Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 13 de 13
Filter
Add more filters










Publication year range
1.
bioRxiv ; 2023 Jun 13.
Article in English | MEDLINE | ID: mdl-37398299

ABSTRACT

Pediatric high-grade gliomas (pHGGs) are diffuse and highly aggressive CNS tumors which remain incurable, with a 5-year overall survival of less than 20%. Within glioma, mutations in the genes encoding the histones H3.1 and H3.3 have been discovered to be age-restricted and specific of pHGGs. This work focuses on the study of pHGGs harboring the H3.3-G34R mutation. H3.3-G34R tumors represent the 9-15% of pHGGs, are restricted to the cerebral hemispheres, and are found predominantly in the adolescent population (median 15.0 years). We have utilized a genetically engineered immunocompetent mouse model for this subtype of pHGG generated via the Sleeping Beauty-transposon system. The analysis of H3.3-G34R genetically engineered brain tumors by RNA-Sequencing and ChIP-Sequencing revealed alterations in the molecular landscape associated to H3.3-G34R expression. In particular, the expression of H3.3-G34R modifies the histone marks deposited at the regulatory elements of genes belonging to the JAK/STAT pathway, leading to an increased activation of this pathway. This histone G34R-mediated epigenetic modifications lead to changes in the tumor immune microenvironment of these tumors, towards an immune-permissive phenotype, making these gliomas susceptible to TK/Flt3L immune-stimulatory gene therapy. The application of this therapeutic approach increased median survival of H3.3-G34R tumor bearing animals, while stimulating the development of anti-tumor immune response and immunological memory. Our data suggests that the proposed immune-mediated gene therapy has potential for clinical translation for the treatment of patients harboring H3.3-G34R high grade gliomas.

2.
STAR Protoc ; 1(3): 100165, 2020 12 18.
Article in English | MEDLINE | ID: mdl-33377059

ABSTRACT

Brainstem gliomas are aggressive tumors that are more prevalent in pediatric patients. The location of these tumors makes them inoperable, and currently there is no effective treatment. Recent genomic data revealed the unique biology of these tumors. The following protocol provides a method to incorporate these specific genetic lesions in a mouse glioma model. Using this model, the effects of these mutations in tumor progression and response to treatments can be studied within a relevant in vivo context. For complete details on the use and execution of this protocol, please refer to Mendez et al. (2020).


Subject(s)
Brain Stem Neoplasms/pathology , Genetic Engineering/methods , Glioma/pathology , Animals , Breeding , Disease Models, Animal , Humans , Mice, Inbred C57BL , Mice, Transgenic , Neoplasm Grading , Spheroids, Cellular/pathology , Transposases/metabolism , Tumor Cells, Cultured
3.
Clin Cancer Res ; 26(15): 4080-4092, 2020 08 01.
Article in English | MEDLINE | ID: mdl-32332014

ABSTRACT

PURPOSE: Diffuse intrinsic pontine glioma (DIPG) bears a dismal prognosis. A genetically engineered brainstem glioma model harboring the recurrent DIPG mutation, Activin A receptor type I (ACVR1)-G328V (mACVR1), was developed for testing an immune-stimulatory gene therapy. EXPERIMENTAL DESIGN: We utilized the Sleeping Beauty transposase system to generate an endogenous mouse model of mACVR1 brainstem glioma. Histology was used to characterize and validate the model. We performed RNA-sequencing analysis on neurospheres harboring mACVR1. mACVR1 neurospheres were implanted into the pons of immune-competent mice to test the therapeutic efficacy and toxicity of immune-stimulatory gene therapy using adenoviruses expressing thymidine kinase (TK) and fms-like tyrosine kinase 3 ligand (Flt3L). mACVR1 neurospheres expressing the surrogate tumor antigen ovalbumin were generated to investigate whether TK/Flt3L treatment induces the recruitment of tumor antigen-specific T cells. RESULTS: Histologic analysis of mACVR1 tumors indicates that they are localized in the brainstem and have increased downstream signaling of bone morphogenetic pathway as demonstrated by increased phospho-smad1/5 and Id1 levels. Transcriptome analysis of mACVR1 neurosphere identified an increase in the TGFß signaling pathway and the regulation of cell differentiation. Adenoviral delivery of TK/Flt3L in mice bearing brainstem gliomas resulted in antitumor immunity, recruitment of antitumor-specific T cells, and increased median survival (MS). CONCLUSIONS: This study provides insights into the phenotype and function of the tumor immune microenvironment in a mouse model of brainstem glioma harboring mACVR1. Immune-stimulatory gene therapy targeting the hosts' antitumor immune response inhibits tumor progression and increases MS of mice bearing mACVR1 tumors.


