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Genetic alterations in signaling pathways that control cell-cycle progression, apoptosis, and cell growth are common hallmarks of cancer, but the extent, mechanisms, and co-occurrence of alterations in these pathways differ between individual tumors and tumor types. Using mutations, copy-number changes, mRNA expression, gene fusions and DNA methylation in 9,125 tumors profiled by The Cancer Genome Atlas (TCGA), we analyzed the mechanisms and patterns of somatic alterations in ten canonical pathways: cell cycle, Hippo, Myc, Notch, Nrf2, PI-3-Kinase/Akt, RTK-RAS, TGFß signaling, p53 and ß-catenin/Wnt. We charted the detailed landscape of pathway alterations in 33 cancer types, stratified into 64 subtypes, and identified patterns of co-occurrence and mutual exclusivity. Eighty-nine percent of tumors had at least one driver alteration in these pathways, and 57% percent of tumors had at least one alteration potentially targetable by currently available drugs. Thirty percent of tumors had multiple targetable alterations, indicating opportunities for combination therapy.
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Bases de Datos Genéticas , Neoplasias/patología , Transducción de Señal/genética , Genes Relacionados con las Neoplasias , Humanos , Neoplasias/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
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.
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Perfilación de la Expresión Génica/métodos , Línea Celular Tumoral , Resistencia a Antineoplásicos , Perfilación de la Expresión Génica/economía , Humanos , Neoplasias/tratamiento farmacológico , Especificidad de Órganos , Preparaciones Farmacéuticas/metabolismo , Análisis de Secuencia de ARN/economía , Análisis de Secuencia de ARN/métodos , Bibliotecas de Moléculas PequeñasRESUMEN
Cuella-Martin et al. (2021) and Hanna et al. (2021) showcase CRISPR base editing in large-scale pooled screens in human cells to discover both loss- and gain-of-function variants, enabling protein structure/function insights and clinical variant interpretation.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , HumanosRESUMEN
Lung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for more than 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole-genome sequence analysis revealed frequent structural rearrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.
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Adenocarcinoma/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Genes Relacionados con las Neoplasias , Secuenciación de Nucleótidos de Alto Rendimiento , Neoplasias Pulmonares/genética , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Pulmón de Células no Pequeñas/patología , Estudios de Cohortes , Exoma , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Mutación , Tasa de MutaciónRESUMEN
BACKGROUND: Alternative RNA splicing is widely dysregulated in cancers including lung adenocarcinoma, where aberrant splicing events are frequently caused by somatic splice site mutations or somatic mutations of splicing factor genes. However, the majority of mis-splicing in cancers is unexplained by these known mechanisms. We hypothesize that the aberrant Ras signaling characteristic of lung cancers plays a role in promoting the alternative splicing observed in tumors. METHODS: We recently performed transcriptome and proteome profiling of human lung epithelial cells ectopically expressing oncogenic KRAS and another cancer-associated Ras GTPase, RIT1. Unbiased analysis of phosphoproteome data identified altered splicing factor phosphorylation in KRAS-mutant cells, so we performed differential alternative splicing analysis using rMATS to identify significantly altered isoforms in lung epithelial cells. To determine whether these isoforms were uniquely regulated by KRAS, we performed a large-scale splicing screen in which we generated over 300 unique RNA sequencing profiles of isogenic A549 lung adenocarcinoma cells ectopically expressing 75 different wild-type or variant alleles across 28 genes implicated in lung cancer. RESULTS: Mass spectrometry data showed widespread downregulation of splicing factor phosphorylation in lung epithelial cells expressing mutant KRAS compared to cells expressing wild-type KRAS. We observed alternative splicing in the same cells, with 2196 and 2416 skipped exon events in KRASG12V and KRASQ61H cells, respectively, 997 of which were shared (p < 0.001 by hypergeometric test). In the high-throughput splicing screen, mutant KRAS induced the greatest number of differential alternative splicing events, second only to the RNA binding protein RBM45 and its variant RBM45M126I. We identified ten high confidence cassette exon events across multiple KRAS variants and cell lines. These included differential splicing of the Myc Associated Zinc Finger (MAZ). As MAZ regulates expression of KRAS, this splice variant may be a mechanism for the cell to modulate wild-type KRAS levels in the presence of oncogenic KRAS. CONCLUSION: Proteomic and transcriptomic profiling of lung epithelial cells uncovered splicing factor phosphorylation and mRNA splicing events regulated by oncogenic KRAS. These data suggest that in addition to widespread transcriptional changes, the Ras signaling pathway can promote post-transcriptional splicing changes that may contribute to oncogenic processes.
