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Medulloblastoma (MB) is the most common malignant brain tumor in children with standard of care consisting of surgery, radiation, and chemotherapy. Recent molecular profiling led to the identification of four molecularly distinct MB subgroups – Wingless (WNT), Sonic Hedgehog (SHH), Group 3, and Group 4. Despite genomic MB characterization and subsequent tumor stratification, clinical treatment paradigms are still largely driven by histology, degree of surgical resection, and presence or absence of metastasis rather than molecular profile. Patients usually undergo resection of their tumor followed by craniospinal radiation (CSI) and a 6 month to one-year multi-agent chemotherapeutic regimen. While there is clearly a need for development of targeted agents specific to the molecular alterations of each patient, targeting proteins responsible for DNA damage repair could have a broader impact regardless of molecular subgrouping. DNA damage response (DDR) protein inhibitors have recently emerged as targeted agents with potent activity as monotherapy or in combination in different cancers. Here we discuss the molecular underpinnings of genomic instability in MB and potential avenues for exploitation through DNA damage response inhibition.

The mesenchymal subtype of glioblastoma is thought to be determined by both cancer cell intrinsic alterations and extrinsic cellular interactions, but remains poorly understood. Here, we dissect glioblastoma-to-microenvironment interactions by single-cell RNA-sequencing analysis of human tumors and model systems, combined with functional experiments. We demonstrate that macrophages induce a transition of glioblastoma cells into mesenchymal-like (MES-like) states. This effect is mediated, both in vitro and in vivo, by macrophage-derived Oncostatin M (OSM) that interacts with its receptors (OSMR or LIFR) in complex with GP130 on glioblastoma cells and activates STAT3. We show that MES-like glioblastoma states are also associated with increased expression of a mesenchymal program in macrophages and with increased cytotoxicity of T cells, highlighting extensive alterations of the immune microenvironment with potential therapeutic implications.

Myeloid cells comprise the majority of immune cells in tumors, where their content and composition is determined by tumor type and driver mutation. While these cells are essential for shaping the tumor microenvironment, promoting tumor growth, and contributing to therapeutic resistance, targeting tumor-associated myeloid cells, including bone-marrow-derived monocytes and neutrophils, has not been successful in the clinics. Monocyte chemoattractant protein (MCP) family, comprising of Ccl2, Ccl7, Ccl8, Ccl12, are essential for monocytes trafficking to the tumor sites. To eliminate monocyte recruitment, we leveraged CRISPR/Cas-9 based gene editing tool to generate a mouse strain that is devoid of all MCP genes, which we termed quadruple MCP knockout (qMCP-/-). Using these mice in combination with genetically engineered mouse models (GEMM) of glioblastoma (GBM), we abolished tumor monocyte infiltration. Due to the functional redundancy of MCP family members, we show that targeting individual MCP genes leads to compensation by other MCPs. In contrast, when all MCPs are genetically deleted and monocyte recruitment is abolished, neutrophil infiltration ensues. Single-cell RNA sequencing revealed that intratumoral neutrophils promoted proneural-to-mesenchymal transition in GBM, and supported tumor aggression by facilitating hypoxia response via TNF production. Remarkably, pharmacologic or genetic interventions that suppress both monocytes and neutrophil infiltration improve the survival of GBM-bearing mice. Taken together, our findings establish that specific subsets of myeloid cells can influence the dynamism of tumor microenvironment, and they emphasize the importance of targeting both monocytes and neutrophils simultaneously for effective GBM immunotherapy.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor. The only intervention that has improved the survival rate of GBM patients over the past several decades has been combining temozolomide (TMZ) with radiotherapy (RT), which increased median survival by only ~2.5 months to where it currently stands at ~15 months. Unfortunately, all GBM patients eventually die due to tumor recurrence. Intrinsic or acquired resistance to TMZ is a significant contributing factor to tumor progression, and various mechanisms have been suggested, including deficiencies in DNA mismatch repair (MMR) genes such as MSH6. To test the biological significance of Msh6 in GBM growth and resistance to TMZ and immunotherapy, we have generated immunocompetent genetically engineered mouse models (GEMMs) of GBM with germline loss of Msh6. To generate tumors with reduced or abrogated Msh6, we utilized GEMMs based on the RCAS/tv-a gene transfer system in combination with mice that exhibited heterozygous and homozygous loss of Msh6. When PDGFB was overexpressed in combination with the silencing of Tp53 to induce tumors, tumor-bearing mice with reduced or deficient Msh6 demonstrated increased tumor growth and shorter survival time compared to wild-type tumor-bearing mice. Our data demonstrate that two weeks of TMZ treatment at a clinically relevant dose of 25 mg/kg provides a significant survival advantage in WT-tumor-bearing mice, while no efficacy was observed in tumor-bearing mice with either heterozygous or homozygous loss of Msh6. We are currently evaluating the mechanism by which of Msh6 confers TMZ-resistance.

BackgroundGlioblastoma (GBM) is a devastating primary brain tumor with a highly immunosuppressive tumor microenvironment, and treatment with oncolytic viruses (OVs) has emerged as a promising strategy for these tumors. Our group constructed a new OV named Delta-24-ACT, which was based on the Delta-24-RGD platform armed with 4-1BB ligand (4-1BBL). In this study, we evaluated the antitumor effect of Delta-24-ACT alone or in combination with an immune checkpoint inhibitor (ICI) in preclinical models of glioma.MethodsThe in vitro effect of Delta-24-ACT was characterized through analyses of its infectivity, replication and cytotoxicity by flow cytometry, immunofluorescence (IF) and MTS assays, respectively. The antitumor effect and therapeutic mechanism were evaluated in vivo using several immunocompetent murine glioma models. The tumor microenvironment was studied by flow cytometry, immunohistochemistry and IF.ResultsDelta-24-ACT was able to infect and exert a cytotoxic effect on murine and human glioma cell lines. Moreover, Delta-24-ACT expressed functional 4-1BBL that was able to costimulate T lymphocytes in vitro and in vivo. Delta-24-ACT elicited a more potent antitumor effect in GBM murine models than Delta-24-RGD, as demonstrated by significant increases in median survival and the percentage of long-term survivors. Furthermore, Delta-24-ACT modulated the tumor microenvironment, which led to lymphocyte infiltration and alteration of their immune phenotype, as characterized by increases in the expression of Programmed Death 1 (PD-1) on T cells and Programmed Death-ligand 1 (PD-L1) on different myeloid cell populations. Because Delta-24-ACT did not induce an immune memory response in long-term survivors, as indicated by rechallenge experiments, we combined Delta-24-ACT with an anti-PD-L1 antibody. In GL261 tumor-bearing mice, this combination showed superior efficacy compared with either monotherapy. Specifically, this combination not only increased the median survival but also generated immune memory, which allowed long-term survival and thus tumor rejection on rechallenge.ConclusionsIn summary, our data demonstrated the efficacy of Delta-24-ACT combined with a PD-L1 inhibitor in murine glioma models. Moreover, the data underscore the potential to combine local immunovirotherapy with ICIs as an effective therapy for poorly infiltrated tumors.

