RESUMO
Patterns of somatic mutations in cancer genes provide information about their functional role in tumourigenesis, and thus indicate their potential for therapeutic exploitation. Yet, the classical distinction between oncogene and tumour suppressor may not always apply. For instance, TP53 has been simultaneously associated with tumour suppressing and promoting activities. Here, we uncover a similar phenomenon for GATA3, a frequently mutated, yet poorly understood, breast cancer gene. We identify two functional classes of frameshift mutations that are associated with distinct expression profiles in tumours, differential disease-free patient survival and gain- and loss-of-function activities in a cell line model. Furthermore, we find an estrogen receptor-independent synthetic lethal interaction between a GATA3 frameshift mutant with an extended C-terminus and the histone methyltransferases G9A and GLP, indicating perturbed epigenetic regulation. Our findings reveal important insights into mutant GATA3 function and breast cancer, provide the first potential therapeutic strategy and suggest that dual tumour suppressive and oncogenic activities are more widespread than previously appreciated.
Assuntos
Neoplasias da Mama/genética , Epigênese Genética , Fator de Transcrição GATA3/genética , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/genética , Neoplasias da Mama/patologia , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/genética , Estrogênios/genética , Estrogênios/metabolismo , Feminino , Mutação da Fase de Leitura , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/uso terapêuticoRESUMO
The great majority of targeted anticancer drugs inhibit mutated oncogenes that display increased activity. Yet many tumors do not contain such actionable aberrations, such as those harboring loss-of-function mutations. The notion of targeting synthetic lethal vulnerabilities in cancer cells has provided an alternative approach to exploiting more of the genetic and epigenetic changes acquired during tumorigenesis. Here, we review synthetic lethality as a therapeutic concept that exploits the inherent differences between normal cells and cancer cells. Furthermore, we provide an overview of the screening approaches that can be used to identify synthetic lethal interactions in human cells and present several recently identified interactions that may be pharmacologically exploited. Finally, we indicate some of the challenges of translating synthetic lethal interactions into the clinic and how these may be overcome.
Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Desenho de Fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Terapia Genética/métodos , Terapia de Alvo Molecular/métodos , Neoplasias/terapia , Animais , Biomarcadores Tumorais/metabolismo , Morte Celular/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais/efeitos dos fármacos , Pesquisa Translacional BiomédicaRESUMO
Near-haploid human cell lines are instrumental for genetic screens and genome engineering as gene inactivation is greatly facilitated by the absence of a second gene copy. However, no completely haploid human cell line has been described, hampering the genetic accessibility of a subset of genes. The near-haploid human cell line HAP1 contains a single copy of all chromosomes except for a heterozygous 30-megabase fragment of Chromosome 15. This large fragment encompasses 330 genes and is integrated on the long arm of Chromosome 19. Here, we employ a CRISPR/Cas9-based genome engineering strategy to excise this sizeable chromosomal fragment and to efficiently and reproducibly derive clones that retain their haploid state. Importantly, spectral karyotyping and single-nucleotide polymorphism (SNP) genotyping revealed that engineered-HAPloid (eHAP) cells are fully haploid with no gross chromosomal aberrations induced by Cas9. Furthermore, whole-genome sequence and transcriptome analysis of the parental HAP1 and an eHAP cell line showed that transcriptional changes are limited to the excised Chromosome 15 fragment. Together, we demonstrate the feasibility of efficiently engineering megabase deletions with the CRISPR/Cas9 technology and report the first fully haploid human cell line.
