RESUMEN
Somatic mutations within non-coding regions and even exons may have unidentified regulatory consequences that are often overlooked in analysis workflows. Here we present RegTools ( www.regtools.org ), a computationally efficient, free, and open-source software package designed to integrate somatic variants from genomic data with splice junctions from bulk or single cell transcriptomic data to identify variants that may cause aberrant splicing. We apply RegTools to over 9000 tumor samples with both tumor DNA and RNA sequence data. RegTools discovers 235,778 events where a splice-associated variant significantly increases the splicing of a particular junction, across 158,200 unique variants and 131,212 unique junctions. To characterize these somatic variants and their associated splice isoforms, we annotate them with the Variant Effect Predictor, SpliceAI, and Genotype-Tissue Expression junction counts and compare our results to other tools that integrate genomic and transcriptomic data. While many events are corroborated by the aforementioned tools, the flexibility of RegTools also allows us to identify splice-associated variants in known cancer drivers, such as TP53, CDKN2A, and B2M, and other genes.
Asunto(s)
Neoplasias , Transcriptoma , Humanos , Transcriptoma/genética , Genómica , Empalme del ARN/genética , Genoma , Neoplasias/genética , Empalme Alternativo/genéticaRESUMEN
PURPOSE: Approximately 10%-40% of patients with lung cancer report no history of tobacco smoking (never-smokers). We analyzed whole-exome and RNA-sequencing data of 160 tumor and normal lung adenocarcinoma (LUAD) samples from never-smokers to identify clinically actionable alterations and gain insight into the environmental and hereditary risk factors for LUAD among never-smokers. METHODS: We performed whole-exome and RNA-sequencing of 88 and 69 never-smoker LUADs. We analyzed these data in conjunction with data from 76 never-smoker and 299 smoker LUAD samples sequenced by The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium. RESULTS: We observed a high prevalence of clinically actionable driver alterations in never-smoker LUADs compared with smoker LUADs (78%-92% v 49.5%; P < .0001). Although a subset of never-smoker samples demonstrated germline alterations in DNA repair genes, the frequency of samples showing germline variants in cancer predisposing genes was comparable between smokers and never-smokers (6.4% v 6.9%; P = .82). A subset of never-smoker samples (5.9%) showed mutation signatures that were suggestive of passive exposure to cigarette smoke. Finally, analysis of RNA-sequencing data showed distinct immune transcriptional subtypes of never-smoker LUADs that varied in their expression of clinically relevant immune checkpoint molecules and immune cell composition. CONCLUSION: In this comprehensive genomic and transcriptome analysis of never-smoker LUADs, we observed a potential role for germline variants in DNA repair genes and passive exposure to cigarette smoke in the pathogenesis of a subset of never-smoker LUADs. Our findings also show that clinically actionable driver alterations are highly prevalent in never-smoker LUADs, highlighting the need for obtaining biopsies with adequate cellularity for clinical genomic testing in these patients.
