RESUMEN
BACKGROUND: Prostate cancer is a heterogeneous disease, but current treatments are not based on molecular stratification. We hypothesized that metastatic, castration-resistant prostate cancers with DNA-repair defects would respond to poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibition with olaparib. METHODS: We conducted a phase 2 trial in which patients with metastatic, castration-resistant prostate cancer were treated with olaparib tablets at a dose of 400 mg twice a day. The primary end point was the response rate, defined either as an objective response according to Response Evaluation Criteria in Solid Tumors, version 1.1, or as a reduction of at least 50% in the prostate-specific antigen level or a confirmed reduction in the circulating tumor-cell count from 5 or more cells per 7.5 ml of blood to less than 5 cells per 7.5 ml. Targeted next-generation sequencing, exome and transcriptome analysis, and digital polymerase-chain-reaction testing were performed on samples from mandated tumor biopsies. RESULTS: Overall, 50 patients were enrolled; all had received prior treatment with docetaxel, 49 (98%) had received abiraterone or enzalutamide, and 29 (58%) had received cabazitaxel. Sixteen of 49 patients who could be evaluated had a response (33%; 95% confidence interval, 20 to 48), with 12 patients receiving the study treatment for more than 6 months. Next-generation sequencing identified homozygous deletions, deleterious mutations, or both in DNA-repair genes--including BRCA1/2, ATM, Fanconi's anemia genes, and CHEK2--in 16 of 49 patients who could be evaluated (33%). Of these 16 patients, 14 (88%) had a response to olaparib, including all 7 patients with BRCA2 loss (4 with biallelic somatic loss, and 3 with germline mutations) and 4 of 5 with ATM aberrations. The specificity of the biomarker suite was 94%. Anemia (in 10 of the 50 patients [20%]) and fatigue (in 6 [12%]) were the most common grade 3 or 4 adverse events, findings that are consistent with previous studies of olaparib. CONCLUSIONS: Treatment with the PARP inhibitor olaparib in patients whose prostate cancers were no longer responding to standard treatments and who had defects in DNA-repair genes led to a high response rate. (Funded by Cancer Research UK and others; ClinicalTrials.gov number, NCT01682772; Cancer Research UK number, CRUK/11/029.).
Asunto(s)
Antineoplásicos/uso terapéutico , Reparación del ADN , Inhibidores Enzimáticos/uso terapéutico , Ftalazinas/uso terapéutico , Piperazinas/uso terapéutico , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Neoplasias de la Próstata/tratamiento farmacológico , Adulto , Anciano , Anemia/inducido químicamente , Proteínas de la Ataxia Telangiectasia Mutada/genética , Reparación del ADN/genética , Resistencia a Antineoplásicos , Inhibidores Enzimáticos/efectos adversos , Fatiga/inducido químicamente , Genes BRCA2 , Genes Supresores de Tumor , Humanos , Masculino , Persona de Mediana Edad , Mutación , Metástasis de la Neoplasia/tratamiento farmacológico , Ftalazinas/efectos adversos , Piperazinas/efectos adversos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patologíaRESUMEN
BACKGROUND: Elevated APOBEC3B expression in tumours correlates with a kataegic pattern of localised hypermutation. We assessed the cellular phenotypes associated with high-level APOBEC3B expression and the influence of p53 status on these phenotypes using an isogenic system. METHODS: We used RNA interference of p53 in cells with inducible APOBEC3B and assessed DNA damage response (DDR) biomarkers. The mutational effects of APOBEC3B were assessed using whole-genome sequencing. In vitro small-molecule inhibitor sensitivity profiling was used to identify candidate therapeutic vulnerabilities. RESULTS: Although APOBEC3B expression increased the incorporation of genomic uracil, invoked DDR biomarkers and caused cell cycle arrest, inactivation of p53 circumvented APOBEC3B-induced cell cycle arrest without reversing the increase in genomic uracil or DDR biomarkers. The continued expression of APOBEC3B in p53-defective cells not only caused a kataegic mutational signature but also caused hypersensitivity to small-molecule DDR inhibitors (ATR, CHEK1, CHEK2, PARP, WEE1 inhibitors) as well as cisplatin/ATR inhibitor and ATR/PARP inhibitor combinations. CONCLUSIONS: Although loss of p53 might allow tumour cells to tolerate elevated APOBEC3B expression, continued expression of this enzyme might impart a number of therapeutic vulnerabilities upon tumour cells.
