RESUMEN
Human Immunodeficiency Viruses (HIV-1 and HIV-2) rely upon host-encoded proteins to facilitate their replication. Here, we combined genome-wide siRNA analyses with interrogation of human interactome databases to assemble a host-pathogen biochemical network containing 213 confirmed host cellular factors and 11 HIV-1-encoded proteins. Protein complexes that regulate ubiquitin conjugation, proteolysis, DNA-damage response, and RNA splicing were identified as important modulators of early-stage HIV-1 infection. Additionally, over 40 new factors were shown to specifically influence the initiation and/or kinetics of HIV-1 DNA synthesis, including cytoskeletal regulatory proteins, modulators of posttranslational modification, and nucleic acid-binding proteins. Finally, 15 proteins with diverse functional roles, including nuclear transport, prostaglandin synthesis, ubiquitination, and transcription, were found to influence nuclear import or viral DNA integration. Taken together, the multiscale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate the early steps of HIV-1 infection.
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Infecciones por VIH/virología , VIH-1/fisiología , Interacciones Huésped-Patógeno , Proteínas/metabolismo , Replicación Viral , Línea Celular , Humanos , Interferencia de ARN , Técnicas del Sistema de Dos HíbridosRESUMEN
Cell fate perturbations underlie many human diseases, including breast cancer. Unfortunately, the mechanisms by which breast cell fate are regulated are largely unknown. The mammary gland epithelium consists of differentiated luminal epithelial and basal myoepithelial cells, as well as undifferentiated stem cells and more restricted progenitors. Breast cancer originates from this epithelium, but the molecular mechanisms that underlie breast epithelial hierarchy remain ill-defined. Here, we use a high-content confocal image-based short hairpin RNA screen to identify tumour suppressors that regulate breast cell fate in primary human breast epithelial cells. We show that ablation of the large tumour suppressor kinases (LATS) 1 and 2 (refs 5, 6), which are part of the Hippo pathway, promotes the luminal phenotype and increases the number of bipotent and luminal progenitors, the proposed cells-of-origin of most human breast cancers. Mechanistically, we have identified a direct interaction between Hippo and oestrogen receptor-α (ERα) signalling. In the presence of LATS, ERα was targeted for ubiquitination and Ddb1-cullin4-associated-factor 1 (DCAF1)-dependent proteasomal degradation. Absence of LATS stabilized ERα and the Hippo effectors YAP and TAZ (hereafter YAP/TAZ), which together control breast cell fate through intrinsic and paracrine mechanisms. Our findings reveal a non-canonical (that is, YAP/TAZ-independent) effect of LATS in the regulation of human breast cell fate.
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Mama/citología , Mama/enzimología , Diferenciación Celular , Linaje de la Célula , Receptor alfa de Estrógeno/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales/agonistas , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Mama/patología , Proteínas Portadoras/metabolismo , Células Cultivadas , Receptor alfa de Estrógeno/agonistas , Femenino , Genes Supresores de Tumor , Humanos , Fosfoproteínas/agonistas , Fosfoproteínas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Serina-Treonina Quinasas/deficiencia , Proteolisis , Transducción de Señal , Factores de Transcripción , Proteínas Supresoras de Tumor/deficiencia , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas , Proteínas Señalizadoras YAPRESUMEN
Diarrhoeal disease is responsible for 8.6% of global child mortality. Recent epidemiological studies found the protozoan parasite Cryptosporidium to be a leading cause of paediatric diarrhoea, with particularly grave impact on infants and immunocompromised individuals. There is neither a vaccine nor an effective treatment. Here we establish a drug discovery process built on scalable phenotypic assays and mouse models that take advantage of transgenic parasites. Screening a library of compounds with anti-parasitic activity, we identify pyrazolopyridines as inhibitors of Cryptosporidium parvum and Cryptosporidium hominis. Oral treatment with the pyrazolopyridine KDU731 results in a potent reduction in intestinal infection of immunocompromised mice. Treatment also leads to rapid resolution of diarrhoea and dehydration in neonatal calves, a clinical model of cryptosporidiosis that closely resembles human infection. Our results suggest that the Cryptosporidium lipid kinase PI(4)K (phosphatidylinositol-4-OH kinase) is a target for pyrazolopyridines and that KDU731 warrants further preclinical evaluation as a drug candidate for the treatment of cryptosporidiosis.
