ABSTRACT
The majority of diffuse midline gliomas, H3 K27-altered (DMG-H3 K27-a), are infiltrating pediatric brain tumors that arise in the pons with no effective treatment. To understand how clonal evolution contributes to the tumor's invasive spread, we performed exome sequencing and SNP array profiling on 49 multi-region autopsy samples from 11 patients with pontine DMG-H3 K27-a enrolled in a phase I clinical trial of PDGFR inhibitor crenolanib. For each patient, a phylogenetic tree was constructed by testing multiple possible clonal evolution models to select the one consistent with somatic mutations and copy number variations across all tumor regions. The tree was then used to deconvolute subclonal composition and prevalence at each tumor region to study convergent evolution and invasion patterns. Somatic variants in the PI3K pathway, a late event, are enriched in our cohort, affecting 70% of patients. Convergent evolution of PI3K at distinct phylogenetic branches was detected in 40% of the patients. 24 (~ 50%) of tumor regions were occupied by subclones of mixed lineages with varying molecular ages, indicating multiple waves of invasion across the pons and extrapontine. Subclones harboring a PDGFRA amplicon, including one that amplified a PDGRFAY849C mutant allele, were detected in four patients; their presence in extrapontine tumor and normal brain samples imply their involvement in extrapontine invasion. Our study expands the current knowledge on tumor invasion patterns in DMG-H3 K27-a, which may inform the design of future clinical trials.
Subject(s)
DNA Copy Number Variations , Glioma , Child , Glioma/drug therapy , Glioma/genetics , Glioma/pathology , Histones/genetics , Humans , Mutation/genetics , Phosphatidylinositol 3-Kinases/genetics , Phylogeny , Protein Kinase InhibitorsABSTRACT
Atypical Teratoid Rhabdoid Tumor (AT/RT) is a rare pediatric central nervous system cancer often characterized by deletion or mutation of SMARCB1, a tumor suppressor gene. In this study, we found that SMARCB1 regulates Human Endogenous Retrovirus K (HERV-K, subtype HML-2) expression. HML-2 is a repetitive element scattered throughout the human genome, encoding several intact viral proteins that have been associated with stem cell maintenance and tumorigenesis. We found HML-2 env expression in both the intracellular and extracellular compartments in all AT/RT cell lines (n = 4) and in 95% of AT/RT patient tissues (n = 37) evaluated. SMARCB1 knock-down in neural stem cells (NSCs) led to an upregulation of HML-2 transcription. We found that SMARCB1 binds adjacent to the HML-2 promoter, repressing its transcription via chromatin immunoprecipitation; restoration of SMARCB1 expression in AT/RT cell lines significantly downregulated HML-2 expression. Further, targeted downregulation of HML-2 transcription via CRISPR-dCas9 coupled with suppressor proteins led to cellular dispersion, decreased proliferation, and cell death in vitro. HML-2 knock-down with shRNA, siRNA, and CRISPR-dCas9 significantly decreased Ras expression as measured by qRT-PCR, suggesting that HML-2 modulates MAPK/ERK signaling in AT/RT cells. Overexpression of NRAS was sufficient to restore cellular proliferation, and MYC, a transcription factor downstream of NRAS, was bound to the HERV-K LTR significantly more in the absence of SMARCB1 expression in AT/RT cells. We show a mechanism by which these undifferentiated tumors remain pluripotent, and we demonstrate that their formation is aided by aberrant HML-2 activation, which is dependent on SMARCB1 and its interaction with MYC.
Subject(s)
Cell Transformation, Neoplastic/genetics , Endogenous Retroviruses/genetics , Rhabdoid Tumor/etiology , Rhabdoid Tumor/pathology , SMARCB1 Protein/deficiency , Sequence Deletion , Virus Activation/genetics , Biomarkers, Tumor , Cell Line, Tumor , Cell Proliferation , Cell-Derived Microparticles/metabolism , Disease Susceptibility , GTP Phosphohydrolases/metabolism , Gene Expression Regulation , Humans , Membrane Proteins/metabolism , Repetitive Sequences, Nucleic Acid , Signal TransductionABSTRACT
Brain tumors are the most common solid tumors of childhood, and the genetic drivers and optimal therapeutic strategies for many of the different subtypes remain unknown. Here, we identify that bithalamic gliomas harbor frequent mutations in the EGFR oncogene, only rare histone H3 mutation (in contrast to their unilateral counterparts), and a distinct genome-wide DNA methylation profile compared to all other glioma subtypes studied to date. These EGFR mutations are either small in-frame insertions within exon 20 (intracellular tyrosine kinase domain) or missense mutations within exon 7 (extracellular ligand-binding domain) that occur in the absence of accompanying gene amplification. We find these EGFR mutations are oncogenic in primary astrocyte models and confer sensitivity to specific tyrosine kinase inhibitors dependent on location within the kinase domain or extracellular domain. We initiated treatment with targeted kinase inhibitors in four children whose tumors harbor EGFR mutations with encouraging results. This study identifies a promising genomically-tailored therapeutic strategy for bithalamic gliomas, a lethal and genetically distinct brain tumor of childhood.
Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Glioma/genetics , Mutation/genetics , Adolescent , Antineoplastic Agents/therapeutic use , Brain Neoplasms/drug therapy , Brain Neoplasms/genetics , Child , Child, Preschool , Epigenesis, Genetic/genetics , ErbB Receptors/genetics , Female , Glioma/drug therapy , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Male , Protein Kinase Inhibitors/pharmacologyABSTRACT
PURPOSE: Atypical teratoid/rhabdoid tumors (AT/RT) are aggressive infantile brain tumors with poor survival. Recent advancements have highlighted significant molecular heterogeneity in AT/RT with an aggressive subgroup featuring overexpression of the MYC proto-oncogene. We perform the first comprehensive metabolic profiling of patient-derived AT/RT cell lines to identify therapeutic susceptibilities in high MYC-expressing AT/RT. EXPERIMENTAL DESIGN: Metabolites were extracted from AT/RT cell lines and separated in ultra-high performance liquid chromatography mass spectrometry. Glutamine metabolic inhibition with 6-diazo-5-oxo-L-norleucine (DON) was tested with growth and cell death assays and survival studies in orthotopic mouse models of AT/RT. Metabolic flux analysis was completed to identify combination therapies to act synergistically to improve survival in high MYC AT/RT. RESULTS: Unbiased metabolic profiling of AT/RT cell models identified a unique dependence of high MYC AT/RT on glutamine for survival. The glutamine analogue, DON, selectively targeted high MYC cell lines, slowing cell growth, inducing apoptosis, and extending survival in orthotopic mouse models of AT/RT. Metabolic flux experiments with isotopically labeled glutamine revealed DON inhibition of glutathione (GSH) synthesis. DON combined with carboplatin further slowed cell growth, induced apoptosis, and extended survival in orthotopic mouse models of high MYC AT/RT. CONCLUSIONS: Unbiased metabolic profiling of AT/RT identified susceptibility of high MYC AT/RT to glutamine metabolic inhibition with DON therapy. DON inhibited glutamine-dependent synthesis of GSH and synergized with carboplatin to extend survival in high MYC AT/RT. These findings can rapidly translate into new clinical trials to improve survival in high MYC AT/RT.
Subject(s)
Diazooxonorleucine/pharmacology , Glutamine/antagonists & inhibitors , Proto-Oncogene Proteins c-myc/metabolism , Rhabdoid Tumor/metabolism , Teratoma/metabolism , Animals , Antimetabolites, Antineoplastic/pharmacology , Apoptosis/drug effects , Cell Proliferation/drug effects , Female , Glutamine/metabolism , Humans , Metabolome/drug effects , Mice , Mice, Nude , Proto-Oncogene Mas , Rhabdoid Tumor/drug therapy , Rhabdoid Tumor/pathology , Teratoma/drug therapy , Teratoma/pathology , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Astroblastoma (AB) is a rare CNS tumor demonstrating abundant astroblastomatous pseudorosettes. Its molecular features have not been comprehensively studied and its status as a tumor entity is controversial. We analyzed a cohort of 27 histologically-defined ABs using DNA methylation profiling, copy number analysis, FISH and site-directed sequencing. Most cases demonstrated mutually exclusive MN1 rearrangements (n = 10) or BRAFV600E mutations (n = 7). Two additional cases harbored RELA rearrangements. Other cases lacked these specific genetic alterations (n = 8). By DNA methylation profiling, tumors with MN1 or RELA rearrangement clustered with high-grade neuroepithelial tumor with MN1 alteration (HGNET-MN1) and RELA-fusion ependymoma, respectively. In contrast, BRAFV600E-mutant tumors grouped with pleomorphic xanthoastrocytoma (PXA). Six additional tumors clustered with either supratentorial pilocytic astrocytoma and ganglioglioma (LGG-PA/GG-ST), normal or reactive cerebrum, or with no defined DNA methylation class. While certain histologic features favored one genetic group over another, no group could be reliably distinguished by histopathology alone. Survival analysis between genetic AB subtypes was limited by sample size, but showed that MN1-rearranged AB tumors were characterized by better overall survival compared to other genetic subtypes, in fact, significantly better than BRAFV600E-mutant tumors (P = 0.013). Our data confirm that histologically-defined ABs are molecularly heterogeneous and do not represent a single entity. They rather encompass several low- to higher-grade glial tumors including neuroepithelial tumors with MN1 rearrangement, PXA-like tumors, RELA ependymomas, and possibly yet uncharacterized lesions. Genetic subtyping of tumors exhibiting AB histology, particularly determination of MN1 and BRAFV600E status, is necessary for important prognostic and possible treatment implications.