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Adenocarcinoma del Pulmón , Neoplasias Pulmonares , Humanos , Empalme Alternativo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Fosforilación , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Proteómica , Línea Celular Tumoral , Neoplasias Pulmonares/patología , Adenocarcinoma del Pulmón/genética , Mutación , Isoformas de Proteínas/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
While advancements in genome sequencing have identified millions of somatic mutations in cancer, their functional impact is poorly understood. We previously developed the expression-based variant impact phenotyping (eVIP) method to use gene expression data to characterize the function of gene variants. The eVIP method uses a decision tree-based algorithm to predict the functional impact of somatic variants by comparing gene expression signatures induced by introduction of wild-type (WT) versus mutant cDNAs in cell lines. The method distinguishes between variants that are gain-of-function, loss-of-function, change-of-function, or neutral. We present eVIP2, software that allows for pathway analysis (eVIP Pathways) and usage with RNA-seq data. To demonstrate the eVIP2 software and approach, we characterized two recurrent frameshift variants in RNF43, a negative regulator of Wnt signaling, frequently mutated in colorectal, gastric, and endometrial cancer. RNF43 WT, RNF43 R117fs, RNF43 G659fs, or GFP control cDNA were overexpressed in HEK293T cells. Analysis with eVIP2 predicted that the frameshift at position 117 was a loss-of-function mutation, as expected. The second frameshift at position 659 has been previously described as a passenger mutation that maintains the RNF43 WT function as a negative regulator of Wnt. Surprisingly, eVIP2 predicted G659fs to be a change-of-function mutation. Additional eVIP Pathways analysis of RNF43 G659fs predicted 10 pathways to be significantly altered, including TNF-α via NFκB signaling, KRAS signaling, and hypoxia, highlighting the benefit of a more comprehensive approach when determining the impact of gene variant function. To validate these predictions, we performed reporter assays and found that each pathway activated by expression of RNF43 G659fs, but not expression of RNF43 WT, was identified as impacted by eVIP2, supporting that RNF43 G659fs is a change-of-function mutation and its effect on the identified pathways. Pathway activation was further validated by Western blot analysis. Lastly, we show primary colon adenocarcinoma patient samples with R117fs and G659fs variants have transcriptional profiles similar to BRAF missense mutations with activated RAS/MAPK signaling, consistent with KRAS signaling pathways being GOF in both variants. The eVIP2 method is an important step towards overcoming the current challenge of variant interpretation in the implementation of precision medicine. eVIP2 is available at https://github.com/BrooksLabUCSC/eVIP2.
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Variación Genética/genética , Genómica/métodos , Transcriptoma/genética , Algoritmos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/genética , Modelos Genéticos , Mutación/genética , Fenotipo , Ubiquitina-Proteína Ligasas/genética , Vía de Señalización Wnt/genéticaRESUMEN
This year, 2011, marks the forty-year anniversary of the statistical analysis of retinoblastoma that provided the first evidence that tumorigenesis can be initiated by as few as two mutations. This work provided the foundation for the two-hit hypothesis that explained the role of recessive tumour suppressor genes (TSGs) in dominantly inherited cancer susceptibility syndromes. However, four decades later, it is now known that even partial inactivation of tumour suppressors can critically contribute to tumorigenesis. Here we analyse this evidence and propose a continuum model of TSG function to explain the full range of TSG mutations found in cancer.
