Diffuse Intrinsic Pontine Glioma Cells Research Articles

DDEL-04. Pharmacokinetic/Pharmacodynamic analysis of the DNA methyltransferase inhibitor 5-azacytidine shows adequate brain tissue penetration with intravenous administration in a DIPG mouse patient-derived xenograft model.

One of the biggest obstacles in developing effective therapies for CNS tumors is drug delivery due to the enigmatic challenge of penetrating the blood-brain barrier. 5-azacytidine is a DNA methyltransferase inhibitor which was the first epigenetic targeting chemotherapeutic approved by the FDA. Altered DNA methylation is a hallmark of many cancers, including diffuse intrinsic pontine glioma (DIPG). 5-azacytidine has been shown to be active in DIPG cell lines, with only modest in-vivo activity. We have previously shown in a non-human primate model that intravenous (IV) administration of 5-azacitidine does not result in measurable CSF penetration, while intrathecal (IT) administration does and is well tolerated. To follow up these studies in a tumor-bearing mouse model (HSJD DIPG007), we performed pharmacokinetic (PK) and pharmacodynamic (PD) analysis following IV vs. IT administration. Forty mice were randomized to receive four weekly doses of IV 5-azacytidine (25 mg/kg), IT 5-azacytidine (40 μg), or corresponding vehicle controls. Four mice from each arm were sacrificed 30 minutes after the last dose and brain tissue was collected for PK/PD analysis. Drug concentration was quantified using ultra-high-performance liquid chromatography with tandem mass spectrometric detection, while pharmacodynamic methylation profiling was performed using the Infinium MethylationEPIC BeadChip (850K). Brain tissue concentrations were comparable between IV (7.6-58.0 pg/mg) and IT (6.9-63.9 pg/mg) dosing. Methylation profiling unexpectedly showed a significantly more pronounced pharmacodynamic effect with IV dosing vs. IT, with a mean decrease of 13.6% vs. 2.6% in global DNA methylation score (GDMS = percentage of highly methylated (beta ≥ 0.7) genomic loci) compared to vehicle controls. For the remaining mice, there was no significant difference in survival. Our results are encouraging that phenotypically relevant demethylating effects can be achieved in the CNS with IV 5-azacytidine administration; however, further research is needed to develop promising combination strategies in DIPG.

DIPG-01. Targeting p300: A master regulator of childhood diffuse intrinsic pontine glioma (DIPG)

Abstract Diffuse intrinsic pontine gliomas (DIPG) are heterogeneous, highly aggressive, accounting for ~10% of all childhood central nervous system tumors that are poorly understood. The heterogeneity of the cell populations could contribute to major variability in tumor growth and chemoresistance followed by inadequate treatment responses. Approximately 80% of DIPG patients harbor mutations on lysine-27 of histone-3 (H3K27M), which forms heterotypic nucleosomes with normal, acetylated-H3K27 (H3K27ac), and colocalizes with bromodomain proteins to sites of active transcription. p300 (or EP300) bromodomain has recently been exploited as a potential therapeutic target in other cancer, with a small molecule inhibitor showing antiproliferative activity. Bromodomain inhibitors (BrDi) have been suggested as a potential therapeutic target for DIPG too which showed antiproliferative activity in vitro. We therefore hypothesize that p300 bromodomain inhibition is a potential therapeutic strategy that must be urgently explored in DIPG. p300 is a multi-domain enhancer protein, expressed in 80% of gliomas, responsible for H3K27ac mark and localizes to sites of H3K27ac enrichment, where it serves as an essential binding partner of histones to various transcription factors. p300 interacts with BMI-1, a polycomb repressive complex 1 (PRC1) group of transcription factor, through an intermediate protein NANOG, which promotes cell proliferation in head-neck squamous cell carcinoma. However, immunohistochemical study demonstrated that there is no NANOG expression found in DIPG patient samples. Therefore, we propose that p300 and BMI-1 interact with each other directly or through an intermediate molecule, essential for tumor growth and thus can be considered as a possible therapeutic target for DIPG. The overall outcome of this research will reveal the unexplored mechanisms of DIPG cell proliferation to overcome chemoresistance in sick children and develop drug/combination against this deadly malignancy, which can be used for future clinical trials.

IMMU-14. SynNotch chimeric antigen receptor (CAR) T-cells as a potential treatment for diffuse intrinsic pontine glioma (DIPG)/diffuse midline glioma (DMG)

BACKGROUND: The development of effective chimeric antigen receptor (CAR) T-cell therapies for malignant pediatric brain tumors remains a challenge due to multiple barriers, including antigenic heterogeneity, on-target off-tumor toxicities, and T cell exhaustion. We have adopted a novel synthetic Notch “synNotch” receptor system and developed innovative T-cell circuits that recognize tumor cells based on the “prime-and-kill” strategy. METHODS: We created a novel synNotch-CAR circuit in which the brain/glioma-specific antigen Brevican (BCAN) primes the T cells to induce expression of a CAR that recognizes interleukin-13 receptor α2 (IL-13Rα2) and ephrin type A receptor (EphA2), thereby eradicating glioma cells expressing either antigen. Immunocompromised mice bearing the SF8628 DIPG cell line in the frontal lobe or brain stem received a single intravenous (IV) infusion of synNotch CAR T-cells (2.5 x 106 each of CD4+ and CD8+ T cells) on day 6 following the tumor inoculation. Mice were monitored for toxicity and tumor growth. RESULTS: Following this synNotch CAR T-cell dose, although tumors in the brainstem did not regress, 3 of the 5 mice with frontal lobe tumors demonstrated complete and sustained remission. Our histological analyses revealed primed CAR T-cells both within and surrounding the tumor in both settings. By flow cytometry, we confirmed the CAR T-cells in the CNS were primed and mostly did not express an exhaustive phenotype. In the spleen, we also found the CAR T-cells in a more naïve and central memory state. CONCLUSIONS: Our work so far has demonstrated that synNotch-CAR T-cells are able to traffic to the tumor microenvironment even in the brainstem, are primed to express the CAR, and most do not express an exhaustive phenotype. Future work will include CAR T-cell dose optimization, continued assessments of the tumor microenvironment, and investigating for antigen escape.

