RESUMEN
Lassa fever virus (LASV) is endemic in West Africa and causes severe hemorrhagic fever and sensorineural hearing loss. We identified a small molecule inhibitor of LASV and used it to analyze the mechanism of entry. Using a photo-reactive analog that retains antiviral activity as a probe, we identified the inhibitor target as lysosome-associated membrane protein 1 (LAMP1), a host factor that binds to the LASV glycoprotein (GP) during infection. We found that LAMP1 binding to LASV GP is cholesterol-dependent, and that the inhibitor blocks infection by competing with cholesterol in LAMP1. Mutational analysis of a docking-based model identified a putative inhibitor binding site in the cholesterol-binding pocket within the LAMP1 domain that binds GP. These findings identify a critical role for cholesterol in LASV entry and a potential target for therapeutic intervention.
Asunto(s)
Colesterol/metabolismo , Virus Lassa/fisiología , Virus Lassa/patogenicidad , Proteínas de Membrana de los Lisosomas/fisiología , Receptores Virales/fisiología , Adamantano/análogos & derivados , Adamantano/química , Adamantano/farmacología , Animales , Antivirales/química , Antivirales/farmacología , Línea Celular , Chlorocebus aethiops , Células HEK293 , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/fisiología , Humanos , Fiebre de Lassa/etiología , Virus Lassa/efectos de los fármacos , Proteínas de Membrana de los Lisosomas/antagonistas & inhibidores , Proteínas de Membrana de los Lisosomas/genética , Modelos Moleculares , Mutación , Estabilidad Proteica , Estructura Terciaria de Proteína , Receptores Virales/antagonistas & inhibidores , Receptores Virales/genética , Células Vero , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/fisiología , Internalización del Virus/efectos de los fármacosRESUMEN
BackgroundMerkel cell carcinoma (MCC) is an aggressive neuroendocrine (NE) skin cancer caused by severe UV-induced mutations or expression of Merkel cell polyomavirus (MCPyV) large and small T antigens (LT and ST). Despite deep genetic differences between MCPyV-positive and -negative subtypes, current clinical diagnostic markers are indistinguishable, and the expression profile of MCC tumors is, to our knowledge, unexplored.MethodsHere, we leveraged bulk and single-cell RNA-Seq of patient-derived tumor biopsies and cell lines to explore the underlying transcriptional environment of MCC.ResultsStrikingly, MCC samples could be separated into transcriptional subtypes that were independent of MCPyV status. Instead, we observed an inverse correlation between a NE gene signature and the Hippo pathway transcription factors Yes1-associated transcriptional regulator (YAP1) and WW domain-containing transcriptional regulator 1 (WWTR1). This inverse correlation was broadly present at the transcript and protein levels in the tumor biopsies as well as in established and patient-derived cell lines. Mechanistically, expression of YAP1 or WWTR1 in a MCPyV-positive MCC cell line induced cell-cycle arrest at least in part through TEA domain-dependent (TEAD-dependent) transcriptional repression of MCPyV LT.ConclusionThese findings identify what we believe to be a previously unrecognized heterogeneity in NE gene expression within MCC and support a model of YAP1/WWTR1 silencing as essential for the development of MCPyV-positive MCC.FundingUS Public Health Service grants R35CA232128, P01CA203655, and P30CA06516.
Asunto(s)
Carcinoma de Células de Merkel , Poliomavirus de Células de Merkel , Infecciones por Polyomavirus , Neoplasias Cutáneas , Infecciones Tumorales por Virus , Humanos , Carcinoma de Células de Merkel/genética , Carcinoma de Células de Merkel/patología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Poliomavirus de Células de Merkel/genética , Péptidos y Proteínas de Señalización Intracelular , Línea Celular , Infecciones por Polyomavirus/genética , Infecciones Tumorales por Virus/genética , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZRESUMEN
Polycomb repressive complex 2 has a critical role in the maintenance of bivalent promoters and is often perturbed in cancer, including neuroendocrine tumors. In this study, we investigated the susceptibility of Merkel cell carcinoma (MCC), a neuroendocrine carcinoma of the skin, to inhibitors of the Polycomb repressive complex 2 catalytic subunit EZH2. We show that a subset of MCC cell lines is sensitive to EZH2 inhibitor-induced cell viability loss. We find that inhibitor treatment of susceptible cells derepresses the Polycomb repressive complex 2 target SIX1, a transcription factor in the PAX-SIX-EYA-DACH network normally involved in inner ear hair cell development, and that PAX-SIX-EYA-DACH network transcription factors are critical contributors to EZH2 inhibitor-induced MCC cell viability loss. Furthermore, we show the EZH2 inhibitor tazemetostat slows the growth of MCC xenografts and derepresses SIX1 and its downstream inner ear transcriptional target MYO6 in vivo. We propose that EZH2 inhibition in MCC leads to SIX1 derepression with dysregulation of hearing-related transcriptional programs and growth inhibition. This study provides evidence that MCC tumors may be specifically susceptible to EZH2 inhibitors, while giving mechanistic insight into the transcriptional programs these inhibitors perturb in MCC, and potentially in other neuroendocrine cancers.
Asunto(s)
Carcinoma de Células de Merkel , Neoplasias Cutáneas , Carcinoma de Células de Merkel/tratamiento farmacológico , Carcinoma de Células de Merkel/genética , Ciclohexilaminas , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Complejo Represivo Polycomb 2/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patologíaRESUMEN
Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I-low, neuroendocrine carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.