Subject(s)
Brain Neoplasms/genetics , Gene Rearrangement/genetics , Genomics/methods , Neoplasms, Neuroepithelial/genetics , Proto-Oncogene Proteins B-raf/genetics , Trans-Activators/genetics , Tumor Suppressor Proteins/genetics , Adolescent , Adult , Aged , Brain Neoplasms/mortality , Brain Neoplasms/pathology , Child , Child, Preschool , Cohort Studies , Female , Humans , Male , Middle Aged , Neoplasms, Neuroepithelial/mortality , Neoplasms, Neuroepithelial/pathology , Prognosis , Survival Rate/trends , Young AdultABSTRACT
Anaplasia may be identified in a subset of tumors with a presumed pilocytic astrocytoma (PA) component or piloid features, which may be associated with aggressive behavior, but the biologic basis of this change remains unclear. Fifty-seven resections from 36 patients (23 M, 13 F, mean age 32 years, range 3-75) were included. A clinical diagnosis of NF1 was present in 8 (22%). Alternative lengthening of telomeres (ALT) was assessed by telomere-specific FISH and/or CISH. A combination of immunohistochemistry, DNA sequencing and FISH were used to study BRAF, ATRX, CDKN2A/p16, mutant IDH1 p.R132H and H3-K27M proteins. ALT was present in 25 (69%) cases and ATRX loss in 20 (57%), mostly in the expected association of ALT+/ATRX- (20/24, 83%) or ALT-/ATRX+ (11/11, 100%). BRAF duplication was present in 8 (of 26) (31%). H3-K27M was present in 5 of 32 (16%) cases, all with concurrent ATRX loss and ALT. ALT was also present in 9 (of 11) cases in the benign PA precursor, 7 of which also had ATRX loss in both the precursor and the anaplastic tumor. In a single pediatric case, ALT and ATRX loss developed in the anaplastic component only, and in another adult case, ALT was present in the PA-A component only, but ATRX was not tested. Features associated with worse prognosis included subtotal resection, adult vs. pediatric, presence of a PA precursor preceding a diagnosis of anaplasia, necrosis, presence of ALT and ATRX expression loss. ALT and ATRX loss, as well as alterations involving the MAPK pathway, are frequent in PA with anaplasia at the time of development of anaplasia or in their precursors. Additionally, a small subset of PA with anaplasia have H3-K27M mutations. These findings further support the concept that PA with anaplasia is a neoplasm with heterogeneous genetic features and alterations typical of both PA and diffuse gliomas.
Subject(s)
Astrocytoma/genetics , Astrocytoma/pathology , Brain Neoplasms/pathology , Adolescent , Adult , Aged , Anaplasia/pathology , Biomarkers, Tumor/genetics , Brain/pathology , Child , Child, Preschool , Female , Glioma/pathology , Histones/genetics , Histones/metabolism , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Male , Middle Aged , Mutation , Nuclear Proteins/genetics , Telomere/genetics , Telomere/physiology , Telomere Homeostasis/genetics , X-linked Nuclear Protein/genetics , X-linked Nuclear Protein/physiologyABSTRACT
This corrects the article DOI: 10.1038/modpathol.2017.12.
ABSTRACT
BACKGROUND: Atypical teratoid/rhabdoid tumors (AT/RTs) are deadly pediatric brain tumors driven by LIN28. Mammalian target of rapamycin (mTOR) is activated in many deadly, drug-resistant cancers and governs important cellular functions such as metabolism and survival. LIN28 regulates mTOR in normal cells. We therefore hypothesized that mTOR is activated downstream of LIN28 in AT/RT, and the brain-penetrating mTOR complex 1 and 2 (mTORC1/2) kinase inhibitor TAK228 would reduce AT/RT tumorigenicity. METHODS: Activation of mTOR in AT/RT was determined by measuring pS6 and pAKT (Ser473) by immunohistochemistry on tissue microarray of 18 primary AT/RT tumors. In vitro growth assays (BrdU and MTS), death assays (CC3, c-PARP by western blot), and survival curves of AT/RT orthotopic xenograft models were used to measure the efficacy of TAK228 alone and in combination with cisplatin. RESULTS: Lentiviral short hairpin RNA-mediated knockdown of LIN28A led to decreased mTOR activation. Primary human AT/RT had high levels of pS6 and pAKT (Ser473) in 21% and 87% of tumors by immunohistochemistry. TAK228 slowed cell growth, induced apoptosis in vitro, and nearly doubled median survival of orthotopic xenograft models of AT/RT. TAK228 combined with cisplatin synergistically slowed cell growth and enhanced cisplatin-induced apoptosis. Suppression of AKT sensitized cells to cisplatin-induced apoptosis and forced activation of AKT protected cells. Combined treatment with TAK228 and cisplatin significantly extended survival of orthotopic xenograft models of AT/RT compared with each drug alone. CONCLUSIONS: TAK228 has efficacy in AT/RT as a single agent and synergizes with conventional chemotherapies by sensitizing tumors to cisplatin-induced apoptosis. These results suggest TAK228 may be an effective new treatment for AT/RT.