DIPG-62. Reducing the levels of genomic 5-hydroxymethylcytosine by inhibiting the TET pathway induces apoptosis and decreases proliferation in Diffuse Intrinsic Pontine Glioma (DIPG)

The H3K27M mutation, observed in 70%-90% of DIPG, causes global hypomethylation. The Ten-Eleven-Translocation enzymes, TETs, which convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) regulate the methylation patterns in the genome. Previously, we observed an increase in the 5hmC levels and altered TET activity in DIPG. We hypothesize that targeting the TETs in DIPG could restore the epigenetic balance. In this study, we target the TET pathway with cell-permeable 2-hydroxyglutarate (2HG) and Bobcat339, a cytosine-based TET inhibitor. We see a dose-dependent decrease in 5hmC (46-96% reduction) and increase in 5mC (~5fold increase) in multiple DIPG cell lines treated with 2HG. We see a corresponding increase in apoptosis and reduction in proliferation (~8fold increase in cleaved-Caspase3 signal (P=0.0001) and ~55% reduction in BrdU-incorporation measured by IF (P=0.0036) in JHH DIPG16A with similar results in JHH DIPG1, HSJD007, and SUDIPG6). We see a 54-90% reduction in 5hmC and ~3fold increase in 5mC with Bobcat 339 treatment in DIPG cell lines JHH DIPG1, JHH DIPG16A, HSJD007, and SUDIPG6. We also see a corresponding increase in apoptosis as measured by cleaved PARP western blot and decrease in proliferation as measured by BrdU incorporation (approximately 32-53% decrease in proliferation). Combined treatment with 2HG and Bobcat339 lead to greater decreases in 5hmC and increases in 5mC compared to single treatment, with synergistic suppression of cell growth as measured by SynergyFinder in HSJD007, JHH DIPG1, and JHH DIPG16A. In vivo studies targeting DIPG orthotopic xenografts are currently underway. These results suggest that TETs contribute to the hypomethylated state observed in DIPG. Thus, inhibiting the TETs can be explored as a potential therapeutic approach to treat DIPG.

DIPG-20. Copper chelation therapy targets S-adenosylmethionine (SAM) metabolism and epigenetic regulators in diffuse intrinsic pontine glioma (DIPG)

DIPG is an incurable pediatric brain cancer of the ventral pons characterized by its complex epigenetic profile. Up to 81% of patients present with mutation H3K27M, resulting in the global reduction of H3K27 trimethylation and increased H3K27 acetylation, deregulating gene expression through an aberrant pattern of epigenetic modification. These alterations affect many cellular and metabolic mechanisms, complicating the search for effective targeted therapeutic strategies. Copper is highly abundant in the pons and is essential for normal brain function and development. However, excess copper accumulation is implicated in several neurological diseases and cancers. Incidentally, recent investigations have indicated the H3-H4 dimer can interact with copper acting as a reductase enzyme. Copper chelation therapy is clinically approved for pediatric patients with Wilson’s Disease, improving their neurological symptoms, and is being trialed in several cancers. We therefore hypothesized copper chelation may represent an effective therapeutic strategy for DIPG. Copper chelator tetraethylenepentamine (TEPA) decreased cell growth and induced apoptosis in DIPG cell lines. To understand these results, unbiased RNA-seq and metabolomics analyses were performed, revealing downregulation of EZH2, DNMT1 and DNMT3B, upregulation of KDM6B and the disruption of key enzymes in the S-adenosylmethionine (SAM)-cycle. Importantly, TEPA downregulated SAM, which donates methyl groups for methylation, S-adenosylhomocysteine, its post-methylation product and α-ketoglutarate, a co-factor for KDM6B. Western blots confirmed the reduced expression of EZH2, DNMT1 and DNMT3B, with further blots examining the chromatin cell fraction revealing modulation of H3K27 trimethylation through copper or TEPA stimulation, and reduction of H3K27 acetylation by TEPA. Importantly, in vitro combinations with Panobinostat were synergistic, while in vivo investigations demonstrated TEPA improved survival in an orthotopic patient derived xenograft (PDX) model, showing complete tumor regression in 25% of treated mice. This study indicates a novel use for copper chelators as epigenetic drugs, and their potential as therapeutics for DIPG.

