RESUMEN
Most human epithelial tumors harbor numerous alterations, making it difficult to predict which genes are required for tumor survival. To systematically identify cancer dependencies, we analyzed 501 genome-scale loss-of-function screens performed in diverse human cancer cell lines. We developed DEMETER, an analytical framework that segregates on- from off-target effects of RNAi. 769 genes were differentially required in subsets of these cell lines at a threshold of six SDs from the mean. We found predictive models for 426 dependencies (55%) by nonlinear regression modeling considering 66,646 molecular features. Many dependencies fall into a limited number of classes, and unexpectedly, in 82% of models, the top biomarkers were expression based. We demonstrated the basis behind one such predictive model linking hypermethylation of the UBB ubiquitin gene to a dependency on UBC. Together, these observations provide a foundation for a cancer dependency map that facilitates the prioritization of therapeutic targets.
Asunto(s)
Neoplasias/genética , Neoplasias/patología , Línea Celular Tumoral , Humanos , Interferencia de ARN , Programas Informáticos , Ubiquitina/genéticaRESUMEN
Wnt/ß-catenin signaling plays a key role in the pathogenesis of colon and other cancers; emerging evidence indicates that oncogenic ß-catenin regulates several biological processes essential for cancer initiation and progression. To decipher the role of ß-catenin in transformation, we classified ß-catenin activity in 85 cancer cell lines in which we performed genome-scale loss-of-function screens and found that ß-catenin active cancers are dependent on a signaling pathway involving the transcriptional regulator YAP1. Specifically, we found that YAP1 and the transcription factor TBX5 form a complex with ß-catenin. Phosphorylation of YAP1 by the tyrosine kinase YES1 leads to localization of this complex to the promoters of antiapoptotic genes, including BCL2L1 and BIRC5. A small-molecule inhibitor of YES1 impeded the proliferation of ß-catenin-dependent cancers in both cell lines and animal models. These observations define a ß-catenin-YAP1-TBX5 complex essential to the transformation and survival of ß-catenin-driven cancers.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Transformación Celular Neoplásica , Neoplasias del Colon/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Dominio T Box/metabolismo , beta Catenina/metabolismo , Animales , Línea Celular Tumoral , Colon/embriología , Colon/metabolismo , Neoplasias del Colon/patología , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Ratones , Ratones Desnudos , Proteínas Proto-Oncogénicas c-yes/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-yes/metabolismo , Survivin , Factores de Transcripción , Transcripción Genética , Proteínas Señalizadoras YAP , Pez Cebra/embriología , Proteína bcl-X/genética , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
Due to genome instability, most cancers exhibit loss of regions containing tumor suppressor genes and collateral loss of other genes. To identify cancer-specific vulnerabilities that are the result of copy number losses, we performed integrated analyses of genome-wide copy number and RNAi profiles and identified 56 genes for which gene suppression specifically inhibited the proliferation of cells harboring partial copy number loss of that gene. These CYCLOPS (copy number alterations yielding cancer liabilities owing to partial loss) genes are enriched for spliceosome, proteasome, and ribosome components. One CYCLOPS gene, PSMC2, encodes an essential member of the 19S proteasome. Normal cells express excess PSMC2, which resides in a complex with PSMC1, PSMD2, and PSMD5 and acts as a reservoir protecting cells from PSMC2 suppression. Cells harboring partial PSMC2 copy number loss lack this complex and die after PSMC2 suppression. These observations define a distinct class of cancer-specific liabilities resulting from genome instability.
Asunto(s)
Genes Esenciales , Inestabilidad Genómica , Neoplasias/genética , ATPasas Asociadas con Actividades Celulares Diversas , Animales , Línea Celular Tumoral , Deleción Cromosómica , Dosificación de Gen , Genes Supresores de Tumor , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Trasplante HeterólogoRESUMEN
Large panels of comprehensively characterized human cancer models, including the Cancer Cell Line Encyclopedia (CCLE), have provided a rigorous framework with which to study genetic variants, candidate targets, and small-molecule and biological therapeutics and to identify new marker-driven cancer dependencies. To improve our understanding of the molecular features that contribute to cancer phenotypes, including drug responses, here we have expanded the characterizations of cancer cell lines to include genetic, RNA splicing, DNA methylation, histone H3 modification, microRNA expression and reverse-phase protein array data for 1,072 cell lines from individuals of various lineages and ethnicities. Integration of these data with functional characterizations such as drug-sensitivity, short hairpin RNA knockdown and CRISPR-Cas9 knockout data reveals potential targets for cancer drugs and associated biomarkers. Together, this dataset and an accompanying public data portal provide a resource for the acceleration of cancer research using model cancer cell lines.
