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SARS-CoV-2 is associated with broad tissue tropism, a characteristic often determined by the availability of entry receptors on host cells. Here, we show that TMEM106B, a lysosomal transmembrane protein, can serve as an alternative receptor for SARS-CoV-2 entry into angiotensin-converting enzyme 2 (ACE2)-negative cells. Spike substitution E484D increased TMEM106B binding, thereby enhancing TMEM106B-mediated entry. TMEM106B-specific monoclonal antibodies blocked SARS-CoV-2 infection, demonstrating a role of TMEM106B in viral entry. Using X-ray crystallography, cryogenic electron microscopy (cryo-EM), and hydrogen-deuterium exchange mass spectrometry (HDX-MS), we show that the luminal domain (LD) of TMEM106B engages the receptor-binding motif of SARS-CoV-2 spike. Finally, we show that TMEM106B promotes spike-mediated syncytium formation, suggesting a role of TMEM106B in viral fusion. Together, our findings identify an ACE2-independent SARS-CoV-2 infection mechanism that involves cooperative interactions with the receptors heparan sulfate and TMEM106B.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Receptores Virales/metabolismo , Internalización del Virus , Unión Proteica , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismoRESUMEN
An enzymatic method has been successfully established enabling the generation of partially base-modified RNA (previously named RZA) constructs, in which all G residues were replaced by isomorphic fluorescent thienoguanosine (thG) analogs, as well as fully modified RZA featuring thG, 5-bromocytosine, 7-deazaadenine and 5-chlorouracil. The transcriptional efficiency of emissive fully modified RZA was found to benefit from the use of various T7 RNA polymerase variants. Moreover, dthG could be incorporated into PCR products by Taq DNA polymerase together with the other three base-modified nucleotides. Notably, the obtained RNA products containing thG as well as thG together with 5-bromocytosine could function as effectively as natural sgRNAs in an in vitro CRISPR-Cas9 cleavage assay. N1-Methylpseudouridine was also demonstrated to be a faithful non-canonical substitute of uridine to direct Cas9 nuclease cleavage when incorporated in sgRNA. The Cas9 inactivation by 7-deazapurines indicated the importance of the 7-nitrogen atom of purines in both sgRNA and PAM site for achieving efficient Cas9 cleavage. Additional aspects of this study are discussed in relation to the significance of sgRNA-protein and PAM--protein interactions that were not highlighted by the Cas9-sgRNA-DNA complex crystal structure. These findings could expand the impact and therapeutic value of CRISPR-Cas9 and other RNA-based technologies.
With the advent of CRISPR-Cas9 gene editing, we now have to hand a simple two-component system amendable to silencing and knock-in editing effectively any gene. Yet we must not forget that the implications of immunotoxicity along with the poor stability and specificity of canonical nucleic acids hold enormous challenges for in vivo applications, especially in gene therapy. Our study endorses the feasibility of the enzymatic approach to incorporate nucleobase modifications into the CRISPR-Cas9 system unveiling the tolerance of Cas9 to N1-methylpseudouridine (m1Ψ)- and emissive thienoguanosine (thG)-modified sgRNA as well as thus far uncharted structural requirements for ensuring proper PAM recognition.
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Sistemas CRISPR-Cas , Ácidos Nucleicos , ADN , Edición Génica/métodos , ARN/química , Fluorescencia , Guanosina/químicaRESUMEN
INTRODUCTION: The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are hexanucleotide repeats in chromosome 9 open reading frame 72 (C9orf72). These repeats produce dipeptide repeat proteins with poly(PR) being the most toxic one. METHODS: We performed a kinome-wide CRISPR/Cas9 knock-out screen in human induced pluripotent stem cell (iPSC) -derived cortical neurons to identify modifiers of poly(PR) toxicity, and validated the role of candidate modifiers using in vitro, in vivo, and ex-vivo studies. RESULTS: Knock-down of NIMA-related kinase 6 (NEK6) prevented neuronal toxicity caused by poly(PR). Knock-down of nek6 also ameliorated the poly(PR)-induced axonopathy in zebrafish and NEK6 was aberrantly expressed in C9orf72 patients. Suppression of NEK6 expression and NEK6 activity inhibition rescued axonal transport defects in cortical neurons from C9orf72 patient iPSCs, at least partially by reversing p53-related DNA damage. DISCUSSION: We identified NEK6, which regulates poly(PR)-mediated p53-related DNA damage, as a novel therapeutic target for C9orf72 FTD/ALS.
