RESUMO
Seminal yeast studies have established the value of comprehensively mapping genetic interactions (GIs) for inferring gene function. Efforts in human cells using focused gene sets underscore the utility of this approach, but the feasibility of generating large-scale, diverse human GI maps remains unresolved. We developed a CRISPR interference platform for large-scale quantitative mapping of human GIs. We systematically perturbed 222,784 gene pairs in two cancer cell lines. The resultant maps cluster functionally related genes, assigning function to poorly characterized genes, including TMEM261, a new electron transport chain component. Individual GIs pinpoint unexpected relationships between pathways, exemplified by a specific cholesterol biosynthesis intermediate whose accumulation induces deoxynucleotide depletion, causing replicative DNA damage and a synthetic-lethal interaction with the ATR/9-1-1 DNA repair pathway. Our map provides a broad resource, establishes GI maps as a high-resolution tool for dissecting gene function, and serves as a blueprint for mapping the genetic landscape of human cells.
Assuntos
Biomarcadores/metabolismo , Colesterol/metabolismo , Epistasia Genética , Redes Reguladoras de Genes , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células Jurkat , Células K562 , Mapeamento de Interação de ProteínasRESUMO
Noncoding mutations in cancer genomes are frequent but challenging to interpret. PVT1 encodes an oncogenic lncRNA, but recurrent translocations and deletions in human cancers suggest alternative mechanisms. Here, we show that the PVT1 promoter has a tumor-suppressor function that is independent of PVT1 lncRNA. CRISPR interference of PVT1 promoter enhances breast cancer cell competition and growth in vivo. The promoters of the PVT1 and the MYC oncogenes, located 55 kb apart on chromosome 8q24, compete for engagement with four intragenic enhancers in the PVT1 locus, thereby allowing the PVT1 promoter to regulate pause release of MYC transcription. PVT1 undergoes developmentally regulated monoallelic expression, and the PVT1 promoter inhibits MYC expression only from the same chromosome via promoter competition. Cancer genome sequencing identifies recurrent mutations encompassing the human PVT1 promoter, and genome editing verified that PVT1 promoter mutation promotes cancer cell growth. These results highlight regulatory sequences of lncRNA genes as potential disease-associated DNA elements.
Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Genes myc , RNA Longo não Codificante/genética , Animais , Neoplasias da Mama/metabolismo , Sistemas CRISPR-Cas , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Cromatina , DNA de Neoplasias/genética , Elementos Facilitadores Genéticos , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Mutação , Transplante de Neoplasias , Regiões Promotoras Genéticas , RNA Longo não Codificante/metabolismo , Transcrição GênicaRESUMO
Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting â¼100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.
Assuntos
Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endorribonucleases , Retroalimentação , Humanos , Modelos Moleculares , Proteínas Serina-Treonina Quinases , RNA Guia de Cinetoplastídeos/metabolismo , Transcrição Gênica , Resposta a Proteínas não DobradasRESUMO
While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%-99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a â¼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways.
Assuntos
Sistemas CRISPR-Cas , Técnicas Genéticas , Transcrição Gênica , Linhagem Celular , Toxina da Cólera/metabolismo , Toxina Diftérica/metabolismo , Genoma Humano , HumanosRESUMO
Genetic interaction (GI) maps, comprising pairwise measures of how strongly the function of one gene depends on the presence of a second, have enabled the systematic exploration of gene function in microorganisms. Here, we present a two-stage strategy to construct high-density GI maps in mammalian cells. First, we use ultracomplex pooled shRNA libraries (25 shRNAs/gene) to identify high-confidence hit genes for a given phenotype and effective shRNAs. We then construct double-shRNA libraries from these to systematically measure GIs between hits. A GI map focused on ricin susceptibility broadly recapitulates known pathways and provides many unexpected insights. These include a noncanonical role for COPI, a previously uncharacterized protein complex affecting toxin clearance, a specialized role for the ribosomal protein RPS25, and functionally distinct mammalian TRAPP complexes. The ability to rapidly generate mammalian GI maps provides a potentially transformative tool for defining gene function and designing combination therapies based on synergistic pairs.