Asunto(s)
Línea Celular Tumoral , Neoplasias/genética , Neoplasias/patología , Antineoplásicos/farmacología , Biomarcadores de Tumor , Metilación de ADN , Resistencia a Antineoplásicos , Etnicidad/genética , Edición Génica , Histonas/metabolismo , Humanos , MicroARNs/genética , Terapia Molecular Dirigida , Neoplasias/metabolismo , Análisis por Matrices de Proteínas , Empalme del ARNRESUMEN
Oncogenic PIK3CA mutations are found in a significant fraction of human cancers, but therapeutic inhibition of PI3K has only shown limited success in clinical trials. To understand how mutant PIK3CA contributes to cancer cell proliferation, we used genome scale loss-of-function screening in a large number of genomically annotated cancer cell lines. As expected, we found that PIK3CA mutant cancer cells require PIK3CA but also require the expression of the TCA cycle enzyme 2-oxoglutarate dehydrogenase (OGDH). To understand the relationship between oncogenic PIK3CA and OGDH function, we interrogated metabolic requirements and found an increased reliance on glucose metabolism to sustain PIK3CA mutant cell proliferation. Functional metabolic studies revealed that OGDH suppression increased levels of the metabolite 2-oxoglutarate (2OG). We found that this increase in 2OG levels, either by OGDH suppression or exogenous 2OG treatment, resulted in aspartate depletion that was specifically manifested as auxotrophy within PIK3CA mutant cells. Reduced levels of aspartate deregulated the malate-aspartate shuttle, which is important for cytoplasmic NAD+ regeneration that sustains rapid glucose breakdown through glycolysis. Consequently, because PIK3CA mutant cells exhibit a profound reliance on glucose metabolism, malate-aspartate shuttle deregulation leads to a specific proliferative block due to the inability to maintain NAD+/NADH homeostasis. Together these observations define a precise metabolic vulnerability imposed by a recurrently mutated oncogene.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase I , Complejo Cetoglutarato Deshidrogenasa , Mutación , Proteínas de Neoplasias , Neoplasias , Animales , Línea Celular Tumoral , Ciclo del Ácido Cítrico/genética , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Glucólisis/genética , Humanos , Complejo Cetoglutarato Deshidrogenasa/biosíntesis , Complejo Cetoglutarato Deshidrogenasa/genética , Ratones , Ratones Desnudos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patologíaRESUMEN
Acute myeloid leukemia (AML) is a heterogeneous disease with complex molecular pathophysiology. To systematically characterize AML's genetic dependencies, we conducted genome-scale short hairpin RNA screens in 17 AML cell lines and analyzed dependencies relative to parallel screens in 199 cell lines of other cancer types. We identified 353 genes specifically required for AML cell proliferation. To validate the in vivo relevance of genetic dependencies observed in human cell lines, we performed a secondary screen in a syngeneic murine AML model driven by the MLL-AF9 oncogenic fusion protein. Integrating the results of these interference RNA screens and additional gene expression data, we identified the transcription factor ZEB2 as a novel AML dependency. ZEB2 depletion impaired the proliferation of both human and mouse AML cells and resulted in aberrant differentiation of human AML cells. Mechanistically, we showed that ZEB2 transcriptionally represses genes that regulate myeloid differentiation, including genes involved in cell adhesion and migration. In addition, we found that epigenetic silencing of the miR-200 family microRNAs affects ZEB2 expression. Our results extend the role of ZEB2 beyond regulating epithelial-mesenchymal transition (EMT) and establish ZEB2 as a novel regulator of AML proliferation and differentiation.
