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Development of effective targeted cancer therapies is fundamentally limited by our molecular understanding of disease pathogenesis. Diffuse intrinsic pontine glioma (DIPG) is a fatal malignancy of the childhood pons characterized by a unique substitution to methionine in histone H3 at lysine 27 (H3K27M) that results in globally altered epigenetic marks and oncogenic transcription. Through primary DIPG tumor characterization and isogenic oncohistone expression, we show that the same H3K27M mutation displays distinct modes of oncogenic reprogramming and establishes distinct enhancer architecture depending upon both the variant of histone H3 and the cell context in which the mutation occurs. Compared with non-malignant pediatric pontine tissue, we identify and functionally validate both shared and variant-specific pathophysiology. Altogether, we provide a powerful resource of epigenomic data in 25 primary DIPG samples and 5 rare normal pediatric pontine tissue samples, revealing clinically relevant functional distinctions previously unidentified in DIPG.
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Glioma Pontino Intrínseco Difuso/genética , Histonas/genética , Encéfalo/patologia , Neoplasias Encefálicas/genética , Reprogramação Celular/genética , Glioma Pontino Intrínseco Difuso/metabolismo , Elementos Facilitadores Genéticos/genética , Epigênese Genética/genética , Epigenômica , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Glioma/genética , Glioma/metabolismo , Humanos , Lisina/genética , Mutação/genética , Ponte/metabolismo , Transdução de Sinais , Transcriptoma/fisiologiaRESUMO
The molecular characteristics of pediatric brain tumors have not only allowed for tumor subgrouping but have led to the introduction of novel treatment options for patients with specific tumor alterations. Therefore, an accurate histologic and molecular diagnosis is critical for optimized management of all pediatric patients with brain tumors, including central nervous system embryonal tumors. We present a case where optical genome mapping identified a ZNF532::NUTM1 fusion in a patient with a unique tumor best characterized histologically as a central nervous system embryonal tumor with rhabdoid features. Additional analyses including immunohistochemistry for NUT protein, methylation array, whole genome, and RNA-sequencing was done to confirm the presence of the fusion in the tumor. This is the first description of a pediatric patient with a ZNF532::NUTM1 fusion, yet the histology of this tumor is similar to that of adult cancers with ZNF::NUTM1 fusions reported in the literature. Although rare, the distinct pathology and underlying molecular characteristics of the ZNF532::NUTM1 tumor separates this from other embryonal tumors. Therefore, screening for this or similar NUTM1 rearrangements should be considered for all patients with unclassified central nervous system tumors with rhabdoid features to ensure accurate diagnosis. Ultimately, with additional cases, we may be able to better inform therapeutic management for these patients. © 2023 The Pathological Society of Great Britain and Ireland.
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Neoplasias Encefálicas , Neoplasias Embrionárias de Células Germinativas , Proteínas de Fusão Oncogênica , Tumor Rabdoide , Criança , Humanos , Neoplasias Encefálicas/genética , Mapeamento Cromossômico , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição/genética , Tumor Rabdoide/genética , Neoplasias Embrionárias de Células Germinativas/genéticaRESUMO
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Diffuse intrinsic pontine glioma (DIPG) is a highly morbid form of pediatric brainstem glioma. Here, we present the first comprehensive protein, mRNA, and methylation profiles of fresh-frozen DIPG specimens (n = 14), normal brain tissue (n = 10), and other pediatric brain tumors (n = 17). Protein profiling identified 2,305 unique proteins indicating distinct DIPG protein expression patterns compared to other pediatric brain tumors. Western blot and immunohistochemistry validated upregulation of Clusterin (CLU), Elongation Factor 2 (EF2), and Talin-1 (TLN1) in DIPGs studied. Comparisons to mRNA expression profiles generated from tumor and adjacent normal brain tissue indicated two DIPG subgroups, characterized by upregulation of Myc (N-Myc) or Hedgehog (Hh) signaling. We validated upregulation of PTCH, a membrane receptor in the Hh signaling pathway, in a subgroup of DIPG specimens. DNA methylation analysis indicated global hypomethylation of DIPG compared to adjacent normal tissue specimens, with differential methylation of 24 genes involved in Hh and Myc pathways, correlating with protein and mRNA expression patterns. Sequencing analysis showed c.83A>T mutations in the H3F3A or HIST1H3B gene in 77 % of our DIPG cohort. Supervised analysis revealed a unique methylation pattern in mutated specimens compared to the wild-type DIPG samples. This study presents the first comprehensive multidimensional protein, mRNA, and methylation profiling of pediatric brain tumor specimens, detecting the presence of two subgroups within our DIPG cohort. This multidimensional analysis of DIPG provides increased analytical power to more fully explore molecular signatures of DIPGs, with implications for evaluating potential molecular subtypes and biomarker discovery for assessing response to therapy.
