Asunto(s)
Neoplasias Encefálicas , Proteínas de Unión al ARN , Humanos , Masculino , Femenino , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/genética , Preescolar , Estudios Retrospectivos , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteína Ácida Fibrilar de la Glía/genética , Nestina/metabolismo , Nestina/genética , Lóbulo Parietal/patología , Lóbulo Parietal/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Neoplasias del Tronco Encefálico/patología , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/metabolismo , Neoplasias del Tronco Encefálico/cirugía , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteína SMARCB1/metabolismo , Proteína SMARCB1/genética , Lóbulo Frontal/patología , Lóbulo Frontal/metabolismo , Formación de Roseta , Estudios de Seguimiento , Antígeno Ki-67/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genéticaRESUMEN
Pediatric high-grade gliomas are a devastating subset of brain tumors, characterized by their aggressive pathophysiology and limited treatment options. Among them, H3 K27-altered diffuse midline gliomas (DMG) of the brainstem stand out due to their distinct molecular features and dismal prognosis. Recent advances in molecular profiling techniques have unveiled the critical role of H3 K27 alterations, particularly a lysine-to-methionine mutation on position 27 (K27M) of the histone H3 tail, in the pathogenesis of DMG. These mutations result in epigenetic dysregulation, which leads to altered chromatin structure and gene expression patterns in DMG tumor cells, ultimately contributing to the aggressive phenotype of DMG. The exploration of targeted therapeutic avenues for DMG has gained momentum in recent years. Therapies, including epigenetic modifiers, kinase inhibitors, and immunotherapies, are under active investigation; these approaches aim to disrupt aberrant signaling cascades and overcome the various mechanisms of therapeutic resistance in DMG. Challenges, including blood-brain barrier penetration and DMG tumor heterogeneity, require innovative approaches to improve drug delivery and personalized treatment strategies. This review aims to provide a comprehensive overview of the evolving understanding of DMG, focusing on the intricate molecular mechanisms driving tumorigenesis/tumor progression and the current landscape of emerging targeted interventions.
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Neoplasias del Tronco Encefálico , Glioma , Histonas , Humanos , Glioma/genética , Glioma/patología , Glioma/metabolismo , Histonas/metabolismo , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/patología , Neoplasias del Tronco Encefálico/metabolismo , Neoplasias del Tronco Encefálico/terapia , Epigénesis Genética , Terapia Molecular Dirigida , Mutación/genética , AnimalesRESUMEN
Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive and fatal pediatric brain cancer. One pre-requisite for tumor cells to infiltrate is adhesion to extracellular matrix (ECM) components. However, it remains largely unknown which ECM proteins are critical in enabling DIPG adhesion and migration and which integrin receptors mediate these processes. Here, we identify laminin as a key ECM protein that supports robust DIPG cell adhesion and migration. To study DIPG infiltration, we developed a DIPG-neural assembloid model, which is composed of a DIPG spheroid fused to a human induced pluripotent stem cell-derived neural organoid. Using this assembloid model, we demonstrate that knockdown of laminin-associated integrins significantly impedes DIPG infiltration. Moreover, laminin-associated integrin knockdown improves DIPG response to radiation and HDAC inhibitor treatment within the DIPG-neural assembloids. These findings reveal the critical role of laminin-associated integrins in mediating DIPG progression and drug response. The results also provide evidence that disrupting integrin receptors may offer a novel therapeutic strategy to enhance DIPG treatment outcomes. Finally, these results establish DIPG-neural assembloid models as a powerful tool to study DIPG disease progression and enable drug discovery.
