RESUMEN
Cancer treatments targeting DNA repair deficiencies often encounter drug resistance, possibly due to alternative metabolic pathways that counteract the most damaging effects. To identify such alternative pathways, we screened for metabolic pathways exhibiting synthetic lethality with inhibition of the DNA damage response kinase Ataxia-telangiectasia-mutated (ATM) using a metabolism-centered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 library. Our data revealed Kelch-like ECH-associated protein 1 (KEAP1) as a key factor involved in desensitizing cancer cells to ATM inhibition both in vitro and in vivo. Cells depleted of KEAP1 exhibited an aberrant overexpression of the cystine transporter SLC7A11, robustly accumulated cystine inducing disulfide stress, and became hypersensitive to ATM inhibition. These hallmarks were reversed in a reducing cellular environment indicating that disulfide stress was a crucial factor. In The Cancer Genome Atlas (TCGA) pan-cancer datasets, we found that ATM levels negatively correlated with KEAP1 levels across multiple solid malignancies. Together, our results unveil ATM and KEAP1 as new targetable vulnerabilities in solid tumors.
Asunto(s)
Ataxia Telangiectasia , Neoplasias Pulmonares , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Cistina/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Neoplasias Pulmonares/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismoRESUMEN
BACKGROUND: AZD0156 is an oral inhibitor of ATM, a serine threonine kinase that plays a key role in DNA damage response (DDR) associated with double-strand breaks. Topoisomerase-I inhibitor irinotecan is used clinically to treat colorectal cancer (CRC), often in combination with 5-fluorouracil (5FU). AZD0156 in combination with irinotecan and 5FU was evaluated in preclinical models of CRC to determine whether low doses of AZD0156 enhance the cytotoxicity of irinotecan in chemotherapy regimens used in the clinic. METHODS: Anti-proliferative effects of single-agent AZD0156, the active metabolite of irinotecan (SN38), and combination therapy were evaluated in 12 CRC cell lines. Additional assessment with clonogenic assay, cell cycle analysis, and immunoblotting were performed in 4 selected cell lines. Four colorectal cancer patient derived xenograft (PDX) models were treated with AZD0156, irinotecan, or 5FU alone and in combination for assessment of tumor growth inhibition (TGI). Immunofluorescence was performed on tumor tissues. The DDR mutation profile was compared across in vitro and in vivo models. RESULTS: Enhanced effects on cellular proliferation and regrowth were observed with the combination of AZD0156 and SN38 in select models. In cell cycle analysis of these models, increased G2/M arrest was observed with combination treatment over either single agent. Immunoblotting results suggest an increase in DDR associated with irinotecan therapy, with a reduced effect noted when combined with AZD0156, which is more pronounced in some models. Increased TGI was observed with the combination of AZD0156 and irinotecan as compared to single-agent therapy in some PDX models. The DDR mutation profile was variable across models. CONCLUSIONS: AZD0156 and irinotecan provide a rational and active combination in preclinical colorectal cancer models. Variability across in vivo and in vitro results may be related to the variable DDR mutation profiles of the models evaluated. Further understanding of the implications of individual DDR mutation profiles may help better identify patients more likely to benefit from treatment with the combination of AZD0156 and irinotecan in the clinical setting.