Background Children with diffuse intrinsic pontine glioma (DIPG) succumb to disease within 2 years of diagnosis despite treatment with ionizing radiation (IR) and/or chemotherapy. Our aim was to determine the role of protein phosphatase, magnesium-dependent 1, delta (PPM1D) mutation, present in up to 25% of cases, in DIPG pathogenesis and treatment. Methods Using genetic and pharmacologic approaches, we assayed effects of PPM1D mutation on DIPG growth and murine survival. We assayed effects of targeting mutated PPM1D alone or with IR on signaling, cell cycle, proliferation, and apoptosis in patient-derived DIPG cells in vitro, in organotypic brain slices, and in vivo. Results PPM1D-mutated DIPG cell lines exhibited increased proliferation in vitro and in vivo, conferring reduced survival in orthotopically xenografted mice, through stabilization of truncated PPM1D protein and inactivation of DNA damage response (DDR) effectors p53 and H2A.X. PPM1D knockdown or treatment with PPM1D inhibitors suppressed growth of PPM1D-mutated DIPGs in vitro. Orthotopic xenografting of PPM1D short hairpin RNA-transduced or PPM1D inhibitor-treated, PPM1D-mutated DIPG cells into immunodeficient mice resulted in reduced tumor proliferation, increased apoptosis, and extended mouse survival. PPM1D inhibition had similar effects to IR alone on DIPG growth inhibition and augmented the anti-proliferative and pro-apoptotic effects of IR in PPM1D-mutated DIPG models. Conclusions PPM1D mutations inactivate DDR and promote DIPG growth. Treatment with PPM1D inhibitors activated DDR pathways and enhanced the anti-proliferative and pro-apoptotic effects of IR in DIPG models. Our results support continued development of PPM1D inhibitors for phase I/II trials in children with DIPG.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem glioma, which carries the deadliest prognosis of all childhood neoplasms. Despite recent advances in molecular profiling and characterization of pediatric high-grade gliomas, patient outcomes have not improved. One critical reason why current therapeutic approaches fail to provide a durable response is the adaptive nature of the highly understudied tumor microenvironment (TME). The most abundant non-neoplastic infiltrates in the DIPG TME are the tumor-associated macrophages (TAMs). There is limited data available on the role of TAMs in DIPG tumor growth and response to therapy, and the chemokines responsible for driving the infiltration of both innate and adaptive immune cells into these tumors remain unidentified.To accurately model DIPGs, we utilized immunocompetent genetically engineered mouse models (GEMMs) based on the RCAS/tv-a gene transfer system. At postnatal days 0-2, mice were injected with retrovirus-producing DF-1 cells directly in the brainstem. Tumorigenesis was driven by PDGFB overexpression and Tp53 silencing, together with human DIPG-specific mutations including: H3 histones with K27M substitution (H3.1K27M/H3.3K27M) and Acvr1R206H. Our data using these GEMMs show that mouse tumors are strikingly similar to their human counterparts as demonstrated by hallmark pathological observations including T2-contrast enhancement, highly infiltrating margins, and pseudopalisading necrosis. Loss of H3K27 trimethylation in histone mutant tumors is also observed in these tumors. Utilizing these GEMMs, we have identified CCL8 and CCL12 as important chemokines in H3.1K27M-DIPGs. Specifically, we observed longer survival in Ccl8/Ccl12 double knockout (DKO) tumor-bearing mice compared to wild-type (WT) mice. Interestingly, spectral flow cytometry analysis revealed no differences in myeloid cell infiltration but an increased number of NK cells, CD4+, and CD8+ T cells in tumors generated in Ccl8/Ccl12 DKO mice compared to WT. We also tested the efficacy of inhibiting two CCL8 receptors using a syngeneic mouse model of DIPG. Pharmacological inhibition of CCR1 and CCR5 rendered a reduction in macrophage presence in the tumors, and resulted in an extension of survival in tumor-bearing mice comparable to the effect of standard of care, radiation therapy. Our ongoing experiments aim to elucidate the mechanisms by which CCL8 and CCL12 modulate immune cell infiltration, and determine whether the effect of CCL8/CCL12 loss on survival seen in H3.1K27M-DIPG holds true for H3.3K27M-DIPG-bearing mice. Citation Format: Gonzalo M. Pinero, Montserrat Puigdelloses Vallcorba, James L. Ross, Zhihong Chen, Tanvi Joshi, Dolores Hambardzumyan. The role of chemokines CCL8 and CCL12 in diffuse intrinsic pontine gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1340.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor. The only intervention that has improved the survival rate of GBM patients over the past several decades has been combining temozolomide (TMZ) with radiotherapy (RT), which increased median survival by only ~2.5 months to where it currently stands at ~15 months. Unfortunately, all GBM patients eventually die due to tumor recurrence. Intrinsic or acquired resistance to TMZ is a significant contributing factor to tumor progression, and various mechanisms have been suggested, including deficiencies in DNA mismatch repair (MMR) genes such as MSH2. Utilizing patient GBM samples from the TCGA, we have identified a correlation between low MSH2 expression and shorter patient survival. To test the biological significance of Msh2 in GBM growth and resistance to TMZ and immunotherapy, we have generated immunocompetent genetically engineered mouse models (GEMMs) of GBM with germline or somatic loss of Msh2. To generate tumors with reduced or abrogated Msh2, we utilized GEMMs based on the RCAS/tv-a gene transfer system in combination with mice that exhibited heterozygous and homozygous loss of Msh2. When PDGFB was overexpressed in combination with the silencing of Tp53 to induce tumors, tumor-bearing mice with reduced or deficient Msh2 demonstrated shorter survival time compared to wild-type tumor-bearing mice. In addition, our results indicate Msh2 deficiency increases the malignancy of gliomas. Our data demonstrate that two weeks of TMZ treatment at a clinically relevant dose of 25 mg/kg provides a significant survival advantage in WT-tumor-bearing mice, while no efficacy was observed in tumor-bearing mice with either heterozygous or homozygous loss of Msh2. We are currently evaluating the role of Msh2 in the efficacy of immune checkpoint inhibitors in glioblastoma. Citation Format: Montserrat Puigdelloses Vallcorba, Alice Buonfiglioli, Gonzalo Piñero, Zhihong Chen, Dolores Hambardzumyan. Reduction or loss of Msh2 confers resistance to temozolomide in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1634.

Diffuse intrinsic pontine glioma (DIPG) is the leading cause of brain tumor-related death in children. It is characterised for having a non-inflammatory microenvironment and be immunologically inert. Therefore, strategies aiming to break the microenvironment status-quo in this disease could provide therapeutic benefit. The complement system promotes tumor progression due to the continuous production of anaphylatoxins leading to the infiltration of myeloid cells, which express high levels of complement receptors (C3aR and C5aR1). We have in silico data showing the high expression of C5aR1 in DIPGs. Thus, we wanted to assess first whether complement C5aR1 could constitute an actionable target, and second whether combining C5aR1 inhibitors with oncolytic virus could result in a superior antitumor immune response than either agent alone in DIPG. In this study, we used two different peptide inhibitors of C5aR1, PMX53 and PMX205 combined with the virus Delta-24-ACT (an oncolytic virus armed with 4-1BBL). We performed in vivo studies to evaluate the efficacy of this combination in immunocompetent DIPG models. Our data showed that the combination Delta-24-ACT/PMX53 significantly extended the median survival of the animals when compared with either agent alone, and led to long-term survivors that generated immune memory. The combination treatment modulated the tumor microenvironment promoting an increase in lymphocytes, mainly CD8+ cells presenting an active phenotype, and a reduction in C5aR1 expression in the myeloid compartment. We are currently evaluating in vivo whether PMX205, which has an improved ability to cross the blood brain barrier, leads to better therapeutic response. In summary, the combination of Delta-24-ACT with a C5aR1 inhibitor showed the capacity to shake the DIPG tumor microenvironment and unleashed an antitumor immune response. These data underscore the possibilities to combine oncolytic virus with targets of the tumor microenvironment to improve their therapeutic benefit in DIPGs.

Tumor hypoxia is linked to poor outcome for glioblastoma (GBM), a highly malignant brain cancer, but the underlying mechanisms and the environmental factors that initiate tumor hypoxia are poorly understood. We tracked tumor hypoxia in GBM in immunocompetent mice with a hypoxia sensitive fluorescent reporter combined with single cell transcriptomics. We found that hypoxic GBM cells are quiescent, immunosuppressive and display a mesenchymal transition, all of which are linked to malignant potency. We also captured in vivo hypoxia gene signature, which is more represented in recurrent GBM and predicts worse outcome. Interestingly, hypoxic GBM cells is a diverse population, consisted of four subclusters, and enriched for immune pathways. Concordantly, our reporter highlighted a distinct geographic pattern of immune cells in hypoxic regions, with phagocytic tumor-associated macrophages (TAMs) and CD8+ cytotoxic T cells (CTLs) congregated in hypoxic cores confined by hypoxic GBM cells in pseudo-palisading patterns. Mechanistically, this is a dynamic temporospatial process, requiring cytokine CCL8. Remarkably, the sequestered TAMs also experience hypoxia, and they are reprogrammed to express immunotolerant markers by factors released from hypoxic GBM cells. Contrary to the conventional viewpoint that hypoxia arises from rapid tumor expansion outstripping vascular supply, we discovered anticancer immunity as an important driving force of tumor hypoxia; attenuating immune responses by implanting GBM in host mice with immunodeficiency or IL1β deletion greatly decreased GBM hypoxia. Analyses of human patient GBM samples highlighted a connection of mesenchymal subtype, immune response, and tumor hypoxia, all contributing to poor survival. Altogether, our study revealed a reciprocal influence of anti-tumor immunity and tumor hypoxia, which has significant ramifications for prognosis and immunotherapy for GBM.

BACKGROUND: Microglia and periphery-derived monocytes infiltrate human and mouse glioblastoma and their density is positively correlated with malignancy. Using microarray and RNA sequencing, we have previously shown that glioblastoma-associated microglia/monocytes (GAMs) express osteopontin/SPP1. METHODS: We used quantitative reverse transcriptase PCR, immunofluorescence stainings, western blot, and flow cytometry to identify the various sources of osteopontin (OPN) expression in human and mouse glioblastoma. We implanted wild type GL261 glioblastoma cells, which do not express significant levels of OPN, into wild type and OPN(−/−) mice to investigate the role of microenvironment-derived OPN on glioblastoma progression. RESULTS: Our data indicate that GAMs are the predominant source of OPN in both human and mouse glioblastoma and express only the secreted form of OPN. Loss of microenvironment-derived OPN enhanced tumor progression. Staining by Ki67 and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling showed no difference in overall cell proliferation but a decreased apoptosis rate in tumors in OPN(−/−) mice. CD31 staining showed a significantly decreased number of microvessels in tumors in OPN(−/−) mice, accompanied by reduced coverage of vessels with platelet derived growth factor receptor β(+) pericytes. Flow cytometry analysis revealed a significant increase of CD11b(+)/CD45(low) microglia but not of CD11b(+)/CD45(high) macrophages/monocytes in tumors in OPN(−/−) mice. Sorted CD11b(+) cells from wild type and OPN(−/−) naïve brains and tumors did not show a significant difference in the expression pattern of activation marker genes. CONCLUSION: Our results show that in tested human and mouse glioblastoma samples, OPN is predominantly expressed and secreted by GAMs and that, in contrast to OPN expression in the tumor cells per se, loss of stroma-derived OPN creates a glioblastoma-promoting microenvironment.