Histone H3.3 K27M chromatin functions implicate a network of neurodevelopmental factors including ASCL1 and NEUROD1 in DIPG

BackgroundThe histone variant H3.3 K27M mutation is a defining characteristic of diffuse intrinsic pontine glioma (DIPG)/diffuse midline glioma (DMG). This histone mutation is responsible for major alterations to histone H3 post-translational modification (PTMs) and subsequent aberrant gene expression. However, much less is known about the effect this mutation has on chromatin structure and function, including open versus closed chromatin regions as well as their transcriptomic consequences.ResultsRecently, we developed isogenic CRISPR-edited DIPG cell lines that are wild-type for histone H3.3 that can be compared to their matched K27M lines. Here we show via ATAC-seq analysis that H3.3K27M glioma cells have unique accessible chromatin at regions corresponding to neurogenesis, NOTCH, and neuronal development pathways and associated genes that are overexpressed in H3.3K27M compared to our isogenic wild-type cell line. As to mechanisms, accessible enhancers and super-enhancers corresponding to increased gene expression in H3.3K27M cells were also mapped to genes involved in neurogenesis and NOTCH signaling, suggesting that these pathways are key to DIPG tumor maintenance. Motif analysis implicates specific transcription factors as central to the neuro-oncogenic K27M signaling pathway, in particular, ASCL1 and NEUROD1.ConclusionsAltogether our findings indicate that H3.3K27M causes chromatin to take on a more accessible configuration at key regulatory regions for NOTCH and neurogenesis genes resulting in increased oncogenic gene expression, which is at least partially reversible upon editing K27M back to wild-type.

Functionalized Macrophage Exosomes with Panobinostat and PPM1D-siRNA for Diffuse Intrinsic Pontine Gliomas Therapy.

Diffuse intrinsic pontine glioma (DIPG) is a rare and fatal pediatric brain tumor. Mutation of p53‐induced protein phosphatase 1 (PPM1D) in DIPG cells promotes tumor cell proliferation, and inhibition of PPM1D expression in DIPG cells with PPM1D mutation effectively reduces the proliferation activity of tumor cells. Panobinostat effectively kills DIPG tumor cells, but its systemic toxicity and low blood–brain barrier (BBB) permeability limits its application. In this paper, a nano drug delivery system based on functionalized macrophage exosomes with panobinostat and PPM1D‐siRNA for targeted therapy of DIPG with PPM1D mutation is prepared. The nano drug delivery system has higher drug delivery efficiency and better therapeutic effect than free drugs. In vivo and in vitro experimental results show that the nano drug delivery system can deliver panobinostat and siRNA across the BBB and achieve a targeted killing effect of DIPG tumor cells, resulting in the prolonged survival of orthotopic DIPG mice. This study provides new ideas for the delivery of small molecule drugs and gene drugs for DIPG therapy.

Exploiting 4-1BB immune checkpoint to enhance the efficacy of oncolytic virotherapy for diffuse intrinsic pontine gliomas.

Diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors, and patient survival has not changed despite many therapeutic efforts, emphasizing the urgent need for effective treatments. Here, we evaluated the anti-DIPG effect of the oncolytic adenovirus Delta-24-ACT, which was engineered to express the costimulatory ligand 4-1BBL to potentiate the antitumor immune response of the virus. Delta-24-ACT induced the expression of functional 4-1BBL on the membranes of infected DIPG cells, which enhanced the costimulation of CD8+ T lymphocytes. In vivo, Delta-24-ACT treatment of murine DIPG orthotopic tumors significantly improved the survival of treated mice, leading to long-term survivors that developed immunological memory against these tumors. In addition, Delta-24-ACT was safe and caused no local or systemic toxicity. Mechanistic studies showed that Delta-24-ACT modulated the tumor-immune content, not only increasing the number, but also improving the functionality of immune cells. All of these data highlight the safety and potential therapeutic benefit of Delta-24-ACT the treatment of patients with DIPG.

An affinity for brainstem microglia in pediatric high-grade gliomas of brainstem origin.

BackgroundHigh-grade gliomas (HGG) in children have a devastating prognosis and occur in a remarkable spatiotemporal pattern. Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), typically occur in mid-childhood, while cortical HGGs are more frequent in older children and adults. The mechanisms behind this pattern are not clear.MethodsWe used mouse organotypic slice cultures and glial cell cultures to test the impact of the microenvironment on human DIPG cells. Comparing the expression between brainstem and cortical microglia identified differentially expressed secreted proteins. The impact of some of these proteins on DIPGs was tested.ResultsDIPGs, pediatric HGGs of brainstem origin, survive and divide more in organotypic slice cultures originating in the brainstem as compared to the cortex. Moreover, brainstem microglia are better able to support tumors of brainstem origin. A comparison between the two microglial populations revealed differentially expressed genes. One such gene, interleukin-33 (IL33), is highly expressed in the pons of young mice and its DIPG receptor is upregulated in this context. Consistent with this observation, the expression levels of IL33 and its receptor, IL1RL1, are higher in DIPG biopsies compared to low-grade cortical gliomas. Furthermore, IL33 can enhance proliferation and clonability of HGGs of brainstem origin, while blocking IL33 in brainstem organotypic slice cultures reduced the proliferation of these tumor cells.ConclusionsCrosstalk between DIPGs and the brainstem microenvironment, in particular microglia, through IL33 and other secreted factors, modulates spatiotemporal patterning of this HGG and could prove to be an important future therapeutic target.

Dual IGF1R/IR inhibitors in combination with GD2-CAR T-cells display a potent anti-tumor activity in diffuse midline glioma H3K27M-mutant.