Asunto(s)
Adenocarcinoma del Pulmón/patología , Biomarcadores de Tumor/genética , Secuenciación del Exoma/métodos , Neoplasias Pulmonares/patología , Mutación , Fumar/tendencias , Adenocarcinoma del Pulmón/epidemiología , Adenocarcinoma del Pulmón/genética , Anciano , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Pulmonares/epidemiología , Neoplasias Pulmonares/genética , Masculino , Pronóstico , Estados Unidos/epidemiologíaRESUMEN
Sex can be an important determinant of cancer phenotype, and exploring sex-biased tumor biology holds promise for identifying novel therapeutic targets and new approaches to cancer treatment. In an established isogenic murine model of glioblastoma (GBM), we discovered correlated transcriptome-wide sex differences in gene expression, H3K27ac marks, large Brd4-bound enhancer usage, and Brd4 localization to Myc and p53 genomic binding sites. These sex-biased gene expression patterns were also evident in human glioblastoma stem cells (GSCs). These observations led us to hypothesize that Brd4-bound enhancers might underlie sex differences in stem cell function and tumorigenicity in GBM. We found that male and female GBM cells exhibited sex-specific responses to pharmacological or genetic inhibition of Brd4. Brd4 knockdown or pharmacologic inhibition decreased male GBM cell clonogenicity and in vivo tumorigenesis while increasing both in female GBM cells. These results were validated in male and female patient-derived GBM cell lines. Furthermore, analysis of the Cancer Therapeutic Response Portal of human GBM samples segregated by sex revealed that male GBM cells are significantly more sensitive to BET (bromodomain and extraterminal) inhibitors than are female cells. Thus, Brd4 activity is revealed to drive sex differences in stem cell and tumorigenic phenotypes, which can be abrogated by sex-specific responses to BET inhibition. This has important implications for the clinical evaluation and use of BET inhibitors.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Glioblastoma/metabolismo , Proteínas Nucleares/metabolismo , Factores Sexuales , Factores de Transcripción/metabolismo , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/genética , Glioblastoma/genética , Histonas/metabolismo , Humanos , Masculino , Ratones , Proteínas Nucleares/fisiología , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Caracteres Sexuales , Factores de Transcripción/fisiología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
PURPOSE: Patients with SCLC rarely undergo biopsies at relapse. When pursued, tissue obtained can be inadequate for molecular testing, posing a challenge in identifying potentially targetable alterations in a clinically meaningful time frame. We examined the feasibility of circulating tumor DNA (ctDNA) testing in identifying potentially targetable alterations in SCLC. EXPERIMENTAL DESIGN: ctDNA test results were prospectively collected from patients with SCLC between 2014 and 2017 and analyzed. ctDNA profiles of SCLC at diagnosis and relapse were also compared. RESULTS: A total of 609 samples collected from 564 patients between 2014 and 2017 were analyzed. The median turnaround time for test results was 14 days. Among patients with data on treatment status, there were 61 samples from 59 patients and 219 samples from 206 patients collected at diagnosis and relapse, respectively. The number of mutations or amplifications detected per sample did not differ by treatment status. Potentially targetable alterations in DNA repair, MAPK and PI3K pathways, and genes such as MYC and ARID1A were identifiable through ctDNA testing. Furthermore, our results support that it may be possible to reconstruct the clonal relationship between detected variants through ctDNA testing. CONCLUSIONS: Patients with relapsed SCLC rarely undergo biopsies for molecular testing and often require prompt treatment initiation. ctDNA testing is less invasive and capable of identifying alterations in relapsed disease in a clinically meaningful timeframe. ctDNA testing on an expanded gene panel has the potential to advance our knowledge of the mechanisms underlying treatment resistance in SCLC and aid in the development of novel treatment strategies.
Asunto(s)
Biomarcadores de Tumor/genética , ADN Tumoral Circulante/genética , Neoplasias Pulmonares/genética , Recurrencia Local de Neoplasia/genética , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/sangre , ADN Tumoral Circulante/sangre , Reparación del ADN/genética , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Estudios de Factibilidad , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Biopsia Líquida/métodos , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Mutación , Recurrencia Local de Neoplasia/sangre , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/patología , Estudios Prospectivos , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/genética , Carcinoma Pulmonar de Células Pequeñas/sangre , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/patología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Adulto JovenRESUMEN
Molecular and behavioral responses to opioids are thought to be primarily mediated by neurons, although there is accumulating evidence that other cell types play a prominent role in drug addiction. To investigate cell-type-specific opioid responses, we performed single-cell RNA sequencing (scRNA-seq) of the nucleus accumbens of mice following acute morphine treatment. Differential expression analysis uncovered unique morphine-dependent transcriptional responses by oligodendrocytes and astrocytes. We examined the expression of selected genes, including Cdkn1a and Sgk1, by FISH, confirming their induction by morphine in oligodendrocytes. Further analysis using RNA-seq of FACS-purified oligodendrocytes revealed a large cohort of morphine-regulated genes. The affected genes are enriched for roles in cellular pathways intimately linked to oligodendrocyte maturation and myelination, including the unfolded protein response. Altogether, our data illuminate the morphine-dependent transcriptional response by oligodendrocytes and offer mechanistic insights into myelination defects associated with opioid abuse.