Assuntos
Sistemas CRISPR-Cas/genética , Haploidia , Deleção de Sequência , Linhagem Celular , Perfilação da Expressão Gênica , Engenharia Genética/métodos , Genômica , Humanos , CariótipoRESUMO
MOTIVATION: Calling changes in DNA, e.g. as a result of somatic events in cancer, requires analysis of multiple matched sequenced samples. Events in low-mappability regions of the human genome are difficult to encode in variant call files and have been under-reported as a result. However, they can be described accurately through thesaurus annotation-a technique that links multiple genomic loci together to explicate a single variant. RESULTS: We here describe software and benchmarks for using thesaurus annotation to detect point changes in DNA from matched samples. In benchmarks on matched normal/tumor samples we show that the technique can recover between five and ten percent more true events than conventional approaches, while strictly limiting false discovery and being fully consistent with popular variant analysis workflows. We also demonstrate the utility of the approach for analysis of de novo mutations in parents/child families. AVAILABILITY AND IMPLEMENTATION: Software performing thesaurus annotation is implemented in java; available in source code on github at GeneticThesaurus (https://github.com/tkonopka/GeneticThesaurus) and as an executable on sourceforge at geneticthesaurus (https://sourceforge.net/projects/geneticthesaurus). Mutation calling is implemented in an R package available on github at RGeneticThesaurus (https://github.com/tkonopka/RGeneticThesaurus). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. CONTACT: tomasz.konopka@ludwig.ox.ac.uk.
Assuntos
Variação Genética , Genoma Humano , Genômica , Humanos , Software , Vocabulário ControladoRESUMO
The upswing in US Food and Drug Administration and European Medicines Agency drug approvals in 2014 may have marked an end to the dry spell that has troubled the pharmaceutical industry over the past decade. Regardless, the attrition rate of drugs in late clinical phases remains high, and a lack of target validation has been highlighted as an explanation. This has led to a resurgence in appreciation of phenotypic drug screens, as these may be more likely to yield compounds with relevant modes of action. However, cell-based screening approaches do not directly reveal cellular targets, and hence target deconvolution and a detailed understanding of drug action are needed for efficient lead optimization and biomarker development. Here, recently developed functional genomics technologies that address this need are reviewed. The approaches pioneered in model organisms, particularly in yeast, and more recently adapted to mammalian systems are discussed. Finally, areas of particular interest and directions for future tool development are highlighted.
Assuntos
Genômica , Preparações Farmacêuticas , Animais , HumanosRESUMO
High-throughput live-cell screens are intricate elements of systems biology studies and drug discovery pipelines. Here, we demonstrate an optogenetics-assisted method that avoids the need for chemical activators and reporters, reduces the number of operational steps and increases information content in a cell-based small-molecule screen against human protein kinases, including an orphan receptor tyrosine kinase. This blueprint for all-optical screening can be adapted to many drug targets and cellular processes.
Assuntos
Ensaios de Triagem em Larga Escala , Luz , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Células HEK293 , Humanos , Inibidores de Proteínas Quinases/química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Detecting genetic variation is one of the main applications of high-throughput sequencing, but is still challenging wherever aligning short reads poses ambiguities. Current state-of-the-art variant calling approaches avoid such regions, arguing that it is necessary to sacrifice detection sensitivity to limit false discovery. We developed a method that links candidate variant positions within repetitive genomic regions into clusters. The technique relies on a resource, a thesaurus of genetic variation, that enumerates genomic regions with similar sequence. The resource is computationally intensive to generate, but once compiled can be applied efficiently to annotate and prioritize variants in repetitive regions. We show that thesaurus annotation can reduce the rate of false variant calls due to mappability by up to three orders of magnitude. We apply the technique to whole genome datasets and establish that called variants in low mappability regions annotated using the thesaurus can be experimentally validated. We then extend the analysis to a large panel of exomes to show that the annotation technique opens possibilities to study variation in hereto hidden and under-studied parts of the genome.
Assuntos
Variação Genética , Genoma Humano , Genômica/métodos , Vocabulário Controlado , Linhagem Celular Tumoral , Exoma , Humanos , Anotação de Sequência Molecular , Sequências Repetitivas de Ácido NucleicoRESUMO
Histone lysine demethylases (KDMs) are of critical importance in the epigenetic regulation of gene expression, yet there are few selective, cell-permeable inhibitors or suitable tool compounds for these enzymes. We describe the discovery of a new class of inhibitor that is highly potent towards the histone lysine demethylases KDM2A/7A. A modular synthetic approach was used to explore the chemical space and accelerate the investigation of key structure-activity relationships, leading to the development of a small molecule with around 75-fold selectivity towards KDM2A/7A versus other KDMs, as well as cellular activity at low micromolar concentrations.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Proteínas F-Box/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Proteínas F-Box/metabolismo , Células HeLa , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Knockout collections are invaluable tools for studying model organisms such as yeast. However, there are no large-scale knockout collections of human cells. Using gene-trap mutagenesis in near-haploid human cells, we established a platform to generate and isolate individual 'gene-trapped cells' and used it to prepare a collection of human cell lines carrying single gene-trap insertions. In most cases, the insertion can be reversed. This growing library covers 3,396 genes, one-third of the expressed genome, is DNA-barcoded and allows systematic screens for a wide variety of cellular phenotypes. We examined cellular responses to TNF-α, TGF-ß, IFN-γ and TNF-related apoptosis-inducing ligand (TRAIL), to illustrate the value of this unique collection of isogenic human cell lines.
