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1.
PLoS Pathog ; 17(11): e1010019, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34780571

RESUMO

Gammaherpesviruses establish life-long infections within their host and have been shown to be the causative agents of devastating malignancies. Chronic infection within the host is mediated through cycles of transcriptionally quiescent stages of latency with periods of reactivation into detectable lytic and productive infection. The mechanisms that regulate reactivation from latency remain poorly understood. Previously, we defined a critical role for the viral cyclin in promoting reactivation from latency. Disruption of the viral cyclin had no impact on the frequency of cells containing viral genome during latency, yet it remains unclear whether the viral cyclin influences latently infected cells in a qualitative manner. To define the impact of the viral cyclin on properties of latent infection, we utilized a viral cyclin deficient variant expressing a LANA-beta-lactamase fusion protein (LANA::ßla), to enumerate both the cellular distribution and frequency of LANA gene expression. Disruption of the viral cyclin did not affect the cellular distribution of latently infected cells, but did result in a significant decrease in the frequency of cells that expressed LANA::ßla across multiple tissues and in both immunocompetent and immunodeficient hosts. Strikingly, whereas the cyclin-deficient virus had a reactivation defect in bulk culture, sort purified cyclin-deficient LANA::ßla expressing cells were fully capable of reactivation. These data emphasize that the γHV68 latent reservoir is comprised of at least two distinct stages of infection characterized by differential LANA expression, and that a primary function of the viral cyclin is to promote LANA expression during latency, a state associated with ex vivo reactivation competence.


Assuntos
Antígenos Virais/metabolismo , Ciclinas/metabolismo , Regulação Viral da Expressão Gênica , Infecções por Herpesviridae/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Virais/metabolismo , Ativação Viral , Replicação Viral , Animais , Antígenos Virais/genética , Ciclinas/genética , Gammaherpesvirinae/fisiologia , Infecções por Herpesviridae/virologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Infecção Persistente , Proteínas Virais/genética , Latência Viral
2.
J Neurooncol ; 165(1): 161-169, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37878192

RESUMO

BACKGROUND: Neurocognitive deficits are common in pediatric brain tumor survivors. The use of single nucleotide polymorphism (SNP) analysis in DNA repair genes may identify children treated with radiation therapy for brain tumors at increased risk for treatment toxicity and adverse neurocognitive outcomes. MATERIALS: The Human 660W-Quad v1.0 DNA BeadChip analysis (Illumina) was used to evaluate 1048 SNPs from 59 DNA repair genes in 46 subjects. IQ testing was measured by the Wechsler Intelligence Scale for Children. Linear regression was used to identify the 10 SNPs with the strongest association with IQ scores while adjusting for radiation type. RESULTS: The low vs high IQ patient cohorts were well matched for time from first treatment to most recent IQ, first treatment age, sex, and treatments received. 5 SNPs on 3 different genes (CYP29, XRCC1, and BRCA1) and on 3 different chromosomes (10, 19, and 17) had the strongest association with most recent IQ score that was not modified by radiation type. Furthermore, 5 SNPs on 4 different genes (WRN, NR3C1, ERCC4, RAD51L1) on 4 different chromosomes (8, 5, 16, 14) had the strongest association with change in IQ independent of radiation type, first IQ, and years between IQ measures. CONCLUSIONS: SNPs offer the potential to predict adverse neurocognitive outcomes in pediatric brain tumor survivors. Our results require validation in a larger patient cohort. Improving the ability to identify children at risk of treatment related neurocognitive deficits could allow for better treatment stratification and early cognitive interventions.


Assuntos
Neoplasias Encefálicas , Criança , Humanos , Neoplasias Encefálicas/complicações , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/radioterapia , Testes de Inteligência , Sobreviventes , Irradiação Craniana/efeitos adversos , Testes Neuropsicológicos , Proteína 1 Complementadora Cruzada de Reparo de Raio-X
3.
J Virol ; 91(20)2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28747504

