Franz Ricklefs

Extracellular Vesicles from High-Grade Glioma Exchange Diverse Pro-oncogenic Signals That Maintain Intratumoral Heterogeneity

A lack of experimental models of tumor heterogeneity limits our knowledge of the complex subpopulation dynamics within the tumor ecosystem. In high-grade gliomas (HGG), distinct hierarchical cell populations arise from different glioma stem-like cell (GSC) subpopulations. Extracellular vesicles (EV) shed by cells may serve as conduits of genetic and signaling communications; however, little is known about how HGG heterogeneity may impact EV content and activity. In this study, we performed a proteomic analysis of EVs isolated from patient-derived GSC of either proneural or mesenchymal subtypes. EV signatures were heterogeneous, but reflected the molecular make-up of the GSC and consistently clustered into the two subtypes. EV-borne protein cargos transferred between proneural and mesenchymal GSC increased protumorigenic behaviors in vitro and in vivo Clinically, analyses of HGG patient data from the The Cancer Genome Atlas database revealed that proneural tumors with mesenchymal EV signatures or mesenchymal tumors with proneural EV signatures were both associated with worse outcomes, suggesting influences by the proportion of tumor cells of varying subtypes in tumors. Collectively, our findings illuminate the heterogeneity among tumor EVs and the complexity of HGG heterogeneity, which these EVs help to maintain. Cancer Res; 76(10); 2876-81. ©2016 AACR.

MicroRNA Signatures and Molecular Subtypes of Glioblastoma: The Role of Extracellular Transfer

Summary Despite the importance of molecular subtype classification of glioblastoma (GBM), the extent of extracellular vesicle (EV)-driven molecular and phenotypic reprogramming remains poorly understood. To reveal complex subpopulation dynamics within the heterogeneous intratumoral ecosystem, we characterized microRNA expression and secretion in phenotypically diverse subpopulations of patient-derived GBM stem-like cells (GSCs). As EVs and microRNAs convey information that rearranges the molecular landscape in a cell type-specific manner, we argue that intratumoral exchange of microRNA augments the heterogeneity of GSC that is reflected in highly heterogeneous profile of microRNA expression in GBM subtypes.

Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles

Glioblastoma can suppress immunity by using surface PD-L1 on extracellular vesicles to block T cell receptor–mediated T cell activation. Binding of programmed death ligand-1 (PD-L1) to programmed cell death protein-1 (PD1) leads to cancer immune evasion via inhibition of T cell function. One of the defining characteristics of glioblastoma, a universally fatal brain cancer, is its profound local and systemic immunosuppression. Glioblastoma has also been shown to generate extracellular vesicles (EVs), which may play an important role in tumor progression. We thus hypothesized that glioblastoma EVs may be important mediators of immunosuppression and that PD-L1 could play a role. We show that glioblastoma EVs block T cell activation and proliferation in response to T cell receptor stimulation. PD-L1 was expressed on the surface of some, but not of all, glioblastoma-derived EVs, with the potential to directly bind to PD1. An anti-PD1 receptor blocking antibody significantly reversed the EV-mediated blockade of T cell activation but only when PD-L1 was present on EVs. When glioblastoma PD-L1 was up-regulated by IFN-γ, EVs also showed some PD-L1–dependent inhibition of T cell activation. PD-L1 expression correlated with the mesenchymal transcriptome profile and was anatomically localized in the perinecrotic and pseudopalisading niche of human glioblastoma specimens. PD-L1 DNA was present in circulating EVs from glioblastoma patients where it correlated with tumor volumes of up to 60 cm3. These results suggest that PD-L1 on EVs may be another mechanism for glioblastoma to suppress antitumor immunity and support the potential of EVs as biomarkers in tumor patients.

Preclinical investigation of combined gene-mediated cytotoxic immunotherapy and immune checkpoint blockade in glioblastoma

Background Combined immunotherapy approaches are promising cancer treatments. We evaluated anti-programmed cell death protein 1 (PD-1) treatment combined with gene-mediated cytotoxic immunotherapy (GMCI) performed by intratumoral injection of a prodrug metabolizing nonreplicating adenovirus (AdV-tk), providing in situ chemotherapy and immune stimulation. Methods The effects of GMCI on PD ligand 1 (PD-L1) expression in glioblastoma were investigated in vitro and in vivo. The efficacy of the combination was investigated in 2 syngeneic mouse glioblastoma models (GL261 and CT-2A). Immune infiltrates were analyzed by flow cytometry. Results GMCI upregulated PD-L1 expression in vitro and in vivo. Both GMCI and anti-PD-1 increased intratumoral T-cell infiltration. A higher percentage of long-term survivors was observed in mice treated with combined GMCI/anti-PD-1 relative to single treatments. Long-term survivors were protected from tumor rechallenge, demonstrating durable memory antitumor immunity. GMCI led to elevated interferon gamma positive T cells and a lower proportion of exhausted double positive PD1+TIM+CD8+ T cells. GMCI also increased PD-L1 levels on tumor cells and infiltrating macrophages/microglia. Our data suggest that anti-PD-1 treatment improves the effectiveness of GMCI by overcoming interferon-induced PD-L1-mediated inhibitory signals, and GMCI improves anti-PD-1 efficacy by increasing tumor-infiltrating T-cell activation. Conclusions Our data show that the GMCI/anti-PD-1 combination is well tolerated and effective in glioblastoma mouse models. These results support evaluation of this combination in glioblastoma patients.