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Neoplasias del Tronco Encefálico , Glioma Pontino Intrínseco Difuso , Integrinas , Laminina , Humanos , Laminina/metabolismo , Integrinas/metabolismo , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/patología , Neoplasias del Tronco Encefálico/metabolismo , Neoplasias del Tronco Encefálico/terapia , Glioma Pontino Intrínseco Difuso/patología , Glioma Pontino Intrínseco Difuso/genética , Adhesión Celular/efectos de los fármacos , Movimiento Celular , Línea Celular Tumoral , Glioma/patología , Glioma/metabolismo , Glioma/genética , Glioma/terapiaRESUMEN
PURPOSE: PNOC003 is a multicenter precision medicine trial for children and young adults with newly diagnosed diffuse intrinsic pontine glioma (DIPG). PATIENTS AND METHODS: Patients (3-25 years) were enrolled on the basis of imaging consistent with DIPG. Biopsy tissue was collected for whole-exome and mRNA sequencing. After radiotherapy (RT), patients were assigned up to four FDA-approved drugs based on molecular tumor board recommendations. H3K27M-mutant circulating tumor DNA (ctDNA) was longitudinally measured. Tumor tissue and matched primary cell lines were characterized using whole-genome sequencing and DNA methylation profiling. When applicable, results were verified in an independent cohort from the Children's Brain Tumor Network (CBTN). RESULTS: Of 38 patients enrolled, 28 patients (median 6 years, 10 females) were reviewed by the molecular tumor board. Of those, 19 followed treatment recommendations. Median overall survival (OS) was 13.1 months [95% confidence interval (CI), 11.2-18.4] with no difference between patients who followed recommendations and those who did not. H3K27M-mutant ctDNA was detected at baseline in 60% of cases tested and associated with response to RT and survival. Eleven cell lines were established, showing 100% fidelity of key somatic driver gene alterations in the primary tumor. In H3K27-altered DIPGs, TP53 mutations were associated with worse OS (TP53mut 11.1 mo; 95% CI, 8.7-14; TP53wt 13.3 mo; 95% CI, 11.8-NA; P = 3.4e-2), genome instability (P = 3.1e-3), and RT resistance (P = 6.4e-4). The CBTN cohort confirmed an association between TP53 mutation status, genome instability, and clinical outcome. CONCLUSIONS: Upfront treatment-naïve biopsy provides insight into clinically relevant molecular alterations and prognostic biomarkers for H3K27-altered DIPGs.
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Astrocitoma , Neoplasias del Tronco Encefálico , ADN Tumoral Circulante , Glioma Pontino Intrínseco Difuso , Glioma , Biología , Biomarcadores , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/metabolismo , Neoplasias del Tronco Encefálico/terapia , Niño , ADN Tumoral Circulante/genética , Glioma Pontino Intrínseco Difuso/genética , Femenino , Inestabilidad Genómica , Glioma/genética , Glioma/metabolismo , Glioma/terapia , Humanos , Adulto JovenRESUMEN
Cancer cells are highly heterogeneous at the transcriptional level and epigenetic state. Methods to study epigenetic heterogeneity are limited in throughput and information obtained per cell. Here, we adapted cytometry by time-of-flight (CyTOF) to analyze a wide panel of histone modifications in primary tumor-derived lines of diffused intrinsic pontine glioma (DIPG). DIPG is a lethal glioma, driven by a histone H3 lysine 27 mutation (H3-K27M). We identified two epigenetically distinct subpopulations in DIPG, reflecting inherent heterogeneity in expression of the mutant histone. These two subpopulations are robust across tumor lines derived from different patients and show differential proliferation capacity and expression of stem cell and differentiation markers. Moreover, we demonstrate the use of these high-dimensional data to elucidate potential interactions between histone modifications and epigenetic alterations during the cell cycle. Our work establishes new concepts for the analysis of epigenetic heterogeneity in cancer that could be applied to diverse biological systems.
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Neoplasias del Tronco Encefálico , Glioma , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/metabolismo , Neoplasias del Tronco Encefálico/patología , Cromatina/genética , Epigénesis Genética , Glioma/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , MutaciónRESUMEN
BACKGROUND: The histone variant H3.3 K27M mutation is a defining characteristic of diffuse intrinsic pontine glioma (DIPG)/diffuse midline glioma (DMG). This histone mutation is responsible for major alterations to histone H3 post-translational modification (PTMs) and subsequent aberrant gene expression. However, much less is known about the effect this mutation has on chromatin structure and function, including open versus closed chromatin regions as well as their transcriptomic consequences. RESULTS: Recently, we developed isogenic CRISPR-edited DIPG cell lines that are wild-type for histone H3.3 that can be compared to their matched K27M lines. Here we show via ATAC-seq analysis that H3.3K27M glioma cells have unique accessible chromatin at regions corresponding to neurogenesis, NOTCH, and neuronal development pathways and associated genes that are overexpressed in H3.3K27M compared to our isogenic wild-type cell line. As to mechanisms, accessible enhancers and super-enhancers corresponding to increased gene expression in H3.3K27M cells were also mapped to genes involved in neurogenesis and NOTCH signaling, suggesting that these pathways are key to DIPG tumor maintenance. Motif analysis implicates specific transcription factors as central to the neuro-oncogenic K27M signaling pathway, in particular, ASCL1 and NEUROD1. CONCLUSIONS: Altogether our findings indicate that H3.3K27M causes chromatin to take on a more accessible configuration at key regulatory regions for NOTCH and neurogenesis genes resulting in increased oncogenic gene expression, which is at least partially reversible upon editing K27M back to wild-type.
