ABSTRACT
Medulloblastoma (MB) encompasses diverse subgroups, and leptomeningeal disease/metastasis (LMD) plays a substantial role in associated fatalities. Despite extensive exploration of canonical genes in MB, the molecular mechanisms underlying LMD and the involvement of the orthodenticle homeobox 2 (OTX2) gene, a key driver in aggressive MB Group 3, remain insufficiently understood. Recognizing OTX2's pivotal role, we investigated its potential as a catalyst for aggressive cellular behaviors, including migration, invasion, and metastasis. OTX2 overexpression heightened cell growth, motility, and polarization in Group 3 MB cells. Orthotopic implantation of OTX2-overexpressing cells in mice led to reduced median survival, accompanied by the development of spinal cord and brain metastases. Mechanistically, OTX2 acted as a transcriptional activator of the Mechanistic Target of Rapamycin (mTOR) gene's promoter and the mTORC2 signaling pathway, correlating with upregulated downstream genes that orchestrate cell motility and migration. Knockdown of mTOR mRNA mitigated OTX2-mediated enhancements in cell motility and polarization. Analysis of human MB tumor samples (N = 952) revealed a positive correlation between OTX2 and mTOR mRNA expression, emphasizing the clinical significance of OTX2's role in the mTORC2 pathway. Our results reveal that OTX2 governs the mTORC2 signaling pathway, instigating LMD in Group 3 MBs and offering insights into potential therapeutic avenues through mTORC2 inhibition.
Subject(s)
Gene Expression Regulation, Neoplastic , Mechanistic Target of Rapamycin Complex 2 , Medulloblastoma , Meningeal Neoplasms , Otx Transcription Factors , Animals , Female , Humans , Male , Mice , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Cerebellar Neoplasms/genetics , Cerebellar Neoplasms/pathology , Cerebellar Neoplasms/metabolism , Mechanistic Target of Rapamycin Complex 2/metabolism , Mechanistic Target of Rapamycin Complex 2/genetics , Medulloblastoma/genetics , Medulloblastoma/pathology , Medulloblastoma/metabolism , Meningeal Neoplasms/genetics , Meningeal Neoplasms/pathology , Meningeal Neoplasms/metabolism , Meningeal Neoplasms/secondary , Otx Transcription Factors/metabolism , Otx Transcription Factors/genetics , Signal TransductionABSTRACT
Glioblastoma is one of the most devastating neoplasms of the central nervous system. This study focused on the development of serum extracellular vesicle (EV)-based glioblastoma tumor marker panels that can be used in a clinic to diagnose glioblastomas and to monitor tumor burden, progression, and regression in response to treatment. RNA sequencing studies were performed using RNA isolated from serum EVs from both patients (n = 85) and control donors (n = 31). RNA sequencing results for preoperative glioblastoma EVs compared to control EVs revealed 569 differentially expressed genes (DEGs, 2XFC, FDR < 0.05). By using these DEGs, we developed serum-EV-based biomarker panels for the following glioblastomas: wild-type IDH1 (96% sensitivity/80% specificity), MGMT promoter methylation (91% sensitivity/73% specificity), p53 gene mutation (100% sensitivity/89% specificity), and TERT promoter mutation (89% sensitivity/100% specificity). This is the first study showing that serum-EV-based biomarker panels can be used to diagnose glioblastomas with a high sensitivity and specificity.
ABSTRACT
Extracellular vesicles (EVs) may be used as a non-invasive screening platform to discover markers associated with early diagnosis, prognosis, and treatment response. Such an approach is invaluable for diseases such as glioblastoma, for which only a few non-invasive diagnostic or prognostic markers are available. We used mass spectrometry to analyze proteomics profiles of EVs derived from four glioblastoma cell lines and human primary astrocytes (HPAs) and found that SRPX is the only protein enriched in the majority of glioblastoma EVs that was absent in the HPA-derived EVs. Then, we evaluated the relationship between SRPX protein expression and tumor grade using immunohistochemical staining (IHC) and performed colony formation and viability assays to analyze the possible function of SRPX in glioblastoma. SRPX mRNA and protein expression were associated with tumor grade. Moreover, temozolomide (TMZ)-resistant tumor tissues showed highly positive SRPX staining, compared to all other tumor grades. Additionally, glioblastoma cells displayed enhanced SRPX gene expression when exposed to TMZ. Knockdown of SRPX gene expression via siRNA inhibited cell viability. Taken together, the results of this study suggest that SRPX can be used as a novel tumor marker for diagnostic and prognostic purposes and can also be a therapeutic target for glioblastomas.
