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BACKGROUND: Significant clinical and research applications are driving large scale adoption of individualized tumor sequencing in cancer in order to identify tumors-specific mutations. When a matched germline sample is available, somatic mutations may be identified using comparative callers. However, matched germline samples are frequently not available such as with archival tissues, which makes it difficult to distinguish somatic from germline variants. While population databases may be used to filter out known germline variants, recent studies have shown private germline variants result in an inflated false positive rate in unmatched tumor samples, and the number germline false positives in an individual may be related to ancestry. METHODS: First, we examined the relationship between the germline false positives and ancestry. Then we developed and implemented a tumor only caller (LumosVar) that leverages differences in allelic frequency between somatic and germline variants in impure tumors. We used simulated data to systematically examine how copy number alterations, tumor purity, and sequencing depth should affect the sensitivity of our caller. Finally, we evaluated the caller on real data. RESULTS: We find the germline false-positive rate is significantly higher for individuals of non-European Ancestry largely due to the limited diversity in public polymorphism databases and due to population-specific characteristics such as admixture or recent expansions. Our Bayesian tumor only caller (LumosVar) is able to greatly reduce false positives from private germline variants, and our sensitivity is similar to predictions based on simulated data. CONCLUSIONS: Taken together, our results suggest that studies of individuals of non-European ancestry would most benefit from our approach. However, high sensitivity requires sufficiently impure tumors and adequate sequencing depth. Even in impure tumors, there are copy number alterations that result in germline and somatic variants having similar allele frequencies, limiting the sensitivity of the approach. We believe our approach could greatly improve the analysis of archival samples in a research setting where the normal is not available.
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Biologia Computacional/métodos , Mutação em Linhagem Germinativa , Neoplasias/genética , Teorema de Bayes , DNA/química , DNA/metabolismo , Variações do Número de Cópias de DNA , Bases de Dados Genéticas , Frequência do Gene , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias/patologia , Medicina de Precisão , Análise de Componente PrincipalRESUMO
BACKGROUND: Primary cardiac angiosarcomas are rare, but they are the most aggressive type of primary cardiac neoplasms. When patients do present, it is with advanced pulmonary and/or cardiac symptoms. Therefore, many times the correct diagnosis is not made at the time of initial presentation. These patients have metastatic disease and the vast majority of these patients die within a few months after diagnosis. Currently the treatment choices are limited and there are no targeted therapies available. CASE PRESENTATION: A 56-year-old male presented with shortness of breath, night sweats, and productive cough for a month. Workup revealed pericardial effusion and multiple bilateral pulmonary nodules suspicious for metastatic disease. Transthoracic echocardiogram showed a large pericardial effusion and a large mass in the base of the right atrium. Results of biopsy of bilateral lung nodules established a diagnosis of primary cardiac angiosarcoma. Aggressive pulmonary disease caused rapid deterioration; the patient went on hospice and subsequently died. Whole exome sequencing of the patient's postmortem tumor revealed a novel KDR (G681R) mutation, and focal high-level amplification at chromosome 1q encompassing MDM4, a negative regulator of TP53. CONCLUSION: Mutations in KDR have been reported previously in angiosarcomas. Previous studies also demonstrated that KDR mutants with constitutive KDR activation could be inhibited with specific KDR inhibitors in vitro. Thus, patients harboring activating KDR mutations could be candidates for treatment with KDR-specific inhibitors.
