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1.
Nat Commun ; 12(1): 6506, 2021 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-34764293

RESUMO

CRISPR knockout fitness screens in cancer cell lines reveal many genes whose loss of function causes cell death or loss of fitness or, more rarely, the opposite phenotype of faster proliferation. Here we demonstrate a systematic approach to identify these proliferation suppressors, which are highly enriched for tumor suppressor genes, and define a network of 145 such genes in 22 modules. One module contains several elements of the glycerolipid biosynthesis pathway and operates exclusively in a subset of acute myeloid leukemia cell lines. The proliferation suppressor activity of genes involved in the synthesis of saturated fatty acids, coupled with a more severe loss of fitness phenotype for genes in the desaturation pathway, suggests that these cells operate at the limit of their carrying capacity for saturated fatty acids, which we confirm biochemically. Overexpression of this module is associated with a survival advantage in juvenile leukemias, suggesting a clinically relevant subtype.

2.
Nucleic Acids Res ; 2021 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-34747468

RESUMO

Network medicine has proven useful for dissecting genetic organization of complex human diseases. We have previously published HumanNet, an integrated network of human genes for disease studies. Since the release of the last version of HumanNet, many large-scale protein-protein interaction datasets have accumulated in public depositories. Additionally, the numbers of research papers and functional annotations for gene-phenotype associations have increased significantly. Therefore, updating HumanNet is a timely task for further improvement of network-based research into diseases. Here, we present HumanNet v3 (https://www.inetbio.org/humannet/, covering 99.8% of human protein coding genes) constructed by means of the expanded data with improved network inference algorithms. HumanNet v3 supports a three-tier model: HumanNet-PI (a protein-protein physical interaction network), HumanNet-FN (a functional gene network), and HumanNet-XC (a functional network extended by co-citation). Users can select a suitable tier of HumanNet for their study purpose. We showed that on disease gene predictions, HumanNet v3 outperforms both the previous HumanNet version and other integrated human gene networks. Furthermore, we demonstrated that HumanNet provides a feasible approach for selecting host genes likely to be associated with COVID-19.

3.
Proc Natl Acad Sci U S A ; 118(36)2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34475205

RESUMO

Prostate cancer is a leading cause of cancer-related mortality in men. The widespread use of androgen receptor (AR) inhibitors has generated an increased incidence of AR-negative prostate cancer, triggering the need for effective therapies for such patients. Here, analysis of public genome-wide CRISPR screens in human prostate cancer cell lines identified histone demethylase JMJD1C (KDM3C) as an AR-negative context-specific vulnerability. Secondary validation studies in multiple cell lines and organoids, including isogenic models, confirmed that small hairpin RNA (shRNA)-mediated depletion of JMJD1C potently inhibited growth specifically in AR-negative prostate cancer cells. To explore the cooperative interactions of AR and JMJD1C, we performed comparative transcriptomics of 1) isogenic AR-positive versus AR-negative prostate cancer cells, 2) AR-positive versus AR-negative prostate cancer tumors, and 3) isogenic JMJD1C-expressing versus JMJD1C-depleted AR-negative prostate cancer cells. Loss of AR or JMJD1C generates a modest tumor necrosis factor alpha (TNFα) signature, whereas combined loss of AR and JMJD1C strongly up-regulates the TNFα signature in human prostate cancer, suggesting TNFα signaling as a point of convergence for the combined actions of AR and JMJD1C. Correspondingly, AR-negative prostate cancer cells showed exquisite sensitivity to TNFα treatment and, conversely, TNFα pathway inhibition via inhibition of its downstream effector MAP4K4 partially reversed the growth defect of JMJD1C-depleted AR-negative prostate cancer cells. Given the deleterious systemic side effects of TNFα therapy in humans and the viability of JMJD1C-knockout mice, the identification of JMJD1C inhibition as a specific vulnerability in AR-negative prostate cancer may provide an alternative drug target for prostate cancer patients progressing on AR inhibitor therapy.