// Yuan Pan 1 , Laura J. Smithson 1 , Yu Ma 1 , Dolores Hambardzumyan 2 , David H. Gutmann 1 1 Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA 2 Department of Oncology, Aflac Cancer and Blood Disorders Center, Emory University, GA, USA Correspondence to: David H. Gutmann, email: gutmannd@wustl.edu Keywords: chemokine, glioma, neurofibromin, mTOR, CD44 Received: October 24, 2016 Accepted: March 13, 2017 Published: March 23, 2017 ABSTRACT Glioblastoma (GBM) is the most common malignant brain tumor in adults, with a median survival of 15 months. These poor clinical outcomes have prompted the development of drugs that block neoplastic cancer cell growth; however, non-neoplastic cell-derived signals (chemokines and cytokines) in the tumor microenvironment may also represent viable treatment targets. One such chemokine, Ccl5, produced by low-grade tumor-associated microglia, is responsible for maintaining neurofibromatosis type 1 (NF1) mouse optic glioma growth in vivo . Since malignant gliomas may achieve partial independence from growth regulatory factors produced by non-neoplastic cells in the tumor microenvironment by producing the same cytokines secreted by the stromal cells in their low-grade counterparts, we tested the hypothesis that CCL5/CCL5-receptor signaling in glioblastoma creates an autocrine circuit important for high-grade glioma growth. Herein, we demonstrate that increased CCL5 expression was restricted to both human and mouse mesenchymal GBM (M-GBM), a molecular subtype characterized by NF1 loss. We further show that the NF1 protein, neurofibromin, negatively regulates Ccl5 expression through suppression of AKT/mTOR signaling. Consistent with its role as a glioblastoma growth regulator, Ccl5 knockdown in M-GBM cells reduces M-GBM cell survival in vitro , and increases mouse glioblastoma survival in vivo . Finally, we demonstrate that Ccl5 operates through an unconventional CCL5 receptor, CD44, to inhibit M-GBM apoptosis. Collectively, these findings reveal an NF1 -dependent CCL5-mediated pathway that regulates M-GBM cell survival, and support the concept that paracrine factors important for low-grade glioma growth can be usurped by high-grade tumors to create autocrine regulatory circuits that maintain malignant glioma survival.

Glioblastoma (GBM) is the most aggressive malignant primary brain tumor in adults, with a median survival of 14.6 months. Recent efforts have focused on identifying clinically relevant subgroups to improve our understanding of pathogenetic mechanisms and patient stratification. Concurrently, the role of immune cells in the tumor microenvironment has received increasing attention, especially T cells and tumor-associated macrophages (TAM). The latter are a mixed population of activated brain-resident microglia and infiltrating monocytes/monocyte-derived macrophages, both of which express ionized calcium-binding adapter molecule 1 (IBA1). This study investigated differences in immune cell subpopulations among distinct transcriptional subtypes of GBM. Human GBM samples were molecularly characterized and assigned to Proneural, Mesenchymal or Classical subtypes as defined by NanoString nCounter Technology. Subsequently, we performed and analyzed automated immunohistochemical stainings for TAM as well as specific T cell populations. The Mesenchymal subtype of GBM showed the highest presence of TAM, CD8+, CD3+ and FOXP3+ T cells, as compared to Proneural and Classical subtypes. High expression levels of the TAM-related gene AIF1, which encodes the TAM-specific protein IBA1, correlated with a worse prognosis in Proneural GBM, but conferred a survival benefit in Mesenchymal tumors. We used our data to construct a mathematical model that could reliably identify Mesenchymal GBM with high sensitivity using a combination of the aforementioned cell-specific IHC markers. In conclusion, we demonstrated that molecularly distinct GBM subtypes are characterized by profound differences in the composition of their immune microenvironment, which could potentially help to identify tumors amenable to immunotherapy.

Glioblastoma is the most common and most aggressive primary brain tumour. Standard of care consists of surgical resection followed by radiotherapy and concomitant and maintenance temozolomide (temozolomide/radiotherapy→temozolomide). Corticosteroids are commonly used perioperatively to control cerebral oedema and are frequently continued throughout subsequent treatment, notably radiotherapy, for amelioration of side effects. The effects of corticosteroids such as dexamethasone on cell growth in glioma models and on patient survival have remained controversial. We performed a retrospective analysis of glioblastoma patient cohorts to determine the prognostic role of steroid administration. A disease-relevant mouse model of glioblastoma was used to characterize the effects of dexamethasone on tumour cell proliferation and death, and to identify gene signatures associated with these effects. A murine anti-VEGFA antibody was used in parallel as an alternative for oedema control. We applied the dexamethasone-induced gene signature to The Cancer Genome Atlas glioblastoma dataset to explore the association of dexamethasone exposure with outcome. Mouse experiments were used to validate the effects of dexamethasone on survival in vivo Retrospective clinical analyses identified corticosteroid use during radiotherapy as an independent indicator of shorter survival in three independent patient cohorts. A dexamethasone-associated gene expression signature correlated with shorter survival in The Cancer Genome Atlas patient dataset. In glioma-bearing mice, dexamethasone pretreatment decreased tumour cell proliferation without affecting tumour cell viability, but reduced survival when combined with radiotherapy. Conversely, anti-VEGFA antibody decreased proliferation and increased tumour cell death, but did not affect survival when combined with radiotherapy. Clinical and mouse experimental data suggest that corticosteroids may decrease the effectiveness of treatment and shorten survival in glioblastoma. Dexamethasone-induced anti-proliferative effects may confer protection from radiotherapy- and chemotherapy-induced genotoxic stress. This study highlights the importance of identifying alternative agents such as vascular endothelial growth factor antagonists for managing oedema in glioblastoma patients. Beyond the established adverse effect profile of protracted corticosteroid use, this analysis substantiates the request for prudent and restricted use of corticosteroids in glioblastoma.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumour, being the leading cause of paediatric death caused by cancer. Despite all the advances made regarding effective therapies, the survival is dismal. Our lab has engineered the oncolytic virus Delta-24-ACT armed with the costimulatory ligand 41BBL in order to increase the antitumoral effect of the adenovirus. 41BB is a costimulatory receptor which promotes the expansion of activated T cells and the generation and maintenance of CD8 T memory cells. Therefore, we propose the use of Delta-24-ACT as a therapeutic approach for DIPG tumours. We observed that Delta-24-ACT is able to infect and replicate in NP53 and PDGFB-driven, two DIPG murine cell lines. Furthermore, 41BBL is expressed in the membranes of the infected cells and results with immunogenic cell death as shown by the different DAMPs. Injection of Delta-24-ACT in DIPG model was safe, showed no sign of toxicity and led to a significantly increase in the median overall survival, generating anti-glioma memory in long-term survivors. Mechanistic experiments, showed an increase of T cell infiltration (mainly CD8), decrease of proliferating cells and a reduction of the number of vessels in FFPE brain samples in the treated mice. We are currently performing nanostring analyses to assess the changes in the transcriptional immune phenotype of treated versus control mice. In summary, our data suggest that Delta-24-ACT is safe and induces a potent antitumor immune response in DIPG models mainly based in the activation of CD8 lymphocytes recruited by the viral activity.

The rise in population aging worldwide is causing an unparalleled increase in death from many cancers, including glioblastoma (GBM). Here, we have explored the impact of aging and rejuvenation on GBM tumorigenesis. Compared with neuro-inflammatory old GBM, young GBM displayed elevated neuronal/synaptic signaling via brain-derived neurotrophic factor (BDNF) and SLIT and NTRK like-family member 6 (SLITRK6), promoting favorable survival rates. These effects were attributed to the rise in nicotinamide adenine dinucleotide (NAD+) levels, as brain rejuvenation by parabiosis or administration of nicotinamide mononucleotide (NMN) in mice elicited a younger phenotype with activated neuronal/synaptic signaling and improved outcomes. Our data indicate that age-associated NAD+ loss contributes to the highly aggressive GBM by the shift from neuronal/synaptic activity to neuro-inflammation in the elderly brain. These findings have therapeutic and preventive implications in GBM and provide mechanistic insights into the exacerbation of GBM tumorigenesis by aging.

Mutation in isocitrate dehydrogenase (mIDH) is the main genetic lesion that defines clinical glioma subtypes and prognosis. This gain of function mutation is associated with the production of the oncometabolite, R-2-hydroxyglutarate, that inhibits α-ketoglutarate dependent enzymes such as TET2 and the Jumonji-C domain containing demethylases. The resultant epigenetic modifications elicit profound effects on the tumor biology and on the glioma-infiltrating immune cells. Here, we report that in genetically engineered mouse glioma models(1), IDH1 mutation caused an expansion of tumor infiltration granulocytes. Upon phenotypic and functional characterization, we uncovered that granulocytes in mIDH1 glioma express low level of immunosuppressive molecules and did not inhibit T-cell function. Single-cell sequencing revealed that these granulocytes are heterogeneous and composed of three distinct populations; neutrophils, pre-neutrophils, and a small fraction of immunosuppressive PMN-MDSCs. Moreover, primary human gliomas showed a higher cellular fraction exhibiting the PMN-MDSCs gene signature in wtIDH1 tumors than the mIDH1 tumors. The mechanism by which mIDH1 mediates non-immune suppressive granulocytes expansion involves epigenetic reprogramming which leads to enhanced expression of granulocyte colony-stimulating factor (G-CSF) in stem-like cells. High G-CSF gene expression is correlated with favorable patient outcome solely in LGG-astrocytoma with mIDH1. Thus, G-CSF represents a potential therapeutic that can be harnessed to improve immunotherapeutic responses in wild type IDH1 glioma patients.

