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Measuring responses in the proteome to various perturbations improves our understanding of biological systems. The value of information gained from such studies is directly proportional to the number of proteins measured. To overcome technical challenges associated with highly multiplexed measurements, we developed an affinity reagent-based method that uses aptamers with protein-like side chains along with an assay that takes advantage of their unique properties. As hybrid affinity reagents, modified aptamers are fully comparable to antibodies in terms of binding characteristics toward proteins, including epitope size, shape complementarity, affinity and specificity. Our assay combines these intrinsic binding properties with serial kinetic proofreading steps to allow highly effective partitioning of stable specific complexes from unstable nonspecific complexes. The use of these orthogonal methods to enhance specificity effectively overcomes the severe limitation to multiplexing inherent to the use of sandwich-based methods. Our assay currently measures half of the unique proteins encoded in the human genome with femtomolar sensitivity, broad dynamic range and exceptionally high reproducibility. Using machine learning to identify patterns of change, we have developed tests based on measurement of multiple proteins predictive of current health states and future disease risk to guide a holistic approach to precision medicine.
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The modern version of the RNA World Hypothesis begins with activated ribonucleotides condensing (nonenzymatically) to make RNA molecules, some of which possess (perhaps slight) catalytic activity. We propose that noncanonical ribonucleotides, which would have been inevitable under prebiotic conditions, might decrease the RNA length required to have useful catalytic function by allowing short RNAs to possess a more versatile collection of folded motifs. We argue that modified versions of the standard bases, some with features that resemble cofactors, could have facilitated that first moment in which early RNA molecules with catalytic capability began their evolutionary path toward self-replication.
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RNA Catalítico/metabolismo , Ribonucleotídeos/metabolismo , Evolução Molecular , RNA/genética , RNA/metabolismo , RNA Catalítico/genéticaRESUMO
Intrauterine growth restriction (IUGR) in premature newborns increases the risk for bronchopulmonary dysplasia, a chronic lung disease characterized by disrupted pulmonary angiogenesis and alveolarization. We previously showed that experimental IUGR impairs angiogenesis; however, mechanisms that impair pulmonary artery endothelial cell (PAEC) function are uncertain. The NF-κB pathway promotes vascular growth in the developing mouse lung, and we hypothesized that IUGR disrupts NF-κB-regulated proangiogenic targets in fetal PAEC. PAECs were isolated from the lungs of control fetal sheep and sheep with experimental IUGR from an established model of chronic placental insufficiency. Microarray analysis identified suppression of NF-κB signaling and significant alterations in extracellular matrix (ECM) pathways in IUGR PAEC, including decreases in collagen 4α1 and laminin α4, components of the basement membrane and putative NF-κB targets. In comparison with controls, immunostaining of active NF-κB complexes, NF-κB-DNA binding, baseline expression of NF-κB subunits p65 and p50, and LPS-mediated inducible activation of NF-κB signaling were decreased in IUGR PAEC. Although pharmacological NF-κB inhibition did not affect angiogenic function in IUGR PAEC, angiogenic function of control PAEC was reduced to a similar degree as that observed in IUGR PAEC. These data identify reductions in endothelial NF-κB signaling as central to the disrupted angiogenesis observed in IUGR, likely by impairing both intrinsic PAEC angiogenic function and NF-κB-mediated regulation of ECM components necessary for vascular development. These data further suggest that strategies that preserve endothelial NF-κB activation may be useful in lung diseases marked by disrupted angiogenesis such as IUGR.