RESUMO

The neurotropic herpesvirus varicella-zoster virus (VZV) establishes a lifelong latent infection in humans following primary infection. The low abundance of VZV nucleic acids in human neurons has hindered an understanding of the mechanisms that regulate viral gene transcription during latency. To overcome this critical barrier, we optimized a targeted capture protocol to enrich VZV DNA and cDNA prior to whole-genome/transcriptome sequence analysis. Since the VZV genome is remarkably stable, it was surprising to detect that VZV32, a VZV laboratory strain with no discernible growth defect in tissue culture, contained a 2,158-bp deletion in open reading frame (ORF) 12. Consequently, ORF 12 and 13 protein expression was abolished and Akt phosphorylation was inhibited. The discovery of the ORF 12 deletion, revealed through targeted genome sequencing analysis, points to the need to authenticate the VZV genome when the virus is propagated in tissue culture.IMPORTANCE Viruses isolated from clinical samples often undergo genetic modifications when cultured in the laboratory. Historically, VZV is among the most genetically stable herpesviruses, a notion supported by more than 60 complete genome sequences from multiple isolates and following multiple in vitro passages. However, application of enrichment protocols to targeted genome sequencing revealed the unexpected deletion of a significant portion of VZV ORF 12 following propagation in cultured human fibroblast cells. While the enrichment protocol did not introduce bias in either the virus genome or transcriptome, the findings indicate the need for authentication of VZV by sequencing when the virus is propagated in tissue culture.


Assuntos
DNA Viral/isolamento & purificação , Genoma Viral , Herpesvirus Humano 3/genética , Fases de Leitura Aberta , Deleção de Sequência , Linhagem Celular , DNA Complementar , Herpesvirus Humano 3/crescimento & desenvolvimento , Humanos , Análise de Sequência de DNA/métodos , Transcriptoma , Proteínas Virais , Vírion , Latência Viral
4.
Pediatr Blood Cancer ; 65(5): e26960, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29350470

RESUMO

BACKGROUND: A desperate need for novel therapies in pediatric ependymoma (EPN) exists, as chemotherapy remains ineffective and radiotherapy often fails. EPN have significant infiltration of immune cells, which correlates with outcome. Immune checkpoint inhibitors provide an avenue for new treatments. This study characterizes tumor-infiltrating immune cells in EPN and aims at predicting candidates for clinical trials using checkpoint inhibitors targeting PD-L1/PD-1 (programmed death ligand 1/programmed death 1). METHODS: The transcriptomic profiles of the primary study cohort of EPN and other pediatric brain tumors were interrogated to identify PD-L1 expression levels. Transcriptomic findings were validated using the western blotting, immunohistochemistry and flow cytometry. RESULTS: We evaluated PD-L1 mRNA expression across four intracranial subtypes of EPN in two independent cohorts and found supratentorial RELA fusion (ST-RELA) tumors to have significantly higher levels. There was a correlation between high gene expression and protein PD-L1 levels in ST-RELA tumors by both the western blot and immunohistochemisty. The investigation of EPN cell populations revealed PD-L1 was expressed on both tumor and myeloid cells in ST-RELA. Other subtypes had little PD-L1 in either tumor or myeloid cell compartments. Lastly, we measured PD-1 levels on tumor-infiltrating T cells and found ST-RELA tumors express PD-1 in both CD4 and CD8 T cells. A functional T-cell exhaustion assay found ST-RELA T cells to be exhausted and unable to secrete IFNγ on stimulation. CONCLUSIONS: These findings in ST-RELA suggest tumor evasion and immunsuppression due to PD-L1/PD-1-mediated T-cell exhaustion. Trials of checkpoint inhibitors in EPN should be enriched for ST-RELA tumors.


Assuntos
Antígeno B7-H1/metabolismo , Biomarcadores Tumorais/metabolismo , Ependimoma/metabolismo , Neoplasias Supratentoriais/metabolismo , Fator de Transcrição RelA/metabolismo , Adolescente , Adulto , Antígeno B7-H1/genética , Biomarcadores Tumorais/genética , Criança , Pré-Escolar , Estudos de Coortes , Ependimoma/genética , Ependimoma/patologia , Feminino , Seguimentos , Perfilação da Expressão Gênica , Humanos , Lactente , Masculino , Terapia de Alvo Molecular , Prognóstico , Neoplasias Supratentoriais/genética , Neoplasias Supratentoriais/patologia , Linfócitos T/metabolismo , Fator de Transcrição RelA/genética , Adulto Jovem
5.
J Virol ; 90(3): 1231-43, 2016 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-26559844