Asunto(s)
Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Proteínas de Homeodominio/genética , Leucemia Mieloide Aguda/genética , MicroARNs/genética , Proteínas Represoras/genética , Animales , Adhesión Celular , Diferenciación Celular , Línea Celular Tumoral , Movimiento Celular , Epigénesis Genética , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , MicroARNs/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/metabolismo , Transducción de Señal , Transcripción Genética , Caja Homeótica 2 de Unión a E-Box con Dedos de ZincRESUMEN
High-grade serous ovarian cancers are characterized by widespread recurrent copy number alterations. Although some regions of copy number change harbor known oncogenes and tumor suppressor genes, the genes targeted by the majority of amplified or deleted regions in ovarian cancer remain undefined. Here we systematically tested amplified genes for their ability to promote tumor formation using an in vivo multiplexed transformation assay. We identified the GRB2-associated binding protein 2 (GAB2) as a recurrently amplified gene that potently transforms immortalized ovarian and fallopian tube secretory epithelial cells. Cancer cell lines overexpressing GAB2 require GAB2 for survival and show evidence of phosphatidylinositol 3-kinase (PI3K) pathway activation, which was required for GAB2-induced transformation. Cell lines overexpressing GAB2 were as sensitive to PI3K inhibition as cell lines harboring mutant PIK3CA. Together, these observations nominate GAB2 as an ovarian cancer oncogene, identify an alternative mechanism to activate PI3K signaling, and underscore the importance of PI3K signaling in this cancer.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Neoplasias Ováricas/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Femenino , Genómica , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Sistemas de Lectura Abierta , Neoplasias Ováricas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de SeñalRESUMEN
Genome-scale RNAi libraries enable the systematic interrogation of gene function. However, the interpretation of RNAi screens is complicated by the observation that RNAi reagents designed to suppress the mRNA transcripts of the same gene often produce a spectrum of phenotypic outcomes due to differential on-target gene suppression or perturbation of off-target transcripts. Here we present a computational method, Analytic Technique for Assessment of RNAi by Similarity (ATARiS), that takes advantage of patterns in RNAi data across multiple samples in order to enrich for RNAi reagents whose phenotypic effects relate to suppression of their intended targets. By summarizing only such reagent effects for each gene, ATARiS produces quantitative, gene-level phenotype values, which provide an intuitive measure of the effect of gene suppression in each sample. This method is robust for data sets that contain as few as 10 samples and can be used to analyze screens of any number of targeted genes. We used this analytic approach to interrogate RNAi data derived from screening more than 100 human cancer cell lines and identified HNF1B as a transforming oncogene required for the survival of cancer cells that harbor HNF1B amplifications. ATARiS is publicly available at http://broadinstitute.org/ataris.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Genómica , Interferencia de ARN , ARN Interferente Pequeño/genética , Programas Informáticos , Animales , Transformación Celular Neoplásica/genética , Biología Computacional/métodos , Perfilación de la Expresión Génica , Genómica/métodos , Factor Nuclear 1-beta del Hepatocito/genética , Humanos , Internet , Ratones , Neoplasias/genética , Fenotipo , Reproducibilidad de los ResultadosRESUMEN
A powerful way to discover key genes with causal roles in oncogenesis is to identify genomic regions that undergo frequent alteration in human cancers. Here we present high-resolution analyses of somatic copy-number alterations (SCNAs) from 3,131 cancer specimens, belonging largely to 26 histological types. We identify 158 regions of focal SCNA that are altered at significant frequency across several cancer types, of which 122 cannot be explained by the presence of a known cancer target gene located within these regions. Several gene families are enriched among these regions of focal SCNA, including the BCL2 family of apoptosis regulators and the NF-kappaBeta pathway. We show that cancer cells containing amplifications surrounding the MCL1 and BCL2L1 anti-apoptotic genes depend on the expression of these genes for survival. Finally, we demonstrate that a large majority of SCNAs identified in individual cancer types are present in several cancer types.