Assuntos
Biblioteca Gênica , Haploidia , Mutagênese Insercional/métodos , Genética Reversa/métodos , Linhagem Celular Tumoral , Genoma Humano , Humanos , Dados de Sequência MolecularRESUMO
Some mutations in cancer cells can be exploited for therapeutic intervention. However, for many cancer subtypes, including triple-negative breast cancer (TNBC), no frequently recurring aberrations could be identified to make such an approach clinically feasible. Characterized by a highly heterogeneous mutational landscape with few common features, many TNBCs cluster together based on their 'basal-like' transcriptional profiles. We therefore hypothesized that targeting TNBC cells on a systems level by exploiting the transcriptional cell state might be a viable strategy to find novel therapies for this highly aggressive disease. We performed a large-scale chemical genetic screen and identified a group of compounds related to the drug PKC412 (midostaurin). PKC412 induced apoptosis in a subset of TNBC cells enriched for the basal-like subtype and inhibited tumor growth in vivo. We employed a multi-omics approach and computational modeling to address the mechanism of action and identified spleen tyrosine kinase (SYK) as a novel and unexpected target in TNBC. Quantitative phosphoproteomics revealed that SYK inhibition abrogates signaling to STAT3, explaining the selectivity for basal-like breast cancer cells. This non-oncogene addiction suggests that chemical SYK inhibition may be beneficial for a specific subset of TNBC patients and demonstrates that targeting cell states could be a viable strategy to discover novel treatment strategies.
Assuntos
Antineoplásicos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Terapia de Alvo Molecular , Proteínas Tirosina Quinases/metabolismo , Fator de Transcrição STAT3/metabolismo , Estaurosporina/análogos & derivados , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Simulação de Acoplamento Molecular , Domínios e Motivos de Interação entre Proteínas , Proteômica/métodos , Análise de Sequência de RNA , Transdução de Sinais , Estaurosporina/farmacologia , Quinase Syk , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Measuring the impact of combinations of genetic or chemical perturbations on cellular fitness, sometimes referred to as synthetic lethal screening, is a powerful method for obtaining novel insights into gene function and drug action. Especially when performed at large scales, gene-gene or gene-drug interaction screens can reveal complex genetic interactions or drug mechanism of action or even identify novel therapeutics for the treatment of diseases.The result of such large-scale screen results can be represented as a matrix with a numeric score indicating the cellular fitness (e.g. viability or doubling time) for each double perturbation. In a typical screen, the majority of combinations do not impact the cellular fitness. Thus, it is critical to first discern true "hits" from noise. Subsequent data exploration and visualization methods can assist to extract meaningful biological information from the data. However, despite the increasing interest in combination perturbation screens, no user friendly open-source program exists that combines statistical analysis, data exploration tools and visualization. RESULTS: We developed TOPS (Tool for Combination Perturbation Screen Analysis), a Java and R-based software tool with a simple graphical user interface that allows the user to import, analyze, filter and plot data from double perturbation screens as well as other compatible data. TOPS was designed in a modular fashion to allow the user to add alternative importers for data formats or custom analysis scripts not covered by the original release.We demonstrate the utility of TOPS on two datasets derived from functional genetic screens using different methods. Dataset 1 is a gene-drug interaction screen and is based on Luminex xMAP technology. Dataset 2 is a gene-gene short hairpin (sh)RNAi screen exploring the interactions between deubiquitinating enzymes and a number of prominent oncogenes using massive parallel sequencing (MPS). CONCLUSIONS: TOPS provides the benchtop scientist with a free toolset to analyze, filter and visualize data from functional genomic gene-gene and gene-drug interaction screens with a flexible interface to accommodate different technologies and analysis algorithms in addition to those already provided here. TOPS is freely available for academic and non-academic users and is released as open source.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Genes , Software , Algoritmos , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Gráficos por Computador , Interpretação Estatística de Dados , Feminino , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Modelos Lineares , Interferência de RNARESUMO
