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We disclose novel amphiphilic ruthenium and osmium complexes that auto-assemble into nanomedicines with potent antiproliferative activity by inhibition of mitochondrial respiration. The self-assembling units were rationally designed from the [M(p-cymene)(1,10-phenanthroline)Cl]PF(6) motif (where M is either Ru(II) or Os(II)) with an appended C(16) fatty chain to achieve high cellular activity, nano-assembling and mitochondrial targeting. These amphiphilic complexes block cell proliferation at the sub-micromolar range and are particularly potent towards glioblastoma neurospheres made from patient-derived cancer stem cells. A subcutaneous mouse model using these glioblastoma stem cells highlights one of our C(16) Os(II) nanomedicines as highly successful in vivo. Mechanistically, we show that they act as metabolic poisons, strongly impairing mitochondrial respiration, corroborated by morphological changes and damage to the mitochondria. A genetic strategy based on RNAi gave further insight on the potential involvement of microtubules as part of the induced cell death. In parallel, we examined the structural properties of these new amphiphilic metal-based constructs, their reactivity and mechanism.

We disclose novel amphiphilic ruthenium and osmium complexes that auto-assemble into nanomedicines with potent antiproliferative activity by inhibition of mitochondrial respiration. The self-assembling units were rationally designed from the [M(p-cymene)(1,10-phenanthroline)Cl]PF6 motif (where M is either RuII or OsII) with an appended C16 fatty chain to achieve high cellular activity, nano-assembling and mitochondrial targeting. These amphiphilic complexes block cell proliferation at the sub-micromolar range and are particularly potent towards glioblastoma neurospheres made from patient-derived cancer stem cells. A subcutaneous mouse model using these glioblastoma stem cells highlights one of our C16 OsII nanomedicines as highly successful in vivo. Mechanistically, we show that they act as metabolic poisons, strongly impairing mitochondrial respiration, corroborated by morphological changes and damage to the mitochondria. A genetic strategy based on RNAi gave further insight on the potential involvement of microtubules as part of the induced cell death. In parallel, we present a careful examination of the structural properties of these new amphiphilic metal-based constructs, their reactivity and mechanism.

The treatment of glioblastoma remains a challenge. Novel therapeutic strategies are urgently needed. Targeting the immunosuppressive glioblastoma-associated microenvironment is an interesting approach in this regard. Tumor-associated macrophages represent a distinct population of tumor-infiltrating immune cells with tumor-promoting features. The colony stimulating factor-1/ colony stimulating factor-1 receptor (CSF-1/CSF1R) axis plays an important role for macrophage differentiation and survival inside the tumor microenvironment. We thus aimed at investigating the antiglioma activity of CSF1R inhibition alone or in combination with blockade of programmed death (PD) 1. We detected CSF1R expression in paired tissue samples of primary and corresponding progressive glioblastoma. We investigated anti-CSF1R treatment alone or in combination with anti-PD1 antibodies in the orthotopic syngeneic VM/Dk SMA560 glioma mouse model, evaluated post-treatment effects and assessed treatment-induced cytotoxicity in a coculture model of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) ex vivo. Anti-CSF1R monotherapy increased the latency until the onset of neurological symptoms. Combinations of anti-CSF1R and anti-PD1 antibodies prolonged the latency until the onset of neurological symptoms and led to long-term survivors in vivo. Immunohistochemical analysis of post-treatment SMA-560 glioma revealed a modulation of the microenvironment, including increased T cell infiltration and reduced numbers of tumor-associated macrophages. Furthermore, we observed treatment-induced cytotoxicity of combined anti-CSF1R and anti-PD1 treatment in the PDM/TILs cocultures ex vivo. Taken together, our data indicates that CSF1R is a promising therapeutic target for glioblastoma, potentially in combination with PD1 inhibition.

