecular aberrations in 117 patients with salivary gland tumors that were, on physician request, tested in a Clinical Laboratory Improvement Amendments (CLIA) laboratory (Foundation Medicine, Cambridge, MA) using next-generation sequencing (182 or 236 genes), and analyzed by N-of-One, Inc. (Lexington, MA). There were 354 total aberrations, with 240 distinct aberrations identified in this patient population. Only 10 individuals (8.5%) had a molecular portfolio that was identical to any other patient (with four different portfolios amongst the ten patients). The most common abnormalities involved the TP53 gene (36/117 [30.8% of patients]), cyclin pathway (CCND1, CDK4/6 or CDKN2A/B) (31/117 [26.5%]) and PI3K pathway (PIK3CA, PIK3R1, PTEN or AKT1/3) (28/117 [23.9%]). In multivariate analysis, statistically significant co-existing aberrations were observed as follows: TP53 and ERBB2 (p = 0.01), cyclin pathway and MDM2 (p = 0.03), and PI3K pathway and HRAS (p = 0.0001). We were able to identify possible cognate targeted therapies in most of the patients (107/117 [91.5%]), including FDA-approved drugs in 80/117 [68.4%]. In conclusion, salivary gland tumors were characterized by multiple distinct aberrations that mostly differed from patient to patient. Significant associations between aberrations in TP53 and ERBB2, the cyclin pathway and MDM2, and HRAS and the PI3K pathway were identified. Most patients had actionable alterations. These results provide a framework for tailored combinations of matched therapies.
Assuntos
Biomarcadores Tumorais/genética , Análise Mutacional de DNA , Perfilação da Expressão Gênica , Genoma Humano , Mutação , Neoplasias das Glândulas Salivares/genética , Antineoplásicos/uso terapêutico , Análise Mutacional de DNA/métodos , Perfilação da Expressão Gênica/métodos , Frequência do Gene , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Modelos Logísticos , Análise Multivariada , Análise de Sequência com Séries de Oligonucleotídeos , Seleção de Pacientes , Fenótipo , Medicina de Precisão , Valor Preditivo dos Testes , Estudos Retrospectivos , Fatores de Risco , Neoplasias das Glândulas Salivares/tratamento farmacológico , Neoplasias das Glândulas Salivares/patologia , Transdução de Sinais/genéticaRESUMO
In order to gain a better understanding of the underlying biology of squamous cell carcinoma (SCC), we tested the hypothesis that SCC originating from different organs may possess common molecular alterations. SCC samples (N = 361) were examined using clinical-grade targeted next-generation sequencing (NGS). The most frequent SCC tumor types were head and neck, lung, cutaneous, gastrointestinal and gynecologic cancers. The most common gene alterations were TP53 (64.5% of patients), PIK3CA (28.5%), CDKN2A (24.4%), SOX2 (17.7%), and CCND1 (15.8%). By comparing NGS results of our SCC cohort to a non-SCC cohort (N = 277), we found that CDKN2A, SOX2, NOTCH1, TP53, PIK3CA, CCND1, and FBXW7 were significantly more frequently altered, unlike KRAS, which was less frequently altered in SCC specimens (all P < 0.05; multivariable analysis). Therefore, we identified "squamousness" gene signatures (TP53, PIK3CA, CCND1, CDKN2A, SOX2, NOTCH 1, and FBXW7 aberrations, and absence of KRAS alterations) that were significantly more frequent in SCC versus non-SCC histologies. A multivariable co-alteration analysis established 2 SCC subgroups: (i) patients in whom TP53 and cyclin pathway (CDKN2A and CCND1) alterations strongly correlated but in whom PIK3CA aberrations were less frequent; and (ii) patients with PIK3CA alterations in whom TP53 mutations were less frequent (all P ≤ 0 .001, multivariable analysis). In conclusion, we identified a set of 8 genes altered with significantly different frequencies when SCC and non-SCC were compared, suggesting the existence of patterns for "squamousness." Targeting the PI3K-AKT-mTOR and/or cyclin pathway components in SCC may be warranted.
Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias Gastrointestinais/patologia , Neoplasias dos Genitais Femininos/patologia , Neoplasias de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Feminino , Neoplasias Gastrointestinais/metabolismo , Neoplasias dos Genitais Femininos/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Estadiamento de Neoplasias , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Análise de Sequência de DNA , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoAssuntos
Adenocarcinoma/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-met/genética , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Crizotinibe , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Mutação , Sítios de Splice de RNA , Resultado do TratamentoRESUMO
During meiosis, two consecutive rounds of chromosome segregation yield four haploid gametes from one diploid cell. The Polo kinase Cdc5 is required for meiotic progression, but how Cdc5 coordinates multiple cell-cycle events during meiosis I is not understood. Here we show that CDC5-dependent phosphorylation of Rec8, a subunit of the cohesin complex that links sister chromatids, is required for efficient cohesin removal from chromosome arms, which is a prerequisite for meiosis I chromosome segregation. CDC5 also establishes conditions for centromeric cohesin removal during meiosis II by promoting the degradation of Spo13, a protein that protects centromeric cohesin during meiosis I. Despite CDC5's central role in meiosis I, the protein kinase is dispensable during meiosis II and does not even phosphorylate its meiosis I targets during the second meiotic division. We conclude that Cdc5 has evolved into a master regulator of the unique meiosis I chromosome segregation pattern.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Meiose/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por SubstratoRESUMO
N-of-One is at the forefront of personalized medicine. The company's mission is to act as a bridge between the research community, diagnostic laboratories, the treating oncologist and the patient to develop customized cancer diagnostic and treatment strategies. N-of-One provides physicians and patients with relevant insight regarding molecular profiling technologies and facilitates rapid, high-quality testing of patient tissues. In addition to expanding treatment options, the services provided by N-of-One potentially provide cost savings by guiding patients and physicians toward more evidence-based, data-driven treatment approaches.
RESUMO
The RAG1 and RAG2 proteins catalyze V(D)J recombination and are essential for generation of the diverse repertoire of antigen receptor genes and effective immune responses. RAG2 is composed of a "core" domain that is required for the recombination reaction and a C-terminal nonessential or "non-core" region. Recent evidence has emerged arguing that the non-core region plays a critical regulatory role in the recombination reaction, and mutations in this region have been identified in patients with immunodeficiencies. Here we present the first structural data for the RAG2 protein, using NMR spectroscopy to demonstrate that the C terminus of RAG2 contains a noncanonical PHD finger. All of the non-core mutations of RAG2 that are implicated in the development of immunodeficiencies are located within the PHD finger, at either zinc-coordinating residues or residues adjacent to an alpha-helix on the surface of the domain that participates in binding to the signaling molecules, phosphoinositides. Functional analysis of disease and phosphoinositide-binding mutations reveals novel intramolecular interactions within the non-core region and suggests that the PHD finger adopts two distinct states. We propose a model in which the equilibrium between these states modulates recombination activity. Together, these data identify the PHD finger as a novel and functionally important domain of RAG2.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Recombinação Genética , VDJ Recombinases/metabolismo , Dedos de Zinco , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência Consenso , Cinética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Control of V(D)J recombination is critical for the generation of a fully developed immune repertoire. The molecular mechanisms underlying the regulation of antigen receptor gene assembly are beginning to be revealed. Here we studied the influence of chromatin modifications on V(D)J cleavage of a polynucleosomal substrate, in which V(D)J cleavage is greatly reduced compared with naked DNA. ATP-dependent remodeling by human SWI/SNF (hSWI/SNF) in the presence of HMG1 led to a substantial increase of cleavage by the recombination activation gene (RAG) proteins. Either BRG1, the ATPase subunit of hSWI/SNF, or SNF2h, the ATPase of human ISWI complexes, was capable of stimulating V(D)J cleavage of the array, although these remodelers act by different mechanisms. No effect of histone hyperacetylation was detectable in this system. As is observed on naked DNA, in the presence of core RAG1, the full-length RAG2 protein proved to be more active than core RAG2 on these polynucleosomal arrays, reinforcing the importance of the RAG2 C-terminal domain for efficient recombination. Comparison of 5 S array cleavage by the RAG proteins or by the restriction enzyme HhaI after remodeling by hSWI/SNF suggested that RAG proteins and HhaI might have different requirements for maximal accessibility of the substrate.
