DIPG Tumors Research Articles

Abstract 6516: Single-cell protein activity inference reveals lymphocytic immune infiltration in diffuse intrinsic pontine glioma induced by focused ultrasound

Abstract Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor, located in the brainstem, and traditionally thought to be immune-excluded with no significant infiltration by cytotoxic T-cells, and therefore not amenable to immunotherapies. Focused ultrasound technology (FUS) for drug delivery is being investigated for DIPG in both preclinical and clinical trial settings. In this study, we sought to study the effects of FUS and radiation therapy (RT) on immune cell infiltration in DIPG tumors to assess their ability to alter the immune microenvironment and potentially enhance combination immunotherapies. Specifically, we generated single-cell RNAseq profiles for 27,780 cells from syngeneic orthotopic DIPG tumors (KPAP cell line containing H3K27M mutation, ATRX and P53 loss, and PDGFRA overexpression) after 14 days of tumor growth across the following treatment arms: vehicle control, FUS, and RT (n=2 each group). FUS was applied by 4-point sonication around the tumor with a pressure of 0.6 Mpa at days 8 and 11 post tumor implantation (PTI). RT was administered in fractions of 3 Gy daily from days 8 to 11 PTI (12Gy total). We leveraged a set of algorithms for network-based inference of regulatory protein activity to infer a set of context-matched gene regulatory networks from which protein activity can be inferred by expression of downstream targets. By this approach, we identified distinct clusters of immune cells segregating by cell type as inferred by singleR and activity of known lineage markers. Strikingly, we found that both RT and FUS significantly depleted a TGFb-expressing population of immune-suppressive macrophages as compared to vehicle controls. Importantly, FUS only additionally increased T-cell infiltration into the tumor micro-environment over three-fold as compared to vehicle controls, which was not observed in response to RT. Further protein validation is underway. This has significant implications for therapeutic combinations of FUS-mediated drug delivery with immune checkpoint inhibitors in DIPG, such that in addition to drug delivery, FUS-mediated immune modulatory effects may provide more proinflammatory response to enhance immunotherapy. Citation Format: Erin White, Hong-Jian Wei, Ester Calvo Fernández, Hanna Minns, Jovana Pavisic, Robyn Gartrell, Cheng-Chia Wu, Andrea Califano, Aleksandar Obradovic. Single-cell protein activity inference reveals lymphocytic immune infiltration in diffuse intrinsic pontine glioma induced by focused ultrasound [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 6516.

IMMU-11. PRECLINICAL HIGH-RISK PEDIATRIC BRAIN TUMOR MODELS FOR IMMUNOTHERAPY: HURDLES AND THE WAY FORWARD

Abstract Despite recent therapeutic advancements in the treatment of pediatric brain tumors, high-risk brain tumors (pHRBT) remain the leading cause of cancer-related deaths in children. In recent years, immunotherapy has become a viable treatment option for several cancers including brain tumors. However, several factors have limited the use of immunotherapy in the treatment of pHRBT. For example, an "immunologically cold" tumor environment has been predominantly implicated in the failure of checkpoint inhibitors (ICIs) as monotherapy in pHRBT. Nevertheless, priming the effects of ICIs with epigenetic modulators through a process called “viral mimicry” that induces the expression of human endogenous retroviruses has the potential to enhance immunotherapy by sparking a T-cell mediated immune response. However, the inadequate understanding of the limitations of the preclinical models has prevented the development of efficient immunotherapy strategies for pHRBT. Although several studies and reviews have compiled some limitations of the available models, a hands-on experience using preclinical models for ICIs testing has not been fully communicated. Here, we share our bedside to bench experience with syngeneic and humanized mouse models of DIPG, ATRT, and medulloblastoma using ICIs therapy in conjunction with an epigenetic alteration. We will present and discuss the limitations of mouse models for the development of immunotherapies for pHRBT. For instance, our results indicated that the baseline levels of MHC-I expression in patients’ DIPG tumors were comparable to matched normal tissue. However, these levels were significantly lower in mouse DIPG tumors. To compensate for this difference, we treated mice with IFN-γ in combination with an epigenetic modulator. Surprisingly, the addition of INF-γ had a negative effect on animal overall survival compared to the treatment with the epigenetic modulator alone. Together, our experience demonstrated the importance of suitability assessment of current models for developing and translating successful immunotherapy strategies to treat pHRBT.