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Displasia Broncopulmonar , Células Endoteliais , Retardo do Crescimento Fetal , Subunidade p50 de NF-kappa B/metabolismo , Artéria Pulmonar , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Animais , Displasia Broncopulmonar/induzido quimicamente , Displasia Broncopulmonar/embriologia , Displasia Broncopulmonar/patologia , Displasia Broncopulmonar/fisiopatologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Retardo do Crescimento Fetal/induzido quimicamente , Retardo do Crescimento Fetal/metabolismo , Retardo do Crescimento Fetal/patologia , Retardo do Crescimento Fetal/fisiopatologia , Lipopolissacarídeos/toxicidade , Gravidez , Artéria Pulmonar/embriologia , Artéria Pulmonar/patologia , Artéria Pulmonar/fisiopatologia , OvinosRESUMO
BACKGROUND: DUF1220 protein domains found primarily in Neuroblastoma BreakPoint Family (NBPF) genes show the greatest human lineage-specific increase in copy number of any coding region in the genome. There are 302 haploid copies of DUF1220 in hg38 (~160 of which are human-specific) and the majority of these can be divided into 6 different subtypes (referred to as clades). Copy number changes of specific DUF1220 clades have been associated in a dose-dependent manner with brain size variation (both evolutionarily and within the human population), cognitive aptitude, autism severity, and schizophrenia severity. However, no published methods can directly measure copies of DUF1220 with high accuracy and no method can distinguish between domains within a clade. RESULTS: Here we describe a novel method for measuring copies of DUF1220 domains and the NBPF genes in which they are found from whole genome sequence data. We have characterized the effect that various sequencing and alignment parameters and strategies have on the accuracy and precision of the method and defined the parameters that lead to optimal DUF1220 copy number measurement and resolution. We show that copy number estimates obtained using our read depth approach are highly correlated with those generated by ddPCR for three representative DUF1220 clades. By simulation, we demonstrate that our method provides sufficient resolution to analyze DUF1220 copy number variation at three levels: (1) DUF1220 clade copy number within individual genes and groups of genes (gene-specific clade groups) (2) genome wide DUF1220 clade copies and (3) gene copy number for DUF1220-encoding genes. CONCLUSIONS: To our knowledge, this is the first method to accurately measure copies of all six DUF1220 clades and the first method to provide gene specific resolution of these clades. This allows one to discriminate among the ~300 haploid human DUF1220 copies to an extent not possible with any other method. The result is a greatly enhanced capability to analyze the role that these sequences play in human variation and disease.
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Dosagem de Genes/genética , Genômica , Proteínas de Neoplasias/genética , Evolução Molecular , Genoma Humano/genética , Genética Humana , Humanos , Proteínas de Neoplasias/química , Domínios Proteicos , Alinhamento de SequênciaRESUMO
Chlamydomonas reinhardtii is well adapted to survive under different environmental conditions due to the unique flexibility of its metabolism. Here we report metabolic pathways that are active during acclimation to anoxia, but were previously not thoroughly studied under dark, anoxic H2-producing conditions in this model green alga. Proteomic analyses, using 2D-differential in-gel electrophoresis in combination with shotgun mass fingerprinting, revealed increased levels of proteins involved in the glycolytic pathway downstream of 3-phosphoglycerate, the glyoxylate pathway, and steps of the tricarboxylic acid (TCA) reactions. Upregulation of the enzyme, isocitrate lyase (ICL), was observed, which was accompanied by increased intracellular succinate levels, suggesting the functioning of glyoxylate pathway reactions. The ICL-inhibitor study revealed presence of reverse TCA reactions under these conditions. Contributions of the serine-isocitrate lyase pathway, glycine cleavage system, and c1-THF/serine hydroxymethyltransferase pathway in the acclimation to dark anoxia were found. We also observed increased levels of amino acids (AAs) suggesting nitrogen reorganization in the form of de novo AA biosynthesis during anoxia. Overall, novel routes for reductant utilization, in combination with redistribution of carbon and nitrogen, are used by this alga during acclimation to O2 deprivation in the dark.
