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Meningiomas are the most common primary intracranial tumors. Although most symptomatic cases can be managed by surgery and/or radiotherapy, a relevant number of patients experience an unfavorable clinical course and additional treatment options are needed. As meningiomas are often perfused by dural branches of the external carotid artery, which is located outside the blood-brain barrier, they might be an accessible target for immunotherapy. However, the landscape of naturally presented tumor antigens in meningioma is unknown. We here provide a T-cell antigen atlas for meningioma by in-depth profiling of the naturally presented immunopeptidome using LC-MS/MS. Candidate target antigens were selected based on a comparative approach using an extensive immunopeptidome data set of normal tissues. Meningioma-exclusive antigens for HLA class I and II are described here for the first time. Top-ranking targets were further functionally characterized by showing their immunogenicity through in vitro T-cell priming assays. Thus, we provide an atlas of meningioma T-cell antigens which will be publicly available for further research. In addition, we have identified novel actionable targets that warrant further investigation as an immunotherapy option for meningioma.
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Neoplasias Meníngeas , Meningioma , Humanos , Meningioma/terapia , Cromatografia Líquida , Espectrometria de Massas em Tandem , Imunoterapia , Linfócitos T , Neoplasias Meníngeas/terapiaRESUMO
BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy has proven to be successful against hematological malignancies. However, exploiting CAR T cells to treat solid tumors is more challenging for various reasons including the lack of suitable target antigens. Here, we identify the transmembrane protein CD317 as a novel target antigen for CAR T cell therapy against glioblastoma, one of the most aggressive solid tumors. METHODS: CD317-targeting CAR T cells were generated by lentivirally transducing human T cells from healthy donors. The anti-glioma activity of CD317-CAR T cells toward various glioma cells was assessed in vitro in cell lysis assays. Subsequently, we determined the efficacy of CD317-CAR T cells to control tumor growth in vivo in clinically relevant mouse glioma models. RESULTS: We generated CD317-specific CAR T cells and demonstrate strong anti-tumor activity against several glioma cell lines as well as primary patient-derived cells with varying CD317 expression levels in vitro. A CRISPR/Cas9-mediated knockout of CD317 protected glioma cells from CAR T cell lysis, demonstrating the target specificity of the approach. Silencing of CD317 expression in T cells by RNA interference reduced fratricide of engineered T cells and further improved their effector function. Using orthotopic glioma mouse models, we demonstrate the antigen-specific anti-tumor activity of CD317-CAR T cells, which resulted in prolonged survival and cure of a fraction of CAR T cell-treated animals. CONCLUSIONS: These data reveal a promising role of CD317-CAR T cell therapy against glioblastoma, which warrants further evaluation to translate this immunotherapeutic strategy into clinical neuro-oncology.
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Glioblastoma , Glioma , Receptores de Antígenos Quiméricos , Camundongos , Animais , Humanos , Receptores de Antígenos Quiméricos/genética , Glioblastoma/patologia , Linfócitos T , Linhagem Celular Tumoral , Imunoterapia Adotiva/métodos , Glioma/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The hepatocyte growth factor (HGF)/MET signaling pathway has been proposed to be involved in the resistance to radiotherapy of glioblastoma via proinvasive and DNA damage response pathways.Here we assessed the role of the MET pathway in the response to radiotherapy in vitro and in vivo in syngeneic mouse glioma models. We find that the murine glioma cell lines GL-261, SMA-497, SMA-540 and SMA-560 express HGF and its receptor MET and respond to exogenous HGF with MET phosphorylation. Glioma cell viability or proliferation are unaffected by genetic or pharmacological MET inhibition using tepotinib or CRISPR/Cas9-engineered Met gene knockout and MET inhibition fails to sensitize glioma cells to irradiation in vitro. In contrast, the combination of tepotinib with radiotherapy prolongs survival of orthotopic SMA-560 or GL-261 glioma-bearing mice compared with radiotherapy or tepotinib treatment alone. Synergy is lost when such experiments are conducted in immunodeficient Rag1-/- mice, and, importantly, also when Met gene expression is disrupted in the tumor cells. Combination therapy suppresses a set of pro-inflammatory mediators including matrix metalloproteases that are upregulated by radiotherapy alone and that have been linked to poor outcome in glioblastoma. Several of these mediators are positively regulated by transforming growth factor (TGF)-ß, and pSMAD2 levels as a surrogate marker of TGF-ß pathway activity are suppressed by combination treatment. We conclude that synergistic suppression of experimental syngeneic glioma growth by irradiation and MET inhibition requires MET expression in the tumor as well as an intact immune system. Clinical evaluation of this combined strategy in newly diagnosed glioblastoma is warranted.
