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Background Diffuse Midline Glioma (DMG) with the H3K27M mutation is a lethal childhood brain cancer, with patients rarely surviving 2 years from diagnosis. Methods We conducted a multi-site Phase 1 trial of the imipridone ONC201 for children with H3K27M-mutant glioma (NCT03416530). Patients enrolled on Arm D of the trial (n = 24) underwent serial lumbar puncture for cell-free tumor DNA (cf-tDNA) analysis and patients on all arms at the University of Michigan underwent serial plasma collection. We performed digital droplet polymerase chain reaction (ddPCR) analysis of cf-tDNA samples and compared variant allele fraction (VAF) to radiographic change (maximal 2D tumor area on MRI). Results Change in H3.3K27M VAF over time (“VAF delta”) correlated with prolonged PFS in both CSF and plasma samples. Nonrecurrent patients that had a decrease in CSF VAF displayed a longer progression free survival (P = .0042). Decrease in plasma VAF displayed a similar trend (P = .085). VAF “spikes” (increase of at least 25%) preceded tumor progression in 8/16 cases (50%) in plasma and 5/11 cases (45.4%) in CSF. In individual cases, early reduction in H3K27M VAF predicted long-term clinical response (>1 year) to ONC201, and did not increase in cases of later-defined pseudo-progression. Conclusion Our work demonstrates the feasibility and potential utility of serial cf-tDNA in both plasma and CSF of DMG patients to supplement radiographic monitoring. Patterns of change in H3K27M VAF over time demonstrate clinical utility in terms of predicting progression and sustained response and possible differentiation of pseudo-progression and pseudo-response.

Background Diffuse midline gliomas (DMG) are highly invasive brain tumors with rare survival beyond two years past diagnosis and limited understanding of the mechanism behind tumor invasion. Previous reports demonstrate upregulation of the protein ID1 with H3K27M and ACVR1 mutations in DMG, but this has not been confirmed in human tumors or therapeutically targeted. Methods Whole exome, RNA, and ChIP-sequencing was performed on the ID1 locus in DMG tissue. Scratch-assay migration and transwell invasion assays of cultured cells were performed following shRNA-mediated ID1-knockdown. In vitro and in vivo genetic and pharmacologic [cannabidiol (CBD)] inhibition of ID1 on DMG tumor growth was assessed. Patient-reported CBD dosing information was collected. Results Increased ID1 expression in human DMG and in utero electroporation (IUE) murine tumors is associated with H3K27M mutation and brainstem location. ChIP-sequencing indicates ID1 regulatory regions are epigenetically active in human H3K27M-DMG tumors and prenatal pontine cells. Higher ID1-expressing astrocyte-like DMG cells share a transcriptional program with oligo/astrocyte-precursor cells (OAPCs) from the developing human brain and demonstrate upregulation of the migration regulatory protein SPARCL1. Genetic and pharmacologic (CBD) suppression of ID1 decreases tumor cell invasion/migration and tumor growth in H3.3/H3.1K27M PPK-IUE and human DIPGXIIIP* in vivo models of pHGG. The effect of CBD on cell proliferation appears to be non-ID1 mediated. Finally, we collected patient-reported CBD treatment data, finding that a clinical trial to standardize dosing may be beneficial. Conclusions H3K27M-mediated re-activation of ID1 in DMG results in a SPARCL1+ migratory transcriptional program that is therapeutically targetable with CBD.