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Neoplasias do Tronco Encefálico/metabolismo , Encéfalo/metabolismo , Glioma/metabolismo , Adolescente , Adulto , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias do Tronco Encefálico/genética , Criança , Pré-Escolar , Estudos de Coortes , Metilação de DNA , Feminino , Perfilação da Expressão Gênica , Glioma/genética , Histonas/genética , Histonas/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Proteômica , RNA Mensageiro/metabolismo , Adulto JovemRESUMO
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 do Tronco Encefálico , DNA Tumoral Circulante , Glioma Pontino Intrínseco Difuso , Glioma , Biologia , Biomarcadores , Neoplasias do Tronco Encefálico/genética , Neoplasias do Tronco Encefálico/metabolismo , Neoplasias do Tronco Encefálico/terapia , Criança , DNA Tumoral Circulante/genética , Glioma Pontino Intrínseco Difuso/genética , Feminino , Instabilidade Genômica , Glioma/genética , Glioma/metabolismo , Glioma/terapia , Humanos , Adulto JovemRESUMO
BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor. Most DIPGs harbor a histone H3 mutation, which alters histone post-translational modification (PTM) states and transcription. Here, we employed quantitative proteomic analysis to elucidate the impact of the H3.3K27M mutation, as well as radiation and bromodomain inhibition (BRDi) with JQ1, on DIPG PTM profiles. METHODS: We performed targeted mass spectrometry on H3.3K27M mutant and wild-type tissues (n = 12) and cell lines (n = 7). RESULTS: We found 29.2 and 26.4% of total H3.3K27 peptides were H3.3K27M in mutant DIPG tumor cell lines and tissue specimens, respectively. Significant differences in modification states were observed in H3.3K27M specimens, including at H3K27, H3K36, and H4K16. In addition, H3.3K27me1 and H4K16ac were the most significantly distinct modifications in H3.3K27M mutant tumors, relative to wild-type. Further, H3.3K36me2 was the most abundant co-occurring modification on the H3.3K27M mutant peptide in DIPG tissue, while H4K16ac was the most acetylated residue. Radiation treatment caused changes in PTM abundance in vitro, including increased H3K9me3. JQ1 treatment resulted in increased mono- and di-methylation of H3.1K27, H3.3K27, H3.3K36 and H4K20 in vitro. CONCLUSION: Taken together, our findings provide insight into the effects of the H3K27M mutation on histone modification states and response to treatment, and suggest that H3K36me2 and H4K16ac may represent unique tumor epigenetic signatures for targeted DIPG therapy.
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Neoplasias do Tronco Encefálico/genética , Glioma Pontino Intrínseco Difuso/genética , Epigenômica/métodos , Perfilação da Expressão Gênica/métodos , Histonas/metabolismo , Neoplasias do Tronco Encefálico/patologia , Glioma Pontino Intrínseco Difuso/patologia , Feminino , Humanos , MasculinoRESUMO
BACKGROUND: Glioblastoma (GBM) remains one of the least successfully treated cancers. It is essential to understand the basic biology of this lethal disease and investigate novel pharmacological targets to treat GBM. The aims of this study were to determine the biological consequences of elevated expression of ΔNp73, an N-terminal truncated isoform of TP73, and to evaluate targeting of its downstream mediators, the angiopoietin 1 (ANGPT1)/tunica interna endothelial cell kinase 2 (Tie2) axis, by using a highly potent, orally available small-molecule inhibitor (rebastinib) in GBM. METHODS: ΔNp73 expression was assessed in glioma sphere cultures, xenograft glioblastoma tumors, and glioblastoma patients by western blot, quantitative reverse transcription PCR, and immunohistochemistry. Immunoprecipitation, chromatin immunoprecipitation (ChiP) and sequential ChIP were performed to determine the interaction between ΔNp73 and E26 transformation-specific (ETS) proto-oncogene 2 (ETS2) proteins. The oncogenic consequences of ΔNp73 expression in glioblastomas were examined by in vitro and in vivo experiments, including orthotopic zebrafish and mouse intracranial-injection models. Effects of rebastinib on growth of established tumors and survival were examined in an intracranial-injection mouse model. RESULTS: ΔNp73 upregulates both ANGPT1 and Tie2 transcriptionally through ETS conserved binding sites on the promoters by interacting with ETS2. Elevated expression of ΔNp73 promotes tumor progression by mediating angiogenesis and survival. Therapeutic targeting of downstream ΔNp73 signaling pathways by rebastinib inhibits growth of established tumors and extends survival in preclinical models of glioblastoma. CONCLUSION: Aberrant expression of ΔNp73 in GBM promotes tumor progression through autocrine and paracrine signaling dependent on Tie2 activation by ANGPT1. Disruption of this signaling by rebastinib improves tumor response to treatment in glioblastoma.
