RESUMO
Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA-repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5'-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.
Assuntos
Caenorhabditis elegans/genética , Reparo do DNA , DNA de Cadeia Simples/genética , DNA/genética , Edição de Genes/métodos , Camundongos/genética , Peixe-Zebra/genética , Animais , Células HEK293 , Humanos , Células K562RESUMO
The EWS/ETS fusion transcription factors drive Ewing sarcoma (EWS) by orchestrating an oncogenic transcription program. Therapeutic targeting of EWS/ETS has been unsuccessful; however, identifying mediators of the EWS/ETS function could offer new therapeutic options. Here, we describe the dependency of EWS/ETS-driven transcription upon chromatin reader BET bromdomain proteins and investigate the potential of BET inhibitors in treating EWS. EWS/FLI1 and EWS/ERG were found in a transcriptional complex with BRD4, and knockdown of BRD2/3/4 significantly impaired the oncogenic phenotype of EWS cells. RNA-seq analysis following BRD4 knockdown or inhibition with JQ1 revealed an attenuated EWS/ETS transcriptional signature. In contrast to previous reports, JQ1 reduced proliferation and induced apoptosis through MYC-independent mechanisms without affecting EWS/ETS protein levels; this was confirmed by depleting BET proteins using PROTAC-BET degrader (BETd). Polycomb repressive complex 2 (PRC2)-associated factor PHF19 was downregulated by JQ1/BETd or BRD4 knockdown in multiple EWS lines. EWS/FLI1 bound a distal regulatory element of PHF19, and EWS/FLI1 knockdown resulted in downregulation of PHF19 expression. Deletion of PHF19 via CRISPR-Cas9 resulted in a decreased tumorigenic phenotype, a transcriptional signature that overlapped with JQ1 treatment, and increased sensitivity to JQ1. PHF19 expression was also associated with worse prognosis in patients with EWS. In vivo, JQ1 demonstrated antitumor efficacy in multiple mouse xenograft models of EWS. Together these results indicate that EWS/ETS requires BET epigenetic reader proteins for its transcriptional program and can be mitigated by BET inhibitors. This study provides a clear rationale for the clinical utility of BET inhibitors in treating EWS.Significance: These findings reveal the dependency of EWS/ETS transcription factors on BET epigenetic reader proteins and demonstrate the potential of BET inhibitors for the treatment of EWS. Cancer Res; 78(16); 4760-73. ©2018 AACR.
Assuntos
Proteínas Nucleares/genética , Proteína Proto-Oncogênica c-fli-1/genética , Sarcoma de Ewing/genética , Fatores de Transcrição/genética , Transcrição Gênica , Animais , Apoptose/genética , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Proteínas de Fusão Oncogênica/genética , Complexo Repressor Polycomb 2/genética , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We have synthesized and studied the biological and biophysical properties of triazole-linked ribo and xylo locked nucleic acid (LNA). The combination of LNA with the Isobe triazole linkage gave high binding affinity when incorporated at the 3' or 5' termini of oligonucleotides, but low binding affinity at internal positions. Antisense oligonucleotides (ASOs) and siRNAs containing triazole dimers were highly active and nuclease resistant. Surprisingly, the xyloLNA-modified siRNA was the most active of the series.
Assuntos
Oligonucleotídeos/química , RNA Interferente Pequeno/química , Triazóis/química , Sítios de Ligação , Conformação Molecular , Oligonucleotídeos/síntese química , Triazóis/síntese químicaRESUMO
UNLABELLED: Next-generation antiandrogen therapies, such as enzalutamide and abiraterone, have had a profound impact on the management of metastatic castration-resistant prostate cancer (mCRPC). However, mCRPC patients invariably develop resistance to these agents. Here, a series of clonal cell lines were developed from enzalutamide-resistant prostate tumor xenografts to study the molecular mechanism of resistance and test their oncogenic potential under various treatment conditions. Androgen receptor (AR) signaling was maintained in these cell lines, which acquired potential resistance mechanisms, including expression of AR-variant 7 (AR-v7) and glucocorticoid receptor. BET bromodomain inhibitors were shown previously to attenuate AR signaling in mCRPC; here, we demonstrate the efficacy of bromodomain and extraterminal (BET) inhibitors in enzalutamide-resistant prostate cancer models. AR antagonists, enzalutamide, and ARN509 exhibit enhanced prostate tumor growth inhibition when combined with BET inhibitors, JQ1 and OTX015, respectively. Taken together, these data provide a compelling preclinical rationale to combine BET inhibitors with AR antagonists to subvert resistance mechanisms. IMPLICATIONS: Therapeutic combinations of BET inhibitors and AR antagonists may enhance the clinical efficacy in the treatment of mCRPC. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/14/4/324/F1.large.jpg
Assuntos
Acetanilidas/administração & dosagem , Antagonistas de Receptores de Andrógenos/administração & dosagem , Azepinas/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Receptores Androgênicos/metabolismo , Triazóis/administração & dosagem , Acetanilidas/farmacologia , Antagonistas de Receptores de Andrógenos/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Azepinas/farmacologia , Benzamidas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Masculino , Camundongos , Nitrilas , Feniltioidantoína/administração & dosagem , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Transdução de Sinais/efeitos dos fármacos , Triazóis/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Resistance to androgen deprivation therapies and increased androgen receptor (AR) activity are major drivers of castration-resistant prostate cancer (CRPC). Although prior work has focused on targeting AR directly, co-activators of AR signaling, which may represent new therapeutic targets, are relatively underexplored. Here we demonstrate that the mixed-lineage leukemia protein (MLL) complex, a well-known driver of MLL fusion-positive leukemia, acts as a co-activator of AR signaling. AR directly interacts with the MLL complex via the menin-MLL subunit. Menin expression is higher in CRPC than in both hormone-naive prostate cancer and benign prostate tissue, and high menin expression correlates with poor overall survival of individuals diagnosed with prostate cancer. Treatment with a small-molecule inhibitor of menin-MLL interaction blocks AR signaling and inhibits the growth of castration-resistant tumors in vivo in mice. Taken together, this work identifies the MLL complex as a crucial co-activator of AR and a potential therapeutic target in advanced prostate cancer.