Oncolytic viruses have become promising therapeutic candidates to treat gliomas. Our group has developed Delta-24-ACT, an oncolytic adenovirus armed with the positive costimulatory ligand 4-1BBL which is capable to trigger the activation of T cells and thereby increase their antitumor immune response. Here we evaluate the anti-glioma effect of Delta-24-ACT alone or in combination with an immune checkpoint inhibitor. We observed that Delta-24-ACT was able to infect and kill murine glioma (GL261-5 and CT-2A) and also human glioma cell lines (U87-MG and U251-MG), while maintaining its replication in the latter. Of importance, Delta-24-ACT infection resulted in 4-1BBL expression on the membrane of glioma cells. Moreover, this ligand was functional and was able to stimulate CD8 lymphocytes in vitro, suggesting the potential of Delta-24-ACT to trigger an effective immune response. Furthermore, in vivo Delta-24-ACT showed anti-tumour effect in two murine glioma models by significantly increasing the median survival time and leading to long-term survivors. Mechanistic studies demonstrated an increase of the T cell infiltration and the activation of different immune cell populations by flow cytometry and a decrease of proliferative cells and tumour vessels by immunohistochemistry on FFPE brain samples. Importantly, the infiltrating lymphocytes also showed signs of exhaustion increasing the amount of IL-10 and the expression of PD-1. To overcome this exhaustion we combined Delta-24-ACT with an anti-PD-1 antibody. Evaluation of this combination in vivo further increased the median survival time of treated tumor-bearing mice and resulted in 50% long-term survivors. Rechallenge studies with the same cell line showed that combination treatment effectively protected these animals of developing tumors and therefore, the acquisition of immune memory. In summary, our data demonstrated that Delta-24-ACT induces a potent anti-tumour effect in vitro and in vivo as a result of the recruitment of immune cell populations modulating the immunosuppressive tumour microenvironment of glioma.

A pathologic hallmark of glioblastoma (GBM), the most common and deadly primary brain tumor in adults, is pseudopalisading necrotic regions in which ischemic conditions are found. Ischemic microenvironments characterized by low oxygen, restricted glucose, and acidic pH due to poor blood supply occurs in both solid tumors and non-neoplastic tissue injury. Modeling these physiologically relevant ischemic conditions within glioblastoma in vitro, we identified chromodomain helicase DNA binding protein 7 (CHD7) as a novel ischemia-regulated gene. CHD7 is an epigenetic reader that controls neural stem cell maintenance and is mutated in CHARGE syndrome, a developmental disorder associated with cranial nerve abnormalities. CHD7 mRNA expression was consistently suppressed in multiple patient-derived xenograft brain tumor lines in response to ischemic conditions, and lower expression of CHD7 correlates with poor GBM patient survival. These microenvironment-mediated decreases in CHD7 protein and mRNA levels observed in glioblastoma were also mirrored in neural progenitor cells in vitro, and CHD7 levels were reduced in the perinecrotic niche of GBM patient and xenograft tissue sections. Genetic targeting of CHD7 increased angiogenesis in vitro in association with differential regulation of angiogenesis associated genes as determined in RNA sequencing analysis. Although targeting CHD7 in orthotopically implanted xenograft brain tumor models did not affect survival outcomes in mice, we observed marked differences in blood vessel architecture and organization, suggesting a major role for CHD7 in regulating vessel formation within these tumors. Together, our data provide insight into the molecular responses to ischemia that regulate angiogenesis.

Glioblastoma (GBM) remains the deadliest of all primary brain tumors with very few effective treatment options. Recently, we reported that high AXL expression is correlated with poor prognosis in GBM patients and demonstrated the therapeutic benefits of targeting AXL, a member of TAM receptor tyrosine kinase family using a novel small molecule inhibitor, BGB324 in immunocompetent mouse GBM models and xenografts of patient-derived glioma stem cells(GSCs). The promise of BGB324 in tumor burden management prompted us to develop a clinical trial with BGB324 as a single agent therapeutic with the goal to extend it as a combinatorial therapy in the future. Our surgical PK/PD clinical trial with BGB324 in recurrent GBM has been approved by the Brain Malignancy Steering Committee at the National Cancer Institute. Study treatment will consist of 2 cohorts of adult GBM patients, one (Group A) receiving the treatment pre-operatively and the other (Group B) receiving no treatment at all prior to surgery. First 5 patients recruited to Group A will be checked for the desired intra-tumoral drug concentration achieved to continue the trial. Group A will be supplemented by an additional 5 patients bringing the number to n=10 in each arm of the trial. Following surgical resection, patients in both cohorts will receive BGB324 daily in 21-day cycles. Treatment will be continued unless patients exhibit significant toxicity or substantial tumor progression. Our preclinical findings show the upregulation of AXL and its role in apoptosis induction in mesenchymal GBM as well as its association with MLK4, a serine threonine kinase we previously characterized as a mesenchymal GSC molecular target. Inhibition of phosphorylation of AXL and concomitant NF-kB activation in mesenchymal GSCs was found to be the nodal target of the drug action. An up-to-date information of the trial will be presented in detail.

While diffuse intrinsic pontine glioma (DIPG), the most common pediatric brainstem tumor, exhibits transient responsiveness to radiation (XRT), overall survival is

Glioblastoma (GBM) is a lethal disease with no effective therapies available. We previously observed upregulation of the TAM (Tyro-3, Axl, and Mer) receptor tyrosine kinase family member AXL in mesenchymal GBM and showed that knockdown of AXL induced apoptosis of mesenchymal, but not proneural, glioma sphere cultures (GSC). In this study, we report that BGB324, a novel small molecule inhibitor of AXL, prolongs the survival of immunocompromised mice bearing GSC-derived mesenchymal GBM-like tumors. We show that protein S (PROS1), a known ligand of other TAM receptors, was secreted by tumor-associated macrophages/microglia and subsequently physically associated with and activated AXL in mesenchymal GSC. PROS1-driven phosphorylation of AXL (pAXL) induced NFκB activation in mesenchymal GSC, which was inhibited by BGB324 treatment. We also found that treatment of GSC-derived mouse GBM tumors with nivolumab, a blocking antibody against the immune checkpoint protein PD-1, increased intratumoral macrophages/microglia and activation of AXL. Combinatorial therapy with nivolumab plus BGB324 effectively prolonged the survival of mice bearing GBM tumors. Clinically, expression of AXL or PROS1 was associated with poor prognosis for patients with GBM. Our results suggest that the PROS1–AXL pathway regulates intrinsic mesenchymal signaling and the extrinsic immune microenvironment, contributing to the growth of aggressive GBM tumors. Significance: These findings suggest that development of combination treatments of AXL and immune checkpoint inhibitors may provide benefit to patients with GBM. Cancer Res; 78(11); 3002–13. ©2018 AACR.

Background Depending on the level, differentiation state, and tumor stage, reactive nitrogen and oxygen species inhibit or increase cancer growth and tumor initiating cell maintenance. The rate-limiting enzyme in a pathway that can regulate reactive species production but has not been thoroughly investigated in glioblastoma (GBM; grade IV astrocytoma) is guanosine triphosphate (GTP) cyclohydrolase 1 (GCH1). We sought to define the role of GCH1 in the regulation of GBM growth and brain tumor initiating cell (BTIC) maintenance. Methods We examined GCH1 mRNA and protein expression in patient-derived xenografts, clinical samples, and glioma gene expression datasets. GCH1 levels were modulated using lentiviral expression systems, and effects on cell growth, self-renewal, reactive species production, and survival in orthotopic patient-derived xenograft models were determined. Results GCH1 was expressed in GBMs with elevated but not exclusive RNA and protein levels in BTICs in comparison to non-BTICs. Overexpression of GCH1 in GBM cells increased cell growth in vitro and decreased survival in an intracranial GBM mouse model. In converse experiments, GCH1 knockdown with short hairpin RNA led to GBM cell growth inhibition and reduced self-renewal in association with decreased CD44 expression. GCH1 was critical for controlling reactive species balance, including suppressing reactive oxygen species production, which mediated GCH1 cell growth effects. In silico analyses demonstrated that higher GCH1 levels in glioma patients correlate with higher glioma grade, recurrence, and worse survival. Conclusions GCH1 expression in established GBMs is pro-tumorigenic, causing increased growth due, in part, to promotion of BTIC maintenance and suppression of reactive oxygen species.

// Xi Feng 1 , Frank Szulzewsky 2,* , Alexan Yerevanian 1,3,* , Zhihong Chen 1 , David Heinzmann 1,4 , Rikke Darling Rasmussen 1 , Virginia Alvarez-Garcia 1 , Yeonghwan Kim 5 , Bingcheng Wang 6 , Ilaria Tamagno 1 , Hao Zhou 7 , Xiaoxia Li 7 , Helmut Kettenmann 2 , Richard M. Ransohoff 1,8 and Dolores Hambardzumyan 1 1 Department of Neurosciences at Cleveland Clinic, Cleveland, Ohio, USA 2 Cellular Neurosciences, Max Delbruck Center for Molecular Medicine, Berlin, Germany 3 Case Western Reserve University School of Medicine, Cleveland, Ohio, USA 4 Department of Cardiology at Tubingen University School of Medicine, Tubingen, Germany 5 Department of Stem Cell Biology and Regenerative Medicine, Cleveland, Ohio, USA 6 Rammelkamp Center for Research, MetroHealth Center, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA 7 Department of Immunology at Cleveland Clinic, Cleveland, Ohio, USA 8 Neuroinflammation Research Center, Cleveland Clinic, Cleveland, Ohio, USA * These authors have contributed equally to this work Correspondence to: Dolores Hambardzumyan, email: // Keywords : CX3CR1/CX3CL1 signaling, glioblastoma, microglia, monocyte Received : January 19, 2015 Accepted : March 10, 2015 Published : March 30, 2015 Abstract The most abundant populations of non-neoplastic cells in the glioblastoma (GBM) microenvironment are resident microglia, macrophages and infiltrating monocytes from the blood circulation. The mechanisms by which monocytes infiltrate into GBM, their fate following infiltration, and their role in GBM growth are not known. Here we tested the hypothesis that loss of the fractalkine receptor CX3CR1 in microglia and monocytes would affect gliomagenesis. Deletion of Cx3cr1 from the microenvironment resulted in increased tumor incidence and shorter survival times in glioma-bearing mice. Loss of Cx3cr1 did not affect accumulation of microglia/macrophages in peri-tumoral areas, but instead indirectly promoted the trafficking of CD11b + CD45 hi CX3CR1 low Ly-6C hi Ly-6G - F4/80 -/low circulating inflammatory monocytes into the CNS, resulting in their increased accumulation in the perivascular area. Cx3cr1-deficient microglia/macrophages and monocytes demonstrated upregulation of IL1β expression that was inversely proportional to Cx3cr1 gene dosage. The Proneural subgroup of the TCGA GBM patient dataset with high IL1β expression showed shorter survival compared to patients with low IL1β. IL1β promoted tumor growth and increased the cancer stem cell phenotype in murine and human Proneural glioma stem cells (GSCs). IL1β activated the p38 MAPK signaling pathway and expression of monocyte chemoattractant protein (MCP-1/CCL2) by tumor cells. Loss of Cx3cr1 in microglia in a monocyte-free environment had no impact on tumor growth and did not alter microglial migration. These data suggest that enhancing signaling to CX3CR1 or inhibiting IL1β signaling in intra-tumoral macrophages can be considered as potential strategies to decrease the tumor-promoting effects of monocytes in Proneural GBM.