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Antineoplásicos/administração & dosagem , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Proteína Proto-Oncogênica c-ets-2/metabolismo , Pirazóis/administração & dosagem , Piridinas/administração & dosagem , Quinolinas/administração & dosagem , Proteína Tumoral p73/metabolismo , Animais , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral/efeitos dos fármacos , Modelos Animais de Doenças , Glioblastoma/tratamento farmacológico , Humanos , Camundongos Transgênicos , Neovascularização Patológica/metabolismo , Proto-Oncogene Mas , Análise de Sobrevida , Peixe-ZebraRESUMO
Children diagnosed with brain tumors have the lowest overall survival of all pediatric cancers. Recent molecular studies have resulted in the discovery of recurrent driver mutations in many pediatric brain tumors. However, despite these molecular advances, the clinical outcomes of high grade tumors, including H3K27M diffuse midline glioma (H3K27M DMG), remain poor. To address the paucity of tissue for biological studies, we have established a comprehensive protocol for the coordination and processing of donated specimens at postmortem. Since 2010, 60 postmortem pediatric brain tumor donations from 26 institutions were coordinated and collected. Patient derived xenograft models and cell cultures were successfully created (76% and 44% of attempts respectively), irrespective of postmortem processing time. Histological analysis of mid-sagittal whole brain sections revealed evidence of treatment response, immune cell infiltration and the migratory path of infiltrating H3K27M DMG cells into other midline structures and cerebral lobes. Sequencing of primary and disseminated tumors confirmed the presence of oncogenic driver mutations and their obligate partners. Our findings highlight the importance of postmortem tissue donations as an invaluable resource to accelerate research, potentially leading to improved outcomes for children with aggressive brain tumors.
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Biomarcadores Tumorais/genética , Neoplasias Encefálicas/patologia , Glioma/patologia , Histonas/genética , Mutação , Adolescente , Adulto , Animais , Autopsia , Neoplasias Encefálicas/genética , Criança , Pré-Escolar , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Humanos , Lactente , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto JovemRESUMO
Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.
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Líquidos Corporais/metabolismo , DNA Tumoral Circulante/metabolismo , Biomarcadores Tumorais/genética , Humanos , Limite de Detecção , Mutação , Reação em Cadeia da PolimeraseRESUMO
Diffuse intrinsic pontine gliomas (DIPGs) are deadly tumors comprising 10%-15% of all childhood CNS cancers. Standard treatment is considered palliative and prognosis is near universal mortality. DIPGs have been classified into genomic subtypes based on histone variants with the lysine to methionine mutation on position 27 of histone tails (K27M). Given the increasing promise of immunotherapy, there have been ongoing efforts to identify tumor-specific antigens to serve as immunologic targets. We evaluated a large cohort of CNS specimens for Wilms' tumor protein (WT1) expression. These specimens include primary pediatric CNS tumors (n = 38 midline gliomas and n = 3 non-midline gliomas; n = 23 DIPG, n = 10 low-grade gliomas, n = 8 high-grade gliomas), and DIPG primary cells. Here, we report the validation of WT1 as a tumor-associated antigen in DIPGs. We further report that WT1 expression is significantly correlated with specific oncohistone variants, with the highest expression detected in the H3.3K27M subgroup. WT1 expression was absent in all control specimens (n = 21). Western blot assays using DIPG primary cells (n = 6) showed a trend of higher WT1 expression in H3.3K27M cells when compared with H3.1 K27M cells and H3 wildtype cells. Our data are the first indication of the association between WT1 and DIPG, with specific upregulation in those harboring oncohistone H3.3K27M.
