Diffuse Intrinsic Pontine Glioma Cell Lines Research Articles

DIPG-72. CREATION, CHARACTERIZATION AND CATALOGUING NEXT-GENERATION DIPG PATIENT-DERIVED CANCER CELL LINES: THE DIPG C3 PROJECT

Abstract BACKGROUND Preclinical studies of diffuse intrinsic pontine glioma (DIPG) have increased significantly over the past 15 years due to the development of DIPG disease-specific tools including cell lines and animal models. We now know that DIPG is a molecularly heterogeneous tumor. While >80% of these tumors have H3K27M alterations, partner mutations can vary. Many DIPG cell lines used for pre-clinical assessments of new therapies have not been fully characterized, therefore, comparing preclinical results across studies has been impeded. We sought to Create, Characterize and Catalogue DIPG cell lines and animal models with the aim of expanding contemporary resources and providing a public database for DIPG researchers. METHODS Existing DIPG cell lines in the Center for Patient-Derived Models (CPDM) were utilized; additional patient-derived cell lines were created in CPDM, and obtained from collaborators. The assembled collection was characterized by whole genome (WGS) and RNA-sequencing (RNAseq). RESULTS To date, we have 21 growth-verified DIPG models with the majority grown as 3D neurospheres in-vitro and capable for long-term culture. Fourteen (14) of these patient-derived models were genomically verified with WGS and RNA-seq platform. Data will be made publicly available to researchers on cBioPortal. CONCLUSIONS Systematic characterization and cataloguing of pre-clinical DIPG models is important to allow more stringent comparison across studies and ensure their utility and impact. These DIPG-specific tools can thus be used in applications such as drug discovery and testing.

DIPG-39. ONCOHISTONE H3.3K27M-DRIVEN CREB5/ID1 AXIS DICTATES THE MALIGNANT CELL STATES OF DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)

Abstract BACKGROUND Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric cancer characterized by the presence of H3K27M-oncohistones, which drive relentless proliferation and differentiation arrest. This study focuses on the H3.3K27M DIPG subtype and investigates the role of the oncohistone-driven CREB5/ID1 axis in promoting malignancy. METHODS We employed a variety of biological in vitro assays and xenograft mouse models utilizing several H3.3K27M and H3.1K27M DIPG cell lines to evaluate the impact of the studied factors on the growth of DIPGs. Additionally, we employed methodologies in molecular biology, bioinformatics, drug screening, and biochemistry to elucidate the underlying mechanisms of tumorigenesis. RESULTS Through inhibition of ID1, this study explores the induction of cellular differentiation and apoptosis in H3.3K27M DIPG, shedding light on the oncogenic mechanisms at play. The intricate regulation of ID1, linked to CREB5—an oncogenic contributor independent of BMP signaling—is elucidated. The involvement of the SWI/SNF complex as a co-regulator of CREB5 is also examined. Moreover, our findings reveal that H3.3K27M-oncohistone triggers CREB5 overexpression, facilitating the formation of a super-enhancer promoter loop at the CREB5 locus. Disruption of this loop demonstrates potent anti-tumor efficacy in treating DIPG. Additionally, the combination of a compound targeting the CREB5/ID1 axis and a BRG1 inhibitor shows promise for DIPG treatment. CONCLUSIONS This research unveils key molecular pathways specific to the H3.3K27M DIPG subtype, offering valuable insights for potential therapeutic interventions against this lethal pediatric cancer. Inhibiting the CREB5/ID1 axis represents a promising strategy for inducing differentiation and apoptosis in DIPG cells, highlighting the potential for targeted therapies in combating this disease.

