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In this review, we discuss the molecular characteristics, development, evolution, and therapeutic perspectives for pediatric high-grade glioma (pHGG) arising in cerebral hemispheres. Recently, the understanding of biology of pHGG experienced a revolution with discoveries arising from genomic and epigenomic high-throughput profiling techniques. These findings led to identification of prevalent molecular alterations in pHGG and revealed a strong connection between epigenetic dysregulation and pHGG development. Although we are only beginning to unravel the molecular biology underlying pHGG, there is a desperate need to develop therapies that would improve the outcome of pHGG patients, as current therapies do not elicit significant improvement in median survival for this patient population. We explore the molecular and cell biology and clinical state-of-the-art of pediatric high-grade gliomas (pHGGs) arising in cerebral hemispheres. We discuss the role of driving mutations, with a special consideration of the role of epigenetic-disrupting mutations. We will also discuss the possibilities of targeting unique molecular vulnerabilities of hemispherical pHGG to design innovative tailored therapies.
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Biomarcadores de Tumor/antagonistas & inhibidores , Neoplasias Encefálicas/tratamiento farmacológico , Glioma/tratamiento farmacológico , Terapia Molecular Dirigida , Biomarcadores de Tumor/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Niño , Glioma/genética , Glioma/patología , Humanos , Clasificación del TumorRESUMEN
Recently, the cancer community has gained a heightened awareness of the roles of extrachromosomal DNA (ecDNA) in cancer proliferation, drug resistance and epigenetic remodeling. However, a hindrance to studying ecDNA is the lack of available cancer model systems that express ecDNA. Increasing our awareness of which model systems express ecDNA will advance our understanding of fundamental ecDNA biology and unlock a wealth of potential targeting strategies for ecDNA-driven cancers. To bridge this gap, we created CytoCellDB, a resource that provides karyotype annotations for cell lines within the Cancer Dependency Map (DepMap) and the Cancer Cell Line Encyclopedia (CCLE). We identify 139 cell lines that express ecDNA, a 200% increase from what is currently known. We expanded the total number of cancer cell lines with ecDNA annotations to 577, which is a 400% increase, covering 31% of cell lines in CCLE/DepMap. We experimentally validate several cell lines that we predict express ecDNA or homogeneous staining regions (HSRs). We demonstrate that CytoCellDB can be used to characterize aneuploidy alongside other molecular phenotypes, (gene essentialities, drug sensitivities, gene expression). We anticipate that CytoCellDB will advance cytogenomics research as well as provide insights into strategies for developing therapeutics that overcome ecDNA-driven drug resistance.
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Epigenetic remodeling is a molecular hallmark of gliomas, and it has been identified as a key mediator of glioma progression. Epigenetic dysregulation contributes to gliomagenesis, tumor progression, and responses to immunotherapies, as well as determining clinical features. This epigenetic remodeling includes changes in histone modifications, chromatin structure, and DNA methylation, all of which are driven by mutations in genes such as histone 3 genes (H3C1 and H3F3A), isocitrate dehydrogenase 1/2 (IDH1/2), α-thalassemia/mental retardation, X-linked (ATRX), and additional chromatin remodelers. Although much of the initial research primarily identified how the epigenetic aberrations impacted glioma progression by solely examining the glioma cells, recent studies have aimed at establishing the role of epigenetic alterations in shaping the tumor microenvironment (TME). In this review, we discuss the mechanisms by which these epigenetic phenomena in glioma remodel the TME and how current therapies targeting epigenetic dysregulation affect the glioma immune response and therapeutic outcomes. Understanding the link between epigenetic remodeling and the glioma TME provides insights into the implementation of epigenetic-targeting therapies to improve the antitumor immune response.