Assuntos
Transporte Biológico , Epistasia Genética , Ricina/toxicidade , Atorvastatina , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Complexo I de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Ácidos Heptanoicos/farmacologia , Humanos , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Pirróis/farmacologia , RNA Interferente Pequeno , Proteínas Ribossômicas/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
We recently used CRISPRi/a-based chemical-genetic screens and cell biological, biochemical, and structural assays to determine that rigosertib, an anti-cancer agent in phase III clinical trials, kills cancer cells by destabilizing microtubules. Reddy and co-workers (Baker et al., 2020, this issue of Molecular Cell) suggest that a contaminating degradation product in commercial formulations of rigosertib is responsible for the microtubule-destabilizing activity. Here, we demonstrate that cells treated with pharmaceutical-grade rigosertib (>99.9% purity) or commercially obtained rigosertib have qualitatively indistinguishable phenotypes across multiple assays. The two formulations have indistinguishable chemical-genetic interactions with genes that modulate microtubule stability, both destabilize microtubules in cells and in vitro, and expression of a rationally designed tubulin mutant with a mutation in the rigosertib binding site (L240F TUBB) allows cells to proliferate in the presence of either formulation. Importantly, the specificity of the L240F TUBB mutant for microtubule-destabilizing agents has been confirmed independently. Thus, rigosertib kills cancer cells by destabilizing microtubules, in agreement with our original findings.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células , Glicina/análogos & derivados , Microtúbulos/efeitos dos fármacos , Neoplasias/patologia , Preparações Farmacêuticas/metabolismo , Sulfonas/farmacologia , Tubulina (Proteína)/metabolismo , Células Cultivadas , Cristalografia por Raios X , Contaminação de Medicamentos , Glicina/farmacologia , Humanos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Preparações Farmacêuticas/química , Conformação Proteica , Tubulina (Proteína)/química , Tubulina (Proteína)/genéticaRESUMO
Long noncoding RNAs (lncRNAs) account for the largest portion of RNA from the transcriptome, yet most of their functions remain unknown. Here, we performed two independent high-throughput CRISPRi screens to understand the role of lncRNAs in monocyte function and differentiation. The first was a reporter-based screen to identify lncRNAs that regulate TLR4-NFkB signaling in human monocytes and the second screen identified lncRNAs involved in monocyte to macrophage differentiation. We successfully identified numerous noncoding and protein-coding genes that can positively or negatively regulate inflammation and differentiation. To understand the functional roles of lncRNAs in both processes, we chose to further study the lncRNA LOUP [lncRNA originating from upstream regulatory element of SPI1 (also known as PU.1)], as it emerged as a top hit in both screens. Not only does LOUP regulate its neighboring gene, the myeloid fate-determining factor SPI1, thereby affecting monocyte to macrophage differentiation, but knockdown of LOUP leads to a broad upregulation of NFkB-targeted genes at baseline and upon TLR4-NFkB activation. LOUP also harbors three small open reading frames capable of being translated and are responsible for LOUP's ability to negatively regulate TLR4/NFkB signaling. This work emphasizes the value of high-throughput screening to rapidly identify functional lncRNAs in the innate immune system.
Assuntos
Diferenciação Celular , Inflamação , Macrófagos , Monócitos , RNA Longo não Codificante , Transdução de Sinais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Humanos , Macrófagos/metabolismo , Macrófagos/citologia , Diferenciação Celular/genética , Monócitos/metabolismo , Monócitos/citologia , Inflamação/genética , Inflamação/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , NF-kappa B/metabolismo , Transativadores/metabolismo , Transativadores/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Sistemas CRISPR-Cas , Regulação da Expressão GênicaRESUMO
Chemical libraries paired with phenotypic screens can now readily identify compounds with therapeutic potential. A central limitation to exploiting these compounds, however, has been in identifying their relevant cellular targets. Here, we present a two-tiered CRISPR-mediated chemical-genetic strategy for target identification: combined genome-wide knockdown and overexpression screening as well as focused, comparative chemical-genetic profiling. Application of these strategies to rigosertib, a drug in phase 3 clinical trials for high-risk myelodysplastic syndrome whose molecular target had remained controversial, pointed singularly to microtubules as rigosertib's target. We showed that rigosertib indeed directly binds to and destabilizes microtubules using cell biological, in vitro, and structural approaches. Finally, expression of tubulin with a structure-guided mutation in the rigosertib-binding pocket conferred resistance to rigosertib, establishing that rigosertib kills cancer cells by destabilizing microtubules. These results demonstrate the power of our chemical-genetic screening strategies for pinpointing the physiologically relevant targets of chemical agents.