Asunto(s)
Analgésicos Opioides/farmacología , Morfina/farmacología , Neuroglía/efectos de los fármacos , Transcriptoma , Animales , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Neuroglía/metabolismo , Núcleo Accumbens/citología , Núcleo Accumbens/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de la Célula IndividualRESUMEN
The piggyBac (PB) transposon has been used in a number of biological applications. The insertion of PB transposons into the genome can disrupt genes or regulatory regions, impacting cellular function, so for many experiments it is important that PB transposition is tightly controlled. Here, we systematically characterize three methods for the post-translational control of the PB transposon in four cell lines. We investigated fusions of the PB transposase with ERT2 and two degradation domains (FKBP-DD, DHFR-DD), in multiple orientations, and determined (i) the fold-induction achieved, (ii) the absolute transposition efficiency of the activated construct and (iii) the effects of two inducer molecules on cellular transcription and function. We found that the FKBP-DD confers the PB transposase with a higher transposition activity and better dynamic range than can be achieved with the other systems. In addition, we found that the FKBP-DD regulates transposon activity in a reversible and dose-dependent manner. Finally, we showed that Shld1, the chemical inducer of FKBP-DD, does not interfere with stem cell differentiation, whereas tamoxifen has significant effects. We believe the FKBP-based PB transposon induction will be useful for transposon-mediated genome engineering, insertional mutagenesis and the genome-wide mapping of transcription factor binding.
Asunto(s)
Elementos Transponibles de ADN , Transposasas/genética , Animales , Diferenciación Celular , Línea Celular , Células Cultivadas , Células HEK293 , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Ligandos , Ratones , Morfolinas/farmacología , Receptores de Estrógenos/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Unión a Tacrolimus/genética , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Tetrahidrofolato Deshidrogenasa/genética , Transcripción Genética , Transposasas/metabolismoRESUMEN
We formulate the thin-film hydrodynamics of a suspension of polar self-driven particles and show that it is prone to several instabilities through the interplay of activity, polarity, and the existence of a free surface. Our approach extends, to self-propelling systems, the work of Ben Amar and Cummings [Phys. Fluids 13 1160 (2001)10.1063/1.1359748] on thin-film nematics. Based on our estimates the instabilities should be seen in bacterial suspensions and the lamellipodium, and are potentially relevant to the morphology of biofilms. We suggest several experimental tests of our theory.
Asunto(s)
Modelos Biológicos , Ósmosis , ViscosidadRESUMEN
We solve a model for the equilibrium folding of DNA via the formation of randomly placed (nonspecific) loops. We find that the loop rearrangement entropy drives a significant reduction in loop size as more loops form. The reduction of the most probable loop size occurs over a wide range of forces and is enhanced by interloop cooperativity, indicating that it should be observable in single DNA experiments.
Asunto(s)
ADN/química , Modelos Químicos , Entropía , Conformación de Ácido NucleicoRESUMEN
We study the formation of loops along a DNA molecule under applied tension, as might occur in single-DNA micromanipulation experiments with proteins which are able to simultaneously bind two DNA sites. We consider the case of "bare" DNA in the loop, which forms a "teardrop" shape, and the case where a single DNA-bending protein produces a "kink" in the middle of the loop; the presence of a right-angle kink in the loop reduces its bending energy by a factor of 3. Using the bending energy plus an estimate of the free energies associated with fluctuations and the elasticity of the extended nonlooped DNA, we obtain a probability distribution for loops as a function of loop size and force. Force strongly suppresses formation of all loops, but suppresses large loops more severely than small ones. This quenching effect of force is reduced in the presence of a kink in the loop. We also calculate the speed at which length is absorbed into loops between arbitrary positions along the DNA (i.e., for non-sequence-specific loop forming proteins). The speed of retraction of the molecule decays as a stretched exponential function of the force with characteristic force scales depending on the geometry of the loops.