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Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Células Madre Pluripotentes Inducidas , Animales , Humanos , Esclerosis Amiotrófica Lateral/genética , Demencia Frontotemporal/genética , Células Madre Pluripotentes Inducidas/metabolismo , Proteína C9orf72/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Sistemas CRISPR-Cas , Pez Cebra/genética , Pez Cebra/metabolismo , Neuronas/metabolismo , Expansión de las Repeticiones de ADN/genética , Quinasas Relacionadas con NIMA/genética , Quinasas Relacionadas con NIMA/metabolismoRESUMEN
While drug resistance mutations provide the gold standard proof for drug target engagement, target deconvolution of inhibitors identified from a phenotypic screen remains challenging. Genetic screening for functional in-frame drug resistance mutations by tiling CRISPR-Cas nucleases across protein coding sequences is a method for identifying a drug's target and binding site. However, the applicability of this approach is constrained by the availability of nuclease target sites across genetic regions that mediate drug resistance upon mutation. In this study, we show that an enhanced AsCas12a variant (enAsCas12a), which harbors an expanded targeting range, facilitates screening for drug resistance mutations with increased activity and resolution in regions that are not accessible to other CRISPR nucleases, including the prototypical SpCas9. Utilizing enAsCas12a, we uncover new drug resistance mutations against inhibitors of NAMPT and KIF11. These findings demonstrate that enAsCas12a is a promising new addition to the CRISPR screening toolbox and allows targeting sites not readily accessible to SpCas9.
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Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Resistencia a Medicamentos/genética , Endonucleasas/metabolismo , Pruebas Genéticas/métodos , Mutación , Sitios de Unión , Unión ProteicaRESUMEN
STK38 (also known as NDR1) is a Hippo pathway serine/threonine protein kinase with multifarious functions in normal and cancer cells. Using a context-dependent proximity-labeling assay, we identify more than 250 partners of STK38 and find that STK38 modulates its partnership depending on the cellular context by increasing its association with cytoplasmic proteins upon nutrient starvation-induced autophagy and with nuclear ones during ECM detachment. We show that STK38 shuttles between the nucleus and the cytoplasm and that its nuclear exit depends on both XPO1 (aka exportin-1, CRM1) and STK38 kinase activity. We further uncover that STK38 modulates XPO1 export activity by phosphorylating XPO1 on serine 1055, thus regulating its own nuclear exit. We expand our model to other cellular contexts by discovering that XPO1 phosphorylation by STK38 regulates also the nuclear exit of Beclin1 and YAP1, key regulator of autophagy and transcriptional effector, respectively. Collectively, our results reveal STK38 as an activator of XPO1, behaving as a gatekeeper of nuclear export. These observations establish a novel mechanism of XPO1-dependent cargo export regulation by phosphorylation of XPO1's C-terminal auto-inhibitory domain.