DIPG-27. DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) CHALLENGE: FROM GENE EXPRESSION PROFILE TO DRUG

Abstract Diffuse intrinsic pontine glioma (DIPG) accounts for 80% of pediatric brainstem tumors and about 10-20% of all central nervous system tumors in childhood. Despite the adoption of aggressive therapeutic approaches, DIPG remains the leading cause of cancer-related mortality in childhood: prognosis is still poor with more than 90% of affected children dying within 18-24 months from the diagnosis.1 Decades of clinical attempts involving more than 200 trials evaluating different conventional cytotoxic drugs, targeted drugs and studies with radiation dose escalating and new radio sensitizing agents, have failed to improve the overall survival of children with DIPG. Since available drugs have proven no benefits hereto for DIPG, it is fundamental to gain insights into the gene expression profile of DIPG patients and speed up the discovery of novel therapeutics for this dismal malignancy. Only the compound ONC201 was discovered to reduce DIPG tumors size in DIPG H3K27M mutated and now is in phase III clinical trials. Given the complex molecular mechanisms underlying DIPG, it would be necessary to fully utilize gene expression profiles to build a fully knowledge of DIPG-related genes and pathways. Preliminarily, the gene expression profile of eight pediatric DIPG samples and two primary pontine tissue samples was downloaded and analyzed starting from the Gene Expression Omnibus (GEO) database2. The GEO data were used to evaluate the differentially expressed genes (DEGs) in the pediatric DIPG samples. Subsequently, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome pathways of DEGs were enriched, integrated, and analyzed to find out genes and signaling pathways involved in the biological and clinic-pathological features of DIPG. More data are being to be added to our meta-analysis.

DIPG-06. PRECLINICAL EVALUATION OF A NOVEL ACVR1 INHIBITOR IN DIFFUSE INTRINSIC PONTINE GLIOMAS

Abstract Diffuse intrinsic pontine glioma (DIPG) is an incurable brain disease in children. DIPG is refractory to current available therapies. Hence, new therapeutic approaches are urgently needed. About 80% of DIPGs harbor histone 3 lysine to methionine (H3K27M) mutation, in H3-3A (encoding H3.3 histone) and H3C2 (encoding H3.1 histone) genes. One quarter of DIPG tumors harbor somatic mutations in ACVR1 gene, encoding the serine/threonine kinase receptor ALK2. ACVR1 mutations co-segregate with H3.1K27M mutation, representing a specific tumour subtype with distinct clinicopathological and molecular features. These mutations are associated with an aberrant activation of SMAD pathway believed to promote cell proliferation in DIPGs. In the present studies, we evaluated the effectiveness of an orally bioavailable ALK inhibitor, TP-0184 (Tolero Pharmaceuticals) in ACVR1 mutated (ACVR1-mut) and wildtype (ACVR1- WT) DIPG models. Our results indicate that TP-0184 effectively inhibited cell proliferation in primary-patient derived DIPG cell lines. However, we did not observe any selectivity toward ACVR1 mutations. TP-0184 does not induce cell death, but rather cell cycle inhibition, arresting cells in G2/M phase. Interestingly, when treated with ionizing radiation (IR), ACVR1-mut cells preferentially activated SMAD signaling compared to ACVR1-WT. We are currently evaluating the combination efficacy of TP-0184 with IR in our in vitro and in vivo DIPG models. Data on tumor growth kinetics, survival and the effect on SMAD signaling pathway will be presented and discussed.