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Análise Mutacional de DNA , Neoplasias Cardíacas/genética , Hemangiossarcoma/genética , Proteínas de Ciclo Celular , Exoma/genética , Evolução Fatal , Amplificação de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaAssuntos
Adenocarcinoma Mucinoso/tratamento farmacológico , Receptores ErbB/genética , Cloridrato de Erlotinib/administração & dosagem , Análise de Sequência de DNA/métodos , Neoplasias Uretrais/tratamento farmacológico , Adenocarcinoma Mucinoso/genética , Idoso , Cloridrato de Erlotinib/uso terapêutico , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Metástase Neoplásica , Medicina de Precisão , Regulação para Cima , Neoplasias Uretrais/genéticaRESUMO
DNA focused panel sequencing has been rapidly adopted to assess therapeutic targets in advanced/refractory cancer. Integrated Genomic Profiling (IGP) utilising DNA/RNA with tumour/normal comparisons in a Clinical Laboratory Improvement Amendments (CLIA) compliant setting enables a single assay to provide: therapeutic target prioritisation, novel target discovery/application and comprehensive germline assessment. A prospective study in 35 advanced/refractory cancer patients was conducted using CLIA-compliant IGP. Feasibility was assessed by estimating time to results (TTR), prioritising/assigning putative therapeutic targets, assessing drug access, ascertaining germline alterations, and assessing patient preferences/perspectives on data use/reporting. Therapeutic targets were identified using biointelligence/pathway analyses and interpreted by a Genomic Tumour Board. Seventy-five percent of cases harboured 1-3 therapeutically targetable mutations/case (median 79 mutations of potential functional significance/case). Median time to CLIA-validated results was 116 days with CLIA-validation of targets achieved in 21/22 patients. IGP directed treatment was instituted in 13 patients utilising on/off label FDA approved drugs (n = 9), clinical trials (n = 3) and single patient IND (n = 1). Preliminary clinical efficacy was noted in five patients (two partial response, three stable disease). Although barriers to broader application exist, including the need for wider availability of therapies, IGP in a CLIA-framework is feasible and valuable in selection/prioritisation of anti-cancer therapeutic targets.
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Testes Diagnósticos de Rotina/métodos , Resistência a Medicamentos , Genômica/métodos , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Humanos , Estudos ProspectivosRESUMO
Glassy cell carcinoma of the cervix (GCCC) is a very rare and aggressive form of cervical cancer. An adolescent female with advanced metastatic disease was enrolled in our genomic profiling research protocol. We identified high-level amplification of epidermal growth factor receptor (EGFR) and Yes-associated protein-1 (YAP1), which led to the addition of EGFR inhibitors to the chemotherapy regimen. Here, we report the first genetically profiled case of GCCC with potential therapeutic implications.
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Receptores ErbB/genética , Biologia Molecular/métodos , Neoplasias do Colo do Útero/genética , Adolescente , Feminino , Humanos , Neoplasias do Colo do Útero/patologiaRESUMO
CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3â Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5â Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.
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Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the FGFR2 locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (in vitro FGFR2 IC50≈350 nM) was noted in a patient with an FGFR2-TACC3 fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (in vitro, FGFR2 IC50≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in ERRFI1, a direct negative regulator of EGFR activation. Rapid and robust disease regression was noted in this ERRFI1 inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. FGFR2 fusions and ERRFI mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations.
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Neoplasias dos Ductos Biliares/tratamento farmacológico , Colangiocarcinoma/tratamento farmacológico , Receptores ErbB/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais/genética , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/patologia , Linhagem Celular Tumoral , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Cloridrato de Erlotinib , Genoma Humano , Humanos , Imidazóis/administração & dosagem , Indazóis , Terapia de Alvo Molecular , Mutação , Prognóstico , Inibidores de Proteínas Quinases , Piridazinas/administração & dosagem , Pirimidinas/administração & dosagem , Quinazolinas/administração & dosagem , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Sulfonamidas/administração & dosagem , TranscriptomaRESUMO
As next-generation sequencing continues to have an expanding presence in the clinic, the identification of the most cost-effective and robust strategy for identifying copy number changes and translocations in tumor genomes is needed. We hypothesized that performing shallow whole genome sequencing (WGS) of 900-1000-bp inserts (long insert WGS, LI-WGS) improves our ability to detect these events, compared with shallow WGS of 300-400-bp inserts. A priori analyses show that LI-WGS requires less sequencing compared with short insert WGS to achieve a target physical coverage, and that LI-WGS requires less sequence coverage to detect a heterozygous event with a power of 0.99. We thus developed an LI-WGS library preparation protocol based off of Illumina's WGS library preparation protocol and illustrate the feasibility of performing LI-WGS. We additionally applied LI-WGS to three separate tumor/normal DNA pairs collected from patients diagnosed with different cancers to demonstrate our application of LI-WGS on actual patient samples for identification of somatic copy number alterations and translocations. With the evolution of sequencing technologies and bioinformatics analyses, we show that modifications to current approaches may improve our ability to interrogate cancer genomes.
