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1.
Cancer Res ; 81(15): 3971-3984, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34099491

RESUMO

Gene fusions frequently result from rearrangements in cancer genomes. In many instances, gene fusions play an important role in oncogenesis; in other instances, they are thought to be passenger events. Although regulatory element rearrangements and copy number alterations resulting from these structural variants are known to lead to transcriptional dysregulation across cancers, the extent to which these events result in functional dependencies with an impact on cancer cell survival is variable. Here we used CRISPR-Cas9 dependency screens to evaluate the fitness impact of 3,277 fusions across 645 cell lines from the Cancer Dependency Map. We found that 35% of cell lines harbored either a fusion partner dependency or a collateral dependency on a gene within the same topologically associating domain as a fusion partner. Fusion-associated dependencies revealed numerous novel oncogenic drivers and clinically translatable alterations. Broadly, fusions can result in partner and collateral dependencies that have biological and clinical relevance across cancer types. SIGNIFICANCE: This study provides insights into how fusions contribute to fitness in different cancer contexts beyond partner-gene activation events, identifying partner and collateral dependencies that may have direct implications for clinical care.

2.
Cell Rep ; 35(13): 109291, 2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34192548

RESUMO

To identify therapeutic targets for KRAS mutant pancreatic cancer, we conduct a druggable genome small interfering RNA (siRNA) screen and determine that suppression of BCAR1 sensitizes pancreatic cancer cells to ERK inhibition. Integrative analysis of genome-scale CRISPR-Cas9 screens also identify BCAR1 as a top synthetic lethal interactor with mutant KRAS. BCAR1 encodes the SRC substrate p130Cas. We determine that SRC-inhibitor-mediated suppression of p130Cas phosphorylation impairs MYC transcription through a DOCK1-RAC1-ß-catenin-dependent mechanism. Additionally, genetic suppression of TUBB3, encoding the ßIII-tubulin subunit of microtubules, or pharmacological inhibition of microtubule function decreases levels of MYC protein in a calpain-dependent manner and potently sensitizes pancreatic cancer cells to ERK inhibition. Accordingly, the combination of a dual SRC/tubulin inhibitor with an ERK inhibitor cooperates to reduce MYC protein and synergistically suppress the growth of KRAS mutant pancreatic cancer. Thus, we demonstrate that mechanistically diverse combinations with ERK inhibition suppress MYC to impair pancreatic cancer proliferation.

3.
Nat Genet ; 53(6): 881-894, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33972779

RESUMO

Esophageal squamous cell carcinomas (ESCCs) harbor recurrent chromosome 3q amplifications that target the transcription factor SOX2. Beyond its role as an oncogene in ESCC, SOX2 acts in development of the squamous esophagus and maintenance of adult esophageal precursor cells. To compare Sox2 activity in normal and malignant tissue, we developed engineered murine esophageal organoids spanning normal esophagus to Sox2-induced squamous cell carcinoma and mapped Sox2 binding and the epigenetic and transcriptional landscape with evolution from normal to cancer. While oncogenic Sox2 largely maintains actions observed in normal tissue, Sox2 overexpression with p53 and p16 inactivation promotes chromatin remodeling and evolution of the Sox2 cistrome. With Klf5, oncogenic Sox2 acquires new binding sites and enhances activity of oncogenes such as Stat3. Moreover, oncogenic Sox2 activates endogenous retroviruses, inducing expression of double-stranded RNA and dependence on the RNA editing enzyme ADAR1. These data reveal SOX2 functions in ESCC, defining targetable vulnerabilities.


Assuntos
Adenosina Desaminase/metabolismo , Epigenoma , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Carcinogênese/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Retrovirus Endógenos/genética , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Humanos , Interferons/metabolismo , Íntrons/genética , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Organoides/patologia , Ligação Proteica , RNA de Cadeia Dupla/metabolismo , Fatores de Transcrição SOXB1/genética , Proteína Supressora de Tumor p53/metabolismo
4.
Nat Commun ; 12(1): 1661, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712601

RESUMO

CRISPR-Cas9 viability screens are increasingly performed at a genome-wide scale across large panels of cell lines to identify new therapeutic targets for precision cancer therapy. Integrating the datasets resulting from these studies is necessary to adequately represent the heterogeneity of human cancers and to assemble a comprehensive map of cancer genetic vulnerabilities. Here, we integrated the two largest public independent CRISPR-Cas9 screens performed to date (at the Broad and Sanger institutes) by assessing, comparing, and selecting methods for correcting biases due to heterogeneous single-guide RNA efficiency, gene-independent responses to CRISPR-Cas9 targeting originated from copy number alterations, and experimental batch effects. Our integrated datasets recapitulate findings from the individual datasets, provide greater statistical power to cancer- and subtype-specific analyses, unveil additional biomarkers of gene dependency, and improve the detection of common essential genes. We provide the largest integrated resources of CRISPR-Cas9 screens to date and the basis for harmonizing existing and future functional genetics datasets.


