RESUMO
As a potent and convenient genome-editing tool, Cas9 has been widely used in biomedical research and evaluated in treating human diseases. Numerous engineered variants of Cas9, dCas9 and other related prokaryotic endonucleases have been identified. However, as these bacterial enzymes are not naturally present in mammalian cells, whether and how bacterial Cas9 proteins are recognized and regulated by mammalian hosts remain poorly understood. Here, we identify Keap1 as a mammalian endogenous E3 ligase that targets Cas9/dCas9/Fanzor for ubiquitination and degradation in an 'ETGE'-like degron-dependent manner. Cas9-'ETGE'-like degron mutants evading Keap1 recognition display enhanced gene editing ability in cells. dCas9-'ETGE'-like degron mutants exert extended protein half-life and protein retention on chromatin, leading to improved CRISPRa and CRISPRi efficacy. Moreover, Cas9 binding to Keap1 also impairs Keap1 function by competing with Keap1 substrates or binding partners for Keap1 binding, while engineered Cas9 mutants show less perturbation of Keap1 biology. Thus, our study reveals a mammalian specific Cas9 regulation and provides new Cas9 designs not only with enhanced gene regulatory capacity but also with minimal effects on disrupting endogenous Keap1 signaling.
Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Edição de Genes , Proteína 1 Associada a ECH Semelhante a Kelch , Ubiquitinação , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Edição de Genes/métodos , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Células HEK293 , Epigenoma , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Engenharia de Proteínas/métodos , Cromatina/metabolismo , Cromatina/genéticaRESUMO
BACKGROUND: In preclinical pancreatic ductal adenocarcinoma (PDAC) models, inhibition of hepatocyte growth factor (HGF) signaling using ficlatuzumab, a recombinant humanized anti-HGF antibody, and gemcitabine reduced tumor burden. METHODS: Patients with previously untreated metastatic PDAC enrolled in a phase Ib dose escalation study with 3 + 3 design of 2 dose cohorts of ficlatuzumab 10 and 20 mg/kg administered intravenously every other week with gemcitabine 1000 mg/m2 and albumin-bound paclitaxel 125 mg/m2 given 3 weeks on and 1 week off. This was followed by an expansion phase at the maximally tolerated dose of the combination. RESULTS: Twenty-six patients (sex, 12 male:14 female; median age, 68 years [range, 49-83 years]) were enrolled, 22 patients were evaluable. No dose-limiting toxicities were identified (N = 7 pts) and ficlatuzumab at 20 mg/kg was chosen as the maximum tolerated dose. Among the 21 patients treated at the MTD, best response by RECISTv1.1: 6 (29%) partial response, 12 (57%) stable disease, 1 (5%) progressive disease, and 2 (9%) not evaluable. Median progression-free survival and overall survival times were 11.0 months (95% CI, 7.6-11.4 months) and 16.2 months (95% CI, 9.1 months to not reached), respectively. Toxicities attributed to ficlatuzumab included hypoalbuminemia (grade 3, 16%; any grade, 52%) and edema (grade 3, 8%; any grade, 48%). Immunohistochemistry for c-Met pathway activation demonstrated higher tumor cell p-Met levels in patients who experienced response to therapy. CONCLUSION: In this phase Ib trial, ficlatuzumab, gemcitabine, and albumin-bound paclitaxel were associated with durable treatment responses and increased rates of hypoalbuminemia and edema.