TMET-35. ONE CARBON AND NUCLEOTIDE METABOLISM ARE NOVEL TARGETS IN PEDIATRIC BRAINSTEM TUMORS

Abstract Diffuse intrinsic pontine glioma (DIPG) are inoperable tumors of the brainstem with no cure. The median survival of children with DIPG is less than 2 years and 5-year overall survival is only 1%. Genomic studies have identified recurrent mutations in histone H3, ACVR and TP53 genes in DIPG. However, there are still no breakthrough drug targets for DIPG. To better understand the disease biology and find new drug targets for DIPG, we recently performed joint pathway analysis of DIPG metabolites (untargeted metabolomics) and differentially expressed genes (RNAseq) relative to normal human neural stem cells. We discovered that specific enzymes of the de novo purine biosynthesis pathway (DNPB), serine synthesis and the associated and one-carbon (1C) metabolism are highly active and the most upregulated in DIPG tumors and cell lines. Gain and loss of function experiments as well as pharmacological studies indicated a mechanistic link between DIPG mutations and DNPB pathway and the dependence of DIPG cells on specific DNPB and 1C metabolism enzymes for in vitro and in vivo growth. The 1C pathway is crucial for rapidly proliferating malignant cells as it senses and regulates cellular nutrient status by allocating and cycling 1C-groups between different acceptor compounds. It controls synthesis of nucleotides, amino acids, glycogen and phosphoglyceride precursors, S-adenosylmethionine (SAM), glutathione and NADPH. Thus, besides dispensing carbon atoms it also regulates cellular epigenetic and redox status. An important carbon donor is serine which is synthesized from glucose at 3-phosphoglycerate step in glycolysis. Untargeted as well as carbon and nitrogen tracing experiments indicated large folate pools in DIPG cells that can serve as a buffer to potentially blunt antifolate therapy. Experiments are underway to understand the importance of the folate cycle in DIPG and design novel and translatable combination therapies for DIPG.

EPCO-17. UNMASKING CLONAL EVOLUTION OF DIFFUSE INTRINSIC GLIOMA USING MULTI-MODAL GENOMIC DATA

Abstract Diffuse intrinsic pontine glioma (DIPG) is an infiltrative incurable tumor affecting children. DIPG tumors often harbor a recurrent H3K27M mutation which leads to a global loss of H3K27me2/3 and overall DNA hypomethylation, suggesting an important role of the epigenome, and consequent transcriptome in DIPG pathogenesis. To thoroughly characterize the clonal evolution of DIPG, we collected 33 samples from 7 DIPG patients using a multi-region sampling strategy and generated whole-exome and transcriptome sequencing, and DNA methylation profiling data. Using our novel bioinformatics approach, 28 distinct tumor sub-clones were identified and characterized in our cohort of DIPG patients whilst simultaneously interrogating the tumor’s ability to migrate and disseminate. When present, initiating tumor clone (Clone 1) exclusively contained H3K27M with significant DNA methylation changes whereas the divergent clones that arise later in DIPG evolutionary trees were typically driven by the copy number aberrations. Further characterization of DIPG sub-clones identified unique gene expression profiles (i.e. cell migration and angiogenesis programs) that support and enable DIPG dissemination. In this study, we uncovered how DIPG evolves at the genetic, epigenetic, and transcriptional levels in parallel and in doing so, reveal novel evolutionary phenotypes at the sub-clonal level related to the tumor’s behavior. We are further validating these results in single-cell RNA sequencing experiments and gaining further insights into the underlying molecular mechanisms responsible for invasive and aggressive DIPG phenotypes.

CSIG-11. HYPOXIA SIGNALING IN ACVR1-MUTANT DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract Diffuse intrinsic pontine glioma (DIPG) is a devastating glial tumor of the brainstem primary occurring in the pediatric population. The majority of DIPG tumors (60-80%) carry lysine- to methionine point mutation in histone H3, resulting in a global reduction of H3K27 trimethylation and subsequently a unique epigenetic landscape. Moreover, a large subset of the DIPG tumors carry activating mutations in ACVR1 – the gene encoding for the BMP receptor ALK2, suggesting an oncogenic role of signaling from this receptor. Importantly potential mechanisms of ACVR1-driven oncogenesis, remain poorly understood. Similar ACVR1 mutations have been described in the rare disorder Fibrodysplasia Ossificans Progressiva (FOP), and evidence from FOP models have suggested several links between hypoxia signaling, BMP signaling, and FOP pathogenesis thus opening up for evaluation of hypoxia signaling as oncogenic effector in ACVR1mutant DIPG. Using cultured DIPG cells and adult glioma cells respectively we have tested the activity of the hypoxia pathway using a hypoxia-responsive elements (HRE)-luciferase reporter assay. Intriguingly, DIPG but not glioma cells overexpressing a mutant ACVR1 construct significantly elevated hypoxia signaling at hypoxia. Similar trends are seen when assaying neuronal stem cell cultures harboring Histone 3.1 mutations along with activated ACVR1/ALK2 signaling. Furthermore, BMP ligand stimulation resulted in a dose-dependent increase of hypoxia signaling in DIPG cells. Through querying clinical gene expression data, we found elevated hypoxia signature scores in pediatric gliomas with mutant ACVR1 as compared to those with wildtype ACVR1. Finally, ACVR1 mutations frequently co-occur with Histone 3.3 and 3.1 mutations, and the hypoxia signature score was significantly higher than baseline only in tumors also harboring these Histone 3.3 or 3.1 mutations. Together, our data suggest that hypoxia signaling is elevated downstream of ACVR1 specifically in pediatric gliomas and prompts for evaluation of hypoxia signaling as potential therapeutical target in DIPG.