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Neoplasias Encefálicas , Glioblastoma , Glioma , Camundongos , Animais , Glioblastoma/genética , Linhagem Celular Tumoral , Glioma/patologia , Transdução de Sinais , Fosforilação , Neoplasias Encefálicas/metabolismoRESUMO
Despite multimodal treatment approaches including surgery, radiotherapy and chemotherapy, the median survival for patients with glioblastoma remains in the range of one year and thus poor. Type I interferons (IFN) are involved in immune responses to viral infection and exhibit anti-tumor activity in certain cancers. Here we explored the biological relevance of constitutive type I IFN signaling in murine glioma models in vitro and in vivo. CT-2A, GL-261, SMA-497, SMA-540 and SMA-560 murine glioma cells expressed IFN type I receptors IFNAR1 and IFNAR2 and were responsive to exogenous IFN stimulation. CRISPR/Cas9-mediated deletion of IFNAR1 decreased the baseline expression of type I IFN response genes in GL-261 cells, but neither in CT-2A nor in SMA-560 cells. IFNAR1 deletion slowed growth in GL-261 and SMA-560, but not in CT-2A cells. However, only the growth of IFNAR1-depleted GL-261 tumors and not that of SMA-560 tumors was delayed in vivo upon orthotopic tumor cell implantation into syngeneic mice. This survival gain was no longer detected when the IFNAR1-depleted GL-261 cells were inoculated into IFNAR1-deficient mice. Altogether these data suggest that constitutive type I IFN signaling in gliomas may be pro-tumorigenic, but only in a microenvironment that is proficient for type I IFN signaling in the host.
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PURPOSE: Glioblastoma is the most common brain tumor in adults and is virtually incurable. Therefore, new therapeutic strategies are urgently needed. Over the last decade, multiple growth-promoting functions have been attributed to CD95, a prototypic death receptor well characterized as an apoptosis mediator upon CD95L engagement. Strategic targeting of non-apoptotic or apoptotic CD95 signaling may hold anti-glioblastoma potential. Due to its antithetic nature, understanding the constitutive role of CD95 signaling in glioblastoma is indispensable. METHODS: We abrogated constitutive Cd95 and Cd95l gene expression by CRISPR/Cas9 in murine glioma models and characterized the consequences of gene deletion in vitro and in vivo. RESULTS: Expression of canonical CD95 but not CD95L was identified in mouse glioma cells in vitro. Instead, a soluble isoform-encoding non-canonical Cd95l transcript variant was detected. In vivo, an upregulation of the membrane-bound canonical CD95L form was revealed. Cd95 or Cd95l gene deletion decreased cell growth in vitro. The growth-supporting role of constitutive CD95 signaling was validated by Cd95 re-transfection, which rescued growth. In vivo, Cd95 or Cd95l gene deletion prolonged survival involving tumor-intrinsic and immunological mechanisms in the SMA-497 model. In the GL-261 model, that expresses no CD95, only CD95L gene deletion prolonged survival, involving a tumor-intrinsic mechanism. CONCLUSION: Non-canonical CD95L/CD95 interactions are growth-promoting in murine glioma models, and glioma growth and immunosuppression may be simultaneously counteracted by Cd95l gene silencing.
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Neoplasias Encefálicas , Glioblastoma , Glioma , Animais , Camundongos , Apoptose/fisiologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Sistemas CRISPR-Cas , Proteína Ligante Fas/genética , Proteína Ligante Fas/metabolismo , Receptor fas/genética , Receptor fas/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioma/metabolismo , Glioma/patologia , Terapia de ImunossupressãoRESUMO
Background: Glioblastoma is the most common brain tumor in adults and virtually incurable. Therefore, new therapeutic strategies are urgently needed. Immune checkpoint inhibition has not shown activity in various phase III trials and intra- as well as intertumoral expression of programmed death ligand 1 (PD-L1) varies in glioblastoma. Methods: We abrogated constitutive PD-L1 gene expression by CRISPR/Cas9 in murine glioma models and characterized the consequences of gene deletion in vitro and in vivo. Results: A heterogeneous expression of Pdl1 mRNA and PD-L1 protein was detected in the glioma cell panel in vitro and in vivo. PD-L1, but not PD-L2, was inducible by interferon ß and γ. Co-culture with splenocytes induced PD-L1 expression in GL-261 and SMA-560, but not in CT-2A cells, in an interferon γ-dependent manner. Conversely, Pdl1 gene silencing conferred a survival benefit in CT-2A, but not in the other 2 models. Accordingly, PD-L1 antibody prolonged survival in CT-2A glioma-bearing mice. This activity required PD-L1 expression on tumor rather than host cells, and the survival gain mediated by PD-L1 loss was reproduced in immune-deficient RAG-/- mice. Conclusions: PD-L1 is expressed and interferon-inducible in murine glioma cell lines. PD-L1 has model-specific roles for tumor growth. Future studies need to determine which subset of glioblastoma patients may benefit from PD-L1 antagonism as part of a multimodality therapeutic approach to glioblastoma.