Assuntos
Neoplasias/genética , Biomarcadores Tumorais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Variações do Número de Cópias de DNA , Genes Essenciais/genética , Genômica/métodos , Humanos , RNA Guia/genética
5.
Br J Cancer ; 125(3): 311-312, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33782565

RESUMO

Cancer cell line models are a cornerstone of cancer research, yet our understanding of how well they represent the molecular features of patient tumours remains limited. Our recent work provides a computational approach to systematically compare large gene expression datasets to better understand which cell lines most closely resemble each tumour type, as well as identify potential gaps in our current cancer models.

6.
Nat Genet ; 53(4): 529-538, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33753930

RESUMO

Exciting therapeutic targets are emerging from CRISPR-based screens of high mutational-burden adult cancers. A key question, however, is whether functional genomic approaches will yield new targets in pediatric cancers, known for remarkably few mutations, which often encode proteins considered challenging drug targets. To address this, we created a first-generation pediatric cancer dependency map representing 13 pediatric solid and brain tumor types. Eighty-two pediatric cancer cell lines were subjected to genome-scale CRISPR-Cas9 loss-of-function screening to identify genes required for cell survival. In contrast to the finding that pediatric cancers harbor fewer somatic mutations, we found a similar complexity of genetic dependencies in pediatric cancer cell lines compared to that in adult models. Findings from the pediatric cancer dependency map provide preclinical support for ongoing precision medicine clinical trials. The vulnerabilities observed in pediatric cancers were often distinct from those in adult cancer, indicating that repurposing adult oncology drugs will be insufficient to address childhood cancers.


Assuntos
Mapeamento Cromossômico/métodos , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Mutação , Proteínas de Neoplasias/genética , Neoplasias/genética , Adulto , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Criança , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Proteínas de Neoplasias/classificação , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , RNA Guia/genética , RNA Guia/metabolismo
7.
Nature ; 590(7846): 486-491, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33505028

RESUMO

Selective targeting of aneuploid cells is an attractive strategy for cancer treatment1. However, it is unclear whether aneuploidy generates any clinically relevant vulnerabilities in cancer cells. Here we mapped the aneuploidy landscapes of about 1,000 human cancer cell lines, and analysed genetic and chemical perturbation screens2-9 to identify cellular vulnerabilities associated with aneuploidy. We found that aneuploid cancer cells show increased sensitivity to genetic perturbation of core components of the spindle assembly checkpoint (SAC), which ensures the proper segregation of chromosomes during mitosis10. Unexpectedly, we also found that aneuploid cancer cells were less sensitive than diploid cells to short-term exposure to multiple SAC inhibitors. Indeed, aneuploid cancer cells became increasingly sensitive to inhibition of SAC over time. Aneuploid cells exhibited aberrant spindle geometry and dynamics, and kept dividing when the SAC was inhibited, resulting in the accumulation of mitotic defects, and in unstable and less-fit karyotypes. Therefore, although aneuploid cancer cells could overcome inhibition of SAC more readily than diploid cells, their long-term proliferation was jeopardized. We identified a specific mitotic kinesin, KIF18A, whose activity was perturbed in aneuploid cancer cells. Aneuploid cancer cells were particularly vulnerable to depletion of KIF18A, and KIF18A overexpression restored their response to SAC inhibition. Our results identify a therapeutically relevant, synthetic lethal interaction between aneuploidy and the SAC.