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Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Perfilação da Expressão Gênica/métodos , Hidrogênio/metabolismo , Metabolômica/métodos , Proteômica/métodos , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Aminoácidos/metabolismo , Anaerobiose , Western Blotting , Carbono/metabolismo , Escuridão , Eletroforese em Gel Bidimensional , Cromatografia Gasosa-Espectrometria de Massas , Espectrometria de Massas , Redes e Vias Metabólicas/genética , Metaboloma , Nitrogênio/metabolismo , Proteoma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Although the reference human genome sequence was declared finished in 2003, some regions of the genome remain incomplete due to their complex architecture. One such region, 1q21.1-q21.2, is of increasing interest due to its relevance to human disease and evolution. Elucidation of the exact variants behind these associations has been hampered by the repetitive nature of the region and its incomplete assembly. This region also contains 238 of the 270 human DUF1220 protein domains, which are implicated in human brain evolution and neurodevelopment. Additionally, examinations of this protein domain have been challenging due to the incomplete 1q21 build. To address these problems, a single-haplotype hydatidiform mole BAC library (CHORI-17) was used to produce the first complete sequence of the 1q21.1-q21.2 region. RESULTS: We found and addressed several inaccuracies in the GRCh37sequence of the 1q21 region on large and small scales, including genomic rearrangements and inversions, and incorrect gene copy number estimates and assemblies. The DUF1220-encoding NBPF genes required the most corrections, with 3 genes removed, 2 genes reassigned to the 1p11.2 region, 8 genes requiring assembly corrections for DUF1220 domains (~91 DUF1220 domains were misassigned), and multiple instances of nucleotide changes that reassigned the domain to a different DUF1220 subtype. These corrections resulted in an overall increase in DUF1220 copy number, yielding a haploid total of 289 copies. Approximately 20 of these new DUF1220 copies were the result of a segmental duplication from 1q21.2 to 1p11.2 that included two NBPF genes. Interestingly, this duplication may have been the catalyst for the evolutionarily important human lineage-specific chromosome 1 pericentric inversion. CONCLUSIONS: Through the hydatidiform mole genome sequencing effort, the 1q21.1-q21.2 region is complete and misassemblies involving inter- and intra-region duplications have been resolved. The availability of this single haploid sequence path will aid in the investigation of many genetic diseases linked to 1q21, including several associated with DUF1220 copy number variations. Finally, the corrected sequence identified a recent segmental duplication that added 20 additional DUF1220 copies to the human genome, and may have facilitated the chromosome 1 pericentric inversion that is among the most notable human-specific genomic landmarks.
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Cromossomos Humanos Par 1 , Genoma Humano , Evolução Biológica , Proteínas de Transporte/genética , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Ligação Genética , Haploidia , Humanos , Estrutura Terciária de Proteína/genética , Duplicações Segmentares GenômicasRESUMO
OBJECTIVE: To determine the extent to which changes in plasma proteins, previously predictive of cardiometabolic outcomes, predict changes in two diabetes remission trials. RESEARCH DESIGN AND METHODS: We applied SomaSignal predictive tests (each derived from â¼5,000 plasma protein measurements using aptamer-based proteomics assay) to baseline and 1-year samples of trial intervention (Diabetes Remission Clinical Trial [DiRECT], n = 118, and Diabetes Intervention Accentuating Diet and Enhancing Metabolism [DIADEM-I], n = 66) and control (DiRECT, n = 144, DIADEM-I, n = 76) group participants. RESULTS: Mean (SD) weight loss in DiRECT (U.K.) and DIADEM-I (Qatar) was 10.2 (7.4) kg and 12.1 (9.5) kg, respectively, vs. 1.0 (3.7) kg and 4.0 (5.4) kg in control groups. Cardiometabolic SomaSignal test results showed significant improvement (Bonferroni-adjusted P < 0.05) in DiRECT and DIADEM-I (expressed as relative difference, intervention minus control) as follows, respectively: liver fat (-26.4%, -37.3%), glucose tolerance (-36.6%, -37.4%), body fat percentage (-8.6%, -8.7%), resting energy rate (-8.0%, -5.1%), visceral fat (-34.3%, -26.1%), and cardiorespiratory fitness (9.5%, 10.3%). Cardiovascular risk (measured with SomaSignal tests) also improved in intervention groups relative to control, but this was significant only in DiRECT (DiRECT, -44.2%, and DIADEM-I, -9.2%). However, weight loss >10 kg predicted significant reductions in cardiovascular risk, -19.1% (95% CI -33.4 to -4.91) in DiRECT and -33.4% (95% CI -57.3, -9.6) in DIADEM-I. DIADEM-I also demonstrated rapid emergence of metabolic improvements at 3 months. CONCLUSIONS: Intentional weight loss in recent-onset type 2 diabetes rapidly induces changes in protein-based risk models consistent with widespread cardiometabolic improvements, including cardiorespiratory fitness. Protein changes with greater (>10 kg) weight loss also predicted lower cardiovascular risk, providing a positive outlook for relevant ongoing trials.