Glioblastoma is an aggressive primary brain tumor with bad prognosis. On the other hand, oncolytic measles virus (MeV) therapy is an experimental glioma treatment strategy with clinical safety and first evidence of anti-tumoral efficacy. Therefore, we investigated the combination of MeV with conventional therapies by cytotoxic survival assays in long-term glioma cell lines LN229, LNZ308, and glioma stem-like GS8 cells, as well as the basal viral infectivity in primary glioblastoma cultures T81/16, T1094/17, and T708/16. We employed Chou-Talalay analysis to identify the synergistic treatment sequence chemotherapy, virotherapy, and finally radiotherapy (CT-VT-RT). RNA sequencing and immunopeptidome analyses were used to delineate treatment-induced molecular and immunological profiles. CT-VT-RT displayed synergistic anti-glioma activity and initiated a type 1 interferon response, along with canonical Janus kinase-signal transducers and activators of transcription (JAK-STAT) signaling, and downstream interferon-stimulated genes were induced, resulting in apoptotic cascades. Furthermore, antigen presentation along with immunostimulatory chemokines was increased in CT-VT-RT-treated glioma cells, indicating a treatment-induced pro-inflammatory phenotype. We identified novel treatment-induced viral and tumor-associated peptides through HLA ligandome analysis. Our data delineate an actionable treatment-induced molecular and immunological signature of CT-VT-RT, and they could be exploited for the design of novel tailored treatment strategies involving virotherapy and immunotherapy. Keywords: glioblastoma, immunopeptidome, oncolytic measles virus, personalized medicine, BATF2, SAMD9

Simple Summary Glioblastomas are incurable tumors of the central nervous system. Currently, treatment strategies combine neurosurgical intervention, radiation therapy, and chemotherapy. Yet, clinical experience shows that tumors acquire escape mechanisms. Furthermore, the tumor-associated microenvironment, including macrophages expressing the receptor CSF1R, promote and nourish tumor cells. The so-called PD1/PDL1 axis is a major reason why tumors can grow with a “magic hat”; i.e., unrecognized from the immune system. The aim of our study was to assess treatment strategies that target macrophages in the microenvironment by blocking CSF1R alone or in combination with PD1 blockade. Using an immune competent mouse model and an ex vivo microtumor model using freshly resected glioblastoma material, we observed prolonged survival of treated mice and an improved “attack” of the immune system. We conclude that targeting CSF1R is a promising strategy that should be explored in clinical trials, potentially in combination with PD1 blockade. Abstract Glioblastoma is an aggressive primary tumor of the central nervous system. Targeting the immunosuppressive glioblastoma-associated microenvironment is an interesting therapeutic approach. Tumor-associated macrophages represent an abundant population of tumor-infiltrating host cells with tumor-promoting features. The colony stimulating factor-1/ colony stimulating factor-1 receptor (CSF-1/CSF1R) axis plays an important role for macrophage differentiation and survival. We thus aimed at investigating the antiglioma activity of CSF1R inhibition alone or in combination with blockade of programmed death (PD) 1. We investigated combination treatments of anti-CSF1R alone or in combination with anti-PD1 antibodies in an orthotopic syngeneic glioma mouse model, evaluated post-treatment effects and assessed treatment-induced cytotoxicity in a coculture model of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) ex vivo. Anti-CSF1R monotherapy increased the latency until the onset of neurological symptoms. Combinations of anti-CSF1R and anti-PD1 antibodies led to longterm survivors in vivo. Furthermore, we observed treatment-induced cytotoxicity of combined anti-CSF1R and anti-PD1 treatment in the PDM/TILs cocultures ex vivo. Our results identify CSF1R as a promising therapeutic target for glioblastoma, potentially in combination with PD1 inhibition.

