RESUMEN
Outcomes for patients with recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) are poor, with median overall survival (OS) ranging from 6 to 18 months. For those who progress on standard-of-care (chemo)immunotherapy, treatment options are limited, necessitating the development of rational therapeutic strategies. Toward this end, we targeted the key HNSCC drivers PI3K-mTOR and HRAS via the combination of tipifarnib, a farnesyltransferase (FTase) inhibitor, and alpelisib, a PI3Kα inhibitor, in multiple molecularly defined subsets of HNSCC. Tipifarnib synergized with alpelisib at the level of mTOR in PI3Kα- or HRAS-dependent HNSCCs, leading to marked cytotoxicity in vitro and tumor regression in vivo. On the basis of these findings, the KURRENT-HN trial was launched to evaluate the effectiveness of this combination in PIK3CA-mutant/amplified and/or HRAS-overexpressing R/M HNSCC. Preliminary evidence supports the clinical activity of this molecular biomarker-driven combination therapy. Combined alpelisib and tipifarnib has potential to benefit >45% of patients with R/M HNSCC. By blocking feedback reactivation of mTORC1, tipifarnib may prevent adaptive resistance to additional targeted therapies, enhancing their clinical utility. SIGNIFICANCE: The mechanistically designed, biomarker-matched strategy of combining alpelisib and tipifarnib is efficacious in PIK3CA- and HRAS-dysregulated head and neck squamous carcinoma and could improve outcomes for many patients with recurrent, metastatic disease. See related commentary by Lee et al., p. 3162.
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Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Humanos , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Recurrencia Local de Neoplasia/tratamiento farmacológico , Serina-Treonina Quinasas TOR/metabolismo , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/genética , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Biomarcadores , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
PURPOSE: FGFR genomic alterations (amplification, mutations, and/or fusions) occur in â¼8% of gliomas, particularly FGFR1 and FGFR3. We conducted a multicenter open-label, single-arm, phase II study of a selective FGFR1-3 inhibitor, infigratinib (BGJ398), in patients with FGFR-altered recurrent gliomas. PATIENTS AND METHODS: Adults with recurrent/progressive gliomas harboring FGFR alterations received oral infigratinib 125 mg on days 1 to 21 of 28-day cycles. The primary endpoint was investigator-assessed 6-month progression-free survival (PFS) rate by Response Assessment in Neuro-Oncology criteria. Comprehensive genomic profiling was performed on available pretreatment archival tissue to explore additional molecular correlations with efficacy. RESULTS: Among 26 patients, the 6-month PFS rate was 16.0% [95% confidence interval (CI), 5.0-32.5], median PFS was 1.7 months (95% CI, 1.1-2.8), and objective response rate was 3.8%. However, 4 patients had durable disease control lasting longer than 1 year. Among these, 3 had tumors harboring activating point mutations at analogous positions of FGFR1 (K656E; n = 2) or FGFR3 (K650E; n = 1) in pretreatment tissue; an FGFR3-TACC3 fusion was detected in the other. Hyperphosphatemia was the most frequently reported treatment-related adverse event (all-grade, 76.9%; grade 3, 3.8%) and is a known on-target toxicity of FGFR inhibitors. CONCLUSIONS: FGFR inhibitor monotherapy with infigratinib had limited efficacy in a population of patients with recurrent gliomas and different FGFR genetic alterations, but durable disease control lasting more than 1 year was observed in patients with tumors harboring FGFR1 or FGFR3 point mutations or FGFR3-TACC3 fusions. A follow-up study with refined biomarker inclusion criteria and centralized FGFR testing is warranted.
