CD95 gene deletion may reduce clonogenic growth and invasiveness of human glioblastoma cells in a CD95 ligand-independent manner

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

Programmed death ligand 1 gene silencing in murine glioma models reveals cell line-specific modulation of tumor growth in vivo

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 β 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

The long non-coding RNA HOTAIRM1 promotes tumor aggressiveness and radiotherapy resistance in glioblastoma

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

Therapeutic Targeting of TGFβ Ligands in Glioblastoma Using Novel Antisense Oligonucleotides Reduces the Growth of Experimental Gliomas

PURPOSE Transforming growth factor (TGF)-β 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 TGFβ inhibition using two novel phosphorothioate-locked nucleic acid (LNA)-modified antisense oligonucleotide gapmers, ISTH1047 and ISTH0047, which specifically target TGFβ and TGFβ. EXPERIMENTAL DESIGN We characterized the effects of ISTH1047 and ISTH0047 on TGFβ expression, downstream signaling and growth of human LN-308, LN-229, and ZH-161 cells as well as murine SMA-560 glioma cells . Furthermore, we assessed their target inhibition and effects on survival in orthotopic xenogeneic and syngeneic rodent glioma models . RESULTS Both antisense oligonucleotides specifically silenced their corresponding target and abrogated SMAD2 phosphorylation in several glioma cell lines. Moreover, inhibition of TGFβ or TGFβ 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 or mRNA expression as well as the expression of the downstream target 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 TGFβ expression with the novel LNA antisense oligonucleotides ISTH1047 or ISTH0047 results in strong antiglioma activity and , which may represent a promising approach to be examined in human patients with glioma

Immunological and tumor-intrinsic mechanisms mediate the synergistic growth suppression of experimental glioblastoma by radiotherapy and MET inhibition

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)-β, and pSMAD2 levels as a surrogate marker of TGF-β 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

Integrin control of the transforming growth factor-β pathway in glioblastoma

Transforming growth factor-β is a central mediator of the malignant phenotype of glioblastoma, the most common and malignant form of intrinsic brain tumours. Transforming growth factor-β promotes invasiveness and angiogenesis, maintains cancer cell stemness and induces profound immunosuppression in the host. Integrins regulate cellular adhesion and transmit signals important for cell survival, proliferation, differentiation and motility, and may be involved in the activation of transforming growth factor-β. We report that αvβ3, αvβ5 and αvβ8 integrins are broadly expressed not only in glioblastoma blood vessels but also in tumour cells. Exposure to αv, β3 or β5 neutralizing antibodies, RNA interference-mediated integrin gene silencing or pharmacological integrin inhibition using the cyclic RGD peptide EMD 121974 (cilengitide) results in reduced phosphorylation of Smad2 in most glioma cell lines, including glioma-initiating cell lines and reduced transforming growth factor-β-mediated reporter gene activity, coinciding with reduced transforming growth factor-β protein levels in the supernatant. Time course experiments indicated that the loss of transforming growth factor-β bioactivity due to integrin inhibition likely results from two distinct mechanisms: an early effect on activation of preformed inactive protein, and second, major effect on transforming growth factor-β gene transcription as confirmed by decreased activity of the transforming growth factor-β gene promoter and decreased transforming growth factor-β1 and transforming growth factor-β2 messenger RNA expression levels. In vivo, EMD 121974 (cilengitide), which is currently in late clinical development as an antiangiogenic agent in newly diagnosed glioblastoma, was a weak antagonist of pSmad2 phosphorylation. These results validate integrin inhibition as a promising strategy not only to inhibit angiogenesis, but also to block transforming growth factor-β-controlled features of malignancy including invasiveness, stemness and immunosuppression in human glioblastom

NKG2D-dependent anti-tumor effects of chemotherapy and radiotherapy against glioblastoma

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

A T-cell antigen atlas for meningioma: novel options for immunotherapy

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

HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy

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