Background Gene-mediated cytotoxic immunotherapy (GMCI) is a local tumor immunotherapy that uses aglatimagene besadenovec (a non-replicating serotype 5 adenovirus, expressing HSV1 thymidine kinase) with the prodrug ganciclovir to induce DNA double strand breaks (DSB), leading to immunogenic tumor cell death and intratumoral immune cell invasion. Here we investigate potential repressors and enhancers of GMCI’s effectiveness. GMCI is currently in clinical trials in combination with immune checkpoint blockade in glioblastoma. Thus we set out to identify potential areas to improve this approach for future application. Dexamethasone is used in symptomatic treatment of glioma patients, although it is known to cause immune suppression. However, the influence of dexamethasone on the efficacy of GMCI has not been explored. In contrast, DNA damage response inhibitors like the ATR inhibitor (ATRi) AZD6738 might not only amend the cytotoxic but also the immunogenic profile of GMCI, rendering it an attractive combination partner. Methods We investigated the effects of ATR-inhibition and dexamethasone on GMCI in vitro using cytotoxicity, flow cytometry and T-cell-killing assays in glioblastoma cell lines. The impact of dexamethasone and ATRi in vivo was assessed in an orthotopic syngeneic murine glioblastoma model. Tumor immune infiltrates were analyzed with flow cytometry. Results Cytotoxicity assays showed that dexamethasone has a slight impact on GMCI in vitro. In T-cell-functional assays, we observed a significantly impaired tumor cell killing. Immune cell response assays revealed a reduced immune cell proliferation after co-culture with supernatant from dexamethasone or combination treated glioblastoma cells. In vivo, while treatment with GMCI alone resulted in longer median symptom-free survival (39.5d) versus no treatment (23d), the combination of GMCI and dexamethasone resulted in the significant reduction of this effect (29d vs 39.5d ; p = 0.0184). The combination of ATRi with GMCI proved to be synergistic in cytotoxicity assays. Flow cytometry revealed a significant increase in DSB-associated H2AX foci as well as an improved immune profile by downregulation of GMCI-induced PD-L1 expression. In vivo, the combination with ATRi led to an increase in long-term surviving animals (66.7%) compared to GMCI (50%) and proved to be highly significant compared to the untreated control (p=0.0022). Conclusions Our data suggest that dexamethasone may decrease the efficacy of immunotherapy for glioma through impaired T cell function: this emphasizes the need in identifying alternatives to dexamethasone to prevent attenuated responses in immunotherapies. The combination of GMCI with ATRi however points to additional therapeutic benefit through enhanced cytotoxic efficacy, improved immunogenicity in vitro and increased long-term survival in vivo, making it a promising future approach for the treatment of glioblastoma. Acknowledgements This was supported by NCI P01CA069246 (Chiocca)

Simple Summary Oncolytic Herpes simplex virus-1 (HSV-1) offers the dual potential of both lytic tumor-specific cell killing and inducing anti-tumor immune responses. The HSV-1 genome can be altered to enhance both components and this may be applicable for the treatment of a broad range of cancers. Several engineered oncolytic viruses based on the HSV-1 backbone are currently under investigation in various clinical trials, both as single agents and in combination with various immunomodulatory drugs. Abstract Herpes simplex virus 1 (HSV-1) provides a genetic chassis for several oncolytic viruses (OVs) currently in clinical trials. Oncolytic HSV1 (oHSV) have been engineered to reduce neurovirulence and enhance anti-tumor lytic activity and immunogenicity to make them attractive candidates in a range of oncology indications. Successful clinical data resulted in the FDA-approval of the oHSV talimogene laherparepvec (T-Vec) in 2015, and several other variants are currently undergoing clinical assessment and may expand the landscape of future oncologic therapy options. This review offers a detailed overview of the latest results from clinical trials as well as an outlook on newly developed HSV-1 oncolytic variants with improved tumor selectivity, replication, and immunostimulatory capacity and related clinical studies.

