RESUMEN
Drug-tolerance has emerged as one of the major non-genetic adaptive processes driving resistance to targeted therapy (TT) in non-small cell lung cancer (NSCLC). However, the kinetics and sequence of molecular events governing this adaptive response remain poorly understood. Here, we combine real-time monitoring of the cell-cycle dynamics and single-cell RNA sequencing in a broad panel of oncogenic addiction such as EGFR-, ALK-, BRAF- and KRAS-mutant NSCLC, treated with their corresponding TT. We identify a common path of drug adaptation, which invariably involves alveolar type 1 (AT1) differentiation and Rho-associated protein kinase (ROCK)-mediated cytoskeletal remodeling. We also isolate and characterize a rare population of early escapers, which represent the earliest resistance-initiating cells that emerge in the first hours of treatment from the AT1-like population. A phenotypic drug screen identify farnesyltransferase inhibitors (FTI) such as tipifarnib as the most effective drugs in preventing relapse to TT in vitro and in vivo in several models of oncogenic addiction, which is confirmed by genetic depletion of the farnesyltransferase. These findings pave the way for the development of treatments combining TT and FTI to effectively prevent tumor relapse in oncogene-addicted NSCLC patients.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Resistencia a Antineoplásicos , Farnesiltransferasa , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Humanos , Farnesiltransferasa/antagonistas & inhibidores , Farnesiltransferasa/metabolismo , Farnesiltransferasa/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Resistencia a Antineoplásicos/genética , Línea Celular Tumoral , Animales , Ratones , Dependencia del Oncogén/genética , Terapia Molecular Dirigida , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Femenino , Ensayos Antitumor por Modelo de Xenoinjerto , Oncogenes/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , QuinolonasRESUMEN
TDP1 removes transcription-blocking topoisomerase I cleavage complexes (TOP1ccs), and its inactivating H493R mutation causes the neurodegenerative syndrome SCAN1. However, the molecular mechanism underlying the SCAN1 phenotype is unclear. Here, we generate human SCAN1 cell models using CRISPR-Cas9 and show that they accumulate TOP1ccs along with changes in gene expression and genomic distribution of R-loops. SCAN1 cells also accumulate transcriptional DNA double-strand breaks (DSBs) specifically in the G1 cell population due to increased DSB formation and lack of repair, both resulting from abortive removal of transcription-blocking TOP1ccs. Deficient TDP1 activity causes increased DSB production, and the presence of mutated TDP1 protein hampers DSB repair by a TDP2-dependent backup pathway. This study provides powerful models to study TDP1 functions under physiological and pathological conditions and unravels that a gain of function of the mutated TDP1 protein, which prevents DSB repair, rather than a loss of TDP1 activity itself, could contribute to SCAN1 pathogenesis.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Mutación , Enfermedades Neurodegenerativas , Hidrolasas Diéster Fosfóricas , Humanos , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Mutación/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo I/genética , Transcripción Genética , Estructuras R-Loop , Sistemas CRISPR-Cas/genéticaRESUMEN
Small GTPases are highly regulated proteins that control essential signaling pathways through the activity of their effector proteins. Among the RHOA subfamily, RHOB regulates peculiar functions that could be associated with the control of the endocytic trafficking of signaling proteins. Here, we used an optimized assay based on tripartite split-GFP complementation to localize GTPase-effector complexes with high-resolution. The detection of RHOB interaction with the Rhotekin Rho binding domain (RBD) that specifically recognizes the active GTP-bound GTPase, is performed in vitro by the concomitant addition of recombinant GFP1-9 and a GFP nanobody. Analysis of RHOB-RBD complexes localization profiles combined with immunostaining and live cell imaging indicated a serum-dependent reorganization of the endosomal and membrane pool of active RHOB. We further applied this technology to the detection of RHO-effector complexes that highlighted their subcellular localization with high resolution among the different cellular compartments.