Subject(s)
Brain Stem Neoplasms/therapy , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Glioma/therapy , Immunotherapy/methods , Activin Receptors, Type I/genetics , Animals , Brain Stem Neoplasms/genetics , Brain Stem Neoplasms/immunology , Brain Stem Neoplasms/pathology , Disease Models, Animal , Female , Genetic Vectors/genetics , Glioma/genetics , Glioma/immunology , Glioma/pathology , Humans , Male , Membrane Proteins/genetics , Mice , Mice, Transgenic , Mutation , Pons/pathology , Primary Cell Culture , RNA-Seq , Signal Transduction/genetics , Signal Transduction/immunology , Spheroids, Cellular , Thymidine Kinase/genetics , Transforming Growth Factor beta/metabolism , Tumor Cells, Cultured/transplantation , Tumor Microenvironment/genetics , Tumor Microenvironment/immunology
4.
Neuro Oncol ; 22(2): 195-206, 2020 02 20.
Article in English | MEDLINE | ID: mdl-32078691

ABSTRACT

Diffuse intrinsic pontine glioma (DIPG) is a rare but deadly pediatric brainstem tumor. To date, there is no effective therapy for DIPG. Transcriptomic analyses have revealed DIPGs have a distinct profile from other pediatric high-grade gliomas occurring in the cerebral hemispheres. These unique genomic characteristics coupled with the younger median age group suggest that DIPG has a developmental origin. The most frequent mutation in DIPG is a lysine to methionine (K27M) mutation that occurs on H3F3A and HIST1H3B/C, genes encoding histone variants. The K27M mutation disrupts methylation by polycomb repressive complex 2 on histone H3 at lysine 27, leading to global hypomethylation. Histone 3 lysine 27 trimethylation is an important developmental regulator controlling gene expression. This review discusses the developmental and epigenetic mechanisms driving disease progression in DIPG, as well as the profound therapeutic implications of epigenetic programming.


Subject(s)
Brain Stem Neoplasms/genetics , Cellular Reprogramming/genetics , Chromatin/genetics , Diffuse Intrinsic Pontine Glioma/genetics , Epigenesis, Genetic/genetics , Animals , Child , Female , Gene Expression Regulation, Neoplastic/genetics , Histones/genetics , Humans , Male , Mutation
5.
Oncotarget ; 11(50): 4607-4612, 2020 Dec 15.
Article in English | MEDLINE | ID: mdl-33400737

ABSTRACT

Diffuse intrinsic pontine glioma (DIPG) is a rare brainstem tumor which carries a dismal prognosis. To date. there are no effective treatments for DIPG. Transcriptomic studies have shown that DIPGs have a distinct profile compared to hemispheric high-grade pediatric gliomas. These specific genomic features coupled with the younger median age group suggest that DIPG is of developmental origin. There is a major unmet need for novel effective therapeutic approaches for DIPG. Clinical and preclinical studies have expanded our understanding of the molecular pathways in this deadly disease. We have developed a genetically engineered brainstem glioma model harboring the recurrent DIPG mutation, activin A receptor type I (ACVR1)-G328V (mACVR1) using the sleeping beauty transposon system. DIPG neurospheres isolated from the genetically engineered mouse model were implanted into the pons of immune-competent mice to assess the therapeutic efficacy and toxicity of immunostimulatory gene therapy using adenoviruses expressing thymidine kinase (TK) and fms-like tyrosine kinase 3 ligand (Flt3L). Immunostimulatory adenoviral-mediated delivery of TK/Flt3L in mice bearing brainstem gliomas resulted in antitumor immunity, recruitment of antitumor-specific T cells, and improved median survival by stimulating the host antitumor immune response. Therapeutic efficacy of the immunostimulatory gene therapy strategy will be tested in the clinical arena in a Phase I clinical trial. We also discuss immunotherapeutic interventions currently being implemented in DIPG patients and discuss the profound therapeutic implications of immunotherapy for this patient populations.