Asunto(s)
Puntos de Control del Ciclo Celular/genética , Citidina Desaminasa/genética , Daño del ADN/genética , Regulación Neoplásica de la Expresión Génica , Antígenos de Histocompatibilidad Menor/genética , Proteína p53 Supresora de Tumor/genética , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Western Blotting , Sistemas CRISPR-Cas , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Quinasa de Punto de Control 2/antagonistas & inhibidores , Cisplatino/farmacología , Citidina Desaminasa/metabolismo , Daño del ADN/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Antígenos de Histocompatibilidad Menor/metabolismo , Mutación , Proteínas Nucleares/antagonistas & inhibidores , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Interferencia de ARN , Uracilo/metabolismoRESUMEN
SF3B1 hotspot mutations are associated with a poor prognosis in several tumor types and lead to global disruption of canonical splicing. Through synthetic lethal drug screens, we identify that SF3B1 mutant (SF3B1MUT) cells are selectively sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi), independent of hotspot mutation and tumor site. SF3B1MUT cells display a defective response to PARPi-induced replication stress that occurs via downregulation of the cyclin-dependent kinase 2 interacting protein (CINP), leading to increased replication fork origin firing and loss of phosphorylated CHK1 (pCHK1; S317) induction. This results in subsequent failure to resolve DNA replication intermediates and G2/M cell cycle arrest. These defects are rescued through CINP overexpression, or further targeted by a combination of ataxia-telangiectasia mutated and PARP inhibition. In vivo, PARPi produce profound antitumor effects in multiple SF3B1MUT cancer models and eliminate distant metastases. These data provide the rationale for testing the clinical efficacy of PARPi in a biomarker-driven, homologous recombination proficient, patient population.
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Neoplasias , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Mutación , Factores de Transcripción/genética , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Proteína BRCA1/genética , Línea Celular Tumoral , Factores de Empalme de ARN/genética , Fosfoproteínas/genéticaRESUMEN
HORMAD1 expression is usually restricted to germline cells, but it becomes mis-expressed in epithelial cells in ~60% of triple-negative breast cancers (TNBCs), where it is associated with elevated genomic instability (1). HORMAD1 expression in TNBC is bimodal with HORMAD1-positive TNBC representing a biologically distinct disease group. Identification of HORMAD1-driven genetic dependencies may uncover novel therapies for this disease group. To study HORMAD1-driven genetic dependencies, we generated a SUM159 cell line model with doxycycline-inducible HORMAD1 that replicated genomic instability phenotypes seen in HORMAD1-positive TNBC (1). Using small interfering RNA screens, we identified candidate genes whose depletion selectively inhibited the cellular growth of HORMAD1-expressing cells. We validated five genes (ATR, BRIP1, POLH, TDP1 and XRCC1), depletion of which led to reduced cellular growth or clonogenic survival in cells expressing HORMAD1. In addition to the translesion synthesis (TLS) polymerase POLH, we identified a HORMAD1-driven dependency upon additional TLS polymerases, namely POLK, REV1, REV3L and REV7. Our data confirms that out-of-context somatic expression of HORMAD1 can lead to genomic instability and reveals that HORMAD1 expression induces dependencies upon replication stress tolerance pathways, such as translesion synthesis. Our data also suggest that HORMAD1 expression could be a patient selection biomarker for agents targeting replication stress.