Glioblastoma (GBM) is a lethal disease without effective therapies. Previously we reported that the TAM receptor tyrosine kinase family member AXL is upregulated in mesenchymal GBM and its knockdown induces apoptosis of mesenchymal but not proneural glioma sphere cultures (GSCs). In this study, we report that BGB324, a novel small molecule inhibitor for AXL, prolongs the survival of immunocompromised mice bearing mesenchymal GSC-derived GBM-like intracranial tumors by inhibition of AXL activation in tumor cells. Mechanistically, we identified that protein S (PROS1), known as a ligand for other TAM receptors, is secreted by tumor-associated macrophages, thereby physically associates and activates AXL in mesenchymal GSCs. The PROS1-driven phosphorylation of AXL resulted in the NF-kB activation in mesenchymal GSCs and this activation was eliminated by BGB324 treatment. In addition, BGB324 downregulated the immune checkpoint protein PD-L1 both in vitro and in vivo. Combination of BGB324 with Nivolumab - the neutralizing antibody for PD-1 (a receptor for PD-L1) was effective to prolong the survival of immunocompetent mice bearing syngeneic GBM models. Clinically, both AXL and PROS1 expression indicated poorer prognosis of GBM patients. These data suggest that the PROS1/AXL pathway regulates intrinsic mesenchymal signaling as well as extrinsic immune microenvironment, contributing to the growth of aggressive GBM tumors. To translate the pre-clinical data to human GBM patients, we designed a surgical PK/PD clinical trial with BGB324 for recurrent GBM and this trial is currently under review by the Brain Malignancy Steering Committee in the National Cancer Institute.

Sir, We appreciate the interest of Drs Clement and Champ in our article, which provided overall compelling evidence for a negative impact of corticosteroid medication on survival in patients with glioblastoma. …

In glioblastoma (GBM), tumor-associated macrophages (TAM) represent up to one half of the cells of the tumor mass, including both infiltrating macrophages and resident brain microglia. In an effort to delineate the temporal and spatial dynamics of TAM composition during gliomagenesis, we used genetically engineered and GL261-induced mouse models in combination with CX3CR1GFP/WT;CCR2RFP/WT double knock-in mice. Using this approach, we demonstrated that CX3CR1LoCCR2Hi monocytes were recruited to the GBM, where they transitioned to CX3CR1HiCCR2Lo macrophages and CX3CR1HiCCR2− microglia-like cells. Infiltrating macrophages/monocytes constituted approximately 85% of the total TAM population, with resident microglia accounting for the approximately 15% remaining. Bone marrow–derived infiltrating macrophages/monocytes were recruited to the tumor early during GBM initiation, where they localized preferentially to perivascular areas. In contrast, resident microglia were localized mainly to peritumoral regions. RNA-sequencing analyses revealed differential gene expression patterns unique to infiltrating and resident cells, suggesting unique functions for each TAM population. Notably, limiting monocyte infiltration via genetic Ccl2 reduction prolonged the survival of tumor-bearing mice. Our findings illuminate the unique composition and functions of infiltrating and resident myeloid cells in GBM, establishing a rationale to target infiltrating cells in this neoplasm. Cancer Res; 77(9); 2266–78. ©2017 AACR.

Purpose: Bevacizumab, a humanized monoclonal antibody to VEGF, is used routinely in the treatment of patients with recurrent glioblastoma (GBM). However, very little is known regarding the effects of bevacizumab on the cells in the perivascular space in tumors. Experimental Design: Established orthotopic xenograft and syngeneic models of GBM were used to determine entry of monoclonal anti-VEGF-A into, and uptake by cells in, the perivascular space. Based on the results, we examined CD133+ cells derived from GBM tumors in vitro. Bevacizumab internalization, trafficking, and effects on cell survival were analyzed using multilabel confocal microscopy, immunoblotting, and cytotoxicity assays in the presence/absence of inhibitors. Results: In the GBM mouse models, administered anti-mouse-VEGF-A entered the perivascular tumor niche and was internalized by Sox2+/CD44+ tumor cells. In the perivascular tumor cells, bevacizumab was detected in the recycling compartment or the lysosomes, and increased autophagy was found. Bevacizumab was internalized rapidly by CD133+/Sox2+-GBM cells in vitro through macropinocytosis with a fraction being trafficked to a recycling compartment, independent of FcRn, and a fraction to lysosomes. Bevacizumab treatment of CD133+ GBM cells depleted VEGF-A and induced autophagy thereby improving cell survival. An inhibitor of lysosomal acidification decreased bevacizumab-induced autophagy and increased cell death. Inhibition of macropinocytosis increased cell death, suggesting macropinocytosis of bevacizumab promotes CD133+ cell survival. Conclusions: We demonstrate that bevacizumab is internalized by Sox2+/CD44+-GBM tumor cells residing in the perivascular tumor niche. Macropinocytosis of bevacizumab and trafficking to the lysosomes promotes CD133+ cell survival, as does the autophagy induced by bevacizumab depletion of VEGF-A. Clin Cancer Res; 23(22); 7059–71. ©2017 AACR.

Glioblastoma is the most common and aggressive primary brain tumor characterized by a dismal prognosis despite aggressive treatment regimens. A hallmark of glioblastoma is aberrant angiogenesis that results in disruption of the blood-brain barrier. This drives glioblastoma-associated cerebral edema caused by leakage of serum from the blood into the brain. Typically, edema is managed by the corticosteroid dexamethasone which is known to normalize the blood brain barrier. Various groups have also demonstrated beneficial effects following normalization of the blood-brain barrier with anti-angiogenic therapies such as cediranib and bevacizumab. Interleukin 1 (IL-1) signaling inhibition, however, has not been investigated in this context despite evidence that it modulates blood-brain barrier integrity in other neuroinflammatory conditions. In this work, we demonstrate that IL-1 signaling does not impact the production of VEGF by bone marrow-derived macrophages and microglia in vitro. Moreover, we demonstrate no effect of dexamethasone treatment on VEGF levels in murine PDGFB-driven glioblastoma generated with RCAS/tv-a technology in vivo. This suggests that the restoration of blood-brain barrier integrity by dexamethasone is VEGF-independent. Dexamethasone is shown here, however, to downregulate both IL-1α and IL-1β expression in vivo and in vitro. Genetic ablation of IL-1β and subsequent MRI analysis is shown to reduce formation of edema in vivo, allowing mice to survive with larger tumors than wildtype controls. Serial sectioning of the tumor, and histological reconstruction of the total volume post-euthanasia confirms these results. Genetic ablation of IL-1R1 in vivo is shown to reduce blood-brain barrier permeability in a Hoechst dye-based vessel leakage assay. Future experiments will elucidate if these effects can be attributed to altered angiogenesis or regulation of endothelial cell junction molecules. Regardless, this work outlines IL-1 signaling as a promising therapeutic target to modulate blood-brain barrier integrity and serve as an alternative to dexamethasone for the treatment of glioblastoma-associated cerebral edema.

The GBM microenvironment is composed of numerous non-neoplastic cells, among which tumor-associated myeloid cells (TAMs) are the most abundant. TAMs are constituted of several different cell types, including brain microglia and bone-marrow-derived infiltrating cells, which can be further divided into inflammatory monocytes, monocyte-derived macrophages, and polymorphnuclear neutrophils. These myeloid cells are believed to promote tumor growth and immune evasion by expressing immune checkpoint proteins. Remarkable progress has been made in treating lung cancer and melanoma by using immune-checkpoint inhibitors; however, mixed results were obtained for its application in GBM. To examine how molecular-subtypes of GBM influence the composition and immunosuppression of tumor-associated myeloid cells, we used the RCAS/tv-a system, a somatic cell-specific gene transfer system, to generate de novo murine GBM (mGBM). By manipulating different known human oncogenic-driver mutations such as overexpressing PDGFB or EGFRVIII, or silencing NF1, we generated Proneural (PN), Classical (CL) and Mesenchymal (MES) GBMs respectively, which phenotypically resemble their human counterparts. We found that the majority (80%) of tumor-infiltrating myeloid cells in PN GBM are bone-marrow derived macrophages, whereas this number is significantly less in CL and MES mGBM. Interestingly, unique to MES, there is a significant presence (16%) of neutrophils in these tumors than in other subtypes (4%). Further characterization for subtype-specific expression of PD-L1, a druggable immune checkpoint molecule, by flow cytometry shows that MES has the highest expression of PD-L1 (Mean Fluorescent Intensity=13,000 ± 3,000), whereas the expression is lower (MFI=8,100 ± 2,000) in PN and lowest (MFI=5,900 ± 2,000) in CL. The current effort is devoted to examining how myeloid cells isolated from different subtypes can differentially suppress T cell functions; and how mice carry these subtypes respond to anti-PD-L1 therapy. Our findings illuminate the unique composition and functions of tumor-associated myeloid cells in different GBM subtypes, establishing a rationale to target these cells in this heterogeneous neoplasm.