Asunto(s)
ADN/análisis , ADN/química , Micromanipulación/métodos , Modelos Químicos , Modelos Moleculares , Conformación de Ácido Nucleico , Estimulación Física/métodos , Simulación por Computador , Elasticidad , Estrés Mecánico , Resistencia a la TracciónRESUMEN
We model the stable self-organized patterns obtained in the nonequilibrium steady states of mixtures of molecular motors and microtubules. In experiments [Nédélec et al., Nature (London) 389, 305 (1997); Surrey et al., Science 292, 1167 (2001)] performed in a quasi-two-dimensional geometry, microtubules are oriented by complexes of motor proteins. This interaction yields a variety of patterns, including arrangements of asters, vortices, and disordered configurations. We model this system via a two-dimensional vector field describing the local coarse-grained microtubule orientation and two scalar density fields associated to molecular motors. These scalar fields describe motors which either attach to and move along microtubules or diffuse freely within the solvent. Transitions between single aster, spiral, and vortex states are obtained as a consequence of confinement, as parameters in our model are varied. For systems in which the effects of confinement can be neglected, we present a map of nonequilibrium steady states, which includes arrangements of asters and vortices separately as well as aster-vortex mixtures and fully disordered states. We calculate the steady state distribution of bound and free motors in aster and vortex configurations of microtubules and compare these to our simulation results, providing qualitative arguments for the stability of different patterns in various regimes of parameter space. We study the role of crowding or "saturation" effects on the density profiles of motors in asters, discussing the role of such effects in stabilizing single asters. We also comment on the implications of our results for experiments.
Asunto(s)
Cristalización/métodos , Microtúbulos/fisiología , Mitosis/fisiología , Modelos Biológicos , Modelos Químicos , Proteínas Motoras Moleculares/fisiología , Movimiento/fisiología , Animales , Mezclas Complejas/química , Simulación por Computador , Dimerización , Humanos , Microtúbulos/química , Proteínas Motoras Moleculares/químicaRESUMEN
We study the linearized hydrodynamics of a two-component fluid membrane near a repulsive wall, using a model that incorporates curvature-concentration coupling as well as hydrodynamic interactions. This model is a simplified version of a recently proposed one [J.-B. Manneville et al., Phys. Rev. E 64, 021908 (2001)] for nonequilibrium force centers embedded in fluid membranes, such as light-activated bacteriorhodopsin pumps incorporated in phospholipid egg phosphatidyl choline (EPC) bilayers. The pump-membrane system is modeled as an impermeable, two-component bilayer fluid membrane in the presence of an ambient solvent, in which one component, representing active pumps, is described in terms of force dipoles displaced with respect to the bilayer midpoint. We first discuss the case in which such pumps are rendered inactive, computing the mode structure in the bulk as well as the modification of hydrodynamic properties by the presence of a nearby wall. These results should apply, more generally, to equilibrium fluid membranes comprised of two components, in which the effects of curvature-concentration coupling are significant, above the threshold for phase separation. We then discuss the fluctuations and mode structure in the steady state of active two-component membranes near a repulsive wall. We find that proximity to the wall smoothens membrane height fluctuations in the stable regime, resulting in a logarithmic scaling of the roughness even for initially tensionless membranes. This explicitly nonequilibrium result is a consequence of the incorporation of curvature-concentration coupling in our hydrodynamic treatment. This result also indicates that earlier scaling arguments which obtained an increase in the roughness of active membranes near repulsive walls upon neglecting the role played by such couplings may need to be reevaluated.