Assuntos
Aneuploidia , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Neoplasias/patologia , Cariótipo Anormal/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Segregação de Cromossomos/efeitos dos fármacos , Diploide , Genes Letais , Humanos , Cinesina/deficiência , Cinesina/genética , Cinesina/metabolismo , Neoplasias/genética , Fuso Acromático/efeitos dos fármacos , Mutações Sintéticas Letais/efeitos dos fármacos , Mutações Sintéticas Letais/genética , Fatores de Tempo
9.
Nat Commun ; 12(1): 22, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397959

RESUMO

Cell lines are key tools for preclinical cancer research, but it remains unclear how well they represent patient tumor samples. Direct comparisons of tumor and cell line transcriptional profiles are complicated by several factors, including the variable presence of normal cells in tumor samples. We thus develop an unsupervised alignment method (Celligner) and apply it to integrate several large-scale cell line and tumor RNA-Seq datasets. Although our method aligns the majority of cell lines with tumor samples of the same cancer type, it also reveals large differences in tumor similarity across cell lines. Using this approach, we identify several hundred cell lines from diverse lineages that present a more mesenchymal and undifferentiated transcriptional state and that exhibit distinct chemical and genetic dependencies. Celligner could be used to guide the selection of cell lines that more closely resemble patient tumors and improve the clinical translation of insights gained from cell lines.


Assuntos
Perfilação da Expressão Gênica , Neoplasias/genética , Linhagem Celular Tumoral , Bases de Dados Genéticas , Transição Epitelial-Mesenquimal/genética , Humanos , Integrinas/metabolismo
10.
Proc Natl Acad Sci U S A ; 117(48): 30566-30576, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33203674

RESUMO

Aneuploidy, defined as whole chromosome gains and losses, is associated with poor patient prognosis in many cancer types. However, the condition causes cellular stress and cell cycle delays, foremost in G1 and S phase. Here, we investigate how aneuploidy causes both slow proliferation and poor disease outcome. We test the hypothesis that aneuploidy brings about resistance to chemotherapies because of a general feature of the aneuploid condition-G1 delays. We show that single chromosome gains lead to increased resistance to the frontline chemotherapeutics cisplatin and paclitaxel. Furthermore, G1 cell cycle delays are sufficient to increase chemotherapeutic resistance in euploid cells. Mechanistically, G1 delays increase drug resistance to cisplatin and paclitaxel by reducing their ability to damage DNA and microtubules, respectively. Finally, we show that our findings are clinically relevant. Aneuploidy correlates with slowed proliferation and drug resistance in the Cancer Cell Line Encyclopedia (CCLE) dataset. We conclude that a general and seemingly detrimental effect of aneuploidy, slowed proliferation, provides a selective benefit to cancer cells during chemotherapy treatment.


Assuntos
Aneuploidia , Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , Genes p53 , Humanos , Paclitaxel/farmacologia , Trissomia/genética
11.
Nat Genet ; 52(11): 1208-1218, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33128048

RESUMO

Cultured cell lines are the workhorse of cancer research, but the extent to which they recapitulate the heterogeneity observed among malignant cells in tumors is unclear. Here we used multiplexed single-cell RNA-seq to profile 198 cancer cell lines from 22 cancer types. We identified 12 expression programs that are recurrently heterogeneous within multiple cancer cell lines. These programs are associated with diverse biological processes, including cell cycle, senescence, stress and interferon responses, epithelial-mesenchymal transition and protein metabolism. Most of these programs recapitulate those recently identified as heterogeneous within human tumors. We prioritized specific cell lines as models of cellular heterogeneity and used them to study subpopulations of senescence-related cells, demonstrating their dynamics, regulation and unique drug sensitivities, which were predictive of clinical response. Our work describes the landscape of heterogeneity within diverse cancer cell lines and identifies recurrent patterns of heterogeneity that are shared between tumors and specific cell lines.


Assuntos
Linhagem Celular Tumoral , Heterogeneidade Genética , Neoplasias/genética , Lesões Pré-Cancerosas/genética , Linhagem Celular Tumoral/efeitos dos fármacos , Senescência Celular/genética , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , RNA-Seq , Estresse Fisiológico/genética , Microambiente Tumoral
12.
Nat Commun ; 11(1): 4296, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855387

RESUMO

Assays to study cancer cell responses to pharmacologic or genetic perturbations are typically restricted to using simple phenotypic readouts such as proliferation rate. Information-rich assays, such as gene-expression profiling, have generally not permitted efficient profiling of a given perturbation across multiple cellular contexts. Here, we develop MIX-Seq, a method for multiplexed transcriptional profiling of post-perturbation responses across a mixture of samples with single-cell resolution, using SNP-based computational demultiplexing of single-cell RNA-sequencing data. We show that MIX-Seq can be used to profile responses to chemical or genetic perturbations across pools of 100 or more cancer cell lines. We combine it with Cell Hashing to further multiplex additional experimental conditions, such as post-treatment time points or drug doses. Analyzing the high-content readout of scRNA-seq reveals both shared and context-specific transcriptional response components that can identify drug mechanism of action and enable prediction of long-term cell viability from short-term transcriptional responses to treatment.