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Glioma , Recurrencia Local de Neoplasia , Adulto , Estudios de Seguimiento , Glioma/tratamiento farmacológico , Glioma/genética , Humanos , Proteínas Asociadas a Microtúbulos , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Compuestos de Fenilurea , Inhibidores de Proteínas Quinasas/efectos adversos , Pirimidinas , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genéticaRESUMEN
BACKGROUND: Treatment options are sparse for patients with advanced cholangiocarcinoma after progression on first-line gemcitabine-based therapy. FGFR2 fusions or rearrangements occur in 10-16% of patients with intrahepatic cholangiocarcinoma. Infigratinib is a selective, ATP-competitive inhibitor of fibroblast growth factor receptors. We aimed to evaluate the antitumour activity of infigratinib in patients with locally advanced or metastatic cholangiocarcinoma, FGFR2 alterations, and previous gemcitabine-based treatment. METHODS: This multicentre, open-label, single-arm, phase 2 study recruited patients from 18 academic centres and hospitals in the USA, Belgium, Spain, Germany, Singapore, Taiwan, and Thailand. Eligible participants were aged 18 years or older, had histologically or cytologically confirmed, locally advanced or metastatic cholangiocarcinoma and FGFR2 fusions or rearrangements, and were previously treated with at least one gemcitabine-containing regimen. Patients received 125 mg of oral infigratinib once daily for 21 days of 28-day cycles until disease progression, intolerance, withdrawal of consent, or death. Radiological tumour evaluation was done at baseline and every 8 weeks until disease progression via CT or MRI of the chest, abdomen, and pelvis. The primary endpoint was objective response rate, defined as the proportion of patients with a best overall response of a confirmed complete or partial response, as assessed by blinded independent central review (BICR) according to Response Evaluation Criteria in Solid Tumors, version 1.1. The primary outcome and safety were analysed in the full analysis set, which comprised all patients who received at least one dose of infigratinib. This trial is registered with ClinicalTrials.gov, NCT02150967, and is ongoing. FINDINGS: Between June 23, 2014, and March 31, 2020, 122 patients were enrolled into our study, of whom 108 with FGFR2 fusions or rearrangements received at least one dose of infigratinib and comprised the full analysis set. After a median follow-up of 10·6 months (IQR 6·2-15·6), the BICR-assessed objective response rate was 23·1% (95% CI 15·6-32·2; 25 of 108 patients), with one confirmed complete response in a patient who only had non-target lesions identified at baseline and 24 partial responses. The most common treatment-emergent adverse events of any grade were hyperphosphataemia (n=83), stomatitis (n=59), fatigue (n=43), and alopecia (n=41). The most common ocular toxicity was dry eyes (n=37). Central serous retinopathy-like and retinal pigment epithelial detachment-like events occurred in 18 (17%) patients, of which ten (9%) were grade 1, seven (6%) were grade 2, and one (1%) was grade 3. There were no treatment-related deaths. INTERPRETATION: Infigratinib has promising clinical activity and a manageable adverse event profile in previously treated patients with locally advanced or metastatic cholangiocarcinoma harbouring FGFR2 gene fusions or rearrangements, and so represents a potential new therapeutic option in this setting. FUNDING: QED Therapeutics and Novartis.
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Colangiocarcinoma/tratamiento farmacológico , Colangiocarcinoma/genética , Metástasis de la Neoplasia/tratamiento farmacológico , Compuestos de Fenilurea/uso terapéutico , Pirimidinas/uso terapéutico , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Administración Oral , Adulto , Anciano , Anciano de 80 o más Años , Alopecia/inducido químicamente , Alopecia/epidemiología , Coriorretinopatía Serosa Central/inducido químicamente , Coriorretinopatía Serosa Central/epidemiología , Colangiocarcinoma/secundario , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapéutico , Progresión de la Enfermedad , Síndromes de Ojo Seco/inducido químicamente , Síndromes de Ojo Seco/epidemiología , Fatiga/inducido químicamente , Fatiga/epidemiología , Femenino , Humanos , Hiperfosfatemia/inducido químicamente , Hiperfosfatemia/epidemiología , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia/patología , Compuestos de Fenilurea/administración & dosificación , Compuestos de Fenilurea/efectos adversos , Pirimidinas/administración & dosificación , Pirimidinas/efectos adversos , Fármacos Sensibilizantes a Radiaciones/administración & dosificación , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Desprendimiento de Retina/inducido químicamente , Desprendimiento de Retina/epidemiología , Seguridad , Estomatitis/inducido químicamente , Estomatitis/epidemiología , Resultado del Tratamiento , GemcitabinaRESUMEN