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Autofagia , Núcleo Celular/metabolismo , Carioferinas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Portadoras/metabolismo , Cromatografía Liquida , Biología Computacional/métodos , Vía de Señalización Hippo , Humanos , Fosforilación , Unión Proteica , Mapeo de Interacción de Proteínas , Transporte de Proteínas , Transducción de Señal , Espectrometría de Masas en Tándem , Proteína Exportina 1RESUMEN
Orthoflaviviruses cause a major threat to global public health, and no antiviral treatment is available yet. Zika virus (ZIKV) entry, together with many other viruses, is known to be enhanced by phosphatidylserine (PS) receptors such as T-cell immunoglobulin mucin domain protein 1 (TIM-1). In this study, we demonstrate for the first time, using cell-based electrical impedance (CEI) biosensing, that ZIKV entry is also enhanced by expression of CD300a, another PS receptor. Furthermore, inhibiting CD300a in immature monocyte-derived dendritic cells partially but significantly inhibits ZIKV replication. As we have previously demonstrated that CEI is a useful tool to study Orthoflavivirus infection in real time, we now use this technology to determine how these PS receptors influence the kinetics of in vitro ZIKV infection. Results show that ZIKV entry is highly sensitive to minor changes in TIM-1 expression, both after overexpression of TIM-1 in infection-resistant HEK293T cells, as well as after partial knockout of TIM-1 in susceptible A549 cells. These results are confirmed by quantification of viral copy number and viral infectivity, demonstrating that CEI is highly suited to study and compare virus-host interactions. Overall, the results presented here demonstrate the potential of targeting this universal viral entry pathway.
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Impedancia Eléctrica , Receptor Celular 1 del Virus de la Hepatitis A , Internalización del Virus , Infección por el Virus Zika , Virus Zika , Humanos , Receptor Celular 1 del Virus de la Hepatitis A/metabolismo , Infección por el Virus Zika/virología , Infección por el Virus Zika/metabolismo , Células HEK293 , Células A549 , Receptores Inmunológicos/metabolismo , Replicación Viral , Técnicas Biosensibles , Lectina 1 Similar a Ig de Unión al Ácido SiálicoRESUMEN
The maintenance of a highly functional metabolic epithelium in vitro is challenging. Metabolic impairments in primary human hepatocytes (PHHs) over time is primarily due to epithelial-to-mesenchymal transitioning (EMT). The immature hepatoma cell line HepG2 was used as an in vitro model to explore strategies for enhancing the hepatic phenotype. The phenotypic characterization includes measuring the urea cycle, lipid storage, tricarboxylic acid-related metabolites, reactive oxygen species, endoplasmic reticulum calcium efflux, mitochondrial membrane potentials, oxygen consumptions rate, and CYP450 biotransformation capacity. Expression studies were performed with transcriptomics, co-immunoprecipitation and proteomics. CRISPR/Cas9 was also employed to genetically engineer HepG2 cells. After confirming that PHHs develop an EMT phenotype, expression of tankyrase1/2 was found to increase over time. EMT was reverted when blocking tankyrases1/2-dependent poly-ADP-ribosylation (PARylation) activity, by biochemical and genetic perturbation. Wnt/ß-catenin inhibitor XAV-939 blocks tankyrase1/2 and treatment elevated several oxygen-consuming reactions (electron-transport chain, OXHPOS, CYP450 mono-oxidase activity, phase I/II xenobiotic biotransformation, and prandial turnover), suggesting that cell metabolism was enhanced. Glutathione-dependent redox homeostasis was also significantly improved in the XAV-939 condition. Oxygen consumption rate and proteomics experiments in tankyrase1/2 double knockout HepG2 cells then uncovered PARylation as master regulator of aerobic-dependent cell respiration. Furthermore, novel tankyrase1/2-dependent PARylation targets, including mitochondrial DLST, and OGDH, were revealed. This work exposed a new mechanistic framework by linking PARylation to respiration and metabolism, thereby broadening the current understanding that underlies these vital processes. XAV-939 poses an immediate and straightforward strategy to improve aerobic activities, and metabolism, in (immature) cell cultures.
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Transición Epitelial-Mesenquimal , Hepatocitos , Tanquirasas , Humanos , Tanquirasas/antagonistas & inhibidores , Tanquirasas/metabolismo , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/fisiología , Poli ADP Ribosilación/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Fenantrenos/farmacologíaRESUMEN
A spontaneously occurring temperature increase in solid tumors has been reported sporadically, but is largely overlooked in terms of cancer biology. Here we show that temperature is increased in tumors of patients with pancreatic ductal adenocarcinoma (PDAC) and explore how this could affect therapy response. By mimicking this observation in PDAC cell lines, we demonstrate that through adaptive changes in lipid metabolism, the temperature increase found in human PDAC confers protection to lipid peroxidation and contributes to gemcitabine resistance. Consistent with the recently uncovered role of p38 MAPK in ferroptotic cell death, we find that the reduction in lipid peroxidation potential following adaptation to tumoral temperature allows for p38 MAPK inhibition, conferring chemoresistance. As an increase in tumoral temperature is observed in several other tumor types, our findings warrant taking tumoral temperature into account in subsequent studies related to ferroptosis and therapy resistance. More broadly, our findings indicate that tumoral temperature affects cancer biology.