Diffuse midline glioma (DMG) with the H3K27M mutation is a lethal childhood brain cancer, with patients rarely surviving 2 years from diagnosis. We conducted a multi-site Phase 1 trial of the imipridone ONC201 for children with H3K27M-mutant glioma ({"type":"clinical-trial","attrs":{"text":"NCT03416530","term_id":"NCT03416530"}}NCT03416530). Patients enrolled on Arm D of the trial (n=24) underwent serial lumbar puncture (baseline, 2, 6 months) for cell-free tumor DNA (cf-tDNA) analysis at time of MRI. Additionally, patients on all arms of the trial at the University of Michigan underwent serial plasma collection. CSF collection was feasible in this cohort, with no procedural complications. We collected 96 plasma samples and 53 CSF samples from 29 patients, including those with H3F3A (H3.3) (n=13), HIST13HB (H3.1) (n= 4), and unknown H3 status/not biopsied (n=12) [range of 0–8 CSF samples and 0–10 plasma samples]. We performed digital droplet polymerase chain reaction (ddPCR) analysis and/or amplicon-based electronic sequencing (Oxford Nanopore) of cf-tDNA samples and compared variant allele fraction (VAF) to radiographic change (maximal 2D tumor area on MRI). Preliminary analysis of samples demonstrates a correlation between changes in tumor size and H3K27M cf-tDNA VAF, when removing samples with concurrent bevacizumab. In multiple cases, early reduction in CSF cf-tDNA predicts long-term clinical response (>1 year) to ONC201, and does not increase in cases of later-defined pseudo-progression (radiation necrosis). For example, a now 9-year old patient with thalamic H3K27M-mutant DMG underwent treatment with ONC201 after initial radiation and developed increase in tumor size at 4 months post-radiation (124% baseline) of unclear etiology at the time. Meanwhile, her ddPCR declined from baseline 6.76% VAF to

Background Glioblastoma (GBM) is an aggressive and highly vascular tumor with median survival below 2 years. Despite advances in surgery, radiotherapy, and chemotherapy, survival has improved modestly. To combat glioma vascular proliferation, anti-angiogenic agents targeting vascular endothelial growth factor (VEGF) were introduced. Preclinically these agents were effective, yet they did not improve overall survival in phase III trials. We tested the hypothesis that ganciclovir (GCV)-mediated killing of proliferating endothelial cells expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK) would have direct antitumor effects, and whether vessel ablation would affect the antitumor activity of anti-VEGF antibodies and radiotherapy. Methods Proliferating endothelial cells were eliminated using GCV-mediated killing of proliferating endothelial cells expressing HSV1-TK (in Tie2-TK-IRES-GFP mice). Syngeneic NRAS/p53 (NP) gliomas were implanted into the brains of Tie2-TK-IRES-GFP mice. Endothelial proliferation activates the Tie2 promoter and HSV1-TK expression. Administration of GCV kills proliferating tumor endothelial cells and slows tumor growth. The effects of endothelial cell ablation on anti-angiogenic therapy were examined using anti-VEGF antibodies or irradiation. Results GCV administration reduced tumor growth and vascular density, increased tumor apoptosis, and prolonged survival. Anti-VEGF antibodies or irradiation also prolonged survival. Surprisingly, combining GCV with irradiation, or with anti-VEGF antibodies, reduced their individual therapeutic effects. Conclusion GCV-mediated killing of proliferating endothelial cells expressing HSV1-TK, anti-VEGF antibodies, or irradiation all reduced growth of a murine glioma. However, elimination of microvascular proliferation decreased the efficacy of anti-VEGF or irradiation therapy. We conclude that, in our model, the integrity of proliferating vessels is necessary for the antiglioma effects of anti-VEGF and radiation therapy.

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor with rare survival beyond two years. This poor prognosis is largely due to the tumor's highly infiltrative and invasive nature. Nearly 80% of DMGs harbor K27M mutation in the genes encoding histone H3.1 (H3F3A) or H3.3 (HISTIH3B), often with concurrent ACVR1 mutation. Inhibitor of DNA-binding (ID) proteins are key transcriptional regulators of genes involved in lineage commitment and are associated with invasiveness and poor clinical outcomes in multiple human cancers. Introduction of H3K27M and ACVR1 mutations increase ID1 expression in cultured astrocytes, but this has not been confirmed in human tumors or targeted therapeutically. We developed an in-utero electroporation (IUE) murine H3K27M-driven tumor model, which demonstrates increased ID1 expression in H3K27M- and ACVR1-mutated tumor cells. Exome and transcriptome sequencing analysis of multi-focal DMG tumors (n=52) and normal brain tissue revealed that increased ID1 expression is associated with H3K27M/ACVR1-mutation and brainstem location, and correlates with poor survival in patients. ChIP-sequencing for H3K27ac and H3K27me3 in multiple DMG tumors (n=5) revealed that the ID1 gene is epigenetically active, which matches the epigenetic state of murine prenatal hindbrain cells. Higher ID1-expressing astrocyte-like DIPG cells share a similar transcriptional program with ID1+/SPARCL1+ positive oligo/astrocyte-precursor (OAPC) cells from the developing human brain and demonstrate upregulation of gene sets involved in regulation of cell migration. Both genetic and pharmacologic [cannabidiol (CBD)] suppression of ID1 result in decreased DIPG cell invasion/migration in vitro and invasion/tumor growth in multiple in vivo models. Mechanistically, CBD reduces proliferation through production of reactive oxygen species. Further, DIPG patients treated off-trial with CBD (n=15) displayed reduced ID1 tumor expression and improved overall survival. In summary, ID1 is upregulated in DIPG through K27M-mediated epigenetic reactivation of a developmental OAPC-like transcriptional state, and ID1-driven invasiveness of DIPG is therapeutically targetable with CBD.