Subject(s)
Antineoplastic Agents/therapeutic use , Benzoxazoles/pharmacology , Cell Proliferation/drug effects , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Rhabdoid Tumor/drug therapy , Teratoma/drug therapy , Animals , Apoptosis/drug effects , Brain Neoplasms/drug therapy , Cell Cycle/drug effects , Cell Line, Tumor , Cisplatin/pharmacology , Humans , Mice , TOR Serine-Threonine Kinases/drug effects , Xenograft Model Antitumor Assays/methodsABSTRACT
Primitive myxoid mesenchymal tumor of infancy is a rare sarcoma that preferentially affects infants. It can be locally aggressive and rarely metastasizes, but the long-term outcome of children with this tumor is mostly unknown. Histologically, it is characterized by primitive cells with abundant myxoid stroma. Internal tandem duplication of B-cell CLL/lymphoma 6 (BCL6)-interacting co-repressor (BCOR) exon 15 has recently been described in clear cell sarcoma of kidney, central nervous system high-grade neuroepithelial tumor with BCOR alteration, and primitive myxoid mesenchymal tumor of infancy. Herein, we report five cases of primitive myxoid mesenchymal tumor of infancy: three girls and two boys with mean age of 6.5 months. The tumors were located in the paraspinal region (n=3), back (n=1), or foot (n=1) and ranged in size from 2.5 to 10.2 cm. BCOR internal tandem duplication was confirmed by PCR and sequencing in all five cases. The minimally duplicated region consisted of nine residues, which is shorter than was previously reported in other BCOR-associated tumors. To assess the clinical value and specificity of the BCOR internal tandem duplication, a group of 11 ETV6-rearranged congenital infantile fibrosarcomas were evaluated and no BCOR internal tandem duplication was identified in any case. Though not detected in congenital infantile fibrosarcomas, BCOR and BCL6 immunoreactivity was present in >90% of the nuclei of tumor cells in each of the five primitive myxoid mesenchymal tumor of infancy. The presence of BCOR internal tandem duplication in all five primitive myxoid mesenchymal tumors of infancy provides evidence that it is a recurrent somatic abnormality and substantiates the concept that this tumor is a unique sarcoma of infancy. Our findings indicate that identification of BCOR internal tandem duplication and/or nuclear immunoreactivity for BCOR or BCL6 can aid in the diagnosis of primitive myxoid mesenchymal tumor of infancy and help to differentiate it from congenital infantile fibrosarcoma.
Subject(s)
Biomarkers, Tumor/analysis , Biomarkers, Tumor/genetics , Fibrosarcoma/chemistry , Fibrosarcoma/congenital , Proto-Oncogene Proteins c-bcl-6/analysis , Proto-Oncogene Proteins/analysis , Proto-Oncogene Proteins/genetics , Repressor Proteins/analysis , Repressor Proteins/genetics , Soft Tissue Neoplasms/chemistry , Soft Tissue Neoplasms/genetics , Tandem Repeat Sequences , Cell Nucleus/chemistry , Diagnosis, Differential , Female , Fibrosarcoma/pathology , Humans , Immunohistochemistry , Infant , Infant, Newborn , Male , Polymerase Chain Reaction , Predictive Value of Tests , Retrospective Studies , Soft Tissue Neoplasms/pathology , Tumor BurdenABSTRACT
UNLABELLED: We sought to determine the possibility of an interrelationship between primary virus replication in the eye, the level of viral DNA in the trigeminal ganglia (TG) during latency, and the amount of virus reactivation following ocular herpes simplex virus type 1 (HSV-1) infection. Mice were infected with virulent (McKrae) or avirulent (KOS and RE) strains of HSV-1, and virus titers in the eyes and TG during primary infection, level of viral gB DNA in TG on day 28 postinfection (p.i.), and virus reactivation on day 28 p.i. as measured by explant reactivation were calculated. Our results suggest that the avirulent strains of HSV-1, even after corneal scarification, had lower virus titers in the eye, had less latency in the TG, and took a longer time to reactivate than virulent strains of HSV-1. The time to explant reactivation of avirulent strains of HSV-1 was similar to that of the virulent LAT((-)) McKrae-derived mutant. The viral dose with the McKrae strain of HSV-1 affected the level of viral DNA and time to explant reactivation. Overall, our results suggest that there is no absolute correlation between primary virus titer in the eye and TG and the level of viral DNA in latent TG and time to reactivation. IMPORTANCE: Very little is known regarding the interrelationship between primary virus replication in the eye, the level of latency in TG, and the time to reactivate in the mouse model. This study was designed to answer these questions. Our results point to the absence of any correlation between the level of primary virus replication and the level of viral DNA during latency, and neither was an indicator of how rapidly the virus reactivated following explant TG-induced reactivation.