DIPG-69. IN-VITRO DETERMINATION OF 5-AMINOLEVULINIC-ACID-TREATMENT EFFICACY IN PATIENT-DERIVED DIPG MODELS

Abstract BACKGROUND Diffuse Intrinsic Pontine Glioma (DIPG) presents a significant clinical challenge due to its inoperable nature and poor response to current treatments. Sonodynamic Therapy (SDT) is a non-invasive therapy that combines low-intensity ultrasound stimulation with a sonosensitizing agent. 5-Aminolevulinic Acid (5-ALA) as a sonosensitizer has yielded positive outcomes in in-vivo glioblastoma models, and a clinical trial (NCT05123534) for DIPG patients is currently ongoing. 5-ALA is a precursor of Protoporphyrin IX (PpIX), which preferentially accumulates in cancer cells and generates cytotoxic reactive oxygen species upon activation by ultrasound or light. Our work is aimed at investigating the accumulation of PpIX in in-vitro DIPG patient-derived cell models to predict the efficacy of 5-ALA SDT treatment. METHODS Patient derived DIPG cell lines (H3K27M, P53 mutant and wild type) were used to create three-dimensional spheroids. The spheroids were dosed with 5-ALA for 4 hours, and PpIX accumulation was quantified by measuring its fluorescence intensity within the cells. The spheroids were then exposed to controlled light stimulation as an in-vitro proxy for SDT. RESULTS We confirmed the uptake of 5-ALA and subsequent PpIX accumulation in all patient-derived DIPG spheroids. Light exposure of 5-ALA treated DIPG spheroids resulted in reduced viability of the tumor models, while 5-ALA itself did not show cytotoxic effects. The extent of PpIX accumulation and cytotoxicity upon PpIX light activation varied among DIPG models with higher sensitivity of p53 wild type cells. Further studies incorporating a larger number of DMG cells with p53 alterations are underway. CONCLUSIONS These findings indicate that SDT using 5-ALA as sonosensitizer may offer clinical benefits for treatment of DIPG, however treatment efficacy could vary considerably between patients. In vitro evaluation of PpIX accumulation and sensitivity to activation could potentially provide a means to stratify patients and to identify those most likely to benefit from 5-ALA-mediated SDT.

DIPG-58. TARGETING DISORDERED DNA METHYLATION IN DIPG TO CONSTRAIN VARIABILITY AND INDUCE IMMUNE SIGNALING

Abstract BACKGROUND Diffuse Intrinsic Pontine Glioma (DIPG) is a universally fatal pediatric brain cancer characterized by the histone H3 K27M mutation. The known downstream consequences of this mutation include loss of repressive chromatin marks, global DNA hypomethylation, and altered gene expression. METHODS We sought to investigate the role of epigenetic variability as a basis of DIPG cellular heterogeneity and plasticity by using Whole Genome Bisulfite Sequencing (WGBS). Existing methods of analysis of WGBS are limited in that they average/smooth methylation data and fail to capture intrinsic variability. We performed (WGBS) on 23 primary patient samples of DIPG and applied an analysis that captures the variability of DNA methylation reads to characterize methylation stochasticity. We then sought to modulate the epigenome pharmacologically using a DNA methyltransferase inhibitor and assessed changes through RNA-Seq and immunopeptidomics. RESULTS We find that DIPG has a marked increase in methylation stochasticity (quantified as methylation entropy). Additionally, while DIPG is globally hypomethylated, it retains DNA methylation at specific regulatory regions, such as bivalent promoters, and key genes, such as FOXG1, CXCR4, and KLF4. We sought to reverse these changes using the DNA methyltransferase inhibitor, decitabine, in 4 DIPG neurosphere cell lines. Treatment with decitabine reversed the entropy seen in DIPG and induced innate immune signaling and interferon signaling in DIPG cells. Using RNA-seq, we found that decitabine treatment dramatically alters gene expression, including activation of endogenous retroviral elements, activation of interferon signaling, de-repression of hypermethylated targets, and expression of putative neoantigens, such as PRAME and DAZL. Immunopeptidomics profiling of MHC Class I bound canonical and non-canonical peptides of DIPG cell lines after decitabine treatment also revealed decitabine-induced differences. CONCLUSIONS Taken together, our study finds that the methylome of DIPG is highly disordered but is also highly responsive to pharmacologic modulation by hypomethylating agents. Furthermore, hypomethylating drugs can increase the immunogenicity of DIPG, thus offering the potential for future combination with immunotherapy.

