RESUMEN
RAD51C is an enigmatic predisposition gene for breast, ovarian, and prostate cancer. Currently, missing structural and related functional understanding limits patient mutation interpretation to homology-directed repair (HDR) function analysis. Here we report the RAD51C-XRCC3 (CX3) X-ray co-crystal structure with bound ATP analog and define separable RAD51C replication stability roles informed by its three-dimensional structure, assembly, and unappreciated polymerization motif. Mapping of cancer patient mutations as a functional guide confirms ATP-binding matching RAD51 recombinase, yet highlights distinct CX3 interfaces. Analyses of CRISPR/Cas9-edited human cells with RAD51C mutations combined with single-molecule, single-cell and biophysics measurements uncover discrete CX3 regions for DNA replication fork protection, restart and reversal, accomplished by separable functions in DNA binding and implied 5' RAD51 filament capping. Collective findings establish CX3 as a cancer-relevant replication stress response complex, show how HDR-proficient variants could contribute to tumor development, and identify regions to aid functional testing and classification of cancer mutations.
Asunto(s)
Neoplasias de la Próstata , Masculino , Humanos , Recombinasa Rad51 , Mutación , Replicación del ADN , Adenosina Trifosfato , Proteínas de Unión al ADNRESUMEN
The prototypic cancer-predisposition disease Fanconi Anemia (FA) is identified by biallelic mutations in any one of twenty-three FANC genes. Puzzlingly, inactivation of one Fanc gene alone in mice fails to faithfully model the pleiotropic human disease without additional external stress. Here we find that FA patients frequently display FANC co-mutations. Combining exemplary homozygous hypomorphic Brca2/Fancd1 and Rad51c/Fanco mutations in mice phenocopies human FA with bone marrow failure, rapid death by cancer, cellular cancer-drug hypersensitivity and severe replication instability. These grave phenotypes contrast the unremarkable phenotypes seen in mice with single gene-function inactivation, revealing an unexpected synergism between Fanc mutations. Beyond FA, breast cancer-genome analysis confirms that polygenic FANC tumor-mutations correlate with lower survival, expanding our understanding of FANC genes beyond an epistatic FA-pathway. Collectively, the data establish a polygenic replication stress concept as a testable principle, whereby co-occurrence of a distinct second gene mutation amplifies and drives endogenous replication stress, genome instability and disease.
Asunto(s)
Neoplasias de la Mama , Anemia de Fanconi , Animales , Femenino , Humanos , Ratones , Proteína BRCA2/genética , Neoplasias de la Mama/genética , Proteínas de Unión al ADN/genética , Anemia de Fanconi/genética , Genotipo , Mutación , FenotipoRESUMEN
PURPOSE: Patients with central nervous system (CNS) tumors are typically treated with radiotherapy, but this is not curative and results in the upregulation of phosphorylated STAT3 (p-STAT3), which drives invasion, angiogenesis, and immune suppression. Therefore, we investigated the combined effect of an inhibitor of STAT3 and whole-brain radiotherapy (WBRT) in a murine model of glioma. EXPERIMENTAL DESIGN: C57BL/6 mice underwent intracerebral implantation of GL261 glioma cells, WBRT, and treatment with WP1066, a blood-brain barrier-penetrant inhibitor of the STAT3 pathway, or the two in combination. The role of the immune system was evaluated using tumor rechallenge strategies, immune-incompetent backgrounds, immunofluorescence, immune phenotyping of tumor-infiltrating immune cells (via flow cytometry), and NanoString gene expression analysis of 770 immune-related genes from immune cells, including those directly isolated from the tumor microenvironment. RESULTS: The combination of WP1066 and WBRT resulted in long-term survivors and enhanced median survival time relative to monotherapy in the GL261 glioma model (combination vs. control P < 0.0001). Immunologic memory appeared to be induced, because mice were protected during subsequent tumor rechallenge. The therapeutic effect of the combination was completely lost in immune-incompetent animals. NanoString analysis and immunofluorescence revealed immunologic reprograming in the CNS tumor microenvironment specifically affecting dendritic cell antigen presentation and T-cell effector functions. CONCLUSIONS: This study indicates that the combination of STAT3 inhibition and WBRT enhances the therapeutic effect against gliomas in the CNS by inducing dendritic cell and T-cell interactions in the CNS tumor.