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Neoplasias do Tronco Encefálico/metabolismo , Glioma Pontino Intrínseco Difuso/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas WT1/biossíntese , Adolescente , Neoplasias do Tronco Encefálico/genética , Neoplasias do Tronco Encefálico/patologia , Criança , Pré-Escolar , Estudos de Coortes , Glioma Pontino Intrínseco Difuso/genética , Glioma Pontino Intrínseco Difuso/patologia , Feminino , Humanos , Lactente , Masculino , Mutação/genética , Proteínas WT1/genética , Adulto JovemRESUMO
Anaplastic astrocytomas are aggressive glial cancers that present poor prognosis and high recurrence. Heterozygous IDH1 R132H mutations are common in adolescent and young adult anaplastic astrocytomas. In a majority of cases, the IDH1 R132H mutation is unique to the tumor, although rare cases of anaplastic astrocytoma have been described in patients with mosaic IDH1 mutations (Ollier disease or Maffucci syndrome). Here, we present two siblings with IDH1 R132H mutant high grade astrocytomas diagnosed at 14 and 26 years of age. Analysis of IDHR132H mutations in the siblings' tumors and non-neoplastic tissues, including healthy regions of the brain, cheek cells, and primary teeth indicate mosaicism of IDHR132H. Whole exome sequencing of the tumor tissue did not reveal any other common mutations between the two siblings. This study demonstrates the first example of IDH1 R132H mosaicism, acquired during early development, that provides an alternative mechanism of cancer predisposition.
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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 do Tronco Encefálico/metabolismo , Glioma Pontino Intrínseco Difuso/metabolismo , Mutação , Proteínas ras/metabolismo , Compostos de Anilina/farmacologia , Animais , Neoplasias do Tronco Encefálico/tratamento farmacológico , Neoplasias do Tronco Encefálico/genética , Neoplasias do Tronco Encefálico/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Glioma Pontino Intrínseco Difuso/tratamento farmacológico , Glioma Pontino Intrínseco Difuso/genética , Glioma Pontino Intrínseco Difuso/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Genes myc , Histonas/genética , Histonas/metabolismo , Humanos , Indóis/farmacologia , Lisina/genética , Lisina/metabolismo , Masculino , Camundongos SCID , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 7 Ativada por Mitógeno/genética , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Células-Tronco Neurais/metabolismo , Proto-Oncogene Mas , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas ras/genéticaRESUMO
PURPOSE: Pediatric diffuse midline glioma (DMG) are highly malignant tumors with poor clinical outcomes. Over 70% of patients with DMG harbor the histone 3 p.K27M (H3K27M) mutation, which correlates with a poorer clinical outcome, and is also used as a criterion for enrollment in clinical trials. Because complete surgical resection of DMG is not an option, biopsy at presentation is feasible, but rebiopsy at time of progression is rare. While imaging and clinical-based disease monitoring is the standard of care, molecular-based longitudinal characterization of these tumors is almost nonexistent. To overcome these hurdles, we examined whether liquid biopsy allows measurement of disease response to precision therapy. EXPERIMENTAL DESIGN: We established a sensitive and specific methodology that detects major driver mutations associated with pediatric DMGs using droplet digital PCR (n = 48 subjects, n = 110 specimens). Quantification of circulating tumor DNA (ctDNA) for H3K27M was used for longitudinal assessment of disease response compared with centrally reviewed MRI data. RESULTS: H3K27M was identified in cerebrospinal fluid (CSF) and plasma in 88% of patients with DMG, with CSF being the most enriched for ctDNA. We demonstrated the feasibility of multiplexing for detection of H3K27M, and additional driver mutations in patient's tumor and matched CSF, maximizing the utility of a single source of liquid biome. A significant decrease in H3K27M plasma ctDNA agreed with MRI assessment of tumor response to radiotherapy in 83% (10/12) of patients. CONCLUSIONS: Our liquid biopsy approach provides a molecularly based tool for tumor characterization, and is the first to indicate clinical utility of ctDNA for longitudinal surveillance of DMGs.