BackgroundDiffuse midline gliomas (DMG) H3K27M-mutant, including diffuse intrinsic pontine glioma (DIPG), are pediatric brain tumors associated with grim prognosis. Although GD2-CAR T-cells demonstrated significant anti-tumor activity against DMG H3K27M-mutant in vivo, a multimodal approach may be needed to more effectively treat patients. We investigated GD2 expression in DMG/DIPG and other pediatric high-grade gliomas (pHGG) and sought to identify chemical compounds that would enhance GD2-CAR T-cell anti-tumor efficacy.MethodsImmunohistochemistry in tumor tissue samples and immunofluorescence in primary patient-derived cell lines were performed to study GD2 expression. We developed a high-throughput cell-based assay to screen 42 kinase inhibitors in combination with GD2-CAR T-cells. Cell viability, western blots, flow-cytometry, real time PCR experiments, DIPG 3D culture models, and orthotopic xenograft model were applied to investigate the effect of selected compounds on DIPG cell death and CAR T-cell function.ResultsGD2 was heterogeneously, but widely, expressed in the tissue tested, while its expression was homogeneous and restricted to DMG/DIPG H3K27M-mutant cell lines. We identified dual IGF1R/IR antagonists, BMS-754807 and linsitinib, able to inhibit tumor cell viability at concentrations that do not affect CAR T-cells. Linsitinib, but not BMS-754807, decreases activation/exhaustion of GD2-CAR T-cells and increases their central memory profile. The enhanced anti-tumor activity of linsitinib/GD2-CAR T-cell combination was confirmed in DIPG models in vitro, ex vivo, and in vivo.ConclusionOur study supports the development of IGF1R/IR inhibitors to be used in combination with GD2-CAR T-cells for treating patients affected by DMG/DIPG and, potentially, by pHGG.

TP53 wild-type/PPM1D mutant diffuse intrinsic pontine gliomas are sensitive to a MDM2 antagonist

Diffuse intrinsic pontine gliomas (DIPGs) are high-grade tumors of the brainstem that often occur in children, with a median overall survival of less than one year. Given the fact that DIPGs are resistant to chemotherapy and are not amenable to surgical resection, it is imperative to develop new therapeutic strategies for this deadly disease. The p53 pathway is dysregulated by TP53 (~ 60%) or PPM1D gain-of-function mutations (~ 30%) in DIPG cases. PPM1D gain-of-function mutations suppress p53 activity and result in DIPG tumorigenesis. While MDM2 is a major negative regulator of p53, the efficacy of MDM2 inhibitor has not been tested in DIPG preclinical models. In this study, we performed a comprehensive validation of MDM2 inhibitor RG7388 in patient-derived DIPG cell lines established from both TP53 wild-type/PPM1D-mutant and TP53 mutant/PPM1D wild-type tumors, as well in TP53 knockout isogenic DIPG cell line models. RG7388 selectively inhibited the proliferation of the TP53 wild-type/PPM1D mutant DIPG cell lines in a dose- and time-dependent manner. The anti-proliferative effects were p53-dependent. RNA-Seq data showed that differential gene expression induced by RG7388 treatment was enriched in the p53 pathways. RG7388 reactivated the p53 pathway and induced apoptosis as well as G1 arrest. In vivo, RG7388 was able to reach the brainstem and exerted therapeutic efficacy in an orthotopic DIPG xenograft model. Hence, this study demonstrates the pre-clinical efficacy potential of RG7388 in the TP53 wild-type/PPM1D mutant DIPG subgroup and may provide critical insight on the design of future clinical trials applying this drug in DIPG patients.

283 TIM-3 blockade modulates the tumor microenvironment improving the outcome of preclinical pediatric diffuse midline glioma models

BackgroundDiffuse Midline Gliomas (DMGs), encompassing Diffuse Intrinsic Pontine Gliomas (DIPGs), are the most aggressive pediatric brain tumors. Their meager survival has not changed despite the combination of radiotherapy with targeted therapies emphasizing the urgent need for effective treatments. Recent research suggested that the DIPG tumor microenvironment is neither highly immunosuppressive nor inflammatory.1 These analyses showed the lack of infiltrating lymphocytes and the abundance of CD11b+ cells. TIM-3 is a member of the T-cell immunoglobulin and mucin domain protein family expressed on multiple immune cell types, including T cells, Treg, NK cells, monocytes, dendritic cells, and microglia, where it potently regulates not only adaptive immunity but also innate immunity.2–3 Therefore, TIM-3 inhibitors could challenge several components in the tumor microenvironment, thereby providing potentially effective treatment for DMGs.MethodsNP53 and XFM murine DIPG cell lines were used for animal experiments in immunocompetent orthotopic models. The tumors were processed by mechanical and enzymatic digestion and immune populations were analyzed by a flow cytometry panel. Antibodies against NK cells (NK1.1), CD4 (GK1.5), CD8 (CD8β) were used for animal depletion experiments alone or in combination.ResultsIn silico assessment of TIM-3 expression in DIPG datasets showed a robust expression of this gene. Moreover, single-cell sequencing analyses of DIPG biopsies uncover its expression in the myeloid compartment (especially in microglia). In vivo efficacy studies showed that treatment with anti-TIM-3 antibody significantly increased the overall survival in two DIPG immunocompetent orthotopic animal models (doubling the median), lead to long-term survivors free of disease (50%) and showed immune memory. Analyses of CD45+ populations in the tumor microenvironment showed a significant increase in microglia, granulocytes, NK and CD8+ cells corresponding with a NK and T-cell activate phenotypes in treated-mice. In addition, we have a substantial decrease in the Treg population, which causes an increase in the CD8/Treg ratio. CD4 and CD8 T-cell depletion led to a significant but not total loss of treatment efficacy. NK cells depletion also reduced the effectiveness of this therapy, albeit to a lesser extent than CD4-CD8 depletion. We are currently investigating the role of microglia in the outcome of the treatment.ConclusionsOur data uncovered TIM-3 as a potential target for the treatment of DIPG tumors. Inhibition of this molecule led to a potent antitumor effect mediated by a profound tumor microenvironment remodelling.ReferencesLieberman NAP, DeGolier K, Kovar HM, et al. Characterization of the immune microenvironment of diffuse intrinsic pontine glioma: implications for development of immunotherapy. Neuro Oncol 2019;21(1):83–94. doi:10.1093/neuonc/noy145.Acharya N, Sabatos-Peyton C, Anderson AC. Tim-3 finds its place in the cancer immunotherapy landscape. J Immunother Cancer 2020;8(1):e000911. doi:10.1136/jitc-2020-000911.Wolf Y, Anderson AC, Kuchroo VK. TIM3 comes of age as an inhibitory receptor. Nat Rev Immunol 2020;20(3):173–185. doi:10.1038/s41577-019-0224-6