Abstract 5227: Combination therapy activating the integrated stress response synergistically suppresses proliferation and induces apoptosis in diffuse intrinsic pontine glioma

Abstract Diffuse intrinsic pontine glioma (DIPG) is a lethal pediatric brain tumor. Radiation, the standard of care, extends life for 6-18 months but has never cured a patient. Therefore, the discovery of novel treatments is imperative. DIPG tumors have elevated baseline activation of the integrated stress response (ISR), an evolutionarily conserved system that allows cells to tolerate various forms of stress. Increased expression of activating transcription factor 4 (ATF4) indicates activation of the ISR. Low levels of ATF4 protect cells from stress, while sustained high-levels of ATF4 result in cell death. Because DIPG has a high baseline level of ATF4, we hypothesized that the ISR activators Sal003 and ONC201 would synergize and kill DIPG cells. After determining the IC25 of Sal003 and ONC201, we treated three patient-derived cell lines: JHH-DIPG1, SF-7761, and JHH-DIPG16A with low micromolar doses. To measure proliferation, we performed immunofluorescence staining for bromodeoxyuridine (BrdU) incorporation. The combination treatment significantly reduced BrdU incorporation (JHH-DIPG1 p=0.0026, SF-7761 p=0.0002, JHH-DIPG16A p<0.0001 by ANOVA and Dunnett’s multiple comparisons test compared to DMSO control). In all three cell lines, the combination also significantly reduced proliferation compared to monotherapy. We next measured apoptosis by staining for cleaved caspase-3 (CC3) and performing western blots for cleaved PARP. The combination of Sal003 and ONC201 significantly increased apoptosis as measured by CC3 immunofluorescence in comparison to DMSO (JHH-DIPG1 p<0.0001, SF-7761 p<0.0001, JHH-DIPG16A p<0.0001 by ANOVA and Dunnett’s multiple comparisons test compared to DMSO control). In all cell lines, combination therapy significantly increased CC3 positivity compared to single treatment. Western blots for cleaved PARP expression detected induction of apoptosis in all three cell lines treated with the combination over DMSO and monotherapy treated cells. In JHH-DIPG1 and SF-7761, the combination increased ATF4 expression. Since Sal003 is not yet available for clinical testing in humans, we will next investigate treatment with ONC201 and the well-tolerated ATF4 inducer fenretinide. The combination of ONC201 with another ISR activating agent has the potential to serve as a therapy for DIPG. Citation Format: Orlandi V. Novak, Antje Arnold, Charles Eberhart, Eric H. Raabe. Combination therapy activating the integrated stress response synergistically suppresses proliferation and induces apoptosis in diffuse intrinsic pontine glioma [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 5227.

DIPG-11. DIPGs show robust MHC class I expression but low lymphoid and high myeloid infiltration partly reversible by DNA methyltransferase inhibition