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Carcinoma Ductal Pancreático , Desoxicitidina , Resistencia a Antineoplásicos , Ferroptosis , Gemcitabina , Metabolismo de los Lípidos , Neoplasias Pancreáticas , Ferroptosis/efectos de los fármacos , Humanos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Resistencia a Antineoplásicos/genética , Línea Celular Tumoral , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/genética , Metabolismo de los Lípidos/efectos de los fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Peroxidación de Lípido/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Temperatura , Animales , RatonesRESUMEN
Despite the success of immunotherapy, overcoming immunoresistance in cancer remains challenging. We identified a unique niche of tumor-associated macrophages (TAMs), coexpressing T cell immunoglobulin and mucin domain-containing 3 (TIM3) and V-domain immunoglobulin suppressor of T cell activation (VISTA), that dominated human and mouse tumors resistant to most of the currently used immunotherapies. TIM3+VISTA+ TAMs were sustained by IL-4-enriching tumors with low (neo)antigenic and T cell-depleted features. TIM3+VISTA+ TAMs showed an anti-inflammatory and protumorigenic phenotype coupled with inability to sense type I interferon (IFN). This was established with cancer cells succumbing to immunogenic cell death (ICD). Dying cancer cells not only triggered autocrine type I IFNs but also exposed HMGB1/VISTA that engaged TIM3/VISTA on TAMs to suppress paracrine IFN-responses. Accordingly, TIM3/VISTA blockade synergized with paclitaxel, an ICD-inducing chemotherapy, to repolarize TIM3+VISTA+ TAMs to proinflammatory TAMs that killed cancer cells via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling. We propose targeting TIM3+VISTA+ TAMs to overcome immunoresistant tumors.
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Receptor 2 Celular del Virus de la Hepatitis A , Inmunoterapia , Macrófagos Asociados a Tumores , Macrófagos Asociados a Tumores/metabolismo , Macrófagos Asociados a Tumores/inmunología , Macrófagos Asociados a Tumores/efectos de los fármacos , Animales , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Humanos , Inmunoterapia/métodos , Ratones , Resistencia a Antineoplásicos , Neoplasias/terapia , Neoplasias/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Línea Celular Tumoral , Microambiente Tumoral/inmunología , Interferón Tipo I/metabolismo , Antígenos B7RESUMEN
Intracellular phase separation is emerging as a universal principle for organizing biochemical reactions in time and space. It remains incompletely resolved how biological function is encoded in these assemblies and whether this depends on their material state. The conserved intrinsically disordered protein PopZ forms condensates at the poles of the bacterium Caulobacter crescentus, which in turn orchestrate cell-cycle regulating signaling cascades. Here we show that the material properties of these condensates are determined by a balance between attractive and repulsive forces mediated by a helical oligomerization domain and an expanded disordered region, respectively. A series of PopZ mutants disrupting this balance results in condensates that span the material properties spectrum, from liquid to solid. A narrow range of condensate material properties supports proper cell division, linking emergent properties to organismal fitness. We use these insights to repurpose PopZ as a modular platform for generating tunable synthetic condensates in human cells.