Asunto(s)
Neoplasias de la Mama Triple Negativas , Proteínas de Ciclo Celular/genética , Daño del ADN/genética , Reparación del ADN , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , ADN Polimerasa Dirigida por ADN/genética , Inestabilidad Genómica/genética , Humanos , Nucleotidiltransferasas/genética , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genéticaRESUMEN
Functional genomic screening with CRISPR has provided a powerful and precise new way to interrogate the phenotypic consequences of gene manipulation in high-throughput, unbiased analyses. However, some experimental paradigms prove especially challenging and require carefully and appropriately adapted screening approaches. In particular, negative selection (or sensitivity) screening, often the most experimentally desirable modality of screening, has remained a challenge in drug discovery. Here we assess whether our new, modular genome-wide pooled CRISPR library can improve negative selection CRISPR screening and add utility throughout the drug development pipeline. Our pooled library is split into three parts, allowing it to be scaled to accommodate the experimental challenges encountered during drug development, such as target identification using unlimited cell numbers compared with target identification studies for cell populations where cell numbers are limiting. To test our new library, we chose to look for drug-gene interactions using a well-described small molecule inhibitor targeting poly(ADP-ribose) polymerase 1 (PARP1), and in particular to identify genes which sensitise cells to this drug. We simulate hit identification and performance using each library partition and support these findings through orthogonal drug combination cell panel screening. We also compare our data with a recently published CRISPR sensitivity dataset obtained using the same PARP1 inhibitor. Overall, our data indicate that generating a comprehensive CRISPR knockout screening library where the number of guides can be scaled to suit the biological question being addressed allows a library to have multiple uses throughout the drug development pipeline, and that initial validation of hits can be achieved through high-throughput cell panels screens where clinical grade chemical or biological matter exist.
Asunto(s)
Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Desarrollo de Medicamentos , Biblioteca de Genes , Proteínas de Unión al ADN , Técnicas de Inactivación de Genes , Células HT29 , Ensayos Analíticos de Alto Rendimiento , Humanos , Preparaciones Farmacéuticas , ARN Guía de Kinetoplastida/genéticaRESUMEN
BACKGROUND: In neuroblastoma, genetic alterations in ATRX, define a distinct poor outcome patient subgroup. Despite the need for new therapies, there is a lack of available models and a dearth of pre-clinical research. METHODS: To evaluate the impact of ATRX loss of function (LoF) in neuroblastoma, we utilized CRISPR-Cas9 gene editing to generate neuroblastoma cell lines isogenic for ATRX. We used these and other models to identify therapeutically exploitable synthetic lethal vulnerabilities associated with ATRX LoF. FINDINGS: In isogenic cell lines, we found that ATRX inactivation results in increased DNA damage, homologous recombination repair (HRR) defects and impaired replication fork processivity. In keeping with this, high-throughput compound screening showed selective sensitivity in ATRX mutant cells to multiple PARP inhibitors and the ATM inhibitor KU60019. ATRX mutant cells also showed selective sensitivity to the DNA damaging agents, sapacitabine and irinotecan. HRR deficiency was also seen in the ATRX deleted CHLA-90 cell line, and significant sensitivity demonstrated to olaparib/irinotecan combination therapy in all ATRX LoF models. In-vivo sensitivity to olaparib/irinotecan was seen in ATRX mutant but not wild-type xenografts. Finally, sustained responses to olaparib/irinotecan therapy were seen in an ATRX deleted neuroblastoma patient derived xenograft. INTERPRETATION: ATRX LoF results in specific DNA damage repair defects that can be therapeutically exploited. In ATRX LoF models, preclinical sensitivity is demonstrated to olaparib and irinotecan, a combination that can be rapidly translated into the clinic. FUNDING: This work was supported by Christopher's Smile, Neuroblastoma UK, Cancer Research UK, and the Royal Marsden Hospital NIHR BRC.
Asunto(s)
Antineoplásicos/farmacología , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Neuroblastoma/genética , Proteína Nuclear Ligada al Cromosoma X/genética , Animales , Antineoplásicos/uso terapéutico , Sistemas CRISPR-Cas , Línea Celular Tumoral , Modelos Animales de Enfermedad , Edición Génica , Técnicas de Inactivación de Genes , Humanos , Inmunohistoquímica , Ratones , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/mortalidad , Neuroblastoma/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Pronóstico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Although PARP inhibitors (PARPi) target homologous recombination defective tumours, drug resistance frequently emerges, often via poorly understood mechanisms. Here, using genome-wide and high-density CRISPR-Cas9 "tag-mutate-enrich" mutagenesis screens, we identify close to full-length mutant forms of PARP1 that cause in vitro and in vivo PARPi resistance. Mutations both within and outside of the PARP1 DNA-binding zinc-finger domains cause PARPi resistance and alter PARP1 trapping, as does a PARP1 mutation found in a clinical case of PARPi resistance. This reinforces the importance of trapped PARP1 as a cytotoxic DNA lesion and suggests that PARP1 intramolecular interactions might influence PARPi-mediated cytotoxicity. PARP1 mutations are also tolerated in cells with a pathogenic BRCA1 mutation where they result in distinct sensitivities to chemotherapeutic drugs compared to other mechanisms of PARPi resistance (BRCA1 reversion, 53BP1, REV7 (MAD2L2) mutation), suggesting that the underlying mechanism of PARPi resistance that emerges could influence the success of subsequent therapies.