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1-Fosfatidilinositol 4-Quinasa/antagonistas & inhibidores , Criptosporidiosis/tratamiento farmacológico , Criptosporidiosis/parasitología , Cryptosporidium/efectos de los fármacos , Cryptosporidium/enzimología , Pirazoles/farmacología , Piridinas/farmacología , Animales , Animales Recién Nacidos , Bovinos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Huésped Inmunocomprometido , Interferón gamma/deficiencia , Interferón gamma/genética , Masculino , Ratones , Ratones Noqueados , Pirazoles/química , Pirazoles/farmacocinética , Piridinas/química , Piridinas/farmacocinética , Ratas , Ratas WistarRESUMEN
Artemisinin (ART) resistance has spread through Southeast Asia, posing a serious threat to the control and elimination of malaria. ART resistance has been associated with mutations in the Plasmodium falciparum kelch-13 (Pfk13) propeller domain. Phenotypically, ART resistance is defined as delayed parasite clearance in patients due to the reduced susceptibility of early ring-stage parasites to the active metabolite of ART dihydroartemisinin (DHA). Early rings can enter a state of quiescence upon DHA exposure and resume growth in its absence. These quiescent rings are referred to as dormant rings or DHA-pretreated rings (here called dormant rings). The imidazolopiperazines (IPZ) are a novel class of antimalarial drugs that have demonstrated efficacy in early clinical trials. Here, we characterized the stage of action of the IPZ GNF179 and evaluated its activity against rings and dormant rings in wild-type and ART-resistant parasites. Unlike DHA, GNF179 does not induce dormancy. We show that GNF179 is more rapidly cidal against schizonts than against ring and trophozoite stages. However, with 12 h of exposure, the compound effectively kills rings and dormant rings of both susceptible and ART-resistant parasites within 72 h. We further demonstrate that in combination with ART, GNF179 effectively prevents recrudescence of dormant rings, including those bearing pfk13 propeller mutations.
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Antimaláricos/farmacología , Artemisininas/farmacología , Imidazoles/farmacología , Piperazinas/farmacología , Plasmodium falciparum/efectos de los fármacos , Pruebas de Sensibilidad Parasitaria , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Esquizontes/efectos de los fármacos , Esquizontes/metabolismo , Trofozoítos/efectos de los fármacos , Trofozoítos/metabolismoRESUMEN
A series of 2-oxopiperazine derivatives were designed from the pyrrolopiperazinone cell-based screening hit 4 as a dengue virus inhibitor. Systematic investigation of the structure-activity relationship (SAR) around the piperazinone ring led to the identification of compound (S)-29, which exhibited potent anti-dengue activity in the cell-based assay across all four dengue serotypes with EC50<0.1µM. Cross-resistant analysis confirmed that the virus NS4B protein remained the target of the new oxopiperazine analogs obtained via scaffold morphing from the HTS hit 4.
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Antivirales/farmacología , Virus del Dengue/efectos de los fármacos , Piperazinas/farmacología , Línea Celular , Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento , Humanos , Relación Estructura-ActividadRESUMEN
Influenza A virus is an RNA virus that encodes up to 11 proteins and this small coding capacity demands that the virus use the host cellular machinery for many aspects of its life cycle. Knowledge of these host cell requirements not only informs us of the molecular pathways exploited by the virus but also provides further targets that could be pursued for antiviral drug development. Here we use an integrative systems approach, based on genome-wide RNA interference screening, to identify 295 cellular cofactors required for early-stage influenza virus replication. Within this group, those involved in kinase-regulated signalling, ubiquitination and phosphatase activity are the most highly enriched, and 181 factors assemble into a highly significant host-pathogen interaction network. Moreover, 219 of the 295 factors were confirmed to be required for efficient wild-type influenza virus growth, and further analysis of a subset of genes showed 23 factors necessary for viral entry, including members of the vacuolar ATPase (vATPase) and COPI-protein families, fibroblast growth factor receptor (FGFR) proteins, and glycogen synthase kinase 3 (GSK3)-beta. Furthermore, 10 proteins were confirmed to be involved in post-entry steps of influenza virus replication. These include nuclear import components, proteases, and the calcium/calmodulin-dependent protein kinase (CaM kinase) IIbeta (CAMK2B). Notably, growth of swine-origin H1N1 influenza virus is also dependent on the identified host factors, and we show that small molecule inhibitors of several factors, including vATPase and CAMK2B, antagonize influenza virus replication.
