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Purpose: One likely cause of treatment failure in glioblastoma is the persistence of glioma stem-like cells (GSLCs) which are highly resistant to therapies currently employed. We found that CXCL12 has highest expression in glioma cells derived from neural progenitor cells (NPC). The development and molecular signature of NPC-derived glioblastomas were analyzed and the therapeutic effect of blocking CXCL12 was tested.Experimental Design: Tumors were induced by injecting DNA into the lateral ventricle of neonatal mice, using the Sleeping Beauty transposase method. Histology and expression of GSLC markers were analyzed during disease progression. Survival upon treatment with pharmacologic (plerixafor) or genetic inhibition of CXCR4 was analyzed. Primary neurospheres were generated and analyzed for proliferation, apoptosis, and expression of proteins regulating survival and cell-cycle progression.Results: Tumors induced from NPCs display histologic features of human glioblastoma and express markers of GSLC. In vivo, inhibiting the CXCL12/CXCR4 signaling axis results in increased survival of tumor-bearing animals. In vitro, CXCR4 blockade induces apoptosis and inhibits cell-cycle progression, downregulates molecules regulating survival and proliferation, and also blocks the hypoxic induction of HIF-1α and CXCL12. Exogenous administration of CXCL12 rescues the drug-induced decrease in proliferation.Conclusions: This study demonstrates that the CXCL12/CXCR4 axis operates in glioblastoma cells under hypoxic stress via an autocrine-positive feedback mechanism, which promotes survival and cell-cycle progression. Our study brings new mechanistic insight and encourages further exploration of the use of drugs blocking CXCL12 as adjuvant agents to target hypoxia-induced glioblastoma progression, prevent resistance to treatment, and recurrence of the disease. Clin Cancer Res; 23(5); 1250–62. ©2016 AACR.

Supplementary methods

Tumors induced from NPCs with NRAS/LgT harbor histological hallmarks of human GBM: S.2 B.Large tumors induced with NRAS/LgT are devoid of patent vascularization.

Genetic knockdown of CXCR4 impairs the growth of NRAS/LgT derived glioma neurospheres in vitro, decreases tumor growth in vivo and increases survival of tumor-bearing animals.

CXCL12 has highest expression in GBM cells induced with NRAS/SV40 LgT.

Comparison of gene expression between the NPC derived glioma cells M7, GL26 and GL26A1 cells (Affymetrix Gene Chip Data).

At 19 days after injection tumor cells induced with NRAS and SV40 LgT express markers characteristic of NPCs and GSLCs: Nestin, GFAP and Olig2.

NPC derived GBM neurospheres harbor the characteristics of Glioms Stem-Like Cells (GSLC)l.

Analysis of tumor area in brains from animals treated for 5 days with either saline or AMD3100.

AMD3100 decreases the proliferation of neurosphere cultures: Magnitude of Change.

Supplementary Figure S7. Anti-CD3ε immunolabeled brain tissue sections from mouse A1 (top image) and mouse N2 (bottom image) shown in Figure 5I.

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.

BackgroundPediatric and adult high-grade glioma (HGG) frequently harbor PDGFRA alterations. We hypothesized that co-treatment with everolimus may improve the efficacy of dasatinib in PDGFRα-driven glioma through combinatorial synergism and increased tumor accumulation of dasatinib.MethodsDose response, synergism studies, P-gp inhibition and pharmacokinetic studies were performed on in vitro and in vivo human and mouse models of HGG. Six patients with recurrent PDGFRα-driven glioma were treated with dasatinib and everolimus.ResultsDasatinib effectively inhibited the proliferation of mouse and human primary HGG cells with a variety of PDGFRA alterations. Dasatinib exhibited synergy with everolimus in the treatment of HGG cells at low nanomolar concentrations of both agents, with reduction in mTOR signaling that persists after dasatinib treatment alone. Prolonged exposure to everolimus significantly improved the CNS retention of dasatinib and extended survival of PPK tumor bearing mice. Pediatric patients (n=6) with glioma tolerated this combination without significant adverse events. Recurrent patients (n=4) demonstrated median overall survival of 8.5 months.ConclusionEfficacy of dasatinib treatment of PDGFRα-driven HGG is improved with everolimus and suggests a promising route for improving targeted therapy for this patient population.Trial RegistrationClinicalTrials.gov NCT03352427FundingThe authors thank the patients and their families for participation in this study. CK is supported by NIH/NINDS K08-NS099427-01, the University of Michigan Chad Carr Pediatric Brain Tumor Center, the Chad Tough Foundation, Hyundai Hope on Wheels, Catching up With Jack, Prayers from Maria Foundation, U CAN-CER VIVE FOUNDATION, Morgan Behen Golf Classic, and the DIPG Collaborative. The PEDS-MIONCOSEQ study was supported by grant 1UM1HG006508 from the National Institutes of Health Clinical Sequencing Exploratory Research Award (PI: Arul Chinnaiyan).