Asunto(s)
Variaciones en el Número de Copia de ADN/genética , Dosificación de Gen/genética , Neoplasias/genética , Apoptosis/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Amplificación de Genes/genética , Genómica , Humanos , Familia de Multigenes/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Neoplasias/clasificación , Neoplasias/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Transducción de Señal , Proteína bcl-X/genéticaRESUMEN
High-grade serous ovarian cancers (HGSCs) are characterized by a high frequency of TP53 mutations, BRCA1/2 inactivation, homologous recombination dysfunction, and widespread copy number changes. Cyclin E1 (CCNE1) gene amplification has been reported to occur independently of BRCA1/2 mutation, and it is associated with primary treatment failure and reduced patient survival. Insensitivity of CCNE1-amplified tumors to platinum cross-linking agents may be partly because of an intact BRCA1/2 pathway. Both BRCA1/2 dysfunction and CCNE1 amplification are known to promote genomic instability and tumor progression. These events may be mutually exclusive, because either change provides a path to tumor development, with no selective advantage to having both mutations. Using data from a genome-wide shRNA synthetic lethal screen, we show that BRCA1 and members of the ubiquitin pathway are selectively required in cancers that harbor CCNE1 amplification. Furthermore, we show specific sensitivity of CCNE1-amplified tumor cells to the proteasome inhibitor bortezomib. These findings provide an explanation for the observed mutual exclusivity of CCNE1 amplification and BRCA1/2 loss in HGSC and suggest a unique therapeutic approach for treatment-resistant CCNE1-amplified tumors.
Asunto(s)
Antineoplásicos/farmacología , Proteína BRCA1/genética , Ácidos Borónicos/farmacología , Ciclina E/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Oncogénicas/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Pirazinas/farmacología , Antineoplásicos/uso terapéutico , Ácidos Borónicos/uso terapéutico , Bortezomib , Ciclina E/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Recombinación Homóloga/efectos de los fármacos , Humanos , Análisis por Micromatrices , Proteínas Oncogénicas/metabolismo , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Pirazinas/uso terapéutico , ARN Interferente Pequeño/genéticaRESUMEN
The proto-oncogene KRAS is mutated in a wide array of human cancers, most of which are aggressive and respond poorly to standard therapies. Although the identification of specific oncogenes has led to the development of clinically effective, molecularly targeted therapies in some cases, KRAS has remained refractory to this approach. A complementary strategy for targeting KRAS is to identify gene products that, when inhibited, result in cell death only in the presence of an oncogenic allele. Here we have used systematic RNA interference to detect synthetic lethal partners of oncogenic KRAS and found that the non-canonical IkappaB kinase TBK1 was selectively essential in cells that contain mutant KRAS. Suppression of TBK1 induced apoptosis specifically in human cancer cell lines that depend on oncogenic KRAS expression. In these cells, TBK1 activated NF-kappaB anti-apoptotic signals involving c-Rel and BCL-XL (also known as BCL2L1) that were essential for survival, providing mechanistic insights into this synthetic lethal interaction. These observations indicate that TBK1 and NF-kappaB signalling are essential in KRAS mutant tumours, and establish a general approach for the rational identification of co-dependent pathways in cancer.
Asunto(s)
Genes ras/genética , Proteína Oncogénica p21(ras)/genética , Proteína Oncogénica p21(ras)/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Alelos , Apoptosis , Línea Celular Tumoral , Supervivencia Celular , Perfilación de la Expresión Génica , Genes Letales , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-rel/metabolismo , Transducción de Señal , Proteína bcl-X/metabolismoRESUMEN
The Alternative Lengthening of Telomeres (ALT) pathway is a telomerase-independent pathway for telomere maintenance that is active in a significant subset of human cancers and in vitro immortalized cell lines. ALT is thought to involve templated extension of telomeres through homologous recombination, but the genetic or epigenetic changes that unleash ALT are not known. Recently, mutations in the ATRX/DAXX chromatin remodeling complex and histone H3.3 were found to correlate with features of ALT in pancreatic neuroendocrine cancers, pediatric glioblastomas, and other tumors of the central nervous system, suggesting that these mutations might contribute to the activation of the ALT pathway in these cancers. We have taken a comprehensive approach to deciphering ALT by applying genomic, molecular biological, and cell biological approaches to a panel of 22 ALT cell lines, including cell lines derived in vitro. Here we show that loss of ATRX protein and mutations in the ATRX gene are hallmarks of ALT-immortalized cell lines. In addition, ALT is associated with extensive genome rearrangements, marked micronucleation, defects in the G2/M checkpoint, and altered double-strand break (DSB) repair. These attributes will facilitate the diagnosis and treatment of ALT positive human cancers.