RESUMO

UNLABELLED: Regulation of gene transcription in varicella-zoster virus (VZV), a ubiquitous human neurotropic alphaherpesvirus, requires coordinated binding of multiple host and virus proteins onto specific regions of the virus genome. Chromatin immunoprecipitation (ChIP) is widely used to determine the location of specific proteins along a genomic region. Since the size range of sheared virus DNA fragments governs the limit of accurate protein localization, particularly for compact herpesvirus genomes, we used a quantitative PCR (qPCR)-based assay to determine the efficiency of VZV DNA shearing before ChIP, after which the assay was used to determine the relationship between transcript abundance and the occupancy of phosphorylated RNA polymerase II (RNAP) on the gene promoter, body, and terminus of VZV genes 9, 51, and 66. The abundance of VZV gene 9, 51, and 66 transcripts in VZV-infected human fetal lung fibroblasts was determined by reverse transcription-linked quantitative PCR. Our results showed that the C-terminal domain of RNAP is hyperphosphorylated at serine 5 (S5(P)) on VZV genes 9, 51, and 66 independently of transcript abundance and the location within the virus gene at both 1 and 3 days postinfection (dpi). In contrast, phosphorylated serine 2 (S2(P))-modified RNAP was not detected at any virus gene location at 3 dpi and was detected at levels only slightly above background levels at 1 dpi. IMPORTANCE: Regulation of herpesvirus gene transcription is an elaborate choreography between proteins and DNA that is revealed by chromatin immunoprecipitation (ChIP). We used a quantitative PCR-based assay to determine fragment size after DNA shearing, a critical parameter in ChIP assays, and exposed a basic difference in the mechanism of transcription between mammalian cells and VZV. We found that hyperphosphorylation at serine 5 of the C-terminal domain of RNAP along the lengths of VZV genes (the promoter, body, and transcription termination site) was independent of mRNA abundance. In contrast, little to no enrichment of serine 3 phosphorylation of RNAP was detected at these virus gene regions. This is distinct from the findings for RNAP at highly regulated host genes, where RNAP S5(P) occupancy decreased and S2(P) levels increased as the polymerase transited through the gene. Overall, these results suggest that RNAP associates with human and virus transcriptional units through different mechanisms.


Assuntos
DNA Viral/química , Herpesvirus Humano 3/fisiologia , RNA Polimerase II/análise , Transcrição Gênica , Células Cultivadas , Imunoprecipitação da Cromatina , Fibroblastos/virologia , Humanos , Fosforilação , Processamento de Proteína Pós-Traducional , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
6.
J Neurovirol ; 23(1): 152-157, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27683235

RESUMO

Varicella zoster virus (VZV), a human neurotropic alphaherpesvirus, becomes latent after primary infection and reactivates to produce zoster. To study VZV latency and reactivation, human trigeminal ganglia removed within 24 h after death were mechanically dissociated, randomly distributed into six-well tissue culture plates and incubated with reagents to inactivate nerve growth factor (NGF) or phosphoinositide 3-kinase (PI3-kinase) pathways. At 5 days, VZV DNA increased in control and PI3-kinase inhibitor-treated cultures to the same extent, but was significantly more abundant in anti-NGF-treated cultures (p = 0.001). Overall, VZV DNA replication is regulated in part by an NGF pathway that is PI3-kinase-independent.


Assuntos
Replicação do DNA , DNA Viral/genética , Herpesvirus Humano 3/genética , Fator de Crescimento Neural/genética , Fosfatidilinositol 3-Quinases/genética , Ativação Viral , Replicação Viral , Adulto , Idoso , Anticorpos Neutralizantes/farmacologia , Autopsia , DNA Viral/biossíntese , Regulação da Expressão Gênica , Herpes Zoster/genética , Herpes Zoster/metabolismo , Herpes Zoster/patologia , Herpes Zoster/virologia , Herpesvirus Humano 3/metabolismo , Herpesvirus Humano 3/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Masculino , Pessoa de Meia-Idade , Fator de Crescimento Neural/antagonistas & inibidores , Fator de Crescimento Neural/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Técnicas de Cultura de Tecidos , Gânglio Trigeminal/efeitos dos fármacos , Gânglio Trigeminal/virologia , Latência Viral
7.
Nat Commun ; 15(1): 4616, 2024 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-38816355

RESUMO

Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. P-TEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates P-TEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of P-TEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for P-TEFb underpinning the early adaptive response to radiotherapy, opening avenues for combinatorial treatment in these lethal malignancies.