Subject(s)
Herpes Simplex/virology , Herpesvirus 1, Human/genetics , Trigeminal Ganglion/virology , Virus Activation/genetics , Virus Latency/genetics , Virus Replication/genetics , Animals , Cornea/virology , DNA, Viral/genetics , Disease Models, Animal , Female , Mice , Mice, Inbred C57BL , Viral Load/methodsABSTRACT
Primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) are highly aggressive, poorly differentiated embryonal tumors occurring predominantly in young children but also affecting adolescents and adults. Herein, we demonstrate that a significant proportion of institutionally diagnosed CNS-PNETs display molecular profiles indistinguishable from those of various other well-defined CNS tumor entities, facilitating diagnosis and appropriate therapy for patients with these tumors. From the remaining fraction of CNS-PNETs, we identify four new CNS tumor entities, each associated with a recurrent genetic alteration and distinct histopathological and clinical features. These new molecular entities, designated "CNS neuroblastoma with FOXR2 activation (CNS NB-FOXR2)," "CNS Ewing sarcoma family tumor with CIC alteration (CNS EFT-CIC)," "CNS high-grade neuroepithelial tumor with MN1 alteration (CNS HGNET-MN1)," and "CNS high-grade neuroepithelial tumor with BCOR alteration (CNS HGNET-BCOR)," will enable meaningful clinical trials and the development of therapeutic strategies for patients affected by poorly differentiated CNS tumors.
Subject(s)
Central Nervous System Neoplasms/genetics , Central Nervous System Neoplasms/pathology , DNA Methylation , Neuroectodermal Tumors/genetics , Neuroectodermal Tumors/pathology , Amino Acid Sequence , Central Nervous System Neoplasms/classification , Central Nervous System Neoplasms/diagnosis , Child , Forkhead Transcription Factors/genetics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Molecular Sequence Data , Neuroectodermal Tumors/classification , Neuroectodermal Tumors/diagnosis , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/genetics , Repressor Proteins/chemistry , Repressor Proteins/genetics , Signal Transduction , Trans-Activators , Tumor Suppressor Proteins/geneticsABSTRACT
PURPOSE: Two phase II studies assessed the efficacy of vismodegib, a sonic hedgehog (SHH) pathway inhibitor that binds smoothened (SMO), in pediatric and adult recurrent medulloblastoma (MB). PATIENTS AND METHODS: Adult patients enrolled onto PBTC-025B and pediatric patients enrolled onto PBTC-032 were treated with vismodegib (150 to 300 mg/d). Protocol-defined response, which had to be sustained for 8 weeks, was confirmed by central neuroimaging review. Molecular tests to identify patterns of response and insensitivity were performed when tissue was available. RESULTS: A total of 31 patients were enrolled onto PBTC-025B, and 12 were enrolled onto PBTC-032. Three patients in PBTC-025B and one in PBTC-032, all with SHH-subgroup MB (SHH-MB), exhibited protocol-defined responses. Progression-free survival (PFS) was longer in those with SHH-MB than in those with non-SHH-MB, and prolonged disease stabilization occurred in 41% of patient cases of SHH-MB. Among those with SHH-MB, loss of heterozygosity of PTCH1 was associated with prolonged PFS, and diffuse staining of P53 was associated with reduced PFS. Whole-exome sequencing identified mutations in SHH genes downstream from SMO in four of four tissue samples from nonresponders and upstream of SMO in two of four patients with favorable responses. CONCLUSION: Vismodegib exhibits activity against adult recurrent SHH-MB but not against recurrent non-SHH-MB. Inadequate accrual of pediatric patients precluded conclusions in this population. Molecular analyses support the hypothesis that SMO inhibitor activity depends on the genomic aberrations within the tumor. Such inhibitors should be advanced in SHH-MB studies; however, molecular and genomic work remains imperative to identify target populations that will truly benefit.
