RESUMO
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.
Assuntos
Colesterol/metabolismo , Vírus Lassa/fisiologia , Vírus Lassa/patogenicidade , Proteínas de Membrana Lisossomal/fisiologia , Receptores Virais/fisiologia , Adamantano/análogos & derivados , Adamantano/química , Adamantano/farmacologia , Animais , Antivirais/química , Antivirais/farmacologia , Linhagem Celular , Chlorocebus aethiops , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Febre Lassa/etiologia , Vírus Lassa/efeitos dos fármacos , Proteínas de Membrana Lisossomal/antagonistas & inibidores , Proteínas de Membrana Lisossomal/genética , Modelos Moleculares , Mutação , Estabilidade Proteica , Estrutura Terciária de Proteína , Receptores Virais/antagonistas & inibidores , Receptores Virais/genética , Células Vero , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/fisiologia , Internalização do Vírus/efeitos dos fármacosRESUMO
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.
Assuntos
Carcinoma de Célula de Merkel , Poliomavírus das Células de Merkel , Infecções por Polyomavirus , Neoplasias Cutâneas , Infecções Tumorais por Vírus , Humanos , Carcinoma de Célula de Merkel/genética , Carcinoma de Célula de Merkel/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Poliomavírus das Células de Merkel/genética , Peptídeos e Proteínas de Sinalização Intracelular , Linhagem Celular , Infecções por Polyomavirus/genética , Infecções Tumorais por Vírus/genética , Proteínas com Motivo de Ligação a PDZ com Coativador TranscricionalRESUMO
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.
Assuntos
Carcinoma de Célula de Merkel , Neoplasias Cutâneas , Carcinoma de Célula de Merkel/tratamento farmacológico , Carcinoma de Célula de Merkel/genética , Cicloexilaminas , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Complexo Repressor Polycomb 2/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologiaRESUMO
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.