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Supplementary Figure 1: Detection of asunercept/ APG101 in brains of treated mice. Supplementary Figure 2: qPCR for MMP2 (A) or MMP9 (B) and representative images of human microglia after 12 h treatment with vehicle (I), 20 µg/ml APG101 (II), 50 U/ml CD95L (III) and both 20 µg/ml APG101 and 50 U/ml CD95L (IV). Supplementary Figure 3: APG101 is effective in murine cells. Supplementary Figure 4: Radiation effects on CD95L expression.

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Purpose: Recent work points out a role of B7H3, a member of the B7-family of costimulatory proteins, in conveying immunosuppression and enforced invasiveness in a variety of tumor entities. Glioblastoma is armed with effective immunosuppressive properties resulting in an impaired recognition and ineffective attack of tumor cells by the immune system. In addition, extensive and diffuse invasion of tumor cells into the surrounding brain tissue limits the efficacy of local therapies. Here, 4IgB7H3 is assessed as diagnostic and therapeutic target for glioblastoma.Experimental Design: To characterize B7H3 in glioblastoma, we conduct analyses not only in glioma cell lines and glioma-initiating cells but also in human glioma tissue specimens.Results: B7H3 expression by tumor and endothelial cells correlates with the grade of malignancy in gliomas and with poor survival. Both soluble 4IgB7H3 in the supernatant of glioma cells and cell-bound 4IgB7H3 are functional and suppress natural killer cell–mediated tumor cell lysis. Gene silencing showed that membrane and soluble 4IgB7H3 convey a proinvasive phenotype in glioma cells and glioma-initiating cells in vitro. These proinvasive and immunosuppressive properties were confirmed in vivo by xenografted 4IgB7H3 gene silenced glioma-initiating cells, which invaded significantly less into the surrounding brain tissue in an orthotopic model and by subcutaneously injected LN-229 cells, which were more susceptible to natural killer cell–mediated cytotoxicity than unsilenced control cells.Conclusions: Because of its immunosuppressive and proinvasive function, 4IgB7H3 may serve as a therapeutic target in the treatment of glioblastoma. Clin Cancer Res; 18(1); 105–17. ©2011 AACR.

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CD95 (Fas/APO-1), a death receptor family member, activity has been linked to tumorigenicity in multiple cancers, including glioblastoma multiforme (GBM). A phase II clinical trial on relapsed glioblastoma patients demonstrated that targeted inhibition of CD95 signaling via the CD95 ligand (CD95L) binding and neutralizing Fc-fusion protein APG101 (asunercept) prolonged patient survival. Although CD95 signaling may be relevant for multiple aspects of tumor growth, the mechanism of action of APG101 in glioblastoma is not clear. APG101 action was examined by in vitro proliferation, apoptosis, and invasion assays with human and murine glioma and human microglial cells, as well as in vivo therapy studies with orthotopic gliomas and clinical data. APG101 inhibits CD95L-mediated invasion of glioma cells. APG101 treatment was effective in glioma-bearing mice, independently of the presence or absence of CD4 and CD8 T lymphocytes, which should be sensitive to CD95L. Combined with radiotherapy, APG101 demonstrated a reduction of tumor growth, fewer tumor satellites, reduced activity of matrix metalloproteinases (MMP) as well as prolonged survival of tumor-bearing mice compared with radiotherapy alone. Inhibiting rather than inducing CD95 activity is a break-of-paradigm therapeutic approach for malignant gliomas. Evidence, both in vitro and in vivo, is provided that CD95L-binding fusion protein treatment enhanced the efficacy of radiotherapy and reduced unwanted proinfiltrative effects by reducing metalloproteinase activity by directly affecting the tumor cells. Implications: APG101 (asunercept) successfully used in a controlled phase II glioblastoma trial (NCT01071837) acts anti-invasively by inhibiting matrix metalloproteinase signaling, resulting in additive effects together with radiotherapy and helping to further develop a treatment for this devastating disease. Mol Cancer Res; 16(5); 767–76. ©2018 AACR.