Subject(s)
Anilides/therapeutic use , Brain Neoplasms/drug therapy , Hedgehog Proteins/metabolism , Medulloblastoma/drug therapy , Pyridines/therapeutic use , Adult , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Female , Hedgehog Proteins/genetics , Humans , Male , Medulloblastoma/genetics , Medulloblastoma/metabolism , Middle Aged , Young AdultABSTRACT
Atypical teratoid rhabdoid tumor (AT/RT) is among the most fatal of all pediatric brain tumors. Aside from loss of function mutations in the SMARCB1 (BAF47/INI1/SNF5) chromatin remodeling gene, little is known of other molecular drivers of AT/RT. LIN28A and LIN28B are stem cell factors that regulate thousands of RNAs and are expressed in aggressive cancers. We identified high-levels of LIN28A and LIN28B in AT/RT primary tumors and cell lines, with corresponding low levels of the LIN28-regulated microRNAs of the let-7 family. Knockdown of LIN28A by lentiviral shRNA in the AT/RT cell lines CHLA-06-ATRT and BT37 inhibited growth, cell proliferation and colony formation and induced apoptosis. Suppression of LIN28A in orthotopic xenograft models led to a more than doubling of median survival compared to empty vector controls (48 vs 115 days). LIN28A knockdown led to increased expression of let-7b and let-7g microRNAs and a down-regulation of KRAS mRNA. AT/RT primary tumors expressed increased mitogen activated protein (MAP) kinase pathway activity, and the MEK inhibitor selumetinib (AZD6244) decreased AT/RT growth and increased apoptosis. These data implicate LIN28/RAS/MAP kinase as key drivers of AT/RT tumorigenesis and indicate that targeting this pathway may be a therapeutic option in this aggressive pediatric malignancy.
Subject(s)
Antineoplastic Agents/pharmacology , Brain Neoplasms/drug therapy , MAP Kinase Signaling System/drug effects , Protein Kinase Inhibitors/pharmacology , RNA-Binding Proteins/metabolism , Rhabdoid Tumor/drug therapy , Teratoma/drug therapy , Animals , Apoptosis , Brain Neoplasms/enzymology , Brain Neoplasms/genetics , Cell Line, Tumor , Cell Proliferation , Dose-Response Relationship, Drug , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , Molecular Targeted Therapy , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , RNA Interference , RNA-Binding Proteins/genetics , Rhabdoid Tumor/enzymology , Rhabdoid Tumor/genetics , Teratoma/enzymology , Teratoma/genetics , Time Factors , Transfection , Tumor Burden , Xenograft Model Antitumor AssaysABSTRACT
Recently, we have reported that CD8α(+) DCs, rather than CD8(+) T cells, are involved in the establishment and maintenance of HSV-1 latency in the trigeminal ganglia (TG) of ocularly infected mice. In the current study, we investigated whether similar results can be obtained using Batf3(-/-) mice that previously were reported to lack CD8α(+) DCs. However, our results demonstrate that Batf3(-/-) mice, without any known infection, express CD8α(+) DCs. Consequently, due to the presence of CD8α(+) DCs, no differences were detected in the level of HSV-1 latency between Batf3(-/-) mice compared with wild type control mice.
Subject(s)
Basic-Leucine Zipper Transcription Factors/deficiency , CD8 Antigens/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Repressor Proteins/deficiency , Animals , CD8 Antigens/genetics , Disease Models, Animal , Gene Knockout Techniques , Herpes Simplex/genetics , Herpes Simplex/immunology , Herpes Simplex/metabolism , Herpesvirus 1, Human/immunology , Immunohistochemistry , Immunophenotyping , Mice , Mice, Knockout , Phenotype , RabbitsABSTRACT
Recently we have shown that the highly conserved herpes simplex virus glycoprotein K (gK) binds to signal peptide peptidase (SPP), also known as minor histocompatibility antigen H13. In this study we have demonstrated for the first time that inhibitors of SPP, such as L685,458, (Z-LL)2 ketone, aspirin, ibuprofen and DAPT, significantly reduced HSV-1 replication in tissue culture. Inhibition of SPP activity via (Z-LL)2 ketone significantly reduced viral transcripts in the nucleus of infected cells. Finally, when administered during primary infection, (Z-LL)2 ketone inhibitor reduced HSV-1 replication in the eyes of ocularly infected mice. Thus, blocking SPP activity may represent a clinically effective and expedient approach to the reduction of viral replication and the resulting pathology.