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Variações do Número de Cópias de DNA , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , Análise de Sequência de DNA/métodos , Translocação Genética , Biblioteca Gênica , Genoma Humano , Genômica/métodos , HumanosRESUMO
BACKGROUND: Maxillary sinus carcinoma (MSC) is a rare cancer of the head and neck region. Patients are treated with surgery, radiation therapy, and chemotherapy and the treatment regimen is based on patient's age, general health condition, disease stage, and its extent of spread. There is very little information available on the genetics of this disease. DNA content based flow sorting of tumor cells followed by array comparative genomic hybridization allows for high definition global assessment of distinct clonal changes within tumor populations. METHODS: We applied this technique to primary and metastatic samples collected from a patient with radio- and chemotherapy refractory maxillary sinus carcinoma to gauge the progression of this disease. RESULTS: A clonal KIT amplicon was present in aneuploid populations sorted from the primary tumor and in divergent subclones arising in metastatic foci found in the brain, lung, and jejunum. The evolution of these subclones was associated with distinct genetic aberrations and DNA ploidies. CONCLUSION: The information presented here paves the path to understanding the development and progression of this disease.
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Neoplasias do Seio Maxilar/genética , Antineoplásicos/uso terapêutico , Hibridização Genômica Comparativa , Humanos , Neoplasias do Seio Maxilar/patologia , Neoplasias do Seio Maxilar/terapia , Metástase Neoplásica , RadioterapiaRESUMO
BACKGROUND: Recent advances in the treatment of cancer have focused on targeting genomic aberrations with selective therapeutic agents. In rare tumors, where large-scale clinical trials are daunting, this targeted genomic approach offers a new perspective and hope for improved treatments. Cancers of the ampulla of Vater are rare tumors that comprise only about 0.2% of gastrointestinal cancers. Consequently, they are often treated as either distal common bile duct or pancreatic cancers. METHODS: We analyzed DNA from a resected cancer of the ampulla of Vater and whole blood DNA from a 63 year-old man who underwent a pancreaticoduodenectomy by whole genome sequencing, achieving 37× and 40× coverage, respectively. We determined somatic mutations and structural alterations. RESULTS: We identified relevant aberrations, including deleterious mutations of KRAS and SMAD4 as well as a homozygous focal deletion of the PTEN tumor suppressor gene. These findings suggest that these tumors have a distinct oncogenesis from either common bile duct cancer or pancreatic cancer. Furthermore, this combination of genomic aberrations suggests a therapeutic context for dual mTOR/PI3K inhibition. CONCLUSIONS: Whole genome sequencing can elucidate an oncogenic context and expose potential therapeutic vulnerabilities in rare cancers.
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BACKGROUND: Olfactory neuroblastoma (ONB) is a rare cancer of the sinonasal tract with little molecular characterization. We performed whole genome sequencing (WGS) on paired normal and tumor DNA from a patient with metastatic-ONB to identify the somatic alterations that might be drivers of tumorigenesis and/or metastatic progression. METHODOLOGY/PRINCIPAL FINDINGS: Genomic DNA was isolated from fresh frozen tissue from a metastatic lesion and whole blood, followed by WGS at >30X depth, alignment and mapping, and mutation analyses. Sanger sequencing was used to confirm selected mutations. Sixty-two somatic short nucleotide variants (SNVs) and five deletions were identified inside coding regions, each causing a non-synonymous DNA sequence change. We selected seven SNVs and validated them by Sanger sequencing. In the metastatic ONB samples collected several months prior to WGS, all seven mutations were present. However, in the original surgical resection specimen (prior to evidence of metastatic disease), mutations in KDR, MYC, SIN3B, and NLRC4 genes were not present, suggesting that these were acquired with disease progression and/or as a result of post-treatment effects. CONCLUSIONS/SIGNIFICANCE: This work provides insight into the evolution of ONB cancer cells and provides a window into the more complex factors, including tumor clonality and multiple driver mutations.