ABSTRACT
Numerous therapies aimed at driving an effective anti-glioma response have been employed over the last decade; nevertheless, survival outcomes for patients remain dismal. This may be due to the expression of immune-checkpoint ligands such as PD-L1 by glioblastoma (GBM) cells which interact with their respective receptors on tumor-infiltrating effector T cells curtailing the activation of anti-GBM CD8+ T cell-mediated responses. Therefore, a combinatorial regimen to abolish immunosuppression would provide a powerful therapeutic approach against GBM. We developed a peptide ligand (CD200AR-L) that binds an uncharacterized CD200 immune-checkpoint activation receptor (CD200AR). We sought to test the hypothesis that CD200AR-L/CD200AR binding signals via he DAP10&12 pathways through in vitro studies by analyzing transcription, protein, and phosphorylation, and in vivo loss of function studies using inhibitors to select signaling molecules. We report that CD200AR-L/CD200AR binding induces an initial activation of the DAP10&12 pathways followed by a decrease in activity within 30 min, followed by reactivation via a positive feedback loop. Further in vivo studies using DAP10&12KO mice revealed that DAP10, but not DAP12, is required for tumor control. When we combined CD200AR-L with an immune-stimulatory gene therapy, in an intracranial GBM model in vivo, we observed increased median survival, and long-term survivors. These studies are the first to characterize the signaling pathway used by the CD200AR, demonstrating a novel strategy for modulating immune checkpoints for immunotherapy currently being analyzed in a phase I adult trial.
Subject(s)
Antigens, CD/metabolism , Brain Neoplasms/metabolism , Glioma/metabolism , Immune Checkpoint Inhibitors/metabolism , Membrane Glycoproteins/metabolism , Receptors, Immunologic/deficiency , Amino Acid Sequence , Animals , Antigens, CD/administration & dosage , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/metabolism , Brain Neoplasms/drug therapy , Brain Neoplasms/genetics , Female , Genetic Therapy/methods , Glioma/drug therapy , Glioma/genetics , Immune Checkpoint Inhibitors/administration & dosage , Membrane Glycoproteins/antagonists & inhibitors , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Structure, Tertiary , Receptors, Immunologic/genetics , Signal Transduction/drug effects , Signal Transduction/physiology , Tumor Microenvironment/drug effects , Tumor Microenvironment/physiologyABSTRACT
PURPOSE: Advances in immunotherapy have revolutionized care for some patients with cancer. However, current checkpoint inhibitors are associated with significant toxicity and yield poor responses for patients with central nervous system tumors, calling into question whether cancer immunotherapy can be applied to glioblastoma multiforme. We determined that targeting the CD200 activation receptors (CD200AR) of the CD200 checkpoint with a peptide inhibitor (CD200AR-L) overcomes tumor-induced immunosuppression. We have shown the clinical efficacy of the CD200AR-L in a trial in companion dogs with spontaneous high-grade glioma. Addition of the peptide to autologous tumor lysate vaccines significantly increased the median overall survival to 12.7 months relative to tumor lysate vaccines alone, 6.36 months. EXPERIMENTAL DESIGN: This study was developed to elucidate the mechanism of the CD200ARs and develop a humanized peptide inhibitor. We developed macrophage cell lines with each of four CD200ARs knocked out to determine their binding specificity and functional response. Using proteomics, we developed humanized CD200AR-L to explore their effects on cytokine/chemokine response, dendritic cell maturation and CMV pp65 antigen response in human CD14+ cells. GMP-grade peptide was further validated for activity. RESULTS: We demonstrated that the CD200AR-L specifically targets a CD200AR complex. Moreover, we developed and validated a humanized CD200AR-L for inducing chemokine response, stimulating immature dendritic cell differentiation and significantly enhanced an antigen-specific response, and determined that the use of the CD200AR-L downregulated the expression of CD200 inhibitory and PD-1 receptors. CONCLUSIONS: These results support consideration of a CD200AR-L as a novel platform for immunotherapy against multiple cancers including glioblastoma multiforme.