BACKGROUND Diffuse intrinsic pontine gliomas (DIPGs) are lethal pediatric brain tumors with no curative therapies. Inhibitor of DNA binding (ID) proteins are key regulators of gene differentiation during embryogenesis. Previous work has shown that H3F3A and ACVR1 mutations increase ID1 expression in cultured astrocytes, but this has not been validated in human DIPG, nor has the regulation and targetability of ID1 been explored in DIPG. RESULTS Analysis of post-mortem tissue and multiple human datasets showed ID1 to be elevated in DIPG, and to correlate with reduced survival. In a multi-focal autopsy of a DIPG case, we also found ID1 expression to be heterogeneous and to correlate with tumor invasion. Chromatin immunoprecipitation qPCR (ChIP-qPCR) revealed elevated H3K27ac and low H3K27me3 at ID1 regulatory regions (enhancers/promoters) in DIPG tissue compared to normal brain, regardless of H3 or ACVR1 mutation status. Analysis of publicly-available ISH and ChIP-sequencing data of developing murine brains revealed H3K27ac at ID1 enhancers to be elevated in the prenatal hindbrain compared to prenatal forebrain and midbrain, and all postnatal brain regions. ID1 shRNA-mediated knockdown of primary human H3K27M DIPG cells (DIPG007) significantly reduced invasion and migration. We also treated DIPG007 cells with cannabidiol (CBD) and found reduced viability at clinically relevant dosing (IC50=2.4 uM) with dose-dependent reduction in ID1 protein. CONCLUSIONS These findings indicate that a multifactorial (genetic and regionally-based) epigenetic upregulation of ID1 drives DIPG invasiveness and is targetable with CBD. ID1 knockdown and CBD treatment experiments in murine models of DIPG are ongoing.

Diffuse intrinsic pontine gliomas (DIPGs) are lethal pediatric brain tumors with no effective therapies beyond radiation. The highly invasive nature of DIPG is key to its aggressive phenotype, but the factors and mechanisms contributing to this aggressive invasion are unknown. Inhibitor of DNA binding (ID) proteins, key regulators of lineage commitment during embryogenesis, are implicated in tumorigenesis in multiple human solid tumors. Prior work showed that recurrent H3F3A and ACVR1 mutations increase ID1 expression in cultured astrocytes. However, the impact and targetability of ID1 have not been explored in human DIPG. Exome and transcriptome sequencing analyses of multi-focal DIPG tumors and normal brain tissue from autopsy (n=52) revealed that ID1 expression is significantly elevated in DIPG samples. Higher ID1 expression correlates with reduced survival in DIPG patients and increased regional invasion in multi-focal autopsy samples. Analyses of developing mouse brain RNA/ChIP-Seq data revealed high ID1 expression and H3K27ac promoter binding in prenatal hindbrain compared to all other prenatal and postnatal brain regions. ChIP-qPCR for H3K27ac and H3K27me3 revealed that ID1 gene regulatory regions are epigenetically poised for upregulation in DIPG tissues compared to normal brain, regardless of H3/ACVR1 mutational status. These data support that the developing pons is regionally poised for ID1 activation. Genetic (shRNA) ID1 knockdown of primary human H3.3K27M-DIPG cells (DIPG007) resulted in significantly reduced invasion/migration and significantly improved survival of K27M-DIPG mice. Knockdown of ID1 in DIPG cells also resulted in down-regulation of the WNK1-NKCC1 pathway, which regulates tumor cell electrolyte homeostasis and migration. Finally, treatment of DIPG007 cells with cannabidiol (CBD) reduced ID1 levels, viability of DIPG cells and significantly improved survival of K27M-DIPG mice. In summary, our findings indicate that multifactorial (genetic and regional) epigenetic upregulation of ID1 drives DIPG invasiveness; and that targeting ID1 with CBD could potentially be an effective therapy for DIPG.