BACKGROUND: Although recent advances in immunotherapy have transformed the treatment landscape for many anatomically defined cancers, these therapies are currently not approved for patients diagnosed with cancer of unknown primary (CUP). Molecular cancer classification using gene expression profiling (GEP) assays has the potential to identify tumor type and putative primary cancers and thereby may allow consideration of immune checkpoint inhibitor (ICI) therapy options for a subset of patients with CUP. Herein, we evaluated and characterized the ability of a 92-gene assay (CancerTYPE ID) to provide a molecular diagnosis and identify putative tumor types that are known to be sensitive to ICI therapies in patients with CUP or uncertain diagnosis. FINDINGS: A total of 24,426 cases from a large-scale research database of 92-gene assay clinical cases were classified, of which 9,350 (38%) were predicted to have an ICI-eligible tumor type. All ICIs with approved indications as of March 2020 were included in the analysis. Non-small cell lung cancer (NSCLC) was the most frequent molecular diagnosis and accounted for 33% of the ICI-eligible tumor types identified and 13% of the overall reportable results. In addition to NSCLC, the assay also frequently identified urothelial carcinomas, gastric cancer, and head and neck squamous cell carcinoma. The distributions of identified tumor types with indications for ICI therapy were similar across age and gender. CONCLUSIONS: Results suggest that molecular profiling with the 92-gene assay identifies a subset of ICI-eligible putative primary cancers in patients with CUP. We propose a treatment strategy based on available tests, including clinicopathologic features, GEP, and ICI biomarkers of response.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Neoplasias Primarias Desconocidas , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Perfilación de la Expresión Génica , Humanos , Inmunoterapia , Neoplasias Primarias Desconocidas/tratamiento farmacológico , Neoplasias Primarias Desconocidas/genéticaRESUMEN
BACKGROUND: Infigratinib (BGJ398) is a potent and selective fibroblast grown factor receptor 1 to 3 (FGFR1-3) inhibitor with significant activity in patients with advanced or metastatic urothelial carcinoma bearing FGFR3 alterations. Given the distinct biologic characteristics of upper tract urothelial carcinoma (UTUC) and urothelial carcinoma of the bladder (UCB), the authors examined whether infigratinib had varying activity in these settings. METHODS: Eligible patients had metastatic urothelial carcinoma with activating FGFR3 mutations and/or fusions. Comprehensive genomic profiling was performed on formalin-fixed, paraffin-embedded tissues. Blood was collected for cell-free DNA analysis using a 600-gene panel. Patients received infigratinib at a dose of 125 mg orally daily (3 weeks on/1 week off) until disease progression or intolerable toxicity occurred. The overall response rate (ORR; partial response [PR] plus complete response [CR]) and disease control rate (DCR; CR plus PR plus stable disease [SD]) were characterized. RESULTS: A total of 67 patients were enrolled; the majority (70.1%) had received ≥2 prior antineoplastic therapies. In 8 patients with UTUC, 1 CR and 3 PRs were observed (ORR, 50%); the remaining patients achieved a best response of SD (DCR, 100%). In patients with UCB, 13 PRs were observed (ORR, 22%), and 22 patients had a best response of SD (DCR, 59.3%). Notable differences in genomic alterations between patients with UTUC and those with UCB included higher frequencies of FGFR3-TACC3 fusions (12.5% vs 6.8%) and FGFR3 R248C mutations (50% vs 11.9%), and a lower frequency of FGFR3 S249C mutations (37.5% vs 59.3%). CONCLUSIONS: Differences in the cumulative genomic profile were observed between patients with UTUC and those with UCB in the current FGFR3-restricted experience, underscoring the distinct biology of these diseases. These results support a planned phase 3 adjuvant study predominantly performed in this population.