4.
Nucleic Acids Res ; 49(14): 8214-8231, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34320214

RESUMO

Because of essential roles of DNA damage response (DDR) in the maintenance of genomic integrity, cellular homeostasis, and tumor suppression, targeting DDR has become a promising therapeutic strategy for cancer treatment. However, the benefits of cancer therapy targeting DDR are limited mainly due to the lack of predictive biomarkers. To address this challenge, we performed CRISPR screens to search for genetic vulnerabilities that affect cells' response to DDR inhibition. By undertaking CRISPR screens with inhibitors targeting key DDR mediators, i.e. ATR, ATM, DNAPK and CHK1, we obtained a global and unbiased view of genetic interactions with DDR inhibition. Specifically, we identified YWHAE loss as a key determinant of sensitivity to CHK1 inhibition. We showed that KLHL15 loss protects cells from DNA damage induced by ATM inhibition. Moreover, we validated that APEX1 loss sensitizes cells to DNAPK inhibition. Additionally, we compared the synergistic effects of combining different DDR inhibitors and found that an ATM inhibitor plus a PARP inhibitor induced dramatic levels of cell death, probably through promoting apoptosis. Our results enhance the understanding of DDR pathways and will facilitate the use of DDR-targeting agents in cancer therapy.


Assuntos
Proteínas 14-3-3/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Quinase 1 do Ponto de Checagem/genética , Dano ao DNA/genética , Proteína Quinase Ativada por DNA/genética , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Sistemas CRISPR-Cas/genética , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Instabilidade Genômica/genética , Humanos , Proteínas dos Microfilamentos/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia
5.
Genome Med ; 13(1): 2, 2021 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-33407829

RESUMO

BACKGROUND: Identifying essential genes in genome-wide loss-of-function screens is a critical step in functional genomics and cancer target finding. We previously described the Bayesian Analysis of Gene Essentiality (BAGEL) algorithm for accurate classification of gene essentiality from short hairpin RNA and CRISPR/Cas9 genome-wide genetic screens. RESULTS: We introduce an updated version, BAGEL2, which employs an improved model that offers a greater dynamic range of Bayes Factors, enabling detection of tumor suppressor genes; a multi-target correction that reduces false positives from off-target CRISPR guide RNA; and the implementation of a cross-validation strategy that improves performance ~ 10× over the prior bootstrap resampling approach. We also describe a metric for screen quality at the replicate level and demonstrate how different algorithms handle lower quality data in substantially different ways. CONCLUSIONS: BAGEL2 substantially improves the sensitivity, specificity, and performance over BAGEL and establishes the new state of the art in the analysis of CRISPR knockout fitness screens. BAGEL2 is written in Python 3 and source code, along with all supporting files, are available on github ( https://github.com/hart-lab/bagel ).

6.
Proc Natl Acad Sci U S A ; 117(52): 33436-33445, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33376220

RESUMO

Fanconi anemia (FA) is caused by defects in cellular responses to DNA crosslinking damage and replication stress. Given the constant occurrence of endogenous DNA damage and replication fork stress, it is unclear why complete deletion of FA genes does not have a major impact on cell proliferation and germ-line FA patients are able to progress through development well into their adulthood. To identify potential cellular mechanisms that compensate for the FA deficiency, we performed dropout screens in FA mutant cells with a whole genome guide RNA library. This uncovered a comprehensive genome-wide profile of FA pathway synthetic lethality, including POLI and CDK4 As little is known of the cellular function of DNA polymerase iota (Pol ι), we focused on its role in the loss-of-function FA knockout mutants. Loss of both FA pathway function and Pol ι leads to synthetic defects in cell proliferation and cell survival, and an increase in DNA damage accumulation. Furthermore, FA-deficient cells depend on the function of Pol ι to resume replication upon replication fork stalling. Our results reveal a critical role for Pol ι in DNA repair and replication fork restart and suggest Pol ι as a target for therapeutic intervention in malignancies carrying an FA gene mutation.


Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Anemia de Fanconi/enzimologia , Estresse Fisiológico , Sistemas CRISPR-Cas/genética , Quinase 4 Dependente de Ciclina , Dano ao DNA , Genoma Humano , Células HCT116 , Humanos , Mutação/genética , Mutações Sintéticas Letais/genética
7.
Genome Biol ; 21(1): 262, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33059726

RESUMO

BACKGROUND: Pooled library CRISPR/Cas9 knockout screening across hundreds of cell lines has identified genes whose disruption leads to fitness defects, a critical step in identifying candidate cancer targets. However, the number of essential genes detected from these monogenic knockout screens is low compared to the number of constitutively expressed genes in a cell. RESULTS: Through a systematic analysis of screen data in cancer cell lines generated by the Cancer Dependency Map, we observe that half of all constitutively expressed genes are never detected in any CRISPR screen and that these never-essentials are highly enriched for paralogs. We investigated functional buffering among approximately 400 candidate paralog pairs using CRISPR/enCas12a dual-gene knockout screening in three cell lines. We observe 24 synthetic lethal paralog pairs that have escaped detection by monogenic knockout screens at stringent thresholds. Nineteen of 24 (79%) synthetic lethal interactions are present in at least two out of three cell lines and 14 of 24 (58%) are present in all three cell lines tested, including alternate subunits of stable protein complexes as well as functionally redundant enzymes. CONCLUSIONS: Together, these observations strongly suggest that functionally redundant paralogs represent a targetable set of genetic dependencies that are systematically under-represented among cell-essential genes in monogenic CRISPR-based loss of function screens.


Assuntos
Sistemas CRISPR-Cas , Genes Essenciais , Neoplasias/genética , Células A549 , Proteína 9 Associada à CRISPR , Células HT29 , Humanos
8.
Nature ; 586(7827): 120-126, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32968282

RESUMO

The genetic circuits that allow cancer cells to evade destruction by the host immune system remain poorly understood1-3. Here, to identify a phenotypically robust core set of genes and pathways that enable cancer cells to evade killing mediated by cytotoxic T lymphocytes (CTLs), we performed genome-wide CRISPR screens across a panel of genetically diverse mouse cancer cell lines that were cultured in the presence of CTLs. We identify a core set of 182 genes across these mouse cancer models, the individual perturbation of which increases either the sensitivity or the resistance of cancer cells to CTL-mediated toxicity. Systematic exploration of our dataset using genetic co-similarity reveals the hierarchical and coordinated manner in which genes and pathways act in cancer cells to orchestrate their evasion of CTLs, and shows that discrete functional modules that control the interferon response and tumour necrosis factor (TNF)-induced cytotoxicity are dominant sub-phenotypes. Our data establish a central role for genes that were previously identified as negative regulators of the type-II interferon response (for example, Ptpn2, Socs1 and Adar1) in mediating CTL evasion, and show that the lipid-droplet-related gene Fitm2 is required for maintaining cell fitness after exposure to interferon-γ (IFNγ). In addition, we identify the autophagy pathway as a conserved mediator of the evasion of CTLs by cancer cells, and show that this pathway is required to resist cytotoxicity induced by the cytokines IFNγ and TNF. Through the mapping of cytokine- and CTL-based genetic interactions, together with in vivo CRISPR screens, we show how the pleiotropic effects of autophagy control cancer-cell-intrinsic evasion of killing by CTLs and we highlight the importance of these effects within the tumour microenvironment. Collectively, these data expand our knowledge of the genetic circuits that are involved in the evasion of the immune system by cancer cells, and highlight genetic interactions that contribute to phenotypes associated with escape from killing by CTLs.


Assuntos
Genoma/genética , Genômica , Neoplasias/genética , Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Evasão Tumoral/genética , Evasão Tumoral/imunologia , Animais , Autofagia , Linhagem Celular Tumoral , Feminino , Genes Neoplásicos/genética , Humanos , Interferon gama/imunologia , Masculino , Camundongos , NF-kappa B/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais
9.
DNA Repair (Amst) ; 95: 102946, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32853826

RESUMO

Ataxia Telangiectasia and Rad3-Related kinase (ATR) is a master regulator of genome maintenance, and participates in DNA replication and various DNA repair pathways. In a genome-wide screen for ATR-dependent fitness genes, we identified a previously uncharacterized gene, C17orf53, whose loss led to hypersensitivity to ATR inhibition. C17orf53 is conserved in vertebrates and is required for efficient cell proliferation. Loss of C17orf53 slowed down DNA replication and led to pronounced interstrand crosslink (ICL) repair defect. We showed that C17orf53 is a ssDNA- and RPA-binding protein and both characteristics are important for its functions in the cell. In addition, using multiple omics methods, we found that C17orf53 works with MCM8/9 to promote cell survival in response to ICL lesions. Taken together, our data suggest that C17orf53 is a novel component involved in ICL repair pathway.