6.
Sci Transl Med ; 11(479)2019 02 13.
Article in English | MEDLINE | ID: mdl-30760578

ABSTRACT

Patients with glioma whose tumors carry a mutation in isocitrate dehydrogenase 1 (IDH1R132H) are younger at diagnosis and live longer. IDH1 mutations co-occur with other molecular lesions, such as 1p/19q codeletion, inactivating mutations in the tumor suppressor protein 53 (TP53) gene, and loss-of-function mutations in alpha thalassemia/mental retardation syndrome X-linked gene (ATRX). All adult low-grade gliomas (LGGs) harboring ATRX loss also express the IDH1R132H mutation. The current molecular classification of LGGs is based, partly, on the distribution of these mutations. We developed a genetically engineered mouse model harboring IDH1R132H, TP53 and ATRX inactivating mutations, and activated NRAS G12V. Previously, we established that ATRX deficiency, in the context of wild-type IDH1, induces genomic instability, impairs nonhomologous end-joining DNA repair, and increases sensitivity to DNA-damaging therapies. In this study, using our mouse model and primary patient-derived glioma cultures with IDH1 mutations, we investigated the function of IDH1R132H in the context of TP53 and ATRX loss. We discovered that IDH1R132H expression in the genetic context of ATRX and TP53 gene inactivation (i) increases median survival in the absence of treatment, (ii) enhances DNA damage response (DDR) via epigenetic up-regulation of the ataxia-telangiectasia-mutated (ATM) signaling pathway, and (iii) elicits tumor radioresistance. Accordingly, pharmacological inhibition of ATM or checkpoint kinases 1 and 2, essential kinases in the DDR, restored the tumors' radiosensitivity. Translation of these findings to patients with IDH1132H glioma harboring TP53 and ATRX loss could improve the therapeutic efficacy of radiotherapy and, consequently, patient survival.


Subject(s)
DNA Damage/genetics , Epigenesis, Genetic , Glioma/genetics , Isocitrate Dehydrogenase/genetics , Mutation/genetics , Tumor Suppressor Proteins/genetics , Up-Regulation/genetics , Animals , Ataxia Telangiectasia Mutated Proteins/metabolism , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Cell Differentiation , DNA Methylation/genetics , DNA Repair/genetics , Disease Models, Animal , Gene Ontology , Genome , Glioma/pathology , Histones/metabolism , Humans , Mice , Oligodendroglia/pathology , Radiation Tolerance , Signal Transduction , Survival Analysis
7.
J Vis Exp ; (143)2019 01 09.
Article in English | MEDLINE | ID: mdl-30688315

ABSTRACT

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as biomarkers with applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. These analyses of biomarkers are also useful to characterize tumor neurospheres (NS) generated from glioma patients and glioma models. Tumor NS provide a valuable in vitro model to assess different features of the tumor from which they are derived and can more accurately mirror glioma biology. Here we describe a detailed method to analyze biomarkers in tumor NS using immunohistochemistry (IHC) on paraffin-embedded tumor NS.


Subject(s)
Biomarkers, Tumor/metabolism , Glioma/immunology , Immunohistochemistry/methods , Paraffin Embedding/methods , Animals , Brain Neoplasms/pathology , Disease Models, Animal , Glioma/pathology , Humans , Mice , Molecular Conformation , Prognosis
8.
Expert Opin Ther Targets ; 22(7): 599-613, 2018 07.
Article in English | MEDLINE | ID: mdl-29889582

ABSTRACT

INTRODUCTION: ATRX is a chromatin remodeling protein whose main function is the deposition of the histone variant H3.3. ATRX mutations are widely distributed in glioma, and correlate with alternative lengthening of telomeres (ALT) development, but they also affect other cellular functions related to epigenetic regulation. Areas covered: We discuss the main molecular characteristics of ATRX, from its various functions in normal development to the effects of its loss in ATRX syndrome patients and animal models. We focus on the salient consequences of ATRX mutations in cancer, from a clinical to a molecular point of view, focusing on both adult and pediatric glioma. Finally, we will discuss the therapeutic opportunities future research perspectives. Expert opinion: ATRX is a major component of various essential cellular pathways, exceeding its functions as a histone chaperone (e.g. DNA replication and repair, chromatin higher-order structure regulation, gene transcriptional regulation, etc.). However, it is unclear how the loss of these functions in ATRX-null cancer cells affects cancer development and progression. We anticipate new treatments and clinical approaches will emerge for glioma and other cancer types as mechanistic and molecular studies on ATRX are only just beginning to reveal the many critical functions of this protein in cancer.