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Ácidos Nucleicos Libres de Células/análisis , Mutación , Compuestos de Fenilurea/uso terapéutico , Pirimidinas/uso terapéutico , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/mortalidadRESUMEN
BACKGROUND: Ampullary adenocarcinoma is a rare gastrointestinal cancer associated with diverse outcomes due to clinical and pathological heterogeneity. Standardized methods to better prognosticate and inform therapeutic selection for ampullary adenocarcinoma are needed. This study explored the novel use and potential prognostic utility of a 92-gene cancer classifier in ampullary adenocarcinomas. METHODS: In this prospectively-defined, blinded study of ampullary adenocarcinoma [N =54; stage T3 or higher (57 %); Grade III (44 %); Node positive (55 %)], the performance of a 92-gene classifier was examined to predict the ampullary subtype that was derived from histomorphological examination of resected ampullary samples. Outcome data for relapse-free survival (RFS) and overall survival (OS) were plotted to compare the prognostic utility of histological subtyping, histomolecular phenotyping, and the 92-gene classifier. Multivariate analysis was used to determine clinicopathological variables that were independently associated with overall survival. RESULTS: The 92-gene classifier demonstrated sensitivities and specificities of 85 % [95 % CI, 66-94] and 68 % [95 % CI, 48-84] and 64 % [95 % CI, 46-79] and 88 % [95 % CI, 70-98] for the pancreaticobiliary and intestinal histological subtypes, respectively. For the 92-gene classifier, improved outcomes were observed for the intestine versus the pancreaticobiliary prediction (median OS 108.1 v 36.4 months; HR, 2.17; 95 % CI, 0.98 to 4.79; P = 0.05). Similar results were seen for ampullary adenocarcinoma stratification by histological subtype (P = 0.04) and histomolecular phenotype (P = 0.02). Within poorly differentiated ampullary adenocarcinomas only the 92-gene classifier demonstrated statistically significant differences in RFS and OS (P < 0.05). CONCLUSIONS: Prognostic stratification of ampullary adenocarcinoma was similar for the 92-gene classifier, histological subtype, and histomolecular phenotype. The 92-gene classifier provides an unbiased standardized molecular-based approach to stratify ampullary tumors.
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Adenocarcinoma/genética , Adenocarcinoma/mortalidad , Ampolla Hepatopancreática/patología , Neoplasias del Conducto Colédoco/genética , Neoplasias del Conducto Colédoco/mortalidad , Adenocarcinoma/diagnóstico , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Neoplasias del Conducto Colédoco/diagnóstico , Neoplasias del Conducto Colédoco/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Estudios ProspectivosRESUMEN
Antisense oligonucleotides (ASOs) are known to trigger mRNA degradation in the nucleus via an RNase H-dependent mechanism. We have now identified a putative cytoplasmic mechanism through which ASO gapmers silence their targets when transfected or delivered gymnotically (i.e. in the absence of any transfection reagent). We have shown that the ASO gapmers can interact with the Ago-2 PAZ domain and can localize into GW-182 mRNA-degradation bodies (GW-bodies). The degradation products of the targeted mRNA, however, are not generated by Ago-2-directed cleavage. The apparent identification of a cytoplasmic pathway complements the previously known nuclear activity of ASOs and concurrently suggests that nuclear localization is not an absolute requirement for gene silencing.
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Citoplasma/metabolismo , Silenciador del Gen , Oligonucleótidos Antisentido , Proteínas Argonautas/metabolismo , Línea Celular , Citoplasma/química , Técnicas de Transferencia de Gen , Oligonucleótidos Antisentido/análisis , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño , TransfecciónRESUMEN
Antisense oligodeoxyribonucleotides have been used for decades to achieve sequence-specific silencing of gene expression. However, all early generation oligonucleotides (e.g., those with no other modifications than the phosphorothioate backbone) are inactive in vitro unless administered using a delivery vehicle. These delivery vehicles are usually lipidic but can also be polyamines or some other particulate reagent. We have found that by employing locked nucleic acid (LNA) phosphorothioate gap-mer nucleic acids of 16 mer or less in length, and by carefully controlling the plating conditions of the target cells and duration of the experiment, sequence-specific gene silencing can be achieved at low micromolar concentrations in vitro in the absence of any delivery vehicle. This process of naked oligonucleotide delivery to achieve gene silencing in vivo, which we have termed gymnosis, has been observed in many both adherent and nonadherent cell lines against several different targets genes.