Patients with diffuse midline glioma (DMG) harboring H3 K27M mutation rarely survive longer than two years and have no proven therapies following first-line radiation. ONC201, a bitopic DRD2 antagonist and allosteric ClpP agonist, has shown encouraging efficacy in early phase studies in H3 K27M-mutant DMG. In order to define response rates in H3 K27M DMG patients and to clarify the genomic, anatomic and molecular predictors of response, we performed an integrated pre-clinical and clinical analysis of ONC201 treatment. ONC201 was effective in intra-uterine electroporation (IUE)-generated H3 K27M-mutant murine glioma models with excellent CNS penetration and survival benefit. Patients with H3 K27M-mutant DMG treated with ONC201 on active clinical trials (n=50, 27 thalamic, 23 brainstem) showed an overall survival (OS) of 28.1 (range: 5.9–105) months from diagnosis (enrollment by 4/29/19, data cut-off 12/28/19), compared to historical median OS of 12 months. Median OS for non-recurrent patients has not been reached (n=16, median follow-up: 16.8 from diagnosis). For non-recurrent thalamic patients (n=8), median PFS is 20.1 (range: 9.3–27.6) months from diagnosis (median time on drug: 14.5 months). Best response for thalamic patients by RANO: 1 CR, 5 PR, 7 SD, 8 PD, 6 not reported. Decreased H3 K27M cell-free tumor DNA in plasma and CSF at 6 months correlated with long-term response. Baseline tumor gene expression profiling in patients treated with ONC201 (n=14) identified EGFR and the cortical developmental transcription factor FOXG1 as the strongest biomarkers of radiographic response to ONC201. Analysis of 541 ONC201-treated human cancer cell lines from DepMap, provided evidence for an EGFR-dependent ONC201 resistance mechanism. Analysis of 38 glioma cell lines further supports FOXG1 as a glioma-specific predictive biomarker of ONC201 response. The unprecedented survival results and radiographic responses to ONC201 in H3K27M DMG make a compelling case for later phase and combinatorial studies.

INTRODUCTION DMG-ACT (DMG- multi-arm Adaptive and Combinatorial Trial) aims to implement a highly innovative clinical trial design of combinatorial arms for patients with diffuse midline gliomas (DMGs) at all disease stages that is adaptive to pre-clinical data generated in eight collaborating institutions. The goals of the team are to: i) rapidly identify and validate promising drugs for clinical use, and ii) predict biomarkers for promising drugs. METHODS In vitro (n=15) and in vivo (n=8) models of DMGs across seven institutions were used to assess single and combination treatments with ONC201, ONC206, marizomib, panobinostat, Val-083, and TAK228. In vivo pharmacokinetic assays using clinically relevant dosing of ONC201, ONC206, and panobinostat were performed. Predictive biomarkers for ONC201 and ONC206 were identified using extensive molecular assays including CRISPR, RNAseq, ELISA, FACS, and IHC. RESULTS Inhibitory concentrations (IC50) were established and validated across participating sites. In vivo validation of single and combination drug assays confirmed drug efficacy as increased survival for: ONC201 (p=0.01), ONC206 (p=0.01), ONC201+ONC206 (p=0.02), and ONC201+panobinostat (p=0.01). Marizomib showed toxicity in murine/zebrafish PDXs models. Murine pharmacokinetic analysis showed peak brain levels of ONC201 and ONC206 above pre-clinical IC50. Molecular testing and analyses of existing drug screen across 537 cancer cell lines validated mitochondrial stress and ATF4 as the main targets induced by ONC201/6. CONCLUSION Thorough preclinical testing in a multi-site laboratory setting is feasible and identified ONC201 in combination with ONC206 as promising therapeutics for DMGs. Preclinical and correlative-clinical studies are ongoing.