Abstract A075: Preclinical analysis of combinatorial glioblastoma therapy with the prodrug-mediated gene therapy vector AdV-TK and immune checkpoint inhibition in GBM therapy

While the brain has traditionally been considered to be an immune-privileged site, evidence supporting the use of immunotherapeutics has been rapidly accumulating. Given that virus-based cancer therapies can be immunostimulatory and immune-checkpoint inhibitors block tumor-induced T-cell exhaustion, the combination of these two approaches offers a potentially synergistic interaction. One of the molecular underpinnings of T-cell exhaustion is the expression of Programmed Death-1 (PD1) on T-cells that recognizes its ligand PD-L1. AdV-tk is an immunostimulatory virus-based approach, known as Gene-Mediated Cytotoxic Immunotherapy (GMCI), that involves the intra-tumoral delivery of a non-replicating adenoviral vector carrying the Herpes virus thymidine kinase gene(TK) followed by administration of an anti-herpetic prodrug(ganciclovir-GCV) and recently showed encouraging results in a Phase II trial in glioblastoma(Wheeler et al.,2016). To provide a rationale for this therapeutic combination we investigated PD-L1 expression during GMCI therapy in human and mouse glioma cells in vitro and found that there was a consistent increase in cell surface PD-L1 levels. Interestingly, this was not associated with an increase of mRNA or protein. We also show that GMCI induces a type-I interferon response, and that the release of IFNβ is at least partially responsible for autocrine/paracrine PD-L1 up-regulation. In vivo studies using an intracranial GL261 model showed high levels of long term survivors in the GMCI/PD1 combination (11/14), compared with GMCI (6/16), anti-PD1 (5/12) and controls (0/11). In addition, tumor infiltrating lymphocytes after GMCI showed an increase in CD8+, CD8+/GranzymeB+, and CD8+/IFNγ+/TNFα+cells suggestive of cytotoxic T-cell activation. However, there was also a significant increase in CD4+, CD4+/FoxP3+, and IL-10 indicating a significant infiltration by Tregs, releasing immunosuppressive cytokines. Additionally, there was a significant increase in PD1+/TIM3+ T-cells, indicative of an immunosuppressive microenvironment. Overall, our data show that GMCI/anti-PD1 combinatorial therapy is effective in a syngeneic tumor model, and strongly support clinical trials of GMCI/checkpoint inhibitor combinations in glioblastoma patients. Citation Format: Maria Carmela Speranza, Kazue Kasai, Franz Ricklefs, Sarah R. Klein, Carmela Passaro, Hiroshi Nakashima, Johanna Kaufmann, Agnieszka Bronisz, Estuardo Aguilar-Cordova, Brian W. Guzik, Gordon J. Freeman, David A. Reardon, Patrick Wen, E. Antonio Chiocca, Sean E. Lawler. Preclinical analysis of combinatorial glioblastoma therapy with the prodrug-mediated gene therapy vector AdV-TK and immune checkpoint inhibition in GBM therapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A075.

Abstract PR04: The role of long noncoding RNA HIF1A-AS2 in hypoxic environment of glioblastoma