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CD95 (Fas/APO-1) is a multifunctional cell surface receptor with antithetic roles. First described to mediate cell death, interactions of CD95 with its natural ligand, CD95L, have also been described to induce tumor-promoting signaling leading to proliferation, invasion and stem cell maintenance, mainly in cancer cells that are resistant to CD95-mediated apoptosis. While activation of CD95-mediated apoptosis in cancer cells may not be clinically practicable due to toxicity, inhibition of tumor-promoting CD95 signaling holds therapeutic potential. In the present study, we characterized CD95 and CD95L expression in human glioma-initiating cells (GIC), a glioblastoma cell population with stem cell features, and investigated the consequences of CRISPR-Cas9-mediated CD95 or CD95L gene deletion. In vitro, GIC expressed CD95 but not CD95L and were sensitive to CD95-mediated apoptosis. Upon genetic deletion of CD95, GIC acquired resistance to CD95L-induced apoptosis but exhibited inferior clonogenic growth, sphere-forming capacity, and invasiveness compared with control cells, suggesting the existence of CD95L-independent constitutive CD95 signaling with tumor-promoting properties in GIC. In vivo, GIC expressed CD95 and a non-canonical form of CD95L lacking the CD95-binding region. CD95 genetic deletion did not prolong survival in immunocompromised GIC-bearing mice. Altogether, these data indicate that canonical CD95L may not be expressed in human GIC and suggest the existence of a CD95L-independent CD95-signaling pathway that maintains some malignancy traits of GIC. The lack of altered survival of tumor-bearing mice after genetic deletion of CD95 suggests that CD95 signaling is not essential to maintain the growth of human GIC xenografted into the brains of nude mice. The ligand-independent tumor-promoting role of constitutive CD95 in our GIC models in vitro highlights the complexity and challenges associated with targeting CD95 with therapeutic intent.
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Glioblastoma is the most common malignant primary brain tumor. To date, clinically relevant biomarkers are restricted to isocitrate dehydrogenase (IDH) gene 1 or 2 mutations and O6-methylguanine DNA methyltransferase (MGMT) promoter methylation. Long non-coding RNAs (lncRNAs) have been shown to contribute to glioblastoma pathogenesis and could potentially serve as novel biomarkers. The clinical significance of HOXA Transcript Antisense RNA, Myeloid-Specific 1 (HOTAIRM1) was determined by analyzing HOTAIRM1 in multiple glioblastoma gene expression data sets for associations with prognosis, as well as, IDH mutation and MGMT promoter methylation status. Finally, the role of HOTAIRM1 in glioblastoma biology and radiotherapy resistance was characterized in vitro and in vivo. We identified HOTAIRM1 as a candidate lncRNA whose up-regulation is significantly associated with shorter survival of glioblastoma patients, independent from IDH mutation and MGMT promoter methylation. Glioblastoma cell line models uniformly showed reduced cell viability, decreased invasive growth and diminished colony formation capacity upon HOTAIRM1 down-regulation. Integrated proteogenomic analyses revealed impaired mitochondrial function and determination of reactive oxygen species (ROS) levels confirmed increased ROS levels upon HOTAIRM1 knock-down. HOTAIRM1 knock-down decreased expression of transglutaminase 2 (TGM2), a candidate protein implicated in mitochondrial function, and knock-down of TGM2 mimicked the phenotype of HOTAIRM1 down-regulation in glioblastoma cells. Moreover, HOTAIRM1 modulates radiosensitivity of glioblastoma cells both in vitro and in vivo. Our data support a role for HOTAIRM1 as a driver of biological aggressiveness, radioresistance and poor outcome in glioblastoma. Targeting HOTAIRM1 may be a promising new therapeutic approach.
