RESUMO
Parent scientists lead a journey to bring surveillance severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing to public schools across the state of Massachusetts and beyond.
Assuntos
COVID-19/diagnóstico , Pessoal de Laboratório , Pais , COVID-19/virologia , Teste para COVID-19 , Comportamento Cooperativo , Educação , Humanos , Massachusetts , SARS-CoV-2/fisiologiaRESUMO
In vitro cancer cultures, including three-dimensional organoids, typically contain exclusively neoplastic epithelium but require artificial reconstitution to recapitulate the tumor microenvironment (TME). The co-culture of primary tumor epithelia with endogenous, syngeneic tumor-infiltrating lymphocytes (TILs) as a cohesive unit has been particularly elusive. Here, an air-liquid interface (ALI) method propagated patient-derived organoids (PDOs) from >100 human biopsies or mouse tumors in syngeneic immunocompetent hosts as tumor epithelia with native embedded immune cells (T, B, NK, macrophages). Robust droplet-based, single-cell simultaneous determination of gene expression and immune repertoire indicated that PDO TILs accurately preserved the original tumor T cell receptor (TCR) spectrum. Crucially, human and murine PDOs successfully modeled immune checkpoint blockade (ICB) with anti-PD-1- and/or anti-PD-L1 expanding and activating tumor antigen-specific TILs and eliciting tumor cytotoxicity. Organoid-based propagation of primary tumor epithelium en bloc with endogenous immune stroma should enable immuno-oncology investigations within the TME and facilitate personalized immunotherapy testing.
Assuntos
Modelos Imunológicos , Neoplasias Experimentais/imunologia , Organoides/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Microambiente Tumoral/imunologia , Animais , Antígeno B7-H1/imunologia , Técnicas de Cocultura , Feminino , Humanos , Imunoterapia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias/imunologia , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Organoides/patologiaRESUMO
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.
Assuntos
Neoplasias/genética , Neoplasias/patologia , Linhagem Celular Tumoral , Humanos , Interferência de RNA , Software , Ubiquitina/genéticaRESUMO
We previously piloted the concept of a Connectivity Map (CMap), whereby genes, drugs, and disease states are connected by virtue of common gene-expression signatures. Here, we report more than a 1,000-fold scale-up of the CMap as part of the NIH LINCS Consortium, made possible by a new, low-cost, high-throughput reduced representation expression profiling method that we term L1000. We show that L1000 is highly reproducible, comparable to RNA sequencing, and suitable for computational inference of the expression levels of 81% of non-measured transcripts. We further show that the expanded CMap can be used to discover mechanism of action of small molecules, functionally annotate genetic variants of disease genes, and inform clinical trials. The 1.3 million L1000 profiles described here, as well as tools for their analysis, are available at https://clue.io.
Assuntos
Perfilação da Expressão Gênica/métodos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Perfilação da Expressão Gênica/economia , Humanos , Neoplasias/tratamento farmacológico , Especificidade de Órgãos , Preparações Farmacêuticas/metabolismo , Análise de Sequência de RNA/economia , Análise de Sequência de RNA/métodos , Bibliotecas de Moléculas PequenasRESUMO
Synthetic lethality-an interaction between two genetic events through which the co-occurrence of these two genetic events leads to cell death, but each event alone does not-can be exploited for cancer therapeutics1. DNA repair processes represent attractive synthetic lethal targets, because many cancers exhibit an impairment of a DNA repair pathway, which can lead to dependence on specific repair proteins2. The success of poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors in cancers with deficiencies in homologous recombination highlights the potential of this approach3. Hypothesizing that other DNA repair defects would give rise to synthetic lethal relationships, we queried dependencies in cancers with microsatellite instability (MSI), which results from deficient DNA mismatch repair. Here we analysed data from large-scale silencing screens using CRISPR-Cas9-mediated knockout and RNA interference, and found that the RecQ DNA helicase WRN was selectively essential in MSI models in vitro and in vivo, yet dispensable in models of cancers that are microsatellite stable. Depletion of WRN induced double-stranded DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models required the helicase activity of WRN, but not its exonuclease activity. These findings show that WRN is a synthetic lethal vulnerability and promising drug target for MSI cancers.