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Neoplasias Encefálicas , Glioma , Humanos , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/tratamiento farmacológico , Glioma/terapia , Glioma/tratamiento farmacológico , Mutación , Cromatina , Isocitrato Deshidrogenasa/genética , Epigénesis Genética , Microambiente Tumoral/genéticaRESUMEN
PURPOSE: Mutant isocitrate dehydrogenase 1 (mIDH1) alters the epigenetic regulation of chromatin, leading to a hypermethylation phenotype in adult glioma. This work focuses on identifying gene targets epigenetically dysregulated by mIDH1 to confer therapeutic resistance to ionizing radiation (IR). EXPERIMENTAL DESIGN: We evaluated changes in the transcriptome and epigenome in a radioresistant mIDH1 patient-derived glioma cell culture (GCC) following treatment with an mIDH1-specific inhibitor, AGI-5198. We identified Zinc Finger MYND-Type Containing 8 (ZMYND8) as a potential target of mIDH1 reprogramming. We suppressed ZMYND8 expression by shRNA knockdown and genetic knockout (KO) in mIDH1 glioma cells and then assessed cellular viability to IR. We assessed the sensitivity of mIDH1 GCCS to pharmacologic inhibition of ZMYND8-interacting partners: HDAC, BRD4, and PARP. RESULTS: Inhibition of mIDH1 leads to an upregulation of gene networks involved in replication stress. We found that the expression of ZMYND8, a regulator of DNA damage response, was decreased in three patient-derived mIDH1 GCCs after treatment with AGI-5198. Knockdown of ZMYND8 expression sensitized mIDH1 GCCs to radiotherapy marked by decreased cellular viability. Following IR, mIDH1 glioma cells with ZMYND8 KO exhibit significant phosphorylation of ATM and sustained γH2AX activation. ZMYND8 KO mIDH1 GCCs were further responsive to IR when treated with either BRD4 or HDAC inhibitors. PARP inhibition further enhanced the efficacy of radiotherapy in ZMYND8 KO mIDH1 glioma cells. CONCLUSIONS: These findings indicate the impact of ZMYND8 in the maintenance of genomic integrity and repair of IR-induced DNA damage in mIDH1 glioma. See related commentary by Sachdev et al., p. 1648.
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Glioma , Isocitrato Deshidrogenasa , Humanos , Isocitrato Deshidrogenasa/metabolismo , Dominios MYND , Epigénesis Genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Glioma/genética , Glioma/radioterapia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMEN
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.
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BACKGROUND: Epinotia aporema (Lepidoptera: Tortricidae) is an important pest of legume crops in South America. Epinotia aporema granulovirus (EpapGV) is a baculovirus that causes a polyorganotropic infection in the host larva. Its high pathogenicity and host specificity make EpapGV an excellent candidate to be used as a biological control agent. RESULTS: The genome of Epinotia aporema granulovirus (EpapGV) was sequenced and analyzed. Its circular double-stranded DNA genome is 119,082 bp in length and codes for 133 putative genes. It contains the 31 baculovirus core genes and a set of 19 genes that are GV exclusive. Seventeen ORFs were unique to EpapGV in comparison with other baculoviruses. Of these, 16 found no homologues in GenBank, and one encoded a thymidylate kinase. Analysis of nucleotide sequence repeats revealed the presence of 16 homologous regions (hrs) interspersed throughout the genome. Each hr was characterized by the presence of 1 to 3 clustered imperfect palindromes which are similar to previously described palindromes of tortricid-specific GVs. Also, one of the hrs (hr4) has flanking sequences suggestive of a putative non-hr ori. Interestingly, two more complex hrs were found in opposite loci, dividing the circular dsDNA genome in two halves. Gene synteny maps showed the great colinearity of sequenced GVs, being EpapGV the most dissimilar as it has a 20 kb-long gene block inversion. Phylogenetic study performed with 31 core genes of 58 baculoviral genomes suggests that EpapGV is the baculovirus isolate closest to the putative common ancestor of tortricid specific betabaculoviruses. CONCLUSIONS: This study, along with previous characterization of EpapGV infection, is useful for the better understanding of the pathology caused by this virus and its potential utilization as a bioinsecticide.
