A De Novo Mouse Model of C11orf95-RELA Fusion-Driven Ependymoma Identifies Driver Functions in Addition to NF-κB.

The majority of supratentorial ependymomas (ST-ependymomas) have few mutations but frequently display chromothripsis of chromosome 11q that generates a fusion between C11orf95 and RELA (RELAFUS). Neural stem cells transduced with RELAFUSex vivo form ependymomas when implanted in the brain. These tumors display enhanced NF-κB signaling, suggesting that this aberrant signal is the principal mechanism of oncogenesis. However, it is not known whether RELAFUS is sufficient to drive de novo ependymoma tumorigenesis in the brain and, if so, whether these tumors also arise from neural stem cells. We show that RELAFUS drives ST-ependymoma formation from periventricular neural stem cells in mice and that RELAFUS-induced tumorigenesis is likely dependent on a series of cell signaling pathways in addition to NF-κB

Untersuchung der Eigenschaften von Gliom-assoziierten Mikroglia/Makrophagen

Malignant glioma belong to the most aggressive neoplasms in humans with no successful treatment available. Patients suffering from glioblastoma multiforme (GBM), the highest-grade glioma, have an average survival time of only 12-15 months after diagnosis. Both microglia and peripheral macrophages/monocytes accumulate within and around glioma, but fail to exert effective anti-tumor activity and even support tumor growth. Here I investigated the properties and functions of glioma-associated microglia/macrophages (GAMs) using different molecular and experimental approaches. In the first project I used microarray analysis to compare the expression profiles of GAMs and naive control cells. Samples were generated from CD11b+ MACS-isolated cells from naïve and GL261-implanted C57BL/6 mouse brains. Around 1000 genes were more than 2-fold up- or downregulated in GAMs when compared to control cells. Comparison to published data sets of M1/M2a,b,c-polarized macrophages revealed a gene expression pattern that has only partial overlap with any of the M1 or M2 gene expression patterns. Samples for the qRT-PCR validation of selected M1 and M2a,b,c-specific genes were generated from two different glioma mouse models and isolated by flow cytometry to distinguish between resident microglia and invading macrophages/monocytes. In both models the unique GAMs phenotype including a mixture of M1 and M2a,b,c-specific genes could be confirmed. To validate the expression of these genes in human samples, I MACS-isolated CD11b+ microglia/macrophages from glioblastoma multiforme (GBM), lower grade brain tumors, and control specimens. Apart from the M1/M2 gene analysis, I demonstrate that Gpnmb and Spp1 are highly expressed in both murine and human GAMs. High expression of these genes has been associated with worsened prognosis in human GBM, as indicated by patient outcome linked to gene expression data. I also show that GAMs are the predominant source of these transcripts in murine and human GBM. In the second project, I investigated the role of Cx3cr1 in the microglia-dependent support of glioma growth by employing a monocyte-free organotypic brain slice model. In Cx3cr1GFP/GFP slices, lacking Cx3cr1 expression, the growth of GL261 tumors was significantly enhanced, when compared to Cx3cr1GFP/wt or Cx3cr1wt/wt slices. In another glioma model the loss of Cx3cr1 did not significantly impact tumor growth when compared to Cx3cr1GFP/wt or Cx3cr1wt/wt slices. In both models a trend toward a higher microglia migration toward tumors could be observed in Cx3cr1GFP/GFP slices when compared to Cx3cr1GFP/wt. This indicates that CX3CR1 signaling attenuates a glioma-directed migration of microglia and subsequent glioma-supporting functions of these cells. The findings of my thesis provide new insights in