High-grade gliomas with arginine or valine substitutions of the histone H3.3 glycine-34 residue (H3.3G34R/V) carry a dismal prognosis, and current treatments, including radiotherapy and chemotherapy, are not curative. Because H3.3G34R/V mutations reprogram epigenetic modifications, we undertook a comprehensive epigenetic approach using ChIP sequencing and ChromHMM computational analysis to define therapeutic dependencies in H3.3G34R/V gliomas. Our analyses revealed a convergence of epigenetic alterations, including (i) activating epigenetic modifications on histone H3 lysine (K) residues such as H3K36 trimethylation (H3K36me3), H3K27 acetylation (H3K27ac), and H3K4 trimethylation (H3K4me3); (ii) DNA promoter hypomethylation; and (iii) redistribution of repressive histone H3K27 trimethylation (H3K27me3) to intergenic regions at the leukemia inhibitory factor (LIF) locus to drive increased LIF abundance and secretion by H3.3G34R/V cells. LIF activated signal transducer and activator of transcription 3 (STAT3) signaling in an autocrine/paracrine manner to promote survival of H3.3G34R/V glioma cells. Moreover, immunohistochemistry and single-cell RNA sequencing from H3.3G34R/V patient tumors revealed high STAT3 protein and RNA expression, respectively, in tumor cells with both inter- and intratumor heterogeneity. We targeted STAT3 using a blood-brain barrier–penetrable small-molecule inhibitor, WP1066, currently in clinical trials for adult gliomas. WP1066 treatment resulted in H3.3G34R/V tumor cell toxicity in vitro and tumor suppression in preclinical mouse models established with KNS42 cells, SJ-HGGx42-c cells, or in utero electroporation techniques. Our studies identify the LIF/STAT3 pathway as a key epigenetically driven and druggable vulnerability in H3.3G34R/V gliomas. This finding could inform development of targeted, combination therapies for these lethal brain tumors.

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.

ATRX, a chromatin remodeler protein, is recurrently mutated in H3F3A-mutant pediatric glioblastoma (GBM) and isocitrate dehydrogenase (IDH)-mutant grade 2/3 adult glioma. Previous work has shown that ATRX-deficient GBM cells show enhanced sensitivity to irradiation, but the etiology remains unclear. We find that ATRX binds the regulatory elements of cell-cycle phase transition genes in GBM cells, and there is a marked reduction in Checkpoint Kinase 1 (CHEK1) expression with ATRX loss, leading to the early release of G2/M entry after irradiation. ATRX-deficient cells exhibit enhanced activation of master cell-cycle regulator ATM with irradiation. Addition of the ATM inhibitor AZD0156 doubles median survival in mice intracranially implanted with ATRX-deficient GBM cells, which is not seen in ATRX-wild-type controls. This study demonstrates that ATRX-deficient high-grade gliomas (HGGs) display Chk1-mediated dysregulation of cell-cycle phase transitions, which opens a window for therapies targeting this phenotype.

