RESUMEN
Inactivation of Polybromo 1 (PBRM1), a specific subunit of the PBAF chromatin remodeling complex, occurs frequently in cancer, including 40% of clear cell renal cell carcinomas (ccRCC). To identify novel therapeutic approaches to targeting PBRM1-defective cancers, we used a series of orthogonal functional genomic screens that identified PARP and ATR inhibitors as being synthetic lethal with PBRM1 deficiency. The PBRM1/PARP inhibitor synthetic lethality was recapitulated using several clinical PARP inhibitors in a series of in vitro model systems and in vivo in a xenograft model of ccRCC. In the absence of exogenous DNA damage, PBRM1-defective cells exhibited elevated levels of replication stress, micronuclei, and R-loops. PARP inhibitor exposure exacerbated these phenotypes. Quantitative mass spectrometry revealed that multiple R-loop processing factors were downregulated in PBRM1-defective tumor cells. Exogenous expression of the R-loop resolution enzyme RNase H1 reversed the sensitivity of PBRM1-deficient cells to PARP inhibitors, suggesting that excessive levels of R-loops could be a cause of this synthetic lethality. PARP and ATR inhibitors also induced cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) innate immune signaling in PBRM1-defective tumor cells. Overall, these findings provide the preclinical basis for using PARP inhibitors in PBRM1-defective cancers. SIGNIFICANCE: This study demonstrates that PARP and ATR inhibitors are synthetic lethal with the loss of PBRM1, a PBAF-specific subunit, thus providing the rationale for assessing these inhibitors in patients with PBRM1-defective cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/11/2888/F1.large.jpg.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/deficiencia , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Renales/patología , Neoplasias Pulmonares/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Mutaciones Letales Sintéticas , Factores de Transcripción/deficiencia , Animales , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Proliferación Celular , Femenino , Humanos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway detects cytosolic DNA to activate innate immune responses. Poly(ADP-ribose) polymerase inhibitors (PARPi) selectively target cancer cells with DNA repair deficiencies such as those caused by BRCA1 mutations or ERCC1 defects. Using isogenic cell lines and patient-derived samples, we showed that ERCC1-defective non-small cell lung cancer (NSCLC) cells exhibit an enhanced type I IFN transcriptomic signature and that low ERCC1 expression correlates with increased lymphocytic infiltration. We demonstrated that clinical PARPi, including olaparib and rucaparib, have cell-autonomous immunomodulatory properties in ERCC1-defective NSCLC and BRCA1-defective triple-negative breast cancer (TNBC) cells. Mechanistically, PARPi generated cytoplasmic chromatin fragments with characteristics of micronuclei; these were found to activate cGAS/STING, downstream type I IFN signaling, and CCL5 secretion. Importantly, these effects were suppressed in PARP1-null TNBC cells, suggesting that this phenotype resulted from an on-target effect of PARPi on PARP1. PARPi also potentiated IFN-γ-induced PD-L1 expression in NSCLC cell lines and in fresh patient tumor cells; this effect was enhanced in ERCC1-deficient contexts. Our data provide a preclinical rationale for using PARPi as immunomodulatory agents in appropriately molecularly selected populations.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Proteínas de Unión al ADN/deficiencia , Endonucleasas/deficiencia , Neoplasias Pulmonares , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Células A549 , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Femenino , Humanos , Interferón gamma/genética , Interferón gamma/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0176578.].
RESUMEN
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
[This corrects the article DOI: 10.1371/journal.pone.0149099.].
RESUMEN
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
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
Activating KRAS mutations are found in approximately 20% of human cancers but no RAS-directed therapies are currently available. Here we describe a novel, robust, KRAS synthetic lethal interaction with the cyclin dependent kinase, CDK1. This was discovered using parallel siRNA screens in KRAS mutant and wild type colorectal isogenic tumour cells and subsequently validated in a genetically diverse panel of 26 colorectal and pancreatic tumour cell models. This established that the KRAS/CDK1 synthetic lethality applies in tumour cells with either amino acid position 12 (p.G12V, pG12D, p.G12S) or amino acid position 13 (p.G13D) KRAS mutations and can also be replicated in vivo in a xenograft model using a small molecule CDK1 inhibitor. Mechanistically, CDK1 inhibition caused a reduction in the S-phase fraction of KRAS mutant cells, an effect also characterised by modulation of Rb, a master control of the G1/S checkpoint. Taken together, these observations suggest that the KRAS/CDK1 interaction is a robust synthetic lethal effect worthy of further investigation.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Mutación , Neoplasias Pancreáticas/metabolismo , Animales , Antineoplásicos/farmacología , Proteína Quinasa CDC2 , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/genética , Relación Dosis-Respuesta a Droga , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Genes Letales , Ensayos Analíticos de Alto Rendimiento , Humanos , Imidazoles/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Pirimidinas/farmacología , Quinolinas/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Análisis de Supervivencia , Tiazoles/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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
Small-molecule inhibitors of PARP1/2, such as olaparib, have been proposed to serve as a synthetic lethal therapy for cancers that harbor BRCA1 or BRCA2 mutations. Indeed, in clinical trials, PARP1/2 inhibitors elicit sustained antitumor responses in patients with germline BRCA gene mutations. In hypothesizing that additional genetic determinants might direct use of these drugs, we conducted a genome-wide synthetic lethal screen for candidate olaparib sensitivity genes. In support of this hypothesis, the set of identified genes included known determinants of olaparib sensitivity, such as BRCA1, RAD51, and Fanconi's anemia susceptibility genes. In addition, the set included genes implicated in established networks of DNA repair, DNA cohesion, and chromatin remodeling, none of which were known previously to confer sensitivity to PARP1/2 inhibition. Notably, integration of the list of candidate sensitivity genes with data from tumor DNA sequencing studies identified CDK12 deficiency as a clinically relevant biomarker of PARP1/2 inhibitor sensitivity. In models of high-grade serous ovarian cancer (HGS-OVCa), CDK12 attenuation was sufficient to confer sensitivity to PARP1/2 inhibition, suppression of DNA repair via homologous recombination, and reduced expression of BRCA1. As one of only nine genes known to be significantly mutated in HGS-OVCa, CDK12 has properties that should confirm interest in its use as a biomarker, particularly in ongoing clinical trials of PARP1/2 inhibitors and other agents that trigger replication fork arrest.