Linking the molecular aberrations of cancer to drug responses could guide treatment choice and identify new therapeutic applications. However, there has been no systematic approach for analyzing gene-drug interactions in human cells. Here we establish a multiplexed assay to study the cellular fitness of a panel of engineered isogenic cancer cells in response to a collection of drugs, enabling the systematic analysis of thousands of gene-drug interactions. Applying this approach to breast cancer revealed various synthetic-lethal interactions and drug-resistance mechanisms, some of which were known, thereby validating the method. NOTCH pathway activation, which occurs frequently in breast cancer, unexpectedly conferred resistance to phosphoinositide 3-kinase (PI3K) inhibitors, which are currently undergoing clinical trials in breast cancer patients. NOTCH1 and downstream induction of c-MYC over-rode the dependency of cells on the PI3K-mTOR pathway for proliferation. These data reveal a new mechanism of resistance to PI3K inhibitors with direct clinical implications.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Fosfoinositídeo-3 Quinase , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Monoubiquitination is a reversible post-translational protein modification that has an important regulatory function in many biological processes, including DNA repair. Deubiquitinating enzymes (DUBs) are proteases that are negative regulators of monoubiquitination, but little is known about their regulation and contribution to the control of conjugated-substrate levels. Here, we show that the DUB ubiquitin specific protease 1 (USP1) deubiquitinates the DNA replication processivity factor, PCNA, as a safeguard against error-prone translesion synthesis (TLS) of DNA. Ultraviolet (UV) irradiation inactivates USP1 through an autocleavage event, thus enabling monoubiquitinated PCNA to accumulate and to activate TLS. Significantly, the site of USP1 cleavage is immediately after a conserved internal ubiquitin-like diglycine (Gly-Gly) motif. This mechanism is reminiscent of the processing of precursors of ubiquitin and ubiquitin-like modifiers by DUBs. Our results define a regulatory mechanism for protein ubiquitination that involves the signal-induced degradation of an inhibitory DUB.
Assuntos
Dano ao DNA/efeitos da radiação , Endopeptidases/metabolismo , Regulação da Expressão Gênica , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ubiquitina/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis , Replicação do DNA , Endopeptidases/química , Endopeptidases/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/fisiologia , Humanos , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Homologia de Sequência de Aminoácidos , Proteases Específicas de Ubiquitina , Raios UltravioletaRESUMO
Systemic mastocytosis is a neoplastic disease of mast cells harboring the activating KIT mutation D816V. In most patients, mast cell infiltration in the bone marrow is accompanied by marked microenvironment alterations, including increased angiogenesis, osteosclerosis, and sometimes fibrosis. Little is known about the mast cell-derived molecules contributing to these bone marrow alterations. We show here that neoplastic mast cells in patients with systemic mastocytosis express oncostatin M (OSM), a profibrogenic and angiogenic modulator. To study the regulation of OSM expression, KIT D816V was inducibly expressed in Ba/F3 cells and was found to up-regulate OSM mRNA and protein levels, suggesting that OSM is a KIT D816V-dependent mediator. Correspondingly, KIT D816V(+) HMC-1.2 cells expressed significantly higher amounts of OSM than the KIT D816V(-) HMC-1.1 subclone. RNA interference-induced knockdown of STAT5, a key transcription factor in KIT D816V(+) mast cells, inhibited OSM expression in HMC-1 cells, whereas a constitutively activated STAT5 mutant induced OSM expression. Finally, OSM secreted from KIT D816V(+) mast cells stimulated growth of endothelial cells, fibroblasts, and osteoblasts, suggesting that mast cell-derived OSM may serve as a key modulator of the marrow microenvironment and thus contribute to the pathology of systemic mastocytosis.