Diffuse intrinsic pontine gliomas (DIPGs) are the most aggressive tumors of the central nervous system in children. Median survival of patients is less than one year post-diagnosis. Radiotherapy remains the only standard treatment but is rarely curative. Immunotherapy is an emerging and promising treatment strategy for children with DIPG. However, general immunotherapy has not lived up to its promise to treat many cancers, including DIPGs, in part due to incomplete understanding of the barriers posed by the tumor microenvironment. We therefore evaluated the immune cell infiltration in a syngeneic mouse model of DIPG and in DIPG tumors collected from patients. We evaluated the expression profiles of T lymphocytes and myeloid cell markers in a cohort of 28 DIPG tumors compared to matched normal tissue specimens. Our data indicate that the expression of MHC I components in DIPG tumors is similar to that of matched normal tissue. Moreover, the well-known immune checkpoint, PD-L1, was not overexpressed in DIPG tumors. Using immunohistochemistry, we demonstrated only rare infiltration of lymphocytes but high enrichment of myeloid cells in tumor tissue. We found similar results in a syngeneic mouse model of DIPG. Collectively, our results indicate that DIPG tumors harbor rare lymphocytes and are enriched in myeloid cells. Recent studies have demonstrated that DNA methyltransferase inhibitors prime some tumors for more effective checkpoint blockade by activating expression of human endogenous retroviruses and sparking a T-cell mediated immune response through a process called “viral mimicry.” We tested the effect of decitabine in a syngeneic mouse model of DIPG. Decitabine reduced the tumor growth kinetics compared to vehicle but was unable to induce the recruitment of lymphoid cells in DIPG tumors. Importantly, we observed a noticeable reduction in the myeloid component of the DIPG microenvironment suggesting a possible role of myeloid cells in tumor growth and progression.

DIPG-13. Immune profiling by RNA-seq deconvolution and single-cell sequencing reveal myeloid cell enrichment in DIPG tumor microenvironment

Abstract Diffuse intrinsic pontine glioma (DIPG) remains an incurable disease with median overall survival <12 months despite decades of clinical trials investigating multimodal therapies. Immunotherapy represents a promising treatment paradigm which has been successfully used in other cancers. An adequate understanding of the tumor microenvironment and immunologic profile is essential to identify potential immunotherapeutic targets to inform immunotherapy design. Previous studies have shown that most DIPG tumors harbor low mutational burden compared to adult cancers and are characterized by a non-inflammatory microenvironment, limiting the development of immunotherapies in this disease. Our team’s prior work similarly demonstrated that most DIPG tumors have an immunologically “cold” microenvironment, but a subset of tumors harbors a more inflammatory gene expression profile and/or higher mutational burden, with trends toward improved survival and favorable radiographic response to radiation. Here, we applied a deconvolution analysis using CIBERSORTx on bulk RNA-seq data from 28 DIPG patients’ tumors paired with matched normal tissue specimens, to profile the immune microenvironment of DIPG and evaluate immune-related gene expression to determine percentages of different types of immune cells. Our results indicate that DIPGs have very limited lymphocyte infiltration. However, the infiltration of macrophages “M2-like” type cells and CD4 memory resting T cells were significantly higher in tumors compared to normal tissue samples. Similar results were found using single-cell RNA sequencing performed on biopsy and autopsy tissue, with less than 5% of total cells identified as immune cells. MHC I components were widely expressed in DIPGs with no significant difference between tumor and normal tissue. Expression of CD11B and CD68 were higher in tumor compared to normal tissue, suggesting enrichment of myeloid cells. Overall, deconvolution analysis of bulk RNA-seq can be used to profile DIPG tumors’ immune microenvironment to aid in the thoughtful design of effective immunotherapeutic strategies for this disease.

IMMU-23. Novel gene-edited CAR-T cell therapy against Diffuse Intrinsic Pontine Glioma

BACKGROUND: We identified high expression of CD99 in DIPG tumors and developed a CAR using our newly identified single chain variable fragment (scFv) targeting CD99 incorporating a 4-1BB co-stimulatory domain. This CD99 CAR demonstrated the ability to dramatically shrink the established orthotopic DIPG tumor, however tumor recurrence remains an obstacle to cure, due to a loss of the CAR-T cells as they also express the target antigen, CD99 (fratricide). To overcome this obstacle, we modified these CAR-T by editing out CD99. METHODS: CD99 was knocked-out from the human T cells using CRISPR-cas9 gene-editing and subsequently transduced with our CD99 CAR-encoding virus, and isolated the pure population of CD99KO T-cells. These novel, gene-edited T-cells expressing CD99 CAR (“CD99KO CARs”) and the un-edited ones (“CD99 CAR”) were tested for tumor-lysis function when co-cultured with DIPG cells. DIPG tumor-bearing mice infused with a one-time dose of CD99KO CAR-T cells or CD99 CAR- or CD19 control CAR-T cells and were monitored for changes in the tumor burden. At the endpoint spleen and bone marrow were isolated to test for CAR+ cell persistence. RESULTS: The CD99KO CAR-T cells demonstrated effective tumor-lysis when co-cultured with DIPG cells. CD99KO CAR-T cells targeting CD99 showed complete clearance of DIPG tumor in orthotopic DIPG mouse models, and no tumor recurrence was seen well-beyond the time frame of expected tumor recurrence after treatment with un-edited CD99 CAR-T cells. There was an un-precedented increase in the xenograft survival, > 200 days, in mice treated with CD99KO CARs and at which time point sustained persistence of CAR+ cells were evident in the animal spleen and bone marrow. CONCLUSIONS: We have generated a new and promising CAR-T cell therapy that is effective against DIPG with enhanced persistence in animal models which is critical for clinical translation.