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Genoma Viral , Granulovirus/genética , Lepidópteros/virología , Nucleósido-Fosfato Quinasa/genética , Filogenia , Secuencia de Aminoácidos , Animales , ADN Viral/genética , Granulovirus/enzimología , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
Pediatric high-grade gliomas (pHGGs) are the leading cause of cancer-related deaths in children in the USA. Sixteen percent of hemispheric pediatric and young adult HGGs encode Gly34Arg/Val substitutions in the histone H3.3 (H3.3-G34R/V). The mechanisms by which H3.3-G34R/V drive malignancy and therapeutic resistance in pHGGs remain unknown. Using a syngeneic, genetically engineered mouse model (GEMM) and human pHGG cells encoding H3.3-G34R, we demonstrate that this mutation led to the downregulation of DNA repair pathways. This resulted in enhanced susceptibility to DNA damage and inhibition of the DNA damage response (DDR). We demonstrate that genetic instability resulting from improper DNA repair in G34R-mutant pHGG led to the accumulation of extrachromosomal DNA, which activated the cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway, inducing the release of immune-stimulatory cytokines. We treated H3.3-G34R pHGG-bearing mice with a combination of radiotherapy (RT) and DNA damage response inhibitors (DDRi) (i.e., the blood-brain barrier-permeable PARP inhibitor pamiparib and the cell-cycle checkpoint CHK1/2 inhibitor AZD7762), and these combinations resulted in long-term survival for approximately 50% of the mice. Moreover, the addition of a STING agonist (diABZl) enhanced the therapeutic efficacy of these treatments. Long-term survivors developed immunological memory, preventing pHGG growth upon rechallenge. These results demonstrate that DDRi and STING agonists in combination with RT induced immune-mediated therapeutic efficacy in G34-mutant pHGG.
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Neoplasias Encefálicas , Citocinas , Reparación del ADN , Glioma , Histonas , Proteínas de la Membrana , Nucleotidiltransferasas , Animales , Niño , Humanos , Ratones , Adulto Joven , Neoplasias Encefálicas/genética , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Glioma/genética , Histonas/genética , Inmunidad , Mutación , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Citocinas/inmunología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéuticoRESUMEN
Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is a radical effector molecule of the innate immune system that can directly inhibit pathogen replication. In order to study subsequent iNOS kidney expression in experimental leptospirosis, Golden Syrian hamsters and C3H/HeJ mice were infected intraperitoneally with 10(2) or 10(7) virulent Leptospira interrogans serovar Copenhageni (LIC) strain Fiocruz L1-130. Results showed increased levels of iNOS mRNA and protein in kidneys of infected animals when compared to that in mock-infected animals. To get a deeper insight into the role of iNOS in experimental leptospirosis, both subject species were treated or not treated with 4-aminopyridine (4-AP, 0.3mg/kg), an iNOS inhibitor. Treatment of infected hamsters with 4-AP accelerated the mortality rate to 100% by one day and increased the mortality rate from 20 to 60% in mice at 14 days post-infection. In kidney tissues, 4-AP treatment increased the bacterial burden, as demonstrated through leptospiral DNA quantification by real-time PCR, and aggravated tubulointerstitial nephritis. In addition, iNOS inhibition reduced the specific humoral response against LIC when compared to that in untreated infected animals. According to these results, iNOS expression and the resulting NO have an important role in leptospirosis.
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Leptospira interrogans/inmunología , Leptospirosis/inmunología , Óxido Nítrico Sintasa de Tipo II/inmunología , Animales , Carga Bacteriana , Cricetinae , Modelos Animales de Enfermedad , Expresión Génica , Perfilación de la Expresión Génica , Riñón/inmunología , Riñón/microbiología , Riñón/patología , Mesocricetus , Ratones , Ratones Endogámicos C3H , Nefritis/inmunología , Nefritis/microbiología , Nefritis/patología , Enfermedades de los Roedores/inmunología , Análisis de Supervivencia , Regulación hacia ArribaRESUMEN
High grade gliomas are malignant brain tumors that arise in the central nervous system, in patients of all ages. Currently, the standard of care, entailing surgery and chemo radiation, exhibits a survival rate of 14-17 months. Thus, there is an urgent need to develop new therapeutic strategies for these malignant brain tumors. Currently, immunotherapies represent an appealing approach to treat malignant gliomas, as the pre-clinical data has been encouraging. However, the translation of the discoveries from the bench to the bedside has not been as successful as with other types of cancer, and no long-lasting clinical benefits have been observed for glioma patients treated with immune-mediated therapies so far. This review aims to discuss our current knowledge about gliomas, their molecular particularities and the impact on the tumor immune microenvironment. Also, we discuss several murine models used to study these therapies pre-clinically and how the model selection can impact the outcomes of the approaches to be tested. Finally, we present different immunotherapy strategies being employed in clinical trials for glioma and the newest developments intended to harness the immune system against these incurable brain tumors.