Background The overexpression of (basic)helix-loop-helix ((b)HLH) transcription factors (TFs) is frequent in malignant glioma. We investigated molecular effects upon disruption of the (b)HLH network by a dominant-negative variant of the E47 protein (dnE47). Our goal was to identify novel molecular subgroup-specific therapeutic strategies. Methods Glioma cell lines LN229, LNZ308, and GS-2/GS-9 were lentivirally transduced. Functional characterization included immunocytochemistry, immunoblots, cytotoxic, and clonogenic survival assays in vitro, and latency until neurological symptoms in vivo. Results of cap analysis gene expression and RNA-sequencing were further validated by immunoblot, flow cytometry, and functional assays in vitro. Results The induction of dnE47-RFP led to cytoplasmic sequestration of (b)HLH TFs and antiglioma activity in vitro and in vivo. Downstream molecular events, ie, alterations in transcription start site usage and in the transcriptome revealed enrichment of cancer-relevant pathways, particularly of the DNA damage response (DDR) pathway. Pharmacologic validation of this result using ataxia telangiectasia and Rad3 related (ATR) inhibition led to a significantly enhanced early and late apoptotic effect compared with temozolomide alone. Conclusions Gliomas overexpressing (b)HLH TFs are sensitive toward inhibition of the ATR kinase. The combination of ATR inhibition plus temozolomide or radiation therapy in this molecular subgroup are warranted.

OBJECTIVE The transcription factor E47 heterodimerizes with helix-loop-helix (HLH) and basic helix-loop-helix transcription (bHLH) factors like ID-1 and Olig2 that are overexpressed in glioblastoma. A dominant-negative variant of the E47 (dnE47) lacking the nuclear translocation signal, leads to cytoplasmatic sequestration of HLH and bHLH transcription factors. Here, we investigated combinations of dnE47-mediated inhibition of the bHLH transcriptional network with temozolomide and irradiation and explored the underlying molecular mechanisms. METHODS Long-term and stem cell glioma lines were transduced with a Doxycycline-inducible dnE47 lentivirus. Functional characterizations included immunocytochemistry, immunoblots, cytotoxicity and clonogenicity assays in vitro and latency until the onset of symptoms in vivo. CAGE and RNASeq were conducted for analyzing the dnE47-induced molecular profile. RESULTS The induction of dnE47 led to cytoplasmatic sequestration of HLH/bHLH transcription, reduced proliferation, increased cytotoxicity and reduced clonogenic survival in vitro and a prolonged latency until the onset of neurological symptoms in vivo. CAGE and RNASeq data revealed alterations in several cancer-relevant pathways. CONCLUSIONS A dnE47-mediated inhibition of the bHLH transcription network induced actionable molecular alterations in glioma cells that could be exploited for the design of novel therapies.

RATIONALE Dexamethasone is frequently used in symptomatic treatment of glioma patients, although it is known to cause immune suppression. Checkpoint inhibitor immunotherapies have not yet been successful in glioma treatments. Gene-mediated cytotoxic immunotherapy (GMCI) is an immunotherapeutic approach that uses aglatimagene besadenovec with an anti-herpetic prodrug to induce immunogenic tumor cell death and immune cell attraction to the tumor site with potent CD8 T cell activation. GMCI is currently in clinical trials for solid tumors including glioblastoma, where it showed encouraging survival results in a Phase 2 study that did not limit the use of dexamethasone. However, the effects of dexamethasone on its efficacy have not been explored. METHODS We investigated the effects of dexamethasone on GMCI in vitro using cytotoxicity and T-cell-killing assays in glioblastoma cell lines. The impact of dexamethasone in vivo was assessed in an orthotopic syngeneic murine glioblastoma model. RESULTS Cyotoxicity assays showed that Dexamethasone has a slight impact on GMCI in vitro. In contrast, we observed a highly significant effect in T-cell-functional assays in which killing was greatly impaired. Immune cell response assays revealed a reduced T-cell proliferation after co-culture with supernatant from dexamethasone or combination treated glioblastoma cells in contrast to GMCI alone. In a murine model, the combination of GMCI and dexamethasone resulted in a significant reduction in median symptom-free survival (29d) in comparison to GMCI alone (39.5d) (P = 0.0184). CONCLUSION Our data suggest that high doses of dexamethasone may negatively impact the efficacy of immunotherapy for glioma, which may be a consequence of impaired T cell function. These results support the idea that there is a need in identifying possible alternatives to dexamethasone to maximize the effectiveness of immunostimulatory therapies such as GMCI.