Asunto(s)
ADN Helicasas/genética , Histonas , Proteínas Nucleares/genética , Homeostasis del Telómero/genética , Telómero/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Ensamble y Desensamble de Cromatina/genética , Proteínas Co-Represoras , Roturas del ADN de Doble Cadena , Daño del ADN/genética , ADN Helicasas/metabolismo , Reparación del ADN/genética , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Inestabilidad Genómica , Células HeLa , Histonas/genética , Histonas/metabolismo , Recombinación Homóloga , Humanos , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Transducción de Señal , Telomerasa/genética , Telómero/metabolismo , Proteína Nuclear Ligada al Cromosoma XRESUMEN
A comprehensive understanding of the molecular vulnerabilities of every type of cancer will provide a powerful roadmap to guide therapeutic approaches. Efforts such as The Cancer Genome Atlas Project will identify genes with aberrant copy number, sequence, or expression in various cancer types, providing a survey of the genes that may have a causal role in cancer. A complementary approach is to perform systematic loss-of-function studies to identify essential genes in particular cancer cell types. We have begun a systematic effort, termed Project Achilles, aimed at identifying genetic vulnerabilities across large numbers of cancer cell lines. Here, we report the assessment of the essentiality of 11,194 genes in 102 human cancer cell lines. We show that the integration of these functional data with information derived from surveying cancer genomes pinpoints known and previously undescribed lineage-specific dependencies across a wide spectrum of cancers. In particular, we found 54 genes that are specifically essential for the proliferation and viability of ovarian cancer cells and also amplified in primary tumors or differentially overexpressed in ovarian cancer cell lines. One such gene, PAX8, is focally amplified in 16% of high-grade serous ovarian cancers and expressed at higher levels in ovarian tumors. Suppression of PAX8 selectively induces apoptotic cell death of ovarian cancer cells. These results identify PAX8 as an ovarian lineage-specific dependency. More generally, these observations demonstrate that the integration of genome-scale functional and structural studies provides an efficient path to identify dependencies of specific cancer types on particular genes and pathways.
Asunto(s)
Neoplasias Ováricas/genética , Oxidorreductasas de Alcohol , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/genética , Femenino , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Oncogenes , Neoplasias Ováricas/patología , Factor de Transcripción PAX8 , Factores de Transcripción Paired Box/genética , ARN Neoplásico/genética , ARN Interferente Pequeño/genéticaRESUMEN
Monocytic acute myeloid leukemia (AML) responds poorly to current treatments, including venetoclax-based therapy. We conducted in vivo and in vitro CRISPR-Cas9 library screenings using a mouse monocytic AML model and identified SETDB1 and its binding partners (ATF7IP and TRIM33) as crucial tumor promoters in vivo. The growth-inhibitory effect of Setdb1 depletion in vivo is dependent mainly on natural killer (NK) cell-mediated cytotoxicity. Mechanistically, SETDB1 depletion upregulates interferon-stimulated genes and NKG2D ligands through the demethylation of histone H3 Lys9 at the enhancer regions, thereby enhancing their immunogenicity to NK cells and intrinsic apoptosis. Importantly, these effects are not observed in non-monocytic leukemia cells. We also identified the expression of myeloid cell nuclear differentiation antigen (MNDA) and its murine counterpart Ifi203 as biomarkers to predict the sensitivity of AML to SETDB1 depletion. Our study highlights the critical and selective role of SETDB1 in AML with granulo-monocytic differentiation and underscores its potential as a therapeutic target for current unmet needs.
RESUMEN
Somatic alterations in cellular DNA underlie almost all human cancers. The prospect of targeted therapies and the development of high-resolution, genome-wide approaches are now spurring systematic efforts to characterize cancer genomes. Here we report a large-scale project to characterize copy-number alterations in primary lung adenocarcinomas. By analysis of a large collection of tumours (n = 371) using dense single nucleotide polymorphism arrays, we identify a total of 57 significantly recurrent events. We find that 26 of 39 autosomal chromosome arms show consistent large-scale copy-number gain or loss, of which only a handful have been linked to a specific gene. We also identify 31 recurrent focal events, including 24 amplifications and 7 homozygous deletions. Only six of these focal events are currently associated with known mutations in lung carcinomas. The most common event, amplification of chromosome 14q13.3, is found in approximately 12% of samples. On the basis of genomic and functional analyses, we identify NKX2-1 (NK2 homeobox 1, also called TITF1), which lies in the minimal 14q13.3 amplification interval and encodes a lineage-specific transcription factor, as a novel candidate proto-oncogene involved in a significant fraction of lung adenocarcinomas. More generally, our results indicate that many of the genes that are involved in lung adenocarcinoma remain to be discovered.