ATRX, a chromatin remodeler protein, is recurrently mutated in H3F3A-mutant pediatric glioblastoma (GBM) and IDH-mutant grade 2/3 adult glioma. Previous work has shown that ATRX-deficient GBM cells show enhanced sensitivity to irradiation, but the etiology remains unclear. We found that ATRX binds regulatory elements of cell cycle phase transition genes in GBM cells, and there is a marked reduction in Checkpoint Kinase 1 (CHEK1) expression with ATRX loss, leading to early release of G2/M entry after irradiation. ATRX-deficient cells exhibit enhanced activation of master cell cycle regulator ATM with irradiation. Addition of the ATM inhibitor AZD0156 doubles median survival in mice intra-cranially implanted with ATRX-deficient GBM cells, which is not seen in ATRX-wildtype controls. This study demonstrates that ATRX-deficient high-grade gliomas (HGGs) display epigenetic dysregulation of cell cycle phase transitions, which opens a new window for therapies targeting this unique phenotype.

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

The original version of this review article unfortunately contained a mistake in the author group section.

PURPOSE OF REVIEW: H3K27M is a frequent histone mutation within diffuse midline gliomas and is associated with a dismal prognosis, so much so that the 2016 CNS WHO classification system created a specific category of “Diffuse Midline Glioma, H3K27M-mutant”. Here we outline the latest pre-clinical data and ongoing current clinical trials that target H3K27M, as well as explore diagnosis and treatment monitoring by serial liquid biopsy. RECENT FINDINGS: Multiple epigenetic compounds have demonstrated efficacy and on-target effects in pre-clinical models. The imipridone ONC201 and the IDO1 inhibitor indoximod have demonstrated early clinical activity against H3K27M-mutant gliomas. Liquid biopsy of cerebrospinal fluid has shown promise for clinical use in H3K27M-mutant tumors for diagnosis and monitoring treatment response. SUMMARY: While H3K27M has elicited a widespread platform of pre-clinical therapies with promise, much progress still needs to be made to improve outcomes for diffuse midline glioma patients. We present current treatment and monitoring techniques as well as novel approaches in identifying and targeting H3K27M-mutant gliomas.

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.

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.

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.

2012 Background: Diffuse midline glioma (DMG) with the H3K27M mutation is a lethal childhood brain cancer, with patients rarely surviving 2 years from diagnosis. There are few available means of monitoring the disease beyond serial MRI scans, making clinical decision making slow, difficult, and often reactive. 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 (baseline, 2 and 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 a total of 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). Results: Preliminary analysis of samples (n=58) demonstrates a correlation between changes in tumor size and H3K27M cf-tDNA VAF, when removing samples with concurrent bevacizumab. Analysis of remaining CSF and plasma samples is ongoing, including analysis of novel biomarkers of response. 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 an 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

// Viveka Nand Yadav 1, 2 , Daniel Zamler 1, 2 , Gregory J. Baker 1, 2, 3 , Padma Kadiyala 1, 2 , Anat Erdreich-Epstein 4 , Ana C. DeCarvalho 5 , Tom Mikkelsen 5 , Maria G. Castro 1, 2 , Pedro R. Lowenstein 1, 2 1 Department of Neurosurgery, University of Michigan Medical School, Ann Arbor, MI 48109, USA 2 Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA 3 Current address: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA 4 Department of Neurosurgery, at the Saban Research Institute at Children’s Hospital Los Angeles, CA 90027, USA 5 Departments of Neurology and Neurosurgery, Henry Ford Hospital, Detroit, MI 48202, USA Correspondence to: Pedro R. Lowenstein, email: pedrol@umich.edu Keywords: CXCR4 knockdown, autovascularization, perivascular invasion, glioma radiotherapy resistance, combination therapies Received: August 31, 2016 Accepted: October 17, 2016 Published: November 11, 2016 ABSTRACT Glioblastoma (GBM) is a highly invasive brain tumor. Perivascular invasion, autovascularization and vascular co-option occur throughout the disease and lead to tumor invasion and progression. The molecular basis for perivascular invasion, i.e., the interaction of glioma tumor cells with endothelial cells is not well characterized. Recent studies indicate that glioma cells have increased expression of CXCR4. We investigated the in-vivo role of CXCR4 in perivascular invasion of glioma cells using shRNA-mediated knock down of CXCR4. We show that primary cultures of human glioma stem cells HF2303 and mouse glioma GL26-Cit cells exhibit significant migration towards human (HBMVE) and mouse (MBVE) brain microvascular endothelial cells. Blocking CXCR4 on tumor cells with AMD3100 in-vitro , inhibits migration of GL26-Cit and HF2303 toward MBVE and HBMVE cells. Additionally, genetic down regulation of CXCR4 in mouse glioma GL26-Cit cells inhibits their in-vitro migration towards MBVE cells; in an in-vivo intracranial mouse model, these cells display reduced tumor growth and perivascular invasion, leading to increased survival. Quantitative analysis of brain sections showed that CXCR4 knockdown tumors are less invasive. Lastly, we tested the effects of radiation on CXCR4 knock down GL26-Cit cells in an orthotopic brain tumor model. Radiation treatment increased apoptosis of CXCR4 downregulated tumor cells and prolonged median survival. In summary, our data suggest that CXCR4 signaling is critical for perivascular invasion of GBM cells and targeting this receptor makes tumors less invasive and more sensitive to radiation therapy. Combination of CXCR4 knock down and radiation treatment might improve the efficacy of GBM therapy.