Abstract 7578: BRD4 protein degradation for p53 mutant diffuse intrinsic pontine glioma (DIPG) via various delivery approaches for ARV-825 by using camel milk exosomes

Abstract DIPGs (diffuse intrinsic pontine gliomas) are a particularly aggressive and malignant type of pediatric brain tumor. Given the tumor's location, surgery has a restricted role, and it is insensitive to most chemotherapies. Although fractionated radiation is the standard of therapy for DIPG, the cancer invariably worsens in 6-12 months. TP53 tumor suppressor protein mutations are found in 70-80% of DIPG tumors. These TP53 mutations have been linked to radiation resistance in DIPG patients. Our goal was to find new pharmacological compounds that would improve the radiation sensitivity of p53 mutant DIPG cell lines and to determine their molecular mechanism of action. Numerous bromodomain-containing protein 4 (BRD4) inhibitors have entered clinical trials in recent years, yielding promising outcomes in tumor treatment since BRD4 expression in DIPG is substantially higher than in neighboring normal brain tissue. So consequently, BRD4 is a target for DIPG treatment. ARV-825 is an emerging PROTAC (Proteolysis Targeting Chimera) compound which can cause rapid and persistent BRD4 protein degradation. However, delivering ARV-825 to pons is a major challenge owing to poor oral bioavailability of PROTACS. In this study, we used camel milk derived exosomes (CME) for delivery of ARV-825 to pons by both nasal and oral routes. To begin with, goat milk exosomes were also considered but CME was chosen because of its innate cytotoxicity against DIPG cell lines. CME were isolated using ultracentrifugation and nanoparticle tracking analysis and revealed a size distribution of 112.5 ± 2.8 nm, zeta potential of -26.38 ± 0.12 mV. CME (1*1011) were cytotoxic to both SF8628 (with a H3.3 K27M mutation) and SF188 (grade IV pediatric glioblastoma with wild type H3.3) cell lines and had 51% and 44% cell viability respectively. ARV-825 showed IC50 of 546.58 ± 22.14 nM and 608.94 ± 30.04 nM in SF8628 cells and SF188 cells respectively. ARV-825’s IC50 was lowered when ARV-825 loaded CME were used. ARV-825 was loaded using sonication and entrapment efficiency was found to be 42.96 ± 2.14 % by HPLC analysis. CME were labelled with Vybrant DiO (CME-DIO) and instilled into each nostril of Sprague Dawley rats daily. Brain sections were viewed with fluorescent microscope and CME-DIO were seen in olfactory region after 3 days and in the pons region after 5 days demonstrating the ability of CME to reach the target site. In-vitro permeability studies using MDCK cells showed approximately 9µg/mL (60%), 15µg/mL (80%) and 16µg/mL (100%) of ARV-825 permeation in 2, 4 and 24 hours respectively when loaded into CME. The results of the present study suggest the possibility of using CME as a viable delivery strategy for ARV-825 against DIPG. Further in vitro and in vivo studies are currently undergoing to validate the results and understand kinetics and the molecular mechanisms of ARV-825 loaded CME. Citation Format: Aakash Nathani, Mounika Aare, Li Sun, Yan Li, Mandip Singh. BRD4 protein degradation for p53 mutant diffuse intrinsic pontine glioma (DIPG) via various delivery approaches for ARV-825 by using camel milk exosomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7578.

Abstract 5108: Immunomodulatory and anti-tumor effect of radiation and induced telomere damage to treat pediatric high-grade gliomas