Asunto(s)
Neoplasias Encefálicas/terapia , Comunicación Celular/inmunología , Quimioradioterapia/métodos , Glioma/terapia , Factor de Transcripción STAT3/antagonistas & inhibidores , Animales , Presentación de Antígeno/efectos de los fármacos , Presentación de Antígeno/efectos de la radiación , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/patología , Comunicación Celular/efectos de los fármacos , Comunicación Celular/efectos de la radiación , Línea Celular Tumoral/ultraestructura , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/efectos de la radiación , Modelos Animales de Enfermedad , Glioma/inmunología , Glioma/patología , Humanos , Memoria Inmunológica/efectos de los fármacos , Ratones , Piridinas/administración & dosificación , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/efectos de la radiación , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Microambiente Tumoral/efectos de la radiación , Tirfostinos/administración & dosificaciónRESUMEN
In order to prioritize available immune therapeutics, immune profiling across glioma grades was conducted, followed by preclinical determinations of therapeutic effect in immune-competent mice harboring gliomas. T cells and myeloid cells were isolated from the blood of healthy donors and the blood and tumors from patients with glioma and profiled for the expression of immunomodulatory targets with an available therapeutic. Murine glioma models were used to assess therapeutic efficacy of agents targeting the most frequently expressed immune targets. In patients with glioma, the A2aR/CD73/CD39 pathway was most frequently expressed, followed by the PD-1 pathway. CD73 expression was upregulated on immune cells by 2-hydroxyglutarate in IDH1 mutant glioma patients. In murine glioma models, adenosine receptor inhibitors demonstrated a modest therapeutic response; however, the addition of other inhibitors of the adenosine pathway did not further enhance this therapeutic effect. Although adenosine receptor inhibitors could recover immunological effector functions in T cells, immune recovery was impaired in the presence of gliomas, indicating that irreversible immune exhaustion limits the effectiveness of adenosine pathway inhibitors in patients with glioma. This study illustrates vetting steps that should be considered before clinical trial implementation for immunotherapy-resistant cancers, including testing an agent's ability to restore immunological function in the context of intended use.
Asunto(s)
Neoplasias Encefálicas/inmunología , Glioma/inmunología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunosupresores/uso terapéutico , 5'-Nucleotidasa/metabolismo , Adulto , Anciano , Animales , Antígenos CD/metabolismo , Apirasa/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Células Cultivadas , Femenino , Glioma/tratamiento farmacológico , Glioma/genética , Glioma/patología , Humanos , Isocitrato Deshidrogenasa/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Clasificación del Tumor , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Adenosina A2A/metabolismoRESUMEN
Classically, p53 tumor suppressor acts in transcription, apoptosis, and cell cycle arrest. Yet, replication-mediated genomic instability is integral to oncogenesis, and p53 mutations promote tumor progression and drug-resistance. By delineating human and murine separation-of-function p53 alleles, we find that p53 null and gain-of-function (GOF) mutations exhibit defects in restart of stalled or damaged DNA replication forks that drive genomic instability, which isgenetically separable from transcription activation. By assaying protein-DNA fork interactions in single cells, we unveil a p53-MLL3-enabled recruitment of MRE11 DNA replication restart nuclease. Importantly, p53 defects or depletion unexpectedly allow mutagenic RAD52 and POLθ pathways to hijack stalled forks, which we find reflected in p53 defective breast-cancer patient COSMIC mutational signatures. These data uncover p53 as a keystone regulator of replication homeostasis within a DNA restart network. Mechanistically, this has important implications for development of resistance in cancer therapy. Combined, these results define an unexpected role for p53-mediated suppression of replication genome instability.