Assuntos
Regulação Neoplásica da Expressão Gênica , Glioma , Fator B de Elongação Transcricional Positiva , Humanos , Glioma/radioterapia , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Animais , Fator B de Elongação Transcricional Positiva/metabolismo , Fator B de Elongação Transcricional Positiva/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Camundongos , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Transcrição Gênica/efeitos da radiação , Apoptose/efeitos da radiação , Apoptose/genética , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Reparo do DNA/efeitos da radiação , Ensaios Antitumorais Modelo de Xenoenxerto
8.
bioRxiv ; 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36747867

RESUMO

Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. PTEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates PTEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of PTEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for PTEFb underpinning the early adaptive response to radiotherapy, opening new avenues for combinatorial treatment in these lethal malignancies.

9.
Res Sq ; 2023 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-37609195

RESUMO

Purpose: Neurocognitive deficits are common in pediatric brain tumor survivors. The use of single nucleotide polymorphism (SNP) analysis in DNA repair genes may identify children treated with radiation therapy for brain tumors at increased risk for treatment toxicity and adverse neurocognitive outcomes. Methods: The Human 660W-Quad v1.0 DNA BeadChip analysis (Illumina) was used to evaluate 1048 SNPs from 59 DNA repair genes in 46 subjects. IQ testing was measured by the Wechsler Intelligence Scale for Children. Linear regression was used to identify the 10 SNPs with the strongest association with IQ scores while adjusting for radiation type. Results: The low vs high IQ patient cohorts were well matched for time from first treatment to most recent IQ, first treatment age, gender, and treatments received. 5 SNPs on 3 different genes (CYP29, XRCC1, and BRCA1) and on 3 different chromosomes (10, 19, and 17) had the strongest association with most recent IQ score that was not modified by radiation type. Furthermore, 5 SNPs on 4 different genes (WRN, NR3C1, ERCC4, RAD51L1) on 4 different chromosomes (8, 5, 16, 14) had the strongest association with change in IQ independent of radiation type, first IQ, and years between IQ measures. Conclusions: SNP polymorphisms offer potential to predict adverse neurocognitive outcomes in pediatric brain tumor survivors. Our results require validation in a larger patient cohort. Improving the ability to identify children at risk of treatment related neurocognitive deficits could allow for better treatment stratification and early cognitive interventions.

10.
Neuro Oncol ; 25(10): 1854-1867, 2023 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-37246777

RESUMO

BACKGROUND: Ependymoma (EPN) posterior fossa group A (PFA) has the highest rate of recurrence and the worst prognosis of all EPN molecular groups. At relapse, it is typically incurable even with re-resection and re-irradiation. The biology of recurrent PFA remains largely unknown; however, the increasing use of surgery at first recurrence has now provided access to clinical samples to facilitate a better understanding of this. METHODS: In this large longitudinal international multicenter study, we examined matched samples of primary and recurrent disease from PFA patients to investigate the biology of recurrence. RESULTS: DNA methylome derived copy number variants (CNVs) revealed large-scale chromosome gains and losses at recurrence in PFA. CNV changes were dominated by chromosome 1q gain and/or 6q loss, both previously identified as high-risk factors in PFA, which were present in 23% at presentation but increased to 61% at first recurrence. Multivariate survival analyses of this cohort showed that cases with 1q gain or 6q loss at first recurrence were significantly more likely to recur again. Predisposition to 1q+/6q- CNV changes at recurrence correlated with hypomethylation of heterochromatin-associated DNA at presentation. Cellular and molecular analyses revealed that 1q+/6q- PFA had significantly higher proportions of proliferative neuroepithelial undifferentiated progenitors and decreased differentiated neoplastic subpopulations. CONCLUSIONS: This study provides clinically and preclinically actionable insights into the biology of PFA recurrence. The hypomethylation predisposition signature in PFA is a potential risk-classifier for trial stratification. We show that the cellular heterogeneity of PFAs evolves largely because of genetic evolution of neoplastic cells.