RATIONALE Herpes virus thymidine kinase (HSV-TK) suicide gene therapy is a well-established approach for in situ tumor cell killing after administration of ganciclovir (GCV), due to the induction of lethal DNA damage in HSV-TK expressing cells. Here we investigated the effects of HSV-TK gene delivery with a non-replicating serotype 5 adenovirus (AdTK) in murine glioblastoma models in combination with the ATR-inhibitor AZD6738 to disrupt the DNA damage response (DDR). METHODS We investigated the effects of disrupted DDR signaling on AdTK therapy in vitro using cytotoxicity, cytokine and flow cytometry assays in glioblastoma cell lines and in vivo with an orthotopic syngeneic murine glioblastoma model. Therapy response was monitored with MRI. Changes in the tumor microenvironment were analyzed with CyTOF. RESULTS The combination of AZD6738 with AdTK was synergistic in cytotoxicity assays, which was complemented by a significant increase of γH2AX foci. Complex modulations of the tumor microenvironment were observed with significantly reduced expression of PD-L1, MICA/B and the pro-tumorigenic cytokines IL1b and IL-4. In vivo, the combination with AZD6738 led to an increase in long-term surviving animals (66.7%) compared to GMCI (50%) and proved to be highly significant in contrast to untreated controls (p=0.0022). However, the combination treatment did not block the growth of tumors upon rechallenge in long-term survivors. CONCLUSION DDR signaling is crucial in the therapeutic efficacy of AdTK/GCV. It significantly enhances cytotoxicity in vitro and in vivo while having complex ramifications at the immunological level, requiring further studies to determine ideal conditions for a maximized therapeutic benefit.

Background The impact of prolonging temozolomide (TMZ) maintenance beyond six cycles in newly diagnosed glioblastoma (GBM) remains a topic of discussion. We investigated the effects of prolonged TMZ maintenance on progression-free survival (PFS) and overall survival (OS). Patients and methods In this retrospective single-center cohort study, we included patients with GBM who were treated with radiation therapy with concomitant and adjuvant TMZ. For analysis, patients were considered who either completed six TMZ maintenance cycles (group B), continued with TMZ therapy beyond six cycles (group C), or stopped TMZ maintenance therapy within the first six cycles (group A). Patients with progression during the first six TMZ maintenance cycles were excluded. Results Clinical data from 107 patients were included for Kaplan-Meier analyses and 102 for Cox regressions. Median PFS times were 8.1 months (95% confidence interval [CI] 6.1-12.4) in group A, 13.7 months (95% CI 10.6-17.5) in group B, and 20.9 months (95% CI 15.2-43.5) in group C. At first progression, response rates of TMZ/lomustine rechallenge were 47% in group B and 13% in group C. Median OS times were 12.7 months (95% CI 10.3-16.8) in group A, 25.2 months (95% CI 17.7-55.5) in group B, and 28.6 months (95% CI 24.4-open) in group C. Nevertheless, multivariate Cox regression for patients in group C compared with group B that accounted for imbalances of other risk factors showed no different relative risk (RR) for OS (RR 0.77, p = .46). Conclusion Our data do not support a general extension of TMZ maintenance therapy beyond six cycles. The Oncologist 2017;22:570-575 IMPLICATIONS FOR PRACTICE: Radiation therapy with concomitant and adjuvant temozolomide (TMZ) maintenance therapy is still the standard of care in patients below the age of 65 years in newly diagnosed glioblastoma. However, in clinical practice, many centers continue TMZ maintenance therapy beyond six cycles. The impact of this continuation is controversial and has not yet been addressed in prospective randomized clinical trials. We compared the effect of more than six cycles of TMZ in comparison with exactly six cycles on overall survival (OS) and progression-free survival (PFS) by multivariate analysis and found a benefit in PFS but not OS. Thus, our data do not suggest prolonging TMZ maintenance therapy beyond six cycles, which should be considered in neurooncological practice.