Assuntos
Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Testes Genéticos/métodos , Glicina/análogos & derivados , Microtúbulos/efeitos dos fármacos , Sulfonas/farmacologia , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/genética , Antineoplásicos/química , Sistemas CRISPR-Cas , Colchicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glicina/química , Glicina/farmacologia , Células HeLa , Humanos , Células K562 , Cinesinas/genética , Cinesinas/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Mutação , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfonas/química , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Vimblastina/farmacologiaRESUMO
Identifying small molecules that inhibit protein synthesis by selectively stalling the ribosome constitutes a new strategy for therapeutic development. Compounds that inhibit the translation of PCSK9, a major regulator of low-density lipoprotein cholesterol, have been identified that reduce LDL cholesterol in preclinical models and that affect the translation of only a few off-target proteins. Although some of these compounds hold potential for future therapeutic development, it is not known how they impact the physiology of cells or ribosome quality control pathways. Here we used a genome-wide CRISPRi screen to identify proteins and pathways that modulate cell growth in the presence of high doses of a selective PCSK9 translational inhibitor, PF-06378503 (PF8503). The two most potent genetic modifiers of cell fitness in the presence of PF8503, the ubiquitin binding protein ASCC2 and helicase ASCC3, bind to the ribosome and protect cells from toxic effects of high concentrations of the compound. Surprisingly, translation quality control proteins Pelota (PELO) and HBS1L sensitize cells to PF8503 treatment. In genetic interaction experiments, ASCC3 acts together with ASCC2, and functions downstream of HBS1L. Taken together, these results identify new connections between ribosome quality control pathways, and provide new insights into the selectivity of compounds that stall human translation that will aid the development of next-generation selective translation stalling compounds to treat disease.
Assuntos
Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/metabolismo , Sequência de Aminoácidos , Sistemas CRISPR-Cas , DNA Helicases/antagonistas & inibidores , DNA Helicases/genética , DNA Helicases/metabolismo , Endonucleases/metabolismo , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Técnicas de Silenciamento de Genes , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Células K562 , Modelos Biológicos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Inibidores de PCSK9 , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , Ribossomos/efeitos dos fármacos , Inibidores de Serina Proteinase/farmacologiaRESUMO
Insufficient or dysregulated energy metabolism may underlie diverse inherited and degenerative diseases, cancer, and even aging itself. ATP is the central energy carrier in cells, but critical pathways for regulating ATP levels are not systematically understood. We combined a pooled clustered regularly interspaced short palindromic repeats interference (CRISPRi) library enriched for mitochondrial genes, a fluorescent biosensor, and fluorescence-activated cell sorting (FACS) in a high-throughput genetic screen to assay ATP concentrations in live human cells. We identified genes not known to be involved in energy metabolism. Most mitochondrial ribosomal proteins are essential in maintaining ATP levels under respiratory conditions, and impaired respiration predicts poor growth. We also identified genes for which coenzyme Q10 (CoQ10) supplementation rescued ATP deficits caused by knockdown. These included CoQ10 biosynthetic genes associated with human disease and a subset of genes not linked to CoQ10 biosynthesis, indicating that increasing CoQ10 can preserve ATP in specific genetic contexts. This screening paradigm reveals mechanisms of metabolic control and genetic defects responsive to energy-based therapies.
