RESUMO
Phenotypic assays have become an established approach to drug discovery. Greater disease relevance is often achieved through cellular models with increased complexity and more detailed readouts, such as gene expression or advanced imaging. However, the intricate nature and cost of these assays impose limitations on their screening capacity, often restricting screens to well-characterized small compound sets such as chemogenomics libraries. Here, we outline a cheminformatics approach to identify a small set of compounds with likely novel mechanisms of action (MoAs), expanding the MoA search space for throughput limited phenotypic assays. Our approach is based on mining existing large-scale, phenotypic high-throughput screening (HTS) data. It enables the identification of chemotypes that exhibit selectivity across multiple cell-based assays, which are characterized by persistent and broad structure activity relationships (SAR). We validate the effectiveness of our approach in broad cellular profiling assays (Cell Painting, DRUG-seq, and Promotor Signature Profiling) and chemical proteomics experiments. These experiments revealed that the compounds behave similarly to known chemogenetic libraries, but with a notable bias toward novel protein targets. To foster collaboration and advance research in this area, we have curated a public set of such compounds based on the PubChem BioAssay dataset and made it available for use by the scientific community.
Assuntos
Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Bibliotecas de Moléculas Pequenas , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Quimioinformática/métodos , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Targeted protein degradation (TPD) mediates protein level through small molecule induced redirection of E3 ligases to ubiquitinate neo-substrates and mark them for proteasomal degradation. TPD has recently emerged as a key modality in drug discovery. So far only a few ligases have been utilized for TPD. Interestingly, the workhorse ligase CRBN has been observed to be downregulated in settings of resistance to immunomodulatory inhibitory drugs (IMiDs). Here we show that the essential E3 ligase receptor DCAF1 can be harnessed for TPD utilizing a selective, non-covalent DCAF1 binder. We confirm that this binder can be functionalized into an efficient DCAF1-BRD9 PROTAC. Chemical and genetic rescue experiments validate specific degradation via the CRL4DCAF1 E3 ligase. Additionally, a dasatinib-based DCAF1 PROTAC successfully degrades cytosolic and membrane-bound tyrosine kinases. A potent and selective DCAF1-BTK-PROTAC (DBt-10) degrades BTK in cells with acquired resistance to CRBN-BTK-PROTACs while the DCAF1-BRD9 PROTAC (DBr-1) provides an alternative strategy to tackle intrinsic resistance to VHL-degrader, highlighting DCAF1-PROTACS as a promising strategy to overcome ligase mediated resistance in clinical settings.
Assuntos
Proteínas de Transporte , Quimera de Direcionamento de Proteólise , Ubiquitina-Proteína Ligases , Proteínas de Transporte/metabolismo , Proteólise , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Suppression of premature termination codons (PTCs) by translational readthrough is a promising strategy to treat a wide variety of severe genetic diseases caused by nonsense mutations. Here, we present two potent readthrough promoters-NVS1.1 and NVS2.1-that restore substantial levels of functional full-length CFTR and IDUA proteins in disease models for cystic fibrosis and Hurler syndrome, respectively. In contrast to other readthrough promoters that affect stop codon decoding, the NVS compounds stimulate PTC suppression by triggering rapid proteasomal degradation of the translation termination factor eRF1. Our results show that this occurs by trapping eRF1 in the terminating ribosome, causing ribosome stalls and subsequent ribosome collisions, and activating a branch of the ribosome-associated quality control network, which involves the translational stress sensor GCN1 and the catalytic activity of the E3 ubiquitin ligases RNF14 and RNF25.
Assuntos
Fibrose Cística , Biossíntese de Proteínas , Humanos , Códon de Terminação/metabolismo , Códon sem Sentido , Ribossomos/metabolismo , Fibrose Cística/genéticaRESUMO
Unbiased transcriptomic RNA-seq data has provided deep insights into biological processes. However, its impact in drug discovery has been narrow given high costs and low throughput. Proof-of-concept studies with Digital RNA with pertUrbation of Genes (DRUG)-seq demonstrated the potential to address this gap. We extended the DRUG-seq platform by subjecting it to rigorous testing and by adding an open-source analysis pipeline. The results demonstrate high reproducibility and ability to resolve the mechanism(s) of action for a diverse set of compounds. Furthermore, we demonstrate how this data can be incorporated into a drug discovery project aiming to develop therapeutics for schizophrenia using human stem cell-derived neurons. We identified both an on-target activation signature, induced by a set of chemically distinct positive allosteric modulators of the N-methyl-d-aspartate (NMDA) receptor, and independent off-target effects. Overall, the protocol and open-source analysis pipeline are a step toward industrializing RNA-seq for high-complexity transcriptomics studies performed at a saturating scale.