Asunto(s)
Técnicas de Silenciamiento del Gen/métodos , Silenciador del Gen , Oligodesoxirribonucleótidos Antisentido/genética , Secuencia de Bases , Western Blotting , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Regulación hacia Abajo , Humanos , Oligonucleótidos/genética , Proteínas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección/métodosRESUMEN
TOK-001 and abiraterone are potent 17-heteroarylsteroid (17-HAS) inhibitors of Cyp17, one of the rate-limiting enzymes in the biosynthesis of testosterone from cholesterol in prostate cancer cells. Nevertheless, the molecular mechanism underlying the prevention of prostate cell growth by 17-HASs still remains elusive. Here, we assess the effects of 17-HASs on androgen receptor (AR) activity in LNCaP and LAPC-4 cells. We demonstrate that both TOK-001 and abiraterone reduced AR protein and mRNA expression, and antagonized AR-dependent promoter activation induced by androgen. TOK-001, but not abiraterone, is an effective apparent competitor of the radioligand [(3)H]R1881 for binding to the wild type and various mutant AR (W741C, W741L) proteins. In agreement with these data, TOK-001 is a consistently superior inhibitor than abiraterone of R1881-induced transcriptional activity of both wild type and mutant AR. However, neither agent was able to trans-activate the AR in the absence of R1881. Our data demonstrate that phospho-4EBP1 levels are significantly reduced by TOK-001 and to a lesser extent by abiraterone alcohol, and suggest a mechanism by which cap-dependent translation is suppressed by blocking assembly of the eIF4F and eIF4G complex to the mRNA 5' cap. Thus, the effects of these 17-HASs on AR signaling are complex, ranging from a decrease in testosterone production through the inhibition of Cyp17 as previously described, to directly reducing both AR protein expression and R1881-induced AR trans-activation.
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Androstadienos/farmacología , Androstenoles/farmacología , Bencimidazoles/farmacología , Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Esteroide 17-alfa-Hidroxilasa/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sustitución de Aminoácidos , Androstenos , Proteínas de Ciclo Celular , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , Mutación Missense , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Unión Proteica , Receptores Androgénicos/genética , Esteroide 17-alfa-Hidroxilasa/genética , Esteroide 17-alfa-Hidroxilasa/metabolismoRESUMEN
For the past 15-20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called 'gymnosis') that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.
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Silenciador del Gen , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos/administración & dosificación , Animales , Línea Celular Tumoral , Humanos , Indicadores y Reactivos , Ratones , Oligonucleótidos/análisis , Oligonucleótidos Antisentido/análisis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , TransfecciónRESUMEN
Efficient delivery of small interfering (si)RNA to specific cell populations in vivo remains a formidable challenge to its successful therapeutic application. We show that siRNA synthetically linked to a CpG oligonucleotide agonist of toll-like receptor (TLR)9 targets and silences genes in TLR9(+) myeloid cells and B cells, both of which are key components of the tumor microenvironment. When a CpG-conjugated siRNA that targets the immune suppressor gene Stat3 is injected in mice either locally at the tumor site or intravenously, it enters tumor-associated dendritic cells, macrophages and B cells. Silencing of Stat3 leads to activation of tumor-associated immune cells and ultimately to potent antitumor immune responses. Our findings demonstrate the potential of TLR agonist-siRNA conjugates for targeted gene silencing coupled with TLR stimulation and immune activation in the tumor microenvironment.
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Técnicas de Transferencia de Gen , Oligodesoxirribonucleótidos/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , Factor de Transcripción STAT3/genética , Receptor Toll-Like 9/agonistas , Análisis de Varianza , Animales , Linfocitos B/fisiología , Línea Celular Tumoral , Quimiocinas/metabolismo , ADN Recombinante/genética , Células Dendríticas/fisiología , Citometría de Flujo , Silenciador del Gen , Inmunidad Innata/efectos de los fármacos , Macrófagos/fisiología , Ratones , Células Mieloides/fisiología , Neoplasias/genética , Neoplasias/inmunología , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/farmacocinética , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacocinéticaRESUMEN
In humans a single species of the RNAseIII enzyme Dicer processes both microRNA precursors into miRNAs and long double-stranded RNAs into small interfering RNAs (siRNAs). An interesting but poorly understood domain of the mammalian Dicer protein is the N-terminal helicase-like domain that possesses a signature DExH motif. Cummins et al. created a human Dicer mutant cell line by inserting an AAV targeting cassette into the helicase domain of both Dicer alleles in HCT116 cells generating an in-frame 43-amino-acid insertion immediately adjacent to the DExH box. This insertion creates a Dicer mutant protein with defects in the processing of most, but not all, endogenous pre-miRNAs into mature miRNA. Using both biochemical and computational approaches, we provide evidence that the Dicer helicase mutant is sensitive to the thermodynamic properties of the stems in microRNAs and short-hairpin RNAs, with thermodynamically unstable stems resulting in poor processing and a reduction in the levels of functional mi/siRNAs. Paradoxically, this mutant exhibits enhanced processing efficiency and concomitant RNA interference when thermodynamically stable, long-hairpin RNAs are used. These results suggest an important function for the Dicer helicase domain in the processing of thermodynamically unstable hairpin structures.