Assuntos
Ependimoma , Neoplasias Infratentoriais , Humanos , Neoplasias Infratentoriais/genética , Aberrações Cromossômicas , Análise de Sobrevida , Ependimoma/genética , Cromossomos
11.
PLoS One ; 17(2): e0263370, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35113934

RESUMO

The cytokine Interferon-γ (IFN-γ) exerts powerful immunoregulatory effects on the adaptive immune system and also enhances functions of the neutrophil (PMN). The clinical use of IFN-γ has been driven by the finding that its administration to patients with chronic granulomatous disease (CGD) results in decreased incidence and severity of infections. However, IFN-γ has no effect on the characteristic defect of CGD, the inability to convert oxygen to microbicidal metabolites including superoxide anion (O2-) during the phagocytosis associated oxidative burst. We administered varying doses of IFN-γ to adult volunteers and studied the effects on plasma drug levels and response molecules and PMNs isolated from blood drawn at intervals over a 96- hour period. Plasma concentrations of IFN-γ, IP-10 and neopterin, and stimulated release of O2- from PMNs exhibited dose- and time-dependent increases after IFN-γ administration. Gene expression in PMNs was altered for 2775 genes; changes occurred rapidly after administration and returned to baseline in 24-36 hours. Several genes involved with neutrophil host defense were upregulated including those for components of the O2- generating NADPH oxidase; innate-immune and Fc receptors; proteins involved in MHCI and II; a regulator of circulating PMN number; guanylate binding proteins; and a key enzyme in synthesis of an essential NOS cofactor. Coordinate changes were detected in protein levels of representative products from several of these genes. Lysates from isolated neutrophils also demonstrated a spike in NO following IFN-γ administration. IFN-γ appears to increase non-oxygen dependent microbicidal functions of PMNs which could provide strategies to compensate for deficiencies, explain its clinical benefit for CGD patients and expand therapeutic applications of IFN-γ to other disorders. Trial registration: Protocol registered in ClinicalTrials.gov, NCT02609932, Effect of IFN-γ on Innate Immune Cells.


Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Doença Granulomatosa Crônica/tratamento farmacológico , Doença Granulomatosa Crônica/metabolismo , Interferon gama/farmacologia , Neutrófilos/efeitos dos fármacos , Adolescente , Adulto , Quimiocina CXCL10/biossíntese , Doença Granulomatosa Crônica/genética , Voluntários Saudáveis , Humanos , Interferon gama/biossíntese , Pessoa de Meia-Idade , NADPH Oxidases/metabolismo , Neopterina/biossíntese , Neutrófilos/metabolismo , Fagocitose , Fenótipo , Explosão Respiratória , Superóxidos , Adulto Jovem
12.
Clin Cancer Res ; 28(11): 2409-2424, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35344040

RESUMO

PURPOSE: Tumor relapse after radiotherapy is a major hurdle in treating pediatric H3K27M-mutant diffuse midline gliomas (DMG). Radiotherapy-induced stress increases association of BCL2 family of proteins with BH3 pro-apoptotic activators preventing apoptosis. We hypothesized that inhibition of radiotherapy-induced BCL2 with a clinically relevant inhibitor, venetoclax, will block BCL2 activity leading to increased apoptosis. BCL2 has never been implicated in DMG as a radiotherapy-induced resistant mechanism. EXPERIMENTAL DESIGN: We performed an integrated genomic analysis to determine genes responsible for radioresistance and a targeted drug screen to identify drugs that synergize with radiation in DMG. Effect of venetoclax on radiation-naïve and 6 Gy radiation on cells was evaluated by studying cell death, changes in BCL2 phosphorylation, reactive oxygen species (ROS), and apoptosis, as well as BCL2 association with BH3 apoptosis initiators. The efficacy of combining venetoclax with radiation was evaluated in vivo using orthotopic xenograft models. RESULTS: BCL2 was identified as a key regulator of tumor growth after radiation in DMGs. Radiation sensitizes DMGs to venetoclax treatment independent of p53 status. Venetoclax as a monotherapy was not cytotoxic to DMG cells. Postradiation venetoclax treatment significantly increased cell death, reduced BCL2-BIM association, and augmented mitochondrial ROS leading to increased apoptosis. Combining venetoclax with radiotherapy significantly enhanced the survival of mice with DMG tumors. CONCLUSIONS: This study shows that venetoclax impedes the antiapoptotic function of radiation-induced BCL2 in DMG, leading to increased apoptosis. Results from these preclinical studies demonstrate the potential use of the BCL2 inhibitor venetoclax combined with radiotherapy for pediatric DMG.