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Neoplasias del Tronco Encefálico , Glioma , Humanos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/metabolismo , Neoplasias del Tronco Encefálico/patología , Cromatina/genética , Glioma/genética , Glioma/metabolismo , Glioma/patología , Histonas/metabolismo , MutaciónRESUMEN
BACKGROUND: Neurodevelopmental disorders increase brain tumor risk, suggesting that normal brain development may have protective properties. Mutations in epigenetic regulators are common in pediatric brain tumors, highlighting a potentially central role for disrupted epigenetic regulation of normal brain development in tumorigenesis. For example, lysine 27 to methionine mutation (H3K27M) in the H3F3A gene occurs frequently in Diffuse Intrinsic Pontine Gliomas (DIPGs), the most aggressive pediatric glioma. As H3K27M mutation is necessary but insufficient to cause DIPGs, it is accompanied by additional mutations in tumors. However, how H3K27M alone increases vulnerability to DIPG tumorigenesis remains unclear. RESULTS: Here, we used human embryonic stem cell models with this mutation, in the absence of other DIPG contributory mutations, to investigate how H3K27M alters cellular proliferation and differentiation. We found that H3K27M increased stem cell proliferation and stem cell properties. It interfered with differentiation, promoting anomalous mesodermal and ectodermal gene expression during both multi-lineage and germ layer-specific cell specification, and blocking normal differentiation into neuroectoderm. H3K27M mutant clones exhibited transcriptomic diversity relative to the more homogeneous wildtype population, suggesting reduced fidelity of gene regulation, with aberrant expression of genes involved in stem cell regulation, differentiation, and tumorigenesis. These phenomena were associated with global loss of H3K27me3 and concordant loss of DNA methylation at specific genes in H3K27M-expressing cells. CONCLUSIONS: Together, these data suggest that H3K27M mutation disrupts normal differentiation, maintaining a partially differentiated state with elevated clonogenicity during early development. This disrupted response to early developmental cues could promote tissue properties that enable acquisition of additional mutations that cooperate with H3K27M mutation in genesis of DMG/DIPG. Therefore, this work demonstrates for the first time that H3K27M mutation confers vulnerability to gliomagenesis through persistent clonogenicity and aberrant differentiation and defines associated alterations of histone and DNA methylation.
Asunto(s)
Neoplasias del Tronco Encefálico , Epigénesis Genética , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/metabolismo , Neoplasias del Tronco Encefálico/patología , Carcinogénesis/genética , Proliferación Celular , Niño , Histonas , Humanos , Mutación , Células Madre/metabolismoRESUMEN
PURPOSE: Crizotinib, a potent oral tyrosine kinase inhibitor, was evaluated in combination with dasatinib in a phase 1 trial (NCT01644773) in children with progressive or recurrent high-grade and diffuse intrinsic pontine gliomas (HGG and DIPG). This study aimed to characterize the pharmacokinetics of crizotinib in this population and identify significant covariates. METHODS: Patients (N = 36, age range 2.9-21.3 years) were treated orally once or twice-daily with 100-215 mg/m2 crizotinib and 50-65 mg/m2 dasatinib. Pharmacokinetic studies were performed for crizotinib alone after the first dose and at steady state, and for the drug combination at steady state. Crizotinib plasma concentrations were measured using a validated LC-MS/MS method. Population modeling was performed (Monolix) and the impact of factors including patient demographics and co-medications were investigated on crizotinib pharmacokinetics. RESULTS: Crizotinib concentrations were described with a linear two-compartment model and absorption lag time. Concomitant dasatinib and overweight/obese status significantly influenced crizotinib pharmacokinetics, resulting in clinically relevant impact (> 20%) on drug exposure. Crizotinib mean apparent clearance (CL/F) was 66.7 L/h/m2 after single-dose and decreased to 26.5 L/h/m2 at steady state when given alone, but not when combined with dasatinib (mean 60.8 L/h/m2). Overweight/obese patients exhibited lower crizotinib CL/F and apparent volume V1/F (mean 46.2 L/h/m2 and 73.3 L/m2) compared to other patients (mean 75.5 L/h/m2 and 119.3 L/m2, p < 0.001). CONCLUSION: A potential pharmacokinetic interaction was observed between crizotinib and dasatinib in children with HGG and DIPG. Further, crizotinib exposure was significantly higher in overweight/obese patients, who may require a dosing adjustment.