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Genes Supresores de Tumor/fisiología , Modelos Genéticos , Neoplasias/genética , Neoplasias/patología , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Predisposición Genética a la Enfermedad/genética , Haploinsuficiencia/genética , Humanos , Neoplasias/metabolismo , Neoplasias/terapiaRESUMEN
After Helicobacter pylori infection in humans, gastric epithelial cells (GECs) undergo apoptosis due to stimulation by the bacteria or inflammatory cytokines. In this study, we assessed the expression and function of brain angiogenesis inhibitor 1 (BAI1) in the engulfment of apoptotic GECs using human tissue and cells. After induction of apoptosis by H. pylori or camptothecin, there was a 5-fold increase in the binding of apoptotic GECs to THP-1 cells or peripheral blood monocyte-derived macrophages as assayed by confocal microscopy or conventional and imaging flow cytometry. Binding was impaired 95% by pretreating apoptotic cells with annexin V, underscoring the requirement for phosphatidylserine recognition. The phosphatidylserine receptor BAI1 was expressed in human gastric biopsy specimens and gastric phagocytes. To confirm the role of BAI1 in apoptotic cell clearance, the functional domain of BAI1 was used as a competitive inhibitor or BAI1 expression was inhibited by small interfering RNA. Both approaches decreased binding and engulfment >40%. Exposing THP-1 cells to apoptotic cells inhibited IL-6 production from 1340 to <364 pg/ml; however, this decrease was independent of phagocytosis. We conclude that recognition of apoptotic cells by BAI1 contributes to their clearance in the human gastric mucosa and this is associated with anti-inflammatory effects.
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Proteínas Angiogénicas/metabolismo , Apoptosis , Células Epiteliales/metabolismo , Infecciones por Helicobacter/metabolismo , Fagocitos/metabolismo , Línea Celular , Técnicas de Cocultivo , Citocinas/metabolismo , Células Epiteliales/microbiología , Mucosa Gástrica/citología , Mucosa Gástrica/microbiología , Gastritis/metabolismo , Regulación de la Expresión Génica , Helicobacter pylori , Humanos , Inflamación , Macrófagos/citología , Macrófagos/metabolismo , Monocitos/citología , Fagocitos/citología , Fagocitosis , Receptores de Superficie Celular/química , Receptores Acoplados a Proteínas G , Estómago/citología , Estómago/microbiologíaRESUMEN
We assessed somatic alleles of six receptor tyrosine kinase genes mutated in lung adenocarcinoma for oncogenic activity. Five of these genes failed to score in transformation assays; however, novel recurring extracellular domain mutations of the receptor tyrosine kinase gene ERBB2 were potently oncogenic. These ERBB2 extracellular domain mutants were activated by two distinct mechanisms, characterized by elevated C-terminal tail phosphorylation or by covalent dimerization mediated by intermolecular disulfide bond formation. These distinct mechanisms of receptor activation converged upon tyrosine phosphorylation of cellular proteins, impacting cell motility. Survival of Ba/F3 cells transformed to IL-3 independence by the ERBB2 extracellular domain mutants was abrogated by treatment with small-molecule inhibitors of ERBB2, raising the possibility that patients harboring such mutations could benefit from ERBB2-directed therapy.
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Adenocarcinoma/enzimología , Neoplasias Pulmonares/enzimología , Mutación/genética , Receptor ErbB-2/genética , Adenocarcinoma/genética , Adenocarcinoma del Pulmón , Alelos , Animales , Movimiento Celular/fisiología , Clonación Molecular , Cartilla de ADN/genética , Dimerización , Immunoblotting , Neoplasias Pulmonares/genética , Ratones , Células 3T3 NIH , Fosforilación , Estructura Terciaria de Proteína/genética , Retroviridae , Espectrometría de Masas en TándemRESUMEN
RIT1 is a rare and understudied oncogene in lung cancer. Despite structural similarity to other RAS GTPase proteins such as KRAS, oncogenic RIT1 activity does not appear to be tightly regulated by nucleotide exchange or hydrolysis. Instead, there is a growing understanding that the protein abundance of RIT1 is important for its regulation and function. We previously identified the deubiquitinase USP9X as a RIT1 dependency in RIT1-mutant cells. Here, we demonstrate that both wild-type and mutant forms of RIT1 are substrates of USP9X. Depletion of USP9X leads to decreased RIT1 protein stability and abundance and resensitizes cells to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in vitro and in vivo. Our work expands upon the current understanding of RIT1 protein regulation and presents USP9X as a key regulator of RIT1-driven oncogenic phenotypes.