Assuntos
Adutos de DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Sequência de Aminoácidos , Sobrevivência Celular , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Humanos , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteína de Replicação A/metabolismo , Alinhamento de Sequência
10.
Nat Commun ; 11(1): 3701, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709883

RESUMO

Despite its importance in human cancers, including colorectal cancers (CRC), oncogenic KRAS has been extremely challenging to target therapeutically. To identify potential vulnerabilities in KRAS-mutated CRC, we characterize the impact of oncogenic KRAS on the cell surface of intestinal epithelial cells. Here we show that oncogenic KRAS alters the expression of a myriad of cell-surface proteins implicated in diverse biological functions, and identify many potential surface-accessible therapeutic targets. Cell surface-based loss-of-function screens reveal that ATP7A, a copper-exporter upregulated by mutant KRAS, is essential for neoplastic growth. ATP7A is upregulated at the surface of KRAS-mutated CRC, and protects cells from excess copper-ion toxicity. We find that KRAS-mutated cells acquire copper via a non-canonical mechanism involving macropinocytosis, which appears to be required to support their growth. Together, these results indicate that copper bioavailability is a KRAS-selective vulnerability that could be exploited for the treatment of KRAS-mutated neoplasms.


Assuntos
Neoplasias Colorretais/metabolismo , Cobre/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Disponibilidade Biológica , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , ATPases Transportadoras de Cobre/metabolismo , Feminino , Humanos , Mucosa Intestinal/patologia , Camundongos , Camundongos Knockout , Camundongos Nus , Camundongos SCID , Mutação
11.
Oncogene ; 39(21): 4312-4322, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32300176

RESUMO

Aurora kinases are a family of serine/threonine kinases vital for cell division. Because of the overexpression of Aurora kinases in a broad range of cancers and their important roles in mitosis, inhibitors targeting Aurora kinases have attracted attention in cancer therapy. VX-680 is an effective pan-Aurora kinase inhibitor; however, its clinical efficacy was not satisfying. In this study, we performed CRISPR/Cas9 screens to identify genes whose depletion shows synthetic lethality with VX-680. The top hit from these screens was GSG2 (also known as Haspin), a serine/threonine kinase that phosphorylates histone H3 at Thr-3 during mitosis. Moreover, both Haspin knockout and Haspin inhibitor-treated HCT116 cells were hypersensitive to VX-680. Furthermore, we showed that the synthetic lethal interaction between Haspin depletion and VX-680 was mediated by the inhibition of Haspin with Aurora kinase B (AURKB), but not with Aurora kinase A (AURKA). Strikingly, combined inhibition of Haspin and AURKB had a better efficacy than single-agent treatment in both head and neck squamous cell carcinoma and non-small cell lung cancer. Taken together, our findings have uncovered a synthetic lethal interaction between AURKB and Haspin, which provides a strong rationale for this combination therapy for cancer patients.


Assuntos
Aurora Quinase B , Sistemas CRISPR-Cas , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Neoplasias , Neoplasias , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Serina-Treonina Quinases , Células A549 , Aurora Quinase B/antagonistas & inibidores , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Estudo de Associação Genômica Ampla , Células HCT116 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo
12.
Neuron ; 106(1): 76-89.e8, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32004439

RESUMO

Unbiased in vivo genome-wide genetic screening is a powerful approach to elucidate new molecular mechanisms, but such screening has not been possible to perform in the mammalian central nervous system (CNS). Here, we report the results of the first genome-wide genetic screens in the CNS using both short hairpin RNA (shRNA) and CRISPR libraries. Our screens identify many classes of CNS neuronal essential genes and demonstrate that CNS neurons are particularly sensitive not only to perturbations to synaptic processes but also autophagy, proteostasis, mRNA processing, and mitochondrial function. These results reveal a molecular logic for the common implication of these pathways across multiple neurodegenerative diseases. To further identify disease-relevant genetic modifiers, we applied our screening approach to two mouse models of Huntington's disease (HD). Top mutant huntingtin toxicity modifier genes included several Nme genes and several genes involved in methylation-dependent chromatin silencing and dopamine signaling, results that reveal new HD therapeutic target pathways.