Anti-angiogenic therapy of glioblastoma with bevacizumab, a vascular endothelial growth factor-A (VEGFA) blocking antibody, may accelerate tumor cell invasion and induce alternative angiogenic pathways. We investigated the roles of the pro-angiogenic receptor APLNR and its cognate ligand apelin in VEGFA/VEGFR2 anti-angiogenic therapy against distinct subtypes of glioblastoma. In proneural glioblastoma, apelin levels were downregulated by VEGFA or VEGFR2 blockade by use of bevacizumab or ramucirumab, respectively. A central role for apelin/APLNR in controlling glioblastoma vascularization was corroborated in a serial implantation model of the angiogenic switch that occurs in human glioblastoma. Apelin and APLNR are broadly expressed in human glioblastoma, and knockdown or knockout of APLN in orthotopic models of proneural or classical glioblastoma subtypes massively reduced glioblastoma vascularization as compared with controls. What is more, direct infusion of the bioactive peptide apelin-13 was able to rescue this vascular loss-of-function phenotype, demonstrating the specific control of tumor vascularization by apelin/APLNR signaling. While high levels of apelin correlated with reduced tumor cell invasiveness, the reduction in apelin expression led to accelerated glioblastoma cell invasion. Analysis of stereotactic glioblastoma biopsies from patients as well as from in vitro and in vivo experiments revealed increased dissemination of APLNR-positive tumor cells when apelin levels were reduced. Most interestingly, application of apelin-F13A, a mutant APLNR ligand, blocked both tumor angiogenesis and glioblastoma cell invasion. Furthermore, co-targeting VEGFR2 and APLNR synergistically improved survival of mice bearing proneural glioblastoma. In summary, we show that apelin/APLNR signaling controls glioblastoma angiogenesis and invasion directly, and that both pathological features are blunted by apelin-F13A. We suggest that apelin-F13A can improve the efficiency and reduce the side effects of established anti-angiogenic treatments for distinct glioblastoma subtypes.

Pediatric high-grade gliomas (pHGG) account for the most cancer-related deaths in children, as there are no effective therapies available. It is known tumor associated macrophages (TAM) can make up 30–40% of the total tumor cell mass in adult high-grade gliomas, promoting tumor growth and immune evasion. This raises the question of whether pHGGs possess a distinct constituency of TAMs due to their unique genetic and epigenetic landscapes. To uncover the composition and behavior of TAMs in pHGG we utilize RCAS/tva, a somatic cell-type specific gene transfer system which allows us to recapitulate all major subtypes of pHGG in newborn immunocompetent mice, including histone wild-type and histone-mutant tumors. We combine RCAS-H3.3K27M, RCAS-H3.3G34R/V, or RCAS-H3.3WT along with their driver mutations such as RCAS-shp53 and RCAS-PDGFA or RCAS-PDGFB. These tumors are induced in Nestin-positive cells, each in their respective locations found in the human population. Tumors driven by PDGFB have a significantly lower median survival compared to PDGFA-driven tumors and have increased infiltration of lymphocytes and TAMs, specifically inflammatory monocytes. In vitro bone marrow derived monocyte and microglial cultures demonstrate the BMDM population is most responsible for the production of inflammatory chemokines and angiogenic factors in the tumor microenvironment. We performed histological analyses on over 40 human patient samples to determine the role of the stromal population in TAM infiltration. Matched human samples were also utilized for pan-cancer immune profiling with NanoString to further characterize the innate and adaptive immune microenvironments. These analyses indicate DIPG/K27M tumors have a higher immune cell infiltrate compared to G34R/V and histone wildtype tumors. Further, we observe higher infiltration of T-cell populations in pHGGs compared to adult HGGs, suggesting these tumors may be amenable to immunotherapy despite being considered “immune cold.” These studies provide the critical foundation needed for the development of novel therapeutics targeting these tumors.

The GBM microenvironment is composed of numerous non-neoplastic cells, among which tumor-associated myeloid cells (TAMs) are the most abundant. TAMs are constituted of several different cell types, including brain microglia and bone-marrow-derived infiltrating cells, which can be further divided into inflammatory monocytes, monocyte-derived macrophages, and polymorphnuclear neutrophils. These myeloid cells are believed to promote tumor growth and immune evasion by expressing immune checkpoint proteins. Remarkable progress has been made in treating lung cancer and melanoma by using immune-checkpoint inhibitors; however, mixed results were obtained for its application in GBM. To examine how molecular-subtypes of GBM influence the composition and immunosuppression of tumor-associated myeloid cells, we used the RCAS_tv-a system, a somatic cell-specific gene transfer system, to generate de novo murine GBM (mGBM). By manipulating different known human oncogenic-driver mutations such as overexpressing PDGFB or EGFRVIII, or silencing NF1, we generated Proneural (PN), Classical (CL) and Mesenchymal (MES) GBMs respectively, which phenotypically resemble their human counterparts. We found that the majority (80%) of tumor-infiltrating myeloid cells in PN GBM are bone-marrow derived macrophages, whereas this number is significantly less in CL and MES mGBM. Interestingly, unique to MES, there is a significant presence (16%) of neutrophils in these tumors than in other subtypes (4%). Further characterization for subtype-specific expression of PD-L1, a druggable immune checkpoint molecule, by flow cytometry shows that MES has the highest expression of PD-L1), whereas the expression is lower in PN and CL subtypes. Treating GBM-bearing mice with anti-PDL1 antibodies did not prolong their survival. Concurrent treatment of irradiation and anti-PDL1 antibody did not confer significant survival advantage either compared to controls. These results reflect the unsuccessful trails of recent human clinical studies; however, it also emphasizes the importance of understanding the mechanism of resistance in GBM immunotherapy before effective immune treatment can be established. Our immunocompetent mouse models provide indispensable tools for these studies. Note: This abstract was not presented at the meeting. Citation Format: Zhihong Chen, Cameron J. Hurting, Ben Gabanic, Jyothi Murukuti, Dolores Hambardzumyan. Genetic driver-mutations define composition and properties of tumor-associated myeloid cells in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1100.

Pediatric high-grade gliomas (pHGG) drastically differ from their adult counterparts in terms of genetic and epigenetic alterations, suggesting they may also differ in the constituency of their tumor microenvironment. It is known tumor associated macrophages (TAMs) can constitute up to 30–40% of the total tumor cell mass in adult high-grade gliomas, yet little is known for pHGGs. This raises the question of whether pHGGs possess a distinct constituency of TAMs due to their unique genetic and epigenetic landscapes. To uncover the composition and behavior of TAMs in pHGG we utilize RCAS/tva, a somatic cell-type specific gene transfer system in which we administer RCAS-PDGFA or RCAS-PDGFB to Nestin-positive cells in the brain of immunocompetent Cdkn2a knockout mice. We observe PDGFB-driven tumors have a significantly lower median survival compared to PDGFA-driven tumors. PDGFB tumors also have increased infiltration of TAMs, made evident by immunohistochemical staining for IBA1. Flow cytometry indicates the increased number of TAMs is due to bone-marrow derived inflammatory monocytes, not the microglial population. Unsupervised clustering and principle component analysis indicates these tumors possess distinct expression profiles. Several chemokines and receptors are highly expressed in PDGFB tumors such as Ccl2, Ccl3, and Ccl7, suggesting chemokine mediated infiltration of TAMs may be occurring. To study PDGF signaling and TAM infiltration in human pHGGs, we stained over 40 tumors for IBA1, PDGF-receptors, and PDGF-ligands. There is a positive correlation between PDGFRβ, PDGFB, and CD31 staining with IBA1, suggesting a stromal role exists for the infiltration of TAMs. Current in vivo studies are being conducted to determine if TAM infiltration can be inhibited by genetically or pharmacologically targeting chemokine signaling pathways. In vivo and in vitro studies are also being performed to determine which cell types are the main sources of chemokines and inflammatory molecules are in the microenvironment.

Glioblastomas (GBM) are one of the most recalcitrant brain tumors because of their aggressive invasive growth and resistance to therapy. They are highly heterogeneous malignancies at both the molecular and histological levels. Specific histological hallmarks including pseudopalisading necrosis and microvascular proliferation distinguish GBM from lower-grade gliomas, and make GBM one of the most hypoxic as well as angiogenic tumors. These microanatomical compartments present specific niches within the tumor microenvironment that regulate metabolic needs, immune surveillance, survival, invasion as well as cancer stem cell maintenance. Here we review features and functions of the distinct GBM niches, detail the different cell constituents and the functional status of the vasculature, and discuss prospects of therapeutically targeting GBM niche constituents.

Within high-grade gliomas, the precise identities and functional roles of stem-like cells remain unclear. In the normal neurogenic niche, ID (Inhibitor of DNA-binding) genes maintain self-renewal and multipotency of adult neural stem cells. Using PDGF- and KRAS-driven murine models of gliomagenesis, we show that high Id1 expression (Id1(high)) identifies tumor cells with high self-renewal capacity, while low Id1 expression (Id1(low)) identifies tumor cells with proliferative potential but limited self-renewal capacity. Surprisingly, Id1(low) cells generate tumors more rapidly and with higher penetrance than Id1(high) cells. Further, eliminating tumor cell self-renewal through deletion of Id1 has modest effects on animal survival, while knockdown of Olig2 within Id1(low) cells has a significant survival benefit, underscoring the importance of non-self-renewing lineages in disease progression.