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Caulobacter crescentus , Proteínas Intrínsecamente Desordenadas , Proteínas Bacterianas/metabolismo , Condensados Biomoleculares , Caulobacter crescentus/metabolismo , División Celular , Humanos , Proteínas Intrínsecamente Desordenadas/metabolismoRESUMEN
The hematopoietic system is highly sensitive to perturbations in the translational machinery, of which an emerging level of regulation lies in the epitranscriptomic modification of transfer RNAs (tRNAs). Here, we interrogate the role of tRNA anticodon modifications in hematopoiesis by using mouse models of conditional inactivation of Elp3, the catalytic subunit of Elongator that modifies wobble uridine in specific tRNAs. Loss of Elp3 causes bone marrow failure by inducing death in committing progenitors and compromises the grafting activity of hematopoietic stem cells. Mechanistically, Elp3 deficiency activates a p53-dependent checkpoint in what resembles a misguided amino acid deprivation response that is accompanied by Atf4 overactivation and increased protein synthesis. While deletion of p53 rescues hematopoiesis, loss of Elp3 prompts the development of p53-mutated leukemia/lymphoma, and inactivation of p53 and Elongator cooperatively promotes tumorigenesis. Specific tRNA-modifying enzymes thus condition differentiation and antitumor fate decisions in hematopoietic stem cells and progenitors.
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Hematopoyesis , Histona Acetiltransferasas/metabolismo , ARN de Transferencia/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Factor de Transcripción Activador 4/metabolismo , Aminoácidos/deficiencia , Animales , Línea Celular , Supervivencia Celular , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/ultraestructura , Ratones Endogámicos C57BL , Biosíntesis de Proteínas , Estrés Fisiológico , Respuesta de Proteína Desplegada , Regulación hacia ArribaRESUMEN
The ongoing COVID-19 pandemic has caused a global economic and health crisis. To identify host factors essential for coronavirus infection, we performed genome-wide functional genetic screens with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human coronavirus 229E. These screens uncovered virus-specific as well as shared host factors, including TMEM41B and PI3K type 3. We discovered that SARS-CoV-2 requires the lysosomal protein TMEM106B to infect human cell lines and primary lung cells. TMEM106B overexpression enhanced SARS-CoV-2 infection as well as pseudovirus infection, suggesting a role in viral entry. Furthermore, single-cell RNA-sequencing of airway cells from patients with COVID-19 demonstrated that TMEM106B expression correlates with SARS-CoV-2 infection. The present study uncovered a collection of coronavirus host factors that may be exploited to develop drugs against SARS-CoV-2 infection or future zoonotic coronavirus outbreaks.
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COVID-19/genética , Sistemas CRISPR-Cas , Genoma Humano/genética , Estudio de Asociación del Genoma Completo/métodos , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Líquido del Lavado Bronquioalveolar/citología , COVID-19/epidemiología , COVID-19/virología , Línea Celular Tumoral , Células Cultivadas , Coronavirus Humano 229E/genética , Epidemias , Células Epiteliales/virología , Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Provirus/fisiología , SARS-CoV-2/fisiología , Internalización del VirusRESUMEN
Increasingly, repeat expansions are being identified as part of the complex genetic architecture of amyotrophic lateral sclerosis. To date, several repeat expansions have been genetically associated with the disease: intronic repeat expansions in C9orf72, polyglutamine expansions in ATXN2 and polyalanine expansions in NIPA1. Together with previously published data, the identification of an amyotrophic lateral sclerosis patient with a family history of spinocerebellar ataxia type 1, caused by polyglutamine expansions in ATXN1, suggested a similar disease association for the repeat expansion in ATXN1. We, therefore, performed a large-scale international study in 11 700 individuals, in which we showed a significant association between intermediate ATXN1 repeat expansions and amyotrophic lateral sclerosis (P = 3.33 × 10-7). Subsequent functional experiments have shown that ATXN1 reduces the nucleocytoplasmic ratio of TDP-43 and enhances amyotrophic lateral sclerosis phenotypes in Drosophila, further emphasizing the role of polyglutamine repeat expansions in the pathophysiology of amyotrophic lateral sclerosis.