Asunto(s)
Transducción de Señal , Proteína de Unión al GTP rhoB , Proteína de Unión al GTP rhoB/genética , Proteína de Unión al GTP rhoB/química , Proteína de Unión al GTP rhoB/metabolismo , GTP Fosfohidrolasas/metabolismo , Membrana Celular/metabolismo , Guanosina Trifosfato/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Cell-free DNA in human blood plasma (cfDNA) is now widely used and studied as a biomarker for several physiological and pathological situations. In addition to genetic and epigenetic alterations that provide information about the presence and the nature of non-constitutive DNA in the body, cfDNA concentration and size distribution may potentially be independent biomarkers suitable for monitoring at-risk patients and therapy efficacy. Here, we describe a simple, in-line, method, which measures cfDNA concentration and size distribution from only a few microliters of plasma without the need to extract and/or concentrate the DNA prior to the analysis. This method is based on a dual hydrodynamic and electrokinetic actuation, adapted for samples containing salts and proteins such as biological fluids. The method provides analytical performances equivalent to those obtained after purification and concentration of cfDNA, with a precision of â¼1% for size features and of 10-20% for the concentrations of the different size fractions. We show that concentration and size distribution of cfDNA analyzed from plasma can differentiate advanced lung cancer patients from healthy controls. This simple and cost-effective method should facilitate further investigations into the potential clinical usefulness of cfDNA size profiling.
Asunto(s)
Ácidos Nucleicos Libres de Células , Neoplasias Pulmonares , Humanos , ADN , Biomarcadores de Tumor , Plasma/químicaRESUMEN
Macrophages are essential for HIV-1 pathogenesis and represent major viral reservoirs. Therefore, it is critical to understand macrophage infection, especially in tissue macrophages, which are widely infected in vivo, but poorly permissive to cell-free infection. Although cell-to-cell transmission of HIV-1 is a determinant mode of macrophage infection in vivo, how HIV-1 transfers toward macrophages remains elusive. Here, we demonstrate that fusion of infected CD4+ T lymphocytes with human macrophages leads to their efficient and productive infection. Importantly, several tissue macrophage populations undergo this heterotypic cell fusion, including synovial, placental, lung alveolar, and tonsil macrophages. We also find that this mode of infection is modulated by the macrophage polarization state. This fusion process engages a specific short-lived adhesion structure and is controlled by the CD81 tetraspanin, which activates RhoA/ROCK-dependent actomyosin contractility in macrophages. Our study provides important insights into the mechanisms underlying infection of tissue-resident macrophages, and establishment of persistent cellular reservoirs in patients.
Asunto(s)
Linfocitos T CD4-Positivos , Fusión Celular , Infecciones por VIH , Macrófagos , Humanos , Linfocitos T CD4-Positivos/metabolismo , Infecciones por VIH/metabolismo , VIH-1/patogenicidad , Macrófagos/metabolismo , Macrófagos/virología , Actomiosina/metabolismoRESUMEN
The protocadherin proteins are cell adhesion molecules at the crossroad of signaling pathways playing a major role in neuronal development. It is now understood that their role as signaling hubs is not only important for the normal physiology of cells but also for the regulation of hallmarks of cancerogenesis. Importantly, protocadherins form a cluster of genes that are regulated by DNA methylation. We have identified for the first time that PCDHB15 gene is DNA-hypermethylated on its unique exon in the metastatic melanoma-derived cell lines and patients' metastases compared to primary tumors. This DNA hypermethylation silences the gene, and treatment with the DNA demethylating agent 5-aza-2'-deoxycytidine reinduces its expression. We explored the role of PCDHB15 in melanoma aggressiveness and showed that overexpression impairs invasiveness and aggregation of metastatic melanoma cells in vitro and formation of lung metastasis in vivo. These findings highlight important modifications of the methylation of the PCDHß genes in melanoma and support a functional role of PCDHB15 silencing in melanoma aggressiveness.