Diffuse intrinsic pontine gliomas (DIPGs) are lethal brain tumors with no effective therapies other than radiation. Inhibitor of DNA binding (ID) proteins, key regulators of lineage commitment during embryogenesis, are implicated in tumorigenesis in multiple human cancers. Prior work showed that recurrent H3F3A and ACVR1 mutations increase ID1 expression in cultured astrocytes. However, this has not been validated in human DIPG. The regulation and targetability of ID1 in DIPG has not been explored either. Exome and transcriptome sequencing analysis of multi-focal DIPG tumors and normal brain tissue from autopsy (n=52) revealed that ID1 expression is significantly elevated in DIPG tissues. Higher ID1 expression correlates with reduced survival in DIPG patients and increased regional invasion in multi-focal autopsy samples. Analyses of developing mouse brain RNA/ChiP-Seq data revealed high ID1 expression and H3K27ac promoter binding in prenatal hind brain compared to all other prenatal and postnatal brain regions. ChIP-qPCR for H3K27ac and H3K27me3 revealed that ID1 gene regulatory regions are epigenetically poised for upregulation in DIPG tissues compared to normal brain, regardless of H3/ACVR1 mutational status. These data support that the developing pons is regionally poised for ID1 activation. Genetic (shRNA) ID1 knockdown in primary human H3.3K27M-DIPG cells (DIPG007) resulted in significantly reduced invasion and migration in vitro. Additionally, DIPG-ID1-KO cells showed improved sensitivity to radiation therapy. Phospho-kinase array analysis of DIPG cells revealed that Akt and WNK1 activity were significantly downregulated upon ID1 knockdown, which was previously shown in lung tumors. Treatment of DIPG007 cells with cannabidiol (CBD) reduced ID1 expression levels and viability/proliferation of DIPG cells in vitro. ID1 knockdown and CBD treatment studies in vivo are ongoing. In summary, our findings indicate that multifactorial (genetic and regional) epigenetic upregulation of ID1 drives DIPG invasiveness and targeting ID1 using CBD may be a potential strategy for the treatment of DIPGs.

Gliomas are a leading cause of cancer mortality in children and adults, and new targeted therapies are desperately needed. ATRX is a chromatin remodeling protein that is recurrently mutated in H3F3A-mutant pediatric glioblastoma (GBM) and IDH-mutant grade 2/3 adult glioma. We previously showed that loss of ATRX in glioma results in tumor growth and additional tumor mutations. However, the mechanism driving these phenotypes has not been fully established. We found that in ChIP-Seq/ChIP-qPCR of mouse neuronal precursor cells (NPCs) and GBM cells with isogenic ATRX loss, ATRX binds regulatory elements for cell cycle phase transition gene sets, and ATRX loss subsequently results in reduced expression. Furthermore, human GBM cells with ATRX knock-out demonstrate higher rates of cells in S and G2/M phases, with clusters of cells demonstrating reduced expression of cell cycle regulatory gene sets by single-cell sequencing (scSeq) analysis. In human and mouse GBM in vitro models, ATRX-deficient cells exhibit a seven-fold increase in mitotic index at 16 hours after sub-lethal radiation and enhanced activation of the master cell cycle regulator ATM with radiation. Treatment of ATRX-deficient gliomas with ATM inhibitors results in a selective increase in dysfunctional cell cycling and increased radio-sensitization in ATRX-deficient glioma cells. Using an ATM-luciferase reporter in orthotopically-implanted human GBM cells, both AZD0156 and AZD1390 demonstrate in vivo pathway inhibition. Mice intra-cranially implanted with ATRX-deficient GBM cells demonstrate a doubling of median survival compared to radiated controls (p=0.0018) when treated with AZD0156 combined with radiation; this is not seen in ATRX-sufficient models. This study demonstrates that ATRX-deficient high-grade gliomas display epigenetic dysregulation of cell cycle phase transitions, which opens a new window for therapies targeting this unique phenotype.