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Factores Biológicos/genética , Factores Biológicos/fisiología , Interacciones Huésped-Patógeno/fisiología , Virus de la Influenza A/crecimiento & desarrollo , Gripe Humana/genética , Gripe Humana/virología , Replicación Viral/fisiología , Animales , Línea Celular , Chlorocebus aethiops , Biblioteca de Genes , Genoma Humano/genética , Interacciones Huésped-Patógeno/genética , Humanos , Subtipo H1N1 del Virus de la Influenza A/clasificación , Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Virus de la Influenza A/clasificación , Interferencia de ARN , Células Vero , Internalización del VirusRESUMEN
Many proteins or other biological macromolecules are localized to more than one subcellular structure. The fraction of a protein in different cellular compartments is often measured by colocalization with organelle-specific fluorescent markers, requiring availability of fluorescent probes for each compartment and acquisition of images for each in conjunction with the macromolecule of interest. Alternatively, tailored algorithms allow finding particular regions in images and quantifying the amount of fluorescence they contain. Unfortunately, this approach requires extensive hand-tuning of algorithms and is often cell type-dependent. Here we describe a machine-learning approach for estimating the amount of fluorescent signal in different subcellular compartments without hand tuning, requiring only the acquisition of separate training images of markers for each compartment. In testing on images of cells stained with mixtures of probes for different organelles, we achieved a 93% correlation between estimated and expected amounts of probes in each compartment. We also demonstrated that the method can be used to quantify drug-dependent protein translocations. The method enables automated and unbiased determination of the distributions of protein across cellular compartments, and will significantly improve imaging-based high-throughput assays and facilitate proteome-scale localization efforts.
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Algoritmos , Inteligencia Artificial , Colorantes Fluorescentes/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Espacio Intracelular/metabolismo , Transporte de Proteínas/fisiología , Proteómica/métodosRESUMEN
Importance: The drug HD201 is a biosimilar candidate for breast cancer treatment as the reference trastuzumab. Objective: To compare the efficacy of HD201 with referent trastuzumab. Design, Setting, and Participants: This randomized clinical trial (TROIKA) included 502 women with ERBB2-positive early breast cancer treated with either HD201 or referent trastuzumab. It was conducted across 70 centers in 12 countries, including Western and Eastern Europe and Asian countries. Randomization was stratified by tumor hormone receptor status, clinical stage, and geographic region of recruitment. This analysis was conducted on February 12, 2021, after the completion of the adjuvant phase at a median of 31 months (IQR, 28-33 months) of follow-up. Interventions: Patients with ERBB2-positive early breast cancer were randomly assigned to receive HD201 or referent trastuzumab in the neoadjuvant setting for 8 cycles, concurrently with 4 cycles of docetaxel, which was followed by 4 cycles of epirubicin and cyclophosphamide. Patients then underwent surgery, which was followed by treatment with 10 cycles of adjuvant HD201 or referent trastuzumab. Main Outcome and Measures: The primary end point was the total pathological complete response (tpCR) assessed after neoadjuvant treatment. Equivalence was concluded if the 95% CI of the absolute difference in tpCR between arms in the per-protocol set was within the margin of more or less than 15%. Other objectives included the breast pathological complete response, overall response, event-free and overall survival, safety, pharmacokinetics, and immunogenicity. Results: A total of 502 female patients (mean [range] age, 53 [26-82] years) were randomized to receive either HD201 or referent trastuzumab, and 474 (94.2%) were eligible for inclusion in the per-protocol set. The baseline characteristics were well balanced between the 2 arms; 195 tumors (38.8%) were hormone receptor-negative , and 213 patients (42.4%) had clinical stage III disease. The tpCR rates were 45% and 48.7% for HD201 and referent trastuzumab, respectively. The difference between the 2 groups was not significant at -3.8% (95% CI, -12.8% to 5.4%) and fell within the predefined equivalence margins. The ratio of the tpCR rates between the 2 arms was 0.92 (95% CI, 0.76 to 1.12). A total of 433 patients (86.1%) presented with 2232 treatment-emergent adverse events of special interest for trastuzumab during the entire treatment period, with 220 (88.0%) and 213 (84.5%) patients in the HD201 and referent trastuzumab groups, respectively. Conclusions and Relevance: The results of this randomized clinical trial found that HD201 demonstrated equivalence to referent trastuzumab in terms of efficacy for the end point of tpCR, with a similar safety profile. Trial Registration: ClinicalTrials.gov Identifier: NCT03013504.