Assuntos
Descoberta de Drogas , Transcriptoma , Descoberta de Drogas/métodos , Humanos , RNA , Reprodutibilidade dos Testes , Análise de Sequência de RNA/métodosRESUMO
The Hippo/YAP pathway controls cell proliferation through sensing physical and spatial organization of cells. How cell-cell contact is sensed by Hippo signaling is poorly understood. Here, we identified the cell adhesion molecule KIRREL1 as an upstream positive regulator of the mammalian Hippo pathway. KIRREL1 physically interacts with SAV1 and recruits SAV1 to cell-cell contact sites. Consistent with the hypothesis that KIRREL1-mediated cell adhesion suppresses YAP activity, knockout of KIRREL1 increases YAP activity in neighboring cells. Analyzing pan-cancer CRISPR proliferation screen data reveals KIRREL1 as the top plasma membrane protein showing strong correlation with known Hippo regulators, highlighting a critical role of KIRREL1 in regulating Hippo signaling and cell proliferation. During liver regeneration in mice, KIRREL1 is upregulated, and its genetic ablation enhances hepatic YAP activity, hepatocyte reprogramming and biliary epithelial cell proliferation. Our data suggest that KIRREL1 functions as a feedback regulator of the mammalian Hippo pathway through sensing cell-cell interaction and recruiting SAV1 to cell-cell contact sites.
Assuntos
Comunicação Celular , Proteínas de Ciclo Celular/metabolismo , Proteínas de Membrana/metabolismo , Adulto , Idoso de 80 Anos ou mais , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Retroalimentação Fisiológica , Feminino , Técnicas de Inativação de Genes , Células HEK293 , Hepatócitos , Via de Sinalização Hippo , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Proteínas de Sinalização YAP/metabolismoRESUMO
Cell-based transcriptional reporters are invaluable in high-throughput compound and CRISPR screens for identifying compounds or genes that can impact a pathway of interest. However, many transcriptional reporters have weak activities and transient responses. This can result in overlooking therapeutic targets and compounds that are difficult to detect, necessitating the resource-consuming process of running multiple screens at various timepoints. Here, we present RADAR, a digitizer circuit for amplifying reporter activity and retaining memory of pathway activation. Reporting on the AP-1 pathway, our circuit identifies compounds with known activity against PKC-related pathways and shows an enhanced dynamic range with improved sensitivity compared to a classical reporter in compound screens. In the first genome-wide pooled CRISPR screen for the AP-1 pathway, RADAR identifies canonical genes from the MAPK and PKC pathways, as well as non-canonical regulators. Thus, our scalable system highlights the benefit and versatility of using genetic circuits in large-scale cell-based screening.
Assuntos
Genômica/métodos , Ensaios de Triagem em Larga Escala/métodos , Sistemas CRISPR-Cas , Genes Reporter , Humanos , Regiões Promotoras Genéticas , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Three limonoid natural products with selective anti-proliferative activity against BRAF(V600E) and NRAS(Q61K)-mutation-dependent melanoma cell lines were identified. Differential transcriptome analysis revealed dependency of compound activity on expression of the mitochondrial cytochrome P450 oxidase CYP27A1, a transcriptional target of melanogenesis-associated transcription factor (MITF). We determined that CYP27A1 activity is necessary for the generation of a reactive metabolite that proceeds to inhibit cellular proliferation. A genome-wide small interfering RNA screen in combination with chemical proteomics experiments revealed gene-drug functional epistasis, suggesting that these compounds target mitochondrial biogenesis and inhibit tumor bioenergetics through a covalent mechanism. Our work suggests a strategy for melanoma-specific targeting by exploiting the expression of MITF target gene CYP27A1 and inhibiting mitochondrial oxidative phosphorylation in BRAF mutant melanomas.
