RESUMO
Merkel cell polyomavirus (MCV) contributes to approximately 80% of all Merkel cell carcinomas (MCCs), a highly aggressive neuroendocrine carcinoma of the skin. MCV-positive MCC expresses small T antigen (ST) and a truncated form of large T antigen (LT) and usually contains wild-type p53 (TP53) and RB (RB1). In contrast, virus-negative MCC contains inactivating mutations in TP53 and RB1. While the MCV-truncated LT can bind and inhibit RB, it does not bind p53. We report here that MCV LT binds to RB, leading to increased levels of ARF, an inhibitor of MDM2, and activation of p53. However, coexpression of ST reduced p53 activation. MCV ST recruits the MYC homologue MYCL (L-Myc) to the EP400 chromatin remodeler complex and transactivates specific target genes. We observed that depletion of EP400 in MCV-positive MCC cell lines led to increased p53 target gene expression. We suspected that the MCV ST-MYCL-EP400 complex could functionally inactivate p53, but the underlying mechanism was not known. Integrated ChIP and RNA-sequencing analysis following EP400 depletion identified MDM2 as well as CK1α, an activator of MDM4, as target genes of the ST-MYCL-EP400 complex. In addition, MCV-positive MCC cells expressed high levels of MDM4. Combining MDM2 inhibitors with lenalidomide targeting CK1α or an MDM4 inhibitor caused synergistic activation of p53, leading to an apoptotic response in MCV-positive MCC cells and MCC-derived xenografts in mice. These results support dual targeting of MDM2 and MDM4 in virus-positive MCC and other p53 wild-type tumors.
Assuntos
Carcinoma de Célula de Merkel/metabolismo , Poliomavírus das Células de Merkel/metabolismo , Proteínas Nucleares/metabolismo , Infecções por Polyomavirus/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Infecções Tumorais por Vírus/metabolismo , Carcinoma de Célula de Merkel/genética , Carcinoma de Célula de Merkel/patologia , Carcinoma de Célula de Merkel/virologia , Proteínas de Ciclo Celular , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Poliomavírus das Células de Merkel/genética , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Infecções por Polyomavirus/genética , Infecções por Polyomavirus/patologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Proteína Supressora de Tumor p53/genética , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/patologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Adeno-associated viral vectors (AAV) are frequently used by neuroscientists to deliver tools, such as biosensors and optogenetic and chemogenetic actuators, in vivo. Despite its widespread use, AAV vector characterization and quality control can vary between labs and viral vector cores leading to variable results and irreproducibility. This protocol describes some of the characterization and quality control assays necessary to confirm an AAV vector's titer, genomic identity, serotype and purity.
RESUMO
Recombinant adeno-associated virus (rAAV) vectors are increasingly popular gene delivery tools in biological systems. They are safe and lead to high-level, long-term transgene expression. rAAV are available in multiple serotypes, natural or engineered, which enable targeting to a wide array of tissues and cell types. In addition, rAAVs are relatively easily produced in a well-equipped lab or obtained from a viral vector core facility. Unfortunately, there is no standardization of quality control assays beyond titering and purity assessments. Next-generation sequencing (NGS) can be used to identify rAAV preparations. Because the rAAV genome is single stranded, previous studies have assumed that rAAV genomes must be converted to double strands before NGS. We demonstrate that rAAV DNA extracts exist primarily as double-stranded species. We hypothesize that these molecules form from the natural base pairing of complementary [+] and [-] strands after DNA extraction and show that rAAV DNA extracts are sufficient templates for downstream NGS without the labor-intensive double-stranding step. Here, we provide a detailed protocol for the simple and rapid NGS of rAAV genomes from DNA extracts. With this protocol, users can quickly confirm the identity of an rAAV preparation and detect the presence of contaminating rAAV DNA. In addition, we share custom Python scripts that allow users to accurately determine the serotype and detect Cre-independent DNA recombination events in rAAV containing Lox sites within minutes. We have used these scripts to analyze more than 100 rAAV preparations. Although we focused on the detection of cross-contaminating rAAV DNA and recombination events, our Python scripts can be customized to detect other sequences or events, such as reverse packaging of plasmid backbone or DNA from the packaging cell line. We find that the NGS of rAAV DNA extracts, termed viral genome sequencing, is a simple and powerful method for rAAV validation.