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Doenças Cardiovasculares , Diabetes Mellitus Tipo 2 , Humanos , Diabetes Mellitus Tipo 2/terapia , Diabetes Mellitus Tipo 2/metabolismo , Ensaios Clínicos Controlados Aleatórios como Assunto , Redução de Peso , Dieta , Proteínas SanguíneasRESUMO
Directional motility in the gliding bacterium Myxococcus xanthus requires controlled cell reversals mediated by the Frz chemosensory system. FrzCD, a cytoplasmic chemoreceptor, does not form membrane-bound polar clusters typical for most bacteria, but rather cytoplasmic clusters that appear helically arranged and span the cell length. The distribution of FrzCD in living cells was found to be dynamic: FrzCD was localized in clusters that continuously changed their size, number, and position. The number of FrzCD clusters was correlated with cellular reversal frequency: fewer clusters were observed in hypo-reversing mutants and additional clusters were observed in hyper-reversing mutants. When moving cells made side-to-side contacts, FrzCD clusters in adjacent cells showed transient alignments. These events were frequently followed by one of the interacting cells reversing. These observations suggest that FrzCD detects signals from a cell contact-sensitive signaling system and then re-localizes as it directs reversals to distributed motility engines.
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Proteínas de Bactérias/metabolismo , Citoplasma/metabolismo , Myxococcus xanthus/citologia , Myxococcus xanthus/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas de Bactérias/química , Proteínas de Fluorescência Verde/metabolismo , Estrutura Secundária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Similarity in facial characteristics between relatives suggests a strong genetic component underlies facial variation. While there have been numerous studies of the genetics of facial abnormalities and, more recently, single nucleotide polymorphism (SNP) genome-wide association studies (GWASs) of normal facial variation, little is known about the role of genetic structural variation in determining facial shape. In a sample of Bantu African children, we found that only 9% of common copy number variants (CNVs) and 10-kb CNV analysis windows are well tagged by SNPs (r2 ≥ 0.8), indicating that associations with our internally called CNVs were not captured by previous SNP-based GWASs. Here, we present a GWAS and gene set analysis of the relationship between normal facial variation and CNVs in a sample of Bantu African children. We report the top five regions, which had p values ≤ 9.35 × 10-6 and find nominal evidence of independent CNV association (p < 0.05) in three regions previously identified in SNP-based GWASs. The CNV region with strongest association (p = 1.16 × 10-6, 55 losses and seven gains) contains NFATC1, which has been linked to facial morphogenesis and Cherubism, a syndrome involving abnormal lower facial development. Genomic loss in the region is associated with smaller average lower facial depth. Importantly, new loci identified here were not identified in a SNP-based GWAS, suggesting that CNVs are likely involved in determining facial shape variation. Given the plethora of SNP-based GWASs, calling CNVs from existing data may be a relatively inexpensive way to aid in the study of complex traits.