Assuntos
Trifosfato de Adenosina/análise , Metabolismo Energético/fisiologia , Ensaios de Triagem em Larga Escala/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Análise de Célula Única/métodos , Ubiquinona/análogos & derivados , Ubiquinona/metabolismoRESUMO
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 activation (CRISPRa) systems have enabled genetic screens in cultured cell lines to discover and characterize drivers and inhibitors of cancer cell growth. We adapted this system for use in vivo to assess whether modulating endogenous gene expression levels can result in functional outcomes in the native environment of the liver. We engineered the catalytically dead CRISPR-associated 9 (dCas9)-positive mouse, cyclization recombination-inducible (Cre) CRISPRa system for cell type-specific gene activation in vivo. We tested the capacity for genetic screening in live animals by applying CRISPRa in a clinically relevant model of liver injury and repopulation. We targeted promoters of interest in regenerating hepatocytes using multiple single guide RNAs (gRNAs), and employed high-throughput sequencing to assess enrichment of gRNA sequences during liver repopulation and to link specific gRNAs to the initiation of carcinogenesis. All components of the CRISPRa system were expressed in a cell type-specific manner and activated endogenous gene expression in vivo. Multiple gRNA cassettes targeting a proto-oncogene were significantly enriched following liver repopulation, indicative of enhanced division of cells expressing the proto-oncogene. Furthermore, hepatocellular carcinomas developed containing gRNAs that activated this oncogene, indicative of cancer initiation events. Also, we employed our system for combinatorial cancer genetics in vivo as we found that while clonal hepatocellular carcinomas were dependent on the presence of the oncogene-inducing gRNAs, they were depleted for multiple gRNAs activating tumor suppressors. CONCLUSION: The in vivo CRISPRa platform developed here allows for parallel and combinatorial genetic screens in live animals; this approach enables screening for drivers and suppressors of cell replication and tumor initiation. (Hepatology 2017).
Assuntos
Carcinogênese/genética , Carcinoma Hepatocelular/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Testes Genéticos/métodos , Neoplasias Hepáticas/genética , Animais , Western Blotting , Regulação Neoplásica da Expressão Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala , Imuno-Histoquímica , Fígado/metabolismo , Fígado/patologia , Camundongos , Oncogenes , RNA Guia de Cinetoplastídeos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Ativação TranscricionalRESUMO
Targeted transcriptional regulation is a powerful tool to study genetic mediators of cellular behavior. Here, we show that catalytically dead Cas9 (dCas9) targeted to genomic regions upstream or downstream of the transcription start site allows for specific and sustainable gene-expression level alterations in tumor cells in vitro and in syngeneic immune-competent mouse models. We used this approach for a high-coverage pooled gene-activation screen in vivo and discovered previously unidentified modulators of tumor growth and therapeutic response. Moreover, by using dCas9 linked to an activation domain, we can either enhance or suppress target gene expression simply by changing the genetic location of dCas9 binding relative to the transcription start site. We demonstrate that these directed changes in gene-transcription levels occur with minimal off-target effects. Our findings highlight the use of dCas9-mediated transcriptional regulation as a versatile tool to reproducibly interrogate tumor phenotypes in vivo.
Assuntos
Proteínas de Bactérias/genética , Endonucleases/genética , Regulação Leucêmica da Expressão Gênica , Técnicas Genéticas , Leucemia Experimental , Animais , Proteína 9 Associada à CRISPR , Dano ao DNA , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Dacarbazina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Camundongos , Análise de Sequência de RNA , Temozolomida , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Broad-spectrum antiviral drugs targeting host processes could potentially treat a wide range of viruses while reducing the likelihood of emergent resistance. Despite great promise as therapeutics, such drugs remain largely elusive. Here we used parallel genome-wide high-coverage short hairpin RNA (shRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screens to identify the cellular target and mechanism of action of GSK983, a potent broad-spectrum antiviral with unexplained cytotoxicity. We found that GSK983 blocked cell proliferation and dengue virus replication by inhibiting the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH). Guided by mechanistic insights from both genomic screens, we found that exogenous deoxycytidine markedly reduced GSK983 cytotoxicity but not antiviral activity, providing an attractive new approach to improve the therapeutic window of DHODH inhibitors against RNA viruses. Our results highlight the distinct advantages and limitations of each screening method for identifying drug targets, and demonstrate the utility of parallel knockdown and knockout screens for comprehensive probing of drug activity.