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Biomarcadores Tumorais , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/epidemiologia , Glioma/diagnóstico , Glioma/epidemiologia , Biópsia Líquida , Fatores Etários , Neoplasias Encefálicas/líquido cefalorraquidiano , Ácidos Nucleicos Livres , DNA Tumoral Circulante , Glioma/líquido cefalorraquidiano , Humanos , Biópsia Líquida/métodos , Imageamento por Ressonância Magnética , Técnicas de Diagnóstico Molecular , Mutação , Estadiamento de Neoplasias , Vigilância em Saúde PúblicaRESUMO
Corticosteroids, such as dexamethasone, are routinely used as palliative care in neuro-oncology for their anti-inflammatory benefits, however many patients experience dose limiting side effects caused by glucocorticoid response element (GRE)-mediated transcription. The purpose of this study was to use a murine model to investigate a new steroid alternative, vamorolone, which promises to reduce side effects through dissociating GRE-mediated transcription and NF-κB -mediated anti-inflammatory actions. To compare vamorolone to dexamethasone in reducing pro-inflammatory signals in vitro, murine glioma cells were treated with dexamethasone, vamorolone or vehicle control. Changes in mRNA expression were assessed using the nanostring inflammatory platform. Furthermore, drug efficacy, post-treatment behavioral activity and side effects were assessed by treating two cohorts of brain tumor bearing mice with dexamethasone, vamorolone, or vehicle control. Our investigation showed that treatment with vamorolone resulted in a reduction of pro-inflammatory signals in tumor cells in vitro similar to treatment with dexamethasone. Treatment with vamorolone resulted in a better safety profile in comparison to dexamethasone treatment. Vamorolone- treated mice showed similar or better activity and survival when compared to dexamethasone-treated mice. Our data indicate vamorolone is a potential steroid-sparing alternative for treating patients with brain tumors.
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Anti-Inflamatórios/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Citocinas/metabolismo , Dexametasona/farmacologia , Glioma/tratamento farmacológico , Mediadores da Inflamação/metabolismo , Esteroides/farmacologia , Animais , Anti-Inflamatórios/toxicidade , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Citocinas/genética , Dexametasona/toxicidade , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Camundongos Endogâmicos NOD , Camundongos SCID , Atividade Motora/efeitos dos fármacos , NF-kappa B/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Diffuse intrinsic pontine glioma (DIPG) is an invasive and treatment-refractory pediatric brain tumor. Primary DIPG tumors harbor a number of mutations including alterations in PTEN, AKT, and PI3K and exhibit activation of mammalian Target of Rapamycin Complex 1 and 2 (mTORC1/2). mTORC1/2 regulate protein translation, cell growth, survival, invasion, and metabolism. Pharmacological inhibition of mTORC1 is minimally effective in DIPG. However, the activity of dual TORC kinase inhibitors has not been examined in this tumor type. Nanomolar levels of the mTORC1/2 inhibitor TAK228 reduced expression of p-AKTS473 and p-S6S240/244 and suppressed the growth of DIPG lines JHH-DIPG1, SF7761, and SU-DIPG-XIII. TAK228 induced apoptosis in DIPG cells and cooperated with radiation to further block proliferation and enhance apoptosis. TAK228 monotherapy inhibited the tumorigenicity of a murine orthotopic model of DIPG, more than doubling median survival (p = 0.0017) versus vehicle. We conclude that dual mTOR inhibition is a promising potential candidate for DIPG treatment.
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Benzoxazóis/farmacologia , Neoplasias do Tronco Encefálico/terapia , Quimiorradioterapia , Glioma/terapia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Neoplasias do Tronco Encefálico/enzimologia , Neoplasias do Tronco Encefálico/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Relação Dose-Resposta a Droga , Glioma/enzimologia , Glioma/patologia , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos Endogâmicos NOD , Camundongos SCID , Complexos Multiproteicos/metabolismo , Invasividade Neoplásica , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
There is no curative treatment for patients with diffuse intrinsic pontine glioma (DIPG). However, with the recent availability of biopsy and autopsy tissue, new data regarding the biologic behavior of this tumor have emerged, allowing greater molecular characterization and leading to investigations which may result in improved therapeutic options. Treatment strategies must address both primary disease sites as well as any metastatic deposits, which may be variably sensitive to a particular approach.In this case report, we present a patient with DIPG treated with irradiation and serial investigational agents. The clinical, pathological and molecular phenotypes of both the progressive primary tumor as well as concomitant metastatic deposits obtained at autopsy are discussed. While some mRNA differences were demonstrated, all analyzed sites of disease shared similar mutational arrangements, suggesting that targeting the mutations of the primary tumor may be effective for all sites of disease.