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A reliable, individualized, and dynamic surrogate of cardiovascular risk, synoptic for key biologic mechanisms, could shorten the path for drug development, enhance drug cost-effectiveness and improve patient outcomes. We used highly multiplexed proteomics to address these objectives, measuring about 5000 proteins in each of 32,130 archived plasma samples from 22,849 participants in nine clinical studies. We used machine learning to derive a 27-protein model predicting 4-year likelihood of myocardial infarction, stroke, heart failure, or death. The 27 proteins encompassed 10 biologic systems, and 12 were associated with relevant causal genetic traits. We independently validated results in 11,609 participants. Compared to a clinical model, the ratio of observed events in quintile 5 to quintile 1 was 6.7 for proteins versus 2.9 for the clinical model, AUCs (95% CI) were 0.73 (0.72 to 0.74) versus 0.64 (0.62 to 0.65), c-statistics were 0.71 (0.69 to 0.72) versus 0.62 (0.60 to 0.63), and the net reclassification index was +0.43. Adding the clinical model to the proteins only improved discrimination metrics by 0.01 to 0.02. Event rates in four predefined protein risk categories were 5.6, 11.2, 20.0, and 43.4% within 4 years; median time to event was 1.71 years. Protein predictions were directionally concordant with changed outcomes. Adverse risks were predicted for aging, approaching an event, anthracycline chemotherapy, diabetes, smoking, rheumatoid arthritis, cancer history, cardiovascular disease, high systolic blood pressure, and lipids. Reduced risks were predicted for weight loss and exenatide. The 27-protein model has potential as a "universal" surrogate end point for cardiovascular risk.
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Doenças Cardiovasculares , Insuficiência Cardíaca , Infarto do Miocárdio , Acidente Vascular Cerebral , Biomarcadores , Insuficiência Cardíaca/tratamento farmacológico , Humanos , Infarto do Miocárdio/tratamento farmacológico , Proteômica , Acidente Vascular Cerebral/complicaçõesRESUMO
BACKGROUND: Copy number variations (CNVs) account for a substantial proportion of inter-individual genomic variation. However, a majority of genomic variation studies have focused on single-nucleotide variations (SNVs), with limited genome-wide analysis of CNVs in large cohorts, especially in populations that are under-represented in genetic studies including people of African descent. METHODS: We carried out a genome-wide copy number analysis in > 3400 healthy Bantu Africans from Tanzania. Signal intensity data from high density (> 2.5 million probes) genotyping arrays were used for CNV calling with three algorithms including PennCNV, DNAcopy and VanillaICE. Stringent quality metrics and filtering criteria were applied to obtain high confidence CNVs. RESULTS: We identified over 400,000 CNVs larger than 1 kilobase (kb), for an average of 120 CNVs (SE = 2.57) per individual. We detected 866 large CNVs (≥ 300 kb), some of which overlapped genomic regions previously associated with multiple congenital anomaly syndromes, including Prader-Willi/Angelman syndrome (Type1) and 22q11.2 deletion syndrome. Furthermore, several of the common CNVs seen in our cohort (≥ 5%) overlap genes previously associated with developmental disorders. CONCLUSIONS: These findings may help refine the phenotypic outcomes and penetrance of variations affecting genes and genomic regions previously implicated in diseases. Our study provides one of the largest datasets of CNVs from individuals of African ancestry, enabling improved clinical evaluation and disease association of CNVs observed in research and clinical studies in African populations.
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Variações do Número de Cópias de DNARESUMO
A draft genome of 906 scaffolds of 115.8 Mb was assembled for Desmodesmus armatus, a diploid, lipid- and storage carbohydrate-accumulating microalga proven relevant for large-scale, outdoor cultivation, and serves as a model alga platform for improving photosynthetic efficiency and carbon assimilation for next-generation bioenergy production.
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Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine; however, their potential clinical application is hampered by the low efficiency of somatic cell reprogramming. Here, we show that the synergistic activity of synthetic modified mRNAs encoding reprogramming factors and miRNA-367/302s delivered as mature miRNA mimics greatly enhances the reprogramming of human primary fibroblasts into iPSCs. This synergistic activity is dependent upon an optimal RNA transfection regimen and culturing conditions tailored specifically to human primary fibroblasts. As a result, we can now generate up to 4,019 iPSC colonies from only 500 starting human primary neonatal fibroblasts and reprogram up to 90.7% of individually plated cells, producing multiple sister colonies. This methodology consistently generates clinically relevant, integration-free iPSCs from a variety of human patient's fibroblasts under feeder-free conditions and can be applicable for the clinical translation of iPSCs and studying the biology of reprogramming.