Assuntos
Hipoalbuminemia , Neoplasias Pancreáticas , Humanos , Masculino , Feminino , Idoso , Gencitabina , Paclitaxel Ligado a Albumina , Hipoalbuminemia/induzido quimicamente , Paclitaxel/efeitos adversos , Neoplasias Pancreáticas/patologia , Albuminas/efeitos adversos , Edema/etiologia , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Neoplasias PancreáticasRESUMO
The interpretation of histone post-translational modifications (PTMs), specifically lysine methylation, by specific classes of "reader" proteins marks an important aspect of epigenetic control of gene expression. Methyl-lysine (Kme) readers often regulate gene expression patterns through the recognition of a specific Kme PTM while participating in or recruiting large protein complexes that contain enzymatic or chromatin remodeling activity. Understanding the composition of these Kme-reader-containing protein complexes can serve to further our understanding of the biological roles of Kme readers, while small molecule chemical tools can be valuable reagents in interrogating novel protein-protein interactions. Here, we describe our efforts to target the chromodomain of M-phase phosphoprotein 8 (MPP8), a member of the human silencing hub (HUSH) complex and a histone 3 lysine 9 trimethyl (H3K9me3) reader that is vital for heterochromatin formation and has specific roles in cancer metastasis. Utilizing a one-bead, one-compound (OBOC) combinatorial screening approach, we identified UNC5246, a peptidomimetic ligand capable of interacting with the MPP8 chromodomain in the context of the HUSH complex. Additionally, a biotinylated derivative of UNC5246 facilitated chemoproteomics studies which revealed hepatoma-derived growth factor-related protein 2 (HRP2) as a novel protein associated with MPP8. HRP2 was further shown to colocalize with MPP8 at the E-cadherin gene locus, suggesting a possible role in cancer cell plasticity.
Assuntos
Proteínas de Ciclo Celular/química , Peptidomiméticos/química , Fosfoproteínas/química , Proteínas de Ciclo Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Histonas/química , Interações Hidrofóbicas e Hidrofílicas , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ligantes , Lisina/química , Espectrometria de Massas , Metilação , Modelos Moleculares , Peptidomiméticos/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Proteômica , Relação Estrutura-AtividadeRESUMO
ATP hydrolysis and downstream signaling pathways in the extracellular space have a major impact upon tumor progression and metastasis. The complexity and interdependence of various cell types in the extracellular space have been increasingly appreciated in recent years. With increased awareness of the importance of this signaling pathway in the pathogenic development and progression of malignancies, there has been attention to therapeutic strategies targeting extracellular adenosine metabolism and signaling. This review summarizes the molecular and physiologic roles of extracellular ATP and adenosine in normal and disease states, and potential therapeutic applications.
Assuntos
Trifosfato de Adenosina/metabolismo , Adenosina/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Linhagem Celular Tumoral , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/imunologia , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Intervalo Livre de Progressão , Antagonistas de Receptores Purinérgicos P1/farmacologia , Antagonistas de Receptores Purinérgicos P1/uso terapêutico , Antagonistas do Receptor Purinérgico P2/farmacologia , Antagonistas do Receptor Purinérgico P2/uso terapêutico , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos P2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Esophageal squamous cell carcinoma (ESCC) is one of the most lethal cancers worldwide and evolves often to lung metastasis. P53R175H (homologous to Trp53R172H in mice) is a common hot spot mutation. How metastasis is regulated by p53R175H in ESCC remains to be investigated. To investigate p53R175H-mediated molecular mechanisms, we used a carcinogen-induced approach in Trp53R172H/- mice to model ESCC. In the primary Trp53R172H/- tumor cell lines, we depleted Trp53R172H (shTrp53) and observed a marked reduction in cell invasion in vitro and lung metastasis burden in a tail-vein injection model in comparing isogenic cells (shCtrl). Furthermore, we performed bulk RNA-seq to compare gene expression profiles of metastatic and primary shCtrl and shTrp53 cells. We identified the YAP-BIRC5 axis as a potential mediator of Trp53R172H -mediated metastasis. We demonstrate that expression of Survivin, an antiapoptotic protein encoded by BIRC5, increases in the presence of Trp53R172H Furthermore, depletion of Survivin specifically decreases Trp53R172H-driven lung metastasis. Mechanistically, Trp53R172H but not wild-type Trp53, binds with YAP in ESCC cells, suggesting their cooperation to induce Survivin expression. Furthermore, Survivin high expression level is associated with increased metastasis in several GI cancers. Taken together, this study unravels new insights into how mutant p53 mediates metastasis.