Assuntos
Medula Óssea/patologia , Mastocitose Sistêmica/metabolismo , Mastocitose Sistêmica/patologia , Oncostatina M/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Western Blotting , Medula Óssea/metabolismo , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Expressão Gênica , Regulação da Expressão Gênica/fisiologia , Humanos , Immunoblotting , Imuno-Histoquímica , Mastócitos/metabolismo , Mastócitos/patologia , Mastocitose Sistêmica/genética , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT5/metabolismoRESUMO
UNLABELLED: Signal transducer and activator of transcription 3 (Stat3) is activated in a variety of malignancies, including hepatocellular carcinoma (HCC). Activation of Ras occurs frequently at advanced stages of HCC by aberrant signaling through growth factor receptors or inactivation of effectors negatively regulating Ras signaling. Here, we addressed the role of Stat3 in Ras-dependent HCC progression in the presence and absence of p19(ARF) /p14(ARF) . We show that constitutive active (ca) Stat3 is tumor suppressive in Ras-transformed p19(ARF-/-) hepatocytes, whereas the expression of Stat3 lacking Tyr(705) phosphorylation (U-Stat3) enhances tumor formation. Accordingly, Ras-transformed Stat3(Δhc) /p19(ARF-/-) hepatocytes (lacking Stat3 and p19(ARF) ) showed increased tumor growth, compared to those expressing Stat3, demonstrating a tumor-suppressor activity of Stat3 in cells lacking p19(ARF) . Notably, endogenous expression of p19(ARF) in Ras-transformed hepatocytes conveyed oncogenic Stat3 functions, resulting in augmented or reduced HCC progression after the expression of caStat3 or U-Stat3, respectively. In accord with these data, the knockdown of p14(ARF) (the human homolog of p19(ARF) ) in Hep3B cells was associated with reduced pY-Stat3 levels during tumor growth to circumvent the tumor-suppressive effect of Stat3. Inhibition of Janus kinases (Jaks) revealed that Jak causes pY-Stat3 activation independently of p14(ARF) levels, indicating that p14(ARF) controls the oncogenic function of pY-Stat3 downstream of Jak. CONCLUSION: These data show evidence that p19(ARF) /p14(ARF) determines the pro- or anti-oncogenic activity of U-Stat3 and pY-Stat3 in Ras-dependent HCC progression.
Assuntos
Carcinoma Hepatocelular/fisiopatologia , Inibidor p16 de Quinase Dependente de Ciclina/fisiologia , Neoplasias Hepáticas/fisiopatologia , Fator de Transcrição STAT3/fisiologia , Animais , Carcinoma Hepatocelular/patologia , Proliferação de Células , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Inibidor p16 de Quinase Dependente de Ciclina/genética , Modelos Animais de Doenças , Progressão da Doença , Hepatócitos/patologia , Janus Quinases/fisiologia , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Knockout , Transdução de Sinais/fisiologiaRESUMO
Protein modification by the conjugation of ubiquitin moieties--ubiquitination--plays a major part in many biological processes, including cell cycle and apoptosis. The enzymes that mediate ubiquitin-conjugation have been well-studied, but much less is known about the ubiquitin-specific proteases that mediate de-ubiquitination of cellular substrates. To study this gene family, we designed a collection of RNA interference vectors to suppress 50 human de-ubiquitinating enzymes, and used these vectors to identify de-ubiquitinating enzymes in cancer-relevant pathways. We report here that inhibition of one of these enzymes, the familial cylindromatosis tumour suppressor gene (CYLD), having no known function, enhances activation of the transcription factor NF-kappaB. We show that CYLD binds to the NEMO (also known as IKKgamma) component of the IkappaB kinase (IKK) complex, and appears to regulate its activity through de-ubiquitination of TRAF2, as TRAF2 ubiquitination can be modulated by CYLD. Inhibition of CYLD increases resistance to apoptosis, suggesting a mechanism through which loss of CYLD contributes to oncogenesis. We show that this effect can be relieved by aspirin derivatives that inhibit NF-kappaB activity, which suggests a therapeutic intervention strategy to restore growth control in patients suffering from familial cylindromatosis.
