RESUMEN
Inhibitors of poly-ADP-ribose polymerase 1 (PARPi) are highly effective in killing cells deficient in homologous recombination (HR); thus, PARPi have been clinically utilized to successfully treat BRCA2-mutant tumors. However, positive response to PARPi is not universal, even among patients with HR-deficiency. Here, we present the results of genome-wide CRISPR knockout and activation screens which reveal genetic determinants of PARPi response in wildtype or BRCA2-knockout cells. Strikingly, we report that depletion of the ubiquitin ligase HUWE1, or the histone acetyltransferase KAT5, top hits from our screens, robustly reverses the PARPi sensitivity caused by BRCA2-deficiency. We identify distinct mechanisms of resistance, in which HUWE1 loss increases RAD51 levels to partially restore HR, whereas KAT5 depletion rewires double strand break repair by promoting 53BP1 binding to double-strand breaks. Our work provides a comprehensive set of putative biomarkers that advance understanding of PARPi response, and identifies novel pathways of PARPi resistance in BRCA2-deficient cells.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Inhibidores de Poli(ADP-Ribosa) Polimerasas/aislamiento & purificación , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/efectos de los fármacos , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Biomarcadores , Daño del ADN , Reparación del ADN , Técnicas de Inactivación de Genes , Células HeLa , Recombinación Homóloga/efectos de los fármacos , Humanos , Lisina Acetiltransferasa 5/metabolismo , Proteínas Mad2/metabolismo , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53 , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The ataxia telangiectasia and Rad3-related (ATR) protein kinase is a key regulator of the cellular response to DNA damage. Due to increased amount of replication stress, cancer cells heavily rely on ATR to complete DNA replication and cell cycle progression. Thus, ATR inhibition is an emerging target in cancer therapy, with multiple ATR inhibitors currently undergoing clinical trials. Here, we describe dual genome-wide CRISPR knockout and CRISPR activation screens employed to comprehensively identify genes that regulate the cellular resistance to ATR inhibitors. Specifically, we investigated two different ATR inhibitors, namely VE822 and AZD6738, in both HeLa and MCF10A cells. We identified and validated multiple genes that alter the resistance to ATR inhibitors. Importantly, we show that the mechanisms of resistance employed by these genes are varied, and include restoring DNA replication fork progression, and prevention of ATR inhibitor-induced apoptosis. In particular, we describe a role for MED12-mediated inhibition of the TGFß signaling pathway in regulating replication fork stability and cellular survival upon ATR inhibition. Our dual genome-wide screen findings pave the way for personalized medicine by identifying potential biomarkers for ATR inhibitor resistance.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Biomarcadores de Tumor/genética , Resistencia a Antineoplásicos/genética , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Biomarcadores de Tumor/metabolismo , Sistemas CRISPR-Cas/genética , Replicación del ADN/efectos de los fármacos , Replicación del ADN/genética , Ensayos de Selección de Medicamentos Antitumorales , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Indoles , Complejo Mediador/genética , Complejo Mediador/metabolismo , Morfolinas , Neoplasias/genética , Neoplasias/patología , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sulfonamidas , Sulfóxidos/farmacología , Sulfóxidos/uso terapéutico , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The DNA damage response is essential to maintain genomic stability, suppress replication stress, and protect against carcinogenesis. The ATR-CHK1 pathway is an essential component of this response, which regulates cell cycle progression in the face of replication stress. PARP14 is an ADP-ribosyltransferase with multiple roles in transcription, signaling, and DNA repair. To understand the biological functions of PARP14, we catalogued the genetic components that impact cellular viability upon loss of PARP14 by performing an unbiased, comprehensive, genome-wide CRISPR knockout genetic screen in PARP14-deficient cells. We uncovered the ATR-CHK1 pathway as essential for viability of PARP14-deficient cells, and identified regulation of DNA replication dynamics as an important mechanistic contributor to the synthetic lethality observed. Our work shows that PARP14 is an important modulator of the response to ATR-CHK1 pathway inhibitors.
Asunto(s)
Replicación del ADN , Poli(ADP-Ribosa) Polimerasas/metabolismo , Mutaciones Letales Sintéticas , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Sistemas CRISPR-Cas , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Humanos , Poli(ADP-Ribosa) Polimerasas/genéticaRESUMEN
Upon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing 293T and RPE1 cells deficient in PCNA ubiquitination, generated through CRISPR/Cas9 gene editing, here, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-dependent but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork and incomplete Okazaki fragment maturation, which interferes with efficient PCNA unloading by ATAD5 and subsequent nucleosome deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and the BRCA pathway results in increased nascent DNA degradation and PARP inhibitor sensitivity. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors.
Asunto(s)
Antígeno Nuclear de Célula en Proliferación/metabolismo , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina/genética , Ensamble y Desensamble de Cromatina/fisiología , Ensayo Cometa , ADN/genética , Daño del ADN/genética , Daño del ADN/fisiología , Reparación del ADN/genética , Reparación del ADN/fisiología , Replicación del ADN/genética , Replicación del ADN/fisiología , Técnica del Anticuerpo Fluorescente , Inestabilidad Genómica/genética , Inestabilidad Genómica/fisiología , Células HEK293 , Células HeLa , Humanos , Antígeno Nuclear de Célula en Proliferación/genética , Unión Proteica , Ubiquitinación/genética , Ubiquitinación/fisiologíaRESUMEN
BRCA proteins are essential for homologous recombination (HR) DNA repair, and their germline or somatic inactivation is frequently observed in human tumors. Understanding the molecular mechanisms underlying the response of BRCA-deficient tumors to chemotherapy is paramount for developing improved personalized cancer therapies. While PARP inhibitors have been recently approved for treatment of BRCA-mutant breast and ovarian cancers, not all patients respond to this therapy, and resistance to these novel drugs remains a major clinical problem. Several mechanisms of chemoresistance in BRCA2-deficient cells have been identified. Rather than restoring normal recombination, these mechanisms result in stabilization of stalled replication forks, which can be subjected to degradation in BRCA2-mutated cells. Here, we show that the transcriptional repressor E2F7 modulates the chemosensitivity of BRCA2-deficient cells. We found that BRCA2-deficient cells are less sensitive to PARP inhibitor and cisplatin treatment after E2F7 depletion. Moreover, we show that the mechanism underlying this activity involves increased expression of RAD51, a target for E2F7-mediated transcriptional repression, which enhances both HR DNA repair, and replication fork stability in BRCA2-deficient cells. Our work describes a new mechanism of therapy resistance in BRCA2-deficient cells, and identifies E2F7 as a putative biomarker for tumor response to PARP inhibitor therapy.