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Glioblastoma/genética , Glioblastoma/radioterapia , MicroRNAs/metabolismo , Tolerância a Radiação/genética , Animais , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Células Clonais , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Humanos , Camundongos Nus , MicroRNAs/genética , Mitocôndrias/metabolismo , Invasividade Neoplásica , Fenótipo , Prognóstico , Proteína 2 Glutamina gama-Glutamiltransferase/metabolismo , Proteogenômica , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: The human leucocyte antigen (HLA) complex controls adaptive immunity by presenting defined fractions of the intracellular and extracellular protein content to immune cells. Understanding the benign HLA ligand repertoire is a prerequisite to define safe T-cell-based immunotherapies against cancer. Due to the poor availability of benign tissues, if available, normal tissue adjacent to the tumor has been used as a benign surrogate when defining tumor-associated antigens. However, this comparison has proven to be insufficient and even resulted in lethal outcomes. In order to match the tumor immunopeptidome with an equivalent counterpart, we created the HLA Ligand Atlas, the first extensive collection of paired HLA-I and HLA-II immunopeptidomes from 227 benign human tissue samples. This dataset facilitates a balanced comparison between tumor and benign tissues on HLA ligand level. METHODS: Human tissue samples were obtained from 16 subjects at autopsy, five thymus samples and two ovary samples originating from living donors. HLA ligands were isolated via immunoaffinity purification and analyzed in over 1200 liquid chromatography mass spectrometry runs. Experimentally and computationally reproducible protocols were employed for data acquisition and processing. RESULTS: The initial release covers 51 HLA-I and 86 HLA-II allotypes presenting 90,428 HLA-I- and 142,625 HLA-II ligands. The HLA allotypes are representative for the world population. We observe that immunopeptidomes differ considerably between tissues and individuals on source protein and HLA-ligand level. Moreover, we discover 1407 HLA-I ligands from non-canonical genomic regions. Such peptides were previously described in tumors, peripheral blood mononuclear cells (PBMCs), healthy lung tissues and cell lines. In a case study in glioblastoma, we show that potential on-target off-tumor adverse events in immunotherapy can be avoided by comparing tumor immunopeptidomes to the provided multi-tissue reference. CONCLUSION: Given that T-cell-based immunotherapies, such as CAR-T cells, affinity-enhanced T cell transfer, cancer vaccines and immune checkpoint inhibition, have significant side effects, the HLA Ligand Atlas is the first step toward defining tumor-associated targets with an improved safety profile. The resource provides insights into basic and applied immune-associated questions in the context of cancer immunotherapy, infection, transplantation, allergy and autoimmunity. It is publicly available and can be browsed in an easy-to-use web interface at https://hla-ligand-atlas.org .
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Antígenos de Neoplasias/imunologia , Antígenos HLA/imunologia , Imunoterapia Adotiva , Neoplasias/terapia , Peptídeos/imunologia , Proteoma , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/transplante , Idoso , Idoso de 80 Anos ou mais , Cromatografia Líquida , Bases de Dados de Proteínas , Feminino , Humanos , Lactente , Recém-Nascido , Ligantes , Masculino , Pessoa de Meia-Idade , Neoplasias/genética , Neoplasias/imunologia , Proteômica , Receptores de Antígenos Quiméricos/genética , Linfócitos T/imunologia , Espectrometria de Massas em TandemRESUMO
Glioblastoma is an invariably deadly disease. A subpopulation of glioma stem-like cells (GSCs) drives tumor progression and treatment resistance. Two recent studies demonstrated that neurons form oncogenic glutamatergic electrochemical synapses with post-synaptic GSCs. This led us to explore whether glutamate signaling through G protein-coupled metabotropic receptors would also contribute to the malignancy of glioblastoma. We found that glutamate metabotropic receptor (Grm)3 is the predominantly expressed Grm in glioblastoma. Associations of GRM3 gene expression levels with survival are confined to the proneural gene expression subtype, which is associated with enrichment of GSCs. Using multiplexed single-cell qRT-PCR, GSC marker-based cell sorting, database interrogations, and functional assays in GSCs derived from patients' tumors, we establish Grm3 as a novel marker and potential therapeutic target in GSCs. We confirm that Grm3 inhibits adenylyl cyclase and regulates extracellular signal-regulated kinase. Targeting Grm3 disrupts self-renewal and promotes differentiation of GSCs. Thus, we hypothesize that Grm3 signaling may complement oncogenic functions of glutamatergic ionotropic receptor activity in neuroglial synapses, supporting a link between neuronal activity and the GSC phenotype. The novel class of highly specific Grm3 inhibitors that we characterize herein have been clinically tested as cognitive enhancers in humans with a favorable safety profile.