Asunto(s)
Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Resistencia a Antineoplásicos , Inestabilidad Genómica , Homeostasis , Humanos , Ratones , Mutación , Proteína p53 Supresora de Tumor/genética , ADN Polimerasa thetaRESUMEN
The DNA repair protein O 6-methylguanine-DNA-methyltransferase (MGMT) is a key determinant of cancer resistance. The MGMT inhibitors O 6-benzylguanine (O6BG) and O 6-(4-bromothenyl)guanine (O6BTG) failed to enhance the therapeutic response due to toxic side effects when applied in combination with alkylating chemotherapeutics, indicating a need of inhibitor targeting. We assessed MGMT targeting that relies on conjugating the inhibitors O6BG and O6BTG to ß-D-glucose, resulting in O6BG-Glu and O6BTG-Glu, respectively. This targeting strategy was selected by taking advantage of high demand of glucose in cancers. Contrary to our expectation, the uptake of O6BG-Glu and O6BTG-Glu was not dependent on glucose transporters. Instead, it seems that after membrane binding the conjugates are taken up via flippases, which normally transport phospholipids. This membrane binding is the consequence of the amphiphilic character of the conjugates, which at higher concentrations lead to the formation of micelle-like particles in aqueous solution. The unusual uptake mechanism of the conjugates highlights the importance of proper linker selection for a successful ligand-based drug delivery strategy. We also demonstrate that proteins of the P4-Type ATPase family are involved in the transport of the glucose conjugates. The findings are not only important for MGMT inhibitor targeting, but also for other amphiphilic drugs.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Metilasas de Modificación del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Glucosa/química , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Transporte Biológico , Línea Celular Tumoral , Membrana Celular/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , HumanosRESUMEN
DNA-damaging anticancer drugs remain a part of metastatic melanoma therapy. Epigenetic reprogramming caused by increased histone deacetylase (HDAC) activity arising during tumor formation may contribute to resistance of melanomas to the alkylating drugs temozolomide, dacarbazine, and fotemustine. Here, we report on the impact of class I HDACs on the response of malignant melanoma cells treated with alkylating agents. The data show that malignant melanomas in situ contain a high level of HDAC1/2 and malignant melanoma cells overexpress HDAC1/2/3 compared with noncancer cells. Furthermore, pharmacologic inhibition of class I HDACs sensitizes malignant melanoma cells to apoptosis following exposure to alkylating agents, while not affecting primary melanocytes. Inhibition of HDAC1/2/3 caused sensitization of melanoma cells to temozolomide in vitro and in melanoma xenografts in vivo HDAC1/2/3 inhibition resulted in suppression of DNA double-strand break (DSB) repair by homologous recombination because of downregulation of RAD51 and FANCD2. This sensitized cells to the cytotoxic DNA lesion O(6)-methylguanine and caused a synthetic lethal interaction with the PARP-1 inhibitor olaparib. Furthermore, knockdown experiments identified HDAC2 as being responsible for the regulation of RAD51. The influence of class I HDACs on DSB repair by homologous recombination and the possible clinical implication on malignant melanoma therapy with temozolomide and other alkylating drugs suggests a combination approach where class I HDAC inhibitors such as valproic acid or MS-275 (entinostat) appear to counteract HDAC- and RAD51/FANCD2-mediated melanoma cell resistance. Cancer Res; 76(10); 3067-77. ©2016 AACR.