Assuntos
Colestanotriol 26-Mono-Oxigenase/metabolismo , Limoninas/farmacologia , Mitocôndrias/efeitos dos fármacos , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colestanotriol 26-Mono-Oxigenase/antagonistas & inibidores , Colestanotriol 26-Mono-Oxigenase/genética , Humanos , Limoninas/química , Limoninas/metabolismo , Limoninas/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
The post-genomic era has seen many advances in our understanding of cancer pathways, yet resistance and tumor heterogeneity necessitate multiple approaches to target even monogenic tumors. Here, we combine phenotypic screening with chemical genetics to identify pre-messenger RNA endonuclease cleavage and polyadenylation specificity factor 3 (CPSF3) as the target of JTE-607, a small molecule with previously unknown target. We show that CPSF3 represents a synthetic lethal node in a subset of acute myeloid leukemia (AML) and Ewing's sarcoma cancer cell lines. Inhibition of CPSF3 by JTE-607 alters expression of known downstream effectors in AML and Ewing's sarcoma lines, upregulates apoptosis and causes tumor-selective stasis in mouse xenografts. Mechanistically, it prevents the release of newly synthesized pre-mRNAs, resulting in read-through transcription and the formation of DNA-RNA hybrid R-loop structures. This study implicates pre-mRNA processing, and specifically CPSF3, as a druggable target providing an avenue to therapeutic intervention in cancer.
Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Leucemia Mieloide Aguda/metabolismo , Precursores de RNA/metabolismo , Sarcoma de Ewing/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sítios de Ligação , Hidrolases de Éster Carboxílico/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Fator de Especificidade de Clivagem e Poliadenilação/genética , Células HEK293 , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Fenótipo , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Piperazinas/farmacologia , Ligação Proteica , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Sarcoma de Ewing/tratamento farmacológicoRESUMO
A hallmark of chronic hepatitis B (CHB) virus infection is the presence of high circulating levels of non-infectious small lipid HBV surface antigen (HBsAg) vesicles. Although rare, sustained HBsAg loss is the idealized endpoint of any CHB therapy. A small molecule, RG7834, has been previously reported to inhibit HBsAg expression by targeting terminal nucleotidyltransferase proteins 4A and 4B (TENT4A and TENT4B). In this study, we describe a genome-wide CRISPR screen to identify other potential host factors required for HBsAg expression and to gain further insights into the mechanism of RG7834. We report more than 60 genes involved in regulating HBsAg and identify additional factors involved in RG7834 activity, including a zinc finger CCHC-type containing 14 (ZCCHC14) protein. We show that ZCCHC14, together with TENT4A/B, stabilizes HBsAg expression through HBV RNA tailing, providing a potential new therapeutic target to achieve functional cure in CHB patients.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/genética , Interações entre Hospedeiro e Microrganismos/genética , Proteínas Nucleares/genética , Antígenos de Superfície/genética , Antivirais/farmacologia , Linhagem Celular Tumoral , DNA Viral/genética , Estudo de Associação Genômica Ampla/métodos , Células Hep G2 , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/virologia , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Humanos , Polinucleotídeo Adenililtransferase/genética , Carga Viral/efeitos dos fármacos , Carga Viral/genéticaRESUMO
Computational approaches have shown promise in contextualizing genes of interest with known molecular interactions. In this work, we evaluate seventeen previously published algorithms based on characteristics of their output and their performance in three tasks: cross validation, prediction of drug targets, and behavior with random input. Our work highlights strengths and weaknesses of each algorithm and results in a recommendation of algorithms best suited for performing different tasks.
Assuntos
Algoritmos , Redes Reguladoras de Genes , Modelos Genéticos , Benchmarking , Biologia Computacional , Bases de Dados Genéticas/estatística & dados numéricos , Bases de Dados de Proteínas/estatística & dados numéricos , Humanos , Mapas de Interação de Proteínas/genéticaRESUMO
EGFR-mutant NSCLCs frequently respond to EGFR tyrosine kinase inhibitors (TKIs). However, the responses are not durable, and the magnitude of tumor regression is variable, suggesting the existence of genetic modifiers of EGFR dependency. Here, we applied a genome-wide CRISPR-Cas9 screening to identify genetic determinants of EGFR TKI sensitivity and uncovered putative candidates. We show that knockout of RIC8A, essential for G-alpha protein activation, enhanced EGFR TKI-induced cell death. Mechanistically, we demonstrate that RIC8A is a positive regulator of YAP signaling, activation of which rescued the EGFR TKI sensitizing phenotype resulting from RIC8A knockout. We also show that knockout of ARIH2, or other components in the Cullin-5 E3 complex, conferred resistance to EGFR inhibition, in part by promoting nascent protein synthesis through METAP2. Together, these data uncover a spectrum of previously unidentified regulators of EGFR TKI sensitivity in EGFR-mutant human NSCLC, providing insights into the heterogeneity of EGFR TKI treatment responses.