Abstract Pediatric high-grade gliomas (pHGGs), including diffuse intrinsic pontine glioma (DIPG) are among the most treatment refractory cancers in children. Despite multimodal therapy, prognosis is dismal with survival of <1 year for DIPG and a 5-year overall survival <10% for other pHGGs. Radiotherapy remains the only standard of care for DIPG that extends the survival by few months. In recent years, immunotherapy is emerging as a potential alternative treatment option. However, several factors have hindered the use of immunotherapy for pHGGs including “cold” immune environment with lack of immune cells infiltration, low tumor mutational burden, and lack of neo-antigen expression that prevents tumor recognition by the immune system. Therefore, novel therapies that activate the immune system while evading the tumor immunosuppression are highly needed. Here, we aim to demonstrate the immunomodulatory anti-tumor effects of THIO (6-thio-2’deoxyguanosine), a nucleoside analog and a telomerase-dependent telomere targeting agent, in DIPG mouse models. Our preclinical in vitro and in vivo data have demonstrated the efficacy of THIO in inducing telomeric damage, G2/M cell cycle arrest, and apoptosis in high-risk medulloblastoma and DIPG. Importantly, we have demonstrated that THIO crosses the blood-brain barrier and specifically targets tumor cells in an orthotopic mouse model of DIPG. THIO was shown to induce anti-tumor immunity through micronuclei formation leading to cGAS-STING pathway mediated activation of the innate and adaptive immune response in colon, lung, and hepatocellular carcinoma tumor models. Our preliminary data demonstrate the THIO-dependent induction of micronuclei formation co-localizing with cGAS and leading to STAT1 activation in DIPG cell lines. We have also shown that THIO synergistically sensitizes DIPG cells to ionizing radiation (IR), significantly decreasing cell proliferation. We are currently testing the combination of THIO+IR in an orthotopic mouse model for DIPG. We will present results on the potential of THIO and IR treatments to stimulate anti-tumor immunity through the activation of STING pathway in a syngeneic mouse model of DIPG. These preclinical studies will support the potential use of THIO and IR to treat children with high-risk pediatric brain tumors. Citation Format: Umaru Banlanjo, Shiva Senthil Kumar, Sergei Gryaznov, Rachid Drissi. Immunomodulatory and anti-tumor effect of radiation and induced telomere damage to treat pediatric high-grade gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5108.

Pharmacological Targeting of Interferon-Related DNA Damage Resistant Signature (IRDS) and XRCC4-Mediated DNA Repair Pathways ss a Novel Therapeutic Approach to DIPG Radio-Sensitization

Despite significant efforts to improve the outcomes of pediatric diffuse midline gliomas, such as diffuse intrinsic pontine glioma (DIPG), prognosis remains dismal with a 5-year survival of <1%. The standard of care treatment for DIPG is fractionated radiation, but the disease inevitably progresses in 6 months or less. There is a significant unmet need to improve the clinical outcomes for pediatric DIPG patients. Approximately 70-80% of DIPG tumors contain mutations in TP53 tumor suppressor protein. These TP53 mutations are associated with resistance to radiation treatments in DIPG patients. The mechanisms of increased radio-resistance of p53-mutant DIPG are poorly understood. The objective of this study was to identify novel pharmacological agents that would augment radiation sensitivity of p53 mutant DIPG cell lines, and to establish their molecular mechanism of action. SF8628 pediatric DIPG cell line harboring p53 mutation was obtained from MilliporeSigma. CellRad benchtop X-ray irradiator (Precision X-Ray) was used for radiation sensitization experiments. Vi-CELL BLU cell viability analyzer was used for high-throughput screening of small molecule compound libraries with and without radiation treatments. Proteomics Core facility was utilized for mass spec analysis of protein targets of Compound-X. To identify novel drug candidates that would sensitize DIPG to therapeutic radiation, we carried out an unbiased screen of curated libraries of small molecules with diverse scaffolds in p53 mutant DIPG cells. This radio-sensitization screen yielded a single molecule, Compound-X, that was found to have profound growth-inhibitory and radiation-sensitizing effects in DIPG cells. Compound-X was found to induce a robust cell cycle arrest of DIPG cells in G2/M, the most radio-sensitive phase of the cell cycle. Furthermore, Compound-X elicited a massive apoptotic cell death of DIPG cells. An unbiased RNA sequencing approach revealed that Compound-X inhibits expression of the Interferon-related DNA damage Resistant Signature (IRDS), a sub-group of interferon-stimulated genes (ISGs) known to promote radiation and chemotherapy resistance in high-grade gliomas. To identify the target of Compound-X, we carried out affinity purification of Compound-X associated complexes from p53 mutant DIPG cell lysates. Mass spectrometry analysis of Compound-X-purified protein complexes identified XRCC4 as a protein that uniquely associated with Compound-X. RNAi knock-down experiments revealed that XRCC4 is required for cytotoxic effects of Compound-X. Importantly, Compound-X-mediated XRCC4 targeting caused a delay in DNA DSBs repair after radiation treatment. An unbiased screen of small molecule drug candidates identified a novel XRCC4-targeting agent, Compound-X, as potent radiation sensitizer in p53 mutant DIPG cells. This work may lead to clinical trials investigating novel XRCC4-targeting agent in pediatric patients with DIPG.