the phenotype and function of GAMs and will help to better understand their role for glioma development and progression and to define new potential targets for future anti-glioma therapy.Maligne Gliome gehören zu den aggressivsten Neoplasmen im Menschen und zur Zeit gibt es für diese Tumore keine wirksame Therapieform. Patienten die an einem Glioblastoma multiforme (GBM), der hochgradigsten Form der Gliome, erkrankt sind besitzen nur eine durchschnittliche Lebenserwartung von 12-15 Monate nach der Diagnose. Sowohl Mikroglia, als auch aus der Periphärie invasierende Makrophagen und Monozyten, sammeln sich in und um die Tumormasse an. Diese Zellen entfalten jedoch keine effektive Immunantwort die sich gegen den Tumor richtet, sondern unterstützen sogar das Tumorwachstum. In dieser Arbeit untersuchte ich die Eigenschaften und Funktionen der Gliom-assoziierten Mikroglia/Makrophagen (GAMs) in zwei verschiedenen Projekten. In dem ersten Projekt habe ich ein Mikroarrayexperiment durchgeführt um die Expressionsprofile von GAMs und naiven Kontrollzellen zu vergleichen. Als Proben wurden CD11b+ Zellen verwendet, welche mittels MACS aus naiven und GL261-implantierten C57BL/6 Maushirnen isoliert wurden. Im Vergleich zu den Kontrollzellen waren in GAMs ungefähr 1000 Gene mehr als 2-fach hoch- oder runterreguliert. Ein Vergleich mit publizierten Datensätzen von M1/M2a,b,c-polarisierten Makrophagen zeigte, dass GAMs ein Genexpressionsmuster besitzen, welches nur eine teilweise Überlappung mit M1 oder M2 Expressionsmustern aufweist. Die Proben für die qRT-PCR Validierung von ausgewählten M1 und M2a,b,c-spezifischen Genen wurden in zwei verschiedenen Gliommodellen erzeugt und mittels Durchflusszytometrie aufgereinigt. Dies ermöglichte es für diese Proben zwischen hirneigenen Mikroglia und invasierenden Makrophagen/Monozyten zu unterscheiden. Ich konnte die Expression ausgewählter M1/M2a,b,c-spezifischer Gene in beiden Tumormodellen in GAMs bestätigen. Desweiteren habe ich mittels MACS CD11b+ Mikroglia/Makrophagen aus humanen Proben (GBM, niedergradige Hirntumore und Kontrollproben) aufgereinigt, um die Expression ausgewählter Gene auch in humanen Proben zu untersuchen. Abgesehen von der M1/M2 Analyse, demonstriere ich in dieser Arbeit, dass die Expression von Gpnmb und Spp1 in murinen und humanen GAMs stark hochreguliert ist. Eine erhöhte Expression dieser Gene ist mit einer schlechteren Lebenserwartung für Patienten mit GBM assoziiert. Ich zeige weiterhin, dass GAMs die Hauptquelle für diese beiden Transkripte in murinen und humanen GBMs sind. In einem zweiten Projekt untersuchte ich mit Hilfe eines Monozyten-freien organotypischen Hirnschnittkulturmodells die Rolle von Cx3cr1 in der Mikroglia-abhängigen Unterstützung des Gliomwachstums. GL261 Tumore die in Cx3cr1GFP/GFP Schnitte injiziert wurden – welche keine Cx3cr1 Expression besitzen – waren signifikant größer als GL261 Tumore welche in Cx3cr1GFP/wt or Cx3cr1wt/wt Schnitte injiziert wurden. In einem weiteren Tumormodell hatte das Fehlen der Cx3cr1 Expression keinen signifikanten Einfluss auf das Tumorwachstum. In beiden Tumormodellen gab es einen Trend hin zu einer höheren Mikrogliamigration in die peritumoralen Bereiche in Cx3cr1GFP/GFP Schnitten (im Vergleich zu Cx3cr1GFP/wt Schnitten). Dies deutet darauf hin, dass der CX3CR1 Signalweg einen inhibierenden Einfluss auf die Gliom-gerichetete Migration und anschließende Gliom-unterstützende Funktionen dieser Zellen hat. Die Ergebnisse meiner Arbeit liefern neue Einsichten in den Phänotyp und die Funktion von GAMs und werden bei der weiteren Charakterisierung dieser Zellen helfen, um neue potentielle Ansatzpunkte für zukünftige Antigliomtherapien zu finden

Visualizing genomic characteristics across an RNA-Seq based reference landscape of normal and neoplastic brain