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Antineoplásicos , Neoplasias de la Mama , Terapia Neoadyuvante , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Persona de Mediana Edad , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Receptor ErbB-2 , Trastuzumab/efectos adversos , Trastuzumab/uso terapéuticoRESUMEN
Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to public health, yet no antiviral drug is available. We performed a high-throughput phenotypic screen using the Novartis compound library and identified candidate chemical inhibitors of DENV. This chemical series was optimized to improve properties such as anti-DENV potency and solubility. The lead compound, NITD-688, showed strong potency against all four serotypes of DENV and demonstrated excellent oral efficacy in infected AG129 mice. There was a 1.44-log reduction in viremia when mice were treated orally at 30 milligrams per kilogram twice daily for 3 days starting at the time of infection. NITD-688 treatment also resulted in a 1.16-log reduction in viremia when mice were treated 48 hours after infection. Selection of resistance mutations and binding studies with recombinant proteins indicated that the nonstructural protein 4B is the target of NITD-688. Pharmacokinetic studies in rats and dogs showed a long elimination half-life and good oral bioavailability. Extensive in vitro safety profiling along with exploratory rat and dog toxicology studies showed that NITD-688 was well tolerated after 7-day repeat dosing, demonstrating that NITD-688 may be a promising preclinical candidate for the treatment of dengue.
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Virus del Dengue , Dengue , Animales , Antivirales/uso terapéutico , Dengue/tratamiento farmacológico , Perros , Ratones , Modelos Animales , Ratas , SerogrupoRESUMEN
We previously demonstrated that mRNAs for the subunits of the Arp2/3 complex localize to protrusions in fibroblasts (Mingle et al. in J Cell Sci 118:2425-2433, 2005). However, the signaling pathway that regulates Arp2/3 complex mRNA localization remains unknown. In this study we have identified lysophosphatidic acid (LPA) as a potent inducer of Arp2 mRNA localization to protrusions in fibroblasts via the RhoA-ROCK pathway. As RhoA is known to be activated locally in the cells, we sought to understand how spatial activation of Rho affects Arp2 mRNA localization. By sequentially performing fluorescence resonance energy transfer (FRET) and fluorescence in situ hybridization (FISH), we have visualized active RhoA and Arp2 mRNA in the same cells. Upon LPA stimulation, approximately two times more cells than those in the serum-free medium showed mutually exclusive localization of active RhoA and Arp2 mRNA. These results demonstrate the importance of localized activation of Rho in Arp2 mRNA localization and provide new insights as to how Rho regulates Arp2/3 complex mRNA localization. To our best knowledge, this is the first report in which FRET and FISH are combined to detect localized protein activity and mRNA in the same cells. This method should be easily adopted for the detection of other fluorescence protein based biosensors and DNA/RNA in the same cells.