Assuntos
Instabilidade de Microssatélites , Repetições de Microssatélites/genética , Neoplasias/genética , Mutações Sintéticas Letais/genética , Helicase da Síndrome de Werner/genética , Apoptose/genética , Sistemas CRISPR-Cas/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Humanos , Modelos Genéticos , Neoplasias/patologia , Interferência de RNA , Proteína Supressora de Tumor p53/metabolismo , Helicase da Síndrome de Werner/deficiênciaRESUMO
An alternative to therapeutic targeting of oncogenes is to perform "synthetic lethality" screens for genes that are essential only in the context of specific cancer-causing mutations. We used high-throughput RNA interference (RNAi) to identify synthetic lethal interactions in cancer cells harboring mutant KRAS, the most commonly mutated human oncogene. We find that cells that are dependent on mutant KRAS exhibit sensitivity to suppression of the serine/threonine kinase STK33 irrespective of tissue origin, whereas STK33 is not required by KRAS-independent cells. STK33 promotes cancer cell viability in a kinase activity-dependent manner by regulating the suppression of mitochondrial apoptosis mediated through S6K1-induced inactivation of the death agonist BAD selectively in mutant KRAS-dependent cells. These observations identify STK33 as a target for treatment of mutant KRAS-driven cancers and demonstrate the potential of RNAi screens for discovering functional dependencies created by oncogenic mutations that may enable therapeutic intervention for cancers with "undruggable" genetic alterations.
Assuntos
Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Camundongos , Mutação , Células NIH 3T3 , Neoplasias/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras) , Interferência de RNA , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismoRESUMO
Mammalian genomes contain thousands of loci that transcribe long noncoding RNAs (lncRNAs), some of which are known to carry out critical roles in diverse cellular processes through a variety of mechanisms. Although some lncRNA loci encode RNAs that act non-locally (in trans), there is emerging evidence that many lncRNA loci act locally (in cis) to regulate the expression of nearby genes-for example, through functions of the lncRNA promoter, transcription, or transcript itself. Despite their potentially important roles, it remains challenging to identify functional lncRNA loci and distinguish among these and other mechanisms. Here, to address these challenges, we developed a genome-scale CRISPR-Cas9 activation screen that targets more than 10,000 lncRNA transcriptional start sites to identify noncoding loci that influence a phenotype of interest. We found 11 lncRNA loci that, upon recruitment of an activator, mediate resistance to BRAF inhibitors in human melanoma cells. Most candidate loci appear to regulate nearby genes. Detailed analysis of one candidate, termed EMICERI, revealed that its transcriptional activation resulted in dosage-dependent activation of four neighbouring protein-coding genes, one of which confers the resistance phenotype. Our screening and characterization approach provides a CRISPR toolkit with which to systematically discover the functions of noncoding loci and elucidate their diverse roles in gene regulation and cellular function.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Loci Gênicos/genética , Genoma Humano/genética , Indóis/farmacologia , Melanoma/genética , RNA Longo não Codificante/genética , Sulfonamidas/farmacologia , Ativação Transcricional/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Loci Gênicos/efeitos dos fármacos , Via de Sinalização Hippo , Humanos , Indóis/uso terapêutico , Melanoma/tratamento farmacológico , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fenótipo , Regiões Promotoras Genéticas/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/uso terapêutico , Sítio de Iniciação de Transcrição , VemurafenibRESUMO
This corrects the article DOI: 10.1038/nature23451.
RESUMO
Genomic analysis of tumours has led to the identification of hundreds of cancer genes on the basis of the presence of mutations in protein-coding regions. By contrast, much less is known about cancer-causing mutations in non-coding regions. Here we perform deep sequencing in 360 primary breast cancers and develop computational methods to identify significantly mutated promoters. Clear signals are found in the promoters of three genes. FOXA1, a known driver of hormone-receptor positive breast cancer, harbours a mutational hotspot in its promoter leading to overexpression through increased E2F binding. RMRP and NEAT1, two non-coding RNA genes, carry mutations that affect protein binding to their promoters and alter expression levels. Our study shows that promoter regions harbour recurrent mutations in cancer with functional consequences and that the mutations occur at similar frequencies as in coding regions. Power analyses indicate that more such regions remain to be discovered through deep sequencing of adequately sized cohorts of patients.
Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/genética , Mutação , Regiões Promotoras Genéticas/genética , Estudos de Coortes , Fatores de Transcrição E2F/metabolismo , Exoma/genética , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ligação Proteica/genética , RNA Longo não Codificante/genética , Receptores de Estrogênio/antagonistas & inibidoresRESUMO
Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness. A high-mesenchymal cell state observed in human tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a non-apoptotic form of cell death induced by the build-up of toxic lipid peroxides. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial-mesenchymal transition in epithelial-derived carcinomas, TGFß-mediated therapy-resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.