Asunto(s)
Adenocarcinoma/genética , Genoma Humano/genética , Neoplasias Pulmonares/genética , Neoplasias/genética , Línea Celular Tumoral , Deleción Cromosómica , Cromosomas Humanos Par 14/genética , Amplificación de Genes/genética , Genómica , Genotipo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Pérdida de Heterocigocidad/genética , Proteínas Nucleares/genética , Polimorfismo de Nucleótido Simple/genética , Proto-Oncogenes Mas , Interferencia de ARN , Factor Nuclear Tiroideo 1 , Factores de Transcripción/genéticaRESUMEN
Aberrant DNA methylation patterns are a prominent feature of cancer. Methylation of DNA is mediated by the DNA methyltransferase (DNMT) protein family, which regulates de novo (DNMT3A and DNMT3B) and maintenance (DNMT1) methylation. Mutations in DNMT3A are observed in approximately 22% of acute myeloid leukemia (AML). We hypothesized that DNMT1 or DNMT3B could function as a synthetic lethal therapeutic strategy for DNMT3A-mutant AML. CRISPR-Cas9 tiling screens were performed to identify functional domains within DNMT1/DNMT3B that exhibited greater dependencies in DNMT3A mutant versus wild-type cell lines. Although increased sensitivity to DNMT1 mutation was observed in some DNMT3A mutant cellular models tested, the subtlety of these results prevents us from basing any conclusions on a synthetic lethal relationship between DNMT1 and DNMT3A. Our data suggests that a therapeutic window for DNMT1 methyltransferase inhibition in DNMT3A-driven AML may exist, but validation in more biologically relevant models is required.
Asunto(s)
Leucemia Mieloide Aguda , Metiltransferasas , Humanos , Metiltransferasas/genética , ADN Metiltransferasa 3A , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Leucemia Mieloide Aguda/genética , Mutación , ADNRESUMEN
More complete knowledge of the molecular mechanisms underlying cancer will improve prevention, diagnosis and treatment. Efforts such as The Cancer Genome Atlas are systematically characterizing the structural basis of cancer, by identifying the genomic mutations associated with each cancer type. A powerful complementary approach is to systematically characterize the functional basis of cancer, by identifying the genes essential for growth and related phenotypes in different cancer cells. Such information would be particularly valuable for identifying potential drug targets. Here, we report the development of an efficient, robust approach to perform genome-scale pooled shRNA screens for both positive and negative selection and its application to systematically identify cell essential genes in 12 cancer cell lines. By integrating these functional data with comprehensive genetic analyses of primary human tumors, we identified known and putative oncogenes such as EGFR, KRAS, MYC, BCR-ABL, MYB, CRKL, and CDK4 that are essential for cancer cell proliferation and also altered in human cancers. We further used this approach to identify genes involved in the response of cancer cells to tumoricidal agents and found 4 genes required for the response of CML cells to imatinib treatment: PTPN1, NF1, SMARCB1, and SMARCE1, and 5 regulators of the response to FAS activation, FAS, FADD, CASP8, ARID1A and CBX1. Broad application of this highly parallel genetic screening strategy will not only facilitate the rapid identification of genes that drive the malignant state and its response to therapeutics but will also enable the discovery of genes that participate in any biological process.