Assuntos
DNA Viral/isolamento & purificação , Dependovirus/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Linhagem Celular , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos , Genoma Viral , Células HEK293 , Humanos , Plasmídeos , TransgenesRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Merkel cell carcinoma (MCC)-a neuroendocrine cancer of the skin-is caused by the integration of Merkel cell polyomavirus and persistent expression of large T antigen and small T antigen. We report that small T antigen in complex with MYCL and the EP400 complex activates the expression of LSD1 (KDM1A), RCOR2 and INSM1 to repress gene expression by the lineage transcription factor ATOH1. LSD1 inhibition reduces the growth of MCC in vitro and in vivo. Through a forward-genetics CRISPR-Cas9 screen, we identified an antagonistic relationship between LSD1 and the non-canonical BAF (ncBAF) chromatin remodelling complex. Changes in gene expression and chromatin accessibility caused by LSD1 inhibition were partially rescued by BRD9 inhibition, revealing that LSD1 and ncBAF antagonistically regulate an overlapping set of genes. Our work provides mechanistic insight into the dependence of MCC on LSD1 and a tumour suppressor role for ncBAF in cancer.
RESUMO
Eradicating tumor dormancy that develops following epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) treatment of EGFR-mutant non-small cell lung cancer, is an attractive therapeutic strategy but the mechanisms governing this process are poorly understood. Blockade of ERK1/2 reactivation following EGFR TKI treatment by combined EGFR/MEK inhibition uncovers cells that survive by entering a senescence-like dormant state characterized by high YAP/TEAD activity. YAP/TEAD engage the epithelial-to-mesenchymal transition transcription factor SLUG to directly repress pro-apoptotic BMF, limiting drug-induced apoptosis. Pharmacological co-inhibition of YAP and TEAD, or genetic deletion of YAP1, all deplete dormant cells by enhancing EGFR/MEK inhibition-induced apoptosis. Enhancing the initial efficacy of targeted therapies could ultimately lead to prolonged treatment responses in cancer patients.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Senescência Celular , Receptores ErbB/metabolismo , Feminino , Deleção de Genes , Humanos , Neoplasias Pulmonares/patologia , MAP Quinase Quinase 1/metabolismo , Masculino , Camundongos , Camundongos Knockout , Mutação , Transdução de Sinais , Transcrição Gênica , Proteínas de Sinalização YAPRESUMO
Renal medullary carcinoma (RMC) is a rare and deadly kidney cancer in patients of African descent with sickle cell trait. We have developed faithful patient-derived RMC models and using whole-genome sequencing, we identified loss-of-function intronic fusion events in one SMARCB1 allele with concurrent loss of the other allele. Biochemical and functional characterization of these models revealed that RMC requires the loss of SMARCB1 for survival. Through integration of RNAi and CRISPR-Cas9 loss-of-function genetic screens and a small-molecule screen, we found that the ubiquitin-proteasome system (UPS) was essential in RMC. Inhibition of the UPS caused a G2/M arrest due to constitutive accumulation of cyclin B1. These observations extend across cancers that harbor SMARCB1 loss, which also require expression of the E2 ubiquitin-conjugating enzyme, UBE2C. Our studies identify a synthetic lethal relationship between SMARCB1-deficient cancers and reliance on the UPS which provides the foundation for a mechanism-informed clinical trial with proteasome inhibitors.
Assuntos
Carcinoma Medular/genética , Neoplasias Renais/genética , Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteassoma/farmacologia , Proteína SMARCB1/genética , Alelos , Animais , Sistemas CRISPR-Cas , Carcinoma Medular/tratamento farmacológico , Ciclo Celular , Linhagem Celular Tumoral , Exoma , Feminino , Humanos , Hibridização in Situ Fluorescente , Rim/metabolismo , Neoplasias Renais/tratamento farmacológico , Camundongos , Camundongos Nus , Mutação , Transplante de Neoplasias , Interferência de RNA , Análise de Sequência de RNA , Ubiquitina/química , Sequenciamento Completo do GenomaRESUMO
To identify novel therapeutic targets in acute myeloid leukemia (AML), we examined kinase expression patterns in primary AML samples. We found that the serine/threonine kinase IKBKE, a noncanonical IkB kinase, is expressed at higher levels in myeloid leukemia cells compared with normal hematopoietic cells. Inhibiting IKBKE, or its close homolog TANK-binding kinase 1 (TBK1), by either short hairpin RNA knockdown or pharmacological compounds, induces apoptosis and reduces the viability of AML cells. Using gene expression profiling and gene set enrichment analysis, we found that IKBKE/TBK1-sensitive AML cells typically possess an MYC oncogenic signature. Consistent with this finding, the MYC oncoprotein was significantly downregulated upon IKBKE/TBK1 inhibition. Using proteomic analysis, we found that the oncogenic gene regulator YB-1 was activated by IKBKE/TBK1 through phosphorylation, and that YB-1 binds to the MYC promoter to enhance MYC gene transcription. Momelotinib (CYT387), a pharmacological inhibitor of IKBKE/TBK1, inhibits MYC expression, reduces viability and clonogenicity of primary AML cells, and demonstrates efficacy in a murine model of AML. Together, these data identify IKBKE/TBK1 as a promising therapeutic target in AML.