Asunto(s)
Resistencia a Antineoplásicos/genética , Neoplasias/tratamiento farmacológico , Poli(ADP-Ribosa) Polimerasa-1/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Anciano , Animales , Proteína BRCA1/genética , Sistemas CRISPR-Cas , Línea Celular Tumoral , Análisis Mutacional de ADN/métodos , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Madre Embrionarias de Ratones , Mutagénesis , Neoplasias/genética , Neoplasias/patología , Ftalazinas/farmacología , Ftalazinas/uso terapéutico , Mutación Puntual , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Medicina de Precisión/métodos , Secuenciación Completa del Genoma/métodos , Ensayos Antitumor por Modelo de Xenoinjerto , Dedos de Zinc/genéticaRESUMEN
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMEN
Osteosarcoma (OS) is an aggressive sarcoma, where novel treatment approaches are required. Genomic studies suggest that a subset of OS, including OS tumour cell lines (TCLs), exhibit genomic loss of heterozygosity (LOH) patterns reminiscent of BRCA1 or BRCA2 mutant tumours. This raises the possibility that PARP inhibitors (PARPi), used to treat BRCA1/2 mutant cancers, could be used to target OS. Using high-throughput drug sensitivity screening we generated chemosensitivity profiles for 79 small molecule inhibitors, including three clinical PARPi. Drug screening was performed in 88 tumour cell lines, including 18 OS TCLs. This identified known sensitivity effects in OS TCLs, such as sensitivity to FGFR inhibitors. When compared to BRCA1/2 mutant TCLs, OS TCLs, with the exception of LM7, were PARPi resistant, including those with previously determined BRCAness LoH profiles. Post-screen validation experiments confirmed PARPi sensitivity in LM7 cells as well as a defect in the ability to form nuclear RAD51 foci in response to DNA damage. LM7 provides one OS model for the study of PARPi sensitivity through a potential defect in RAD51-mediated DNA repair. The drug sensitivity dataset we generated in 88 TCLs could also serve as a resource for the study of drug sensitivity effects in OS.
Asunto(s)
Proteína BRCA1/genética , Proteína BRCA2/genética , Resistencia a Antineoplásicos/genética , Osteosarcoma/tratamiento farmacológico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Conjuntos de Datos como Asunto , Ensayos de Selección de Medicamentos Antitumorales/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Mutagénesis , Mutación , Osteosarcoma/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Recombinasa Rad51/metabolismoRESUMEN
The cell adhesion glycoprotein E-cadherin (CDH1) is commonly inactivated in breast tumors. Precision medicine approaches that exploit this characteristic are not available. Using perturbation screens in breast tumor cells with CRISPR/Cas9-engineered CDH1 mutations, we identified synthetic lethality between E-cadherin deficiency and inhibition of the tyrosine kinase ROS1. Data from large-scale genetic screens in molecularly diverse breast tumor cell lines established that the E-cadherin/ROS1 synthetic lethality was not only robust in the face of considerable molecular heterogeneity but was also elicited with clinical ROS1 inhibitors, including foretinib and crizotinib. ROS1 inhibitors induced mitotic abnormalities and multinucleation in E-cadherin-defective cells, phenotypes associated with a defect in cytokinesis and aberrant p120 catenin phosphorylation and localization. In vivo, ROS1 inhibitors produced profound antitumor effects in multiple models of E-cadherin-defective breast cancer. These data therefore provide the preclinical rationale for assessing ROS1 inhibitors, such as the licensed drug crizotinib, in appropriately stratified patients.Significance: E-cadherin defects are common in breast cancer but are currently not targeted with a precision medicine approach. Our preclinical data indicate that licensed ROS1 inhibitors, including crizotinib, should be repurposed to target E-cadherin-defective breast cancers, thus providing the rationale for the assessment of these agents in molecularly stratified phase II clinical trials. Cancer Discov; 8(4); 498-515. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 371.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Cadherinas/deficiencia , Crizotinib/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Anilidas/farmacología , Anilidas/uso terapéutico , Animales , Antígenos CD/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/metabolismo , Cadherinas/genética , Línea Celular Tumoral , Crizotinib/uso terapéutico , Femenino , Humanos , Ratones , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinolinas/farmacología , Quinolinas/uso terapéuticoRESUMEN
The failure to develop effective therapies for pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG) is in part due to their intrinsic heterogeneity. We aimed to quantitatively assess the extent to which this was present in these tumors through subclonal genomic analyses and to determine whether distinct tumor subpopulations may interact to promote tumorigenesis by generating subclonal patient-derived models in vitro and in vivo. Analysis of 142 sequenced tumors revealed multiple tumor subclones, spatially and temporally coexisting in a stable manner as observed by multiple sampling strategies. We isolated genotypically and phenotypically distinct subpopulations that we propose cooperate to enhance tumorigenicity and resistance to therapy. Inactivating mutations in the H4K20 histone methyltransferase KMT5B (SUV420H1), present in <1% of cells, abrogate DNA repair and confer increased invasion and migration on neighboring cells, in vitro and in vivo, through chemokine signaling and modulation of integrins. These data indicate that even rare tumor subpopulations may exert profound effects on tumorigenesis as a whole and may represent a new avenue for therapeutic development. Unraveling the mechanisms of subclonal diversity and communication in pGBM and DIPG will be an important step toward overcoming barriers to effective treatments.
Asunto(s)
Neoplasias del Tronco Encefálico/patología , Glioblastoma/patología , Animales , Neoplasias del Tronco Encefálico/genética , Carcinogénesis/patología , Separación Celular , Niño , Células Clonales , Genotipo , Glioblastoma/genética , Humanos , Ratones Desnudos , Fenotipo , Células Tumorales CultivadasRESUMEN
Although PARP inhibitors target BRCA1- or BRCA2-mutant tumor cells, drug resistance is a problem. PARP inhibitor resistance is sometimes associated with the presence of secondary or "revertant" mutations in BRCA1 or BRCA2 Whether secondary mutant tumor cells are selected for in a Darwinian fashion by treatment is unclear. Furthermore, how PARP inhibitor resistance might be therapeutically targeted is also poorly understood. Using CRISPR mutagenesis, we generated isogenic tumor cell models with secondary BRCA1 or BRCA2 mutations. Using these in heterogeneous in vitro culture or in vivo xenograft experiments in which the clonal composition of tumor cell populations in response to therapy was monitored, we established that PARP inhibitor or platinum salt exposure selects for secondary mutant clones in a Darwinian fashion, with the periodicity of PARP inhibitor administration and the pretreatment frequency of secondary mutant tumor cells influencing the eventual clonal composition of the tumor cell population. In xenograft studies, the presence of secondary mutant cells in tumors impaired the therapeutic effect of a clinical PARP inhibitor. However, we found that both PARP inhibitor-sensitive and PARP inhibitor-resistant BRCA2 mutant tumor cells were sensitive to AZD-1775, a WEE1 kinase inhibitor. In mice carrying heterogeneous tumors, AZD-1775 delivered a greater therapeutic benefit than olaparib treatment. This suggests that despite the restoration of some BRCA1 or BRCA2 gene function in "revertant" tumor cells, vulnerabilities still exist that could be therapeutically exploited. Mol Cancer Ther; 16(9); 2022-34. ©2017 AACR.