Asunto(s)
Genómica/métodos , Neoplasias/patología , Oncogenes/fisiología , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Proliferación Celular , Homólogo de la Proteína Chromobox 5 , Genoma Humano , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Oncogenes/genética , Farmacogenética , ARN Interferente Pequeño , Células Tumorales Cultivadas , Receptor fas/metabolismoRESUMEN
The majority of lung adenocarcinomas express the lineage-specific thyroid transcription factor-1 (TTF-1). We recently reported that in a subset of lung adenocarcinomas the TTF-1 gene is amplified. Although the prognostic significance of TTF-1 expression has been previously investigated, the significance of TTF-1 amplification has not been established. We studied 89 consecutive patients with lung adenocarcinomas treated by surgery at Brigham and Women's Hospital between 1997 and 1999 and performed immunohistochemical analysis for TTF-1 expression and fluorescence in situ hybridization for TTF-1 amplification. We investigated associations between clinical-pathological characteristics, TTF-1 expression, TTF-1 amplification and overall survival. TTF-1 expression was categorized as high (48%), low (24%) or absent (28%). TTF-1 was amplified in 7% of cases. Patients with adenocarcinomas with low or high TTF-1 expression had a significantly better outcome than those with absent TTF-1 expression (median overall survival times of 72.4, 77.8 and 30.5 months, respectively, P = 0.002). In contrast, patients with adenocarcinomas with TTF-1 expression had a worse outcome if TTF-1 was amplified (median overall survival time 39.5 versus 87.5 months). In multivariate analysis, improved overall survival was independently predicted by TTF-1 expression in combination with no TTF-1 amplification (P < 0.001). In patients with lung adenocarcinoma, TTF-1 expression is a predictor of good outcome. Patients with no TTF-1 expression or TTF-1 expression and TTF-1 gene amplification tend to have a significantly worse prognosis than patients with TTF-1 expression and no TTF-1 gene amplification.
Asunto(s)
Adenocarcinoma/metabolismo , Amplificación de Genes , Neoplasias Pulmonares/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Adenocarcinoma/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Neoplasias Pulmonares/genética , Masculino , Persona de Mediana Edad , Proteínas Nucleares/genética , Factor Nuclear Tiroideo 1 , Factores de Transcripción/genéticaRESUMEN
Cancer is often seen as a disease of mutations and chromosomal abnormalities. However, some cancers, including pediatric rhabdoid tumors (RTs), lack recurrent alterations targetable by current drugs and need alternative, informed therapeutic options. To nominate potential targets, we performed a high-throughput small-molecule screen complemented by a genome-scale CRISPR-Cas9 gene-knockout screen in a large number of RT and control cell lines. These approaches converged to reveal several receptor tyrosine kinases (RTKs) as therapeutic targets, with RTK inhibition effective in suppressing RT cell growth in vitro and against a xenograft model in vivo. RT cell lines highly express and activate (phosphorylate) different RTKs, creating dependency without mutation or amplification. Downstream of RTK signaling, we identified PTPN11, encoding the pro-growth signaling protein SHP2, as a shared dependency across all RT cell lines. This study demonstrates that large-scale perturbational screening can uncover vulnerabilities in cancers with "quiet" genomes.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Tumor Rabdoide/genética , Animales , Antineoplásicos/uso terapéutico , Sistemas CRISPR-Cas , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Ratones , Ratones Desnudos , Mutación , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Tumor Rabdoide/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Chordoma is a primary bone cancer with no approved therapy1. The identification of therapeutic targets in this disease has been challenging due to the infrequent occurrence of clinically actionable somatic mutations in chordoma tumors2,3. Here we describe the discovery of therapeutically targetable chordoma dependencies via genome-scale CRISPR-Cas9 screening and focused small-molecule sensitivity profiling. These systematic approaches reveal that the developmental transcription factor T (brachyury; TBXT) is the top selectively essential gene in chordoma, and that transcriptional cyclin-dependent kinase (CDK) inhibitors targeting CDK7/12/13 and CDK9 potently suppress chordoma cell proliferation. In other cancer types, transcriptional CDK inhibitors have been observed to downregulate highly expressed, enhancer-associated oncogenic transcription factors4,5. In chordoma, we find that T is associated with a 1.5-Mb region containing 'super-enhancers' and is the most highly expressed super-enhancer-associated transcription factor. Notably, transcriptional CDK inhibition leads to preferential and concentration-dependent downregulation of cellular brachyury protein levels in all models tested. In vivo, CDK7/12/13-inhibitor treatment substantially reduces tumor growth. Together, these data demonstrate small-molecule targeting of brachyury transcription factor addiction in chordoma, identify a mechanism of T gene regulation that underlies this therapeutic strategy, and provide a blueprint for applying systematic genetic and chemical screening approaches to discover vulnerabilities in genomically quiet cancers.