Assuntos
Antineoplásicos , Glioma , Animais , Antineoplásicos/farmacologia , Apoptose , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Linhagem Celular Tumoral , Glioma/tratamento farmacológico , Glioma/genética , Glioma/radioterapia , Humanos , Camundongos , Recidiva Local de Neoplasia/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2 , Radiação Ionizante , Espécies Reativas de Oxigênio , Sulfonamidas
13.
Neuro Oncol ; 24(2): 273-286, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34077540

RESUMO

BACKGROUND: Medulloblastoma (MB) is a heterogeneous disease in which neoplastic cells and associated immune cells contribute to disease progression. We aimed to determine the influence of neoplastic and immune cell diversity on MB biology in patient samples and animal models. METHODS: To better characterize cellular heterogeneity in MB we used single-cell RNA sequencing, immunohistochemistry, and deconvolution of transcriptomic data to profile neoplastic and immune populations in patient samples and animal models across childhood MB subgroups. RESULTS: Neoplastic cells cluster primarily according to individual sample of origin which is influenced by chromosomal copy number variance. Harmony alignment reveals novel MB subgroup/subtype-associated subpopulations that recapitulate neurodevelopmental processes, including photoreceptor and glutamatergic neuron-like cells in molecular subgroups GP3 and GP4, and a specific nodule-associated neuronally differentiated subpopulation in the sonic hedgehog subgroup. We definitively chart the spectrum of MB immune cell infiltrates, which include subpopulations that recapitulate developmentally related neuron-pruning and antigen-presenting myeloid cells. MB cellular diversity matching human samples is mirrored in subgroup-specific mouse models of MB. CONCLUSIONS: These findings provide a clearer understanding of the diverse neoplastic and immune cell subpopulations that constitute the MB microenvironment.


Assuntos
Neoplasias Cerebelares , Meduloblastoma , Animais , Neoplasias Cerebelares/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/genética , Humanos , Meduloblastoma/genética , Camundongos , Transcriptoma , Microambiente Tumoral/genética
14.
J Cereb Blood Flow Metab ; 41(4): 761-770, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-32501156

RESUMO

Stroke-induced cerebral ischemia is a major cause of death and disability. The disruption of blood flow results in neuronal and glial cell death leading to brain injury. Reperfusion restores oxygen to the affected tissue, but can also cause damage through an enhanced oxidative stress and inflammatory response. This study examines mitochondrial transfer from MSC to neurons and the role it plays in neuronal preservation after oxidant injury. We observed the transfer of mitochondria from MSC to mouse neurons in vitro following hydrogen peroxide exposure. The observed transfer was dependent on cell-to-cell contact and led to increased neuronal survival and improved metabolism. A number of pro-inflammatory and mitochondrial motility genes were upregulated in neurons after hydrogen peroxide exposure. This included Miro1 and TNFAIP2, linking inflammation and mitochondrial transfer to oxidant injury. Increasing Miro1 expression in MSC improved the metabolic benefit of mitochondrial transfer after neuronal oxidant injury. Decreasing Miro1 expression had the opposite effect, decreasing the metabolic benefit of MSC co-culture. MSC transfer of mitochondria to oxidant-damaged neurons may help improve neuronal preservation and functional recovery after stroke.


Assuntos
Transplante de Células-Tronco Mesenquimais/métodos , Mitocôndrias/transplante , Neurônios/metabolismo , Oxidantes/toxicidade , Traumatismo por Reperfusão/prevenção & controle , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Sobrevivência Celular , Técnicas de Cocultura , Técnicas de Silenciamento de Genes , Peróxido de Hidrogênio/toxicidade , Inflamação/genética , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Traumatismo por Reperfusão/metabolismo , Fatores de Necrose Tumoral/genética
15.
J Neuropathol Exp Neurol ; 80(4): 345-353, 2021 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-33749791

RESUMO

Diffuse midline gliomas (DMGs) are incurable pediatric tumors with extraordinarily limited treatment options. Decades of clinical trials combining conventional chemotherapies with radiation therapy have failed to improve these outcomes, demonstrating the need to identify and validate druggable biologic targets within this disease. NTRK1/2/3 fusions are found in a broad range of pediatric cancers, including high-grade gliomas and a subset of DMGs. Phase 1/2 studies of TRK inhibitors have demonstrated good tolerability, effective CNS penetration, and promising objective responses across all patients with TRK fusion-positive cancers, but their use has not been explored in TRK fusion-positive DMG. Here, we report 3 cases of NTRK fusions co-occurring within H3K27M-positive pontine diffuse midline gliomas. We employ a combination of single-cell and bulk transcriptome sequencing from TRK fusion-positive DMG to describe the phenotypic consequences of this co-occurring alteration. We then use ex vivo short-culture assays to evaluate the potential response to TRK inhibition in this disease. Together, these data highlight the importance of routine molecular characterization of these highly aggressive tumors and identify a small subset of patients that may benefit from currently available targeted therapies.