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Neoplasias Encefálicas/patologia , Glioma/patologia , Autopsia , Biópsia , Neoplasias do Tronco Encefálico/patologia , Criança , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Mutação , Metástase Neoplásica , Fenótipo , Ponte/patologia , Análise de Componente Principal , RNA Mensageiro/metabolismo , Resultado do TratamentoRESUMO
Diffuse Intrinsic Pontine Gliomas (DIPGs) are deadly paediatric brain tumours where needle biopsies help guide diagnosis and targeted therapies. To address spatial heterogeneity, here we analyse 134 specimens from various neuroanatomical structures of whole autopsy brains from nine DIPG patients. Evolutionary reconstruction indicates histone 3 (H3) K27M--including H3.2K27M--mutations potentially arise first and are invariably associated with specific, high-fidelity obligate partners throughout the tumour and its spread, from diagnosis to end-stage disease, suggesting mutual need for tumorigenesis. These H3K27M ubiquitously-associated mutations involve alterations in TP53 cell-cycle (TP53/PPM1D) or specific growth factor pathways (ACVR1/PIK3R1). Later oncogenic alterations arise in sub-clones and often affect the PI3K pathway. Our findings are consistent with early tumour spread outside the brainstem including the cerebrum. The spatial and temporal homogeneity of main driver mutations in DIPG implies they will be captured by limited biopsies and emphasizes the need to develop therapies specifically targeting obligate oncohistone partnerships.
Assuntos
Neoplasias do Tronco Encefálico/genética , Carcinogênese/genética , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Histonas/genética , Mutação , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Autopsia , Mapeamento Encefálico , Tronco Encefálico/metabolismo , Tronco Encefálico/patologia , Neoplasias do Tronco Encefálico/metabolismo , Neoplasias do Tronco Encefálico/patologia , Carcinogênese/metabolismo , Carcinogênese/patologia , Cérebro/metabolismo , Cérebro/patologia , Criança , Classe Ia de Fosfatidilinositol 3-Quinase , Evolução Clonal , Glioma/metabolismo , Glioma/patologia , Histonas/metabolismo , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Proteína Fosfatase 2C , Transdução de Sinais , Técnicas Estereotáxicas , Fatores de Tempo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Diffuse intrinsic pontine glioma (DIPG) is one of the least understood and most deadly childhood cancers. Historically, there has been a paucity of DIPG specimens for molecular analysis. However, due to the generous participation of DIPG families in programs for postmortem specimen donation, there has been a recent surge in molecular analysis of newly available tumor specimens. Collaborative efforts to share data and tumor specimens have resulted in rapid discoveries in other pediatric brain tumors, such as medulloblastoma, and therefore have the potential to shed light on the biology of DIPG. Given the generous gift of postmortem tissue donation from DIPG patients, there is a need for standardized postmortem specimen accrual to facilitate rapid and effective multi-institutional molecular studies.We developed and implemented an autopsy protocol for rapid procurement, documenting and storing these specimens. Sixteen autopsies were performed throughout the United States and Canada and processed using a standard protocol and inventory method, including specimen imaging, fixation, snap freezing, orthotopic injection, or preservation. This allowed for comparative clinical and biological studies of rare postmortem DIPG tissue specimens, generation of in vivo and in vitro models of DIPG, and detailed records to facilitate collaborative analysis.
Assuntos
Autopsia/normas , Neoplasias do Tronco Encefálico/patologia , Glioma/patologia , Ponte/patologia , Manejo de Espécimes/normas , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Manejo de Espécimes/métodos , Obtenção de Tecidos e Órgãos/métodos , Obtenção de Tecidos e Órgãos/normasRESUMO
NOTCH regulates stem cells during normal development and stemlike cells in cancer, but the roles of NOTCH in the lethal pediatric brain tumor diffuse intrinsic pontine glioma (DIPG) remain unknown. Because DIPGs express stem cell factors such as SOX2 and MYCN, we hypothesized that NOTCH activity would be critical for DIPG growth. We determined that primary DIPGs expressed high levels of NOTCH receptors, ligands, and downstream effectors. Treatment of the DIPG cell lines JHH-DIPG1 and SF7761 with the γ-secretase inhibitor MRK003 suppressed the level of the NOTCH effectors HES1, HES4, and HES5; inhibited DIPG growth by 75%; and caused a 3-fold induction of apoptosis. Short hairpin RNAs targeting the canonical NOTCH pathway caused similar effects. Pretreatment of DIPG cells with MRK003 suppressed clonogenic growth by more than 90% and enhanced the efficacy of radiation therapy. The high level of MYCN in DIPG led us to test sequential therapy with the bromodomain inhibitor JQ1 and MRK003, and we found that JQ1 and MRK003 inhibited DIPG growth and induced apoptosis. Together, these results suggest that dual targeting of NOTCH and MYCN in DIPG may be an effective therapeutic strategy in DIPG and that adding a γ-secretase inhibitor during radiation therapy may be efficacious initially or during reirradiation.