Subject(s)
Glioma/genetics , Mental Retardation, X-Linked/genetics , X-linked Nuclear Protein/genetics , alpha-Thalassemia/genetics , Adult , Animals , Child , Chromatin Assembly and Disassembly/genetics , Epigenesis, Genetic , Glioma/pathology , Glioma/therapy , Humans , Mental Retardation, X-Linked/physiopathology , Mutation , Telomere Homeostasis , alpha-Thalassemia/physiopathology
9.
J Vis Exp ; (131)2018 01 29.
Article in English | MEDLINE | ID: mdl-29443090

ABSTRACT

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory regions of oncogenes and tumor suppressors can lead to changes in gene expression and play an important role in the pathogenesis of brain tumors. Native chromatin immunoprecipitation (ChIP) is a popular technique that allows the detection of modifications or other proteins tightly bound to DNA. In contrast to cross-linked ChIP, in native ChIP, cells are not treated with formaldehyde to covalently link protein to DNA. This is advantageous because sometimes crosslinking may fix proteins that only transiently interact with DNA and do not have functional significance in gene regulation. In addition, antibodies are generally raised against unfixed peptides. Therefore, antibody specificity is increased in native ChIP. However, it is important to keep in mind that native ChIP is only applicable to study histones or other proteins that bind tightly to DNA. This protocol describes the native chromatin immunoprecipitation on murine brain tumor neurospheres.


Subject(s)
Brain Neoplasms/immunology , Chromatin Immunoprecipitation/methods , Chromatin/immunology , DNA/immunology , Epigenomics/methods , Histone Code/genetics , Animals , DNA/metabolism , Humans , Mice
10.
Cancer Res ; 77(2): 227-233, 2017 01 15.
Article in English | MEDLINE | ID: mdl-28062403

ABSTRACT

Genes encoding proteins that regulate chromatin structure and DNA modifications [i.e., chromatin regulatory factors (CRF)] and genes encoding histone proteins harbor recurrent mutations in most human cancers. These mutations lead to modifications in tumor chromatin and DNA structure and an altered epigenetic state that contribute to tumorigenesis. Mutated CRFs have now been identified in most types of cancer and are increasingly regarded as novel therapeutic targets. In this review, we discuss DNA alterations in CRFs and how these influence tumor chromatin structure and function, which in turn leads to tumorigenesis. We also discuss the clinical implications and review concepts of targeted treatments for these mutations. Continued research on CRF mutations will be critical for our future understanding of cancer biology and the development and implementation of novel cancer therapies. Cancer Res; 77(2); 227-33. ©2017 AACR.


Subject(s)
Cell Transformation, Neoplastic/genetics , Chromatin/genetics , Epigenesis, Genetic/genetics , Gene Expression Regulation, Neoplastic/physiology , Neoplasms/genetics , Animals , Chromatin Assembly and Disassembly/genetics , Histones , Humans , Neoplasms/pathology
11.
Gut ; 66(1): 124-136, 2017 01.
Article in English | MEDLINE | ID: mdl-27402485

ABSTRACT

BACKGROUND: Pancreatic cancer is characterised by the accumulation of a fibro-inflammatory stroma. Within this stromal reaction, myeloid cells are a predominant population. Distinct myeloid subsets have been correlated with tumour promotion and unmasking of anti-tumour immunity. OBJECTIVE: The goal of this study was to determine the effect of myeloid cell depletion on the onset and progression of pancreatic cancer and to understand the relationship between myeloid cells and T cell-mediated immunity within the pancreatic cancer microenvironment. METHODS: Primary mouse pancreatic cancer cells were transplanted into CD11b-diphtheria toxin receptor (DTR) mice. Alternatively, the iKras* mouse model of pancreatic cancer was crossed into CD11b-DTR mice. CD11b+ cells (mostly myeloid cell population) were depleted by diphtheria toxin treatment during tumour initiation or in established tumours. RESULTS: Depletion of myeloid cells prevented KrasG12D-driven pancreatic cancer initiation. In pre-established tumours, myeloid cell depletion arrested tumour growth and in some cases, induced tumour regressions that were dependent on CD8+ T cells. We found that myeloid cells inhibited CD8+ T-cell anti-tumour activity by inducing the expression of programmed cell death-ligand 1 (PD-L1) in tumour cells in an epidermal growth factor receptor (EGFR)/mitogen-activated protein kinases (MAPK)-dependent manner. CONCLUSION: Our results show that myeloid cells support immune evasion in pancreatic cancer through EGFR/MAPK-dependent regulation of PD-L1 expression on tumour cells. Derailing this crosstalk between myeloid cells and tumour cells is sufficient to restore anti-tumour immunity mediated by CD8+ T cells, a finding with implications for the design of immune therapies for pancreatic cancer.