Asunto(s)
Neoplasias Colorrectales , Fluorouracilo , Humanos , Irinotecán/uso terapéutico , Fluorouracilo/farmacología , Fluorouracilo/uso terapéutico , Apoptosis , Línea Celular Tumoral , Puntos de Control de la Fase G2 del Ciclo Celular , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Camptotecina , Proteínas de la Ataxia Telangiectasia Mutada/genéticaRESUMEN
BACKGROUND: Personalised medicine strategies may improve outcomes in pancreatic ductal adenocarcinoma (PDAC), but validation of predictive biomarkers is required. Having developed a clinical trial to assess the ATR inhibitor, AZD6738, in combination with gemcitabine (ATRi/gem), we investigated ATM loss as a predictive biomarker of response to ATRi/gem in PDAC. METHODS: Through kinase inhibition, siRNA depletion and CRISPR knockout of ATM, we assessed how ATM targeting affected the sensitivity of PDAC cells to ATRi/gem. Using flow cytometry, immunofluorescence and immunoblotting, we investigated how ATRi/gem synergise in ATM-proficient and ATM-deficient cells, before assessing the impact of ATM loss on ATRi/gem sensitivity in vivo. RESULTS: Complete loss of ATM function (through pharmacological inhibition or CRISPR knockout), but not siRNA depletion, sensitised to ATRi/gem. In ATM-deficient cells, ATRi/gem-induced replication catastrophe was augmented, while phospho-Chk2-T68 and phospho-KAP1-S824 persisted via DNA-PK activity. ATRi/gem caused growth delay in ATM-WT xenografts in NSG mice and induced regression in ATM-KO xenografts. CONCLUSIONS: ATM loss augments replication catastrophe-mediated cell death induced by ATRi/gem and may predict clinical responsiveness to this combination. ATM status should be carefully assessed in tumours from patients with PDAC, since distinction between ATM-low and ATM-null could be critical in maximising the success of clinical trials using ATM expression as a predictive biomarker.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Carcinoma Ductal Pancreático/tratamiento farmacológico , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamiento farmacológico , Piridinas/farmacología , Pirimidinas/farmacología , Quinolinas/farmacología , Sulfóxidos/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/fisiología , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Sinergismo Farmacológico , Femenino , Técnicas de Inactivación de Genes , Humanos , Indoles , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Morfolinas , Neoplasias Pancreáticas/patología , Piridinas/administración & dosificación , Pirimidinas/administración & dosificación , Quinolinas/administración & dosificación , ARN Interferente Pequeño/farmacología , Sulfonamidas , Sulfóxidos/administración & dosificación , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
The structure-specific nuclease human flap endonuclease-1 (hFEN1) plays a key role in DNA replication and repair and may be of interest as an oncology target. We present the crystal structure of inhibitor-bound hFEN1, which shows a cyclic N-hydroxyurea bound in the active site coordinated to two magnesium ions. Three such compounds had similar IC50 values but differed subtly in mode of action. One had comparable affinity for protein and protein-substrate complex and prevented reaction by binding to active site catalytic metal ions, blocking the necessary unpairing of substrate DNA. Other compounds were more competitive with substrate. Cellular thermal shift data showed that both inhibitor types engaged with hFEN1 in cells, and activation of the DNA damage response was evident upon treatment with inhibitors. However, cellular EC50 values were significantly higher than in vitro inhibition constants, and the implications of this for exploitation of hFEN1 as a drug target are discussed.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Endonucleasas de ADN Solapado/antagonistas & inhibidores , Endonucleasas de ADN Solapado/metabolismo , Dominio Catalítico/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Endonucleasas de ADN Solapado/química , Humanos , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , TemperaturaRESUMEN
The inhibition of ataxia-telangiectasia mutated (ATM) has been shown to chemo- and radio-sensitize human glioma cells in vitro and therefore might provide an exciting new paradigm in the treatment of glioblastoma multiforme (GBM). The effective treatment of GBM will likely require a compound with the potential to efficiently cross the blood-brain barrier (BBB). Starting from clinical candidate AZD0156, 4, we investigated the imidazoquinolin-2-one scaffold with the goal of improving likely CNS exposure in humans. Strategies aimed at reducing hydrogen bonding, basicity, and flexibility of the molecule were explored alongside modulating lipophilicity. These studies identified compound 24 (AZD1390) as an exceptionally potent and selective inhibitor of ATM with a good preclinical pharmacokinetic profile. 24 showed an absence of human transporter efflux in MDCKII-MDR1-BCRP studies (efflux ratio <2), significant BBB penetrance in nonhuman primate PET studies (Kp,uu 0.33) and was deemed suitable for development as a clinical candidate to explore the radiosensitizing effects of ATM in intracranial malignancies.