Assuntos
Sistemas CRISPR-Cas , Carcinoma Pulmonar de Células não Pequenas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas Culina , Receptores ErbB/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Humanos , Metionil Aminopeptidases/metabolismo , Camundongos , Camundongos Nus , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcriptoma , Ubiquitina-Proteína Ligases/genética , Proteínas de Sinalização YAP , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Axin is a key scaffolding protein responsible for the formation of the ß-catenin destruction complex. Stability of Axin protein is regulated by the ubiquitin-proteasome system, and modulation of cellular concentration of Axin protein has a profound effect on Wnt/ß-catenin signaling. Although E3s promoting Axin ubiquitination have been identified, the deubiquitinase responsible for Axin deubiquitination and stabilization remains unknown. Here, we identify USP7 as a potent negative regulator of Wnt/ß-catenin signaling through CRISPR screens. Genetic ablation or pharmacological inhibition of USP7 robustly increases Wnt/ß-catenin signaling in multiple cellular systems. USP7 directly interacts with Axin through its TRAF domain, and promotes deubiquitination and stabilization of Axin. Inhibition of USP7 regulates osteoblast differentiation and adipocyte differentiation through increasing Wnt/ß-catenin signaling. Our study reveals a critical mechanism that prevents excessive degradation of Axin and identifies USP7 as a target for sensitizing cells to Wnt/ß-catenin signaling.
Assuntos
Proteína Axina/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo , beta Catenina/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Proteína Axina/genética , Linhagem Celular , Linhagem Celular Tumoral , Citometria de Fluxo , Células HCT116 , Humanos , Imunoprecipitação , Camundongos , Osteoblastos/metabolismo , Estabilidade Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Peptidase 7 Específica de Ubiquitina/genética , Ubiquitinação/genética , Ubiquitinação/fisiologia , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia , beta Catenina/genéticaRESUMO
Biliary epithelial cells (BECs) form bile ducts in the liver and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells that can form BEC-like organoids, suggesting that RSPO-LGR4/5-mediated WNT/ß-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPR-based loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found that BECs lack and do not require LGR4/5-mediated WNT/ß-catenin signaling during a DR, whereas YAP and mTORC1 signaling are required for this process. Upregulation of AXIN2 and LGR5 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool, delineate signaling pathways involved in a DR, and clarify the identity and roles of injury-induced periportal LGR5+ cells.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ductos Biliares/patologia , Proteínas de Ciclo Celular/metabolismo , Células Epiteliais/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteína Axina/genética , Proteína Axina/metabolismo , Proteínas de Ciclo Celular/genética , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Modelos Animais de Doenças , Humanos , Regeneração Hepática , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Piridinas/toxicidade , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Trombospondinas/genética , Trombospondinas/metabolismo , Via de Sinalização Wnt , Proteínas de Sinalização YAPRESUMO
The identification of activating mutations in NOTCH1 in 50% of T cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic transformation and in targeting the pathway. A phenotypic screen identified compounds that interfere with trafficking of Notch and induce apoptosis via an endoplasmic reticulum (ER) stress mechanism. Target identification approaches revealed a role for SLC39A7 (ZIP7), a zinc transport family member, in governing Notch trafficking and signaling. Generation and sequencing of a compound-resistant cell line identified a V430E mutation in ZIP7 that confers transferable resistance to the compound NVS-ZP7-4. NVS-ZP7-4 altered zinc in the ER, and an analog of the compound photoaffinity labeled ZIP7 in cells, suggesting a direct interaction between the compound and ZIP7. NVS-ZP7-4 is the first reported chemical tool to probe the impact of modulating ER zinc levels and investigate ZIP7 as a novel druggable node in the Notch pathway.