Assuntos
Estesioneuroblastoma Olfatório/genética , Variação Genética , Genoma/genética , Cavidade Nasal , Metástase Neoplásica/genética , Neoplasias Nasais/genética , Adulto , Sequência de Bases , Variações do Número de Cópias de DNA , Biblioteca Gênica , Genes Neoplásicos/genética , Humanos , Masculino , Dados de Sequência Molecular , Mutação/genética , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNARESUMO
Constitutive activation of STAT3 is a common feature in many solid tumors including non-small cell lung carcinoma (NSCLC). While activation of STAT3 is commonly achieved by somatic mutations to JAK2 in hematologic malignancies, similar mutations are not often found in solid tumors. Previous work has instead suggested that STAT3 activation in solid tumors is more commonly induced by hyperactive growth factor receptors or autocrine cytokine signaling. The interplay between STAT3 activation and other well-characterized oncogenic "driver" mutations in NSCLC has not been fully characterized, though constitutive STAT3 activation has been proposed to play an important role in resistance to various small-molecule therapies that target these oncogenes. In this study we demonstrate that STAT3 is constitutively activated in human NSCLC samples and in a variety of NSCLC lines independent of activating KRAS or tyrosine kinase mutations. We further show that genetic or pharmacologic inhibition of the gp130/JAK2 signaling pathway disrupts activation of STAT3. Interestingly, treatment of NSCLC cells with the JAK1/2 inhibitor ruxolitinib has no effect on cell proliferation and viability in two-dimensional culture, but inhibits growth in soft agar and xenograft assays. These data demonstrate that JAK2/STAT3 signaling operates independent of known driver mutations in NSCLC and plays critical roles in tumor cell behavior that may not be effectively inhibited by drugs that selectively target these driver mutations.
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Carcinoma Pulmonar de Células não Pequenas/metabolismo , Janus Quinase 2/metabolismo , Neoplasias Pulmonares/metabolismo , Mutação/fisiologia , Fator de Transcrição STAT3/metabolismo , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Humanos , Neoplasias Pulmonares/genética , Nitrilas , Pirazóis/farmacologia , Pirimidinas , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Previous studies have now demonstrated that both genic and global hypomethylation characterizes the multiple myeloma (MM) epigenome. Whether these methylation changes are associated with global and corresponding increases (or decreases) in transcriptional activity are poorly understood. The purpose of our current study was to correlate DNA methylation levels in MM to gene expression. We analyzed matching datasets generated by the GoldenGate methylation BeadArray and Affymetrix gene expression platforms in 193 MM samples. We subsequently utilized two independent statistical approaches to identify methylation-expression correlations. In the first approach, we used a linear correlation parameter by computing a Pearson correlation coefficient. In the second approach, we discretized samples into low and high methylation groups and then compared the gene expression differences between the groups. Only methylation of 2.1% and 25.3% of CpG sites on the methylation array correlated to gene expression by Pearson correlation or the discretization method, respectively. Among the genes with methylation-expression correlations were IGF1R, DLC1, p16, and IL17RB. In conclusion, DNA methylation may directly regulate relatively few genes and suggests that additional studies are needed to determine the effects of genome-wide methylation changes in MM.
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Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Mieloma Múltiplo/genética , Transcrição Gênica , Aberrações Cromossômicas , Biologia Computacional/métodos , Ilhas de CpG , Epigênese Genética , Epigenômica , Perfilação da Expressão Gênica , Humanos , Reprodutibilidade dos TestesRESUMO
INTRODUCTION: The Hedgehog Signaling Pathway (HHSP) has been implicated in the development of multiple cancers. HHSP activation may primarily be hedgehog ligand-dependent in non-small cell lung cancer (NSCLC); while a subset may be ligand-independent. In this study NSCLC primary tumors were used to identify correlations between multiple biomarkers thought to be involved in the HHSP and the clinical outcomes of patients with NSCLC. Identification of such correlations could be used to aid in NSCLC treatment and predicting patient prognosis. METHODS: A tissue microarray representing 248 clinically annotated stage I-II NSCLC cases was stained using immunohistochemistry (IHC) and scored for HHSP proteins namely, SHH, PTCH1, SMO, GLI1, and GLI2; as well as, ALDH1A1, a putative cancer stem cell marker. Data was analyzed for correlation between IHC staining, EGFR and KRAS mutations, and clinical characteristics including relapse-free survival (RFS) and overall survival (OS). RESULTS: In adenocarcinoma, there were significant correlations of IHC expression between SHH and downstream HHSP receptor SMO (p=0.017) and transcription factor GLI1 (p=0.001), while SMO correlated with GLI1 (p=0.007). In squamous cell carcinoma, SHH significantly correlated with GLI2 protein expression (p=0.003). After multiple testing correction, there was no significant correlation between any of the six markers and RFS or OS. CONCLUSIONS: Key downstream components of the HHSP show correlation with sonic hedgehog ligand (SHH) expression, suggesting that ligand-dependent signaling is more prevalent in primary NSCLC tumors. Surprisingly, in early-stage NSCLC, there were no significant correlations between HHSP proteins or ALDH1A1 and RFS or OS.