Assuntos
Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Recombinação Genética , Fatores de Transcrição/genética , Adenosina Trifosfatases/metabolismo , Animais , DNA Helicases , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Fatores de Transcrição/metabolismoRESUMO
Following V(D)J cleavage, the newly liberated DNA signal ends can be either fused together into a signal joint or used as donor DNA in RAG-mediated transposition. We find that both V(D)J cleavage and release of flanking coding DNA occur before the target capture step of transposition can proceed; no coding DNA is ever detected in the target capture complex. Separately from its role in V(D)J cleavage, the DDE motif of the RAG1/2 active site is specifically required for target DNA capture. The requirement for cleavage and release of coding DNA prior to either physical target binding or functional target commitment suggests that the RAG1/2 transposase contains a single binding site for non-RSS DNA that can accommodate either target DNA or coding DNA, but not both together. Perhaps the presence of coding DNA may aid in preventing transpositional resolution of V(D)J recombination intermediates.
Assuntos
DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Recombinação Genética , Sítios de Ligação , DNA/química , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Proteínas Nucleares , Conformação de Ácido NucleicoRESUMO
Although costimulation plays an important role in activating naive T cells, its role in negative selection is controversial. By following thymocyte deletion induced by endogenous superantigens in mice lacking B7-1 and/or B7-2, we have identified a role for both B7-1 and B7-2 in negative selection. Studies using CD28-deficient and CD28/CTLA-4-double-deficient mice have revealed that either CD28 or another as yet undefined coreceptor can mediate these B7-dependent signals that promote negative selection. Finally, CTLA-4 delivers signals that inhibit selection, suggesting that CTLA-4 and CD28 have opposing functions in thymic development. Combined, the data demonstrate that B7-1/B7-2-dependent signals help shape the T cell repertoire.
Assuntos
Antígenos CD/fisiologia , Antígenos de Diferenciação/fisiologia , Antígeno B7-1/fisiologia , Antígenos CD28/fisiologia , Deleção Clonal/imunologia , Regulação para Baixo/imunologia , Glicoproteínas de Membrana/fisiologia , Transdução de Sinais/imunologia , Animais , Antígenos CD/genética , Antígenos de Diferenciação/genética , Antígeno B7-1/genética , Antígeno B7-2 , Antígenos CD28/genética , Antígenos CD40/genética , Antígenos CD40/fisiologia , Antígeno CTLA-4 , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Deleção Clonal/genética , Regulação para Baixo/genética , Enterotoxinas/biossíntese , Enterotoxinas/fisiologia , Depleção Linfocítica , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Transdução de Sinais/genética , Staphylococcus aureus/imunologia , Superantígenos/biossíntese , Superantígenos/fisiologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
The assembly of antigen receptor genes by V(D)J recombination is initiated by the RAG1/RAG2 protein complex, which introduces double-strand breaks between recombination signal sequences and their coding DNA. Truncated forms of RAG1 and RAG2 are functional in vivo and have been used to study V(D)J cleavage, hybrid joint formation and transposition in vitro. Here we have characterized the activities of the full-length proteins. Unlike core RAG2, which supports robust transposition in vitro, full-length RAG2 blocks transposition of signal ends following V(D)J cleavage. Thus, one role of this non-catalytic domain may be to prevent transposition in developing lymphoid cells. Although full-length RAG1 and RAG2 proteins rarely form hybrid joints in vivo in the absence of non-homologous end-joining factors, we show that the full-length proteins alone can catalyze this reaction in vitro.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Recombinação Genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/isolamento & purificação , Hibridização Genética , Conformação de Ácido Nucleico , Estrutura Terciária de ProteínaRESUMO
The recombination-activating gene (RAG)1 and RAG2 proteins comprise the lymphocyte-specific components of the V(D)J recombinase and are required for the assembly of antigen-receptor variable-region genes. A mutant truncated RAG2 protein ("core" RAG2) lacking the C-terminal 144 amino acids, together with core RAG1, is able to mediate the basic biochemical steps required for V(D)J recombination in vitro and in transfected cell lines. Here we examine the effect of replacing the endogenous RAG2 locus in mice with core RAG2. These mice generate substantial numbers of B and T cells, demonstrating that the core RAG2 protein retains significant in vivo function. However, core RAG2 mice display a reduction in the total number of B and T cells, reflecting impaired lymphocyte development at the progenitor stage associated with reduced chromosomal V(D)J recombination. We discuss potential roles of the RAG2 C terminus in mediating rearrangement of endogenous antigen-receptor loci.