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Técnicas de Reprogramação Celular , Linhagem Celular , Fibroblastos , Humanos , Células-Tronco Pluripotentes Induzidas , RNARESUMO
OBJECTIVES: Neuromyelitis optica spectrum disorder (NMOSD) is a severe inflammatory disorder of the central nervous system (CNS) targeted against aquaporin-4 (AQP4). The origin and trafficking of AQP4-specific B cells in NMOSD remains unknown. METHODS: Peripheral (n = 7) and splenic B cells (n = 1) recovered from seven NMOSD patients were sorted into plasmablasts, naïve, memory, and CD27-IgD- double negative (DN) B cells, and variable heavy chain (VH) transcriptome sequences were generated by deep sequencing. Peripheral blood (PB) VH repertoires were compared to the same patient's single-cell cerebrospinal fluid (CSF) plasmablast (PB) VH transcriptome, CSF immunoglobulin (Ig) proteome, and serum Ig proteome. Recombinant antibodies were generated from paired CSF heavy- and light chains and tested for AQP4 reactivity. RESULTS: Approximately 9% of the CSF VH sequences aligned with PB memory B cells, DN B cells, and plasmablast VH sequences. AQP4-specific VH sequences were observed in each peripheral B-cell compartment. Lineage analysis of clonally related VH sequences indicates that CSF AQP4-specific B cells are closely related to an expanded population of DN B cells that may undergo antigen-specific B-cell maturation within the CNS. CSF and serum Ig proteomes overlapped with the VH sequences from each B-cell compartment; the majority of matches occurring between the PB VH sequences and serum Ig proteome. INTERPRETATION: During an acute NMOSD relapse, a dynamic exchange of B cells occurs between the periphery and CNS with AQP4-specific CSF B cells emerging from postgerminal center memory B cells and plasmablasts. Expansion of the PB DN B-cell compartment may be a potential biomarker of NMOSD activity.
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The initiation of DNA replication is typically tightly regulated by proteins that form initiation complexes at specific sequences known as replication origins. In Archaea and Eukaryotes, Cdc6, a near-universally conserved protein binds and facilitates the origin-dependent assembly of the replicative apparatus. TK1901 encodes Cdc6 in Thermococcus kodakarensis but, as we report here, TK1901 and the presumed origin of replication can be deleted from the genome of this hyperthermophilic Archaeon without any detectable effects on growth, genetic competence or the ability to support autonomous plasmid replication. All regions of the genome were equally represented in the sequences generated by whole genome sequencing of DNA isolated from T. kodakarensis strains with or without TK1901, inconsistent with DNA initiation occurring at one or few origins, and instead suggestive of replication initiating at many sites distributed throughout the genome. We were unable to generate strains lacking the recombination factors, RadA or RadB, consistent with T. kodakarensis cells, that are oligoploid (7-19 genomes per cell), employing a recombination-based mechanism of DNA replication. Deletion of the previously presumed origin region reduced the long-term viability of cultures supporting the possibility that retaining an origin-based mechanism of DNA initiation provides a survival mechanism for stationary phase cells with only one genome.
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PURPOSE: To determine the pharmacokinetics (PK), maximum tolerated dose (MTD), safety, and antitumor activity of an oral formulation of rigosertib, a dual phosphoinositide 3-kinase (PI3K) and polo-like kinase 1 (Plk1) pathway inhibitor, in patients with advanced solid malignancies. EXPERIMENTAL DESIGN: Patients with advanced solid malignancies received rigosertib twice daily continuously in 21-day cycles. Doses were escalated until intolerable grade ≥2 toxicities, at which point the previous dose level was expanded to define the MTD. All patients were assessed for safety, PK, and response. Urinary PK were performed at the MTD. Archival tumors were assessed for potential molecular biomarkers with multiplex mutation testing. A subset of squamous cell carcinomas (SCC) underwent exome sequencing. RESULTS: Forty-eight patients received a median of 2 cycles of therapy at 5 dose levels. Rigosertib exposure increased with escalating doses. Dose-limiting toxicities were hematuria and dysuria. The most common grade ≥2 drug-related toxicities involved urothelial irritation. The MTD is 560 mg twice daily. Activity was seen in head and neck SCCs (1 complete response, 1 partial response) and stable disease for ≥12 weeks was observed in 8 additional patients. Tumors experiencing ≥partial response had PI3K pathway activation, inactivated p53, and unique variants in ROBO3 and FAT1, two genes interacting with the Wnt/ß-catenin pathway. CONCLUSIONS: The recommended phase II dose of oral rigosertib is 560 mg twice daily given continuously. Urinary toxicity is the dose-limiting and most common toxicity. Alterations in PI3K, p53, and Wnt/ß-catenin pathway signaling should be investigated as potential biomarkers of response in future trials.