Oligodendroglioma is an indolent, chemosensitive glioma characterized by 1p/19q co-deletion. This loss of genetic information may be responsible for its unique treatment sensitivity. Our preliminary data suggests a role for the 1p gene, isoprenylcysteine carboxylmethyltransferase (ICMT). ICMT is the only enzyme to methylate prenylcysteine subtrates and has broad influence on lamin A function in the nucleus. We hypothesized that ICMT may be responsible for the unique treatment sensitivity of oligodendroglioma. An in vivo brain tumor model was established in ICMT knock-down transgenic mice whereby subventricular zone cells of neonates were transfected with oncogenic plasmids and a transposase to generate a tumor, recapitulating de novo glial oncogenesis. A Kaplan-Meier analysis was performed for mice treated with CCNU chemotherapy grouped according to ICMT expression levels from low to high by genotype (hypomorphic, heteromorphic, or wildtype). In vitro RNA interference studies were performed in glioma cell lines expressing a short-hairpin against ICMT. Procarbazine, carmustine, vincristine (PCV) and radiosensitivity assays were performed. The impact of ICMT inhibition on lamin A function was characterized by immunofluorescence. There was a significant difference in the median overall survival of CCNU treated transgenic mice by ICMT genotype. Hypomorphic mice had the longest median survival (91±29 days,n=9) compared to heteromorphs (78±19,n=14) and wildtype (72±44,n=16). In vitro, cells with ICMT inhibited demonstrated increased sensitivity to PCV and radiation. Lamin A immunofluorescence revealed disrupted nuclear membranes in ICMT-inhibited cells, suggesting potential increased sensitivity to DNA damaging agents. The 1p/19q co-deletion that is a characteristic of oligodendroglioma may be related to its unique treatment sensitivity. Preliminary in vivo and in vitro data implicate ICMT as a gene on chromosome 1p that may play an important role. Further characterization of the involved molecular mechanisms may lead to novel therapeutic strategies that exploit associated vulnerabilities in oligodendroglioma and other glial neoplasms. MEDULLOBLASTOMA

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, lowered tumor cell proliferation, and 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. Citation Format: Chan Chung, Stefan Sweha, Drew Pratt, Benita Tamrazi, Pooja Panwalkar, Adam Banda, Jill Bayliss, Debra Hawes, Fusheng Yang, Ho-Joon Lee, Mengrou Shan, Marcin Cieslik, Tingting Qin, Christian Werner, Daniel Wahl, Costas Lyssiotis, Viveka Nand Yadav, Carl Koschmann, Arul Chinnaiyan, Stefan Blüml, Alexander Judkins, Sriram Venneti. Integrated metabolic and epigenomic reprograming by H3K27M mutations in diffuse intrinsic pontine gliomas [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PR02.

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.