A combined immunopeptidomics, proteomics, and cell surface proteomics approach to identify immunotherapy targets for diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG), recently reclassified as a subtype of diffuse midline glioma, is a highly aggressive brainstem tumor affecting children and young adults, with no cure and a median survival of only 9 months. Conventional treatments are ineffective, highlighting the need for alternative therapeutic strategies such as cellular immunotherapy. However, identifying unique and tumor-specific cell surface antigens to target with chimeric antigen receptor (CAR) or T-cell receptor (TCR) therapies is challenging. In this study, a multi-omics approach was used to interrogate patient-derived DIPG cell lines and to identify potential targets for immunotherapy. Through immunopeptidomics, a range of targetable peptide antigens from cancer testis and tumor-associated antigens as well as peptides derived from human endogenous retroviral elements were identified. Proteomics analysis also revealed upregulation of potential drug targets and cell surface proteins such as Cluster of differentiation 27 (CD276) B7 homolog 3 protein (B7H3), Interleukin 13 alpha receptor 2 (IL-13Rα2), Human Epidermal Growth Factor Receptor 3 (HER2), Ephrin Type-A Receptor 2 (EphA2), and Ephrin Type-A Receptor 3 (EphA3). The results of this study provide a valuable resource for the scientific community to accelerate immunotherapeutic approaches for DIPG. Identifying potential targets for CAR and TCR therapies could open up new avenues for treating this devastating disease.

Reversal of cancer gene expression identifies repurposed drugs for diffuse intrinsic pontine glioma

Diffuse intrinsic pontine glioma (DIPG) is an aggressive incurable brainstem tumor that targets young children. Complete resection is not possible, and chemotherapy and radiotherapy are currently only palliative. This study aimed to identify potential therapeutic agents using a computational pipeline to perform an in silico screen for novel drugs. We then tested the identified drugs against a panel of patient-derived DIPG cell lines. Using a systematic computational approach with publicly available databases of gene signature in DIPG patients and cancer cell lines treated with a library of clinically available drugs, we identified drug hits with the ability to reverse a DIPG gene signature to one that matches normal tissue background. The biological and molecular effects of drug treatment was analyzed by cell viability assay and RNA sequence. In vivo DIPG mouse model survival studies were also conducted. As a result, two of three identified drugs showed potency against the DIPG cell lines Triptolide and mycophenolate mofetil (MMF) demonstrated significant inhibition of cell viability in DIPG cell lines. Guanosine rescued reduced cell viability induced by MMF. In vivo, MMF treatment significantly inhibited tumor growth in subcutaneous xenograft mice models. In conclusion, we identified clinically available drugs with the ability to reverse DIPG gene signatures and anti-DIPG activity in vitro and in vivo. This novel approach can repurpose drugs and significantly decrease the cost and time normally required in drug discovery.

Abstract 5716: Using copper chelating agents to simultaneously target epigenetic mechanisms and receptor tyrosine kinase (RTK) signaling in diffuse intrinsic pontine glioma (DIPG)