Asunto(s)
Resistencia a Antineoplásicos , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Melanoma/patología , Recombinasa Rad51/metabolismo , Neoplasias Cutáneas/patología , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Proliferación Celular/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/antagonistas & inhibidores , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Técnica del Anticuerpo Fluorescente , Histona Desacetilasas/química , Histona Desacetilasas/genética , Recombinación Homóloga/efectos de los fármacos , Humanos , Técnicas para Inmunoenzimas , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , ARN Mensajero/genética , Recombinasa Rad51/antagonistas & inhibidores , Recombinasa Rad51/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/efectos de los fármacos , Piel/metabolismo , Piel/patología , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Melanoma Cutáneo MalignoRESUMEN
Various mechanisms of drug resistance attenuate the effectiveness of cancer therapeutics, including drug transport and DNA repair. The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is a key factor determining the resistance against alkylating anticancer drugs inducing the genotoxic DNA lesions O(6)-methylguanine and O(6)-chloroethylguanine, and MGMT inactivation or depletion renders cells more susceptible to treatment with methylating and chloroethylating agents. Highly specific and efficient inhibitors of the repair protein MGMT were designed, including O(6)-benzylguanine (O(6)BG) and O(6)-(4-bromothenyl)guanine (O(6)BTG) that are nontoxic on their own. Unfortunately, these inhibitors do not select between MGMT in normal and cancer cells, causing nontarget effects in the healthy tissue. Therefore, a targeting strategy for MGMT inhibitors is required. Here, we used O(6)BG and O(6)BTG conjugated to ß-d-glucose (O(6)BG-Glu and O(6)BTG-Glu, respectively) in order to selectively inhibit MGMT in tumors, harnessing their high demand for glucose. Both glucose conjugates efficiently inhibited MGMT in several cancer cell lines, but with different extents of sensitization to DNA alkylating agents, with lomustine being more effective than temozolomide. We further show that the glucose conjugates are subject to ATP-binding cassette (ABC) transporter mediated efflux, involving P-glycoprotein, MRP1, and BCRP, which impacts the efficiency of MGMT inhibition. Surprisingly, also O(6)BG and O(6)BTG were subject to an active transport out of the cell. We also show that pharmacological inhibition of efflux transporters increases the induction of cell death following treatment with these MGMT inhibitors and temozolomide. We conclude that strategies of attenuating the efflux by ABC transporters are required for achieving successful MGMT targeting.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Metilasas de Modificación del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Dacarbazina/análogos & derivados , Resistencia a Antineoplásicos , Inhibidores Enzimáticos/farmacología , Neoplasias/tratamiento farmacológico , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Antineoplásicos Alquilantes/farmacología , Apoptosis , Western Blotting , Proliferación Celular , Dacarbazina/farmacología , Técnica del Anticuerpo Fluorescente , Glucosa/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Temozolomida , Células Tumorales CultivadasRESUMEN
In both pro- and eukaryotes, the mutagenic and toxic DNA adduct O(6)-methylguanine (O(6)MeG) is subject to repair by alkyltransferase proteins via methyl group transfer. In addition, in prokaryotes, there are proteins with sequence homology to alkyltransferases, collectively designated as alkyltransferase-like (ATL) proteins, which bind to O(6)-alkylguanine adducts and mediate resistance to alkylating agents. Whether such proteins might enable similar protection in higher eukaryotes is unknown. Here we expressed the ATL protein of Escherichia coli (eATL) in mammalian cells and addressed the question whether it is able to protect them against the cytotoxic effects of alkylating agents. The Chinese hamster cell line CHO-9, the nucleotide excision repair (NER) deficient derivative 43-3B and the DNA mismatch repair (MMR) impaired derivative Tk22-C1 were transfected with eATL cloned in an expression plasmid and the sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was determined in reproductive survival, DNA double-strand break (DSB) and apoptosis assays. The results indicate that eATL expression is tolerated in mammalian cells and conferes protection against killing by MNNG in both wild-type and 43-3B cells, but not in the MMR-impaired cell line. The protection effect was dependent on the expression level of eATL and was completely ablated in cells co-expressing the human O(6)-methylguanine-DNA methyltransferase (MGMT). eATL did not protect against cytotoxicity induced by the chloroethylating agent lomustine, suggesting that O(6)-chloroethylguanine adducts are not target of eATL. To investigate the mechanism of protection, we determined O(6)MeG levels in DNA after MNNG treatment and found that eATL did not cause removal of the adduct. However, eATL expression resulted in a significantly lower level of DSBs in MNNG-treated cells, and this was concomitant with attenuation of G2 blockage and a lower level of apoptosis. The results suggest that eATL confers protection against methylating agents by masking O(6)MeG/thymine mispaired adducts, preventing them from becoming a substrate for mismatch repair-mediated DSB formation and cell death.