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Proteína 2 Relacionada con la Actina/metabolismo , Fibroblastos/metabolismo , Lisofosfolípidos/farmacología , ARN Mensajero/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Núcleo Celular/metabolismo , Células Cultivadas , Embrión de Pollo , Citoplasma/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Transferencia Resonante de Energía de Fluorescencia , Hibridación Fluorescente in SituRESUMEN
Cryptosporidiosis is a diarrheal disease predominantly caused by Cryptosporidium parvum ( Cp) and Cryptosporidium hominis ( Ch), apicomplexan parasites which infect the intestinal epithelial cells of their human hosts. The only approved drug for cryptosporidiosis is nitazoxanide, which shows limited efficacy in immunocompromised children, the most vulnerable patient population. Thus, new therapeutics and in vitro infection models are urgently needed to address the current unmet medical need. Toward this aim, we have developed novel cytopathic effect (CPE)-based Cp and Ch assays in human colonic tumor (HCT-8) cells and compared them to traditional imaging formats. Further model validation was achieved through screening a collection of FDA-approved drugs and confirming many previously known anti- Cryptosporidium hits as well as identifying a few novel candidates. Collectively, our data reveals this model to be a simple, functional, and homogeneous gain of signal format amenable to high throughput screening, opening new avenues for the discovery of novel anticryptosporidials.
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Antiprotozoarios/aislamiento & purificación , Cryptosporidium parvum/efectos de los fármacos , Cryptosporidium parvum/crecimiento & desarrollo , Evaluación Preclínica de Medicamentos/métodos , Células Epiteliales/parasitología , Antiprotozoarios/farmacología , Línea Celular , HumanosAsunto(s)
Hidrazonas/farmacología , Hidrocarburos Clorados/farmacología , Mitocondrias/metabolismo , Dinámicas Mitocondriales/efectos de los fármacos , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , GTP Fosfohidrolasas/deficiencia , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Humanos , Hidrazonas/química , Hidrocarburos Clorados/química , Ratones , Microscopía Confocal , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Factores de Acoplamiento de la Fosforilación Oxidativa/metabolismoRESUMEN
The intramembrane protease signal peptide peptidase-like 2a (SPPL2a) is a potential drug target for the treatment of autoimmune diseases due to an essential role in B cells and dendritic cells. To screen a library of 1.4 million compounds for inhibitors of SPPL2a, we developed an imaging assay detecting nuclear translocation of the proteolytically released cytosolic substrate fragment. The state-of-the-art hit calling approach based on nuclear translocation resulted in numerous false-positive hits, mainly interrupting intracellular protein trafficking. To filter the false positives, we extracted 340 image-based readouts and developed a novel multiparametric analysis method that successfully triaged the primary hit list. The identified scaffolds were validated by demonstrating activity on endogenous SPPL2a and substrate CD74/p8 in B cells. The multiparametric analysis discovered diverse cellular phenotypes and provided profiles for the whole library. The principle of the presented imaging assay, the screening strategy, and multiparametric analysis are potentially applicable in future screening campaigns.
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Flavivirus infections by Zika and dengue virus impose a significant global healthcare threat with no US Food and Drug Administration (FDA)-approved vaccination or specific antiviral treatment available. Here, we present the discovery of an anti-flaviviral natural product named cavinafungin. Cavinafungin is a potent and selectively active compound against Zika and all four dengue virus serotypes. Unbiased, genome-wide genomic profiling in human cells using a novel CRISPR/Cas9 protocol identified the endoplasmic-reticulum-localized signal peptidase as the efficacy target of cavinafungin. Orthogonal profiling in S. cerevisiae followed by the selection of resistant mutants pinpointed the catalytic subunit of the signal peptidase SEC11 as the evolutionary conserved target. Biochemical analysis confirmed a rapid block of signal sequence cleavage of both host and viral proteins by cavinafungin. This study provides an effective compound against the eukaryotic signal peptidase and independent confirmation of the recently identified critical role of the signal peptidase in the replicative cycle of flaviviruses.