// Tobias Kessler 1, * , Felix Sahm 3, 6, * , Jonas Blaes 1 , Matthias Osswald 1, 4 , Petra Rubmann 1 , David Milford 7 , Severino Urban 8 , Leonie Jestaedt 7 , Sabine Heiland 7 , Martin Bendszus 7 , Anne Hertenstein 2, 4 , Philipp-Niclas Pfenning 1 , Carmen Ruiz de Almodovar 8 , Antje Wick 4 , Frank Winkler 1, 4 , Andreas von Deimling 3, 6 , Michael Platten 2, 4 , Wolfgang Wick 1, 4 , Markus Weiler 1, 4, 5 1 Clinical Cooperation Unit Neurooncology, German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany 2 Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology and German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany 3 Clinical Cooperation Unit Neuropathology, German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany 4 Department of Neurooncology at the National Center for Tumor Diseases, Heidelberg University Hospital, Heidelberg, Germany 5 Department of General Neurology, Heidelberg University Hospital, Heidelberg, Germany 6 Department of Neuropathology, Heidelberg University Hospital, Heidelberg, Germany 7 Department of Neuroradiology, Heidelberg University Hospital, Heidelberg, Germany 8 Biochemistry Center Heidelberg University, Heidelberg, Germany * These authors have contributed equally to this work Correspondence to: Markus Weiler, e-mail: m.weiler@dkfz.de Wolfgang Wick, e-mail: wolfgang.wick@med.uni-heidelberg.de Keywords: angiogenesis, glioblastoma, invasion, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), vascular endothelial growth factor receptor (VEGFR)-2 Received: October 26, 2014 Accepted: December 14, 2014 Published: February 19, 2015 ABSTRACT Loss of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a prerequisite for tumor cell-specific expression of vascular endothelial growth factor receptor (VEGFR)-2 in glioblastoma defining a subgroup prone to develop evasive resistance towards antiangiogenic treatments. Immunohistochemical analysis of human tumor tissues showed VEGFR-2 expression in glioma cells in 19% of specimens examined, mainly in the infiltration zone. Glioma cell VEGFR-2 positivity was restricted to PTEN-deficient tumor specimens. PTEN overexpression reduced VEGFR-2 expression in vitro , as well as knock-down of raptor or rictor . Genetic interference with VEGFR-2 revealed proproliferative, antiinvasive and chemoprotective functions for VEGFR-2 in glioma cells. VEGFR-2-dependent cellular effects were concomitant with activation of ’kappa-light-chain-enhancer’ of activated B-cells, protein kinase B, and N-myc downstream regulated gene 1. Two-photon in vivo microscopy revealed that expression of VEGFR-2 in glioma cells hampers antiangiogenesis. Bevacizumab induces a proinvasive response in VEGFR-2-positive glioma cells. Patients with PTEN-negative glioblastomas had a shorter survival after initiation of bevacizumab therapy compared with PTEN-positive glioblastomas. Conclusively, expression of VEGFR-2 in glioma cells indicates an aggressive glioblastoma subgroup developing early resistance to temozolomide or bevacizumab. Loss of PTEN may serve as a biomarker identifying those tumors upfront by routine neuropathological methods.