Asunto(s)
Antineoplásicos/farmacología , Proteína BRCA1/genética , Proteína BRCA2/genética , Resistencia a Antineoplásicos/genética , Mutación , Animales , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular Tumoral , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Proteínas Nucleares/antagonistas & inhibidores , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Pirimidinonas , Selección Genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We describe a screen for cellular response to drugs that makes use of haploid embryonic stem cells. We generated ten libraries of mutants with piggyBac gene trap transposon integrations, totalling approximately 100,000 mutant clones. Random barcode sequences were inserted into the transposon vector to allow the number of cells bearing each insertion to be measured by amplifying and sequencing the barcodes. These barcodes were associated with their integration sites by inverse PCR. We exposed these libraries to commonly used cancer drugs and profiled changes in barcode abundance by Ion Torrent sequencing in order to identify mutations that conferred sensitivity. Drugs tested included conventional chemotherapeutics as well as targeted inhibitors of topoisomerases, poly(ADP-ribose) polymerase (PARP), Hsp90 and WEE1.
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Elementos Transponibles de ADN , Células Madre Embrionarias de Ratones , Neoplasias , Animales , Antineoplásicos/farmacología , Estudio de Asociación del Genoma Completo , Haploidia , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/genéticaRESUMEN
Synovial sarcoma (SS) is an aggressive soft-tissue malignancy characterized by expression of SS18-SSX fusions, where treatment options are limited. To identify therapeutically actionable genetic dependencies in SS, we performed a series of parallel, high-throughput small interfering RNA (siRNA) screens and compared genetic dependencies in SS tumor cells with those in >130 non-SS tumor cell lines. This approach revealed a reliance of SS tumor cells upon the DNA damage response serine/threonine protein kinase ATR. Clinical ATR inhibitors (ATRi) elicited a synthetic lethal effect in SS tumor cells and impaired growth of SS patient-derived xenografts. Oncogenic SS18-SSX family fusion genes are known to alter the composition of the BAF chromatin-remodeling complex, causing ejection and degradation of wild-type SS18 and the tumor suppressor SMARCB1. Expression of oncogenic SS18-SSX fusion proteins caused profound ATRi sensitivity and a reduction in SS18 and SMARCB1 protein levels, but an SSX18-SSX1 Δ71-78 fusion containing a C-terminal deletion did not. ATRi sensitivity in SS was characterized by an increase in biomarkers of replication fork stress (increased γH2AX, decreased replication fork speed, and increased R-loops), an apoptotic response, and a dependence upon cyclin E expression. Combinations of cisplatin or PARP inhibitors enhanced the antitumor cell effect of ATRi, suggesting that either single-agent ATRi or combination therapy involving ATRi might be further assessed as candidate approaches for SS treatment. Cancer Res; 77(24); 7014-26. ©2017 AACR.
Asunto(s)
Antineoplásicos/uso terapéutico , Terapia Molecular Dirigida/métodos , Sarcoma Sinovial/tratamiento farmacológico , Animales , Antineoplásicos/administración & dosificación , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/fisiología , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Interferencia de ARN/fisiología , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/uso terapéutico , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/uso terapéutico , Sarcoma Sinovial/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cancer reflects the progressive accumulation of genetic alterations and subsequent genetic instability of cells. Cytogenetic studies have demonstrated the importance of aneuploidy in differentiated thyroid cancer development. The pituitary tumour transforming gene (PTTG), also known as securin, is a mitotic checkpoint protein which inhibits sister chromatid separation during mitosis. PTTG is highly expressed in many cancers and overexpression of PTTG induces aneuploidy in vitro. Using fluorescent intersimple sequence repeat PCR (FISSR-PCR), we investigated the relationship between PTTG expression and the degree of genetic instability in normal and tumorous thyroid samples. The genomic instability index (GI index) was 6.7-72.7% higher in cancers than normal thyroid tissues. Follicular thyroid tumours exhibited greater genetic instability than papillary tumours (27.6% (n=9) versus 14.5% (n=10), P=0.03). We also demonstrated a strong relationship between PTTG expression and the degree of genetic instability in thyroid cancers (R2=0.80, P=0.007). To further investigate PTTG's role in genetic instability, we transfected FTC133 thyroid follicular cells and observed increased genetic instability in cells overexpressing PTTG compared with vector-only-transfected controls (n=3, GI Index VO=29.7+/-5.2 versus PTTG=63.7+/-6.4, P=0.013). Further, we observed a dose response in genetic instability and PTTG expression (GI Index low dose (0.5 microg DNA/ six-well plate) PTTG=15.3%+/-1.7 versus high dose (3 microg DNA) PTTG=50.8%+/-3.3, P=0.006). Overall, we describe the first use of FISSR-PCR in human cancers, and demonstrate that PTTG expression correlates with genetic instability in vivo, and induces genetic instability in vitro. We conclude that PTTG may be an important gene in the mutator phenotype development in thyroid cancer.