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Neoplasias Pulmonares , Melanoma , Humanos , Metilación de ADN , Melanoma/genética , Transducción de Señal , Exones , Neoplasias Pulmonares/genéticaRESUMEN
Strategies based on intracellular expression of artificial binding domains present several advantages over manipulating nucleic acid expression or the use of small molecule inhibitors. Intracellularly-functional nanobodies can be considered as promising macrodrugs to study key signaling pathways by interfering with protein-protein interactions. With the aim of studying the RAS-related small GTPase RHOA family, we previously isolated, from a synthetic phage display library, nanobodies selective towards the GTP-bound conformation of RHOA subfamily proteins that lack selectivity between the highly conserved RHOA-like and RAC subfamilies of GTPases. To identify RHOA/ROCK pathway inhibitory intracellular nanobodies, we implemented a stringent, subtractive phage display selection towards RHOA-GTP followed by a phenotypic screen based on F-actin fiber loss. Intracellular interaction and intracellular selectivity between RHOA and RAC1 proteins was demonstrated by adapting the sensitive intracellular protein-protein interaction reporter based on the tripartite split-GFP method. This strategy led us to identify a functional intracellular nanobody, hereafter named RH28, that does not cross-react with the close RAC subfamily and blocks/disrupts the RHOA/ROCK signaling pathway in several cell lines without further engineering or functionalization. We confirmed these results by showing, using SPR assays, the high specificity of the RH28 nanobody towards the GTP-bound conformation of RHOA subfamily GTPases. In the metastatic melanoma cell line WM266-4, RH28 expression triggered an elongated cellular phenotype associated with a loss of cellular contraction properties, demonstrating the efficient intracellular blocking of RHOA/B/C proteins downstream interactions without the need of manipulating endogenous gene expression. This work paves the way for future therapeutic strategies based on protein-protein interaction disruption with intracellular antibodies.
Asunto(s)
Anticuerpos de Dominio Único , Actinas/metabolismo , Guanosina Trifosfato , Transducción de Señal , Anticuerpos de Dominio Único/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteínas ras/metabolismoRESUMEN
Aberrant DNA methylation is a well-known feature of tumours and has been associated with metastatic melanoma. However, since melanoma cells are highly heterogeneous, it has been challenging to use affected genes to predict tumour aggressiveness, metastatic evolution, and patients' outcomes. We hypothesized that common aggressive hypermethylation signatures should emerge early in tumorigenesis and should be shared in aggressive cells, independent of the physiological context under which this trait arises. We compared paired melanoma cell lines with the following properties: (i) each pair comprises one aggressive counterpart and its parental cell line and (ii) the aggressive cell lines were each obtained from different host and their environment (human, rat, and mouse), though starting from the same parent cell line. Next, we developed a multi-step genomic pipeline that combines the DNA methylome profile with a chromosome cluster-oriented analysis. A total of 229 differentially hypermethylated genes was commonly found in the aggressive cell lines. Genome localization analysis revealed hypermethylation peaks and clusters, identifying eight hypermethylated gene promoters for validation in tissues from melanoma patients. Five Cytosine-phosphate-Guanine (CpGs) identified in primary melanoma tissues were transformed into a DNA methylation score that can predict survival (log-rank test, p=0.0008). This strategy is potentially universally applicable to other diseases involving DNA methylation alterations.
Asunto(s)
Melanoma , Neoplasias Cutáneas , Animales , Cromosomas , Islas de CpG , Citosina , Metilación de ADN , Epigénesis Genética , Epigenoma , Regulación Neoplásica de la Expresión Génica , Guanina , Humanos , Melanoma/genética , Melanoma/patología , Ratones , Fosfatos , Ratas , Neoplasias Cutáneas/genética , Melanoma Cutáneo MalignoRESUMEN
Lung cancer is the leading cause of cancer-related deaths among men and women worldwide. Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are effective therapies for advanced non-small-cell lung cancer (NSCLC) patients harbouring EGFR-activating mutations, but are not curative due to the inevitable emergence of resistances. Recent in vitro studies suggest that resistance to EGFR-TKI may arise from a small population of drug-tolerant persister cells (DTP) through non-genetic reprogramming, by entering a reversible slow-to-non-proliferative state, before developing genetically derived resistances. Deciphering the molecular mechanisms governing the dynamics of the drug-tolerant state is therefore a priority to provide sustainable therapeutic solutions for patients. An increasing number of molecular mechanisms underlying DTP survival are being described, such as chromatin and epigenetic remodelling, the reactivation of anti-apoptotic/survival pathways, metabolic reprogramming, and interactions with their micro-environment. Here, we review and discuss the existing proposed mechanisms involved in the DTP state. We describe their biological features, molecular mechanisms of tolerance, and the therapeutic strategies that are tested to target the DTP.