91 Impact of ultra-fast ‘FLASH’ radiotherapy on single cell immunogenomics in diffuse intrinsic pontine glioma (DIPG)

BackgroundDiffuse intrinsic pontine gliomas (DIPG’s) are immunologically inert tumors with a median survival of 9–15 months. Radiation therapy (RT) is the mainstay treatment for DIPG but is associated with immunodepletion of the tumor microenvironment (TME) at high dose ranges. FLASH, or ultra-fast dose rate RT, represents a novel ablative technique that may spare TME immune responses while decreasing tumor burden. Here, we present single-cell immune profiling of DIPG tumors treated with FLASH, conventional dose rate RT (CONV) or no RT (SHAM).MethodsMurine H3.3K27M mutant DIPG cells were stereotactically injected and tumor induction confirmed by magnetic resonance imaging (MRI) 15 days later. DIPG-bearing mice were randomly assigned to one of three treatment groups (n=4/group), FLASH, CONV or SHAM. A fourth group with no tumor (NML) was included as a negative biological control. A modified linear accelerator was used to deliver 15 Gy of electron RT to the brainstem at dose rates of 90 Gy/second and 2 Gy/minute, for the FLASH and CONV groups, respectively. Four days post-RT, mice brainstems were harvested, homogenized, stained for CD45 and tagged with a hashtag antibody specific to each group. CD45+ immune cells were isolated and sequenced using the 10X Genomics chromium single-cell 3’ platform. After processing and alignment of the reads using CellRanger with default parameters, the data was quality checked and filtered before hashtag demultiplexing, unsupervised clustering and downstream analysis was implemented following the Seurat R package. Differential expression evaluated based on the non-parametric Wilcoxon rank sum test. Key genes determine by an adjusted p value of < 0.05 based on bonferroni correction and |avg log2FC| > 0.8.ResultsPreliminary analysis identifies 15 clusters with distinct CD45 immune phenotypes (figure 1). Differential gene expression analysis by hashtag antibody (treatment group) reveals 14 clusters differentially expressing key genes, including 3 clusters upregulated in DIPG compared to NML, and 2 clusters upregulated in irradiated tumors compared to SHAM and NML (figure 2). Notably, analysis demonstrates an individual cluster upregulated in FLASH versus all other groups (p = 3.07E-171). Further deconvolution of specific immune phenotypes represented by each cluster is ongoing.Abstract 91 Figure 1tSNE plot based on clustering of RNA signatures, grouped by RNAAbstract 91 Figure 2tSNE plot based on clustering of RNA signatures, grouped by hashtag antibodyConclusionsOur preliminary analysis shows differential immune responses among DIPG tumors compared to NML. We also find several immune cell subsets that are unique to DIPG treated with CONV or FLASH compared to unirradiated samples. Most notably, we identify a single immune cell subset that is exclusive to FLASH alone, indicating that FLASH elicits a unique immune response in murine DIPG.

Focused Ultrasound-Mediated Blood-Brain Barrier Opening Enhances Panobinostat Efficacy in a Murine Diffuse Intrinsic Pontine Glioma Model

Purpose/Objective(s)Diffuse intrinsic pontine glioma (DIPG) is the most lethal brain tumor in children with no effective treatment options to date. The prognosis remains poor with a median overall survival of less than 1 year. Recent in vitro drug screening studies found the histone deacetylase (HDAC) inhibitor, panobinostat, to be highly effective against DIPG. However, as panobinostat has poor blood-brain barrier (BBB) penetration, phase 1 clinical trials using systemic administration of panobinostat have been unsuccessful. Therefore, improved efficacy will require enhancement of delivery into the brain. Focused ultrasound (FUS), a non-ionizing acoustic form of radiation, has been shown to safely and non-invasively open the BBB, which can increase drug delivery. In this study, we hypothesize that FUS-mediated BBB opening (BBBO) can enhance the delivery of panobinostat for a therapeutic benefit in DIPG.Materials/MethodsA syngeneic DIPG model was established by injecting mouse DIPG cells (PDGFB+, H3.3K27M, p53−/−) intracranially. A single-element, spherical-segment FUS transducer (center frequency: 0.5 MHz) driven by a function generator through a power amplifier was used with concurrent microbubble injection to perform BBBO. We then utilized magnetic resonance imaging to evaluate BBBO and tumor progression.ResultsWe first demonstrated that FUS-mediated BBBO is safe and feasible in mice with DIPG tumors by MRI and passive cavitation detection. Our DIPG cell line was shown to be very sensitive to panobinostat in cell viability assay (IC50 = 0.012 μM). The combined treatment of FUS-mediated BBBO and panobinostat showed benefits in both local control and overall survival.ConclusionThe current results demonstrate that FUS could increase the treatment efficacy of panobinostat in a DIPG animal model due to the improved targeted dose of systemic panobinostat in the DIPG tumors. We believe these data establish a strong preclinical rationale to assess the combination of FUS-mediated BBB opening with panobinostat in pediatric patients with DIPG. Diffuse intrinsic pontine glioma (DIPG) is the most lethal brain tumor in children with no effective treatment options to date. The prognosis remains poor with a median overall survival of less than 1 year. Recent in vitro drug screening studies found the histone deacetylase (HDAC) inhibitor, panobinostat, to be highly effective against DIPG. However, as panobinostat has poor blood-brain barrier (BBB) penetration, phase 1 clinical trials using systemic administration of panobinostat have been unsuccessful. Therefore, improved efficacy will require enhancement of delivery into the brain. Focused ultrasound (FUS), a non-ionizing acoustic form of radiation, has been shown to safely and non-invasively open the BBB, which can increase drug delivery. In this study, we hypothesize that FUS-mediated BBB opening (BBBO) can enhance the delivery of panobinostat for a therapeutic benefit in DIPG. A syngeneic DIPG model was established by injecting mouse DIPG cells (PDGFB+, H3.3K27M, p53−/−) intracranially. A single-element, spherical-segment FUS transducer (center frequency: 0.5 MHz) driven by a function generator through a power amplifier was used with concurrent microbubble injection to perform BBBO. We then utilized magnetic resonance imaging to evaluate BBBO and tumor progression. We first demonstrated that FUS-mediated BBBO is safe and feasible in mice with DIPG tumors by MRI and passive cavitation detection. Our DIPG cell line was shown to be very sensitive to panobinostat in cell viability assay (IC50 = 0.012 μM). The combined treatment of FUS-mediated BBBO and panobinostat showed benefits in both local control and overall survival. The current results demonstrate that FUS could increase the treatment efficacy of panobinostat in a DIPG animal model due to the improved targeted dose of systemic panobinostat in the DIPG tumors. We believe these data establish a strong preclinical rationale to assess the combination of FUS-mediated BBB opening with panobinostat in pediatric patients with DIPG.