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Glioblastoma is a poorly immunogenic cancer, and the successes with recent immunotherapies in extracranial malignancies have, so far, not been translated to this devastating disease. Therefore, there is an urgent need for new strategies to convert the immunologically cold glioma microenvironment into a hot one to enable effective antitumor immunity. Using the L19 antibody, which is specific to a tumor-associated epitope of extracellular fibronectin, we developed antibody-cytokine fusions-immunocytokines-with interleukin-2 (IL2), IL12, or tumor necrosis factor (TNF). We showed that L19 accumulated in the tumor microenvironment of two orthotopic immunocompetent mouse glioma models. Furthermore, intravenous administration of L19-mIL12 or L19-mTNF cured a proportion of tumor-bearing mice, whereas L19-IL2 did not. This therapeutic activity was abolished in RAG-/- mice or upon depletion of CD4 or CD8 T cells, suggesting adaptive immunity. Mechanistically, both immunocytokines promoted tumor-infiltrating lymphocytes and increased the amounts of proinflammatory cytokines within the tumor microenvironment. In addition, L19-mTNF induced tumor necrosis. Systemic administration of the fully human L19-TNF fusion protein to patients with glioblastoma (NCT03779230) was safe, decreased regional blood perfusion within the tumor, and was associated with increasing tumor necrosis and an increase in tumor-infiltrating CD4 and CD8 T cells. The extensive preclinical characterization and subsequent clinical translation provide a robust basis for future studies with immunocytokines to treat malignant brain tumors.
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Glioblastoma , Animais , Linfócitos T CD8-Positivos , Citocinas , Glioblastoma/terapia , Humanos , Imunoterapia , Interleucina-2 , Camundongos , Microambiente TumoralRESUMO
BACKGROUND: Type I interferons (IFN-α/ß) are cytokines that are typically expressed in response to double-stranded RNA associated with viral infections. Glioblastomas are the most common malignant primary brain tumors, characterized by an infiltrative growth pattern and prominent angiogenic activity, and thought to be maintained by a subpopulation of glioma-initiating (stem-like) cells (GICs). The growth of human GIC lines is highly sensitive to IFN-ß. METHODS: Repetitive pulse stimulation with IFN-ß1a (IS) was used to generate IS sublines that had acquired resistance to IFN-ß-induced suppression of sphere formation. These cell lines were characterized by analyses of type 1 IFN signaling, growth patterns, and transcriptomic profiles. RESULTS: Here we report that repetitive IFN-ß1a stimulation (IS) induces a stable phenotype (referred to as IS) at the level of maintaining sphere formation, although classical IFN signaling defined by the expression of both IFN receptors, myxovirus resistance protein A (MxA) accumulation, and STAT1 induction is unaffected. Furthermore, this stably altered IS phenotype is characterized by constitutively decreased sphere formation capacity and morphological features of senescence and autophagy. Transcriptional profiling reveals increased type I IFN signaling in these IS cells, but decreased expression of genes involved in receptor signaling and cell migration. CONCLUSIONS: Altogether, these data suggest a role for promoting IFN-ß signaling in glioblastoma and might provide clues to design future therapeutic approaches.
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Glioblastomas commonly (40%) exhibit epidermal growth factor receptor (EGFR) amplification; half of these tumors carry the EGFRvIII deletion variant characterized by an in-frame deletion of exons 2-7, resulting in constitutive EGFR activation. Although EGFR tyrosine kinase inhibitors had only modest effects in glioblastoma, novel therapeutic agents targeting amplified EGFR or EGFRvIII continue to be developed.Depatuxizumab mafodotin (ABT-414) is an EGFR-targeting antibody-drug conjugate consisting of the mAb 806 and a toxic payload, monomethyl auristatin F. Because glioma cell lines and patient-derived glioma-initiating cell models expressed too little EGFR in vitro to be ABT-414-sensitive, we generated glioma sublines overexpressing EGFR or EGFRvIII to explore determinants of ABT-414-induced cell death.Overexpression of EGFRvIII induces sensitization to ABT-414 more readily than overexpression of EGFR in vitro and in vivo Exposure to ABT-414 in vivo eliminated EGFRvIII-expressing tumor cells, and recurrent tumors were devoid of EGFRvIII expression. There is no bystander killing of cells devoid of EGFR expression. Surprisingly, either exposure to EGF or to EGFR tyrosin kinase inhibitors reduce EGFR protein levels and are thus not strategies to promote ABT-414-induced cell killing. Furthermore, glioma cells overexpressing kinase-dead EGFR or EGFRvIII retain binding of mAb 806 and sensitivity to ABT-414, allowing to dissociate EGFR phosphorylation from the emergence of the "active" EGFR conformation required for ABT-414 binding.The combination of EGFR-targeting antibody-drug conjugates with EGFR tyrosine kinase inhibitors carries a high risk of failure. Promoting EGFR expression rather than phosphorylation should result in glioblastoma cell sensitization to ABT-414.