DIPG-12. Induced mitotic abnormalities associated with BMI-1 modulation sensitize diffuse intrinsic pontine glioma cells to ionizing radiation

Abstract Diffuse intrinsic pontine glioma (DIPG) remains an incurable childhood brain cancer with a median overall survival of less than 12 months, affecting 200-300 children annually in the United States. Hence, there is an unmet need for the development of novel and effective targeted therapies. BMI-1 is a subunit of the multimeric protein complex Polycomb repressor complex 1 (PRC1) implicated in self-renewal of normal and cancer cells, and in DNA damage signaling. We have previously identified BMI-1 as a potential therapeutic target in DIPG and have shown that BMI-1 is highly expressed in DIPG tumors regardless of H3K27 mutational status. Treatment of DIPG cells with PTC596, a small molecule initially identified as a BMI-1 modulator, and ionizing radiation (IR) impairs the kinetics of DNA damage response. in vivo, treatment with PTC596 alone delayed tumor growth kinetics and induced in-tumor apoptosis. However, we observed tumor regrowth once PTC596 treatment is completed or discontinued. In the present study, we evaluated the use of PTC596 in combination with IR. Our in vivo results indicate that PTC596 sensitizes DIPG cells to IR inducing a prolonged cell growth arrest 14 days post-treatment compared to IR or PTC596 alone. The effectiveness of this combination is currently evaluated in murine orthotopic DIPG models and the results will be presented. PTC596 is being tested in newly diagnosed children with DIPG and high-grade gliomas (NCT03605550). Data collected from this study will support the development of a novel therapy including PTC596 in combination with radiotherapy to treat children with DIPG.

DIPG-16. Evaluation of myeloid component of DIPG microenvironment

Diffuse intrinsic pontine glioma (DIPG) is an aggressive and incurable disease of the central nervous system in children with median overall survival of less than one year. In recent years, several immunotherapy strategies have emerged as an option to treat DIPG. However, the low mutational burden and rare infiltration of T lymphocytes, render these tumors immunologically “cold” and therefore pose challenges for general immunotherapy. The myeloid component was implicated in the immunosuppression in other solid tumors. Previous data have shown that DIPG tumors are enriched in macrophages, but their role in tumor growth and progression have not been elucidated. Specifically, it remains unclear whether the myeloid cells are recruited to the tumor microenvironment from the peripheral circulation. Here, we examined the recruitment of myeloid cell populations to the tumor microenvironment and further delineated their role in tumor progression in a syngeneic mouse model of DIPG. We showed that this DIPG mouse model displays an immune microenvironment similar to that of patients’ DIPGs. DIPG tumors harbored rare tumor infiltrating lymphocytes and are enriched in myeloid cells. To further characterize the phenotype and functions of these myeloid populations, we evaluated the changes in proportions of myeloid cell subsets using flow cytometry (CD11b, Ly6c, Ly6G, MHCII, F4/80, CD206, Arg1) in the bone marrow, peripheral blood, and in the tumor microenvironment during tumor progression. Also, we investigated the role of these myeloid cells in angiogenesis and immune suppression by performing histological and expression analyses of endothelial markers and chemokines (CD31, CD34, KDR, IL-10, IL-13, IL-4, CCL2, CCL5). Furthermore, decitabine (DNA methyltransferase inhibitor) treated tumors showed a decrease in myeloid population associated with a reduction in tumor growth, suggesting an important role of myeloid populations in tumor growth and progression.