The Long Non-Coding RNA H19 Drives the Proliferation of Diffuse Intrinsic Pontine Glioma with H3K27 Mutation.

Diffuse intrinsic pontine glioma (DIPG) is an incurable paediatric malignancy. Identifying the molecular drivers of DIPG progression is of the utmost importance. Long non-coding RNAs (lncRNAs) represent a large family of disease- and tissue-specific transcripts, whose functions have not yet been elucidated in DIPG. Herein, we studied the oncogenic role of the development-associated H19 lncRNA in DIPG. Bioinformatic analyses of clinical datasets were used to measure the expression of H19 lncRNA in paediatric high-grade gliomas (pedHGGs). The expression and sub-cellular location of H19 lncRNA were validated in DIPG cell lines. Locked nucleic acid antisense oligonucleotides were designed to test the function of H19 in DIPG cells. We found that H19 expression was higher in DIPG vs. normal brain tissue and other pedHGGs. H19 knockdown resulted in decreased cell proliferation and survival in DIPG cells. Mechanistically, H19 buffers let-7 microRNAs, resulting in the up-regulation of oncogenic let-7 target (e.g., SULF2 and OSMR). H19 is the first functionally characterized lncRNA in DIPG and a promising therapeutic candidate for treating this incurable cancer.

Abstract 3025: PRMT5 inhibition reduces viability and stemness of pediatric high grade glioma

Abstract No current therapies halt the progression of Diffuse Intrinsic Pontine Glioma (DIPG), a pediatric brainstem tumor, with a median survival of only 6-9 months. DIPG cells carry characteristic missense histone H3 K27M mutations and activating ACVR1 mutations in 80% and 25% of cases respectively, and have transcriptomes similar to that of oligodendrocyte precursor cells (OPCs). We aim to find novel therapeutic targets for DIPG cells carrying these ACVR1 and H3 mutations. We performed parallel adherent and spheroid in vitro viability screens in patient derived DIPG cells using a small molecule epigenetic library generated by the SGC (https://www.thesgc.org/chemical-probes/epigenetics). 16/51 probes significantly reduced the viability of HSJD-DIPG-007 cells (ACVR1 R206H, H3.3 K27M) grown adherently for 7 days, of which 6 compounds inhibit the protein arginine methyltransferase (PRMT) family. The 2 most effective compounds inhibit PRMT5 (GSK591 and LLY-283) reducing viability >50% in adherent and spheroid screens. Dose response curves in 5 DIPG and 1 glioblastoma (GBM) cell line confirmed a potent reduction of viability in ACVR1 mutant cells, with GI50s of 13, 57 and 79nM after GSK591 inhibition in HSJD-DIPG-007, SU-DIPG-XXI and SU-DIPG-IV cells respectively. In comparison ACVR1 WT cells were at least 10x less sensitive to GSK591 with GI50s of 2.9, >10, >10μM in HSJD-DIPG-011, HSJD-GBM-002 and SU-DIPG-VI cells. To provide mechanistic insight as to how PRMT5 inhibition reduced viability of DIPG cells RNA-sequencing was performed at 5 time points, over a 7 day treatment with LLY-283 (GI40). Transcriptomic analysis found PRMT5 inhibition significantly enriched neural differentiation genes (including NLGN3, MDK, APOE and DKK1) suggesting a reduction in stemness. To explore this further we performed an in vitro limiting dilution assay after 7 days pre-treatment with the LLY-283 GI80, and confirmed that PRMT5 inhibition reduced single cell colony formation. This reduction of stemness could represent the conversion of DIPG cells from their typical OPC-like state into a more differentiated, less malignant phenotype. Following in vitro efficacy of PRMT5 inhibition, orthotopic patient derived DIPG xenografts (PDX) were treated with LLY-283 over 28 days (50mg/kg, 3 days on 4 days off). Like many single agent therapies tested in this highly aggressive DIPG PDX model, PRMT5 did not improve survival. However a reduction in stemness, as seen in vitro¸ does not always reduce primary tumor burden. Therefore proteomic characterization of neural differentiation in the primary PDX samples is underway, and investigation into secondary tumor initiation would be warranted. Our data shows PRMT5 inhibitors are a promising new target for DIPG, specifically in ACVR1 mutant DIPG, and justifies further in vivo investigations into changes in tumor initiation capacity and exploration of rational combinations to improve survival outcome. Citation Format: Elizabeth J. Brown, Leire Balaguer-Lluna, Adam Cribbs, Martin Philpott, Ángel M. Carcaboso, Gillian Farnie, Alex N. Bullock. PRMT5 inhibition reduces viability and stemness of pediatric high grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3025.