Purpose: While multiple protein-coding genes are known to play a crucial role in the formation and progression of glioblastoma multiforme (GBM), the role of long-non-coding RNAs (lncRNAs) in these cascades remains to be fully characterized. Considering the fundamental roles of hypoxia in the cellular and microenvironmental complexity and unexplored function of lncRNAs in GBM pathobiology, the identification of novel lncRNAs and their target pathways that drive the adaptation to hypoxic niche is crucial for better understanding of the development and progression of this highly heterogeneous brain tumor. Transcriptome profiling of GBM have revealed the presence of four clearly distinguishable subtypes, variably expressed in individual cells within a tumor. This finding may have potential clinical implications, including subtype-specific rearrangements of transcriptional programs related to oncogenic signaling, growth and hypoxia. However, the subtype-specific role of lncRNAs in tumorigenic potential of GBM subtypes remains largely unknown. Materials and Methods: The brain tissue samples including GBM tumors and non-pathological tissue adjacent to the tumor were collected. GBM-derived primary stem cells (GSCs) classified by transcriptome analysis as proneural (P) and mesenchymal (M), were collected and exposed to hypoxic conditions. Using custom designed Nanostring platform analysis followed by qPCR, the expression patterns of 70 cancer-related lncRNAs were characterized. The in situ hybridization and qPCR analysis was used to validate sub-cellular localization of selected lncRNA. RNA immunoprecipitation (RIP) and mass-spectroscopy (MS) was performed to map RNA-protein interaction and identified targets were validated by Western blotting. The global GSC transcriptome profiling upon lncRNA de-regulation was performed using Pan Cancer Nanostring platform. The knock-down and overexpression strategies in GSC were used to characterize cellular and molecular phenotypes in vitro and in in vivo intracranial GBM model. Results: We identified lncRNA HIF1A-AS2 (Hypoxia Inducible Factor 1 alpha - antisense 2) as one of the most deregulated in GBM comparing to the matched adjacent tissue. Despite lack of difference in the hypoxia-dependent activation of HIF1A protein between P and M GSC, HIF1A-AS2 was specifically upregulated in M GSC in vitro and in vivo. The deregulation of expression of HIF1A-AS2 had a broad effect on autophagy-related signaling network regulated in response to hypoxia in M but not P GSC. The IGF2BP2 and DHX9 were identified as direct protein partners of this lncRNA. The deregulation of HIF1A-AS2 affected the recruitment of IGF2BP2 to its mRNA targets (e.g. HMG1) and stability of DHX9 protein (but not mRNA), resulting in deregulation of its target genes (e.g FOSL1). Downregulation of HIF1A-AS2 in vivo led to de-regulation of its downstream effectors and resulted in survival benefit of mice bearing M GSC-originated tumors. Conclusion: P and M GSC respond differently to hypoxic stress by induction of autophagy to maintain their homeostasis and viability and this mechanism is controlled by HIF1A-AS2. This abstract is also presented as Poster A16. Citation Format: Marco Mineo, Franz Ricklefs, Shawn S. Lyons, Pavel Ivanov, E. Antonio Chiocca, Jakub Godlewski, Agnieszka Bronisz. The role of long noncoding RNA HIF1A-AS2 in hypoxic environment of glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr PR04.

Abstract 1929: The novel role of microRNA-128 in proneural to mesenchymal subtype transition in glioblastoma stem cells by targeting components of pro-oncogenic Polycomb Repressor Complex

Heterogeneous glioblastoma multiforme (GBM) was categorized based on transcriptional signatures into four subtypes (proneural (PN), neural, classical, and mesenchymal (MES)). In order to develop effective targeted therapeutic strategies, understanding the heterogeneous gene expression and molecular features of these subtypes is crucial. De-regulation of microRNA expression and activity has been shown to play an important role in tumor initiation and progression, including gliomagenesis. We have previously reported that expression of microRNA-128 (miR-128) is significantly down regulated in GBM and it diminishes self-renewal of GBM stem-like cells (GSCs) and sensitizes them to irradiation. Proneural-to-mesenchymal transition (PMT) manifested by concomitant up regulation of MES markers and down regulation of PN markers, is associated with increased malignancy, therapy-resistance and worse prognosis, but the underlying causes of PMT have not been convincingly characterized yet. In this study, we have demonstrated that miR-128 can regulate the PMT in GSC subsets. We showed that the expression of miR-128 in PN GSCs was significantly higher compared to MES subtype. As a tumor suppressive microRNA, miR-128 inhibited the MES GSC-specific high expression of Bmi1 and Suz12, two components of Polycomb Repressor Complexes (PRC) 1 and 2, respectively. In both GSC subtypes, miR-128 driven targeting of PRCs suppressed their epigenetic activity measured by ubiquitination of H2AK119 and tri-methylation of H3K27. Stable down regulation of miR-128 in PN GSCs significantly increased the expression of MES-specific gene signature (BCL2A1, CD44, WT-1, LYN, and MET) while its stable up regulation in MES GSCs resulted in the restoration of PN specific gene signature (CD133, SOX2, NES, OLIG2, and NOTCH1). We also showed that stable expression of miR-128 in GSCs could regulate the process of irradiation-induced PMT. Our in vivo studies showed the anti-tumorigenic role of miR-128 in both PN and MES GSC-derived intracranial tumor models. Taken together, we demonstrated that altering levels of miR-128 was sufficient to cause or reverse PMT, most likely by targeting the level/functions of PRCs and their target genes in GBM. Citation Format: Arun K. Rooj, Marco Mineo, Franz Ricklefs, Agnieszka Bronisz, Ennio Chiocca, Jakub Godlewski. The novel role of microRNA-128 in proneural to mesenchymal subtype transition in glioblastoma stem cells by targeting components of pro-oncogenic Polycomb Repressor Complex. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1929.