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Aldeído Desidrogenase/biossíntese , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas Hedgehog/metabolismo , Neoplasias Pulmonares/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Família Aldeído Desidrogenase 1 , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Intervalo Livre de Doença , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Proteínas Hedgehog/genética , Humanos , Imuno-Histoquímica/métodos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Ligantes , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Retinal Desidrogenase , Transdução de Sinais , Receptor Smoothened , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Gli2 com Dedos de Zinco , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
miRNAs play an important role in the regulation of a wide assortment of cellular processes by sequestering target mRNAs and inhibiting translation of the proteins that they encode. Multiple miRNAs can regulate single mRNA molecules and, alternatively, a single miRNA can act on a number of mRNA targets. Dysfunctional miRNAs are commonly found in a variety of solid cancers and are attractive candidates for next-generation therapeutics. This article highlights miRNA signatures proposed for lung cancer classification and diagnosis, chemo- and radio-therapy resistance, metastasis and prediction of treatment outcome and survival.
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Neoplasias Pulmonares/genética , MicroRNAs/análise , Biomarcadores Tumorais/genética , Transformação Celular Neoplásica/genética , Resistencia a Medicamentos Antineoplásicos/genética , Detecção Precoce de Câncer , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/terapia , MicroRNAs/metabolismo , MicroRNAs/uso terapêutico , Metástase Neoplásica , Prognóstico , Tolerância a Radiação/genéticaRESUMO
Tyrosine kinase inhibitors (TKIs) have been in use as cancer therapeutics for nearly a decade, and their utility in targeting specific malignancies with defined genetic lesions has proven to be remarkably effective. Recent efforts to characterize the spectrum of genetic lesions found in non-small cell lung carcinoma (NSCLC) have provided important insights into the molecular basis of this disease and have also revealed a wide array of tyrosine kinases that might be effectively targeted for rationally designed therapies. The findings of these studies, however, also provide a cautionary tale about the limitations of single-agent therapies, which fail to account for the genetic heterogeneity and pathway redundancy that characterize advanced NSCLC. Emergence of drug resistance mechanisms to specific TKIs, such as gefitinib and erlotinib, suggests that more sophisticated chemotherapeutic paradigms that target multiple pathways at the same time will be required to effectively treat this disease.
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The concept of using cholinesterase bioscavengers for prophylaxis against organophosphorous nerve agents and pesticides has progressed from the bench to clinical trial. However, the supply of the native human proteins is either limited (e.g., plasma-derived butyrylcholinesterase and erythrocytic acetylcholinesterase) or nonexisting (synaptic acetylcholinesterase). Here we identify a unique form of recombinant human butyrylcholinesterase that mimics the native enzyme assembly into tetramers; this form provides extended effective pharmacokinetics that is significantly enhanced by polyethylene glycol conjugation. We further demonstrate that this enzyme (but not a G117H/E197Q organophosphorus acid anhydride hydrolase catalytic variant) can prevent morbidity and mortality associated with organophosphorous nerve agent and pesticide exposure of animal subjects of two model species.
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Butirilcolinesterase/farmacologia , Substâncias para a Guerra Química/toxicidade , Fármacos Neuroprotetores/farmacologia , Nicotiana/metabolismo , Compostos Organofosforados/toxicidade , Praguicidas/toxicidade , Animais , Butirilcolinesterase/metabolismo , Butirilcolinesterase/farmacocinética , Substâncias para a Guerra Química/metabolismo , Cromatografia Líquida de Alta Pressão , Cobaias , Humanos , Immunoblotting , Cinética , Camundongos , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacocinética , Compostos Organofosforados/metabolismo , Praguicidas/metabolismo , Polietilenoglicóis/metabolismo , Engenharia de ProteínasRESUMO
Organophosphorous pesticides and nerve agents inhibit the enzyme acetylcholinesterase at neuronal synapses and in neuromuscular junctions. The resulting accumulation of acetylcholine overwhelms regulatory mechanisms, potentially leading to seizures and death from respiratory collapse. While current therapies are only capable of reducing mortality, elevation of the serum levels of the related enzyme butyrylcholinesterase (BChE) by application of the purified protein as a bioscavenger of organophosphorous compounds is effective in preventing all symptoms associated with poisoning by these toxins. However, BChE therapy requires large quantities of enzyme that can easily overwhelm current sources. Here, we report genetic optimization, cloning and high-level expression of human BChE in plants. Plant-derived BChE is shown to be biochemically similar to human plasma-derived BChE in terms of catalytic activity and inhibitor binding. We further demonstrate the ability of the plant-derived bioscavenger to protect animals against an organophosphorous pesticide challenge.