Abstract In order to better understand the relationship between normal and neoplastic brain, we combined five publicly available large-scale datasets, correcting for batch effects and applying Uniform Manifold Approximation and Projection (UMAP) to RNA-Seq data. We assembled a reference Brain-UMAP including 702 adult gliomas, 802 pediatric tumors and 1409 healthy normal brain samples, which can be utilized to investigate the wealth of information obtained from combining several publicly available datasets to study a single organ site. Normal brain regions and tumor types create distinct clusters and because the landscape is generated by RNA-Seq, comparative gene expression profiles and gene ontology patterns are readily evident. To our knowledge, this is the first meta-analysis that allows for comparison of gene expression and pathways of interest across adult gliomas, pediatric brain tumors, and normal brain regions. We provide access to this resource via the open source, interactive online tool Oncoscape, where the scientific community can readily visualize clinical metadata, gene expression patterns, gene fusions, mutations, and copy number patterns for individual genes and pathway over this reference landscape

Arming oHSV with ULBP3 drives abscopal immunity in lymphocyte-depleted glioblastoma

Oncolytic viruses induce local tumor destruction and inflammation. Whether virotherapy can also overcome immunosuppression in noninfected tumor areas is under debate. To address this question, we have explored immunologic effects of oncolytic herpes simplex viruses (oHSVs) in a genetically engineered mouse model of isocitrate dehydrogenase (IDH) wild-type glioblastoma, the most common and most malignant primary brain tumor in adults. Our model recapitulates the genomics, the diffuse infiltrative growth pattern, and the extensive macrophage-dominant immunosuppression of human glioblastoma. Infection with an oHSV that was armed with a UL16-binding protein 3 (ULBP3) expression cassette inhibited distant tumor growth in the absence of viral spreading (abscopal effect) and yielded accumulation of activated macrophages and T cells. There was also abscopal synergism of oHSVULBP3 with anti-programmed cell death 1 (anti-PD-1) against distant, uninfected tumor areas; albeit consistent with clinical trials in patients with glioblastoma, monotherapy with anti-PD-1 was ineffective in our model. Arming oHSV with ULBP3 led to upregulation of antigen processing and presentation gene sets in myeloid cells. The cognate ULBP3 receptor NKG2D, however, is not present on myeloid cells, suggesting a noncanonical mechanism of action of ULBP3. Overall, the myeloid-dominant, anti-PD-1-sensitive abscopal effect of oHSVULBP3 warrants further investigation in patients with IDH wild-type glioblastoma

Phase separation of YAP-MAML2 differentially regulates the transcriptome

Phase separation (PS) drives the formation of biomolecular condensates that are emerging biological structures involved in diverse cellular processes. Recent studies have unveiled PS-induced formation of several transcriptional factor (TF) condensates that are transcriptionally active, but how strongly PS promotes gene activation remains unclear. Here, we show that the oncogenic TF fusion Yes-associated protein 1-Mastermind like transcriptional coactivator 2 (YAP-MAML2) undergoes PS and forms liquid-like condensates that bear the hallmarks of transcriptional activity. Furthermore, we examined the contribution of PS to YAP-MAML2-mediated gene expression by developing a chemogenetic tool that dissolves TF condensates, allowing us to compare phase-separated and non-phase-separated conditions at identical YAP-MAML2 protein levels. We found that a small fraction of YAP-MAML2-regulated genes is further affected by PS, which include the canonical YAP target genes CTGF and CYR61, and other oncogenes. On the other hand, majority of YAP-MAML2-regulated genes are not affected by PS, highlighting that transcription can be activated effectively by diffuse complexes of TFs with the transcriptional machinery. Our work opens new directions in understanding the role of PS in selective modulation of gene expression, suggesting differential roles of PS in biological processes

Epigenomic analysis of formalin-fixed paraffin-embedded samples by CUT&Tag

Abstract For more than a century, formalin-fixed paraffin-embedded (FFPE) sample preparation has been the preferred method for long-term preservation of biological material. However, the use of FFPE samples for epigenomic studies has been difficult because of chromatin damage from long exposure to high concentrations of formaldehyde. Previously, we introduced Cleavage Under Targeted Accessible Chromatin (CUTAC), an antibody-targeted chromatin accessibility mapping protocol based on CUT&Tag. Here we show that simple modifications of our CUTAC protocol either in single tubes or directly on slides produce high-resolution maps of paused RNA Polymerase II at enhancers and promoters using FFPE samples. We find that transcriptional regulatory element differences produced by FFPE-CUTAC distinguish between mouse brain tumors and identify and map regulatory element markers with high confidence and precision, including microRNAs not detectable by RNA-seq. Our simple workflows make possible affordable epigenomic profiling of archived biological samples for biomarker identification, clinical applications and retrospective studies