Asunto(s)
Antineoplásicos/farmacocinética , Neoplasias del Tronco Encefálico/tratamiento farmacológico , Crizotinib/farmacocinética , Glioma Pontino Intrínseco Difuso/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Adolescente , Adulto , Antineoplásicos/administración & dosificación , Neoplasias del Tronco Encefálico/metabolismo , Neoplasias del Tronco Encefálico/patología , Niño , Preescolar , Crizotinib/administración & dosificación , Glioma Pontino Intrínseco Difuso/metabolismo , Glioma Pontino Intrínseco Difuso/patología , Femenino , Estudios de Seguimiento , Humanos , Masculino , Dosis Máxima Tolerada , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Pronóstico , Distribución Tisular , Adulto JovenRESUMEN
Diffuse intrinsic pontine glioma (DIPG) is an incurable paediatric malignancy. Identifying the molecular drivers of DIPG progression is of the utmost importance. Long non-coding RNAs (lncRNAs) represent a large family of disease- and tissue-specific transcripts, whose functions have not yet been elucidated in DIPG. Herein, we studied the oncogenic role of the development-associated H19 lncRNA in DIPG. Bioinformatic analyses of clinical datasets were used to measure the expression of H19 lncRNA in paediatric high-grade gliomas (pedHGGs). The expression and sub-cellular location of H19 lncRNA were validated in DIPG cell lines. Locked nucleic acid antisense oligonucleotides were designed to test the function of H19 in DIPG cells. We found that H19 expression was higher in DIPG vs. normal brain tissue and other pedHGGs. H19 knockdown resulted in decreased cell proliferation and survival in DIPG cells. Mechanistically, H19 buffers let-7 microRNAs, resulting in the up-regulation of oncogenic let-7 target (e.g., SULF2 and OSMR). H19 is the first functionally characterized lncRNA in DIPG and a promising therapeutic candidate for treating this incurable cancer.
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Neoplasias del Tronco Encefálico/genética , Proliferación Celular , Glioma/genética , ARN Largo no Codificante/metabolismo , Neoplasias del Tronco Encefálico/metabolismo , Neoplasias del Tronco Encefálico/patología , Células Cultivadas , Regulación Neoplásica de la Expresión Génica , Glioma/metabolismo , Glioma/patología , Histonas/metabolismo , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Mutación , ARN Largo no Codificante/genéticaRESUMEN
Diffuse intrinsic pontine glioma (DIPG), a rare pediatric brain tumor, afflicts approximately 350 new patients each year in the United States. DIPG is noted for its lethality, as fewer than 1% of patients survive to five years. Multiple clinical trials involving chemotherapy, radiotherapy, and/or targeted therapy have all failed to improve clinical outcomes. Recently, high-throughput sequencing of a cohort of DIPG samples identified potential therapeutic targets, including interleukin 13 receptor subunit alpha 2 (IL13Rα2) which was expressed in multiple tumor samples and comparably absent in normal brain tissue, identifying IL13Rα2 as a potential therapeutic target in DIPG. In this work, we investigated the role of IL13Rα2 signaling in progression and invasion of DIPG and viability of IL13Rα2 as a therapeutic target through the use of immunoconjugate agents. We discovered that IL13Rα2 stimulation via canonical ligands demonstrates minimal impact on both the cellular proliferation and cellular invasion of DIPG cells, suggesting IL13Rα2 signaling is non-essential for DIPG progression in vitro. However, exposure to an anti-IL13Rα2 antibody-drug conjugate demonstrated potent pharmacological response in DIPG cell models both in vitro and ex ovo in a manner strongly associated with IL13Rα2 expression, supporting the potential use of targeting IL13Rα2 as a DIPG therapy. However, the tested ADC was effective in most but not all cell models, thus selection of the optimal payload will be essential for clinical translation of an anti-IL13Rα2 ADC for DIPG.
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Neoplasias del Tronco Encefálico/metabolismo , Glioma Pontino Intrínseco Difuso/metabolismo , Diseño de Fármacos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/metabolismo , Subunidad alfa2 del Receptor de Interleucina-13/metabolismo , Animales , Neoplasias del Tronco Encefálico/tratamiento farmacológico , Neoplasias del Tronco Encefálico/patología , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Coturnix , Glioma Pontino Intrínseco Difuso/tratamiento farmacológico , Glioma Pontino Intrínseco Difuso/patología , Relación Dosis-Respuesta a Droga , Humanos , Interleucina-13/administración & dosificación , Interleucina-13/metabolismo , Subunidad alfa2 del Receptor de Interleucina-13/antagonistas & inhibidores , Invasividad Neoplásica/patología , Células Tumorales CultivadasRESUMEN
Asparagine synthetase (ASNS) is a gene on the long arm of chromosome 7 that is copy-number amplified in the majority of glioblastomas. ASNS copy-number amplification is associated with a significantly decreased survival. Using patient-derived glioma stem cells (GSC), we showed that significant metabolic alterations occur in gliomas when perturbing the expression of ASNS, which is not merely restricted to amino acid homeostasis. ASNS-high GSCs maintained a slower basal metabolic profile yet readily shifted to a greatly increased capacity for glycolysis and oxidative phosphorylation when needed. This led ASNS-high cells to a greater ability to proliferate and spread into brain tissue. Finally, we demonstrate that these changes confer resistance to cellular stress, notably oxidative stress, through adaptive redox homeostasis that led to radiotherapy resistance. Furthermore, ASNS overexpression led to modifications of the one-carbon metabolism to promote a more antioxidant tumor environment revealing a metabolic vulnerability that may be therapeutically exploited. IMPLICATIONS: This study reveals a new role for ASNS in metabolic control and redox homeostasis in glioma stem cells and proposes a new treatment strategy that attempts to exploit one vulnerable metabolic node within the larger multilayered tumor network.