Assuntos
Neoplasias Pulmonares/fisiopatologia , Survivina/genética , Survivina/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Pulmonares/genética , Camundongos , Mutação , Metástase Neoplásica , Transcriptoma , Proteína Supressora de Tumor p53/metabolismoRESUMO
Pancreatic cancer metastasis is a leading cause of cancer-related deaths, yet very little is understood regarding the underlying biology. As a result, targeted therapies to inhibit metastasis are lacking. Here, we report that the parathyroid hormone-related protein (PTHrP encoded by PTHLH) is frequently amplified as part of the KRAS amplicon in patients with pancreatic cancer. PTHrP upregulation drives the growth of both primary and metastatic tumors in mice and is highly enriched in pancreatic ductal adenocarcinoma metastases. Loss of PTHrP-either genetically or pharmacologically-dramatically reduces tumor burden, eliminates metastasis, and enhances overall survival. These effects are mediated in part through a reduction in epithelial-to-mesenchymal transition, which reduces the ability of tumor cells to initiate metastatic cascade. Spp1, which encodes osteopontin, is revealed to be a downstream effector of PTHrP. Our results establish a new paradigm in pancreatic cancer whereby PTHrP is a driver of disease progression and emerges as a novel therapeutic vulnerability. SIGNIFICANCE: Pancreatic cancer often presents with metastases, yet no strategies exist to pharmacologically inhibit this process. Herein, we establish the oncogenic and prometastatic roles of PTHLH, a novel amplified gene in pancreatic ductal adenocarcinoma. We demonstrate that blocking PTHrP activity reduces primary tumor growth, prevents metastasis, and prolongs survival in mice.This article is highlighted in the In This Issue feature, p. 1601.
Assuntos
Neoplasias Pancreáticas/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Metástase Neoplásica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteína Relacionada ao Hormônio Paratireóideo/antagonistas & inibidores , Proteína Relacionada ao Hormônio Paratireóideo/genéticaRESUMO
Cancer cell identity and plasticity are required in transition states, such as epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET), in primary tumor initiation, progression, and metastasis. The functional roles of EMT, MET, and the partial state (referred to as pEMT) may vary based on the type of tumor, the state of dissemination, and the degree of metastatic colonization. Herein, we review EMT, MET, pEMT, and plasticity in the context of tumor metastasis.
Assuntos
Plasticidade Celular , Transição Epitelial-Mesenquimal , Neoplasias/patologia , Animais , Modelos Animais de Doenças , Humanos , Modelos Biológicos , Metástase NeoplásicaRESUMO
Proper regulation of the chromatin landscape is essential for maintaining eukaryotic cell identity and diverse cellular processes. The importance of the epigenome comes, in part, from the ability to influence gene expression through patterns in DNA methylation, histone tail modification, and chromatin architecture. Decades of research have associated this process of chromatin regulation and gene expression with human diseased states. With the goal of understanding how chromatin dysregulation contributes to disease, as well as preventing or reversing this type of dysregulation, a multidisciplinary effort has been launched to control the epigenome. Chemicals that alter the epigenome have been used in labs and in clinics since the 1970s, but more recently there has been a shift in this effort towards manipulating the chromatin landscape in a locus-specific manner. This review will provide an overview of chromatin biology to set the stage for the type of control being discussed, evaluate the recent technological advances made in controlling specific regions of chromatin, and consider the translational applications of these works.
Assuntos
Cromatina/genética , Epigênese Genética , Metilação de DNA , Regulação da Expressão Gênica , Histonas/metabolismo , HumanosRESUMO
Gene expression can be activated or suppressed using CRISPR--Cas9 systems. However, tools that enable dose-dependent activation of gene expression without the use of exogenous transcription regulatory proteins are lacking. Here we describe chemical epigenetic modifiers (CEMs) designed to activate the expression of target genes by recruiting components of the endogenous chromatin-activating machinery, eliminating the need for exogenous transcriptional activators. The system has two parts: catalytically inactive Cas9 (dCas9) in complex with FK506-binding protein (FKBP) and a CEM consisting of FK506 linked to a molecule that interacts with cellular epigenetic machinery. We show that CEMs upregulate gene expression at target endogenous loci up to 20-fold or more depending on the gene. We also demonstrate dose-dependent control of transcriptional activation, function across multiple diverse genes, reversibility of CEM activity and specificity of our best-in-class CEM across the genome.