H3K27M-midline gliomas are fatal tumors that mainly harbor H3.3K27M mutations resulting in global H3K27me3 reduction that impacts neuroglial-differentiation. However, the exact mechanisms by which H3.3K27M mutations promote cancer are poorly understood. Metabolic reprogramming is a hallmark of cancer and we hypothesized that H3.3K27M mutations can reprogram metabolism to support uncontrolled growth. We demonstrate that H3.3K27M-mutant cells show elevated levels of critical enzymes related to glycolysis and TCA cycle metabolism including hexokinase-2, isocitrate dehydrogenase (IDH)-1 and glutamate dehydrogenase. H3.3K27M cells also demonstrated enhanced glycolysis, glutamine and TCA-cycle metabolism accompanied by increased alpha-ketoglutarate (α-KG) production. Mutant IDH (mIDH)1/2 converts α-KG to D-2-hydroxyglutarate (D-2HG). D-2HG increases H3K27me3 by inhibiting α-KG’s function to drive H3K27-demethylases. We discovered that H3.3K27M cells use α-KG in an opposing manner to maintain low H3K27me3. Inhibiting enzymes related to α-KG generation including hexokinase-2, glutamate-dehydrogenase and wild type-IDH1 increased global H3K27me3, altered chromatin accessibility at neuroglial-differentiation factors and lowered tumor cell proliferation. In vivo inhibition of glutamine metabolism and/ or wild type-IDH1 using blood-brain barrier penetrant small molecule inhibitors increased overall survival in vivo in two independent H3.3K27M animal models (p< 0.0001). H3K27M and mIDH1 were mutually exclusive in patient tumor samples and D-2HG treatment or forced-mIDH1 expression in H3.3K27M cells increased global H3K27me3 and cell death. Finally H3.3K27M and mIDH1 were synthetic lethal in vitro. Our data suggest that H3.3K27M and mIDH1 hijack a critical and conserved metabolic pathway in opposing manners to regulate global H3K27me3. These results have implications for understanding the pathogenesis of fatal H3K27M-gliomas and for developing therapeutic strategies by disruption of an integrated metabolic/epigenetic-axis.

Gliomas are a leading cause of cancer mortality in children and adults and new targeted therapies are desperately needed. ATRX is a chromatin remodeling protein that is recurrently mutated in H3F3A-mutant pediatric GBM and IDH-mutant grade 2/3 adult glioma. We previously showed that loss of ATRX in glioma results in tumor growth and additional tumor mutations. However, the mechanism driving these phenotypes has not been fully established. We found that in ChIP-Seq datasets of mouse neuronal precursor cells (NPCs) and experimental models of human glioma cells, ATRX binds and regulates the chromatin state of promoters and enhancers for gene sets associated with regulation of the cell cycle G2/M checkpoint. In line with this, analysis of single-cell seq (sc-seq) data from IDH-mutant gliomas (n=16) shows that ATRX-mutant tumors (IDH-A) demonstrate a population of cycling cells with dysregulated cell cycle phase gene set expression when compared to ATRX-wildtype tumors (IDH-O). In glioma models, ATRX-deficient cells exhibit a seven-fold increase in mitotic index at 16 hours after sub-lethal radiation and enhanced activation of the master cell cycle regulator ATM with radiation. Treatment of ATRX-deficient gliomas with ATM inhibitors results in a selective increase in dysfunctional cell cycling and increased radio-sensitization in ATRX-deficient glioma cells. Using an ATM-luciferase reporter in orthotopically-implanted human GBM cells, both AZD0156 and AZD1390 demonstrate in vivo pathway inhibition. Mice intra-cranially implanted with ATRX-deficient GBM cells demonstrate a doubling of median survival compared to radiated controls (p=0.0018) when treated with AZD0156 combined with radiation. This study demonstrates that ATRX-deficient glioma display epigenetic dysregulation of the G2/M checkpoint, which opens a new window for therapies targeting this unique phenotype.