Assuntos
Sobrevivência Celular/genética , Proteína Huntingtina/genética , Doença de Huntington/genética , Neostriado/metabolismo , Neurônios/metabolismo , Animais , Comportamento Animal , Sistemas CRISPR-Cas , Técnicas de Silenciamento de Genes , Biblioteca Gênica , Genes Essenciais/genética , Camundongos , Camundongos Transgênicos , Nucleosídeo NM23 Difosfato Quinases/genética , Nucleosídeo Difosfato Quinase D/genética , Agregados Proteicos , Interferência de RNA , RNA Guia , RNA Interferente Pequeno , Receptores de Dopamina D2/genética , Análise de Sequência de RNA
13.
DNA Repair (Amst) ; 87: 102803, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31991288

RESUMO

DNA damage response (DDR) is critically important for cell survival, genome maintenance, and its defect has been exploited therapeutically in cancer treatment. Many DDR-targeting agents have been generated and have entered the clinic and/or clinical trials. In order to provide a global and unbiased view of DDR network, we designed a focused CRISPR library targeting 365 DDR genes and performed CRISPR screens on the responses to several DDR inhibitors and DNA-damaging agents in 293A cells. With these screens, we determined responsive pathways enriched under treatment with different types of small-molecule agents. Additionally, we showed that POLE3/4-deficient cells displayed enhanced sensitivity to an ATR inhibitor, a PARP inhibitor, and camptothecin. Moreover, by performing DDR screens in isogenic TP53 wild-type and TP53 knock-out cell lines, our results suggest that the performance of our CRISPR DDR dropout screens is independent of TP53 status. Collectively, our findings indicate that CRISPR DDR screens can be used to identify potential targets of small-molecule drugs and reveal that TP53 status does not affect the outcome of these screens.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Dano ao DNA/genética , Resistência a Medicamentos/genética , Bibliotecas de Moléculas Pequenas/farmacologia , DNA Polimerase III/genética , Proteínas de Ligação a DNA/genética , Biblioteca Gênica , Genes p53/genética , Células HEK293 , Humanos , Nucleoproteínas/genética , Inibidores de Poli(ADP-Ribose) Polimerases/metabolismo
14.
J Cell Biol ; 219(2)2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31881079

RESUMO

Activation of Wnt signaling entails ßcatenin protein stabilization and translocation to the nucleus to regulate context-specific transcriptional programs. The majority of colorectal cancers (CRCs) initiate following APC mutations, resulting in Wnt ligand-independent stabilization and nuclear accumulation of ßcatenin. The mechanisms underlying ßcatenin nucleocytoplasmic shuttling remain incompletely defined. Using a novel, positive selection, functional genomic strategy, DEADPOOL, we performed a genome-wide CRISPR screen and identified IPO11 as a required factor for ßcatenin-mediated transcription in APC mutant CRC cells. IPO11 (Importin-11) is a nuclear import protein that shuttles cargo from the cytoplasm to the nucleus. IPO11-/- cells exhibit reduced nuclear ßcatenin protein levels and decreased ßcatenin target gene activation, suggesting IPO11 facilitates ßcatenin nuclear import. IPO11 knockout decreased colony formation of CRC cell lines and decreased proliferation of patient-derived CRC organoids. Our findings uncover a novel nuclear import mechanism for ßcatenin in cells with high Wnt activity.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais/genética , beta Catenina/genética , beta Carioferinas/genética , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Humanos , Mutação , Via de Sinalização Wnt/genética
15.
Open Biol ; 9(9): 190156, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31506018