Subject(s)
Aspartic Acid Endopeptidases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Herpesvirus 1, Human/physiology , Herpesvirus 1, Human/pathogenicity , Keratitis, Herpetic/virology , Virus Replication/drug effects , Animals , Aspirin/pharmacology , Carbamates/pharmacology , Cell Fractionation , Cells, Cultured , DNA, Viral/genetics , Dipeptides/pharmacology , Disease Models, Animal , Female , Gene Expression Regulation, Viral/physiology , Herpesvirus 1, Human/genetics , Ibuprofen/pharmacology , Keratitis, Herpetic/prevention & control , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Ophthalmic Solutions , RNA, Messenger/metabolism , Rabbits , Real-Time Polymerase Chain Reaction , Skin/cytology , Skin/virologyABSTRACT
It is generally accepted that CD8 T cells play the key role to maintain HSV-1 latency in trigeminal ganglia of ocularly infected mice. Yet, comparably little is known about the role of innate immunity in establishment of viral latency. In the current study, we investigated whether CD8α DCs impact HSV-1 latency by examining latency in the trigeminal ganglia (TG) of wild-type (WT) C57BL/6 versus CD8α-/- (lack functional CD8 T cells and CD8α+ DCs), CD8ß-/- (have functional CD8α+ T cells and CD8α+ DCs), and ß2m-/- (lack functional CD8 T cells but have CD8α+ DCs) mice as well as BXH2 (have functional CD8 T cells but lack CD8α+ DCs) versus WT C3H (have functional CD8α T cells and CD8α+ DCs) mice. We also determined whether the phenotype of CD8α-/- and BXH2 mice could be restored to that of WT mice by adoptive transfer of WT CD8+ T cells or bone marrow (BM) derived CD8α+ DCs. Our results clearly demonstrate that CD8α DCs, rather than CD8 T cells, are responsible for enhanced viral latency and recurrences.
Subject(s)
CD8 Antigens/immunology , CD8-Positive T-Lymphocytes/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Herpesvirus 1, Human/immunology , Virus Latency/immunology , Adoptive Transfer/methods , Animals , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/virology , Dendritic Cells/virology , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Knockout , Recurrence , Trigeminal Ganglion/immunology , Trigeminal Ganglion/metabolism , Trigeminal Ganglion/virologyABSTRACT
PURPOSE: We have shown previously that HSV-1 glycoprotein K (gK) exacerbates corneal scarring (CS) in mice and rabbits. Here, we investigated the relative impact of gK overexpression on host responses during primary corneal infection and latency in trigeminal ganglia (TG) of infected mice. METHODS: Mice were infected ocularly with HSV-gK(3) (expressing two extra copies of gK replacing latency associated transcript [LAT]), HSV-gK(3) revertant (HSV-gK(3)R), or wild-type HSV-1 strain McKrae. Individual corneas on day 5 post infection (PI) and TG on day 28 PI were isolated and used for detection of gB DNA in the TG, HSV-1 receptors in the cornea and TG, and inflammatory infiltrates in TG. RESULTS: During primary HSV-1 infection, gK overexpression resulted in altered expression of herpesvirus entry mediator (HVEM), 3-O-sulfated heparin sulfate (3-OS-HS), paired immunoglobulin-like type 2 receptor-α (PILR-α), nectin-1, and nectin-2 in cornea of BALB/c, but not C57BL/6 mice. However, gK overexpression did have an effect on 3-OS-HS, PILR-α, nectin-1, and nectin-2 expression (but not HVEM expression) in TG of C57BL/6 mice during latency. These differences did not affect the level of latency, but instead were correlated with the presence of CS. The presence of LAT increased HVEM expression and this effect was enhanced further by the presence of CS in latently-infected mice. Finally, the presence of LAT, but not overexpression of gK, affected CD4, CD8, TNF-α, Tim-3, PD-1, IL-21, IL-2, and IFN-γ expression in TG. CONCLUSIONS: We demonstrate a novel link between gK exacerbation of CS and HSV-1 receptors, suggesting a gK-induced molecular route for the pathogenesis as well as selective advantage of these entry routes for the pathogen during latency-reactivation cycle.