Subject(s)
Antigens, CD/metabolism , Dendritic Cells/immunology , Glioblastoma/drug therapy , Immunotherapy/methods , Orexin Receptors/metabolism , Peptide Fragments/pharmacology , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Animals , Antigens, CD/chemistry , Cells, Cultured , Glioblastoma/immunology , Glioblastoma/metabolism , Humans , Immune Tolerance , Macrophages/immunology , Macrophages/metabolism , Mice , Orexin Receptors/chemistry , Peptide Fragments/chemical synthesis , Programmed Cell Death 1 Receptor/immunologyABSTRACT
Recent advances in immunotherapy have included inhibition of immune checkpoint proteins in the tumor microenvironment and tumor lysate-based vaccination strategies. We combined these approaches in pet dogs with high-grade glioma. Administration of a synthetic peptide targeting the immune checkpoint protein, CD200, enhanced the capacity of antigen-presenting cells to prime T-cells to mediate an anti-glioma response. We found that in canine spontaneous gliomas, local injection of a canine-specific, CD200-directed peptide before subcutaneous delivery of an autologous tumor lysate vaccine prolonged survival relative to a historical control treated with autologous tumor lysate alone (median survivals of 12.7 months and 6.36 months, respectively). Antigen-presenting cells and T-lymphocytes primed with this peptide suppressed their expression of the inhibitory CD200 receptor, thereby enhancing their ability to initiate immune reactions in a glioblastoma microenvironment replete with the immunosuppressive CD200 protein. These results support consideration of a CD200 ligand as a novel glioblastoma immunotherapeutic agent.
ABSTRACT
There are over 400 ongoing clinical trials using tumor-derived vaccines. This approach is especially attractive for many types of brain tumors, including glioblastoma, yet so far the clinical response is highly variable. One contributor to poor response is CD200, which acts as a checkpoint blockade, inducing immune tolerance. We demonstrate that, in response to vaccination, glioma-derived CD200 suppresses the anti-tumor immune response. In contrast, a CD200 peptide inhibitor that activates antigen-presenting cells overcomes immune tolerance. The addition of the CD200 inhibitor significantly increased leukocyte infiltration into the vaccine site, cytokine and chemokine production, and cytolytic activity. Our data therefore suggest that CD200 suppresses the immune system's response to vaccines, and that blocking CD200 could improve the efficacy of cancer immunotherapy.
Subject(s)
Antigens, CD/metabolism , Antigens, Neoplasm/metabolism , Central Nervous System Neoplasms/therapy , Dendritic Cells/immunology , Glioblastoma/therapy , Immunotherapy/methods , Leukocytes/immunology , Animals , Antigen Presentation , Antigens, CD/genetics , Antigens, CD/immunology , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , Cell Movement , Cells, Cultured , Central Nervous System Neoplasms/immunology , Cytokines/metabolism , Cytotoxicity, Immunologic , Glioblastoma/immunology , Humans , Immune Tolerance , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA, Small Interfering/genetics , Tumor EscapeABSTRACT
Bajo entornos naturales, las plantas de maracuyá amarillo frecuentemente se enfrentan a condiciones de anegamiento, un factor limitante para la producción generalizada de su cultivo, especialmente en tierras bajas inundables. El presente estudio se encargó de identificar la activación de la enzima alcohol deshidrogenasa (ADH) durante la inundación, como un posible mecanismo de sobrevivencia de plántulas de maracuyá amarillo. Se evaluó la actividad de la enzima ADH durante 0, 1, 3, 7, 9 y 14 días de tratamiento con inundación y sin inundación, en raíces de plántulas de maracuyá amarillo de tres meses y medio de germinadas. En las raíces de plántulas de maracuyá amarillo en condiciones de inundación, la actividad de la enzima ADH presentó un aumento significativo respecto a las plántulas en condiciones normales de riego (sin inundación). Esto sugiere que la actividad de la enzima alcohol deshidrogenasa, implicada en el metabolismo anaeróbico, es un posible mecanismo de supervivencia al anegamiento de plántulas de maracuyá amarillo en periodos cortos de inundación.
Under natural environments, yellow passion fruit plants often face flooded conditions as a limiting factor for the widespread production of the crop, especially in flood-prone lowlands. This study was carried out to identify the activation of the enzyme alcohol dehydrogenase (ADH) during the flood, as a possible mechanism for the survival of yellow passion fruit seedlings. The activity of the ADH enzyme was assessed on days 0, 1, 3, 7, 9 and 14, with and without flooding, on rooted yellow passion fruit seedlings germinated during three and a half months. In the roots of the yellow passion fruit seedlings in flood conditions, the activity of the ADH enzyme showed a significant increase compared to seedlings under normal irrigation (no flooding). These results suggest that the activity of the alcohol dehydrogenase enzyme involved in anaerobic metabolism, is a possible mechanism for survival of yellow passion fruit seedlings waterlogged in short periods of flooding.