RESUMEN
Patient mortality rates have remained stubbornly high (40%) for the past 35 years in head and neck squamous cell carcinoma (HNSCC) due to inherent or acquired drug resistance. Thus, a critical issue in advanced SCC is to identify and target the mechanisms that contribute to therapy resistance. We report that the transcriptional inhibitor, E2F7, is mislocalized to the cytoplasm in >80% of human HNSCCs, whereas the transcriptional activator, E2F1, retains localization to the nucleus in SCC. This results in an imbalance in the control of E2F-dependent targets such as SPHK1, which is derepressed and drives resistance to anthracyclines in HNSCC. Specifically, we show that (i) E2F7 is subject to exportin 1 (XPO1)-dependent nuclear export, (ii) E2F7 is selectively mislocalized in most of SCC and multiple other tumor types, (iii) mislocalization of E2F7 in HNSCC causes derepression of Sphk1 and drives anthracycline resistance, and (iv) anthracycline resistance can be reversed with a clinically available inhibitor of XPO1, selinexor, in xenotransplant models of HNSCC. Thus, we have identified a strategy to repurpose anthracyclines for use in SCC. More generally, we provide a strategy to restore the balance of E2F1 (activator) and E2F7 (inhibitor) activity in cancer.
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Antraciclinas/farmacología , Núcleo Celular/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Factor de Transcripción E2F7/metabolismo , Carioferinas/antagonistas & inhibidores , Terapia Molecular Dirigida , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Doxorrubicina/farmacología , Factor de Transcripción E2F1/metabolismo , Humanos , Carioferinas/metabolismo , Ratones Endogámicos NOD , Ratones SCID , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteína Exportina 1RESUMEN
Unraveling the mechanism of action and molecular target of small molecules remains a major challenge in drug discovery. While many cancer drugs target genetic vulnerabilities, loss-of-function screens fail to identify essential genes in drug mechanism of action. Here, we report CRISPRres, a CRISPR-Cas-based genetic screening approach to rapidly derive and identify drug resistance mutations in essential genes. It exploits the local genetic variation created by CRISPR-Cas-induced non-homologous end-joining (NHEJ) repair to generate a wide variety of functional in-frame mutations. Using large sgRNA tiling libraries and known drug-target pairs, we validate it as a target identification approach. We apply CRISPRres to the anticancer agent KPT-9274 and identify nicotinamide phosphoribosyltransferase (NAMPT) as its main target. These results present a powerful and simple genetic approach to create many protein variants that, in combination with positive selection, can be applied to reveal the cellular target of small-molecule inhibitors.
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Sistemas CRISPR-Cas , Genes Esenciales/genética , Terapia Molecular Dirigida/métodos , Mutagénesis Sitio-Dirigida/métodos , Bibliotecas de Moléculas Pequeñas/farmacología , Acrilamidas/farmacología , Aminopiridinas/farmacología , Antineoplásicos/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Resistencia a Medicamentos/genética , Células HCT116 , Células HL-60 , Humanos , Células K562 , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Nicotinamida Fosforribosiltransferasa/genéticaRESUMEN
Purpose: Human exportin-1 (XPO1) is the key nuclear-cytoplasmic transport protein that exports different cargo proteins out of the nucleus. Inducing nuclear accumulation of these proteins by inhibiting XPO1 causes cancer cell death. First clinical validation of pharmacological inhibition of XPO1 was obtained with the Selective Inhibitor of Nuclear Export (SINE) compound selinexor (KPT-330) demonstrating activity in phase-II/IIb clinical trials when dosed 1 to 3 times weekly. The second-generation SINE compound KPT-8602 shows improved tolerability and can be dosed daily. Here, we investigate and validate the drug-target interaction of KPT-8602 and explore its activity against acute lymphoblastic leukemia (ALL).Experimental Design: We examined the effect of KPT-8602 on XPO1 function and XPO1-cargo as well as on a panel of leukemia cell lines. Mutant XPO1 leukemia cells were designed to validate KPT-8602's drug-target interaction. In vivo, anti-ALL activity was measured in a mouse ALL model and patient-derived ALL xenograft models.Results: KPT-8602 induced caspase-dependent apoptosis in a panel of leukemic cell lines in vitro Using CRISPR/Cas9 genome editing, we demonstrated the specificity of KPT-8602 for cysteine 528 in the cargo-binding groove of XPO1 and validated the drug target interaction. In vivo, KPT-8602 showed potent anti-leukemia activity in a mouse ALL model as well as in patient-derived T- and B-ALL xenograft models without affecting normal hematopoiesis.Conclusions: KPT-8602 is highly specific for XPO1 inhibition and demonstrates potent anti-leukemic activity supporting clinical application of the second-generation SINE compound for the treatment of ALL. Clin Cancer Res; 23(10); 2528-41. ©2016 AACR.