Subject(s)
B7-H1 Antigen/metabolism , CD8-Positive T-Lymphocytes/immunology , Carcinoma, Pancreatic Ductal/immunology , Myeloid Cells/immunology , Pancreatic Neoplasms/immunology , Programmed Cell Death 1 Receptor/metabolism , Tumor Microenvironment/immunology , Animals , CD11b Antigen/analysis , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Cell Cycle Checkpoints , Cell Line, Tumor , Cell Transformation, Neoplastic/immunology , Cell Transformation, Neoplastic/metabolism , ErbB Receptors/metabolism , Humans , Immune Tolerance , Immunity, Cellular , Lymphocyte Activation , Lymphocytes, Tumor-Infiltrating , MAP Kinase Signaling System , Mice , Mitogen-Activated Protein Kinases/metabolism , Myeloid Cells/chemistry , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Proto-Oncogene Proteins p21(ras)/genetics , Tumor Escape
12.
Dev Dyn ; 246(1): 7-27, 2017 01.
Article in English | MEDLINE | ID: mdl-27761977

ABSTRACT

BACKGROUND: To send meaningful information to the brain, an inner ear cochlear implant (CI) must become closely coupled to as large and healthy a population of remaining spiral ganglion neurons (SGN) as possible. Inner ear gangliogenesis depends on macrophage migration inhibitory factor (MIF), a directionally attractant neurotrophic cytokine made by both Schwann and supporting cells (Bank et al., 2012). MIF-induced mouse embryonic stem cell (mESC)-derived "neurons" could potentially substitute for lost or damaged SGN. mESC-derived "Schwann cells" produce MIF, as do all Schwann cells (Huang et al., a; Roth et al., 2007; Roth et al., 2008) and could attract SGN to a "cell-coated" implant. RESULTS: Neuron- and Schwann cell-like cells were produced from a common population of mESCs in an ultra-slow-flow microfluidic device. As the populations interacted, "neurons" grew over the "Schwann cell" lawn, and early events in myelination were documented. Blocking MIF on the Schwann cell side greatly reduced directional neurite outgrowth. MIF-expressing "Schwann cells" were used to coat a CI: Mouse SGN and MIF-induced "neurons" grew directionally to the CI and to a wild-type but not MIF-knockout organ of Corti explant. CONCLUSIONS: Two novel stem cell-based approaches for treating the problem of sensorineural hearing loss are described. Developmental Dynamics 246:7-27, 2017. © 2016 Wiley Periodicals, Inc.


Subject(s)
Cell Differentiation , Lab-On-A-Chip Devices/standards , Mouse Embryonic Stem Cells/cytology , Neurons/cytology , Schwann Cells/cytology , Animals , Cochlear Implants/standards , Hearing Loss/therapy , Intramolecular Oxidoreductases/physiology , Macrophage Migration-Inhibitory Factors/physiology , Mice , Myelin Sheath/metabolism , Spiral Ganglion
13.
Sci Transl Med ; 8(328): 328ra28, 2016 Mar 02.
Article in English | MEDLINE | ID: mdl-26936505

ABSTRACT

Recent work in human glioblastoma (GBM) has documented recurrent mutations in the histone chaperone protein ATRX. We developed an animal model of ATRX-deficient GBM and showed that loss of ATRX reduces median survival and increases genetic instability. Further, analysis of genome-wide data for human gliomas showed that ATRX mutation is associated with increased mutation rate at the single-nucleotide variant (SNV) level. In mouse tumors, ATRX deficiency impairs nonhomologous end joining and increases sensitivity to DNA-damaging agents that induce double-stranded DNA breaks. We propose that ATRX loss results in a genetically unstable tumor, which is more aggressive when left untreated but is more responsive to double-stranded DNA-damaging agents, resulting in improved overall survival.


Subject(s)
Brain Neoplasms/pathology , DNA End-Joining Repair , DNA Helicases/deficiency , Glioma/pathology , Nuclear Proteins/deficiency , Animals , Brain Neoplasms/genetics , Cell Proliferation , Chromosomes, Mammalian/genetics , DNA Copy Number Variations/genetics , DNA Damage , DNA Helicases/genetics , DNA Helicases/metabolism , Disease Models, Animal , Glioma/genetics , Humans , Mice , Microsatellite Instability , Mutation/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Polymorphism, Single Nucleotide/genetics , Survival Analysis , Telomere Homeostasis , Transposases/metabolism , X-linked Nuclear Protein
SELECTION OF CITATIONS
SEARCH DETAIL
...