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Antineoplásicos/administração & dosagem , Glicina/análogos & derivados , Neoplasias/tratamento farmacológico , Sulfonas/administração & dosagem , Administração Oral , Adulto , Idoso , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Proteínas de Ciclo Celular/antagonistas & inibidores , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/efeitos adversos , Feminino , Glicina/administração & dosagem , Glicina/efeitos adversos , Glicina/farmacocinética , Humanos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Sulfonas/efeitos adversos , Sulfonas/farmacocinética , Adulto Jovem , Quinase 1 Polo-LikeRESUMO
The targeting of oncogenic 'driver' kinases with small molecule inhibitors has proven to be a highly effective therapeutic strategy in selected non-small cell lung cancer (NSCLC) patients. However, acquired resistance to targeted therapies invariably arises and is a major limitation to patient care. ROS1 fusion proteins are a recently described class of oncogenic driver, and NSCLC patients that express these fusions generally respond well to ROS1-targeted therapy. In this study, we sought to determine mechanisms of acquired resistance to ROS1 inhibition. To accomplish this, we analyzed tumor samples from a patient who initially responded to the ROS1 inhibitor crizotinib but eventually developed acquired resistance. In addition, we generated a ROS1 inhibition-resistant derivative of the initially sensitive NSCLC cell line HCC78. Previously described mechanisms of acquired resistance to tyrosine kinase inhibitors including target kinase-domain mutation, target copy number gain, epithelial-mesenchymal transition, and conversion to small cell lung cancer histology were found to not underlie resistance in the patient sample or resistant cell line. However, we did observe a switch in the control of growth and survival signaling pathways from ROS1 to EGFR in the resistant cell line. As a result of this switch, ROS1 inhibition-resistant HCC78 cells became sensitive to EGFR inhibition, an effect that was enhanced by co-treatment with a ROS1 inhibitor. Our results suggest that co-inhibition of ROS1 and EGFR may be an effective strategy to combat resistance to targeted therapy in some ROS1 fusion-positive NSCLC patients.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Transdução de Sinais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Crizotinibe , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/antagonistas & inibidores , Amplificação de Genes/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacosRESUMO
The dual pathway inhibitor rigosertib inhibits phosphoinositide 3-kinase (PI3K) pathway activation as well as polo-like kinase 1 (PLK1) activity across a broad spectrum of cancer cell lines. The importance of PIK3CA alterations in squamous cell carcinoma of the head and neck (HNSCC) has raised interest in exploring agents targeting PI3K, the product of PIK3CA. The genetic and molecular basis of rigosertib treatment response was investigated in a panel of 16 HNSCC cell lines, and direct patient tumor xenografts from eight patients with HNSCC [four HPV-serotype16 (HPV16)-positive]. HNSCC cell lines and xenografts were characterized by pathway enrichment gene expression analysis, exon sequencing, gene copy number, Western blotting, and immunohistochemistry (IHC). Rigosertib had potent antiproliferative effects on 11 of 16 HPV(-) HNSCC cell lines. Treatment sensitivity was confirmed in two cell lines using an orthotopic in vivo xenograft model. Growth reduction after rigosertib treatment was observed in three of eight HNSCC direct patient tumor lines. The responsive tumor lines carried a combination of a PI3KCA-activating event (amplification or mutation) and a p53-inactivating event (either HPV16- or mutation-mediated TP53 inactivation). In this study, we evaluated the in vitro and in vivo efficacy of rigosertib in both HPV(+) and HPV(-) HNSCCs, focusing on inhibition of the PI3K pathway. Although consistent inhibition of the PI3K pathway was not evident in HNSCC, we identified a combination of PI3K/TP53 events necessary, but not sufficient, for rigosertib sensitivity.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Glicina/análogos & derivados , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Sulfonas/administração & dosagem , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/virologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Classe I de Fosfatidilinositol 3-Quinases , Glicina/administração & dosagem , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Mutação , Papillomaviridae/efeitos dos fármacos , Infecções por Papillomavirus/tratamento farmacológico , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Quinase 1 Polo-LikeRESUMO