Asunto(s)
Ataxia Telangiectasia , Glioblastoma , Piridinas , Quinolonas , Animales , Humanos , Barrera Hematoencefálica/metabolismo , Ataxia Telangiectasia/tratamiento farmacológico , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Neoplasias , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Glioblastoma/tratamiento farmacológicoRESUMEN
Histone H2AX plays a key role in DNA damage signalling in the surrounding regions of DNA double-strand breaks (DSBs). In response to DNA damage, H2AX becomes phosphorylated on serine residue 139 (known as γH2AX), resulting in the recruitment of the DNA repair effectors 53BP1 and BRCA1. Here, by studying resistance to poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1/2-deficient mammary tumours, we identify a function for γH2AX in orchestrating drug-induced replication fork degradation. Mechanistically, γH2AX-driven replication fork degradation is elicited by suppressing CtIP-mediated fork protection. As a result, H2AX loss restores replication fork stability and increases chemoresistance in BRCA1/2-deficient tumour cells without restoring homology-directed DNA repair, as highlighted by the lack of DNA damage-induced RAD51 foci. Furthermore, in the attempt to discover acquired genetic vulnerabilities, we find that ATM but not ATR inhibition overcomes PARP inhibitor (PARPi) resistance in H2AX-deficient tumours by interfering with CtIP-mediated fork protection. In summary, our results demonstrate a role for H2AX in replication fork biology in BRCA-deficient tumours and establish a function of H2AX separable from its classical role in DNA damage signalling and DSB repair.
Asunto(s)
Proteína BRCA1 , Proteína BRCA2 , Replicación del ADN , Resistencia a Antineoplásicos , Histonas , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Animales , Femenino , Humanos , Ratones , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteína BRCA1/metabolismo , Proteína BRCA1/deficiencia , Proteína BRCA1/genética , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/deficiencia , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/tratamiento farmacológico , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Daño del ADN , Reparación del ADN , Replicación del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Histonas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Recombinasa Rad51/metabolismo , Recombinasa Rad51/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Ratones DesnudosRESUMEN
PURPOSE: Limited effective treatments are currently available for central nervous system (CNS) metastasis (CM). This is largely driven by the inability of current therapeutics to penetrate the blood brain barrier (BBB) and the lack of preclinical models for testing new therapies. Here we study the efficacy of AZD1390, a BBB penetrating ataxia-telangiectasia mutated inhibitor, as a radiosensitizer for breast cancer CM treatment. EXPERIMENTAL DESIGN: Three patient-derived xenograft (PDX) tumors including 2 HER2+ and 1 triple-negative breast cancer harboring DNA damage response (DDR) gene mutations, were implanted subcutaneously in the flank of mice to assess tumor growth inhibition by AZD1390 combined with radiation. Animal survival was further assessed by implanting the best responding PDX model orthotopically in the brain. RESULTS: Pretreatment with AZD1390 followed by radiation therapy inhibited growth of PDX tumors implanted in the flank, and improved survival in orthotopic models with average survival of 222 days compared with 123 days in controls. Administration of AZD1390 posttreatment for 21 days had no further benefits. While the combination therapy resulted in sustained tumor inhibition, sporadic regrowth was observed in some mice 50 to 100 days posttreatment in all models. Gene expression comparing these tumors with complete responders demonstrated changes in upregulation of oncogenic proteins, which are potential drivers of tumor growth after treatment. CONCLUSIONS: Our results demonstrate that AZD1390 effectively sensitizes breast cancer CM to radiation therapy in DDR mutant tumors. This study demonstrates the potential of using AZD1390 as a novel therapeutic agent for patients with breast cancer CM.
Asunto(s)
Neoplasias del Sistema Nervioso Central , Neoplasias Primarias Secundarias , Fármacos Sensibilizantes a Radiaciones , Neoplasias de la Mama Triple Negativas , Humanos , Ratones , Animales , Fármacos Sensibilizantes a Radiaciones/farmacología , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Neoplasias del Sistema Nervioso Central/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Modelos Animales de Enfermedad , Neoplasias Primarias Secundarias/tratamiento farmacológico , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Melanoma Cutáneo MalignoRESUMEN
Metnase is a human protein with methylase (SET) and nuclease domains that is widely expressed, especially in proliferating tissues. Metnase promotes non-homologous end-joining (NHEJ), and knockdown causes mild hypersensitivity to ionizing radiation. Metnase also promotes plasmid and viral DNA integration, and topoisomerase IIα (TopoIIα)-dependent chromosome decatenation. NHEJ factors have been implicated in the replication stress response, and TopoIIα has been proposed to relax positive supercoils in front of replication forks. Here we show that Metnase promotes cell proliferation, but it does not alter cell cycle distributions, or replication fork progression. However, Metnase knockdown sensitizes cells to replication stress and confers a marked defect in restart of stalled replication forks. Metnase promotes resolution of phosphorylated histone H2AX, a marker of DNA double-strand breaks at collapsed forks, and it co-immunoprecipitates with PCNA and RAD9, a member of the PCNA-like RAD9-HUS1-RAD1 intra-S checkpoint complex. Metnase also promotes TopoIIα-mediated relaxation of positively supercoiled DNA. Metnase is not required for RAD51 focus formation after replication stress, but Metnase knockdown cells show increased RAD51 foci in the presence or absence of replication stress. These results establish Metnase as a key factor that promotes restart of stalled replication forks, and implicate Metnase in the repair of collapsed forks.