A hypoxic microenvironment induces resistance to alkylating agents by activating targets in the mammalian target of rapamycin (mTOR) pathway. The molecular mechanisms involved in this mTOR-mediated hypoxia-induced chemoresistance, however, are unclear. Here we identify the mTOR target N-myc downstream regulated gene 1 (NDRG1) as a key determinant of resistance toward alkylating chemotherapy, driven by hypoxia but also by therapeutic measures such as irradiation, corticosteroids, and chronic exposure to alkylating agents via distinct molecular routes involving hypoxia-inducible factor (HIF)-1alpha, p53, and the mTOR complex 2 (mTORC2)/serum glucocorticoid-induced protein kinase 1 (SGK1) pathway. Resistance toward alkylating chemotherapy but not radiotherapy was dependent on NDRG1 expression and activity. In posttreatment tumor tissue of patients with malignant gliomas, NDRG1 was induced and predictive of poor response to alkylating chemotherapy. On a molecular level, NDRG1 bound and stabilized methyltransferases, chiefly O6-methylguanine-DNA methyltransferase (MGMT), a key enzyme for resistance to alkylating agents in glioblastoma patients. In patients with glioblastoma, MGMT promoter methylation in tumor tissue was not more predictive for response to alkylating chemotherapy in patients who received concomitant corticosteroids.

Purpose: To compare conventional magnetic resonance imaging (MRI) techniques (T2-w and Gadolinium-DTPA-enhanced T1-w images) and Gadofluorine-M (GfM), a novel contrast agent in MRI, in murine gliomas. Materials and Methods: Growth monitoring of murine gliomas (induced in mice) was performed on a 2.3 Tesla Bruker Biospec MRI unit. First all animals were investigated with conventional MRI techniques. In group I GfM was applied at an early stage of disease, in group II at a later stage. After injection of GfM follow-up MRI was performed without further injection of contrast agent. On MR images tumor size and signal intensities were assessed. Animals were killed for histological evaluation. Results: In both groups GfM delineated tumor extents larger and more precisely than conventional MRI techniques. The difference between GfM and conventional MRI techniques reached level of significance at both tumor stages. Follow-up MRI after singular injection of GfM showed persistence of GfM in tumor tissue. On tissue sections GfM-enhancing areas corresponded closely to vital tumor tissue. GfM showed a mainly intracellular accumulation. Conclusion: Application of GfM resulted in superior delineation of experimental glioma compared with conventional MRI techniques. Thus, GfM bears a high potential in clinical application. J. Magn. Reson. Imaging 2012;35:551-560. © 2011 Wiley Periodicals, Inc.

An essential mode of acquired resistance to radiotherapy (RT) appears to be promotion of tumor cell motility and invasiveness in various cancer types, including glioblastoma, a process resembling 'evasive resistance'. Hence, a logical advancement of RT would be to identify suitable complementary treatment strategies, ideally targeting cell motility. Here we report that the combination of focal RT and mammalian target of rapamycin (mTOR) inhibition using clinically relevant concentrations of temsirolimus (CCI-779) prolongs survival in a syngeneic mouse glioma model through additive cytostatic effects. In vitro, the mTOR inhibitor CCI-779 exerted marked anti-invasive effects, irrespective of the phosphatase and tensin homolog deleted on chromosome 10 status and counteracted the proinvasive effect of sublethal irradiation. Mechanistically, we identified regulator of G-protein signaling 4 (RGS4) as a novel target of mTOR inhibition and a key driver of glioblastoma invasiveness, sensitive to the anti-invasive properties of CCI-779. Notably, suppression of RGS4-dependent glioma cell invasion was signaled through both mTOR complexes, mTORC1 and mTORC2, in a concentration-dependent manner, indicating that high doses of CCI-779 may overcome tumor-cell resistance associated with the sole inhibition of mTORC1. We conclude that combined RT and mTOR inhibition is a promising therapeutic option that warrants further clinical investigation in upfront glioblastoma therapy.