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Antineoplásicos/administración & dosificación , Carioferinas/antagonistas & inhibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Tiazoles/administración & dosificación , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Sistemas CRISPR-Cas , Línea Celular Tumoral , Edición Génica , Humanos , Carioferinas/genética , Ratones , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Receptores Citoplasmáticos y Nucleares/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína Exportina 1Asunto(s)
Apoptosis/efectos de los fármacos , Hidrazinas/farmacología , Carioferinas/antagonistas & inhibidores , Leucemia-Linfoma de Células T del Adulto/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Triazoles/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Antineoplásicos/farmacología , Línea Celular Tumoral , Humanos , Carioferinas/metabolismo , Leucemia-Linfoma de Células T del Adulto/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteína Exportina 1RESUMEN
Validation of drug-target interaction is essential in drug discovery and development. The ultimate proof for drug-target validation requires the introduction of mutations that confer resistance in cells, an approach that is not straightforward in mammalian cells. Using CRISPR/Cas9 genome editing, we show that a homozygous genomic C528S mutation in the XPO1 gene confers cells with resistance to selinexor (KPT-330). Selinexor is an orally bioavailable inhibitor of exportin-1 (CRM1/XPO1) with potent anticancer activity and is currently under evaluation in human clinical trials. Mutant cells were resistant to the induction of cytotoxicity, apoptosis, cell cycle arrest, and inhibition of XPO1 function, including direct binding of the drug to XPO1. These results validate XPO1 as the prime target of selinexor in cells and identify the selectivity of this drug toward the cysteine 528 residue of XPO1. Our findings demonstrate that CRISPR/Cas9 genome editing enables drug-target validation and drug-target selectivity studies in cancer cells.
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Antineoplásicos/química , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Hidrazinas/química , Carioferinas/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Triazoles/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Secuencia de Bases , Puntos de Control del Ciclo Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Recombinación Homóloga , Humanos , Hidrazinas/metabolismo , Hidrazinas/farmacología , Células Jurkat , Carioferinas/genética , Carioferinas/metabolismo , Cinética , Mutación , Unión Proteica , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Triazoles/metabolismo , Triazoles/farmacología , Proteína Exportina 1RESUMEN
Infection with HIV ultimately leads to advanced immunodeficiency resulting in an increased incidence of cancer. For example primary effusion lymphoma (PEL) is an aggressive non-Hodgkin lymphoma with very poor prognosis that typically affects HIV infected individuals in advanced stages of immunodeficiency. Here we report on the dual anti-HIV and anti-PEL effect of targeting a single process common in both diseases. Inhibition of the exportin-1 (XPO1) mediated nuclear transport by clinical stage orally bioavailable small molecule inhibitors (SINE) prevented the nuclear export of the late intron-containing HIV RNA species and consequently potently suppressed viral replication. In contrast, in CRISPR-Cas9 genome edited cells expressing mutant C528S XPO1, viral replication was unaffected upon treatment, clearly demonstrating the anti-XPO1 mechanism of action. At the same time, SINE caused the nuclear accumulation of p53 tumor suppressor protein as well as inhibition of NF-κB activity in PEL cells resulting in cell cycle arrest and effective apoptosis induction. In vivo, oral administration arrested PEL tumor growth in engrafted mice. Our findings provide strong rationale for inhibiting XPO1 as an innovative strategy for the combined anti-retroviral and anti-neoplastic treatment of HIV and PEL and offer perspectives for the treatment of other AIDS-associated cancers and potentially other virus-related malignancies.