Asunto(s)
Reparación del ADN , Replicación del ADN , N-Metiltransferasa de Histona-Lisina/fisiología , Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular/aislamiento & purificación , Proliferación Celular , Supervivencia Celular , ADN-Topoisomerasas de Tipo II/metabolismo , ADN Superhelicoidal/metabolismo , Proteínas de Unión al ADN/metabolismo , N-Metiltransferasa de Histona-Lisina/aislamiento & purificación , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Humanos , Inmunoprecipitación , Antígeno Nuclear de Célula en Proliferación/aislamiento & purificación , Recombinasa Rad51/análisisRESUMEN
Myofibroblastic cancer-associated fibroblast (myoCAF)-rich tumors generally contain few T cells and respond poorly to immune-checkpoint blockade. Although myoCAFs are associated with poor outcome in most solid tumors, the molecular mechanisms regulating myoCAF accumulation remain unclear, limiting the potential for therapeutic intervention. Here, we identify ataxia-telangiectasia mutated (ATM) as a central regulator of the myoCAF phenotype. Differentiating myofibroblasts in vitro and myoCAFs cultured ex vivo display activated ATM signaling, and targeting ATM genetically or pharmacologically could suppress and reverse differentiation. ATM activation was regulated by the reactive oxygen species-producing enzyme NOX4, both through DNA damage and increased oxidative stress. Targeting fibroblast ATM in vivo suppressed myoCAF-rich tumor growth, promoted intratumoral CD8 T-cell infiltration, and potentiated the response to anti-PD-1 blockade and antitumor vaccination. This work identifies a novel pathway regulating myoCAF differentiation and provides a rationale for using ATM inhibitors to overcome CAF-mediated immunotherapy resistance. SIGNIFICANCE: ATM signaling supports the differentiation of myoCAFs to suppress T-cell infiltration and antitumor immunity, supporting the potential clinical use of ATM inhibitors in combination with checkpoint inhibition in myoCAF-rich, immune-cold tumors.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada , Fibroblastos Asociados al Cáncer , Inmunoterapia , Neoplasias , Humanos , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Diferenciación Celular , Miofibroblastos/metabolismo , Resistencia a AntineoplásicosRESUMEN
Cancers overcome replicative immortality by activating either telomerase or an alternative lengthening of telomeres (ALT) mechanism. ALT occurs in ~25% of high-risk neuroblastomas, and progression in patients with ALT neuroblastoma during or after front-line therapy is frequent and often fatal. Temozolomide + irinotecan is commonly used as salvage therapy for neuroblastoma. Patient-derived cell lines and xenografts established from patients with relapsed ALT neuroblastoma demonstrated de novo resistance to temozolomide + irinotecan [SN-38 in vitro, P < 0.05; in vivo mouse event-free survival (EFS), P < 0.0001] vs. telomerase-positive neuroblastomas. We observed that ALT neuroblastoma cells manifested constitutive ataxia-telangiectasia mutated (ATM) activation due to spontaneous telomere dysfunction which was not observed in telomerase-positive neuroblastoma cells. We demonstrated that induction of telomere dysfunction resulted in ATM activation that, in turn, conferred resistance to temozolomide + SN-38 (4.2-fold change in IC50, P < 0.001). ATM knockdown (shRNA) or inhibition using a clinical-stage small-molecule inhibitor (AZD0156) reversed resistance to temozolomide + irinotecan in ALT neuroblastoma cell lines in vitro (P < 0.001) and in four ALT xenografts in vivo (EFS, P < 0.0001). AZD0156 showed modest to no enhancement of temozolomide + irinotecan activity in telomerase-positive neuroblastoma cell lines and xenografts. Ataxia telangiectasia and Rad3 related (ATR) inhibition using AZD6738 did not enhance temozolomide + SN-38 activity in ALT neuroblastoma cells. Thus, ALT neuroblastoma chemotherapy resistance occurs via ATM activation and is reversible with ATM inhibitor AZD0156. Combining AZD0156 with temozolomide + irinotecan warrants clinical testing for neuroblastoma.