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Asparagina/biosíntesis , Neoplasias del Tronco Encefálico/metabolismo , Encéfalo/metabolismo , Glioma/metabolismo , Células Madre Neoplásicas/metabolismo , Estrés Oxidativo/fisiología , Animales , Aspartatoamoníaco Ligasa/metabolismo , Células HEK293 , Humanos , Ratones , Estudios RetrospectivosAsunto(s)
Biomarcadores de Tumor/biosíntesis , Neoplasias del Tronco Encefálico/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Glioma/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Receptores de Superficie Celular/biosíntesis , Biomarcadores de Tumor/genética , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/patología , Línea Celular Tumoral , Movimiento Celular/fisiología , Niño , Preescolar , Femenino , Glioma/genética , Glioma/patología , Humanos , Masculino , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Proteínas del Tejido Nervioso/genética , Receptores de Superficie Celular/genéticaRESUMEN
Diffuse intrinsic pontine glioma (DIPG) kills more children than any other type of brain tumor. Despite clinical trials testing many chemotherapeutic agents, palliative radiotherapy remains the standard treatment. Here, we utilized Cre/loxP technology to show that deleting Ataxia telangiectasia mutated (Atm) in primary mouse models of DIPG can enhance tumor radiosensitivity. Genetic deletion of Atm improved survival of mice with p53-deficient but not p53 wild-type gliomas after radiotherapy. Similar to patients with DIPG, mice with p53 wild-type tumors had improved survival after radiotherapy independent of Atm deletion. Primary p53 wild-type tumor cell lines induced proapoptotic genes after radiation and repressed the NRF2 target, NAD(P)H quinone dehydrogenase 1 (Nqo1). Tumors lacking p53 and Ink4a/Arf expressed the highest level of Nqo1 and were most resistant to radiation, but deletion of Atm enhanced the radiation response. These results suggest that tumor genotype may determine whether inhibition of ATM during radiotherapy will be an effective clinical approach to treat DIPGs.
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Neoplasias del Tronco Encefálico , Eliminación de Gen , Genotipo , Glioma , Tolerancia a Radiación , Animales , Proteínas de la Ataxia Telangiectasia Mutada/deficiencia , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/metabolismo , Neoplasias del Tronco Encefálico/radioterapia , Línea Celular Tumoral , Pollos , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Glioma/genética , Glioma/metabolismo , Glioma/radioterapia , Ratones , Ratones Transgénicos , NAD(P)H Deshidrogenasa (Quinona)/genética , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
H3K27M diffuse intrinsic pontine gliomas (DIPGs) are fatal and lack treatments. They mainly harbor H3.3K27M mutations resulting in H3K27me3 reduction. Integrated analysis in H3.3K27M cells, tumors, and in vivo imaging in patients showed enhanced glycolysis, glutaminolysis, and tricarboxylic acid cycle metabolism with high alpha-ketoglutarate (α-KG) production. Glucose and/or glutamine-derived α-KG maintained low H3K27me3 in H3.3K27M cells, and inhibition of key enzymes in glycolysis or glutaminolysis increased H3K27me3, altered chromatin accessibility, and prolonged survival in animal models. Previous studies have shown that mutant isocitrate-dehydrogenase (mIDH)1/2 glioma cells convert α-KG to D-2-hydroxyglutarate (D-2HG) to increase H3K27me3. Here, we show that H3K27M and IDH1 mutations are mutually exclusive and experimentally synthetic lethal. Overall, we demonstrate that H3.3K27M and mIDH1 hijack a conserved and critical metabolic pathway in opposing ways to maintain their preferred epigenetic state. Consequently, interruption of this metabolic/epigenetic pathway showed potent efficacy in preclinical models, suggesting key therapeutic targets for much needed treatments.