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Anticorpos Monoclonais Humanizados/farmacologia , Glioblastoma/patologia , Imunoconjugados/farmacologia , Animais , Apoptose , Proliferação de Células , Receptores ErbB/genética , Receptores ErbB/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Camundongos , Camundongos Nus , Mutação , Fosforilação , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Novel treatments for glioblastoma, the most common malignant primary brain tumor, are urgently required. Type I interferons (IFN) are natural cytokines primarily involved in the defense against viral infections, which may also serve a role in the control of cancer, notably in the suppression of the cancer stem cell phenotype. TG02 is a novel orally available cyclin-dependent kinase 9 inhibitor which induces glioma cell apoptosis without profound caspase activation, which is currently explored in early clinical trials in newly diagnosed and recurrent glioblastoma. In the present study, human glioma-initiating cell line models were used to explore whether IFN-ß modulates the anti-glioma activity of TG02. The present study employed immunoblotting to assess protein levels, several viability assays and gene silencing strategies to assess gene function. Pre-exposure to IFN-ß sensitized human glioma models to a subsequent exposure to TG02. Combination treatment was associated with increased DEVD-amc cleaving caspase activity that was blocked by the anti-apoptotic protein, BCL2. However, BCL2 did not protect from the synergistic effects of IFN and TG02 on glioma cell growth. Furthermore, although IFN strongly induced pro-apoptotic XIAP-associated factor (XAF) expression, disrupting XAF expression did not abrogate the synergy with TG02. Consistent with that, caspase 3 gene silencing did not abrogate the effects of TG02 or IFN-ß alone or in combination. Finally, it was observed that IFN-ß may indeed modulate the effects of TG02 upstream in the signaling cascade since inhibition of RNA polymerase II phosphorylation, a direct readout of the pharmacodynamic activity of TG02, was facilitated when glioma cells were pre-exposed to IFN-ß. In summary, these data suggest that type I IFN may be combined with TG02 to limit glioblastoma growth, but that the well characterized effects of IFN and TG02 on apoptotic signaling are dispensable for synergistic tumor growth inhibition. Instead, exploring how IFN signaling primes glioma cells for TG02-mediated direct target inhibition may help to design novel and effective pharmacological approaches to glioblastoma.
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PURPOSE: Transforming growth factor (TGF)-ß is expressed at high levels by glioma cells and contributes to the malignant phenotype of glioblastoma. However, its therapeutic targeting remains challenging. Here, we examined an alternative therapeutic approach of TGFß inhibition using two novel phosphorothioate-locked nucleic acid (LNA)-modified antisense oligonucleotide gapmers, ISTH1047 and ISTH0047, which specifically target TGFß1 and TGFß2. EXPERIMENTAL DESIGN: We characterized the effects of ISTH1047 and ISTH0047 on TGFß1/2 expression, downstream signaling and growth of human LN-308, LN-229, and ZH-161 cells as well as murine SMA-560 glioma cells in vitro. Furthermore, we assessed their target inhibition and effects on survival in orthotopic xenogeneic and syngeneic rodent glioma models in vivo. RESULTS: Both antisense oligonucleotides specifically silenced their corresponding target and abrogated SMAD2 phosphorylation in several glioma cell lines. Moreover, inhibition of TGFß1 or TGFß2 expression by ISTH1047 or ISTH0047 reduced the migration and invasiveness of LN-308 and SMA-560 glioma cells. Systemic antisense oligonucleotide administration to glioma-bearing mice suppressed TGFß1 or TGFß2 mRNA expression as well as the expression of the downstream target PAI-1 in orthotopic gliomas. Glioma-bearing mice had significantly prolonged survival upon systemic treatment with ISTH1047 or ISTH0047, which was associated with a reduction of intratumoral SMAD2 phosphorylation and, in a fully immunocompetent model, with increased immune cell infiltration. CONCLUSIONS: Targeting TGFß expression with the novel LNA antisense oligonucleotides ISTH1047 or ISTH0047 results in strong antiglioma activity in vitro and in vivo, which may represent a promising approach to be examined in human patients with glioma.