Asunto(s)
Ataxia Telangiectasia , Neuroblastoma , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Resistencia a Antineoplásicos , Humanos , Ratones , Recurrencia Local de Neoplasia , Neuroblastoma/tratamiento farmacológico , Piridinas , Quinolinas , Telómero , Homeostasis del TelómeroRESUMEN
Ionizing radiation induces delayed genomic instability in human cells, including chromosomal abnormalities and hyperrecombination. Here, we investigate delayed genome instability of cells exposed to UV radiation. We examined homologous recombination-mediated reactivation of a green fluorescent protein (GFP) gene in p53-proficient human cells. We observed an approximately 5-fold enhancement of delayed hyperrecombination (DHR) among cells surviving a low dose of UV-C (5 J/m2), revealed as mixed GFP+/- colonies. UV-B did not induce DHR at an equitoxic (75 J/m2) dose or a higher dose (150 J/m2). UV is known to induce delayed hypermutation associated with increased oxidative stress. We found that hypoxanthine phosphoribosyltransferase (HPRT) mutation frequencies were approximately 5-fold higher in strains derived from GFP+/- (DHR) colonies than in strains in which recombination was directly induced by UV (GFP+ colonies). To determine whether hypermutation was directly caused by hyperrecombination, we analyzed hprt mutation spectra. Large-scale alterations reflecting large deletions and insertions were observed in 25% of GFP+ strains, and most mutants had a single change in HPRT. In striking contrast, all mutations arising in the hypermutable GFP+/- strains were small (1- to 2-base) changes, including substitutions, deletions, and insertions (reminiscent of mutagenesis from oxidative damage), and the majority were compound, with an average of four hprt mutations per mutant. The absence of large hprt deletions in DHR strains indicates that DHR does not cause hypermutation. We propose that UV-induced DHR and hypermutation result from a common source, namely, increased oxidative stress. These two forms of delayed genome instability may collaborate in skin cancer initiation and progression.
Asunto(s)
Mutagénesis/efectos de la radiación , Mutación Puntual/genética , Recombinación Genética/efectos de la radiación , Rayos Ultravioleta , Muerte Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Exones/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Modelos Biológicos , Mutagénesis/genética , Recombinación Genética/genética , Células Tumorales CultivadasRESUMEN
Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase I inhibitor topotecan. Thus, we here establish that inactivating terminal components of the non-homologous end-joining (NHEJ) machinery or of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib reflects delayed engagement of homologous recombination at DNA-replication-fork associated single-ended double-strand breaks (DSBs), allowing some to be subject to toxic NHEJ. Preventing DSB ligation by NHEJ, or enhancing homologous recombination by BRCA1-A complex disruption, suppresses this toxicity, highlighting a crucial role for ATM in preventing toxic LIG4-mediated chromosome fusions. Notably, suppressor mutations in ATM-mutant backgrounds are different to those in BRCA1-mutant scenarios, suggesting new opportunities for patient stratification and additional therapeutic vulnerabilities for clinical exploitation.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/genética , Reparación del ADN por Unión de Extremidades/genética , Resistencia a Antineoplásicos/genética , Animales , Antineoplásicos/uso terapéutico , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1/metabolismo , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Roturas del ADN de Doble Cadena/efectos de los fármacos , ADN Ligasa (ATP)/metabolismo , Replicación del ADN/efectos de los fármacos , Replicación del ADN/genética , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Células Madre Embrionarias de Ratones , Mutación , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Ftalazinas/farmacología , Ftalazinas/uso terapéutico , Piperazinas/farmacología , Piperazinas/uso terapéutico , Topotecan/farmacología , Topotecan/uso terapéuticoRESUMEN
We report the discovery of a novel series of 3-cinnoline carboxamides as highly potent and selective ataxia telangiectasia mutated (ATM) kinase inhibitors. Optimization of this series focusing on potency and physicochemical properties (especially permeability) led to the identification of compound 21, a highly potent ATM inhibitor (ATM cell IC50 0.0028 µM) with excellent kinase selectivity and favorable physicochemical and pharmacokinetics properties. In vivo, 21 in combination with irinotecan showed tumor regression in the SW620 colorectal tumor xenograft model, superior inhibition to irinotecan alone. Compound 21 was selected for preclinical evaluation alongside AZD0156.