RESUMO

The response to DNA replication stress in eukaryotes is under the control of the ataxia-telangiectasia and Rad3-related (ATR) kinase. ATR responds to single-stranded (ss) DNA to stabilize distressed DNA replication forks, modulate DNA replication firing and prevent cells with damaged DNA or incomplete DNA replication from entering into mitosis. Furthermore, inhibitors of ATR are currently in clinical development either as monotherapies or in combination with agents that perturb DNA replication. To gain a genetic view of the cellular pathways requiring ATR kinase function, we mapped genes whose mutation causes hypersensitivity to ATR inhibitors with genome-scale CRISPR/Cas9 screens. We delineate a consensus set of 117 genes enriched in DNA replication, DNA repair and cell cycle regulators that promote survival when ATR kinase activity is suppressed. We validate 14 genes from this set and report genes not previously described to modulate response to ATR inhibitors. In particular we found that the loss of the POLE3/POLE4 proteins, which are DNA polymerase ε accessory subunits, results in marked hypersensitivity to ATR inhibition. We anticipate that this 117-gene set will be useful for the identification of genes involved in the regulation of genome integrity and the characterization of new biological processes involving ATR, and may reveal biomarkers of ATR inhibitor response in the clinic.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Variação Genética , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Sistemas CRISPR-Cas , Linhagem Celular , Edição de Genes , Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Marcação de Genes , Genes Reporter , Estudos de Associação Genética , Humanos , Interferência de RNA , RNA Guia
16.
Genome Med ; 11(1): 52, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31439014

RESUMO

BACKGROUND: Chemogenetic profiling enables the identification of gene mutations that enhance or suppress the activity of chemical compounds. This knowledge provides insights into drug mechanism of action, genetic vulnerabilities, and resistance mechanisms, all of which may help stratify patient populations and improve drug efficacy. CRISPR-based screening enables sensitive detection of drug-gene interactions directly in human cells, but until recently has primarily been used to screen only for resistance mechanisms. RESULTS: We present drugZ, an algorithm for identifying both synergistic and suppressor chemogenetic interactions from CRISPR screens. DrugZ identifies synthetic lethal interactions between PARP inhibitors and both known and novel members of the DNA damage repair pathway, confirms KEAP1 loss as a resistance factor for ERK inhibitors in oncogenic KRAS backgrounds, and defines the genetic context for temozolomide activity. CONCLUSIONS: DrugZ is an open-source Python software for the analysis of genome-scale drug modifier screens. The software accurately identifies genetic perturbations that enhance or suppress drug activity. Interestingly, analysis of new and previously published data reveals tumor suppressor genes are drug-agnostic resistance genes in drug modifier screens. The software is available at github.com/hart-lab/drugz .


Assuntos
Algoritmos , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Descoberta de Drogas/métodos , Farmacogenética/métodos , Variantes Farmacogenômicos , Software , Predisposição Genética para Doença , Humanos , Mutação
17.
Nat Commun ; 10(1): 3144, 2019 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-31316073

RESUMO

Capitalizing on the inherent multiplexing capability of AsCpf1, we developed a multiplexed, high-throughput screening strategy that minimizes library size without sacrificing gene targeting efficiency. We demonstrated that AsCpf1 can be used for functional genomics screenings and that an AsCpf1-based multiplexed library performs similarly as compared to currently available monocistronic CRISPR/Cas9 libraries, with only one vector required for each gene. We construct the smallest whole-genome CRISPR knock-out library, Mini-human, for the human genome (n = 17,032 constructs targeting 16,977 protein-coding genes), which performs favorably compared to conventional Cas9 libraries.


Assuntos
Sistemas CRISPR-Cas/genética , Biblioteca Gênica , Proteína 9 Associada à CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes , Humanos , RNA Guia/química
18.
Cell Rep ; 27(3): 971-986.e9, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30995489

RESUMO

Glioblastoma therapies have remained elusive due to limitations in understanding mechanisms of growth and survival of the tumorigenic population. Using CRISPR-Cas9 approaches in patient-derived GBM stem cells (GSCs) to interrogate function of the coding genome, we identify actionable pathways responsible for growth, which reveal the gene-essential circuitry of GBM stemness and proliferation. In particular, we characterize members of the SOX transcription factor family, SOCS3, USP8, and DOT1L, and protein ufmylation as important for GSC growth. Additionally, we reveal mechanisms of temozolomide resistance that could lead to combination strategies. By reaching beyond static genome analysis of bulk tumors, with a genome-wide functional approach, we reveal genetic dependencies within a broad range of biological processes to provide increased understanding of GBM growth and treatment resistance.