Anti-PD-L1 antibody direct activation of macrophages contributes to a radiation-induced abscopal response in glioblastoma

BACKGROUND Most glioblastoma recurrences occur near prior radiation treatment sites. Future clinical success will require achieving and optimizing an 'abscopal effect', whereby un-irradiated neoplastic cells outside treatment sites are recognized and attacked by the immune system. Radiation combined with anti-PD-L1 demonstrated modest efficacy in phase II human glioblastoma clinical trials, but the mechanism and relevance of the abscopal effect during this response remains unknown. METHODS We modified an immune-competent, genetically-driven mouse glioma model (forced PDGF expression + PTEN loss) where a portion of the tumor burden is irradiated (PDGF) and another un-irradiated luciferase expressing tumor (PDGF+Luciferase) is used as a readout of the abscopal effect following systemic anti-PD-L1 immunotherapy. We assessed relevance of tumor neoepitope during the abscopal response by inducing expression of EGFRvIII (PDGF+EGFRvIII). Statistical tests were two-sided. RESULTS Following radiation of one lesion, anti-PD-L1 immunotherapy enhanced the abscopal response to the un-irradiated lesion. In PDGF-driven gliomas without tumor neoepitope (PDGF+Luciferase, n=8), the abscopal response occurred via anti-PD-L1-driven, ERK-mediated, bone marrow-derived macrophage phagocytosis of adjacent un-irradiated tumor cells, with modest survival implications (median survival 41 days vs. radiation alone 37.5 days, P=.03). In PDGF-driven gliomas with tumor neoepitope (PDGF+EGFRvIII, n=8), anti-PD-L1-enhanced abscopal response was associated with macrophage and T-cell infiltration and increased survival benefit (median survival 36 days vs. radiation alone 28 days, P=.001). CONCLUSION Our results indicate that anti-PD-L1 immunotherapy enhances a radiation induced abscopal response via canonical T-cell activation and direct macrophage activation in glioblastoma

Glioma-Associated Microglia/Macrophages Display an Expression Profile Different from M1 and M2 Polarization and Highly Express Gpnmb and Spp1

Malignant glioma belong to the most aggressive neoplasms in humans with no successful treatment available. Patients suffering from glioblastoma multiforme (GBM), the highest-grade glioma, have an average survival time of only around one year after diagnosis. Both microglia and peripheral macrophages/monocytes accumulate within and around glioma, but fail to exert effective anti-tumor activity and even support tumor growth. Here we use microarray analysis to compare the expression profiles of glioma-associated microglia/macrophages and naive control cells. Samples were generated from CD11b(+) MACS-isolated cells from naive and GL261-implanted C57BL/6 mouse brains. Around 1000 genes were more than 2-fold up-or downregulated in glioma-associated microglia/macrophages when compared to control cells. A comparison with published data sets of M1, M2a,b,c-polarized macrophages revealed a gene expression pattern that has only partial overlap with any of the M1 or M2 gene expression patterns. Samples for the qRT-PCR validation of selected M1 and M2a,b,c-specific genes were generated from two different glioma mouse models and isolated by flow cytometry to distinguish between resident microglia and invading macrophages. We confirmed in both models the unique glioma-associated microglia/macrophage phenotype including a mixture of M1 and M2a,b,c-specific genes. To validate the expression of these genes in human we MACS-isolated CD11b(+) microglia/macrophages from GBM, lower grade brain tumors and control specimens. Apart from the M1/M2 gene analysis, we demonstrate that the expression of Gpnmb and Spp1 is highly upregulated in both murine and human glioma-associated microglia/macrophages. High expression of these genes has been associated with poor prognosis in human GBM, as indicated by patient survival data linked to gene expression data. We also show that microglia/macrophages are the predominant source of these transcripts in murine and human GBM. Our findings provide new potential targets for future anti-glioma therapy