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Neoplasias del Tronco Encefálico/genética , Glioma Pontino Intrínseco Difuso/genética , Epigenómica/métodos , Histonas/genética , Mutación , Animales , Neoplasias del Tronco Encefálico/metabolismo , Línea Celular Tumoral , Glioma Pontino Intrínseco Difuso/metabolismo , Regulación Neoplásica de la Expresión Génica , Glucólisis , Histonas/metabolismo , Humanos , Lisina/genética , Lisina/metabolismo , Metilación , Ratones Endogámicos NOD , Ratones Noqueados , Ratones Desnudos , Ratones SCID , Trasplante HeterólogoRESUMEN
Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor with a 5-year survival of <1%. Up to 80% of the DIPG tumors contain a specific K27M mutation in one of the two genes encoding histone H3 (H3K27M). Furthermore, p53 mutations found in >70-80% of H3K27M DIPG, and mutant p53 status is associated with a decreased response to radiation treatment and worse overall prognosis. Recent evidence indicates that H3K27M mutation disrupts tri-methylation at H3K27 leading to aberrant gene expression. Jumonji family histone demethylases collaborates with H3K27 mutation in DIPG by erasing H3K27 trimethylation and thus contributing to derepression of genes involved in tumorigenesis. Since the first line of treatment for pediatric DIPG is fractionated radiation, we investigated the effects of Jumonji demethylase inhibition with GSK-J4, and mutant p53 targeting/oxidative stress induction with APR-246, on radio-sensitization of human H3K27M DIPG cells. Both APR-246 and GSK-J4 displayed growth inhibitory effects as single agents in H3K27M DIPG cells. Furthermore, both of these agents elicited mild radiosensitizing effects in human DIPG cells (sensitizer enhancement ratios (SERs) of 1.12 and 1.35, respectively; p < 0.05). Strikingly, a combination of APR-246 and GSK-J4 displayed a significant enhancement of radiosensitization, with SER of 1.50 (p < 0.05) at sub-micro-molar concentrations of the drugs (0.5 µM). The molecular mechanism of the observed radiosensitization appears to involve DNA damage repair deficiency triggered by APR-246/GSK-J4, leading to the induction of apoptotic cell death. Thus, a therapeutic approach of combined targeting of mutant p53, oxidative stress induction, and Jumonji demethylase inhibition with radiation in DIPG warrants further investigation.
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Neoplasias del Tronco Encefálico , Fraccionamiento de la Dosis de Radiación , Glioma , Mutación Missense , Quinuclidinas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Proteína p53 Supresora de Tumor , Sustitución de Aminoácidos , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/metabolismo , Neoplasias del Tronco Encefálico/patología , Neoplasias del Tronco Encefálico/terapia , Línea Celular Tumoral , Glioma/genética , Glioma/metabolismo , Glioma/patología , Glioma/terapia , Histona Demetilasas/antagonistas & inhibidores , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Humanos , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: An impermeable blood-brain barrier and drug efflux via ATP-binding cassette (ABC) transporters such as p-glycoprotein may contribute to underwhelming efficacy of peripherally delivered agents to treat diffuse intrinsic pontine glioma (DIPG). OBJECTIVE: To explore the pharmacological augmentation of convection-enhanced delivery (CED) infusate for DIPG. METHODS: The efficacy of CED dasatinib, a tyrosine kinase inhibitor, in a transgenic H3.3K27M mutant murine model was assessed. mRNA expression of ABCB1 (p-glycoprotein) was analyzed in 14 tumor types in 274 children. In Vitro viability studies of dasatinib, the p-glycoprotein inhibitor, tariquidar, and dexamethasone were performed in 2 H3.3K27M mutant cell lines. Magnetic resonance imaging (MRI) was used to evaluate CED infusate (gadolinium/dasatinib) distribution in animals pretreated with tariquidar and dexamethasone. Histological assessment of apoptosis was performed. RESULTS: Continuous delivery CED dasatinib improved median overall survival (OS) of animals harboring DIPG in comparison to vehicle (39.5 and 28.5 d, respectively; P = .0139). Mean ABCB1 expression was highest in K27M gliomas. In Vitro, the addition of tariquidar and dexamethasone further enhanced the efficacy of dasatinib (P < .001). In Vivo, MRI demonstrated no difference in infusion dispersion between animals pretreated with dexamethasone plus tariquidar prior to CED dasatinib compared to the CED dasatinib. However, tumor apoptosis was the highest in the pretreatment group (P < .001). Correspondingly, median OS was longer in the pretreatment group (49 d) than the dasatinib alone group (39 d) and no treatment controls (31.5 d, P = .0305). CONCLUSION: ABC transporter inhibition plus dexamethasone enhances the efficacy of CED dasatinib, resulting in enhanced tumor cellular apoptosis and improved survival in H3.3K27M mutant DIPG.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Antineoplásicos/administración & dosificación , Neoplasias del Tronco Encefálico , Dasatinib/administración & dosificación , Glioma Pontino Intrínseco Difuso , Animales , Apoptosis/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/metabolismo , Convección , Dexametasona/farmacología , Glioma Pontino Intrínseco Difuso/genética , Glioma Pontino Intrínseco Difuso/metabolismo , Histonas/genética , Humanos , Imagen por Resonancia Magnética , Ratones , Ratones Transgénicos , Quinolinas/farmacologíaRESUMEN