Abstract DIPG is an incurable and inoperable pediatric brain cancer in the ventral pons characterized by its complex mutational profile. Epigenetic mutations to H3K27M are seen in up to 81% of patients, resulting in the global loss of the repressive H3K27 trimethylation marker, increasing chromatin accessibility and deregulating gene expression. However, these epigenetic changes are often partnered by secondary mutations, with activating mutations and copy number gains observed in a wide variety of RTK genes and their associated signaling pathways. The combination of these two mutational paradigms results in the broad alteration of cellular and metabolic mechanisms, complicating the search for effective targeted therapeutic strategies. Copper, a metal ion essential for normal cellular function is known to be highly expressed in the pons, and influence ERK and PI3K/AKT signaling. Importantly, excess copper accumulation has been implicated in the pathogenesis of multiple neurological diseases and cancers. Copper chelation therapy is a clinically approved strategy for pediatric patients with Wilson’s Disease, known to improve their neurological symptoms, and is under investigation for use in a number of cancers. Therefore, we hypothesized copper chelation may be an effective therapeutic strategy for DIPG. Cytotoxicity assays using copper chelator tetraethylenepentamine (TEPA) demonstrated millimolar efficacy and synergized with mTOR inhibitor everolimus in a panel of DIPG cell lines, with H3-K27M mutant cell lines found to be more sensitive. Western blots were used to identify copper integrated pathways, with DIPG cell lines stimulated with sub-lethal copper concentrations demonstrating increased phosphorylated expression of ERK1/2, ERK5, AKT and p70SK61. TEPA reduced the phosphorylation of these markers compared to controls. Importantly, epigenetic mechanisms were sensitive to copper, as both copper stimulation and TEPA modulated H3K27 trimethylation, and TEPA decreased pro-expression H3K27 acetylation. In further blots examining epigenetic regulators, TEPA reduced the expression of EZH2, DNMT3B and DNMT1. RNA-sequencing analysis revealed TEPA downregulated genes involved in S-adenosylmethionine production metabolism, DNA regulation, cell cycle and mTORC signaling and specifically, EZH2, DNMT3B and DNMT1 expression. In vivo investigations demonstrated TEPA’s ability to improve survival in an orthotopic xenograft model, and via xenogen luminescence imaging complete tumor regression in 25% of treated mice. This study indicates copper directly impacts epigenetic and RTK mechanisms, and that their targeting through copper chelation agents represents a potential therapeutic strategy for DIPG, both as a single agent and in combination. Citation Format: Filip Michniewicz, Federica Saletta, Tayla Watkinson, Daniele Mercatelli, Federico M. Giorgi, Jessica Bell, Maria Tsoli, David Ziegler, Orazio Vittorio. Using copper chelating agents to simultaneously target epigenetic mechanisms and receptor tyrosine kinase (RTK) signaling in diffuse intrinsic pontine glioma (DIPG) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5716.

Abstract 1671: Engineering new cellular models to decipher H3.3K27M mutation role in DIPGs' resistance to therapies

Abstract Among pediatric brain tumors, Diffuse Intrinsic Pontine Gliomas (DIPGs) display a particularly dismal prognosis, highlighted by their median survival lower than one year. Indeed, DIPGs’ location and infiltrative properties preclude their surgical resection. Moreover, DIPGs poorly respond to chemotherapeutic agents. In this context, the only treatment for these tumors remains palliative radiotherapy, systematically followed by tumor progression. In addition to their resistance to therapies, DIPGs are characterized by recurrent histone H3 mutations. The H3.3K27M mutation is the most frequent and results from a heterozygous single nucleotide variant in the H3F3A gene, inducing the lysine 27 substitution by a methionine. Although H3.3K27M’s driver role in DIPGs tumorigenesis is now established, its role in their chemo- and radioresistance remains unclear. Aiming to decipher the potential role of this mutation in pediatric gliomas’ resistance to therapies, we established isogenic cellular models of H3.3K27M induction and reversion.We first induced H3.3K27M mutation in three initially non-mutated supratentorial pediatric glioma cell lines. Thus, we generated models that stably expressed the dominant-negative H3.3K27M or the wild type H3.3 as controls. Complementarily, to study H3.3K27M roles in a DIPG cell context, we also developed H3.3K27M reversion models in two initially mutated DIPG cell lines by applying a gene-editing strategy based on the combinatorial use of the CRISPR/Cas9 technology and an insert.We showed that H3.3K27M induction in Res259 and KNS42 cells conferred a radioresistant phenotype to a fractionated radiotherapy schedule. Besides, we performed a screening of 80 anti-cancer drugs, which revealed a differential impact of the mutation on the drug sensitivity profiles of our three H3.3K27M-induced cell lines. These results indicate that H3.3K27M can control pediatric glioma cells’ resistance to therapies, but in a heterogeneous way depending on the cellular context. Along this line, we are currently characterizing the chemo- and radiotherapy response of our new DIPG H3.3K27M-reversed models. Altogether, our first results support a role for H3.3K27M in pediatric gliomas resistance to treatments, and our complementary models pave the way for identifying new H3.3K27M-dependent mechanisms and promising targets to sensitize DIPGs to therapies. Citation Format: Andria Rakotomalala, Paul Lewandowski, Quentin Bailleul, Clara Savary, Mélanie Arcicasa, Christine Bal, Maud Hamadou, Paul Huchedé, Audrey Restouin, Remy Castellano, Yves Collette, Audrey Vincent, Pierre-Olivier Angrand, Eric Adriaenssens, Xuefen Le Bourhis, Pierre Leblond, Marie Castets, Eddy Pasquier, Alessandro Furlan, Samuel Meignan. Engineering new cellular models to decipher H3.3K27M mutation role in DIPGs' resistance to therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1671.