RESUMEN
SIGNIFICANCE: We propose a technique devoted to real-time high-resolution imaging of skin microvascularization. AIM: The process utilizes the temporal variation of the spatially depolarized optical speckle field generated by moving red blood cells when illuminated with fully polarized coherent light. APPROACH: Polarimetric filtering prevents the contribution of surface scattering from reaching the camera and thus favors the detection of multiscattered photons from the deeper layers of the skin. RESULTS: Full-field images reveal the microvasculature with a spatial resolution of 80 µm. The acquisition speed allows for real-time applications. CONCLUSIONS: We demonstrate the ability of this method to determine in 1 s a stable and reliable microvascular activity, enabling numerous clinical applications that require quantitative measurements.
Asunto(s)
Diagnóstico por Imagen , Piel , Recuento de Eritrocitos , Eritrocitos , Piel/diagnóstico por imagenRESUMEN
As key regulators of the actin cytoskeleton, RHO GTPase expression and/or activity are deregulated in tumorigenesis and metastatic progression. Nevertheless, the vast majority of experiments supporting this conclusion was conducted on cell lines but not on human tumor samples that were mostly studied at the expression level only. Up to now, the activity of RHO proteins remains poorly investigated in human tumors. In this article, we present the development of a robust nanobody-based ELISA assay, with a high selectivity that allows an accurate quantification of RHO protein GTP-bound state in the nanomolar range (1 nM; 20 µg/L), not only in cell lines after treatment but also in tumor samples. Of note, we present here a fine analysis of RHOA-like and RAC1 active state in tumor samples with the most comprehensive study of RHOA-GTP and RHOC-GTP levels performed on human breast tumor samples. We revealed increased GTP-bound RHOA and RHOC protein activities in tumors compared to normal tissue counterparts, and demonstrated that the RHO active state and RHO expression are two independent parameters among different breast cancer subtypes. Our results further highlight the regulation of RHO protein activation in tumor samples and the relevance of directly studying RHO GTPase activities involvement in molecular pathways.
Asunto(s)
Neoplasias de la Mama , Proteína de Unión al GTP rhoA , Proteína rhoC de Unión a GTP , Transformación Celular Neoplásica , Femenino , Guanosina Trifosfato , Humanos , Proteína de Unión al GTP rhoA/metabolismo , Proteína rhoC de Unión a GTP/metabolismoRESUMEN
Here, we provide a protocol for the selection of conformation-specific intracellular antibody degraders using a cell-based screening method. We applied this protocol to select antibody-based degraders targeting the active form of the small GTPase RHOB (i.e., RHOB-GTP) using an engineered H2882 cell line. The protocol can be used to study the function of RHOB active conformation in various cellular settings. This protocol can be broadly applied to select any kind of intracellular antibody degraders, regardless of conformational state. For complete details on the use and execution of this protocol, please refer to Bery et al. (2019).