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Productos Biológicos/farmacología , Virus del Dengue/fisiología , Lipopéptidos/farmacología , Replicación Viral/efectos de los fármacos , Virus Zika/fisiología , Productos Biológicos/química , Sistemas CRISPR-Cas/genética , Virus del Dengue/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Genoma Humano , Genómica , Células HCT116 , Humanos , Lipopéptidos/química , Proteínas de la Membrana , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Serina Endopeptidasas , Proteínas Virales/metabolismo , Virus Zika/efectos de los fármacosRESUMEN
Plasmodium liver hypnozoites, which cause disease relapse, are widely considered to be the last barrier towards malaria eradication. The biology of this quiescent form of the parasite is poorly understood which hinders drug discovery. We report a comparative transcriptomic dataset of replicating liver schizonts and dormant hypnozoites of the relapsing parasite Plasmodium cynomolgi. Hypnozoites express only 34% of Plasmodium physiological pathways, while 91% are expressed in replicating schizonts. Few known malaria drug targets are expressed in quiescent parasites, but pathways involved in microbial dormancy, maintenance of genome integrity and ATP homeostasis were robustly expressed. Several transcripts encoding heavy metal transporters were expressed in hypnozoites and the copper chelator neocuproine was cidal to all liver stage parasites. This transcriptomic dataset is a valuable resource for the discovery of vaccines and effective treatments to combat vivax malaria.
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Perfilación de la Expresión Génica , Hígado/parasitología , Macaca mulatta/parasitología , Plasmodium cynomolgi/crecimiento & desarrollo , Plasmodium cynomolgi/genética , Esquizontes/crecimiento & desarrollo , Esquizontes/genética , Animales , Femenino , MasculinoRESUMEN
The retroviral v-ErbA oncoprotein is a highly mutated variant of the thyroid hormone receptor alpha (TRalpha), which is unable to bind T(3) and interferes with the action of TRalpha in mammalian and avian cancer cells. v-ErbA dominant-negative activity is attributed to competition with TRalpha for T(3)-responsive DNA elements and/or auxiliary factors involved in the transcriptional regulation of T(3)-responsive genes. However, competition models do not address the altered subcellular localization of v-ErbA and its possible implications in oncogenesis. Here, we report that v-ErbA dimerizes with TRalpha and the retinoid X receptor and sequesters a significant fraction of the two nuclear receptors in the cytoplasm. Recruitment of TRalpha to the cytoplasm by v-ErbA can be partially reversed in the presence of ligand and when chromatin is disrupted by the histone deacetylase inhibitor trichostatin A. These results define a new mode of action of v-ErbA and illustrate the importance of cellular compartmentalization in transcriptional regulation and oncogenesis.
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Neoplasias/metabolismo , Proteínas Oncogénicas v-erbA/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Citoplasma/metabolismo , Dimerización , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Carioferinas/metabolismo , Ligandos , Ratones , Células 3T3 NIH , Proteínas Oncogénicas v-erbA/genética , Transporte de Proteínas/fisiología , Receptor beta X Retinoide/metabolismo , Receptores alfa de Hormona Tiroidea/genética , Receptores alfa de Hormona Tiroidea/metabolismo , Proteína Exportina 1RESUMEN
A molecular understanding of drug resistance mechanisms enables surveillance of the effectiveness of new antimicrobial therapies during development and deployment in the field. We used conventional drug resistance selection as well as a regime of limiting dilution at early stages of drug treatment to probe two antimalarial imidazolopiperazines, KAF156 and GNF179. The latter approach permits the isolation of low-fitness mutants that might otherwise be out-competed during selection. Whole-genome sequencing of 24 independently derived resistant Plasmodium falciparum clones revealed four parasites with mutations in the known cyclic amine resistance locus (pfcarl) and a further 20 with mutations in two previously unreported P. falciparum drug resistance genes, an acetyl-CoA transporter (pfact) and a UDP-galactose transporter (pfugt). Mutations were validated both in vitro by CRISPR editing in P. falciparum and in vivo by evolution of resistant Plasmodium berghei mutants. Both PfACT and PfUGT were localized to the endoplasmic reticulum by fluorescence microscopy. As mutations in pfact and pfugt conveyed resistance against additional unrelated chemical scaffolds, these genes are probably involved in broad mechanisms of antimalarial drug resistance.