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MicroARNs/química , MicroARNs/metabolismo , ARN Helicasas/química , ARN no Traducido/química , ARN no Traducido/metabolismo , Ribonucleasa III/química , Línea Celular , Humanos , Mutación , Estructura Terciaria de Proteína , ARN Helicasas/genética , ARN Helicasas/metabolismo , Precursores del ARN/química , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , TermodinámicaRESUMEN
Dicer-substrate small interfering RNAs (DsiRNAs) are synthetic RNA duplexes that are processed by Dicer into 21-mer species and show improved potency as triggers of RNA interference, particularly when used at low dose. Chemical modification patterns that are compatible with high potency 21-mer small interfering RNAs have been reported by several groups. However, modification patterns have not been studied for Dicer-substrate duplexes. We therefore synthesized a series of chemically modified 27-mer DsiRNAs and correlated modification patterns with functional potency. Some modification patterns profoundly reduced function although other patterns maintained high potency. Effects of sequence context were observed, where the relative potency of modification patterns varied between sites. A modification pattern involving alternating 2'-O-methyl RNA bases was developed that generally retains high potency when tested in different sites in different genes, evades activation of the innate immune system, and improves stability in serum.
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ARN Helicasas DEAD-box/metabolismo , Endorribonucleasas/metabolismo , ARN Interferente Pequeño/genética , Secuencia de Bases , Células Cultivadas , ARN Helicasas DEAD-box/análisis , Endorribonucleasas/análisis , Genes Reporteros , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Células HCT116 , Células HeLa , Humanos , Interferón-alfa/análisis , Interferón-alfa/metabolismo , Cinética , Leucocitos Mononucleares/metabolismo , Luciferasas de Renilla/metabolismo , Datos de Secuencia Molecular , Plásmidos , ARN/genética , Interferencia de ARN , ARN Bicatenario/genética , ARN Interferente Pequeño/síntesis química , ARN Interferente Pequeño/química , Estándares de Referencia , Ribonucleasa III , Sensibilidad y Especificidad , Especificidad por Sustrato , TransfecciónRESUMEN
MicroRNAs (miRNAs) are 21-24 nucleotide (nt) duplex RNAs that are created from precursor transcripts by subsequent processing steps mediated by members of the RNAseIII family, Drosha and Dicer. One of the two strands is incorporated into the active sites of the Argonaute family of proteins, where it serves as a guide for Watson-Crick base pairing with complementary sequences in target messenger RNAs (mRNAs). In mammals, the majority of miRNAs guide the RNA-induced silencing complex (RISC) to the 3' untranslated regions (UTRs) of mRNA targets, with the consequence that translation of the target mRNAs is inhibited. The importance of miRNAs in normal cellular development and metabolism is only now being realized. miRNA deficiencies or excesses have been correlated with a number of clinically important diseases ranging from myocardial infarction to cancers. The loss or gain of miRNA function can be caused by a single point mutation in either the miRNA or its target or by epigenetic silencing of primary miRNA transcription units. This review summarizes miRNA biogenesis and biology, explores the potential roles miRNAs can play in a variety of diseases, and suggests some therapeutic applications for restoring or inhibiting miRNA function.