Assuntos
Glutationa Peroxidase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Caderinas/metabolismo , Morte Celular , Linhagem Celular Tumoral , Linhagem da Célula , Transdiferenciação Celular , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal , Humanos , Ferro/metabolismo , Peróxidos Lipídicos/metabolismo , Masculino , Melanoma/tratamento farmacológico , Melanoma/enzimologia , Melanoma/metabolismo , Melanoma/patologia , Mesoderma/efeitos dos fármacos , Mesoderma/enzimologia , Mesoderma/metabolismo , Mesoderma/patologia , Neoplasias/genética , Neoplasias/patologia , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteômica , Proteínas Proto-Oncogênicas B-raf/genética , Reprodutibilidade dos Testes , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
BACKGROUND: Primary and metastatic prostate cancers have low mutation rates and recurrent alterations in a small set of genes, enabling targeted sequencing of prostate cancer-associated genes as an efficient approach to characterizing patient samples (compared to whole-exome and whole-genome sequencing). For example, targeted sequencing provides a flexible, rapid, and cost-effective method for genomic assessment of patient-derived cell lines to evaluate fidelity to initial patient tumor samples. METHODS: We developed a prostate cancer-specific targeted next-generation sequencing (NGS) panel to detect alterations in 62 prostate cancer-associated genes as well as recurring gene fusions with ETS family members, representing the majority of common alterations in prostate cancer. We tested this panel on primary prostate cancer tissues and blood biopsies from patients with metastatic prostate cancer. We generated patient-derived cell lines from primary prostate cancers using conditional reprogramming methods and applied targeted sequencing to evaluate the fidelity of these cell lines to the original patient tumors. RESULTS: The prostate cancer-specific panel identified biologically and clinically relevant alterations, including point mutations in driver oncogenes and ETS family fusion genes, in tumor tissues from 29 radical prostatectomy samples. The targeted panel also identified genomic alterations in cell-free DNA and circulating tumor cells (CTCs) from patients with metastatic prostate cancer, and in standard prostate cancer cell lines. We used the targeted panel to sequence our set of patient-derived cell lines; however, no prostate cancer-specific mutations were identified in the tumor-derived cell lines, suggesting preferential outgrowth of normal prostate epithelial cells. CONCLUSIONS: We evaluated a prostate cancer-specific targeted NGS panel to detect common and clinically relevant alterations (including ETS family gene fusions) in prostate cancer. The panel detected driver mutations in a diverse set of clinical samples of prostate cancer, including fresh-frozen tumors, cell-free DNA, CTCs, and cell lines. Targeted sequencing of patient-derived cell lines highlights the challenge of deriving cell lines from primary prostate cancers and the importance of genomic characterization to credential candidate cell lines. Our study supports that a prostate cancer-specific targeted sequencing panel provides an efficient, clinically feasible approach to identify genetic alterations across a spectrum of prostate cancer samples and cell lines.
Assuntos
Ácidos Nucleicos Livres , Neoplasias da Próstata , Linhagem Celular , Credenciamento , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Mutação , Neoplasias da Próstata/genéticaAssuntos
Pesquisa Biomédica/organização & administração , Pesquisa Biomédica/tendências , Objetivos , Cooperação Internacional , Terapia de Alvo Molecular/tendências , Neoplasias/tratamento farmacológico , Neoplasias/genética , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Pesquisa Biomédica/economia , Sistemas CRISPR-Cas/genética , Sobrevivência Celular/efeitos dos fármacos , Análise Mutacional de DNA , Conjuntos de Dados como Assunto , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Disseminação de Informação , Aprendizado de Máquina , Mutação , Neoplasias/metabolismo , Neoplasias/patologia , Projetos Piloto , Medicina de Precisão , Reprodutibilidade dos TestesRESUMO
Methods that integrate molecular network information and tumor genome data could complement gene-based statistical tests to identify likely new cancer genes; but such approaches are challenging to validate at scale, and their predictive value remains unclear. We developed a robust statistic (NetSig) that integrates protein interaction networks with data from 4,742 tumor exomes. NetSig can accurately classify known driver genes in 60% of tested tumor types and predicts 62 new driver candidates. Using a quantitative experimental framework to determine in vivo tumorigenic potential in mice, we found that NetSig candidates induce tumors at rates that are comparable to those of known oncogenes and are ten-fold higher than those of random genes. By reanalyzing nine tumor-inducing NetSig candidates in 242 patients with oncogene-negative lung adenocarcinomas, we find that two (AKT2 and TFDP2) are significantly amplified. Our study presents a scalable integrated computational and experimental workflow to expand discovery from cancer genomes.