Summary H3K27M diffuse intrinsic pontine gliomas (DIPGs) are fatal and lack treatments. They mainly harbor H3.3K27M mutations resulting in H3K27me3 reduction. Integrated analysis in H3.3K27M cells, tumors, and in vivo imaging in patients showed enhanced glycolysis, glutaminolysis, and tricarboxylic acid cycle metabolism with high alpha-ketoglutarate (α-KG) production. Glucose and/or glutamine-derived α-KG maintained low H3K27me3 in H3.3K27M cells, and inhibition of key enzymes in glycolysis or glutaminolysis increased H3K27me3, altered chromatin accessibility, and prolonged survival in animal models. Previous studies have shown that mutant isocitrate-dehydrogenase (mIDH)1/2 glioma cells convert α-KG to D-2-hydroxyglutarate (D-2HG) to increase H3K27me3. Here, we show that H3K27M and IDH1 mutations are mutually exclusive and experimentally synthetic lethal. Overall, we demonstrate that H3.3K27M and mIDH1 hijack a conserved and critical metabolic pathway in opposing ways to maintain their preferred epigenetic state. Consequently, interruption of this metabolic/epigenetic pathway showed potent efficacy in preclinical models, suggesting key therapeutic targets for much needed treatments.

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.

Type I or invariant natural killer T cells belong to a unique lineage of innate T cells, which express markers of both T lymphocytes and NK cells, namely T cell receptor (TCR) and NK1.1 (CD161C), respectively. Thus, apart from direct killing of target cells like NK cells, and they also produce a myriad of cytokines which modulate the adaptive immune responses. Unlike traditional T cells which carry a conventional αβ TCR, NKT cells express semi-invariant TCR – Vα14-Jα18, coupled with Vβ8, Vβ7 and Vβ2 in mice. In humans, the invariant TCR is composed of Vα24-Jα18, coupled with Vβ11.

Whether tumors in general and gliomas in particular are self-organized has not been determined. Brain tumor self-organization would be revealed through individual recognizable structures. Here we demonstrate that brain tumors are indeed self-organized. Brain tumor self-organization is evidenced by the existence of oncostreams, multicellular structures composed of elongated glioma cells. Oncostream structure, function and molecular makeup was studied in several glioma models. In experimental mouse models, and in human GBM tumors, oncostreams were demarcated anatomically as multicellular arrangements of fusiform cells, 10–20 cells wide, of various lengths and distributed throughout the tumors. Using genetically engineered glioma models we found a negative correlation between oncostream density and survival, suggesting that oncostreams contribute to tumor aggressiveness. As oncostreams also reached tumor borders, it is likely that they participate in glioma invasion. Further, by placing slow moving glioma stem cells into oncostreams we detected that these function as streams that distribute glioma cells throughout the tumors. Importantly, we performed the molecular analysis of oncostreams using a Laser Microdissection Microscope to dissect oncostreams from surrounding tumor. The transcriptome landscape was analyzed by next generation sequencing using RNA-Seq and advanced bioinformatics algorithms. The molecular analysis showed that oncostreams are defined by a distinctive transcriptome landscape. We hereby show that oncostreams: (i) are anatomically discrete structures that reveal the existence of glioma self-organization, (ii) are functionally unique as they regulate glioma growth, invasion and spread, and (iii) molecularly distinct, their genetic landscape and associated gene networks differentiate oncostreams from surrounding glioma. Understanding glioma self-organization and its molecular basis will contribute to uncover novel targets for future translational development.