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Gliomas are one of the most lethal types of cancers accounting for â¼80% of all central nervous system (CNS) primary malignancies. Among gliomas, glioblastomas (GBM) are the most aggressive, characterized by a median patient survival of fewer than 15 months. Recent molecular characterization studies uncovered the genetic signatures and methylation status of gliomas and correlate these with clinical prognosis. The most relevant molecular characteristics for the new glioma classification are IDH mutation, chromosome 1p/19q deletion, histone mutations, and other genetic parameters such as ATRX loss, TP53, and TERT mutations, as well as DNA methylation levels. Similar to other solid tumors, glioma progression is impacted by the complex interactions between the tumor cells and immune cells within the tumor microenvironment. The immune system's response to cancer can impact the glioma's survival, proliferation, and invasiveness. Salient characteristics of gliomas include enhanced vascularization, stimulation of a hypoxic tumor microenvironment, increased oxidative stress, and an immune suppressive milieu. These processes promote the neuro-inflammatory tumor microenvironment which can lead to the loss of blood-brain barrier (BBB) integrity. The consequences of a compromised BBB are deleteriously exposing the brain to potentially harmful concentrations of substances from the peripheral circulation, adversely affecting neuronal signaling, and abnormal immune cell infiltration; all of which can lead to disruption of brain homeostasis. In this review, we first describe the unique features of inflammation in CNS tumors. We then discuss the mechanisms of tumor-initiating neuro-inflammatory microenvironment and its impact on tumor invasion and progression. Finally, we also discuss potential pharmacological interventions that can be used to target neuro-inflammation in gliomas.
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Glioblastoma (GBM) is the most common and aggressive primary brain tumor in the adult population and it carries a dismal prognosis. Inefficient drug delivery across the blood brain barrier (BBB), an immunosuppressive tumor microenvironment (TME) and development of drug resistance are key barriers to successful glioma treatment. Since gliomas occur through sequential acquisition of genetic alterations, gene therapy, which enables to modification of the genetic make-up of target cells, appears to be a promising approach to overcome the obstacles encountered by current therapeutic strategies. Gene therapy is a rapidly evolving field with the ultimate goal of achieving specific delivery of therapeutic molecules using either viral or non-viral delivery vehicles. Gene therapy can also be used to enhance immune responses to tumor antigens, reprogram the TME aiming at blocking glioma-mediated immunosuppression and normalize angiogenesis. Nano-particles-mediated gene therapy is currently being developed to overcome the BBB for glioma treatment. Another approach to enhance the anti-glioma efficacy is the implementation of viro-immunotherapy using oncolytic viruses, which are immunogenic. Oncolytic viruses kill tumor cells due to cancer cell-specific viral replication, and can also initiate an anti-tumor immunity. However, concerns still remain related to off target effects, and therapeutic and transduction efficiency. In this review, we describe the rationale and strategies as well as advantages and disadvantages of current gene therapy approaches against gliomas in clinical and preclinical studies. This includes different delivery systems comprising of viral, and non-viral delivery platforms along with suicide/prodrug, oncolytic, cytokine, and tumor suppressor-mediated gene therapy approaches. In addition, advances in glioma treatment through BBB-disruptive gene therapy and anti-EGFRvIII/VEGFR gene therapy are also discussed. Finally, we discuss the results of gene therapy-mediated human clinical trials for gliomas. In summary, we highlight the progress, prospects and remaining challenges of gene therapies aiming at broadening our understanding and highlighting the therapeutic arsenal for GBM.