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Alquilantes/toxicidad , Aductos de ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Guanina/análogos & derivados , Transferasas Alquil y Aril/genética , Animales , Apoptosis , Células CHO , Cricetinae , Cricetulus , ADN/efectos de los fármacos , ADN/metabolismo , Roturas del ADN de Doble Cadena , Metilasas de Modificación del ADN/metabolismo , Reparación del ADN , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Escherichia coli/genética , Guanina/metabolismo , Humanos , Metilnitronitrosoguanidina/toxicidad , Transgenes , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Apoptosis, autophagy, necrosis and cellular senescence are key responses of cells that were exposed to genotoxicants. The types of DNA damage triggering these responses and their interrelationship are largely unknown. Here we studied these responses in glioma cells treated with the methylating agent temozolomide (TMZ), which is a first-line chemotherapeutic for this malignancy. We show that upon TMZ treatment cells undergo autophagy, senescence and apoptosis in a specific time-dependent manner. Necrosis was only marginally induced. All these effects were completely abrogated in isogenic glioma cells expressing O(6)-methylguanine-DNA methyltransferase (MGMT), indicating that a single type of DNA lesion, O(6)-methylguanine (O(6)MeG), is able to trigger all these responses. Studies with mismatch repair mutants and MSH6, Rad51 and ATM knockdowns revealed that autophagy induced by O(6)MeG requires mismatch repair and ATM, and is counteracted by homologous recombination. We further show that autophagy, which precedes apoptosis, is a survival mechanism as its inhibition greatly ameliorated the level of apoptosis following TMZ at therapeutically relevant doses (<100 µM). Cellular senescence increases with post-exposure time and, similar to autophagy, precedes apoptosis. If autophagy was abrogated, TMZ-induced senescence was reduced. Therefore, we propose that autophagy triggered by O(6)MeG adducts is a survival mechanism that stimulates cells to undergo senescence rather than apoptosis. Overall, the data revealed that a specific DNA adduct, O(6)MeG, has the capability of triggering autophagy, senescence and apoptosis and that the decision between survival and death is determined by the balance of players involved. The data also suggests that inhibition of autophagy may ameliorate the therapeutic outcome of TMZ-based cancer therapy.
Asunto(s)
Antineoplásicos Alquilantes/toxicidad , Daño del ADN/efectos de los fármacos , Dacarbazina/análogos & derivados , Glioma/genética , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas de la Ataxia Telangiectasia Mutada , Autofagia/efectos de los fármacos , Autofagia/genética , Proteínas de Ciclo Celular/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Senescencia Celular/genética , Reparación de la Incompatibilidad de ADN , Metilasas de Modificación del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Dacarbazina/toxicidad , Glioma/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Recombinación Homóloga , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Temozolomida , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Recent evidence has challenged the default assumption that all DNA-reactive alkylating agents exhibit a linear dose-response. Emerging evidence suggests that the model alkylating agents methyl- and ethylmethanesulfonate and methylnitrosourea (MNU) and ethylnitrosourea observe a nonlinear dose-response with a no observed genotoxic effect level (NOGEL). Follow-up mechanistic studies are essential to understand the mechanism of cellular tolerance and biological relevance of such NOGELs. MNU is one of the most mutagenic simple alkylators. Therefore, understanding the mechanism of mutation induction, following low-dose MNU treatment, sets precedence for weaker mutagenic alkylating agents. Here, we tested MNU at 10-fold lower concentrations than a previous study and report a NOGEL of 0.0075 µg/ml (72.8nM) in human lymphoblastoid cells, quantified through the hypoxanthine (guanine) phosphoribosyltransferase assay (OECD 476). Mechanistic studies reveal that the NOGEL is dependent upon repair of O(6)-methylguanine (O(6)MeG) by the suicide enzyme O(6)MeG-DNA methyltransferase (MGMT). Inactivation of MGMT sensitizes cells to MNU-induced mutagenesis and shifts the NOGEL to the left on the dose axis.