RESUMEN
ATM inhibitors, such as 7, have demonstrated the antitumor potential of ATM inhibition when combined with DNA double-strand break-inducing agents in mouse xenograft models. However, the properties of 7 result in a relatively high predicted clinically efficacious dose. In an attempt to minimize attrition during clinical development, we sought to identify ATM inhibitors with a low predicted clinical dose (<50 mg) and focused on strategies to increase both ATM potency and predicted human pharmacokinetic half-life (predominantly through the increase of volume of distribution). These efforts resulted in the discovery of 64 (AZD0156), an exceptionally potent and selective inhibitor of ATM based on an imidazo[4,5- c]quinolin-2-one core. 64 has good preclinical phamacokinetics, a low predicted clinical dose, and a high maximum absorbable dose. 64 has been shown to potentiate the efficacy of the approved drugs irinotecan and olaparib in disease relevant mouse models and is currently undergoing clinical evaluation with these agents.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Diseño de Fármacos , Piridinas/farmacocinética , Quinolinas/farmacocinética , Quinolonas/farmacología , Quinolonas/farmacocinética , Administración Oral , Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Disponibilidad Biológica , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Conformación Proteica , Inhibidores de Proteínas Quinasas , Piridinas/administración & dosificación , Piridinas/química , Quinolinas/administración & dosificación , Quinolinas/química , Quinolonas/administración & dosificación , Quinolonas/química , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Inhibition of ataxia-telangiectasia mutated (ATM) during radiotherapy of glioblastoma multiforme (GBM) may improve tumor control by short-circuiting the response to radiation-induced DNA damage. A major impediment for clinical implementation is that current inhibitors have limited central nervous system (CNS) bioavailability; thus, the goal was to identify ATM inhibitors (ATMi) with improved CNS penetration. Drug screens and refinement of lead compounds identified AZ31 and AZ32. The compounds were then tested in vivo for efficacy and impact on tumor and healthy brain. Both AZ31 and AZ32 blocked the DNA damage response and radiosensitized GBM cells in vitro AZ32, with enhanced blood-brain barrier (BBB) penetration, was highly efficient in vivo as radiosensitizer in syngeneic and human, orthotopic mouse glioma model compared with AZ31. Furthermore, human glioma cell lines expressing mutant p53 or having checkpoint-defective mutations were particularly sensitive to ATMi radiosensitization. The mechanism for this p53 effect involves a propensity to undergo mitotic catastrophe relative to cells with wild-type p53. In vivo, apoptosis was >6-fold higher in tumor relative to healthy brain after exposure to AZ32 and low-dose radiation. AZ32 is the first ATMi with oral bioavailability shown to radiosensitize glioma and improve survival in orthotopic mouse models. These findings support the development of a clinical-grade, BBB-penetrating ATMi for the treatment of GBM. Importantly, because many GBMs have defective p53 signaling, the use of an ATMi concurrent with standard radiotherapy is expected to be cancer-specific, increase the therapeutic ratio, and maintain full therapeutic effect at lower radiation doses. Mol Cancer Ther; 17(8); 1637-47. ©2018 AACR.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Glioma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Administración Oral , Animales , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Línea Celular Tumoral , Humanos , Ratones , Ratones Desnudos , Inhibidores de Proteínas Quinasas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacologíaRESUMEN
Poor survival rates of patients with tumors arising from or disseminating into the brain are attributed to an inability to excise all tumor tissue (if operable), a lack of blood-brain barrier (BBB) penetration of chemotherapies/targeted agents, and an intrinsic tumor radio-/chemo-resistance. Ataxia-telangiectasia mutated (ATM) protein orchestrates the cellular DNA damage response (DDR) to cytotoxic DNA double-strand breaks induced by ionizing radiation (IR). ATM genetic ablation or pharmacological inhibition results in tumor cell hypersensitivity to IR. We report the primary pharmacology of the clinical-grade, exquisitely potent (cell IC50, 0.78 nM), highly selective [>10,000-fold over kinases within the same phosphatidylinositol 3-kinase-related kinase (PIKK) family], orally bioavailable ATM inhibitor AZD1390 specifically optimized for BBB penetration confirmed in cynomolgus monkey brain positron emission tomography (PET) imaging of microdosed 11C-labeled AZD1390 (Kp,uu, 0.33). AZD1390 blocks ATM-dependent DDR pathway activity and combines with radiation to induce G2 cell cycle phase accumulation, micronuclei, and apoptosis. AZD1390 radiosensitizes glioma and lung cancer cell lines, with p53 mutant glioma cells generally being more radiosensitized than wild type. In in vivo syngeneic and patient-derived glioma as well as orthotopic lung-brain metastatic models, AZD1390 dosed in combination with daily fractions of IR (whole-brain or stereotactic radiotherapy) significantly induced tumor regressions and increased animal survival compared to IR treatment alone. We established a pharmacokinetic-pharmacodynamic-efficacy relationship by correlating free brain concentrations, tumor phospho-ATM/phospho-Rad50 inhibition, apoptotic biomarker (cleaved caspase-3) induction, tumor regression, and survival. On the basis of the data presented here, AZD1390 is now in early clinical development for use as a radiosensitizer in central nervous system malignancies.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidad , Inhibidores de Proteínas Quinasas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Animales , Apoptosis/efectos de los fármacos , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Humanos , Ratones , Fosforilación , Inhibidores de Proteínas Quinasas/química , Tolerancia a Radiación/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/química , Transducción de Señal/efectos de los fármacos , Resultado del Tratamiento , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Rayos X , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Flap endonuclease 1 (FEN1) is a structure selective endonuclease required for proficient DNA replication and the repair of DNA damage. Cellularly active inhibitors of this enzyme have previously been shown to induce a DNA damage response and, ultimately, cell death. High-throughput screens of human cancer cell-lines identify colorectal and gastric cell-lines with microsatellite instability (MSI) as enriched for cellular sensitivity to N-hydroxyurea series inhibitors of FEN1, but not the PARP inhibitor olaparib or other inhibitors of the DNA damage response. This sensitivity is due to a synthetic lethal interaction between FEN1 and MRE11A, which is often mutated in MSI cancers through instabilities at a poly(T) microsatellite repeat. Disruption of ATM is similarly synthetic lethal with FEN1 inhibition, suggesting that disruption of FEN1 function leads to the accumulation of DNA double-strand breaks. These are likely a result of the accumulation of aberrant replication forks, that accumulate as a consequence of a failure in Okazaki fragment maturation, as inhibition of FEN1 is toxic in cells disrupted for the Fanconi anemia pathway and post-replication repair. Furthermore, RAD51 foci accumulate as a consequence of FEN1 inhibition and the toxicity of FEN1 inhibitors increases in cells disrupted for the homologous recombination pathway, suggesting a role for homologous recombination in the resolution of damage induced by FEN1 inhibition. Finally, FEN1 appears to be required for the repair of damage induced by olaparib and cisplatin within the Fanconi anemia pathway, and may play a role in the repair of damage associated with its own disruption.