Asunto(s)
Inestabilidad Genómica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Glándula Tiroides/metabolismo , Glándula Tiroides/patología , Neoplasias de la Tiroides/genética , Caspasa 3 , Caspasa 7 , Caspasas/metabolismo , Línea Celular , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Securina , Neoplasias de la Tiroides/patologíaRESUMEN
Identifying genetic biomarkers of synthetic lethal drug sensitivity effects provides one approach to the development of targeted cancer therapies. Mutations in ARID1A represent one of the most common molecular alterations in human cancer, but therapeutic approaches that target these defects are not yet clinically available. We demonstrate that defects in ARID1A sensitize tumour cells to clinical inhibitors of the DNA damage checkpoint kinase, ATR, both in vitro and in vivo. Mechanistically, ARID1A deficiency results in topoisomerase 2A and cell cycle defects, which cause an increased reliance on ATR checkpoint activity. In ARID1A mutant tumour cells, inhibition of ATR triggers premature mitotic entry, genomic instability and apoptosis. The data presented here provide the pre-clinical and mechanistic rationale for assessing ARID1A defects as a biomarker of single-agent ATR inhibitor response and represents a novel synthetic lethal approach to targeting tumour cells.
Asunto(s)
Puntos de Control del Ciclo Celular/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Antineoplásicos/farmacología , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proteínas de Unión al ADN , Humanos , Interferencia de ARNRESUMEN
One approach to identifying cancer-specific vulnerabilities and therapeutic targets is to profile genetic dependencies in cancer cell lines. Here, we describe data from a series of siRNA screens that identify the kinase genetic dependencies in 117 cancer cell lines from ten cancer types. By integrating the siRNA screen data with molecular profiling data, including exome sequencing data, we show how vulnerabilities/genetic dependencies that are associated with mutations in specific cancer driver genes can be identified. By integrating additional data sets into this analysis, including protein-protein interaction data, we also demonstrate that the genetic dependencies associated with many cancer driver genes form dense connections on functional interaction networks. We demonstrate the utility of this resource by using it to predict the drug sensitivity of genetically or histologically defined subsets of tumor cell lines, including an increased sensitivity of osteosarcoma cell lines to FGFR inhibitors and SMAD4 mutant tumor cells to mitotic inhibitors.
Asunto(s)
Neoplasias/enzimología , Neoplasias/genética , Proteínas Quinasas/metabolismo , Línea Celular Tumoral , Perfilación de la Expresión Génica , Humanos , Mutación , Neoplasias/patología , Proteínas Quinasas/química , Proteínas Quinasas/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Proteína Smad4/antagonistas & inhibidores , Proteína Smad4/genética , Proteína Smad4/metabolismoRESUMEN
New targeted approaches to ovarian clear cell carcinomas (OCCC) are needed, given the limited treatment options in this disease and the poor response to standard chemotherapy. Using a series of high-throughput cell-based drug screens in OCCC tumor cell models, we have identified a synthetic lethal (SL) interaction between the kinase inhibitor dasatinib and a key driver in OCCC, ARID1A mutation. Imposing ARID1A deficiency upon a variety of human or mouse cells induced dasatinib sensitivity, both in vitro and in vivo, suggesting that this is a robust synthetic lethal interaction. The sensitivity of ARID1A-deficient cells to dasatinib was associated with G1-S cell-cycle arrest and was dependent upon both p21 and Rb. Using focused siRNA screens and kinase profiling, we showed that ARID1A-mutant OCCC tumor cells are addicted to the dasatinib target YES1. This suggests that dasatinib merits investigation for the treatment of patients with ARID1A-mutant OCCC. Mol Cancer Ther; 15(7); 1472-84. ©2016 AACR.