Imaged-guided focused ultrasound in combination with various formulations of doxorubicin for the treatment of diffuse intrinsic pontine glioma

BackgroundDiffuse intrinsic pontine glioma (DIPG) is a notoriously difficult tumor to treat, with an overall survival of DIPG patients being only 11 months. One of the major obstacles for the effective treatment of DIPG is the blood–brain barrier (BBB). In order to circumvent the BBB, drug delivery methods are needed that target the pontine area. One such approach is microbubble-mediated focused ultrasound (FUS)—a non-invasive method that can temporarily and locally open the BBB. Previously, it was shown that FUS is safe with minimal side effects and rapid recovery times in preclinical animal models with different DIPG tumors. However, recent studies have shown that combining FUS with a single treatment of the chemotherapeutic drug doxorubicin did not improve survival in a DIPG xenograft model. As the duration of doxorubicin exposure might play a role in tumor response, we hypothesized that the use of a long-circulation (PEGylated) liposomal formulation of doxorubicin could lead to improved overall survival through a longer exposure time to the tumor.MethodDIPG xenograft models were established with orthotopic injections of HSJD-DIPG-07 tumor cells into the pontine area of female athymic nude-foxn1nu mice. Tumor engraftment was confirmed with bioluminescence imaging (BLI) 40 days post-inoculation. Mice were randomized into groups receiving either liposomal formulations of doxorubicin (2B3-101 or Caelyx®) or free doxorubicin in combination with or without FUS treatment. Treatment groups received 5 mg/kg 2B3-101 or Caelyx® 1 h before FUS treatment or 5 mg/kg free doxorubicin immediately after FUS.ResultsHistological analysis, however, revealed liposome extravasation in healthy controls but not in HSJD-DIPG-07 xenograft 24 h after treatment. Furthermore, BLI monitoring did not show reduced signal after treatment, which was further illustrated with a survival analysis, showing no significant difference between treated and control animals (p = 0.3).ConclusionWe did not observe a treatment effect after a single dose of free doxorubicin or the liposomal formulations 2B3-101 or Caelyx® in combination with FUS in DIPG-bearing mice.

Targeting of the alphav beta3 integrin complex by CAR-T cells leads to rapid regression of diffuse intrinsic pontine glioma and glioblastoma

BackgroundDiffuse intrinsic pontine glioma (DIPG) and glioblastoma (GBM) are two highly aggressive and generally incurable gliomas with little therapeutic advancements made in the past several decades. Despite immense initial success...

EXTH-15. MULTI-FACETED INHIBITION OF TET PATHWAY WITH CELL-PERMEABLE 2HG AND BOBCAT 339 REDUCES PROLIFERATION AND INDUCES APOPTOSIS IN DIPG

Abstract Diffuse Intrinsic Pontine Glioma (DIPG) is one of the worst forms of pediatric brain tumors, with a 100% mortality-rate. A prominent mutation observed in them is a lysine to methionine change in histone3 which causes trapping of the repressive-chromatin-complex, leading to genome-level hypomethylation, affecting global epigenetic-regulation. Ten-Eleven Translocation (TET) proteins are alpha-ketoglutarate (α-KG) dependent dioxygenases that mediate the conversion of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), a key-step in removing DNA methylation-marks. We previously identified increased levels of 5hmC and TETs in DIPG. In IDH mutant glioma, the R132H-IDH produces an oncometabolite, 2-hydroxyglutarate (2HG), instead of α-KG. 2HG competitively inhibits TET function, resulting in a hypermethylated genome. Bobcat339, a cytosine-analog targets TETs by blocking the cytosine binding-site on TETs. We hypothesized that cell-permeable 2HG and Bobcat339 would synergize to block TET activity in DIPG, restore epigenetic-balance by increasing genomic methylation, and induce cell-death. 2HG induced apoptosis in DIPG cells at concentrations ranging from 100-300uM, as measured by cleaved-PARP western and cleaved-caspase3 immunofluorescence (2-7fold increase in CC3, depending on cell line). Cell-permeable 2HG also decreased proliferation, measured by phospho-RB western in DIPG cells (30-50% reduction in pRB band intensity, depending on cell line). Similarly, Bobcat339 suppressed proliferation at concentrations from 10-50uM, measured by BrdU incorporation (25% reduction in BrdU+ at 50uM, p= 0.02 compared to control). Bobcat339 induced apoptosis, measured by cPARP western and CC3 immunofluorescence (4-10fold more of CC3+ compared to control p< 0.0004). In combination, cell-permeable 2HG (100-200uM) and Bobcat339 (10-20uM) combined to suppress cell-viability, measured by CellTiterBlue assay, producing ZIP scores of ~20 in DIPG cells (indicating high-level synergy). In combination, 2HG and Bobcat339 increased apoptosis in DIPG (3-fold increase in cleaved-PARP band intensity in JHH-DIPG1 cells treated with 20uM Bobcat339 + 100uM 2HG, compared to single-treated cells). Our results may lead to new approaches that target TET pathway in DIPG tumors.