In a strategy to identify novel genes involved in glioma pathogenesis by molecular characterization of chromosomal translocation breakpoints, we identified the KIAA1797 gene, encoding a protein with an as yet undefined function, to be disrupted by a 7;9 translocation in a primary glioblastoma culture. Array-based comparative genomic hybridization detected deletions involving KIAA1797 in around half of glioblastoma cell lines and glioblastomas investigated. Quantification of messenger RNA levels in human tissues demonstrated highest KIAA1797 expression in brain, reduced levels in all glioblastoma cell lines and most glioblastomas and similar levels in glial and neuronal cells by analysis of different hippocampal regions from murine brain. Antibodies against KIAA1797 were generated and showed similar protein levels in cortex and subcortical white matter of human brain, while levels were significantly reduced in glioblastomas with KIAA1797 deletion. By immunofluorescence of astrocytoma cells, KIAA1797 co-localized with vinculin in focal adhesions. Physical interaction between KIAA1797 and vinculin was demonstrated via co-immunoprecipitation. Functional in vitro assays demonstrated a significant decrease in colony formation, migration and invasion capacity of LN18 and U87MG glioma cells carrying a homozygous KIAA1797 deletion ectopically expressing KIAA1797 compared with mock-transduced cells. In an in vivo orthotopic xenograft mouse model, U87MG tumour lesions expressing KIAA1797 had a significantly reduced volume compared to tumours not expressing KIAA1797. In summary, the frequently deleted KIAA1797 gene encodes a novel focal adhesion complex protein with tumour suppressor function in gliomas, which we name 'focadhesin'. Since KIAA1797 genetic variation has been implicated in Alzheimer's disease, our data are also relevant for neurodegeneration.

Previous findings suggest an angiogenesis-regulating function of the p53 tumor suppressor protein in various malignancies. With several antiangiogenic agents entering the clinic, we assessed the value of the TP53 status in predicting angiogenesis in glioblastoma in vivo and examined underlying angiogenic-signaling pathways in vitro. We identified 26 TP53 wild-type and 9 TP53 mutated treatment-naive, primary, isocitrate dehydrogenase 1 (IDH1) wild-type glioblastoma specimens by sequence analysis and quantified vascularization. P53 responsiveness of the angiogenesis-related target genes, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), thrombospondin 1 (TSP-1), brain-specific angiogenesis inhibitor 1 (BAI1), and collagen prolyl-4-hydroxylase alpha 2 (P4HA2), was evaluated by (i) overexpression of wild-type p53 in homozygously TP53-deleted LN-308 cells; (ii) shRNA-mediated p53 knockdown in the TP53 wild-type LNT-229 cells; and (iii) chemical induction of wild-type p53 expression in LNT-229 cells by camptothecin. Irrespective of the TP53 status, vascularization did not differ significantly between the two groups of glioblastoma specimens. Of all target genes, only P4HA2 mRNA was upregulated through wild-type p53. As opposed to several nonglial tumors, in glioblastoma cells, p53-mediated transcriptional induction of P4HA2 mRNA neither resulted in increased levels of P4HA2 protein or antiangiogenic endostatin nor did it influence endothelial cell sprouting, viability, or transmigration in vitro. Moreover, p53-uncoupled stable overexpression of P4HA2 in LN-308 cells did not affect endothelial cell viability. These data challenge the view of p53 as an angiogenesis-regulator in glioblastoma in that relevant signaling pathways are silenced, potentially contributing to the angiogenic switch during malignant progression.

Podoplanin (PDPN) is a mucin like type-1 transmembrane protein consisting of 162 amino acids, which comprises a highly glycosylated extracellular domain and a nine amino acid intracellular domain. The specific expression of PDPN on lymphatic but not on blood vascular endothelial cells has established application as a histological marker for lymphangiogenesis and lymphatic vessels in different species. PDPN is highly expressed in tumors of different origin, including squamous cell cancers, germinal neoplasia and tumors of the central nervous system. Recently, we found strong overexpression of PDPN in human astrocytic brain tumors, specifically in primary glioblastoma multiforme (GB). Regulation of PDPN expression involves increased PI3 kinase activation, subsequent activation of protein kinaseB/Akt and the downstream transcription factor AP-1. Activation of this pathway is mediated by loss of PTEN function, consistently, mice lacking Pten exhibit elevated Pdpn protein levels in the subventricular zone of the brain tissue. Additionally, PDPN expression is subject to negative transcriptional regulation by promoter methylation in human GB and in glioma cell lines. In patients high expression of PDPN is associated with poor prognosis. Silencing of PDPN results in a decrease in proliferation, migration and invasion of glioma cell lines in vitro and slows down the migration in orthotopic brain slices. Consistently, tumor formation after injection of PDPN knock-down cells into SCID mice is significantly reduced compared to tumors after injection of parental cells. These data highlight a functional role of PDPN in glioma progression and suggest targeting of PDPN expression as a promising strategy for the treatment of patients with primary GB. Citation Format: Heike Peterziel, Julia Müller, Danner Andreas, Sebastian Barbus, Haikun Liu, Philipp Pfenning, Patrick N. Harter, Michel Mittelbronn, Wolfgang Wick, Günther Schuetz, Bernhard Radlwimmer, Peter Lichter, Jochen Hess, Peter Angel. Regulation and function of the mucin-like glycoprotein podoplanin in glioma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3797. doi:10.1158/1538-7445.AM2013-3797