Assuntos
Sistemas CRISPR-Cas/genética , Cromatina/metabolismo , Regulação da Expressão Gênica , Proteínas de Ciclo Celular/metabolismo , Epigênese Genética , Genoma Humano , Células HEK293 , Humanos , RNA Guia de Cinetoplastídeos/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismoRESUMO
Core regulatory transcription factors (CR TFs) orchestrate the placement of super-enhancers (SEs) to activate transcription of cell-identity specifying gene networks, and are critical in promoting cancer. Here, we define the core regulatory circuitry of rhabdomyosarcoma and identify critical CR TF dependencies. These CR TFs build SEs that have the highest levels of histone acetylation, yet paradoxically the same SEs also harbor the greatest amounts of histone deacetylases. We find that hyperacetylation selectively halts CR TF transcription. To investigate the architectural determinants of this phenotype, we used absolute quantification of architecture (AQuA) HiChIP, which revealed erosion of native SE contacts, and aberrant spreading of contacts that involved histone acetylation. Hyperacetylation removes RNA polymerase II (RNA Pol II) from core regulatory genetic elements, and eliminates RNA Pol II but not BRD4 phase condensates. This study identifies an SE-specific requirement for balancing histone modification states to maintain SE architecture and CR TF transcription.
Assuntos
Histonas/metabolismo , Rabdomiossarcoma/genética , Fatores de Transcrição/genética , Acetilação , Benzamidas/farmacologia , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Elementos Facilitadores Genéticos , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/genética , Humanos , Piridinas/farmacologia , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Estabilidade de RNA , Fatores de Transcrição SOXE/genética , Análise de Célula ÚnicaRESUMO
Adeno-associated viruses (AAV) are Dependoparvoviruses that have shown promise as recombinant vectors for gene therapy. While infectious pathways of AAV are well studied, gaps remain in our understanding of host factors affecting vector genome expression. Here, we map the role of ring finger protein 121 (RNF121), an E3 ubiquitin ligase, as a key regulator of AAV genome transcription. CRISPR-mediated knockout of RNF121 (RNF121 KO) in different cells markedly decreased AAV transduction regardless of capsid serotype or vector dose. Recombinant AAV transduction is partially rescued by overexpressing RNF121, but not by co-infection with helper Adenovirus. Major steps in the AAV infectious pathway including cell surface binding, cellular uptake, nuclear entry, capsid uncoating and second strand synthesis are unaffected. While gene expression from transfected plasmids or AAV genomes is unaffected, mRNA synthesis from AAV capsid-associated genomes is markedly decreased in RNF121 KO cells. These observations were attributed to transcriptional arrest as corroborated by RNAPol-ChIP and mRNA half-life measurements. Although AAV capsid proteins do not appear to be direct substrates of RNF121, the catalytic domain of the E3 ligase appears essential. Inhibition of ubiquitin-proteasome pathways revealed that blocking Valosin Containing Protein (VCP/p97), which targets substrates to the proteasome, can selectively and completely restore AAV-mediated transgene expression in RNF121 KO cells. Expanding on this finding, transcriptomic and proteomic analysis revealed that the catalytic subunit of DNA PK (DNAPK-Cs), a known activator of VCP, is upregulated in RNF121 KO cells and that the DNA damage machinery is enriched at sites of stalled AAV genome transcription. We postulate that a network of RNF121, VCP and DNA damage response elements function together to regulate transcriptional silencing and/or activation of AAV vector genomes.
Assuntos
Carcinoma Hepatocelular/virologia , Proteína Quinase Ativada por DNA/metabolismo , Dependovirus/genética , Genoma Viral , Proteínas de Membrana/metabolismo , Transdução Genética , Proteína com Valosina/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proteína Quinase Ativada por DNA/genética , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteoma , Transcriptoma , Células Tumorais Cultivadas , Ubiquitina/metabolismo , Ubiquitinação , Proteína com Valosina/genética , Internalização do VírusRESUMO
Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.