Asunto(s)
Reparación del ADN/efectos de los fármacos , Endonucleasas de ADN Solapado/metabolismo , Hidroxiurea/toxicidad , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cisplatino/toxicidad , ADN/efectos de los fármacos , ADN/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Endonucleasas de ADN Solapado/antagonistas & inhibidores , Endonucleasas de ADN Solapado/genética , Humanos , Hidroxiurea/química , Proteína Homóloga de MRE11 , Inestabilidad de Microsatélites/efectos de los fármacos , Mutación , Ftalazinas/toxicidad , Piperazinas/toxicidad , Inhibidores de Poli(ADP-Ribosa) Polimerasas/toxicidad , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Recombinasa Rad51/genéticaRESUMEN
Non-homologous DNA end-joining (NHEJ) is a major pathway of double strand break (DSB) repair in human cells. Here we show that vanillin (3-methoxy-4-hydroxybenzaldehyde)--a naturally occurring food component and an acknowledged antimutagen, anticlastogen and anticarcinogen--is an inhibitor of NHEJ. Vanillin blocked DNA end-joining by human cell extracts by directly inhibiting the activity of DNA-PK, a crucial NHEJ component. Inhibition was selective and vanillin had no detectable effect on other steps of the NHEJ process, on an unrelated protein kinase or on DNA mismatch repair by cell extracts. Subtoxic concentrations of vanillin did not affect the ATM/ATR-dependent phosphorylation of Chk2 or the S-phase checkpoint response after ionising radiation. They significantly potentiated the cytotoxicity of cisplatin, but did not affect sensitivity to UVC. A limited screen of structurally related compounds identified two substituted vanillin derivatives that were 100- and 50-fold more potent than vanillin as DNA-PK inhibitors. These compounds also sensitised cells to cisplatin. The inhibition of NHEJ is consistent with the antimutagenic and other biological properties of vanillin, possibly altering the balance between DSB repair by NHEJ and homologous recombination.
Asunto(s)
Antimutagênicos/farmacología , Benzaldehídos/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Antimutagênicos/química , Antimutagênicos/toxicidad , Antineoplásicos/toxicidad , Benzaldehídos/química , Benzaldehídos/toxicidad , Línea Celular , Cisplatino/toxicidad , Reparación del ADN/efectos de los fármacos , Proteína Quinasa Activada por ADN , Proteínas de Unión al ADN/análisis , Sinergismo Farmacológico , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/toxicidad , Humanos , Proteínas Nucleares , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Recombinasa Rad51 , Recombinación Genética/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
After a DNA damage signal multiple polymers of ADP ribose attached to poly(ADP) ribose (PAR) polymerases (PARPs) are broken down by the enzyme poly(ADP) ribose glycohydrolase (PARG). Inhibition of PARG leads to a failure of DNA repair and small molecule inhibition of PARG has been a goal for many years. To determine whether biochemical inhibitors of PARG are active in cells we have designed an immunofluorescence assay to detect nuclear PAR after DNA damage. This 384-well assay is suitable for medium throughput high-content screening and can detect cell-permeable inhibitors of PARG from nM to µM potency. In addition, the assay has been shown to work in murine cells and in a variety of human cancer cells. Furthermore, the assay is suitable for detecting the DNA damage response induced by treatment with temozolomide and methylmethane sulfonate (MMS). Lastly, the assay has been shown to be robust over a period of several years.
RESUMEN
MSH2 and MLH1 have a central role in correcting mismatches in DNA occurring during DNA replication and have been implicated in the engagement of apoptosis induced by a number of cytotoxic anticancer agents. The function of MLH1 is not clearly defined, although it is required for mismatch repair (MMR) and engagement of apoptosis after certain types of DNA damage. In order to identify other partners of MLH1 that may be involved in signalling MMR or apoptosis, we used human MLH1 in yeast two-hybrid screens of normal human breast and ovarian cDNA libraries. As well as known partners of MLH1 such as PMS1, MLH3 and MBD4, we identified the carboxy terminus of the human c-MYC proto-oncogene as an interacting sequence. We demonstrate, both in vitro by yeast two-hybrid and GST-fusion pull-down experiments, as well as in vivo by coimmunoprecipitation from human tumour cell extracts, that MLH1 interacts with the c-MYC protein. We further demonstrate that the heterodimeric partner of c-MYC, MAX, interacts with a different MMR protein, MSH2, both in vitro and in vivo. Using an inducible c-MYC-ER fusion gene, we show that elevated c-MYC expression leads to an increased HGPRT mutation rate of Rat1 cells and an increase in the number of frameshift mutants at the HGPRT locus. The effect on HGPRT mutation rate is small (2-3-fold), but is consistent with deregulated c-MYC expression partially inhibiting MMR activity.