Assuntos
Perfilação da Expressão Gênica/métodos , Neoplasias/genética , Análise de Célula Única/métodos , Antineoplásicos/farmacologia , Sequência de Bases , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Modelos Estatísticos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Polimorfismo de Nucleotídeo Único , Piridonas/farmacologia , Pirimidinonas/farmacologia
13.
Nat Cancer ; 1(2): 235-248, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32613204

RESUMO

Anti-cancer uses of non-oncology drugs have occasionally been found, but such discoveries have been serendipitous. We sought to create a public resource containing the growth inhibitory activity of 4,518 drugs tested across 578 human cancer cell lines. We used PRISM, a molecular barcoding method, to screen drugs against cell lines in pools. An unexpectedly large number of non-oncology drugs selectively inhibited subsets of cancer cell lines in a manner predictable from the cell lines' molecular features. Our findings include compounds that killed by inducing PDE3A-SLFN12 complex formation; vanadium-containing compounds whose killing depended on the sulfate transporter SLC26A2; the alcohol dependence drug disulfiram, which killed cells with low expression of metallothioneins; and the anti-inflammatory drug tepoxalin, which killed via the multi-drug resistance protein ABCB1. The PRISM drug repurposing resource (https://depmap.org/repurposing) is a starting point to develop new oncology therapeutics, and more rarely, for potential direct clinical translation.

14.
Nat Genet ; 52(2): 219-230, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32025000

RESUMO

Somatic alterations in cancer genes are being detected in normal and premalignant tissue, thus placing greater emphasis on gene-environment interactions that enable disease phenotypes. By combining early genetic alterations with disease-relevant exposures, we developed an integrative mouse model to study gastric premalignancy. Deletion of Trp53 in gastric cells confers a selective advantage and promotes the development of dysplasia in the setting of dietary carcinogens. Organoid derivation from dysplastic lesions facilitated genomic, transcriptional and functional evaluation of gastric premalignancy. Cell cycle regulators, most notably Cdkn2a, were upregulated by p53 inactivation in gastric premalignancy, serving as a barrier to disease progression. Co-deletion of Cdkn2a and Trp53 in dysplastic gastric organoids promoted cancer phenotypes but also induced replication stress, exposing a susceptibility to DNA damage response inhibitors. These findings demonstrate the utility of mouse models that integrate genomic alterations with relevant exposures and highlight the importance of gene-environment interactions in shaping the premalignant state.


Assuntos
Lesões Pré-Cancerosas/patologia , Neoplasias Gástricas/etiologia , Proteína Supressora de Tumor p53/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Esôfago de Barrett/genética , Esôfago de Barrett/patologia , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/genética , Exposição Ambiental/efeitos adversos , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Humanos , Metilnitrosoureia/toxicidade , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutação , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Organoides/patologia , Lesões Pré-Cancerosas/genética , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia
15.
Nature ; 569(7757): 503-508, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31068700

RESUMO

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.


Assuntos
Linhagem Celular Tumoral , Neoplasias/genética , Neoplasias/patologia , Antineoplásicos/farmacologia , Biomarcadores Tumorais , Metilação de DNA , Resistencia a Medicamentos Antineoplásicos , Grupos Étnicos/genética , Edição de Genes , Histonas/metabolismo , Humanos , MicroRNAs/genética , Terapia de Alvo Molecular , Neoplasias/metabolismo , Análise Serial de Proteínas , Splicing de RNA
16.
Nature ; 568(7753): 551-556, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30971823

RESUMO

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/deficiência
17.
Cancer Res ; 79(10): 2564-2579, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30898838

RESUMO

We hypothesized that candidate dependencies for which there are small molecules that are either approved or in advanced development for a nononcology indication may represent potential therapeutic targets. To test this hypothesis, we performed genome-scale loss-of-function screens in hundreds of cancer cell lines. We found that knockout of EGLN1, which encodes prolyl hydroxylase domain-containing protein 2 (PHD2), reduced the proliferation of a subset of clear cell ovarian cancer cell lines in vitro. EGLN1-dependent cells exhibited sensitivity to the pan-EGLN inhibitor FG-4592. The response to FG-4592 was reversed by deletion of HIF1A, demonstrating that EGLN1 dependency was related to negative regulation of HIF1A. We also found that ovarian clear cell tumors susceptible to both genetic and pharmacologic inhibition of EGLN1 required intact HIF1A. Collectively, these observations identify EGLN1 as a cancer target with therapeutic potential. SIGNIFICANCE: These findings reveal a differential dependency of clear cell ovarian cancers on EGLN1, thus identifying EGLN1 as a potential therapeutic target in clear cell ovarian cancer patients.