Subject(s)
Cornea/virology , Gene Expression Regulation , Herpesvirus 1, Human/metabolism , Keratitis, Herpetic/metabolism , RNA, Viral/genetics , Receptors, Virus/genetics , Viral Proteins/genetics , Animals , Cells, Cultured , Cornea/metabolism , Cornea/pathology , Disease Models, Animal , Female , Herpesvirus 1, Human/genetics , Keratitis, Herpetic/genetics , Keratitis, Herpetic/virology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Rabbits , Real-Time Polymerase Chain Reaction , Receptors, Virus/biosynthesis , Viral Proteins/biosynthesis , Virus ReplicationABSTRACT
UNLABELLED: The latency-associated transcript (LAT) of herpes simplex virus 1 (HSV-1), CD8α(+) dendritic cells (DCs), and programmed death 1 (PD-1) have all been implicated in the HSV-1 latency-reactivation cycle. It is not known, however, whether an interaction between LAT and CD8α(+) DCs regulates latency and T-cell exhaustion. To address this question, we used LAT-expressing [LAT(+)] and LAT-negative [LAT(-)] viruses. Depletion of DCs in mice ocularly infected with LAT(+) virus resulted in a reduction in the number of T cells expressing PD-1 in the trigeminal ganglia (TG), whereas depletion of DCs in mice similarly infected with LAT(-) virus did not alter PD-1 expression. CD8α(+) DCs, but not CD4(+) DCs, infected with LAT(+) virus had higher levels of ICP0, ICP4, thymidine kinase (TK), and PD-1 ligand 1 (PD-L1) transcripts than those infected with LAT(-) virus. Coculture of infected bone marrow (BM)-derived DCs from wild-type (WT) mice, but not infected DCs from CD8α(-/-) mice, with WT naive T cells contributed to an increase in PD-1 expression. Transfer of bone marrow from WT mice but not CD8α(-/-) mice to recipient Rag1(-/-) mice increased the number of latent viral genomes in reconstituted mice infected with the LAT(+) virus. Collectively, these data indicated that a reduction in latency correlated with a decline in the levels of CD8α(+) DCs and PD-1 expression. In summary, our results demonstrate an interaction among LAT, PD-1, and CD11c CD8α(+) cells that regulates latency in the TG of HSV-1-infected mice. IMPORTANCE: Very little is known regarding the interrelationship of LAT, PD-1, and CD8α(+) DCs and how such interactions might contribute to relative numbers of latent viral genomes. We show here that (i) in both in vivo and in vitro studies, deficiency of CD8α(+) DCs significantly reduced T-cell exhaustion in the presence of LAT(+) virus but not LAT(-) virus; (ii) HSV-1 infectivity was significantly lower in LAT(-)-infected DCs than in their LAT(+)-infected counterparts; and (iii) adoptive transfer of bone marrow (BM) from WT but not CD8α(-/-) mice to recipient Rag1(-/-) mice restored latency to the level in WT mice following infection with LAT(+) virus. These studies point to a key role for CD8α(+) DCs in T-cell exhaustion in the presence of LAT, which leads to larger numbers of latent viral genomes. Thus, altering this negative function of CD8α(+) DCs can potentially be used to generate a more effective vaccine against HSV infection.
Subject(s)
CD8 Antigens/metabolism , Herpes Simplex/metabolism , Herpesvirus 1, Human/physiology , MicroRNAs/metabolism , Programmed Cell Death 1 Receptor/metabolism , Virus Latency , Animals , CD8 Antigens/genetics , Cells, Cultured , Dendritic Cells/metabolism , Dendritic Cells/virology , Herpes Simplex/genetics , Herpes Simplex/virology , Herpesvirus 1, Human/genetics , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , MicroRNAs/genetics , Programmed Cell Death 1 Receptor/genetics , Trigeminal Ganglion/metabolism , Trigeminal Ganglion/virologyABSTRACT
Glycoprotein K (gK) is a virion envelope protein of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), which plays important roles in virion entry, morphogenesis and egress. Two-hybrid and pull-down assays were utilized to demonstrate that gK and no other HSV-1 genes specifically binds to signal peptide peptidase (SPP), also known as minor histocompatibility antigen H13. SPP dominant negative mutants, shRNA against SPP significantly reduced HSV-1 replication in vitro. SPP also affected lysosomes and ER responses to HSV-1 infection. Thus, in this study we have shown for the first time that gK, despite its role in fusion and egress, is also involved in binding the cytoplasmic protein SPP. These results also suggest that SPP plays an important role in viral replication and possibly virus pathogenesis. This makes SPP unique in that its function appears to be required by the virus as no other protein can compensate its loss in terms of viral replication.
Subject(s)
Aspartic Acid Endopeptidases/metabolism , Herpesvirus 1, Human/metabolism , Herpesvirus 1, Human/physiology , Viral Proteins/metabolism , Animals , Aspartic Acid Endopeptidases/genetics , Chlorocebus aethiops , HeLa Cells , Herpesvirus 1, Human/genetics , Humans , Lysosomes/metabolism , Protein Binding , RNA, Small Interfering , Two-Hybrid System Techniques , Vero Cells , Viral Proteins/genetics , Virus Replication/genetics , Virus Replication/physiologyABSTRACT
CD80 plays a critical role in stimulation of T cells and subsequent control of infection. To investigate the effect of CD80 on HSV-1 infection, we constructed a recombinant HSV-1 virus that expresses two copies of the CD80 gene in place of the latency associated transcript (LAT). This mutant virus (HSV-CD80) expressed high levels of CD80 and had similar virus replication kinetics as control viruses in rabbit skin cells. In contrast to parental virus, this CD80 expressing recombinant virus replicated efficiently in immature dendritic cells (DCs). Additionally, the susceptibility of immature DCs to HSV-CD80 infection was mediated by CD80 binding to PD-L1 on DCs. This interaction also contributed to a significant increase in T cell activation. Taken together, these results suggest that inclusion of CD80 as a vaccine adjuvant may promote increased vaccine efficacy by enhancing the immune response directly and also indirectly by targeting to DC.