ATRX is a histone chaperone protein recurrently mutated in pediatric GBM. We previously confirmed its role in tumor progression and mutational burden in glioma. However, the mechanism which mediates the proliferative advantage of ATRX loss in pediatric GBM remains unexplained. Recent data revealed a distinct pattern of DNA binding sites of the ATRX protein using ChIP-seq in mouse neuronal precursor cells (mNPCs). Using the ATRX peaks identified in p53(-/-) mNPCs, we confirmed that ATRX binding sites were significantly enriched in gene promoters (p

Pediatric high-grade glioma (HGG) and diffuse intrinsic pontine glioma (DIPG) frequently harbor alterations in PDGFRA. The CNS penetration of PDGFRA inhibitors, such as dasatinib, is limited by the tumor-efflux protein P-glycoprotein (P-gp). We hypothesized that co-treatment with everolimus, which has been shown to block P-gp in non-tumor models, would increase CNS penetration and efficacy of dasatinib in PDGFRA-driven HGG and DIPG. Dasatinib effectively treated mouse DIPG cells generated from an intra-uterine electroporation (IUE) model (TP53, PDGFRA and H3K27M mutations), with an IC(50) of 100 nM and a dose-dependent reduction in PDGFRA and pPDGFRA by western blot. Using an in vitro P-gp inhibitor assay, we confirmed that everolimus strongly blocks P-gp activity at 1 uM (p=0.0028 vs untreated, and NS vs complete P-gp block). Treatment studies using the IUE model are ongoing. Brief treatment with everolimus resulted in sub-IC(50) dasatinib average mouse cortex (23 nM) and tumor (65 nM) concentrations by mass spectroscopy, but prolonged (>24 hours) everolimus exposure resulted in improved average cortex (288 nM) and brainstem tumor (360 nM) concentrations. Based on this promising pre-clinical data, we established a phase 2 trial employing dasatinib and everolimus in children with HGG and DIPG that contain PDGFRA alterations (NCT03352427). The first two patients (a recurrent PDGFRA-amplified HGG and a recurrent PDGFRA D842V-mutated DIPG) treated with dasatinib and everolimus after re-irradiation survived 6 months and 9 months (ongoing), respectively, after progression, which compares very favorably to historical controls. Paired CSF samples (before and after addition of everolimus) from the PDGFRA-amplified patient showed a 50% increase in CSF dasatinib level after addition of everolimus. In summary, we demonstrate that dasatinib treatment of PDGFRA-driven pediatric HGG and DIPG is improved with everolimus blockade of P-gp. This represents a novel route for improving the CNS penetration and efficacy of precision therapies for pediatric HGG.

Glioblastoma is the most vascularized and aggressive glioma with a median survival of less than 2 years. Despite promising clinical trials inhibiting vascular endothelial growth factor (VEGF) failed to improve patient survival. We used Tie2p/e LV [Tie2-TK-IRIS-GFP] mice which express the conditionally cytotoxic gene thymidine kinase (TK) under the control of endothelial-specific Tie2 promoter. Administration of gancylovir (GCV) for 7 days disrupts reactive, proliferating blood vessels within the tumor. We implanted mice with NRAS/shp53 (NP) GBM neurospheres. Three days later mice were treated with GCV to kill reactive tumor vessels. GCV prolonged survival by delaying tumor proliferation and increasing tumor necrosis. GCV administration decreased blood vessel density in the center but not in the tumor peripheral. GCV mediated blood vessel disruption resulted in tumor cell death. To further increase tumor cell death and develop anti-tumor immune responses, we used TK+Flt3L adenoviruses mediated immune therapy to increase mouse survival. We found that combination of tumor blood vessel disruption by GCV, and TK/Flt3L immune therapy, significantly extended the survival of mice compared to GCV therapy alone. Furthermore, adding radiation therapy to the GCV/TK+Flt3L immune therapy further enhanced mouse survival. We assumed that if anti-VEGF therapy effectively induces tumor blood vessel normalization then this could further increase the efficacy of TK+Flt3L therapy by increasing immune cells infiltration into the tumor. However, combining anti-VEGF therapy with TK+Flt3L did not improve survival compared to each therapy alone suggesting that blood vessel disruption may actually decrease the efficacy of anti-VEGF therapy. Our results provide evidence that combining direct tumor blood vessel disruption, and TK/Flt3L immune therapy, represents a novel therapeutic approach for the treatment of GBM. We also demonstrate that blood vessel disruption limits the anti-tumor effect of anti-VEGF therapy. This could explain the recently described failure of anti-VEGF therapy in human GBM patients.