Assuntos
Antivirais/farmacologia , Sistemas CRISPR-Cas/genética , Carbazóis/farmacologia , Lentivirus/efeitos dos fármacos , RNA Interferente Pequeno/genética , Carbazóis/química , Linhagem Celular Tumoral , Clonagem Molecular , Humanos , Lentivirus/fisiologiaRESUMO
Genetic screening based on loss-of-function phenotypes is a powerful discovery tool in biology. Although the recent development of clustered regularly interspaced short palindromic repeats (CRISPR)-based screening approaches in mammalian cell culture has enormous potential, RNA interference (RNAi)-based screening remains the method of choice in several biological contexts. We previously demonstrated that ultracomplex pooled short-hairpin RNA (shRNA) libraries can largely overcome the problem of RNAi off-target effects in genome-wide screens. Here, we systematically optimize several aspects of our shRNA library, including the promoter and microRNA context for shRNA expression, selection of guide strands, and features relevant for postscreen sample preparation for deep sequencing. We present next-generation high-complexity libraries targeting human and mouse protein-coding genes, which we grouped into 12 sublibraries based on biological function. A pilot screen suggests that our next-generation RNAi library performs comparably to current CRISPR interference (CRISPRi)-based approaches and can yield complementary results with high sensitivity and high specificity.
Assuntos
Genoma , Interferência de RNA , Animais , Inteligência Artificial , Humanos , Camundongos , RNA Interferente Pequeno/genéticaRESUMO
The genetic principle of synthetic lethality is clinically validated in cancers with loss of specific DNA damage response (DDR) pathway genes (i.e. BRCA1/2 tumor suppressor mutations). The broader question of whether and how oncogenes create tumor-specific vulnerabilities within DDR networks remains unanswered. Native FET protein family members are among the earliest proteins recruited to DNA double-strand breaks (DSBs) during the DDR, though the function of both native FET proteins and FET fusion oncoproteins in DSB repair remains poorly defined. Here we focus on Ewing sarcoma (ES), an EWS-FLI1 fusion oncoprotein-driven pediatric bone tumor, as a model for FET rearranged cancers. We discover that the EWS-FLI1 fusion oncoprotein is recruited to DNA DSBs and interferes with native EWS function in activating the DNA damage sensor ATM. Using preclinical mechanistic approaches and clinical datasets, we establish functional ATM deficiency as a principal DNA repair defect in ES and the compensatory ATR signaling axis as a collateral dependency and therapeutic target in FET rearranged cancers. Thus, aberrant recruitment of a fusion oncoprotein to sites of DNA damage can disrupt normal DSB repair, revealing a mechanism for how oncogenes can create cancer-specific synthetic lethality within DDR networks.
RESUMO
While oncogenes promote cancer cell growth, unrestrained proliferation represents a significant stressor to cellular homeostasis networks such as the DNA damage response (DDR). To enable oncogene tolerance, many cancers disable tumor suppressive DDR signaling through genetic loss of DDR pathways and downstream effectors (e.g., ATM or p53 tumor suppressor mutations). Whether and how oncogenes can help "self-tolerize" by creating analogous functional deficiencies in physiologic DDR networks is not known. Here we focus on Ewing sarcoma, a FET fusion oncoprotein (EWS-FLI1) driven pediatric bone tumor, as a model for the class of FET rearranged cancers. Native FET protein family members are among the earliest factors recruited to DNA double-strand breaks (DSBs) during the DDR, though the function of both native FET proteins and FET fusion oncoproteins in DNA repair remains to be defined. Using preclinical mechanistic studies of the DDR and clinical genomic datasets from patient tumors, we discover that the EWS-FLI1 fusion oncoprotein is recruited to DNA DSBs and interferes with native FET (EWS) protein function in activating the DNA damage sensor ATM. As a consequence of FET fusion-mediated interference with the DDR, we establish functional ATM deficiency as the principal DNA repair defect in Ewing sarcoma and the compensatory ATR signaling axis as a collateral dependency and therapeutic target in multiple FET rearranged cancers. More generally, we find that aberrant recruitment of a fusion oncoprotein to sites of DNA damage can disrupt physiologic DSB repair, revealing a mechanism for how growth-promoting oncogenes can also create a functional deficiency within tumor suppressive DDR networks.