Assuntos
Estudo de Associação Genômica Ampla , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Neoplasias Ovarianas/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Ovarianas/patologia , Interferência de RNA
18.
Nat Commun ; 9(1): 4610, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30389920

RESUMO

The availability of multiple datasets comprising genome-scale RNAi viability screens in hundreds of diverse cancer cell lines presents new opportunities for understanding cancer vulnerabilities. Integrated analyses of these data to assess differential dependency across genes and cell lines are challenging due to confounding factors such as batch effects and variable screen quality, as well as difficulty assessing gene dependency on an absolute scale. To address these issues, we incorporated cell line screen-quality parameters and hierarchical Bayesian inference into DEMETER2, an analytical framework for analyzing RNAi screens ( https://depmap.org/R2-D2 ). This model substantially improves estimates of gene dependency across a range of performance measures, including identification of gold-standard essential genes and agreement with CRISPR/Cas9-based viability screens. It also allows us to integrate information across three large RNAi screening datasets, providing a unified resource representing the most extensive compilation of cancer cell line genetic dependencies to date.


Assuntos
Testes Genéticos , Modelos Genéticos , Neoplasias/genética , Interferência de RNA , Genes Essenciais , Humanos , Software
19.
Nat Genet ; 50(10): 1381-1387, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30224644

RESUMO

Unlike most tumor suppressor genes, the most common genetic alterations in tumor protein p53 (TP53) are missense mutations1,2. Mutant p53 protein is often abundantly expressed in cancers and specific allelic variants exhibit dominant-negative or gain-of-function activities in experimental models3-8. To gain a systematic view of p53 function, we interrogated loss-of-function screens conducted in hundreds of human cancer cell lines and performed TP53 saturation mutagenesis screens in an isogenic pair of TP53 wild-type and null cell lines. We found that loss or dominant-negative inhibition of wild-type p53 function reliably enhanced cellular fitness. By integrating these data with the Catalog of Somatic Mutations in Cancer (COSMIC) mutational signatures database9,10, we developed a statistical model that describes the TP53 mutational spectrum as a function of the baseline probability of acquiring each mutation and the fitness advantage conferred by attenuation of p53 activity. Collectively, these observations show that widely-acting and tissue-specific mutational processes combine with phenotypic selection to dictate the frequencies of recurrent TP53 mutations.


Assuntos
Mutagênese/fisiologia , Mutação , Neoplasias/genética , Proteína Supressora de Tumor p53/genética , Células A549 , Alelos , Sistemas CRISPR-Cas , Células Cultivadas , Análise Mutacional de DNA , Bases de Dados Genéticas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias/patologia , Análise de Sequência de DNA
20.
Nat Genet ; 49(12): 1779-1784, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29083409

RESUMO

The CRISPR-Cas9 system has revolutionized gene editing both at single genes and in multiplexed loss-of-function screens, thus enabling precise genome-scale identification of genes essential for proliferation and survival of cancer cells. However, previous studies have reported that a gene-independent antiproliferative effect of Cas9-mediated DNA cleavage confounds such measurement of genetic dependency, thereby leading to false-positive results in copy number-amplified regions. We developed CERES, a computational method to estimate gene-dependency levels from CRISPR-Cas9 essentiality screens while accounting for the copy number-specific effect. In our efforts to define a cancer dependency map, we performed genome-scale CRISPR-Cas9 essentiality screens across 342 cancer cell lines and applied CERES to this data set. We found that CERES decreased false-positive results and estimated sgRNA activity for both this data set and previously published screens performed with different sgRNA libraries. We further demonstrate the utility of this collection of screens, after CERES correction, for identifying cancer-type-specific vulnerabilities.


Assuntos
Sistemas CRISPR-Cas , Biologia Computacional/métodos , Variações do Número de Cópias de DNA , Dosagem de Genes/genética , Predisposição Genética para Doença/genética , Algoritmos , Linhagem Celular Tumoral , Humanos , Modelos Genéticos , Neoplasias/diagnóstico , Neoplasias/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
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