Like other solid tumors, glioblastomas (GB) feature vast hypoxic areas that are thought to promote tumor growth, angiogenesis and even drug resistance and hence facilitate an aggressive growth and recurrence after treatment. Elucidation of hypoxia-signalling and identification of the major regulators of hypoxia-dependent chemoprotection seems logical for the optimization of current treatment regimens. Using comparative 2D gel electrophoresis and mass spectrometry analysis, we here identify N-myc downstream regulated gene 1 (NDRG1) as upregulated by chronic sublethal hypoxia in GB cell lines. Immunohistochemical analysis of human astrocytoma specimens shows a marked increase in expression from WHO °II to °IV and a preferential perinecrotic localization of NDRG1 in GB. In vitro, besides the hypoxia-driven induction of NDRG1, which is mediated by Hif1α and Hif2α, NDRG1 is also induced by irradiation. NDRG1 limits glioma cell invasiveness in vitro and reduces proliferation in vitro and in vivo. Furthermore NDRG1 acts antiangiogenic with reduced endothelial cell migration and sprouting, and interferes with the expression of multiple angiogenesis-related signalling molecules. Most interestingly NDRG1 protects from temozolomide (TMZ)-induced G2/M-arrest and subsequent reduction of tumor cell proliferation. Analyzing pairs of GB tissue samples of relapsed glioblastoma patients reveals a dramatic increase of NDRG1-positive cells suggesting a treatment-related selection for NDRG1 expressing populations and a potential role for NDRG1 in the recurrence of GB. In line with these findings, quantifying the NDRG1 status in tissue specimens of the UKT05 GB trial, which looked at a dose-intensified TMZ therapy plus standard radiotherapy, reveals a negative correlation between NDRG1 status and overall survival. In the present work we identify hypoxia-driven NDRG1 as a modulator of tumor growth, invasion, angiogenesis and as a novel mediator of chemoresistance towards TMZ in GB. Our findings shed light on the role of hypoxia in adaption to chemotherapeutic treatment and introduce NDRG1 as a promising candidate for targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4736. doi:1538-7445.AM2012-4736