PURPOSE: Diffuse intrinsic pontine gliomas (DIPG) are the most severe pediatric brain tumors. Although accepted as the standard therapeutic, radiotherapy is only efficient transiently and not even in every patient. The goal of the study was to identify the underlying molecular determinants of response to radiotherapy in DIPG. EXPERIMENTAL DESIGN: We assessed in vitro response to ionizing radiations in 13 different DIPG cellular models derived from treatment-naïve stereotactic biopsies reflecting the genotype variability encountered in patients at diagnosis and correlated it to their principal molecular alterations. Clinical and radiologic response to radiotherapy of a large cohort of 73 DIPG was analyzed according to their genotype. Using a kinome-wide synthetic lethality RNAi screen, we further identified target genes that can sensitize DIPG cells to ionizing radiations. RESULTS: We uncover TP53 mutation as the main driver of increased radioresistance and validated this finding in four isogenic pairs of TP53WT DIPG cells with or without TP53 knockdown. In an integrated clinical, radiological, and molecular study, we show that TP53MUT DIPG patients respond less to irradiation, relapse earlier after radiotherapy, and have a worse prognosis than their TP53WT counterparts. Finally, a kinome-wide synthetic lethality RNAi screen identifies CHK1 as a potential target, whose inhibition increases response to radiation specifically in TP53MUT cells. CONCLUSIONS: Here, we demonstrate that TP53 mutations are driving DIPG radioresistance both in patients and corresponding cellular models. We suggest alternative treatment strategies to mitigate radioresistance with CHK1 inhibitors. These findings will allow to consequently refine radiotherapy schedules in DIPG.
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Neoplasias del Tronco Encefálico/metabolismo , Glioma Pontino Intrínseco Difuso/metabolismo , Tolerancia a Radiación , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/mortalidad , Neoplasias del Tronco Encefálico/radioterapia , Ciclo Celular/genética , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Supervivencia Celular/efectos de la radiación , Glioma Pontino Intrínseco Difuso/genética , Glioma Pontino Intrínseco Difuso/mortalidad , Glioma Pontino Intrínseco Difuso/radioterapia , Relación Dosis-Respuesta en la Radiación , Técnicas de Silenciamiento del Gen , Histonas/genética , Histonas/metabolismo , Humanos , Mutación , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/efectos de la radiación , Pronóstico , Interferencia de ARN , ARN Interferente Pequeño/genética , Tolerancia a Radiación/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Diffuse intrinsic pontine glioma (DIPG) is an untreatable, heterogeneous high-grade glioma (HGG) of the brainstem. This highly aggressive cancer affects mostly young children and is uniformly fatal. Genomic studies show that DIPG is driven by somatic mutations to histone H3, either H3.1 or H3.3 variants (HIST1H3B/C and H3F3A), altering the epigenetic landscape of primitive oligodendrocyte or astrocyte precursor cells of the pontine region of the brainstem. Lysine-to-methionine point mutations at amino acid 27 (H3K27M) co-occur with alterations in signaling genes, including the receptor tyrosine kinases (PDGFR/KIT/VEGFR/MET/EGFR), activin A receptor (ACVR1), intracellular kinases (PI3K/AKT/mTOR), cyclin-dependent kinases (CDKs1/4/6), transcriptional regulators (MYCN), and tumor suppressors (PTEN/TP53). This cooperation drives gene expression signatures that inhibit cellular differentiation (ID1/2, Hedgehog) and promotes malignant transformation. Unique to DIPG, is the frequency of co-occurring sets of genomic insults. However, mapping of the oncogenic signaling pathways activated in response to recurring mutations is unresolved. Herein, known oncogenic signal pathways activated in response to recurring somatic mutations and gene amplifications in DIPG are reviewed. Additionally, an important role for high-resolution quantitative proteomics/phosphoproteomics in the characterization of signaling cascades are highlighted. These regulate the cell cycle, epigenetics and anti-apoptotic processes, information critical for the development of improved treatment strategies for DIPG.