Asunto(s)
Adenocarcinoma de Células Claras/genética , Antineoplásicos/farmacología , Dasatinib/farmacología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Neoplasias Ováricas/genética , Inhibidores de Proteínas Quinasas/farmacología , Mutaciones Letales Sintéticas , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Adenocarcinoma de Células Claras/tratamiento farmacológico , Adenocarcinoma de Células Claras/patología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Proteínas de Unión al ADN , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Perfilación de la Expresión Génica , Humanos , Ratones , Terapia Molecular Dirigida , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The nuclear receptor NR2E1 (also known as TLX or tailless) controls the self-renewal of neural stem cells (NSCs) and has been implied as an oncogene which initiates brain tumors including glioblastomas. Despite NR2E1 regulating targets like p21(CIP1) or PTEN we still lack a full explanation for its role in NSC self-renewal and tumorigenesis. We know that polycomb repressive complexes also control stem cell self-renewal and tumorigenesis, but so far, no formal connection has been established between NR2E1 and PRCs. In a screen for transcription factors regulating the expression of the polycomb protein CBX7, we identified NR2E1 as one of its more prominent regulators. NR2E1 binds at the CBX7 promoter, inducing its expression. Notably CBX7 represses NR2E1 as part of a regulatory loop. Ectopic NR2E1 expression inhibits cellular senescence, extending cellular lifespan in fibroblasts via CBX7-mediated regulation of p16(INK4a) and direct repression of p21(CIP1). In addition NR2E1 expression also counteracts oncogene-induced senescence. The importance of NR2E1 to restrain senescence is highlighted through the process of knocking down its expression, which causes premature senescence in human fibroblasts and epithelial cells. We also confirmed that NR2E1 regulates CBX7 and restrains senescence in NSCs. Finally, we observed that the expression of NR2E1 directly correlates with that of CBX7 in human glioblastoma multiforme. Overall we identified control of senescence and regulation of polycomb action as two possible mechanisms that can join those so far invoked to explain the role of NR2E1 in control of NSC self-renewal and cancer.
Asunto(s)
Senescencia Celular/genética , Receptores Citoplasmáticos y Nucleares/fisiología , Animales , Células Cultivadas , Retroalimentación Fisiológica , Regulación de la Expresión Génica , Células HEK293 , Humanos , Masculino , Ratones , Células-Madre Neurales/fisiología , Receptores Nucleares Huérfanos , Complejo Represivo Polycomb 1/genética , Regiones Promotoras Genéticas , Unión Proteica , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
BACKGROUND: Polycomb group proteins form multicomponent complexes that are important for establishing lineage-specific patterns of gene expression. Mammalian cells encode multiple permutations of the prototypic Polycomb repressive complex 1 (PRC1) with little evidence for functional specialization. An aim of this study is to determine whether the multiple orthologs that are co-expressed in human fibroblasts act on different target genes and whether their genomic location changes during cellular senescence. RESULTS: Deep sequencing of chromatin immunoprecipitated with antibodies against CBX6, CBX7, CBX8, RING1 and RING2 reveals that the orthologs co-localize at multiple sites. PCR-based validation at representative loci suggests that a further six PRC1 proteins have similar binding patterns. Importantly, sequential chromatin immunoprecipitation with antibodies against different orthologs implies that multiple variants of PRC1 associate with the same DNA. At many loci, the binding profiles have a distinctive architecture that is preserved in two different types of fibroblast. Conversely, there are several hundred loci at which PRC1 binding is cell type-specific and, contrary to expectations, the presence of PRC1 does not necessarily equate with transcriptional silencing. Interestingly, the PRC1 binding profiles are preserved in senescent cells despite changes in gene expression. CONCLUSIONS: The multiple permutations of PRC1 in human fibroblasts congregate at common rather than specific sites in the genome and with overlapping but distinctive binding profiles in different fibroblasts. The data imply that the effects of PRC1 complexes on gene expression are more subtle than simply repressing the loci at which they bind.