Assuntos
Apoptose , NF-kappa B/metabolismo , Proteínas Supressoras de Tumor/deficiência , Apoptose/efeitos dos fármacos , Aspirina/análogos & derivados , Aspirina/farmacologia , Linhagem Celular , Enzima Desubiquitinante CYLD , Humanos , Quinase I-kappa B , NF-kappa B/antagonistas & inibidores , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Interferência de RNA , Fator 2 Associado a Receptor de TNF , Transfecção , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina/metabolismoRESUMO
Intratumor heterogeneity is a key hallmark of cancer that contributes to progression and therapeutic resistance. Phenotypic heterogeneity is in part caused by Darwinian selection of subclones that arise by random (epi)genetic aberrations. In addition, cancer cells are endowed with increased cellular plasticity compared with their normal counterparts, further adding to their heterogeneous behavior. However, the molecular mechanisms underpinning cancer cell plasticity are incompletely understood. Here, I outline the hypothesis that cancer-associated perturbations collectively disrupt normal gene regulatory networks (GRNs) by increasing their entropy. Importantly, in this model both somatic driver and passenger alterations contribute to 'perturbation-driven entropy', thereby increasing phenotypic heterogeneity and evolvability. This additional layer of heterogeneity may contribute to our understanding of cancer evolution and therapeutic resistance.
Assuntos
Evolução Molecular , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Heterogeneidade Genética , Neoplasias/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Plasticidade Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Entropia , Epigênese Genética , Humanos , Modelos Genéticos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Células-Tronco Neoplásicas/patologiaRESUMO
Forward genetic screens in mammalian cell lines, such as RNAi and CRISPR-Cas9 screens, have made major contributions to the elucidation of diverse signaling pathways. Here, we exploited human haploid cells as a robust comparative screening platform and report a set of quantitative forward genetic screens for identifying regulatory mechanisms of mTORC1 signaling, a key growth control pathway that senses diverse metabolic states. Selected chemical and genetic perturbations in this screening platform, including rapamycin treatment and genetic ablation of the Ragulator subunit LAMTOR4, revealed the known core mTORC1 regulatory signaling complexes and the intimate interplay of the mTORC1 pathway with lysosomal function, validating the approach. In addition, we identified a differential requirement for LAMTOR4 and LAMTOR5 in regulating the mTORC1 pathway under fed and starved conditions. Furthermore, we uncovered a previously unknown "synthetic-sick" interaction between the tumor suppressor folliculin and LAMTOR4, which may have therapeutic implications in cancer treatment. Together, our study demonstrates the use of iterative "perturb and observe" genetic screens to uncover regulatory mechanisms driving complex mammalian signaling networks.
Assuntos
Retroalimentação Fisiológica , Testes Genéticos/métodos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Haploidia , Células HeLa , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genéticaAssuntos
Sistema do Grupo Sanguíneo Rh-Hr/genética , Animais , Técnicas de Cultura de Células/métodos , Divisão Celular , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Perfilação da Expressão Gênica , Humanos , RNA de Cadeia Dupla/genética , Sistema do Grupo Sanguíneo Rh-Hr/fisiologiaRESUMO
Lung cancer is the leading cause of cancer deaths, and effective treatments are urgently needed. Loss-of-function mutations in the DNA damage response kinase ATM are common in lung adenocarcinoma but directly targeting these with drugs remains challenging. Here we report that ATM loss-of-function is synthetic lethal with drugs inhibiting the central growth factor kinases MEK1/2, including the FDA-approved drug trametinib. Lung cancer cells resistant to MEK inhibition become highly sensitive upon loss of ATM both in vitro and in vivo. Mechanistically, ATM mediates crosstalk between the prosurvival MEK/ERK and AKT/mTOR pathways. ATM loss also enhances the sensitivity of KRAS- or BRAF-mutant lung cancer cells to MEK inhibition. Thus, ATM mutational status in lung cancer is a mechanistic biomarker for MEK inhibitor response, which may improve patient stratification and extend the applicability of these drugs beyond RAS and BRAF mutant tumours.