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Proliferação de Células , Glioblastoma/terapia , Oligonucleotídeos Antissenso/farmacologia , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta2/antagonistas & inibidores , Animais , Apoptose , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Ligantes , Camundongos , Camundongos Nus , Invasividade Neoplásica , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta2/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
TG02 is a novel cyclin-dependent kinase (CDK) inhibitor and thought to act mainly via CDK-9 inhibition-dependent depletion of short-lived oncoproteins such as MCL-1 or c-MYC. We studied the activity of TG02 in 9 human long-term glioma cell lines (LTC) and 5 glioma-initiating cell lines (GIC) using various cell death assays in vitro and in the LN-229 LTC and ZH-161 GIC models in vivo. TG02 exhibits strong anti-tumor cell activity with EC50 concentrations in the nanomolar range. Median survival in the LN-229 and ZH-161 models was moderately prolonged by TG02. Neither constitutive CDK levels nor those of MCL-1 or c-MYC correlated with sensitivity to TG02. Cdk-9 or cdk-5 gene silencing alone did not fully reproduce the effects of TG02. C-myc gene silencing inhibited cell growth, but did not modulate TG02 activity. Electron microscopy revealed cell death to be essentially apoptotic. High concentrations of TG02 induced annexin V binding and minor caspase 3 cleavage, but the pan-caspase inhibitor, zVAD-fmk, or BCL-2 or MCL-1 gene transfer only moderately attenuated TG02-induced cell death, and caspase inhibition did not prevent loss of MCL-1 or c-MYC. TG02 activity was independent of O6 -methylguanine DNA methyltransferase expression. Repetitive exposure to TG02 did not generate an acquired TG02 resistance phenotype, but accumulation of MCL-1, loss of c-MYC, or senescence. TG02 is a highly potent apoptosis-inducing agent in glioma cells in vitro. Caspase inhibition does not rescue TG02-treated cells and repetitive exposure fails to confer acquired resistance, supporting the clinical evaluation of TG02 in glioblastoma.
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Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Feminino , Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacocinética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Tecidual , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In glioblastoma, the benefit from temozolomide chemotherapy is largely limited to a subgroup of patients (30-35%) with tumors exhibiting methylation of the promoter region of the O6-methylguanine-DNA methyltransferase (MGMT) gene. In order to allow more patients to benefit from this treatment, we explored magnetic resonance image-guided microbubble-enhanced low-intensity pulsed focused ultrasound (LIFU) to transiently open the blood-brain barrier and deliver a first-in-class liposome-loaded small molecule MGMT inactivator in mice bearing temozolomide-resistant gliomas. We demonstrate that a liposomal O6-(4-bromothenyl)guanine (O6BTG) derivative can efficiently target MGMT, thereby sensitizing murine and human glioma cells to temozolomide in vitro. Furthermore, we report that image-guided LIFU mediates the delivery of the stable liposomal MGMT inactivator in the tumor region resulting in potent MGMT depletion in vivo. Treatment with this new liposomal MGMT inactivator facilitated by LIFU-mediated blood-brain barrier opening reduced tumor growth and significantly prolonged survival of glioma-bearing mice, when combined with temozolomide chemotherapy. Exploring this novel combined approach in the clinic to treat glioblastoma patients with MGMT promoter-unmethylated tumors is warranted.