TSPO PET as a pharmacodynamic biomarker to image a rat model of diffuse intrinsic pontine human glioma and assess ketogenic diet efficacy

Diffuse intrinsic pontine gliomas (DIPG) are highly infiltrative paediatric gliomas. It remains the first cause of death linked to cerebral tumor in children. There is an urgent need to find new therapeutic strategies to increase the survival of these patients. In this context, specific pharmacodynamic (PD) biomarkers are needed to accurately assess therapeutic response and ease therapies’ validation. Molecular imaging (MI), especially positron emission tomography (PET) offers the possibility to quantify biologic processes taking place at cellular and subcellular levels. MI can define the real extent of tumors and treatment response on highly infiltrative tumor such as DIPG. 15% of DIPG's tumors present a dysregulated PIK3 pathway which promotes tumor growth. Ketogenic diet (KD), a metabolic therapy, consisting in a high intake of fat and a low intake of carbohydrates can increase the vulnerability of tumors cells. It could act as a tumor suppressor via the inhibition of PI3 K pathway. Here, we investigated the interest of using 18F-DPA-714 (i.e. ligand of the translocator protein of 18 kDa, TSPO) as a PD PET ligand to evaluate the extent of DIPG tumors. We explored via TSPO PET, the benefits of ketogenic diet (4:1) on human DIPG cells (HSJD-DIPG-007, PI3KCA-mutated, established at the Sant Joan de Déu Hospital (Barcelona, Spain)). In vivo study was performed on nude rats orthotopically grafted. KD was introduced one week after the tumor implementation in relevant groups. Ketosis was followed by dosage of beta-hydroxybutyrate and glucose blood concentrations. Four groups were imaged at 5 weeks post-implementation: sham with and without KD ( n = 4, for each condition), grafted with and without KD ( n = 8, for each condition). Brains of the animals were collected after imaging sessions to correlate 18F-DPA-714 PET signal with immunofluorescence biomarkers distribution. TSPO is highly expressed on this cell line, that opens up an opportunity for PET imaging. Ketosis was successfully induced in the rat groups receiving KD (corroborated by an increased brain uptake of 18F-FDG in treated groups). An increased 18F-DPA-714 uptake is observed in the pons and ex vivo immunofluorescence revealed a strong correlation between TSPO signaling and DIPG cells presence. We are the first team to prove that TSPO can be a potent biomarker to follow DIPG progression. Further analyses are ongoing to statute about the KD efficacy.

Abstract 635: Response to novel imipridone combination therapies targeting H3K27M mutant diffuse midline glioma (DMG)

Abstract ONC201, a first-in-class, small molecule, imipridone therapy, is known to induce apoptosis via upregulation of TNF-related apoptosis inducing ligand (TRAIL) and its proapoptotic death receptor (DR5), independent of p53. Current trials have been exploring ONC201 as a monotherapy or in conjunction with radiotherapy, in treatment of patients with newly diagnosed and refractory diffuse intrinsic pontine gliomas (DIPG), a disease which remains incurable at present. We sought to identify synergy between ONC201 or second generation impridones (ONC206 and ONC212), and other chemotherapeutics with known promising pre-clinical activity against DMGs. Seven patient derived H3K27M mutant DIPG cell lines (SU-DIPG-IV, SU-DIPG-13, SU-DIPG-25, SUDIPG-27, SU-DIPG-29, SU-DIPG-36, SF8628) were grown in culture and exposed to ONC201, ONC206, or ONC212 either as monotherapy or in combination with other FDA approved chemotherapeutics (histone de-acetylase inhibitors [HDACi], marizomib, etoposide, and temozolomide). Dose dependent response to imipridone therapy was noted in all cell lines, with mean half maximal inhibitory concentration (IC50) of 1.46 µM in ONC201, 0.11 µM in ONC206, and 0.03 µM in ONC212. Synergy was identified via combination studies, with combination indices of <1 indicating some degree of synergy, and <0.5 indicating very strong synergy. We identified strong synergy between ONC201 and HDAC inhibitors: Panobinostat (CI 0.01) and Romidepsin (CI 0.08), with lesser synergy detected with Entinostat (CI 0.59). Romidepsin similarly showed robust synergy with ONC206 (CI 0.1), as did proteasome inhibitor Marizomib (CI 0.1). Combination of ONC212 and Panobinostat demonstrated induction of apoptosis as measured by the induction of poly (ADP-ribose) polymerase (PARP) cleavage. Our results suggest an increased sensitivity of H3K27M DIPG cell lines to second generation imipridones as compared to ONC201. Additionally, we have shown promising synergism between imipridones with Panobinostat or Romidepsin, which should be considered for clinical translation. Citation Format: Robyn Borsuk, Lanlan Zhou, Yiqun Zhang, Varun V. Prabhu, Joshua E. Allen, Nikos Tapinos, Rishi Lulla, Wafik S. El-Deiry. Response to novel imipridone combination therapies targeting H3K27M mutant diffuse midline glioma (DMG) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 635.