Asunto(s)
Ingeniería Celular , Proteolisis , Anticuerpos de Cadena Única/metabolismo , Proteína de Unión al GTP rhoB/metabolismo , Línea Celular , Humanos , Conformación Proteica , Anticuerpos de Cadena Única/genética , Proteína de Unión al GTP rhoB/genéticaRESUMEN
Liquid biopsy is a rapidly emerging field due to an increasing number of oncogenic drivers and a better understanding of resistance mechanisms to targeted therapies in non-small cell lung cancer (NSCLC). The sensitivity of the most widely used blood-based assays is, however, limited in particular in cases of low tumor volume where shed of tumor-derived material can be limited. A negative result thus requires biopsy confirmation using minimally invasive sampling procedures that can result in small specimens, which are often not suitable for genotyping. Liquid biopsy is not limited to plasma, and tumor DNA circulating in other body fluids such as urine, pleural fluid, cerebrospinal fluid, or cytology specimen-derived supernatant can be exploited. In comparison to cell blocks, these fluids in close contact to the tumor may contain a more abundant and less analytically demanding tumor DNA. In this review, we discuss the potential applications of circulating tumor DNA derived from cytology samples in NSCLC, from early stage (screening, nodule characterization) to metastatic disease.
Asunto(s)
Biomarcadores de Tumor/sangre , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Biopsia Líquida , Neoplasias Pulmonares/metabolismo , Biopsia/métodos , Carcinoma de Pulmón de Células no Pequeñas/genética , ADN Tumoral Circulante/genética , Humanos , Biopsia Líquida/métodos , Neoplasias Pulmonares/genéticaRESUMEN
Vγ9Vδ2 T cells are known to be efficient anti-tumor effectors activated through phosphoantigens (PAg) that are naturally expressed by tumor cells or induced by amino bisphosphonates treatment. This PAg-activation which is TCR and butyrophilin BTN3A dependent can be modulated by NKG2D ligands, immune checkpoint ligands, adhesion molecules, and costimulatory molecules. This could explain the immune-resistance observed in certain clinical trials based on Vγ9Vδ2 T cells therapies. In NSCLC, encouraging responses were obtained with zoledronate administrations for 50% of patients. According to the in vivo results, we showed that the in vitro Vγ9Vδ2 T cell reactivity depends on the NSCLC cell line considered. If the PAg-pretreated KRAS mutated A549 is highly recognized and killed by Vγ9Vδ2 T cells, the EGFR mutated PC9 remains resistant to these killers despite a pre-treatment either with zoledronate or with exogenous BrHPP. The immune resistance of PC9 was shown not to be due to immune checkpoint ligands able to counterbalance NKG2D ligands or adhesion molecules such as ICAM-1 highly expressed by PC9. RHOB has been shown to be involved in the Vγ9Vδ2 TCR signaling against these NSCLC cell lines, in this study we therefore focused on its intracellular behavior. In comparison to a uniform distribution of RHOB in endosomes and at the plasma membrane in A549, the presence of large endosomal clusters of RHOB was visualized by a split-GFP system, suggesting that RHOB rerouting in the PC9 tumor cell could impair the reactivity of the immune response.
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Antígenos de Neoplasias/inmunología , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Proteína de Unión al GTP rhoB/metabolismo , Antígenos de Neoplasias/metabolismo , Línea Celular Tumoral , Células Cultivadas , Endosomas/inmunología , Endosomas/metabolismo , Humanos , Neoplasias Pulmonares/etiología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , FosforilaciónAsunto(s)
Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/terapia , ADN Tumoral Circulante/sangre , Análisis Mutacional de ADN/métodos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/terapia , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/sangre , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/genética , ADN Tumoral Circulante/análisis , ADN de Neoplasias/análisis , ADN de Neoplasias/sangre , Pruebas Genéticas/métodos , Humanos , Inmunoterapia/métodos , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/genética , Mutación , Pronóstico , Receptor de Muerte Celular Programada 1/análisis , Receptor de Muerte Celular Programada 1/sangre , Receptor de Muerte Celular Programada 1/genética , Resultado del TratamientoRESUMEN
Liquid biopsy refers to the analysis of any tumour-derived material circulating in the blood or any other body fluid. This concept is particularly relevant in lung cancer as the tumour is often difficult to reach and may need an invasive and potentially harmful procedure. Moreover, the multitude of anticancer drugs and their sequential use underline the importance of conducting an iterative assessment of tumour biology. Liquid biopsies can noninvasively detect any targetable genomic alteration and guide corresponding targeted therapy, in addition to monitoring response to treatment and exploring the genetic changes at resistance, overcoming spatial and temporal heterogeneity.In this article, we review the available data in the field, which suggest the potential of liquid biopsy in the area of lung cancer, with a particular focus on cell-free DNA and circulating tumour cells. We discuss their respective applications in patient selection and monitoring through targeted therapy, as well as immune checkpoint inhibitors. The current data and future applications of liquid biopsy in the early stage setting are also investigated.Liquid biopsy has the potential to help manage nonsmall cell lung cancer throughout all stages of lung cancer: screening, minimal residual disease detection to guide adjuvant treatment, early detection of relapse, systemic treatment initiation and monitoring of response (targeted or immune therapy), and resistance genotyping.