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Emerging evidence suggests that some cancers contain a population of stem-like TICs (tumor-initiating cells) and eliminating TICs may offer a new strategy to develop successful anti-cancer therapies. As molecular mechanisms underlying the maintenance of the TIC pool are poorly understood, the development of TIC-specific therapeutics remains a major challenge. We first identified and characterized TICs and non-TICs isolated from a mouse breast cancer model. TICs displayed increased tumorigenic potential, self-renewal, heterogeneous differentiation, and bipotency. Gene expression analysis and immunostaining of TICs and non-TICs revealed that FGFR2 was preferentially expressed in TICs. Loss of FGFR2 impaired self-renewal of TICs, thus resulting in marked decreases in the TIC population and tumorigenic potential. Restoration of FGFR2 rescued the defects in TIC pool maintenance, bipotency, and breast tumor growth driven by FGFR2 knockdown. In addition, pharmacological inhibition of FGFR2 kinase activity led to a decrease in the TIC population which resulted in suppression of breast tumor growth. Moreover, human breast TICs isolated from patient tumor samples were found enriched in a FGFR2+ population that was sufficient to initiate tumor growth. Our data suggest that FGFR2 is essential in sustaining the breast TIC pool through promotion of self-renewal and maintenance of bipotent TICs, and raise the possibility of FGFR2 inhibition as a strategy for anti-cancer therapy by eradicating breast TICs.
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Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Mamarias Animales/metabolismo , Células Madre Neoplásicas/inmunología , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Antígeno CD24/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular , Proliferación Celular , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Integrina beta1/metabolismo , Ratones , Ratones Endogámicos NOD , Microscopía Fluorescente , Trasplante de Neoplasias , Células Madre Neoplásicas/metabolismo , Transducción de SeñalRESUMEN
Hippo signaling represents a tumor suppressor pathway that regulates organ size and tumorigenesis through phosphorylation and inhibition of the transcription coactivator YAP. Here, we show that serum deprivation dramatically induces YAP Ser127 phosphorylation and cytoplasmic retention, independent of cell-cell contact. Through chemical isolation and activity profiling, we identified serum-derived sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) as small molecule activators of YAP. S1P induces YAP nuclear localization through S1P(2) receptor, Rho GTPase activation, and F-actin polymerization, independent of the core Hippo pathway kinases. Bioinformatics studies also showed that S1P stimulation induces YAP target gene expression in mouse liver and human embryonic stem cells. These results revealed potent small molecule regulators of YAP and suggest that S1P and LPA might modulate cell proliferation and tumorigenesis through YAP activation.
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Lisofosfolípidos/farmacología , Proteínas Nucleares/metabolismo , Esfingosina/análogos & derivados , Factores de Transcripción/metabolismo , Actinas/metabolismo , Animales , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Lisofosfolípidos/sangre , Lisofosfolípidos/química , Lisofosfolípidos/aislamiento & purificación , Ratones , Proteínas Nucleares/química , Fosforilación/efectos de los fármacos , Receptores de Lisoesfingolípidos/metabolismo , Transducción de Señal/efectos de los fármacos , Esfingosina/sangre , Esfingosina/aislamiento & purificación , Esfingosina/farmacología , Factores de Transcripción/química , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Pathogens commonly utilize endocytic pathways to gain cellular access. The endosomal pattern recognition receptors TLR7 and TLR9 detect pathogen-encoded nucleic acids to initiate MyD88-dependent proinflammatory responses to microbial infection. Using genome-wide RNAi screening and integrative systems-based analysis, we identify 190 cofactors required for TLR7- and TLR9-directed signaling responses. A set of cofactors were crossprofiled for their activities downstream of several immunoreceptors and then functionally mapped based on the known architecture of NF-κB signaling pathways. Protein complexes and pathways involved in ubiquitin-protein ligase activities, sphingolipid metabolism, chromatin modifications, and ancient stress responses were found to modulate innate recognition of endosomal nucleic acids. Additionally, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) was characterized as necessary for ubiquitin-dependent TLR9 targeting to the endolysosome. Proteins and pathways identified here should prove useful in delineating strategies to manipulate innate responses for treatment of autoimmune disorders and microbial infection.