Abstract 318: Multi-omics to edge into precision medicine for DIPG

Abstract Effective targeted cancer therapies are limited due to lack of our understanding of disease pathogenesis at the molecular level. Diffuse intrinsic pontine glioma (DIPG) is an incurable pediatric brain tumor with 80% of DIPGs harboring H3F3A (H3.3) mutation that lead to a substitution of methionine for lysine at position 27 (K27M). The tumors bearing H3.3K27M mutation arise throughout the midline structures with global depletion of H3.3K27 me3 (trimethylation). These histone mutations modify the epigenome and alter oncogenic transcription, causing oncogenic insults to progenitor cells in early neurodevelopment (PMID: 30453529). To determine the reprogramming pathways in the cell context of H3.3K27M tumors, we conducted liquid chromatography-mass spectrometry-based proteomic and phosphoproteomic analysis on seven patient-derived DIPG cell lines that can be stably passaged in serum-free neural stem cell media and displayed distinct morphologies, growth rates and chromosome abnormalities (PMID: 21368213). Three normal neuronal stem cell lines were included as non-tumor brain cells for comparison. Pathway analysis identified 29 pathways that are significantly altered in DIPG compared to normal brain cells at both the protein abundance and phosphosite level. Notably, AKT and MAPK associated PI3K signaling, VEGF signaling, mTOR signaling, and HIF1a signaling were differentially active in H3.3K27M tumors compared to healthy control cell lines. We saw significantly higher activity of multiple kinases involved in axon guidance and cytoskeletal remodeling in DIPG, such as PTK2B, DYRK2, TTBK2 and MARK2. This is the first time to report an increased abundance and kinase activity of Pyk2 protein (coded by PTK2B), a close homologue of FAK and its associated signaling in DIPG. Overexpression and autophosphorylation of Pyk2 are required to stimulate glioma cell migration (PMIDs: 15967096, 18648907). Pyk2 has also been proposed to act in concert with Src to link Gi- or Gq-coupled receptors with the mitogen-activated protein (MAP) kinase signaling pathway (PMID:12960403). Because of the shared signaling across kinase pathways, targeting activated Pyk2 in DIPG may complement inhibitors of other dysregulated signaling networks in DIPG such as MAPK2, VEGFR, PI3K and Src. Our data also found that IL13RA2 was upregulated in DIPG; others have shown that increased expression of IL13RA2 is associated with poor prognosis in GBM (PMIDs: 32913543, 30366424). We conclude that for H3 K27M DIPG tumors, campaigns to target Pyk2, MAPK2, VEGFR, PI3K, Src and IL13Ra2 using small molecules that traverse the blood brain barrier loom as promising opportunities for drug development. Citation Format: Nanyun Tang, Kristin Leskoske, Krystine Garcia-Mansfield, Ritin Sharma, Hannah Tolson, Patrick Pirrotte, Michael E. Berens. Multi-omics to edge into precision medicine for DIPG [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 318.

HGG-05. NEURONAL ACTIVITY PROMOTES PEDIATRIC HIGH-GRADE GLIOMA GROWTH THROUGH A NLGN3-CSPG4 SIGNALING AXIS

High-grade gliomas, including diffuse intrinsic pontine glioma (DIPG), are a lethal group of cancers whose progression is strongly regulated by neuronal activity {Venkatesh 2015}{Venkatesh 2017}{Venkatesh 2019}. One way in which glioma cells sense neuronal activity is via interaction with the ectodomain of post-synaptic adhesion protein neuroligin-3 (NLGN3), which is cleaved and released into the tumor microenvironment (TME) by the sheddase ADAM10. This interaction drives glioma growth, but the relevant binding partner of shed NLGN3 (sNLGN3) on glioma cells is currently unknown. Here, we report that sNLGN3 binds to chondroitin sulfate proteoglycan 4 (CSPG4), in turn inducing regulated intramembrane proteolysis (RIP) of CSPG4, and initiating a signaling cascade within DIPG cells to promote tumor growth. CSPG4 RIP involves activity-regulated ectodomain shedding by ADAM10 and subsequent gamma secretase-mediated release of the intracellular domain in healthy oligodendroglial precursor cells (OPCs), putative cells of origin for several forms of high-grade glioma {Sakry 2014}{Nayak 2018}. Incubation of high-grade glioma cells or healthy OPCs with recombinant NLGN3 is sufficient to augment ADAM10-mediated ectodomain release of CSPG4 and subsequent gamma secretase-mediated cleavage of the CSPG4 intracellular domain (ICD). Pre-treatment of glioma cells or OPCs with an ADAM10 inhibitor entirely blocks NLGN3-induced CSPG4 shedding. Acute depletion of CSPG4 via CRISPR gene editing renders glioma cells insensitive to the growth-promoting effects of NLGN3 application in vitro. We are now performing experiments to better discern how the CSPG4 ICD regulates signaling consequences downstream of sNLGN3 binding. In addition, we are using surface plasmon resonance to investigate whether the shed ectodomains of NLGN3 and CSPG4 remain in complex or only transiently interact. Altogether, our data form a critical missing link in understanding how glioma cells sense, translate and respond to neuronal activity in the TME and identify a new therapeutic target to disrupt neuron-glioma interactions.