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Carcinoma de Pulmón de Células no Pequeñas/patología , Biopsia Líquida/tendencias , Neoplasias Pulmonares/patología , Células Neoplásicas Circulantes/patología , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/terapia , Ácidos Nucleicos Libres de Células/sangre , Ácidos Nucleicos Libres de Células/genética , Toma de Decisiones Clínicas , Predicción , Humanos , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Estadificación de Neoplasias , Células Neoplásicas Circulantes/metabolismo , Medicina de Precisión , Valor Predictivo de las Pruebas , Resultado del TratamientoRESUMEN
EGFR-mutated lung adenocarcinoma patients treated with gefitinib and osimertinib show a therapeutic benefit limited by the appearance of secondary mutations, such as EGFRT790M and EGFRC797S. It is generally assumed that these secondary mutations render EGFR completely unresponsive to the inhibitors, but contrary to this, we uncovered here that gefitinib and osimertinib increased STAT3 phosphorylation (p-STAT3) in EGFRT790M and EGFRC797S tumoral cells. Interestingly, we also found that concomitant Notch inhibition with gefitinib or osimertinib treatment induced a p-STAT3-dependent strong reduction in the levels of the transcriptional repressor HES1. Importantly, we showed that tyrosine kinase inhibitor-resistant tumors, with EGFRT790M and EGFRC797S mutations, were highly responsive to the combined treatment of Notch inhibitors with gefitinib or osimertinib, respectively. Finally, in patients with EGFR mutations treated with tyrosine kinase inhibitors, HES1 protein levels increased during relapse and correlated with shorter progression-free survival. Therefore, our results offer a proof of concept for an alternative treatment to chemotherapy in lung adenocarcinoma osimertinib-treated patients after disease progression.
Asunto(s)
Acrilamidas/farmacología , Adenocarcinoma del Pulmón , Compuestos de Anilina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Receptores ErbB , Gefitinib/farmacología , Neoplasias Pulmonares , Mutación Missense , Proteínas de Neoplasias , Inhibidores de Proteínas Quinasas/farmacología , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Sustitución de Aminoácidos , Animales , Resistencia a Antineoplásicos/genética , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Transgénicos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción HES-1/genética , Factor de Transcripción HES-1/metabolismoRESUMEN
The selective downregulation of activated intracellular proteins is a key challenge in cell biology. RHO small GTPases switch between a guanosine diphosphate (GDP)-bound and a guanosine triphosphate (GTP)-bound state that drives downstream signaling. At present, no tool is available to study endogenous RHO-GTPinduced conformational changes in live cells. Here, we established a cell-based screen to selectively degrade RHOB-GTP using F-box-intracellular single-domain antibody fusion. We identified one intracellular antibody (intrabody) that shows selective targeting of endogenous RHOB-GTP mediated by interactions between the CDR3 loop of the domain antibody and the GTP-binding pocket of RHOB. Our results suggest that, while RHOB is highly regulated at the expression level, only the GTP-bound pool, but not its global expression, mediates RHOB functions in genomic instability and in cell invasion. The F-box/intrabody-targeted protein degradation represents a unique approach to knock down the active form of small GTPases or other proteins with multiple cellular activities.