Introduction: Malignant gliomas are highly invasive tumors, associated with a dismal survival rate despite standard of care, which includes surgical resection, radiotherapy and chemotherapy with temozolomide (TMZ). Precision immunotherapies or combinations of immunotherapies that target unique tumor-specific features may substantially improve upon existing treatments. Areas covered: Clinical trials of single immunotherapies have shown therapeutic potential in high-grade glioma patients, and emerging preclinical studies indicate that combinations of immunotherapies may be more effective than monotherapies. In this review, the authors discuss emerging combinations of immunotherapies and compare efficacy of single vs. combined therapies tested in preclinical brain tumor models. Expert opinion: Malignant gliomas are characterized by a number of factors which may limit the success of single immunotherapies including inter-tumor and intra-tumor heterogeneity, intrinsic resistance to traditional therapies, immunosuppression, and immune selection for tumor cells with low antigenicity. Combination of therapies which target multiple aspects of tumor physiology are likely to be more effective than single therapies. While a limited number of combination immunotherapies are described which are currently being tested in preclinical and clinical studies, the field is expanding at an astounding rate, and endless combinations remain open for exploration. Fil: Chandran, Mayuri. University of Michigan; Estados Unidos Fil: Candolfi, Marianela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina Fil: Shah, Diana. University of Michigan; Estados Unidos Fil: Mineharu, Yohei. Kyoto University; Japón Fil: Yadav, Viveka Nand. University of Michigan; Estados Unidos Fil: Koschmann, Carl. University of Michigan; Estados Unidos Fil: Asad, Antonela Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina Fil: Lowenstein, Pedro R.. University of Michigan; Estados Unidos Fil: Castro, Maria Gabriela. University of Michigan; Estados Unidos

Poxviruses display species tropism—variola virus is a human-specific virus, while vaccinia virus causes repeated outbreaks in dairy cattle. Consistent with this, variola virus complement regulator SPICE (smallpox inhibitor of complement enzymes) exhibits selectivity in inhibiting the human alternative complement pathway and vaccinia virus complement regulator VCP (vaccinia virus complement control protein) displays selectivity in inhibiting the bovine alternative complement pathway. In the present study, we examined the species specificity of VCP and SPICE for the classical pathway (CP). We observed that VCP is ∼43-fold superior to SPICE in inhibiting bovine CP. Further, functional assays revealed that increased inhibitory activity of VCP for bovine CP is solely due to its enhanced cofactor activity, with no effect on decay of bovine CP C3-convertase. To probe the structural basis of this specificity, we utilized single- and multi-amino-acid substitution mutants wherein 1 or more of the 11 variant VCP residues were substituted in the SPICE template. Examination of these mutants for their ability to inhibit bovine CP revealed that E108, E120, and E144 are primarily responsible for imparting the specificity and contribute to the enhanced cofactor activity of VCP. Binding and functional assays suggested that these residues interact with bovine factor I but not with bovine C4(H 2 O) (a moiety conformationally similar to C4b). Mapping of these residues onto the modeled structure of bovine C4b-VCP-bovine factor I supported the mutagenesis data. Taken together, our data help explain why the vaccine strain of vaccinia virus was able to gain a foothold in domesticated animals. IMPORTANCE Vaccinia virus was used for smallpox vaccination. The vaccine-derived virus is now circulating and causing outbreaks in dairy cattle in India and Brazil. However, the reason for this tropism is unknown. It is well recognized that the virus is susceptible to neutralization by the complement classical pathway (CP). Because the virus encodes a soluble complement regulator, VCP, we examined whether this protein displays selectivity in targeting bovine CP. Our data show that it does exhibit selectivity in inhibiting the bovine CP and that this is primarily determined by its amino acids E108, E120, and E144, which interact with bovine serine protease factor I to inactivate bovine C4b—one of the two subunits of CP C3-convertase. Of note, the variola complement regulator SPICE contains positively charged residues at these positions. Thus, these variant residues in VCP help enhance its potency against the bovine CP and thereby the fitness of the virus in cattle.

Natural killer (NK) cells safeguard against early tumor formation by destroying transformed target cells in a process referred to as NK immune surveillance. However, the immune escape mechanisms used by malignant brain tumors to subvert this innate type of immune surveillance remain unclear. Here we show that malignant glioma cells suppress NK immune surveillance by overexpressing the β-galactoside–binding lectin galectin-1. Conversely, galectin-1–deficient glioma cells could be eradicated by host NK cells before the initiation of an antitumor T-cell response. In vitro experiments demonstrated that galectin-1–deficient GL26-Cit glioma cells are ∼3-fold more sensitive to NK-mediated tumor lysis than galectin-1–expressing cells. Our findings suggest that galectin-1 suppression in human glioma could improve patient survival by restoring NK immune surveillance that can eradicate glioma cells. Cancer Res; 74(18); 5079–90. ©2014 AACR.