CD95 is a prototype death receptor that regulates the induction of apoptosis, upon binding of its ligand CD95L. Therefore, activation of the CD95L/CD95 system has been regarded an excellent target for treatment of various malignancies. In contrast to the aim of promoting CD95 activation, there is compelling evidence that CD95 is able to promote tumor growth through intracellular non-apoptotic signalling mechanisms. This mechanism has recently been described for glioblastoma, in which activation of CD95 by CD95L leads to invasive growth and glioma cell migration. This is signalled through increased activity of pivotal glioblastoma invasion-related proteases, that is matrix metalloproteinases (MMP). With this shift of paradigms, blocking of the CD95L mediated invasion of tumor cells and subsequent tumor progression may therefore be a promising approach in anti-glioma therapy. APG101 is a fusion protein, consisting of the extracellular domain of human CD95 and the Fc-region of human immunoglobulin G. Hence, it acts as a soluble CD95 receptor trapping the CD95 ligand. The presented project aims at analyzing the clinical relevance of blocking CD95 signalling, given the fact that glioma patients with upregulated CD95 expression have worse survival rates than those with intermediate expression levels. In proof-of principle experiments APG101 inhibits CD95L-mediated invasion of glioma cells. More importantly, APG101-treatment (100 mg/kg body weight) resulted in significantly prolonged survival of SMA560-tumor bearing Vm/Dk mice, less glioma cell satellites in the surrounding tissue and reduced activity of MMP. APG101 in combination with focal irradiation at 6 Gy demonstrated a remarkable reduction of tumor growth with a significantly prolonged survival compared with irradiation treatment alone and inhibition of the proinvasive properties of radiotherapy as demonstrated by magnetic resonance imaging and histology. Surprisingly, APG-101 added to the vascular endothelial growth factor receptor (VEGFR) inhibitor cediranib (AZD2171) did not increase the survival of SMA-560-tumor bearing mice as compared to cediranib alone nor did it impair the proinvasive consequences of cediranib. Our data strongly support the potential use and clinical evaluation of APG101 in combination with radiotherapy in the treatment of malignant glioma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-382. doi:10.1158/1538-7445.AM2011-LB-382

Two key properties of glioblastomas are vascular proliferation and invasion of tumor cells into the surrounding tissues, both limiting the efficacy of surgery and radiochemotherapy treatment regimens. Inhibition of mammalian target of rapamycin (mTOR) by Temsirolimus (CCI-779) only has modest single compound activity in recurrent glioma. Given the fact that postoperative radiochemotherapy is the standard of care in the first-line treatment of newly diagnosed glioblastoma, the first part of the presented project aims at analyzing combined mTOR/radiotherapy in the syngeneic, orthotopic VM/Dk/SMA-560 mouse glioma model. The combined treatment of CCI-779, a small-molecule inhibitor of the mTOR kinase complex 1 (mTORC1) approved for advanced renal cell carcinoma and mantle cell lymphoma, at 20 mg/KG from day 3 until day 17 and focal irradiation at 6 Gy on day 5 after tumor inocculation demonstrated remarkable antiangiogenic and antitumoral activity as well as prolonged survival of tumor bearing animals of 9 days, significant compared with irradiation- or CCI-779-treatment alone. Loss of phosphatase and tensin homologue on chromosome ten (PTEN), which is a common event in glioblastoma, results in activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mTOR signaling pathway, leading to neovascularisation, cell cycle progression and escape from apoptosis. So far, conflicting data on the sensitivity of PTEN wild-type (wt) versus mutant cells exist. Similarly, the relevance of the feedback activation of Akt by mTOR inhibition is debated. Here, analysis of PTEN on mRNA, promoter methylation as well as protein levels clearly demonstrates for cell lines as well as primary glioma cells that proliferation of PTEN wt cells is also sensitive to mTOR inhibition albeight at higher concentrations. We further demonstrate by differential effects on the mTOR complexes (mTORC) 1 and 2 by shRNA as compared to sole mTORC 1 inhibition by CCI-779 that feedback activation of Akt, which is more prominent in PTEN mutant than in wild-type cells, may in fact have additional therapeutic antiinvasive and angiogenic effects via inhibition of a G-protein-interacting protein and vascular endothelial growth factor receptor (VEGFR)-2, respectively. CCI-779 exerted marked anti-angiogenic effects both by reducing levels of VEGFR and by inhibiting radiation-enhanced proliferation of brain endothelial cells. Moreover, CCI-779 applied after radiosensibilization inhibited glioma invasiveness in a supra-additive way and reverted the proinvasive effect of sublethal irradiation alone. The results support the clinical evaluation of combined targeted mTOR inhibition with CCI-779 and radiotherapy in patients with newly diagnosed glioblastomas that is going to be conducted in the European Organization for Research and Treatment of Cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1308A.