Medulloblastoma (MB), the most common pediatric brain tumor, has a 70% survival rate but standard treatments often lead to devastating life-long side effects, and recurrence is fatal. MB was classified based on molecular and genetic profiles and resulted in four distinct subgroups. The most common subclass of MB is SHH, which accounts for approximately 30% of cases. This class has been successfully modeled in vivo in murine models, which closely recapitulate human disease, providing a convenient and relevant model system for analyzing the SHH MB subclass in vivo. To better understand the mechanisms of tumor growth and recurrence, recent attention has been focused on determining the composition and role of non-tumor cells comprising the tumor microenvironment (TME). Tumor-associated macrophages (TAM) are a key component of the TME that could have two opposite effects on tumor. TAMs can help tumors to evade the immune system by suppressing other immune cell functions, and contribute to tumor growth by promoting angiogenesis. On the other hand, they could suppress tumor growth and delay tumor development. Recently, it was reported that of the subgroups, human SHH MB has the greatest number of TAMs, as well as increased expression of macrophage-associated genes. However, to date, there are no studies addressing the functional role of TAMs in SHH MB. In this work, we demonstrate a functional role of TAMs in both in vitro and in vivo models. We show that reduction of macrophage numbers leads to increased animal mortality in a murine model of SHH MB. Further investigation of the TME and TAMs in MB has the potential to elucidate immune system involvement in the disease and lead to development of novel treatment options.

Glioblastoma-associated microglia and macrophages/monocytes (GAMs) constitute the most abundant type of immune cells in glioblastoma. To date there is still insufficient knowledge about the expression profiles and activation states of these cells. We have previously performed RNA-Seq and microarray on CD11b+ freshly isolated human and mouse GAMs. Here we combine this data with analysis from published whole tumor TCGA data to identify new myeloid-derived transcripts in glioblastoma. We first re-analyzed our previously published human GAMs RNA-Seq dataset for genes that are at least 4-fold up regulated in comparison to naïve microglia and displayed an absolute log2 expression greater than 7. Subsequently, we queried the TCGA glioblastoma dataset to identify genes that showed a high correlation to the myeloid markers CD68, CD14, and AIF1. We found that the expression of IBSP, FCGBP, MARCO, RNASE1, CTSL, SPP1, and GPNMB significantly correlated with the expression of the different myeloid markers. In addition, the correlation was the highest in the proneural subtype samples, which have previously been shown to harbor the highest percentage of myeloid cells. Three of these genes (Spp1, Ctsl, and Gpnmb) were also highly expressed in murine GAMs. Finally, we performed immunofluorescence stainings for CTSL and GPNMB in human GBM samples and PDGF-driven mouse tumors and could show that both proteins were highly abundant in IBA1+ myeloid cells in both human and mouse samples. Conclusively, we have identified several new myeloid-specific genes in glioblastoma that might pose potential new targets. Subsequent studies will be necessary to evaluate the role of these genes in glioblastoma.

Pediatric high-grade gliomas (pHGG) account for the most cancer-related deaths in children under the age of 19 years old. Recent advances have demonstrated that pHGGs drastically differ from their adult counterparts in terms of genetic and epigenetic alterations, suggesting they may also differ in the constituency of their tumor microenvironment. It is now known tumor associated macrophages (TAMs) can make up 30–40% of the total tumor cell mass in adult high-grade gliomas and play important roles in immune suppression and tumor promotion. This raises the question of whether pHGGs possess a distinct constituency of TAMs due to their unique genetic and epigenetic landscapes. To uncover the composition and behavior of TAMs in pHGG we utilize RCAS/tva, a somatic cell-type specific gene transfer system in which we administer RCAS-PDGFA or RCAS-PDGFB to simulate PDGF receptor activation in immune-competent new-born mice. We found that PDGFB-driven tumors have a significantly lower median survival compared to PDGFA-driven tumors. PDGFB-driven tumors also have increased infiltration of TAMs, made evident by immunohistochemical staining for IBA1. Flow cytometry analysis indicates the increased number of TAMs is due to an increased inflammatory monocyte population, but not due to the microglial population. We hypothesize these findings are attributable to the additional activation of the stromal population by PDGFB, as it can activate both PDGFRα and PDGFRβ while PDGFA only activates PDGFRα. Gene expression analysis indicates that several chemokines, chemokine receptors, macrophage, and immune markers are differentially expressed between PDGFB and -A tumor types. To establish correlations between PDGF signaling and TAM infiltration in human pediatric tumor samples, we stained 37 tumors for IBA1, PDGF-receptors, and PDGF-ligands. A positive correlation exists between high PDGFRb and PDGFB staining with high IBA1. Further studies will be done to discern the effects of increased TAM infiltration and the mechanisms driving enhanced tumor malignancy.

Diffuse intrinsic pontine glioma (DIPG), the most common pediatric brainstem tumor, is associated with a dismal prognosis with almost no survivors within two years of diagnosis. It is unamenable to surgical resection, is not responsive to conventional chemotherapy, and responds only temporarily to radiation therapy (XRT). Recent publications identified carboxy (C)-terminal truncating mutations of PPM1D, which encodes the protein WIP1, in up to 25% of DIPGs. WIP1 is a serine/threonine protein phosphatase that plays an important role in DNA damage response (DDR). Prior publications show that most, but not all, PPM1D C-terminal mutants function in a dominant negative fashion to promote tumor growth in cancer models. We hypothesized that PPM1D mutation promotes DIPG growth, and WIP1 inhibition suppresses tumor proliferation. We further hypothesized that, due to its key role in DDR, WIP1 inhibition enhances the antitumor effects of XRT. Using Sanger sequencing, we identified de novo mutation of PPM1D in the C-terminus of multiple patient-derived DIPG cell lines. We also stably introduced clinically relevant PPM1D mutations, which we identified in DIPGs resected from patients, into a PPM1D wild-type DIPG cell line, DIPG VI, as well as into DIPG cells derived from a DIPG mouse model. Using trypan blue exclusion, bioluminescence, and immunofluorescence detection of markers of proliferation and cell death, in the presence or absence of PPM1D knockdown or pharmacologic WIP1 inhibition, and/or following XRT exposure, we measured proliferation of DIPG VI, DIPG VI L513X, a cell line with stable expression of a mutant PPM1D, and DIPG7, a cell line with a de novo PPM1D mutation. We also assayed proliferation in organotypic brain slice cultures derived from symptomatic immunocompromised mice orthotopically xenografted with DIPG cells and of DIPG cells embedded within the brainstem of organotypic brain slices derived from immunocompetent mice. Finally, we orthotopically xenografted DIPG cells into the early post-natal (P0-2) brainstem of immunodeficient mice, treated mice with vehicle or with a small-molecule WIP1 inhibitor, and measured effects on survival, as well as proliferation in tumor cells post-necropsy. Under cell culture conditions, expression of PPM1D mutants promoted tumor cell proliferation in wild-type PPM1D murine and human DIPG cells. Conversely, PPM1D knockdown significantly suppressed DIPG cell proliferation, especially in PPM1D mutated DIPG cells. Similarly, treatment with a small-molecule WIP1 inhibitor suppressed the growth of PPM1D-mutated, but not PPM1D wild-type DIPG cells, in neurosphere cultures, on immunocompetent organotypic brain slices, and on organotypic brain slices derived from symptomatic, orthotopic, xenografted mice. WIP1 inhibition had similar effects on growth inhibition to XRT alone and augmented the antiproliferative and proapoptotic effects of XRT in vitro. Mice bearing an orthotopic xenograft of PPM1D-mutated human DIPG cells, DIPG7, died of disease earlier than mice xenografted with PPM1D wild-type human DIPG cells, DIPG VI. Treatment of mice bearing orthotopically, xenografted DIPG7 cells with a WIP1 inhibitor via a continuous 14-day infusion into the lateral ventricles resulted in a significant reduction in proliferation of tumor cells. In summary, our results show that C-terminal mutated PPM1D is oncogenic in DIPG, promoting proliferation and reduced survival in vivo. WIP1 inhibition suppresses growth and increases the radiosensitivity of PPM1D-mutated DIPG. These results suggest that WIP1 is a druggable target alone and in combination with XRT in DIPG. Further studies will examine the PPM1D-mediated mechanisms of tumorigenesis and treatment responsiveness in DIPG in order to develop clinically viable treatment paradigms that target PPM1D-mutated tumors. Citation Format: Mwangala P. Akamandisa, Kai Nie, Jing Wen, Rita Nahta, Dolores Hambardzumyan, Robert C. Castellino. Mechanisms of PPM1D-mediated tumorigenesis in diffuse intrinsic pontine glioma (DIPG) [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B05.