Assuntos
Cromatina/metabolismo , Epigenômica , Regulação da Expressão Gênica , Histonas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Acetilação , Animais , Imunoprecipitação da Cromatina , DNA/metabolismo , Citometria de Fluxo , Células HEK293 , Humanos , Camundongos , Microscopia de Fluorescência , Transcrição GênicaRESUMO
One of the most sensitive, time-consuming, and variable steps of chromatin immunoprecipitation (ChIP) is chromatin sonication. Traditionally, this process can take hours to properly sonicate enough chromatin for multiple ChIP assays. Further, the length of sheared DNA is often inconsistent. In order to faithfully measure chemical and structural changes at the chromatin level, sonication needs to be reliable. Thus, chromatin fragmentation by sonication represents a significant bottleneck to downstream quantitative analysis. To improve the consistency and efficiency of chromatin sonication, we developed and tested a cavitation enhancing reagent based on sonically active nanodroplets. Here, we show that nanodroplets increase sonication efficiency by 16-fold and provide more consistent levels of chromatin fragmentation. Using the previously characterized chromatin in vivo assay (CiA) platform, we generated two distinct chromatin states in order to test nanodroplet-assisted sonication sensitivity in measuring post-translational chromatin marks. By comparing euchromatin to chemically induced heterochromatin at the same CiA:Oct4 locus, we quantitatively measure the capability of our new sonication technique to resolve differences in chromatin structure. We confirm that nanodroplet-assisted sonication results are indistinguishable from those of samples processed with traditional sonication in downstream applications. While the processing time for each sample was reduced from 38.4 to 2.3 min, DNA fragment distribution sizes were significantly more consistent with a coefficient of variation 2.7 times lower for samples sonicated in the presence of nanodroplets. In conclusion, sonication utilizing the nanodroplet cavitation enhancement reagent drastically reduces the amount of processing time and provides consistently fragmented chromatin of high quality for downstream applications.
Assuntos
Imunoprecipitação da Cromatina/métodos , Fragmentação do DNA/efeitos da radiação , DNA/genética , Sonicação/métodos , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cromatina/efeitos da radiação , DNA/química , DNA/efeitos da radiação , Eucromatina/efeitos da radiação , Heterocromatina/efeitos da radiação , Camundongos , Nanopartículas/química , Fator 3 de Transcrição de Octâmero/química , Fator 3 de Transcrição de Octâmero/genéticaRESUMO
Epigenome editing is a powerful method for life science research and could give rise to new therapies for diseases initiated or maintained by epigenetic dysregulation, including several types of cancers and autoimmune disorders. In addition, much is still unknown about the mechanisms by which histone-modifying proteins work in concert to properly regulate gene expression. To investigate and manipulate complex epigenetic interactions in live cells, we have developed a small molecule platform for specifically inducing gene repression and histone deacetylation at a reporter gene. We synthesized bifunctional ligands, or chemical epigenetic modifiers (CEMs), that contain two functional groups: a FK506 derivative capable of binding to a FKBP-Gal4 fusion transcription factor, and a histone deacetylase (HDAC) inhibitor that recruits HDAC-containing corepressor complexes. In our reporter cell line, which contains a GFP reporter allele upstream of a Gal4 DNA binding array in the murine Oct4 locus, our lead CEM repressed GFP expression by 50%. We also show that CEM recruitment of deacetylation activity causes marked deacetylation along our target loci. This system allowed us to detail the direct results of deacetylation to chromatin and measure the resulting gene expression in a chemically dependent and reversible manner. The CEMs system provides new insights into epigenetic gene regulation and has the potential to control disease-relevant gene regulation. The CEMs are derived from FDA-approved epigenetic modulator drugs, and use their pharmacology in a gene-specific way that avoids the toxicities and off-target effects caused by whole-cell application of these drugs.