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Butirilcolinesterase/biossíntese , Plantas Geneticamente Modificadas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Butirilcolinesterase/química , Butirilcolinesterase/genética , Eletroforese em Gel de Poliacrilamida , Humanos , Camundongos , Compostos Organofosforados/antagonistas & inibidores , Compostos Organofosforados/toxicidade , Plantas Geneticamente Modificadas/genética , Alinhamento de SequênciaRESUMO
Plants are potentially the most economical platforms for the large-scale production of recombinant proteins. Thus, plant-based expression of subunit human immunodeficiency virus type 1 (HIV-1) vaccines provides an opportunity for their global use against the acquired immunodeficiency syndrome pandemic. CTB-MPR(649-684)[CTB, cholera toxin B subunit; MPR, membrane proximal (ectodomain) region of gp41] is an HIV-1 vaccine candidate that has been shown previously to induce antibodies that block a pathway of HIV-1 mucosal transmission. In this article, the molecular characterization of CTB-MPR(649-684) expressed in transgenic Nicotiana benthamiana plants is reported. Virtually all of the CTB-MPR(649-684) proteins expressed in the selected line were shown to have assembled into pentameric, GM1 ganglioside-binding complexes. Detailed biochemical analyses on the purified protein revealed that it was N-glycosylated, predominantly with high-mannose-type glycans (more than 75%), as predicted from a consensus asparagine-X-serine/threonine (Asn-X-Ser/Thr) N-glycosylation sequon on the CTB domain and an endoplasmic reticulum retention signal attached at the C-terminus of the fusion protein. Despite this modification, the plant-expressed protein retained the nanomolar affinity to GM1 ganglioside and the critical antigenicity of the MPR(649-684) moiety. Furthermore, the protein induced mucosal and serum anti-MPR(649-684) antibodies in mice after mucosal prime-systemic boost immunization. Our data indicate that plant-based expression can be a viable alternative for the production of this subunit HIV-1 vaccine candidate.
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Vacinas contra a AIDS/imunologia , HIV-1/imunologia , Nicotiana/imunologia , Plantas Geneticamente Modificadas/imunologia , Proteínas Recombinantes de Fusão/imunologia , Vacinas contra a AIDS/biossíntese , Animais , Toxina da Cólera/genética , Toxina da Cólera/imunologia , Feminino , Glicosilação , Anticorpos Anti-HIV/sangue , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/imunologia , Humanos , Imunidade nas Mucosas , Camundongos , Camundongos Endogâmicos BALB C , Plantas Geneticamente Modificadas/genética , Proteínas Recombinantes de Fusão/genética , Nicotiana/genética , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Therapeutically valuable proteins are often rare and/or unstable in their natural context, calling for production solutions in heterologous systems. A relevant example is that of the stress-induced, normally rare, and naturally unstable "read-through" human acetylcholinesterase variant, AChE-R. AChE-R shares its active site with the synaptic AChE-S variant, which is the target of poisonous organophosphate anticholinesterase insecticides such as the parathion metabolite paraoxon. Inherent AChE-R overproduction under organophosphate intoxication confers both short-term protection (as a bioscavenger) and long-term neuromuscular damages (as a regulator). Here we report the purification, characterization, and testing of human, endoplasmic reticulum-retained AChE-R(ER) produced from plant-optimized cDNA in Nicotiana benthamiana plants. AChE-R(ER) purified to homogeneity showed indistinguishable biochemical properties, with IC50 = 10(-7) M for the organophosphate paraoxon, similar to mammalian cell culture-derived AChE. In vivo titration showed dose-dependent protection by intravenously injected AChE-R(ER) of FVB/N male mice challenged with a lethal dose of paraoxon, with complete elimination of short-term clinical symptoms at near molar equivalence. By 10 days postexposure, AChE-R prophylaxis markedly limited postexposure increases in plasma murine AChE-R levels while minimizing the organophosphate-induced neuromuscular junction dismorphology. Our findings present plant-produced AChE-R(ER) as a bimodal agent, conferring both short- and long-term protection from organophosphate intoxication.