Assuntos
Proteínas de Transporte de Cátions/genética , Estresse do Retículo Endoplasmático/fisiologia , Receptor Notch1/genética , Animais , Apoptose , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/fisiologia , Linhagem Celular , Transformação Celular Neoplásica , Retículo Endoplasmático/fisiologia , Humanos , Mutação , Transporte Proteico , Receptor Notch1/fisiologia , Transdução de Sinais , Zinco/metabolismoRESUMO
The most frequent genetic alterations in melanoma are gain-of-function (GOF) mutations in BRAF, which result in RAF-MEK-ERK signaling pathway addiction. Despite therapeutic success of RAF and MEK inhibitors in treating BRAFV600-mutant tumors, a major challenge is the inevitable emergence of drug resistance, which often involves reactivation of the MAPK pathway. Interestingly, resistant tumors are often sensitive to drug withdrawal, suggesting that hyperactivation of the MAPK pathway is not tolerated. To further characterize this phenomenon, isogenic models of inducible MAPK hyperactivation in BRAFV600E melanoma cells were generated by overexpression of ERK2. Using this model system, supraphysiologic levels of MAPK signaling led to cell death, which was reversed by MAPK inhibition. Furthermore, complete tumor regression was observed in an ERK2-overexpressing xenograft model. To identify mediators of MAPK hyperactivation-induced cell death, a large-scale pooled shRNA screen was conducted, which revealed that only shRNAs against BRAF and MAP2K1 rescued loss of cell viability. This suggested that no single downstream ERK2 effector was required, consistent with pleiotropic effects on multiple cellular stress pathways. Intriguingly, the detrimental effect of MAPK hyperactivation could be partially attributed to secreted factors, and more than 100 differentially secreted proteins were identified. The effect of ERK2 overexpression was highly context dependent, as RAS/RAF mutant but not RAS/RAF wild-type melanoma were sensitive to this perturbation. IMPLICATIONS: This vulnerability to MAPK hyperactivation raises the possibility of novel therapeutic approaches for RAS/RAF-mutant cancers.
Assuntos
Sistema de Sinalização das MAP Quinases , Melanoma/genética , Melanoma/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas ras/metabolismo , Animais , Apoptose/fisiologia , Linhagem Celular Tumoral , Feminino , Xenoenxertos , Humanos , Melanoma/patologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/biossíntese , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas ras/genéticaRESUMO
Transcriptional repression of ubiquitin B (UBB) is a cancer-subtype-specific alteration that occurs in a substantial population of patients with cancers of the female reproductive tract. UBB is 1 of 2 genes encoding for ubiquitin as a polyprotein consisting of multiple copies of ubiquitin monomers. Silencing of UBB reduces cellular UBB levels and results in an exquisite dependence on ubiquitin C (UBC), the second polyubiquitin gene. UBB is repressed in approximately 30% of high-grade serous ovarian cancer (HGSOC) patients and is a recurrent lesion in uterine carcinosarcoma and endometrial carcinoma. We identified ovarian tumor cell lines that retain UBB in a repressed state, used these cell lines to establish orthotopic ovarian tumors, and found that inducible expression of a UBC-targeting shRNA led to tumor regression, and substantial long-term survival benefit. Thus, we describe a recurrent cancer-specific lesion at the level of ubiquitin production. Moreover, these observations reveal the prognostic value of UBB repression and establish UBC as a promising therapeutic target for ovarian cancer patients with recurrent UBB silencing.
Assuntos
Inativação Gênica , Proteínas de Neoplasias/biossíntese , Neoplasias Ovarianas/metabolismo , Ubiquitina C/biossíntese , Ubiquitina/biossíntese , Linhagem Celular Tumoral , Feminino , Humanos , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Ubiquitina/genética , Ubiquitina C/genéticaRESUMO
RNAi is broadly used to map gene regulatory networks, but the identification of genes that are responsible for the observed phenotypes is challenging, as small interfering RNAs (siRNAs) simultaneously downregulate the intended on targets and many partially complementary off targets. Additionally, the scarcity of publicly available control datasets hinders the development and comparative evaluation of computational methods for analyzing the data. Here, we introduce PheLiM (https://github.com/andreariba/PheLiM), a method that uses predictions of siRNA on- and off-target downregulation to infer gene-specific contributions to phenotypes. To assess the performance of PheLiM, we carried out siRNA- and CRISPR/Cas9-based genome-wide screening of two well-characterized pathways, bone morphogenetic protein (BMP) and nuclear factor κB (NF-κB), and we reanalyzed publicly available siRNA screens. We demonstrate that PheLiM has the overall highest accuracy and most reproducible results compared to other available methods. PheLiM can accommodate various methods for predicting siRNA off targets and is broadly applicable to the identification of genes underlying complex phenotypes.