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Over 200,000 individuals are diagnosed with lung cancer in the U.S. every year, with a growing proportion of cases, especially lung adenocarcinoma, occurring in individuals who have never smoked. Women over the age of 50 comprise the largest affected demographic. To understand the genomic drivers of lung adenocarcinoma and therapeutic response in this population, we performed whole genome and/or whole exome sequencing on 73 matched lung tumor/normal pairs from post-menopausal women who participated in the Women's Health Initiative. Somatic copy number alterations showed little variation by smoking status, suggesting that aneuploidy may be a general characteristic of lung cancer regardless of smoke exposure. Similarly, clock-like and APOBEC mutation signatures were prevalent but did not differ in tumors from smokers and never-smokers. However, mutations in both EGFR and KRAS showed unique allelic differences determined by smoking status that are known to alter tumor response to targeted therapy. Mutations in the MYC-network member MGA were more prevalent in tumors from smokers. Fusion events in ALK, RET, and ROS1 were absent, likely due to age-related differences in fusion prevalence. Our work underscores the profound impact of smoking status, age, and sex on the tumor mutational landscape and identifies areas of unmet medical need.
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Publishing in Science, Girish and colleagues achieve chromosome-level genome editing to reveal a requirement for aneuploidy in breast and melanoma cancers. Authors developed and leveraged ReDACT (restoring disomy in aneuploid cells using CRISPR targeting) to generate isogenic models of aneuploidy and demonstrate that some cancers are addicted to increased copy number of specific chromosome arms.
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Edición Génica , Neoplasias , Humanos , Aneuploidia , Neoplasias/genética , CariotipoRESUMEN
We developed pgMAP, an analysis pipeline to map gRNA sequencing reads from dual-targeting CRISPR screens. pgMAP output includes a dual gRNA read counts table and quality control metrics including the proportion of correctly-paired reads and CRISPR library sequencing coverage across all time points and samples. pgMAP is implemented using Snakemake and is available open-source under the MIT license at https://github.com/fredhutch/pgmap_pipeline.
RESUMEN
RIT1 is a rare and understudied oncogene in lung cancer. Despite structural similarity to other RAS GTPase proteins such as KRAS, oncogenic RIT1 activity does not appear to be tightly regulated by nucleotide exchange or hydrolysis. Instead, there is a growing understanding that the protein abundance of RIT1 is important for its regulation and function. We previously identified the deubiquitinase USP9X as a RIT1 dependency in RIT1-mutant cells. Here, we demonstrate that both wild-type and mutant forms of RIT1 are substrates of USP9X. Depletion of USP9X leads to decreased RIT1 protein stability and abundance and resensitizes cells to EGFR tyrosine kinase inhibitors. Our work expands upon the current understanding of RIT1 protein regulation and presents USP9X as a key regulator of RIT1-driven oncogenic phenotypes.
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Lung cancer in never-smokers disproportionately affects older women. To understand the mutational landscape of this cohort, we performed detailed genome characterization of 73 lung adenocarcinomas from participants of the Womenâ™s Health Initiative (WHI). We find enrichment of EGFR mutations in never-/light-smokers and KRAS mutations in heavy smokers as expected, but we also show that the specific variants of these genes differ by smoking status, with important therapeutic implications. Mutational signature analysis revealed signatures of clock, APOBEC, and DNA repair deficiency in never-/light-smokers; however, the mutational load of these signatures did not differ significantly from those found in smokers. Last, tumors from both smokers and never-/light-smokers shared copy number subtypes, with no significant differences in aneuploidy. Thus, the genomic landscape of lung cancer in never-/light-smokers and smokers is predominantly differentiated by somatic mutations and not copy number alterations.