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Anticuerpos de Dominio Único/metabolismo , Proteína de Unión al GTP rhoB/metabolismo , Sitios de Unión , Movimiento Celular/efectos de los fármacos , Cristalografía por Rayos X , Doxiciclina/farmacología , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Expresión Génica/efectos de los fármacos , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Mutagénesis , Estructura Terciaria de Proteína , Interferencia de ARN , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/genética , Proteína de Unión al GTP rhoB/antagonistas & inhibidores , Proteína de Unión al GTP rhoB/genéticaRESUMEN
Although accumulation of DNA damage and genomic instability in resting cells can cause neurodegenerative disorders, our understanding of how transcription produces DNA double-strand breaks (DSBs) is limited. Transcription-blocking topoisomerase I cleavage complexes (TOP1ccs) are frequent events that prime DSB production in non-replicating cells. Here, we report a mechanism of their formation by showing that they arise from two nearby single-strand breaks (SSBs) on opposing DNA strands: one SSB from the removal of transcription-blocking TOP1ccs by the TDP1 pathway and the other from the cleavage of R-loops by endonucleases, including XPF, XPG, and FEN1. Genetic defects in TOP1cc removal (TDP1, PNKP, and XRCC1) or in the resolution of R-loops (SETX) enhance DSB formation and prevent their repair. Such deficiencies cause neurological disorders. Owing to the high frequency of TOP1cc trapping and the widespread distribution of R-loops, these persistent transcriptional DSBs could accumulate over time in neuronal cells, contributing to the neurodegenerative diseases.
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Roturas del ADN de Doble Cadena , Roturas del ADN de Cadena Simple , ADN-Topoisomerasas de Tipo I/metabolismo , Estructuras R-Loop , Línea Celular , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Endonucleasas de ADN Solapado/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismoRESUMEN
OBJECTIVES: Tumor mutational burden is an emerging biomarker of response to immune checkpoint inhibitors (ICI), whose clinical adoption is challenging. We hypothesized that targeting limited but relevant genetic alterations in plasma cell-free DNA along with early monitoring may non-invasively predict response to ICI in advanced non-small cell lung cancer (NSCLC). MATERIAL AND METHODS: Plasma samples from patients with progressive NSCLC collected before ICI initiation and at 1 month were profiled from responders (R: PFSâ¯>â¯6 months) and non-responders (NR: progressive disease at first evaluation) using amplicon sequencing of hotspots and coding regions from 36 genes. The molecular profile of ctDNA, and its early kinetics were analyzed. RESULTS: 97 patients were analyzed, of which 86 (39 R, 47 NR) were evaluable. Alterations in ctDNA were detectable in 67/86 baseline samples (78%). The detection of a targetable oncogenic driver was associated with a 2 months PFS. The presence of a PTEN or STK11 mutation was correlated with early progression (HR 8.9, pâ¯=â¯0.09 for PTEN, HR 4.7, pâ¯=â¯0.003 for STK11), while transversion mutations (Tv) in KRAS and TP53 predicted better outcomes (HR 0.36, pâ¯=â¯0.011 for TP53 Tv; HR 0.46, pâ¯=â¯0.11 for KRAS Tv). Patients with a low "immune score" (driver and/or PTEN or STK11 mutation and/or without KRAS or TP53 Tv) derived poor outcomes (median PFS 2 months), compared with patients with a high immune score (no driver, no PTEN or STK11 and with KRAS or TP53 Tv (median PFS 14 months, pâ¯=â¯0.0001, HR 2.96). Early changes in the ctDNA allele fraction (AF) of 65 specimens were correlated with clinical outcomes (14 months PFS if AF decreases vs. 2 months if AF increases, pâ¯<â¯0.0001). CONCLUSION: Targeted sequencing of plasma ctDNA and monitoring its early variations can predict response to ICI.