Assuntos
Neoplasias do Tronco Encefálico/genética , Glioma/genética , Histona Desmetilases com o Domínio Jumonji/genética , Glicoproteínas de Membrana/genética , Mutação/genética , Receptor trkB/genética , Receptor trkC/genética , Neoplasias do Tronco Encefálico/diagnóstico por imagem , Criança , Pré-Escolar , Evolução Fatal , Feminino , Glioma/diagnóstico por imagem , Humanos , Masculino
16.
Sci Rep ; 11(1): 8786, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33888746

RESUMO

Cardiovascular disease (CVD) is the most common cause of death in patients with native and post-transplant chronic kidney disease (CKD). To identify new biomarkers of vascular injury and inflammation, we analyzed the proteome of plasma and circulating extracellular vesicles (EVs) in native and post-transplant CKD patients utilizing an aptamer-based assay. Proteins of angiogenesis were significantly higher in native and post-transplant CKD patients versus healthy controls. Ingenuity pathway analysis (IPA) indicated Ephrin receptor signaling, serine biosynthesis, and transforming growth factor-ß as the top pathways activated in both CKD groups. Pro-inflammatory proteins were significantly higher only in the EVs of native CKD patients. IPA indicated acute phase response signaling, insulin-like growth factor-1, tumor necrosis factor-α, and interleukin-6 pathway activation. These data indicate that pathways of angiogenesis and inflammation are activated in CKD patients' plasma and EVs, respectively. The pathways common in both native and post-transplant CKD may signal similar mechanisms of CVD.


Assuntos
Indutores da Angiogênese/metabolismo , Inflamação/metabolismo , Falência Renal Crônica/metabolismo , Biomarcadores/metabolismo , Humanos , Projetos Piloto
17.
J Neuropathol Exp Neurol ; 80(12): 1099-1107, 2021 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-34850053

RESUMO

Brain tumors are the most common solid tumor in children, and low-grade gliomas (LGGs) are the most common childhood brain tumor. Here, we report on 3 patients with LGG harboring previously unreported or rarely reported RAF fusions: FYCO1-RAF1, CTTNBP2-BRAF, and SLC44A1-BRAF. We hypothesized that these tumors would show molecular similarity to the canonical KIAA1549-BRAF fusion that is the most widely seen alteration in pilocytic astrocytoma (PA), the most common pediatric LGG variant, and that this similarity would include mitogen-activated protein kinase (MAPK) pathway activation. To test our hypothesis, we utilized immunofluorescent imaging and RNA-sequencing in normal brain, KIAA1549-BRAF-harboring tumors, and our 3 tumors with novel fusions. We performed immunofluorescent staining of ERK and phosphorylated ERK (p-ERK), identifying increased p-ERK expression in KIAA1549-BRAF fused PA and the novel fusion samples, indicative of MAPK pathway activation. Geneset enrichment analysis further confirmed upregulated downstream MAPK activation. These results suggest that MAPK activation is the oncogenic mechanism in noncanonical RAF fusion-driven LGG. Similarity in the oncogenic mechanism suggests that LGGs with noncanonical RAF fusions are likely to respond to MEK inhibitors.


Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Fusão Oncogênica/genética , Quinases raf/genética , Adolescente , Neoplasias Encefálicas/metabolismo , Criança , Feminino , Glioma/metabolismo , Humanos , Masculino
18.
Clin Cancer Res ; 27(22): 6197-6208, 2021 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-34433654