Assuntos
Antineoplásicos Alquilantes/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Dacarbazina/administração & dosagem , Glioblastoma/tratamento farmacológico , Guanina/análogos & derivados , Lipossomos/administração & dosagem , Animais , Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Dacarbazina/uso terapêutico , Sistemas de Liberação de Medicamentos/métodos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/uso terapêutico , Glioblastoma/diagnóstico por imagem , Glioblastoma/metabolismo , Guanina/administração & dosagem , Guanina/uso terapêutico , Lipossomos/uso terapêutico , Imageamento por Ressonância Magnética/métodos , Camundongos , O(6)-Metilguanina-DNA Metiltransferase/antagonistas & inibidores , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Ondas UltrassônicasRESUMO
Glioblastoma is the most frequent malignant primary brain tumor. In a hierarchical tumor model, glioblastoma stem-like cells (GSC) play a major role in tumor initiation and maintenance as well as in therapy resistance and recurrence. Thus, targeting this cellular subset may be key to effective immunotherapy. Here, we present a mass spectrometry-based analysis of HLA-presented peptidomes of GSC and glioblastoma patient specimens. Based on the analysis of patient samples (n = 9) and GSC (n = 3), we performed comparative HLA peptidome profiling against a dataset of normal human tissues. Using this immunopeptidome-centric approach we could clearly delineate a subset of naturally presented, GSC-associated HLA ligands, which might serve as highly specific targets for T cell-based immunotherapy. In total, we identified 17 antigens represented by 41 different HLA ligands showing natural and exclusive presentation both on GSC and patient samples. Importantly, in vitro immunogenicity and antigen-specific target cell killing assays suggest these peptides to be epitopes of functional CD8+ T cell responses, thus rendering them prime candidates for antigen-specific immunotherapy of glioblastoma.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Antígenos HLA/metabolismo , Células-Tronco Neoplásicas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Criança , Estudos de Coortes , Feminino , Glioblastoma/genética , Glioblastoma/patologia , Glioblastoma/terapia , Humanos , Imunoterapia/métodos , Isocitrato Desidrogenase/genética , Ligantes , Masculino , Pessoa de Meia-IdadeRESUMO
O6 -methylguanine DNA methyltransferase (MGMT) promoter methylation is a predictive biomarker for benefit from alkylating chemotherapy, specifically temozolomide (TMZ), in glioblastoma, the most common malignant intrinsic brain tumor. Glioma-initiating cells (GIC) with stem-like properties have been associated with resistance to therapy and progression. We assessed the levels of MGMT mRNA and MGMT protein by real-time PCR and immunoblot and evaluated the impact of MGMT on TMZ sensitivity in clonogenicity assays in GIC sphere cultures (S) or differentiated adherent monolayer cultures (M). Nuclear factor kappa B (NF-κB) signaling was assessed by reporter assay and immunoblot. Compared to M cells, S cells expressed higher levels of MGMT. Differentiation of GIC induced by S-to-M transition resulted in a gradual loss of MGMT expression and increased TMZ sensitivity. This transcriptional regulation of MGMT was restricted to cell lines without MGMT promoter methylation and was not coupled to any specific neurobasal (NB) stem cell medium supplement or loss of cell adhesion. Expression levels of p50/p65 subunits of NF-κB, a transcriptional regulator of MGMT, were increased in S cells. Inhibition of NF-κB by the small molecule inhibitor, BAY 11-7082, or siRNA-mediated gene silencing, reduced MGMT levels. In summary, alkylator resistance of S cells is mainly promoted by over-expression of MGMT which results from increased activity of the NF-κB pathway in this cell culture model of glioma stem-like cells. Read the Editorial Highlight for this article on page 688.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Temozolomida/farmacologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , NF-kappa B/metabolismo , RNA Mensageiro/metabolismoRESUMO
Purpose: NKG2D is a potent activating immune cell receptor, and glioma cells express the cognate ligands (NKG2DL). These ligands are inducible by cellular stress and temozolomide (TMZ) or irradiation (IR), the standard treatment of glioblastoma, could affect their expression. However, a role of NKG2DL for the efficacy of TMZ and IR has never been addressed.Experimental Design: We assessed the effect of TMZ and IR on NKG2DL in vitro and in vivo in a variety of murine and human glioblastoma models, including glioma-initiating cells, and a cohort of paired glioblastoma samples from patients before and after therapy. Functional effects were studied with immune cell assays. The relevance of the NKG2D system for the efficacy of TMZ and IR was assessed in vivo in syngeneic orthotopic glioblastoma models with blocking antibodies and NKG2D knockout mice.Results: TMZ or IR induced NKG2DL in vitro and in vivo in all glioblastoma models, and glioblastoma patient samples had increased levels of NKG2DL after therapy with TMZ and IR. This enhanced the immunogenicity of glioma cells in a NGK2D-dependent manner, was independent from cytotoxic or growth inhibitory effects, attenuated by O6-methylguanine-DNA-methyltransferase (MGMT), and required the DNA damage response. The survival benefit afforded by TMZ or IR relied on an intact NKG2D system and was decreased upon inhibition of the NKG2D pathway.Conclusions: The immune system may influence the activity of convential cancer treatments with particular importance of the NKG2D pathway in glioblastoma. Our data provide a rationale to combine NKG2D-based immunotherapies with TMZ and IR. Clin Cancer Res; 24(4); 882-95. ©2017 AACR.