As reliable biomarkers of disease activity are lacking, monitoring of therapeutic response in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) remains a challenge. We sought to determine whether nerve ultrasound and electrophysiology scoring could close this gap. In CIDP patients (fulfilling EFNS/PNS criteria), we performed high-resolution nerve ultrasound to determine ultrasound pattern sum scores (UPSS) and predominant echotexture nerve conduction study scores (NCSS) as well as Medical Research Council sum scores (MRCSS) and inflammatory neuropathy cause and treatment disability scores (INCAT) at baseline and after 12 months of standard treatment. We retrospectively correlated ultrasound morphology with nerve histology when available. 72/80 CIDP patients featured multifocal nerve enlargement, and 35/80 were therapy-naïve. At baseline, clinical scores correlated with NCSS (r(2) = 0.397 and r(2) = 0.443, p 50% of measured segments, possibly reflecting axonal degeneration; and 3) almost no enlargement, reflecting “burned-out” or “cured” disease without active inflammation. Clinical improvement after 12 months was best in patients with pattern 1 (up to 75% vs up to 43% in pattern 2/3, Fisher’s exact test p

Objective Polyneuropathies are increasingly analyzed by ultrasound. Summarizing, diffuse enlargement is typical in Charcot-Marie Tooth type 1 (CMT1a), regional enlargement occurs in inflammatory neuropathies. However, a distinction of subtypes is still challenging. Therefore, this study focused on fascicle size and pattern in controls and distinct neuropathies. Methods Cross-sectional area (CSA) of the median, ulnar and peroneal nerve (MN, UN, PN) was measured at predefined landmarks in 50 healthy controls, 15 CMT1a and 13 MMN patients. Additionally, largest fascicle size and number of visible fascicles was obtained at the mid-upper arm cross-section of the MN and UN and in the popliteal fossa cross-section of the PN. Results Cut-off normal values for fascicle size in the MN, UN and PN were defined ( 2 , 2 and 2 ). In CMT1a CSA and fascicle values are significantly enlarged in all nerves, while in MMN CSA and fascicles are regionally enlarged with predominance in the upper arm nerves. The ratio of enlarged fascicles and all fascicles was significantly increased in CMT1a (>50%) in all nerves (p 20%), representing differential fascicle enlargement (enlarged and normal fascicles at the same location) sparing the peroneal nerve (regional fascicle enlargement). Based on these findings distinct fascicle patterns were defined. Conclusion Normal values for fascicle size could be evaluated; while CMT1a features diffuse fascicle enlargement, MMN shows regional and differential predominance with enlarged fascicles as single pathology. Significance Pattern analysis of fascicles might facilitate distinction of several otherwise similar neuropathies.

Aims Previous data suggest that expression of transcription factors FoxG1 and Olig-2 can separate hotspot H3F3A-mutant tumors in pediatric glioma. We evaluated their prognostic potential and feasibility for identifying H3F3A-mutant tumors among IDH-mutant/wildtype gliomas. Methods Immunohistochemistry of FoxG1/Olig-2 and ATRX in 471 cases of diffuse gliomas and molecular determination of IDH, H3F3A, MGMT and 1p/19 codeletion status. Results Mean percentage of FoxG1 positive tumor cells increased from 17% in WHO grade II to over 21% in grade III to 37% in grade IV tumors, while mean Olig-2 indices decreased from 29% to 28% to 17% respectively. FoxG1 indices were similar in astrocytic and oligodendroglial tumors, while Olig-2 indices were increased in oligodendrogliomas compared to astrocytic tumors (n=451, p

Die Versorgung des Frontalhirns erfolgt aus der Halsschlagader, der Arteria carotis interna (ACI), die sich intrakraniell in die Arteria cerebri anterior (ACA) und Arteria cerebri media (ACM) aufteilt. Abb. 29.1 und Abb. 29.2 zeigen die arteriellen Versorgungsgebiete der Arterien.