As glioma cells infiltrate the brain they become associated with various microanatomic brain structures such as blood vessels, white matter tracts, and brain parenchyma. How these distinct invasion patterns coordinate tumor growth and influence clinical outcomes remain poorly understood. We have investigated how perivascular growth affects glioma growth patterning and response to antiangiogenic therapy within the highly vascularized brain. Orthotopically implanted rodent and human glioma cells are shown to commonly invade and proliferate within brain perivascular space. This form of brain tumor growth and invasion is also shown to characterize de novo generated endogenous mouse brain tumors, biopsies of primary human glioblastoma (GBM), and peripheral cancer metastasis to the human brain. Perivascularly invading brain tumors become vascularized by normal brain microvessels as individual glioma cells use perivascular space as a conduit for tumor invasion. Agent-based computational modeling recapitulated biological perivascular glioma growth without the need for neoangiogenesis. We tested the requirement for neoangiogenesis in perivascular glioma by treating animals with angiogenesis inhibitors bevacizumab and DC101. These inhibitors induced the expected vessel normalization, yet failed to reduce tumor growth or improve survival of mice bearing orthotopic or endogenous gliomas while exacerbating brain tumor invasion. Our results provide compelling experimental evidence in support of the recently described failure of clinically used antiangiogenics to extend the overall survival of human GBM patients.

e13508 Background: Pediatric and adult high-grade glioma (HGG) frequently harbor PDGFRA alterations. 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, will increase CNS penetration and efficacy of dasatinib in in vitro and in vivo models as well as in human PDGFRA-driven glioma. Methods: Tumors were generated in mice using an intra-uterine electroporation (IUE) model [introduction of TP53, PDGFRA and H3K27M mutations in pre-natal cortex]. Dose response, synergism studies, P-GP inhibition and pharmacodynamics/pharmacokinetic studies were then performed on in vitro and in vivo models employing this IUE system. A phase 2 trial employing dasatinib and everolimus was established for children with HGG and diffuse intrinsic pontine glioma (DIPG) that contain PDGFRA alterations (NCT03352427). Paired CSF/plasma samples (before and after addition of everolimus) were collected from enrolled patients. Results: Dasatinib effectively treated mouse HGG cells with an IC50 of 100 nM. Dose-dependent reduction in PDGFRA and pPDGFRA was found. P-gp inhibitor assay confirmed that everolimus strongly blocks P-gp activity at 1 uM (p = 0.0028 vs untreated). Mice treated with dasatinib and everolimus had extended survival as compared to control. Two-hour exposure to everolimus resulted in sub-IC50 dasatinib concentration in cortex (23 nM) and tumor (65 nM). 24-hour exposure to everolimus resulted in greater cortex (235 nM) and tumor (509 nM) concentrations. Two trial patients, recurrent HGG ( PDGFRA-amplified) and recurrent DIPG ( PDGFRA D842V) respectively, survived 6 months and 9 months (ongoing) after progression, which compares very favorably to historical controls. A paired CSF sample from the PDGFRA-amplified patient showed a 50% increase in CSF dasatinib level after addition of everolimus. Conclusions: Dasatinib treatment of PDGFRA-driven HGG is improved with everolimus blockade of P-gp and represents a novel route for improving CNS penetration and efficacy of therapies for HGG. Clinical trial information: NCT03352427.

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.

Variola and vaccinia viruses, the two most important members of the family Poxviridae, are known to encode homologs of the human complement regulators named smallpox inhibitor of complement enzymes (SPICE) and vaccinia virus complement control protein (VCP), respectively, to subvert the host complement system. Intriguingly, consistent with the host tropism of these viruses, SPICE has been shown to be more human complement-specific than VCP, and in this study we show that VCP is more bovine complement-specific than SPICE. Based on mutagenesis and mechanistic studies, we suggest that the major determinant for the switch in species selectivity of SPICE and VCP is the presence of oppositely charged residues in the central complement control modules, which help enhance their interaction with factor I and C3b, the proteolytically cleaved form of C3. Thus, our results provide a molecular basis for the species selectivity in poxviral complement regulators.