Glioblastoma (GBM), or grade IV astrocytoma, is a deadly disease due in part to the high degree of intratumoral heterogeneity that contributes to treatment failures. Previous studies have shown the importance of reactive species balances, partially controlled by the coupling of nitric oxide synthases (NOS) with their cofactor, in maintenance of glioma stem cell (GSC) phenotype as well as survival of cancer cells in general. In this study, we investigated the roles of GTP cyclohydrolase 1 (GCH1), which is the first and rate-limiting enzyme of the pathway producing of NOS cofactor producing pathway, in GBM stem cell phenotypes via redox balances. We found that GCH1 RNA and protein expression were increased in GSCs in comparison to non-GSCs, but that GCH1 was not exclusive to the GSC fraction. Indeed, GCH1 was elevated in GBM in comparison to normal brain. Overexpression of GCH1 in GBM cells increased cell growth in vitro and neurosphere-forming capability and decreased survival in an intracranial GBM mouse model. In contrast, GCH1 knockdown with short hairpin RNA in GBM cells led to growth inhibition in vitro as well as increased survival in animal models. GCH1 increased CD44 expression and was upregulated in the detrimental mesenchymal GBM subtype in which CD44 served as a marker. Mechanistically, we found that the expression of GCH1 increased BH4 production, as well as augmented multiple antioxidant pathways, including the expression of PARK7, was critical for controlling reactive species balance, including suppressing reactive oxygen species production. In silico analyses demonstrated that higher GCH1 levels in glioma patients correlate with higher glioma grade, recurrence and worse survival. Together, our data suggest that upregulation of GCH1 in GSCs promotes tumor maintenance and is a key regulator of reactive oxygen species in GBM, and GCH1 pathway is a potential target for therapy. Citation Format: Anh N. Tran, Kiera Walker, David G. Harrison, Wei Chen, James Mobley, Lauren Hocevar, James R. Hackney, Randee Sedaka, Jennifer Pollock, Matthew S. Goldberg, Dolores Hambardzumyan, Sara J. Cooper, G Yancey Gillespie, Anita B. Hjelmeland. Glioblastoma, cancer stem cells, and reactive species balances: A case for GTP cyclohydrolase 1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 163.

Brainstem gliomas (BSG) are a rare group of central nervous system tumors that arise mostly in children and usually portend a particularly poor prognosis. We report the development of a genetically engineered mouse model of BSG using the RCAS/tv-a system and its implementation in preclinical trials. Using immunohistochemistry, we found that platelet-derived growth factor (PDGF) receptor α is overexpressed in 67% of pediatric BSGs. Based on this observation, we induced low-grade BSGs by overexpressing PDGF-B in the posterior fossa of neonatal nestin tv-a mice. To generate high-grade BSGs, we overexpressed PDGF-B in combination with Ink4a-ARF loss, given that this locus is commonly lost in high-grade pediatric BSGs. We show that the likely cells of origin for these mouse BSGs exist on the floor of the fourth ventricle and cerebral aqueduct. Irradiation of these high-grade BSGs shows that although single doses of 2, 6, and 10 Gy significantly increased the percent of terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL)–positive nuclei, only 6 and 10 Gy significantly induce cell cycle arrest. Perifosine, an inhibitor of AKT signaling, significantly induced TUNEL-positive nuclei in this high-grade BSG model, but in combination with 10 Gy, it did not significantly increase the percent of TUNEL-positive nuclei relative to 10 Gy alone at 6, 24, and 72 hours. Survival analysis showed that a single dose of 10 Gy significantly prolonged survival by 27% (P = 0.0002) but perifosine did not (P = 0.92). Perifosine + 10 Gy did not result in a significantly increased survival relative to 10 Gy alone (P = 0.23). This PDGF-induced BSG model can serve as a preclinical tool for the testing of novel agents. Cancer Res; 70(6); 2548–57

During development, proliferation of cerebellar granule neuron precursors (CGNP), candidate cells-of-origin for the pediatric brain tumor medulloblastoma, requires signaling by Sonic hedgehog (Shh) and insulin-like growth factor (IGF), the pathways of which are also implicated in medulloblastoma. One of the consequences of IGF signaling is inactivation of the mammalian target of rapamycin (mTOR)–suppressing tuberous sclerosis complex (TSC), comprised of TSC1 and TSC2, leading to increased mRNA translation. We show that mice, in which TSC function is impaired, display increased mTOR pathway activation, enhanced CGNP proliferation, glycogen synthase kinase-3α/β (GSK-3α/β) inactivation, and cytoplasmic localization of the cyclin-dependent kinase inhibitor p27Kip1, which has been proposed to cause its inactivation or gain of oncogenic functions. We observed the same characteristics in wild-type primary cultures of CGNPs in which TSC1 and/or TSC2 were knocked down, and in mouse medulloblastomas induced by ectopic Shh pathway activation. Moreover, Shh-induced mouse medulloblastomas manifested Akt-mediated TSC2 inactivation, and the mutant TSC2 allele synergized with aberrant Shh signaling to increase medulloblastoma incidence in mice. Driving exogenous TSC2 expression in Shh-induced medulloblastoma cells corrected p27Kip1 localization and reduced proliferation. GSK-3α/β inactivation in the tumors in vivo and in primary CGNP cultures was mTOR-dependent, whereas p27Kip1 cytoplasmic localization was regulated upstream of mTOR by TSC2. These results indicate that a balance between Shh mitogenic signaling and TSC function regulating new protein synthesis and cyclin-dependent kinase inhibition is essential for the normal development and prevention of tumor formation or expansion. [Cancer Res 2009;69(18):7224–34]

Gli signaling is critical for central nervous system development and is implicated in tumorigenesis. To monitor Gli signaling in gliomas in vivo, we created platelet-derived growth factor–induced gliomas in a Gli-luciferase reporter mouse. We find that Gli activation is found in gliomas and correlates with grade. In addition, we find that sonic hedgehog (SHH) is expressed in these tumors and also correlates with grade. We identify microvascular proliferation and pseudopalisades, elements that define high-grade gliomas as SHH-producing microenvironments. We describe two populations of SHH-producing stromal cells that reside in perivascular niche (PVN), namely low-cycling astrocytes and endothelial cells. Using the Ptc-LacZ knock-in mouse as a second Gli responsive reporter, we show β-galactosidase activity in the PVN and in some tumors diffusely throughout the tumor. Lastly, we observe that SHH is similarly expressed in human gliomas and note that an intact tumor microenvironment or neurosphere conditions in vitro are required for Gli activity. [Cancer Res 2008;68(7):2241–49]

Purpose: The median survival for patients diagnosed with glioblastoma multiforme, the most common type of brain tumor, is less than 1 year. Animal glioma models that are more predictive of therapeutic response in human patients than traditional models and that are genetically and histologically accurate are an unmet need. The nestin tv-a (Ntv-a) genetically engineered mouse spontaneously develops glioma when infected with ALV-A expressing platelet-derived growth factor, resulting in autocrine platelet-derived growth factor signaling. Experimental Design: In the Ntv-a genetically engineered mouse model, T2-weighted and T1-weighted, contrast-enhanced magnetic resonance images were correlated with histology, glioma grade (high or low), and survival. Magnetic resonance imaging (MRI) was therefore used to enroll mice with high-grade gliomas into a second study that tested efficacy of the current standard of care for glioma, temozolomide (100 mg/kg qdx5 i.p., n = 13). Results: The Ntv-a model generated a heterogeneous group of gliomas, some with high-grade growth rate and histologic characteristics and others with characteristics of lower-grade gliomas. We showed that MRI could be used to predict tumor grade and survival. Temozolomide treatment of high-grade tv-a gliomas provided a 14-day growth delay compared with vehicle controls. Diffusion MRI measurement of the apparent diffusion coefficient showed an early decrease in cellularity with temozolomide, similar to that observed in humans. Conclusions: The use of MRI in the Ntv-a model allows determination of glioma grade and survival prediction, distribution of mice with specific tumor types into preclinical trials, and efficacy determination both by tumor growth and early apparent diffusion coefficient response.

Glioblastoma multiforme (GBM) is the most belligerent adult brain tumor, which is defined by its aggressive growth due to uncontrolled cellular proliferation, resistance to apoptosis, diffuse infiltration and invasion, propensity for necrosis and hypoxia, and vigorous angiogenesis. Recurrence of these invasive tumors is inevitable despite advanced multimodal therapy with surgery followed by radiation and temozolomide treatment resulting in a dismal median survival of beyond one year. A recent milestone in glioma research has been the revelation that therapies targeting other cell types besides tumor cells; i.e., the cell constituents of the vascular niche can convey substantial improvements in radiographic response, progression-free survival, and quality of life. The changing perspective on the environment as a functional contributor represents a profound shift in the approach to biology and therapeutic decisions in GBM. Here, by studying paracrine signaling cues of intratumoral myeloid cells in the perivascular tumor niche and microglial cells in the invading tumor niche we found that these two immune cell populations have distinct and different functions with microglial cells playing a pivotal role in tumor cell invasiveness while intratumoral myeloid cells regulate angiogenesis and tumor survival. Importantly, blocking immune cell-tumor cell paracrine signaling cues endorsed an immunestimulatory and angiostatic environment and ceased tumor invasiveness.

High-grade gliomas (HGGs), including glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG), are the most aggressive malignant high-grade gliomas in children, and are incurable with current therapies. The most frequent genetic lesions that drive formation of HGGs include amplification/overexpression, mutation, and/or activation of receptor tyrosine kinases (RTK), such as PDGFRA, and K27M mutations in histone H3 proteins. We have discovered that the Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) transcriptional activators, which encode paralogous transcriptional activators, are frequently overexpressed in RTK-mutant/overexpressing pediatric HGGs. We have also discovered that YAP/TAZ activity is required for to promote stemness and self-renewal in tumor cells. We hypothesize that, because of oncogenic RTK alterations, YAP/TAZ become constitutively activated to drive a gene expression program that promotes uncontrolled expansion of neural stem/progenitor cells to create malignant tumors, and that this can be exacerbated by H3-K27M epigenetic dysregulation. In testing this hypothesis, we have generated compelling preliminary data showing that pharmacologic inhibition of YAP/TAZ activity specifically provokes growth arrest and cell death of RTK-overexpressing HGG cells, particularly in the context of H3-K27M mutations. Our ongoing research is focused on identifying the YAP/TAZ transcriptional program that drives tumorigenesis. Our research indicates that YAP/TAZ inhibition may be a promising therapeutic strategy for pediatric HGGs.