RESUMO

PURPOSE: Selective RAF-targeted therapy is effective in some patients with BRAFV600E-mutated glioma, though emergent and adaptive resistance occurs through ill-defined mechanisms. EXPERIMENTAL DESIGN: Paired pre-/post- RAF inhibitor (RAFi)-treated glioma samples (N = 15) were obtained and queried for treatment-emergent genomic alterations using DNA and RNA sequencing (RNA-seq). Functional validation of putative resistance mechanisms was performed using established and patient-derived BRAFV600E-mutant glioma cell lines. RESULTS: Analysis of 15 tissue sample pairs identified 13 alterations conferring putative resistance were identified among nine paired samples (including mutations involving ERRFI1, BAP1, ANKHD1, and MAP2K1). We performed functional validation of mechanisms of resistance, including loss of NF1, PTEN, or CBL, in BRAFV600E-mutant glioma lines, and demonstrate they are capable of conferring resistance in vitro. Knockdown of CBL resulted in increased EGFR expression and phosphorylation, a possible mechanism for maintaining ERK signaling within the cell. Combination therapy with a MEKi or EGFR inhibitor was able to overcome resistance to BRAFi, in NF1 knockdown and CBL knockdown, respectively. Restoration of wild-type PTEN in B76 cells (PTEN-/-) restored sensitivity to BRAFi. We identified and validated CRAF upregulation as a mechanism of resistance in one resistant sample. RNA-seq analysis identified two emergent expression patterns in resistant samples, consistent with expression patterns of known glioma subtypes. CONCLUSIONS: Resistance mechanisms to BRAFi in glioma are varied and may predict effective precision combinations of targeted therapy, highlighting the importance of a personalized approach.


Assuntos
Glioma , Proteínas Proto-Oncogênicas B-raf , Glioma/tratamento farmacológico , Glioma/genética , Humanos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas de Ligação a RNA , Transdução de Sinais , Proteínas Supressoras de Tumor , Ubiquitina Tiolesterase
19.
Nat Commun ; 12(1): 5531, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34545084

RESUMO

Radiation-induced high-grade gliomas (RIGs) are an incurable late complication of cranial radiation therapy. We performed DNA methylation profiling, RNA-seq, and DNA sequencing on 32 RIG tumors and an in vitro drug screen in two RIG cell lines. We report that based on DNA methylation, RIGs cluster primarily with the pediatric receptor tyrosine kinase I high-grade glioma subtype. Common copy-number alterations include Chromosome (Ch.) 1p loss/1q gain, and Ch. 13q and Ch. 14q loss; focal alterations include PDGFRA and CDK4 gain and CDKN2A and BCOR loss. Transcriptomically, RIGs comprise a stem-like subgroup with lesser mutation burden and Ch. 1p loss and a pro-inflammatory subgroup with greater mutation burden and depleted DNA repair gene expression. Chromothripsis in several RIG samples is associated with extrachromosomal circular DNA-mediated amplification of PDGFRA and CDK4. Drug screening suggests microtubule inhibitors/stabilizers, DNA-damaging agents, MEK inhibition, and, in the inflammatory subgroup, proteasome inhibitors, as potentially effective therapies.


Assuntos
Glioma/genética , Glioma/patologia , Radiação , Adolescente , Criança , Estudos de Coortes , Simulação por Computador , Variações do Número de Cópias de DNA/genética , Metilação de DNA/genética , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Humanos , Gradação de Tumores , Transcriptoma/genética , Adulto Jovem
20.
Neurooncol Adv ; 2(1): vdaa051, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32642704

RESUMO

BACKGROUND: Atypical teratoid/thabdoid tumor (AT/RT) remains a difficult-to-treat tumor with a 5-year overall survival rate of 15%-45%. Proteasome inhibition has recently been opened as an avenue for cancer treatment with the FDA approval of bortezomib (BTZ) in 2003 and carfilzomib (CFZ) in 2012. The aim of this study was to identify and characterize a pre-approved targeted therapy with potential for clinical trials in AT/RT. METHODS: We performed a drug screen using a panel of 134 FDA-approved drugs in 3 AT/RT cell lines. Follow-on in vitro studies used 6 cell lines and patient-derived short-term cultures to characterize selected drug interactions with AT/RT. In vivo efficacy was evaluated using patient derived xenografts in an intracranial murine model. RESULTS: BTZ and CFZ are highly effective in vitro, producing some of the strongest growth-inhibition responses of the evaluated 134-drug panel. Marizomib (MRZ), a proteasome inhibitor known to pass the blood-brain barrier (BBB), also strongly inhibits AT/RT proteasomes and generates rapid cell death at clinically achievable doses in established cell lines and freshly patient-derived tumor lines. MRZ also significantly extends survival in an intracranial mouse model of AT/RT. CONCLUSIONS: MRZ is a newer proteasome inhibitor that has been shown to cross the BBB and is already in phase II clinical trials for adult high-grade glioma (NCT NCT02330562 and NCT02903069). MRZ strongly inhibits AT/RT cell growth both in vitro and in vivo via a moderately well-characterized mechanism and has direct translational potential for patients with AT/RT.

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