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-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.

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.

127 CAR T cells targeting the integrin alpha v beta 3 exhibit robust anti-tumor responses against diffuse intrinsic pontine glioma and glioblastoma

BackgroundDiffuse intrinsic pontine glioma (DIPG) and glioblastoma (GBM) are two highly aggressive and mostly incurable gliomas with little therapeutic advancements made in the past several decades. Despite immense initial success of chimeric antigen receptor (CAR) T cells for the treatment of leukemia and lymphoma, significant headway into the application of CAR T cells against solid tumors, including gliomas, is still forthcoming. The integrin complex alpha v beta 3 (avb3) is present on multiple and diverse solid tumor types and tumor vasculature with limited expression throughout most normal tissues, qualifying it as an appealing target for CAR T cell-mediated immunotherapy.MethodsPatient-derived diffuse intrinsic pontine glioma (DIPG) cells and glioblastoma (GBM) cell lines were evaluated by flow cytometry for surface expression of avb3. Second-generation CAR T cells expressing an anti-avb3 scFv were generated by retroviral transduction containing either a CD28 or 4-1BB co-stimulatory domain and CD3zeta. CAR T cells were evaluated by flow cytometry for CAR expression, memory phenotype distribution, and inhibitory receptor profile. DIPG and GBM cell lines were orthotopically implanted into NSG mice via stereotactic injection and monitored with bioluminescent imaging to evaluate avb3 CAR T cell-mediated anti-tumor responses.ResultsWe found that patient-derived DIPG cells and GBM cell lines express high levels of surface avb3 by flow cytometry, while avb3 is minimally expressed on normal tissues by RNA sequencing and protein microarray. Second-generation CAR T cells expressing an anti-avb3 single-chain variable fragment (scFv) were designed and generated by retroviral transduction containing either a CD28 or 4-1BB co-stimulatory domain and CD3zeta. avb3 CAR T cells demonstrated efficient, antigen-specific tumor cell killing in both cytotoxicity assays and in in vivo models of orthotopically and stereotactically implanted DIPG and GBM tumors into relevant locations in the brain of NSG mice. Tumor responses were rapid and robust with systemic CAR T cell proliferation and long-lived persistence associated with long-term survival.ConclusionsThese results highlight the potential of avb3 CAR T cells for immunotherapeutic treatment of deadly brain tumors with reduced risk of on-target, off-tumor mediated toxicity due to the restricted nature of avb3 expression in normal tissues.AcknowledgementsWe would like to acknowledge the following core facilities at the University of Virginia: The Research Histology Core, the Biorepository and Tissue Research Facility, and the Molecular Imaging Core which are supported by the University of Virginia School of Medicine and through the University of Virginia Cancer Center National Cancer Institute P30 Center Grant. We would also like to acknowledge the University of Virginia Center for Comparative Medicine for providing animal care and services.

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

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 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 &amp;lt;1 indicating some degree of synergy, and &amp;lt;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.