Assuntos
Neoplasias Encefálicas/patologia , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Glioblastoma/patologia , Células-Tronco Neoplásicas/metabolismo , Temozolomida/farmacologia , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/mortalidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Endopeptidases/genética , Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Biblioteca Gênica , Glioblastoma/tratamento farmacológico , Glioblastoma/mortalidade , Histona Metiltransferases/metabolismo , Humanos , Camundongos , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteína 3 Supressora da Sinalização de Citocinas/genética , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Análise de Sobrevida , Temozolomida/uso terapêutico , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo
19.
Life Sci Alliance ; 2(2)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30979825

RESUMO

Genetic interactions mediate the emergence of phenotype from genotype. The systematic survey of genetic interactions in yeast showed that genes operating in the same biological process have highly correlated genetic interaction profiles, and this observation has been exploited to infer gene function in model organisms. Such assays of digenic perturbations in human cells are also highly informative, but are not scalable, even with CRISPR-mediated methods. As an alternative, we developed an indirect method of deriving functional interactions. We show that genes having correlated knockout fitness profiles across diverse, non-isogenic cell lines are analogous to genes having correlated genetic interaction profiles across isogenic query strains and similarly imply shared biological function. We constructed a network of genes with correlated fitness profiles across 276 high-quality CRISPR knockout screens in cancer cell lines into a "coessentiality network," with up to 500-fold enrichment for co-functional gene pairs, enabling strong inference of gene function and highlighting the modular organization of the cell.


Assuntos
Técnicas de Inativação de Genes , Redes Reguladoras de Genes/genética , Neoplasias/genética , Neoplasias/patologia , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Bases de Dados Genéticas , Genes Neoplásicos/genética , Genótipo , Humanos , Fenótipo , Biossíntese de Proteínas , RNA Interferente Pequeno/genética , Saccharomyces cerevisiae/genética , Transdução de Sinais/genética
20.
Sci Rep ; 9(1): 842, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30696911

RESUMO

Cell surface antigen discovery is of great interest for biomedical research both for isolation of rare cell populations and therapeutic targeting. We developed a rapid, cost-effective, fully in vitro technology which facilities the simultaneous target discovery and human antibody generation on the surface of virtually any cell population of interest. We apply our technique to human colorectal cancer-initiating cells (CICs) and identify hundreds of unique human antibodies. We characterized the top three antibody candidates targeting these CICs and identify their protein targets as integrin α7 (ITGA7), HLA-A1 and integrin ß6 (ITGB6). We demonstrate that these antibodies can be used to isolate self-renewing colorectal CICs, and that the integrin α7 antibody can prospectively identify glioblastoma brain tumor initiating cells as well as human muscle stem cells. We also demonstrate that genetic ablation of integrin ß6 impedes colorectal CIC function. The methodology can be readily applied to other cell populations including stem cells, cancer, or immune cells to facilitate the rapid identification of novel targets and simultaneous generation of potent and specific antibodies with therapeutic potential.


Assuntos
Anticorpos/imunologia , Antígenos de Superfície/imunologia , Biomarcadores Tumorais/imunologia , Neoplasias Colorretais/imunologia , Glioblastoma/imunologia , Antígenos CD/imunologia , Células CACO-2 , Células HCT116 , Células HEK293 , Antígeno HLA-A1/imunologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Cadeias alfa de Integrinas/imunologia , Cadeias beta de Integrinas/genética , Cadeias beta de Integrinas/imunologia , Células MCF-7 , Células PC-3 , Interferência de RNA , RNA Interferente Pequeno/genética , Células Tumorais Cultivadas
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