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Mutant isocitrate dehydrogenase 1 (IDH1-R132H; mIDH1) is a hallmark of adult gliomas. Lower grade mIDH1 gliomas are classified into 2 molecular subgroups: 1p/19q codeletion/TERT-promoter mutations or inactivating mutations in α-thalassemia/mental retardation syndrome X-linked (ATRX) and TP53. This work focuses on glioma subtypes harboring mIDH1, TP53, and ATRX inactivation. IDH1-R132H is a gain-of-function mutation that converts α-ketoglutarate into 2-hydroxyglutarate (D-2HG). The role of D-2HG within the tumor microenvironment of mIDH1/mATRX/mTP53 gliomas remains unexplored. Inhibition of D-2HG, when used as monotherapy or in combination with radiation and temozolomide (IR/TMZ), led to increased median survival (MS) of mIDH1 glioma-bearing mice. Also, D-2HG inhibition elicited anti-mIDH1 glioma immunological memory. In response to D-2HG inhibition, PD-L1 expression levels on mIDH1-glioma cells increased to similar levels as observed in WT-IDH gliomas. Thus, we combined D-2HG inhibition/IR/TMZ with anti-PDL1 immune checkpoint blockade and observed complete tumor regression in 60% of mIDH1 glioma-bearing mice. This combination strategy reduced T cell exhaustion and favored the generation of memory CD8+ T cells. Our findings demonstrate that metabolic reprogramming elicits anti-mIDH1 glioma immunity, leading to increased MS and immunological memory. Our preclinical data support the testing of IDH-R132H inhibitors in combination with IR/TMZ and anti-PDL1 as targeted therapy for mIDH1/mATRX/mTP53 glioma patients.
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Reprogramación Celular , Glioma/terapia , Glutaratos/farmacología , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Línea Celular Tumoral , Reprogramación Celular/efectos de los fármacos , Reprogramación Celular/genética , Reprogramación Celular/inmunología , Quimioradioterapia , Mutación con Ganancia de Función , Glioma/genética , Glioma/inmunología , Glioma/patología , Humanos , Memoria Inmunológica/efectos de los fármacos , Memoria Inmunológica/genética , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/inmunología , Ratones , Temozolomida/farmacología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/inmunología , Proteína Nuclear Ligada al Cromosoma X/genética , Proteína Nuclear Ligada al Cromosoma X/inmunologíaRESUMEN
Mutant isocitrate-dehydrogenase 1 (mIDH1) synthesizes the oncometabolite 2-hydroxyglutarate (2HG), which elicits epigenetic reprogramming of the glioma cells' transcriptome by inhibiting DNA and histone demethylases. We show that the efficacy of immune-stimulatory gene therapy (TK/Flt3L) is enhanced in mIDH1 gliomas, due to the reprogramming of the myeloid cells' compartment infiltrating the tumor microenvironment (TME). We uncovered that the immature myeloid cells infiltrating the mIDH1 TME are mainly nonsuppressive neutrophils and preneutrophils. Myeloid cell reprogramming was triggered by granulocyte colony-stimulating factor (G-CSF) secreted by mIDH1 glioma stem/progenitor-like cells. Blocking G-CSF in mIDH1 gliomabearing mice restores the inhibitory potential of the tumor-infiltrating myeloid cells, accelerating tumor progression. We demonstrate that G-CSF reprograms bone marrow granulopoiesis, resulting in noninhibitory myeloid cells within mIDH1 glioma TME and enhancing the efficacy of immune-stimulatory gene therapy.
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Polyhedron envelope protein (PEP) is the major component of the calyx that surrounds the baculovirus occlusion body (OB). PEP has been associated with the stabilization and resistance of polyhedra in the environment. Due to the abundant levels of PEP in OBs, we decided to use this protein as a fusion partner to redirect foreign proteins to baculovirus polyhedra. In this study we developed a strategy that involves the generation of a monoclonal transformed insect cell line expressing a protein of interest fused to the the Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) N-terminus of PEP that enables the packaging of foreign proteins into the OBs without generating a recombinant baculovirus. This proved to be an efficient platform that could be exploited to improve wild type baculovirus for their use as bioinsecticides without facing the concerns of releasing genetically modified DNA to the environment and bypassing the associated regulatory issues. We demonstrated, using immunological, proteomic and microscopy techniques, that the envelope of AgMNPV OBs can effectively trap chimeric proteins in an infected insect cell line expressing AgMNPV PEP fused to the enhanced green fluorescent protein (eGFP). Furthermore, packaging of chimeric PEP also took place with heterologous OBs such as those of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), another group I alphabaculovirus.