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It was originally proposed that tumour suppressor genes (TSGs) act in a recessive manner. Instead, numerous TSGs, including p53 and PTEN, exhibit haplo-insufficiency as a consequence of the dose-dependency of TSG function. Due to the challenges of identifying haplo-insufficient TSGs by human genetics analysis alone, mouse models play a pivotal role in firmly establishing the haplo-insufficiency of a gene, as in the recent identification of DOK2 as a haplo-insufficient lung TSG. In many cases, TSGs exhibit conditional or compound haplo-insufficiency, in which loss of one TSG allele is functionally important only in certain settings or after compound loss of other genes. The 5q deletion syndrome (5q(-) ) is a paradigm of compound haplo-insufficiency and demonstrates the importance of combinatorial interactions to elicit specific phenotypes. These concepts must be integrated into basic science studies to avoid delay in the identification of important TSGs. In the clinical realm, the challenges for molecular pathologists are the development of quantitative measures that can accurately and systematically ascertain the status of haplo-insufficient genes in tumour biopsies, and the use of this information to accurately predict prognosis and response to therapy.
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Haploinsuficiencia/genética , Neoplasias/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Genes Supresores de Tumor , Genes p53 , Humanos , Ratones , Neoplasias Experimentales/genética , Fosfohidrolasa PTEN/genética , Fosfoproteínas/genéticaRESUMEN
Lung cancers in never- and light-smokers often harbor targetable oncogenic mutations in Ras pathway genes. Here, a novel OCLN-RASGRF1 fusion is identified in an otherwise Ras wild-type lung tumor. Studying this and other RASGRF1 fusions, the authors show that these fusions lead to malignant phenotypes that can be reversed by MEK inhibition. See related article by Hunihan et al., p. 3091.
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Neoplasias Pulmonares , ras-GRF1 , Carcinogénesis/genética , Genes ras , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Oncogenes , ras-GRF1/genéticaRESUMEN
Most variants in most genes across most organisms have an unknown impact on the function of the corresponding gene. This gap in knowledge is especially acute in cancer, where clinical sequencing of tumors now routinely reveals patient-specific variants whose functional impact on the corresponding genes is unknown, impeding clinical utility. Transcriptional profiling was able to systematically distinguish these variants of unknown significance as impactful vs. neutral in an approach called expression-based variant-impact phenotyping. We profiled a set of lung adenocarcinoma-associated somatic variants using Cell Painting, a morphological profiling assay that captures features of cells based on microscopy using six stains of cell and organelle components. Using deep-learning-extracted features from each cell's image, we found that cell morphological profiling (cmVIP) can predict variants' functional impact and, particularly at the single-cell level, reveals biological insights into variants that can be explored at our public online portal. Given its low cost, convenient implementation, and single-cell resolution, cmVIP profiling therefore seems promising as an avenue for using non-gene specific assays to systematically assess the impact of variants, including disease-associated alleles, on gene function.
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Adenocarcinoma del Pulmón , Neoplasias Pulmonares , Adenocarcinoma del Pulmón/genética , Alelos , Humanos , Neoplasias Pulmonares/genética , Microscopía , FenotipoRESUMEN
Genome sequencing studies have identified millions of somatic variants in cancer, but it remains challenging to predict the phenotypic impact of most. Experimental approaches to distinguish impactful variants often use phenotypic assays that report on predefined gene-specific functional effects in bulk cell populations. Here, we develop an approach to functionally assess variant impact in single cells by pooled Perturb-seq. We measured the impact of 200 TP53 and KRAS variants on RNA profiles in over 300,000 single lung cancer cells, and used the profiles to categorize variants into phenotypic subsets to distinguish gain-of-function, loss-of-function and dominant negative variants, which we validated by comparison with orthogonal assays. We discovered that KRAS variants did not merely fit into discrete functional categories, but spanned a continuum of gain-of-function phenotypes, and that their functional impact could not have been predicted solely by their frequency in patient cohorts. Our work provides a scalable, gene-agnostic method for coding variant impact phenotyping, with potential applications in multiple disease settings.
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Neoplasias Pulmonares , Proteínas Proto-Oncogénicas p21(ras) , Mapeo Cromosómico , Humanos , Neoplasias Pulmonares/genética , Fenotipo , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
In recent work, we performed CRISPR/Cas9 screening in RIT1 (Ras-like in all tissues)-mutant cancer cells. We found that RIT1-mutant cells are vulnerable to loss of mitotic regulators, and mutant RIT1 synergizes with YAP1 (yes-associated protein 1) in oncogenesis. These findings can be leveraged to identify targeted therapies for RIT1-mutant cancer.