RESUMO
Cis-regulatory elements control gene expression and are dynamic in their structure, reflecting changes to the composition of diverse effector proteins over time1-3. Here we sought to connect the structural changes at cis-regulatory elements to alterations in cellular fate and function. To do this we developed PRINT, a computational method that uses deep learning to correct sequence bias in chromatin accessibility data and identifies multi-scale footprints of DNA-protein interactions. We find that multi-scale footprints enable more accurate inference of TF and nucleosome binding. Using PRINT with single-cell multi-omics, we discover wide-spread changes to the structure and function of candidate cis-regulatory elements (cCREs) across hematopoiesis, wherein nucleosomes slide, expose DNA for TF binding, and promote gene expression. Activity segmentation using the co-variance across cell states identifies "sub-cCREs" as modular cCRE subunits of regulatory DNA. We apply this single-cell and PRINT approach to characterize the age-associated alterations to cCREs within hematopoietic stem cells (HSCs). Remarkably, we find a spectrum of aging alterations among HSCs corresponding to a global gain of sub-cCRE activity while preserving cCRE accessibility. Collectively, we reveal the functional importance of cCRE structure across cell states, highlighting changes to gene regulation at single-cell and single-base-pair resolution.
RESUMO
Oncogenic FOXO1 gene fusions drive a subset of rhabdomyosarcoma (RMS) with poor survival; to date, these cancer drivers are therapeutically intractable. To identify new therapies for this disease, we undertook an isogenic CRISPR-interference screen to define PAX3-FOXO1-specific genetic dependencies and identified genes in the GATOR2 complex. GATOR2 loss in RMS abrogated aa-induced lysosomal localization of mTORC1 and consequent downstream signaling, slowing G1-S cell cycle transition. In vivo suppression of GATOR2 impaired the growth of tumor xenografts and favored the outgrowth of cells lacking PAX3-FOXO1. Loss of a subset of GATOR2 members can be compensated by direct genetic activation of mTORC1. RAS mutations are also sufficient to decouple mTORC1 activation from GATOR2, and indeed, fusion-negative RMS harboring such mutations exhibit aa-independent mTORC1 activity. A bisteric, mTORC1-selective small molecule induced tumor regressions in fusion-positive patient-derived tumor xenografts. These findings highlight a vulnerability in FOXO1 fusion-positive RMS and provide rationale for the clinical evaluation of bisteric mTORC1 inhibitors, currently in phase I testing, to treat this disease. Isogenic genetic screens can, thus, identify potentially exploitable vulnerabilities in fusion-driven pediatric cancers that otherwise remain mostly undruggable.
Assuntos
Neoplasias , Criança , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Proteína Forkhead Box O1/genéticaRESUMO
Human chromosomes are pervasively transcribed, but systematic understanding of coding and lncRNA genome function in cell differentiation is lacking. Using CRISPR interference (CRISPRi) in human induced pluripotent stem cells, we performed dual genome-wide screens - assessing 18,905 protein-coding and 10,678 lncRNA loci - and identified 419 coding and 201 lncRNA genes that regulate neural induction. Integrative analyses revealed distinct properties of coding and lncRNA genome function, including a 10-fold enrichment of lncRNA genes for roles in differentiation compared to proliferation. Further, we applied Perturb-seq to obtain granular insights into neural induction phenotypes. While most coding hits stalled or aborted differentiation, lncRNA hits were enriched for the genesis of diverse cellular states, including those outside the neural lineage. In addition to providing a rich resource (danlimlab.shinyapps.io/dualgenomewide) for understanding coding and lncRNA gene function in development, these results indicate that the lncRNA genome regulates lineage commitment in a manner fundamentally distinct from coding genes.
RESUMO
CRISPR-mediated interference (CRISPRi), a robust and specific system for programmably repressing transcription, provides a versatile tool for systematically characterizing the function of long noncoding RNAs (lncRNAs). When used with highly parallel, lentiviral pooled screening approaches, CRISPRi enables the targeted knockdown of tens of thousands of lncRNA-expressing loci in a single screen. Here we describe the use of CRISPRi to target lncRNA loci in a pooled screen, using cell growth and proliferation as an example of a phenotypic readout. Considerations for custom lncRNA-targeting libraries, alternative phenotypic readouts, and orthogonal validation approaches are also discussed.