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Baculoviridae/metabolismo , Nucleopoliedrovirus/metabolismo , Cuerpos de Oclusión Viral/metabolismo , Proteínas de la Matriz de Cuerpos de Oclusión/metabolismo , Proteómica , Animales , Baculoviridae/genética , Línea Celular , Genes Reporteros , Insectos , Nucleopoliedrovirus/genética , Cuerpos de Oclusión Viral/genética , Proteínas de la Matriz de Cuerpos de Oclusión/genética , Proteínas RecombinantesRESUMEN
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).
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Neoplasias del Tronco Encefálico/patología , Ingeniería Genética/métodos , Glioma/patología , Animales , Cruzamiento , Modelos Animales de Enfermedad , Humanos , Ratones Endogámicos C57BL , Ratones Transgénicos , Clasificación del Tumor , Esferoides Celulares/patología , Transposasas/metabolismo , Células Tumorales CultivadasRESUMEN
Gliomas are the most common malignant brain tumors in the pediatric population. Even though great efforts have been made to understand their distinctive molecular characteristics, there has not been any improvements in the median survival in decades. In children, high-grade glial tumors have a median survival of 9-15 months. It has recently been demonstrated that pediatric high-grade gliomas (pHGG) are biologically and molecularly different from the adult counterparts, which could explain why conventional treatments universally fail. The development of an in vivo pHGG model harboring the specific genetic alterations encountered in pediatric gliomas is imperative in order to study the molecular basis that drives the progression and aggressiveness of these tumors. It would also enable harnessing these results for the development of novel therapeutic approaches. Our lab has implemented a method to induce brain tumors using transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mouse brain. One of the main advantages of this method is that tumors are induced by altering the genome of the host cells, allowing us to recapitulate the salient features of the human disease. In this chapter we describe a method to isolate two cell populations from tumors generated in situ in mice, i.e., one population enriched in tumor cells and another population enriched in CD45+ cells. We also present methodologies as to how tumor infiltrating immune cells can be phenotypically characterized using flow cytometry.
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Neoplasias Encefálicas/inmunología , Citometría de Flujo/métodos , Glioma/inmunología , Sistema Inmunológico/citología , Microambiente Tumoral , Animales , Neoplasias Encefálicas/genética , Niño , Modelos Animales de Enfermedad , Ingeniería Genética/métodos , Glioma/genética , Humanos , Sistema Inmunológico/inmunología , Separación Inmunomagnética/métodos , Antígenos Comunes de Leucocito/análisis , Antígenos Comunes de Leucocito/inmunología , Ratones , Transposasas/genéticaRESUMEN
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.
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Neoplasias del Tronco Encefálico/terapia , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Glioma/terapia , Inmunoterapia/métodos , Receptores de Activinas Tipo I/genética , Animales , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/inmunología , Neoplasias del Tronco Encefálico/patología , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos/genética , Glioma/genética , Glioma/inmunología , Glioma/patología , Humanos , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Mutación , Puente/patología , Cultivo Primario de Células , RNA-Seq , Transducción de Señal/genética , Transducción de Señal/inmunología , Esferoides Celulares , Timidina Quinasa/genética , Factor de Crecimiento Transformador beta/metabolismo , Células Tumorales Cultivadas/trasplante , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
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.
Asunto(s)
Neoplasias del Tronco Encefálico/genética , Reprogramación Celular/genética , Cromatina/genética , Glioma Pontino Intrínseco Difuso/genética , Epigénesis Genética/genética , Animales , Niño , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Histonas/genética , Humanos , Masculino , MutaciónRESUMEN
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.