Targeted therapy development in head and neck squamous cell carcinoma (HNSCC) is challenging given the rarity of activating mutations. Additionally, HNSCC incidence is increasing related to human papillomavirus (HPV). We sought to develop an in vivo model derived from patients reflecting the evolving HNSCC epidemiologic landscape, and use it to identify new therapies. Primary and relapsed tumors from HNSCC patients, both HPV+ and HPV-, were implanted on mice, giving rise to 25 strains. Resulting xenografts were characterized by detecting key mutations, measuring protein expression by IHC and gene expression/pathway analysis by mRNA-sequencing. Drug efficacy studies were run with representative xenografts using the approved drug cetuximab as well as the new PI3K inhibitor PX-866. Tumors maintained their original morphology, genetic profiles and drug susceptibilities through serial passaging. The genetic makeup of these tumors was consistent with known frequencies of TP53, PI3KCA, NOTCH1 and NOTCH2 mutations. Because the EGFR inhibitor cetuximab is a standard HNSCC therapy, we tested its efficacy and observed a wide spectrum of efficacy. Cetuximab-resistant strains had higher PI3K/Akt pathway gene expression and protein activation than cetuximab-sensitive strains. The PI3K inhibitor PX-866 had anti-tumor efficacy in HNSCC models with PIK3CA alterations. Finally, PI3K inhibition was effective in two cases with NOTCH1 inactivating mutations. In summary, we have developed an HNSCC model covering its clinical spectrum whose major genetic alterations and susceptibility to anticancer agents represent contemporary HNSCC. This model enables to prospectively test therapeutic-oriented hypotheses leading to personalized medicine.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Alphapapillomavirus/patogenicidade , Animais , Western Blotting , Carcinoma de Células Escamosas/virologia , Cetuximab , Biologia Computacional , Receptores ErbB/genética , Receptores ErbB/metabolismo , Gonanos/farmacologia , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Imuno-Histoquímica , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Obese postmenopausal women have increased risk of breast cancers with poorer clinical outcomes than their lean counterparts. However, the mechanisms underlying these associations are poorly understood. Rodent model studies have recently identified a period of vulnerability for mammary cancer promotion, which emerges during weight gain after the loss of ovarian function (surgical ovariectomy; OVX). Thus, a period of transient weight gain may provide a life cycle-specific opportunity to prevent or treat postmenopausal breast cancer. We hypothesized that a combination of impaired metabolic regulation in obese animals prior to OVX plus an OVX-induced positive energy imbalance might cooperate to drive tumor growth and progression. To determine if lean and obese rodents differ in their metabolic response to OVX-induced weight gain, and whether this difference affects later mammary tumor metabolism, we performed a nutrient tracer study during the menopausal window of vulnerability. Lean animals preferentially deposited excess nutrients to mammary and peripheral tissues rather than to the adjacent tumors. Conversely, obese animals deposited excess nutrients into the tumors themselves. Notably, tumors from obese animals also displayed increased expression of the progesterone receptor (PR). Elevated PR expression positively correlated with tumor expression of glycolytic and lipogenic enzymes, glucose uptake, and proliferation markers. Treatment with the antidiabetic drug metformin during ovariectomy-induced weight gain caused tumor regression and downregulation of PR expression in tumors. Clinically, expression array analysis of breast tumors from postmenopausal women revealed that PR expression correlated with a similar pattern of metabolic upregulation, supporting the notion that PR+ tumors have enhanced metabolic capacity after menopause. Our findings have potential explanative power in understanding why obese, postmenopausal women display an increased risk of breast cancer.