GBM remains the deadliest primary malignant brain tumor. Given the importance of invading cells, less attention has been paid to the tumor mass, even if such a mass eventually kills the patient. We previously demonstrated that human and mouse transplantable or GEMM gliomas display regular anatomical multicellular structures containing elongated cells which we named ‘oncostreams’. Oncostreams are 10–20 cells wide, 2–400 um long, and are distributed throughout the tumors. Furthermore, we uncovered a negative correlation between oncostream density and animal survival suggesting that oncostreams play a role in tumor malignancy. Further data indicate that oncostreams aid local invasion of normal brain. Co-implantation experiments demonstrated that oncostreams facilitate the intratumoral spread of slow migrating cells. To determine a possible molecular distinctiveness of oncostreams we used laser capture microdissection followed by RNA-Seq, bioinformatics and network analysis. Evaluation of the transcriptome demonstrated differential expression (DE) of genes between oncostreams and adjacent tumor. Functional enrichment of DE genes showed that “collagen catabolic processes”, “positive regulation of cell migration”, and “extracellular matrix organization” were the most over-represented gene ontologies. Network analysis indicated that Col1a1, ACTA2, MMP9, MMP10 and ADAMTS2, genes important for cell migration and ECM interactions, are part of these networks. IHC and PCR were used to validate RNA-Seq expression changes. To understand the cellular dynamics in our system we used time lapse imagining and evaluated the results using high level statistical analyses. The following tests, i.e., velocity distribution, pair-wise correlation of local position, velocity correlation, etc. were used to characterize the dynamics of cellular intratumoral motility. These data demonstrate the existence of collective motion within glioma tumors. Taken together, our results show that oncostreams, move by collective motion, are anatomically and molecularly distinct, reveal the existence of glioma self-organization, and regulate glioma growth and invasion. Targeting oncostreams is a candidate therapeutic strategy.

ANGI-16. IN VIVO AND IN VITRO STUDIES SHOW THAT BRAIN DERIVED ENDOTHELIAL CELLS STIMULATE MIGRATION OF HUMAN, MOUSE AND RAT GLIOMA CELLS Viveka Nand Yadav1,2, Gregory J. Baker1,2, Daniel A. Orringer1, Jason A. Heth1, Shawn Hervey-Jumper1, Oren Sagher1, Maria G. Castro1,2, and Pedro R. Lowenstein1,2; Department of Neurosurgery, University of Michigan Medical School, Ann Arbor, MI, USA; Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA Glioblastoma multiforme (GBM) is the most lethal of human brain tumors. Glioma cells invade the brain by active migration along blood vessels, white matter tracts, interstitially, or surrounding the meninges. Molecular determinants of glioma migration patterns, i.e., those that attract glioma cells to blood vessels remain partially understood. We observed that several mouse and human glioma cells migrate primarily along the blood vasculature. Here, we tested the ability of human and mouse brain-derived endothelial (MBVE) cells to stimulate the migration of human, mouse and rat-derived glioma cell lines in an in vitro migration assay. Among different primary human glioma cell lines, HF2303 and MSP-12 cells showed significant directional migration while MGG8, IN859, IN2045 and U251 cells did not. Further, Gl26-cit, a mouse glioma and CNS1-cit a rat cell line also exhibited significant migration towards MBVE. These results were in line with our in vivo data wherein HF2303, and Gl26-cit, but not MGG8 cells, invaded mainly along blood vessels. Differential migration of various human glioma cell lines in response to MBVE cells in vitro is probably related to their growth patterns in vivo. To decipher the mechanism of GBM cell migration, we tested the role of various chemokines using in vitro migration assays. The migration of Gl26-cit and HF2303 towards MBVE were significantly inhibited by AMD3100, an inhibitor of CXCR4. Lastly,MBVE together with mouse astrocytes promote the in vitro migration of Gl26-cit cells to a greater extent than MBVE alone. In summary, the behavior of glioma cells in vitro is consistent with their migration patterns in vivo and therefore, using in vitro migration models are a good tool to identify molecular mechanisms responsible for glioma cell growth towards blood vessels. The CXCL12-CXCR4 signaling was found to be critical for the migration of both mouse and human glioma cell lines. Neuro-Oncology 17:v41–v44, 2015. doi:10.1093/neuonc/nov207.16 Published by Oxford University Press on behalf of the Society for Neuro-Oncology 2015.