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MicroARNs/fisiología , Humanos , MicroARNs/genética , Interferencia de ARN , ARN Mensajero/genéticaRESUMEN
Long interspersed elements (LINE-1 or L1) are the most active transposable elements in the human genome. Due to their high copy number and ability to sponsor retrotransposition of nonautonomous RNA sequences, unchecked L1 activity can negatively impact the genome by a number of means. Substantial evidence in lower eukaryotes demonstrates that the RNA interference (RNAi) machinery plays a major role in containing transposon activity. Despite extensive analysis in other eukaryotes, no experimental evidence has been presented that L1-derived siRNAs exist, or that the RNAi plays a significant role in restricting L1 activity in the human genome. This review will present evidence showing a direct role for RNAi in suppressing the movement of transposable elements in other eukaryotes, as well as speculate on the role RNAi might play in protecting the human genome from LINE-1 activity.
RESUMEN
Whether long interspersed element-1 (L1 or LINE-1) retrotransposition can occur in quiescent, nondividing, and/or terminally differentiated somatic cells has remained an unanswered fundamental question in human genetics. Here, we used a ubiquitously active phosphoglycerate kinase-1 promoter to drive the expression of a highly active human L1 element from an adenovirus-L1 hybrid vector. This vector system achieved retrotransposition in up to 91% of actively growing immortalized cells, and we demonstrated that L1 retrotransposition can be suppressed by the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine. This adenovirus vector enabled efficient delivery of the L1 element into differentiated primary human somatic cells and G1/S-arrested cells, resulting in retrotransposition in both cases; however, it was not detected in G0-arrested cells. Thus, these data indicate that L1 retrotransposition can occur in nondividing somatic cells.
Asunto(s)
Retroelementos , Adenoviridae/genética , Secuencia de Bases , Ciclo Celular , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Vectores Genéticos , Humanos , Elementos de Nucleótido Esparcido Largo , Modelos Genéticos , Datos de Secuencia Molecular , Fosfoglicerato Quinasa/metabolismo , TransfecciónRESUMEN
Long interspersed nuclear elements (LINE-1 or L1) comprise 17% of the human genome, although only 80-100 L1s are considered retrotransposition-competent (RC-L1). Despite their small number, RC-L1s are still potential hazards to genome integrity through insertional mutagenesis, unequal recombination and chromosome rearrangements. In this study, we provide several lines of evidence that the LINE-1 retrotransposon is susceptible to RNA interference (RNAi). First, double-stranded RNA (dsRNA) generated in vitro from an L1 template is converted into functional short interfering RNA (siRNA) by DICER, the RNase III enzyme that initiates RNAi in human cells. Second, pooled siRNA from in vitro cleavage of L1 dsRNA, as well as synthetic L1 siRNA, targeting the 5'-UTR leads to sequence-specific mRNA degradation of an L1 fusion transcript. Finally, both synthetic and pooled siRNA suppressed retrotransposition from a highly active RC-L1 clone in cell culture assay. Our report is the first to demonstrate that a human transposable element is subjected to RNAi.
Asunto(s)
Elementos de Nucleótido Esparcido Largo , Interferencia de ARN , Regiones no Traducidas 5'/metabolismo , Línea Celular , Genes Reporteros , Células HeLa , Humanos , Luciferasas/análisis , Luciferasas/genética , ARN Bicatenario/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Ribonucleasa III/metabolismoRESUMEN
The crystal structure based model of the catalytic center of Ago2 revealed that the siRNA and the mRNA must be able to form an A-helix for correct positing of the scissile phosphate bond for cleavage in RNAi. This suggests that base pairing of the target mRNA with itself, i.e. secondary structure, must be removed before cleavage. Early on in the siRNA design, GC-rich target sites were avoided because of their potential to be involved in strong secondary structure. It is still unclear how important a factor mRNA secondary structure is in RNAi. However, it has been established that a difference in the thermostability of the ends of an siRNA duplex dictate which strand is loaded into the RNA-induced silencing complex. Here, we use a novel secondary structure prediction method and duplex-end differential calculations to investigate the importance of a secondary structure in the siRNA design. We found that the differential duplex-end stabilities alone account for functional prediction of 60% of the 80 siRNA sites examined, and that secondary structure predictions improve the prediction of site efficacy. A total of 80% of the non-functional sites can be eliminated using secondary structure predictions and duplex-end differential.