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Neoplasias del Tronco Encefálico/metabolismo , Glioma Pontino Intrínseco Difuso/metabolismo , Transducción de Señal , Animales , Neoplasias del Tronco Encefálico/genética , Glioma Pontino Intrínseco Difuso/genética , Código de Histonas , Humanos , Mutación/genética , Proteómica , Transducción de Señal/genéticaRESUMEN
Diffuse intrinsic pontine gliomas (DIPG) are incurable brain tumors with an aggressive onset. Apart from irradiation, there are currently no effective therapies available for patients with DIPG, who have a median survival time of less than one year. Most DIPG cells harbor mutations in genes encoding histone H3 (H3K27M) proteins, resulting in a global reduction of H3K27 trimethylation and activation of oncogenic signaling pathways. Here we show that the H3K27M mutations contribute to RAS pathway signaling, which is augmented by additional RAS activators including PDGFRA. H3K27M mutation led to increased expression of receptor tyrosine kinases (RTK). A RAS pathway functional screen identified ERK5, but not ERK1/2, as a RAS pathway effector important for DIPG growth. Suppression of ERK5 decreased DIPG cell proliferation and induced apoptosis in vitro and in vivo. In addition, depletion or inhibition of ERK5 significantly increased survival of mice intracranially engrafted with DIPG cells. Mechanistically, ERK5 directly stabilized the proto-oncogene MYC at the protein level. Collectively, our data demonstrate an underappreciated role of H3K27M in RAS activation and reveal novel therapeutic targets for treating DIPG tumors. SIGNIFICANCE: These findings identify the H3K27M mutation as an enhancer of RAS activation in DIPG and ERK5 as a novel, immediately actionable molecular target. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/16/4026/F1.large.jpg.
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Neoplasias del Tronco Encefálico/metabolismo , Glioma Pontino Intrínseco Difuso/metabolismo , Mutación , Proteínas ras/metabolismo , Compuestos de Anilina/farmacología , Animales , Neoplasias del Tronco Encefálico/tratamiento farmacológico , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/patología , Línea Celular Tumoral , Proliferación Celular/genética , Glioma Pontino Intrínseco Difuso/tratamiento farmacológico , Glioma Pontino Intrínseco Difuso/genética , Glioma Pontino Intrínseco Difuso/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Genes myc , Histonas/genética , Histonas/metabolismo , Humanos , Indoles/farmacología , Lisina/genética , Lisina/metabolismo , Masculino , Ratones SCID , Proteína Quinasa 7 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 7 Activada por Mitógenos/genética , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Células-Madre Neurales/metabolismo , Proto-Oncogenes Mas , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas ras/genéticaRESUMEN
Diffuse intrinsic pontine glioma (DIPG), an infiltrative, high grade glioma (HGG) affecting young children, has the highest mortality rate of all pediatric cancers. Despite treatment, average survival is less than twelve months, and five-year survival under 5%. We previously detected increased expression of Tenascin-C (TNC) protein in DIPG cerebrospinal fluid and tumor tissue relative to normal specimens. TNC is an extracellular matrix (ECM) glycoprotein that mediates cell-matrix interactions, guides migrating neurons during normal brain development and is thought to maintain the periventricular stem cell niche in the developing brain. Tumor TNC expression is reported in adult glioma and other cancers. However, the pattern and effects of TNC expression in DIPG has not been previously explored. Here, we characterize TNC expression in patient derived pediatric supratentorial HGG (n = 3) and DIPG (n = 6) cell lines, as well as pediatric glioma tumor (n = 50) and normal brain tissue specimens (n = 3). We found tumor specific TNC gene and protein overexpression that directly correlated with higher tumor grade (WHO III and IV, p = 0.05), H3K27 M mutation (p = 0.012), shorter progression free survival (p = 0.034), and poorer overall survival (0.041) in association with these factors. TNC knockdown via lentiviral shRNA transfection of HGG (n = 1) and DIPG (n = 3) cell lines resulted in decreased cell proliferation, migration, and invasion in vitro (p < 0.01), while TNC cDNA transfection resulted in increased cell migration, invasion and proliferation (p < 0.01) as well as altered cell morphology in H3K27 M mutant DIPG lines. Whole transcriptome sequencing analysis (RNA-Seq) on DIPG (n = 3) and HGG (n = 2) cell lines after TNC cDNA, shRNA, and empty vector control transfection revealed the effects of TNC expression level on global gene expression profiles. Together, our findings reveal TNC expression in DIPG in association with H3K27 M mutation and VEGF signaling, and suggest that TNC may contribute to DIPG tumor phenotype, and serve as a clinically detectable biomarker for DIPG.