Assuntos
Carcinogênese/genética , Biologia Computacional/métodos , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Neoplasias/genética , Neoplasias/genética , Humanos , MutaçãoRESUMO
How do small molecules exert their effects in mammalian cells? This seemingly simple question continues to represent one of the fundamental challenges of modern translational science and as such has long been the subject of intense scientific scrutiny. In their recent study, Garnett and colleagues (Gonçalves et al, 2020) demonstrate proof-of-concept for a new way to attack this problem systematically for Oncology drugs, by identifying correlated CRISPR- and drug-killing profiles in the Cancer Dependency Map dataset.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Neoplasias , Animais , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
Jesse Boehm and Todd Golub call for an international effort to establish >10,000 cancer cell line models as a community resource. Cancer cell line factories will facilitate the creation of a cancer dependency map, connecting cancer genomics to therapeutic dependencies.
Assuntos
Antineoplásicos/farmacologia , Ecossistema , Redes Reguladoras de Genes , Genômica/métodos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Linhagem Celular Tumoral , HumanosRESUMO
Renal angiomyolipoma is a kidney tumor in the perivascular epithelioid (PEComa) family that is common in patients with Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM) but occurs rarely sporadically. Though histologically benign, renal angiomyolipoma can cause life-threatening hemorrhage and kidney failure. Both angiomyolipoma and LAM have mutations in TSC2 or TSC1. However, the frequency and contribution of other somatic events in tumor development is unknown. We performed whole exome sequencing in 32 resected tumor samples (n = 30 angiomyolipoma, n = 2 LAM) from 15 subjects, including three with TSC. Two germline and 22 somatic inactivating mutations in TSC2 were identified, and one germline TSC1 mutation. Twenty of 32 (62%) samples showed copy neutral LOH (CN-LOH) in TSC2 or TSC1 with at least 8 different LOH regions, and 30 of 32 (94%) had biallelic loss of either TSC2 or TSC1. Whole exome sequencing identified a median of 4 somatic non-synonymous coding region mutations (other than in TSC2/TSC1), a mutation rate lower than nearly all other cancer types. Three genes with mutations were known cancer associated genes (BAP1, ARHGAP35 and SPEN), but they were mutated in a single sample each, and were missense variants with uncertain functional effects. Analysis of sixteen angiomyolipomas from a TSC subject showed both second hit point mutations and CN-LOH in TSC2, many of which were distinct, indicating that they were of independent clonal origin. However, three tumors had two shared mutations in addition to private somatic mutations, suggesting a branching evolutionary pattern of tumor development following initiating loss of TSC2. Our results indicate that TSC2 and less commonly TSC1 alterations are the primary essential driver event in angiomyolipoma/LAM, whereas other somatic mutations are rare and likely do not contribute to tumor development.
Assuntos
Angiomiolipoma/genética , Neoplasias Renais/genética , Linfangioleiomiomatose/genética , Proteínas Supressoras de Tumor/genética , Adulto , Angiomiolipoma/patologia , Carcinogênese/genética , Exoma/genética , Feminino , Mutação em Linhagem Germinativa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Renais/patologia , Perda de Heterozigosidade/genética , Linfangioleiomiomatose/patologia , Masculino , Mutação , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose TuberosaRESUMO
Whole-genome approaches to identify genetic and epigenetic alterations in cancer genomes have begun to provide new insights into the range of molecular events that occurs in human tumours. Although in some cases this knowledge immediately illuminates a path towards diagnostic or therapeutic implementation, the bewildering lists of mutations in each tumour make it clear that systematic functional approaches are also necessary to obtain a comprehensive molecular understanding of cancer. Here we review the current range of methods, assays and approaches for genome-scale interrogation of gene function in cancer. We also discuss the integration of functional-genomics approaches with the outputs from cancer genome sequencing efforts.
Assuntos
Genoma Humano/genética , Genoma Humano/fisiologia , Genômica/tendências , Neoplasias/genética , Algoritmos , Animais , Bases de Dados Genéticas/tendências , Humanos , Modelos Biológicos , Projetos de Pesquisa/tendênciasRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Feminino , Genômica , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Fases de Leitura Aberta , Neoplasias Ovarianas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de SinaisRESUMO
Epigenetic control of genome function is an important regulatory mechanism in diverse processes such as lineage commitment and environmental sensing, and in disease etiologies ranging from neuropsychiatric disorders to cancer. Here we report a robust, high-throughput targeted, quantitative mass spectrometry (MS) method to rapidly profile modifications of the core histones of chromatin that compose the epigenetic landscape, enabling comparisons among cells with differing genetic backgrounds, genomic perturbations, and drug treatments.
Assuntos
Cromatina/química , Epigenômica/métodos , Espectrometria de Massas/métodos , Genômica , Histonas/metabolismo , Humanos , Processamento de Proteína Pós-Traducional , ProteômicaRESUMO
Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. Identification of resistance mechanisms in a manner that elucidates alternative 'druggable' targets may inform effective long-term treatment strategies. Here we expressed â¼600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.