CSIG-31. MULTI-OMICS TO EDGE INTO PRECISION MEDICINE FOR DIPG

Abstract DIPG is an incurable pediatric brain tumor with 80% of patients harboring H3F3A (H3.3) mutation that substitutes methionine for lysine at position 27 (K27M), resulting in 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 (1). To determine the reprogramming pathways in the cell context of H3.3K27M tumors, we conducted LC-MS based proteomic and phosphoproteomic analysis on seven patient-derived DIPG cell lines. 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. PYK2 has been proposed to act in concert with Src to link Gi- or Gq-coupled receptors with the mitogen-activated protein (MAP) kinase signaling pathway (2). 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. 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.

TAMI-80. CELLULAR METABOLISM IN DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract Diffuse intrinsic pontine glioma (DIPG) is an aggressive form of brain tumor in children, comprising >10% of all pediatric brain tumors. The median survival after diagnosis is < 1 year. Since DIPG tumors infiltrate brainstem and pons, they are inoperable. Currently radiotherapy is the mainstay of treatment, and there is a great need for novel therapies for the treatment of DIPG. Cellular metabolism plays a key role in carcinogenesis, unravelling active metabolic pathways in DIPG would help in developing targeted therapies. Glucose and glutamine are the two major nutrients necessary for the growth and proliferation of cancer cells. In this study, we have investigated the glucose and glutamine metabolism in SF8628 DIPG cells using 1H/13C NMR and GC-MS based metabolic flux analysis. SF8628 cells were grown in DMEM containing 11.0 mM glucose, supplemented with 10% FBS, and 2.0 mM glutamine at 37 °C under humidified air and 5% CO2. When cells reached confluency (replicates = 4), treated with 11.0 mM [U-13C]glucose or 4.0 mM glutamine in DMEM (supplemented with 10% FBS). After 24 h, cells were harvested for NMR/GC-MS analysis. The 13C-isotopomer analysis revealed that SF8628 cells produced 25.26 ± 10.63% acetyl-CoA from [U-13C]glucose which is ~3.7 times higher than that produced from GBM cells (6.83 ± 0.76%; our previous work), suggesting that DIPGs are metabolically very active. [U-13C]glutamine metabolism showed that DIPG cells also have an active TCA cycle metabolism (citrate M+4; 40.07 ± 1.06%) and moderately active reductive carboxylation pathway (citrate M+5; 10.59 ± 1.13%). Inhibition of both glycolytic and glutaminolysis pathways will be valuable in developing treatment strategies for DIPGs and these studies are in progress.

CSIG-12. THE ROLE OF HYPOXIA SIGNALING AS AN ONCOGENIC EFFECTOR IN ACVR1-MUTANT DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract Diffuse intrinsic pontine gliomas (DIPG) are aggressive and highly treatment resistant pediatric tumors of the brainstem. The majority of the DIPG tumors (60-80%) carry somatic histone H3 mutations resulting in a global reduction of H3K27 trimethylation and subsequently a unique epigenetic landscape. Moreover, activating mutations in ACVR1 – the gene encoding for the BMP receptor ALK2 – were recently described in a large subset of DIPGs, suggestive of an oncogenic role of signaling from this receptor. Potential mechanisms of ACVR1-driven oncogenesis, however, remain poorly understood. Similar and overlapping ACVR1 mutations have been described in the rare disorder Fibrodysplasia Ossificans Progressiva (FOP), and evidence from FOP models have suggested several links between hypoxia signaling, BMP signaling, and FOP pathogenesis. We used cultured DIPG cells and adult glioma cells respectively to test the activity of the hypoxia pathway using a hypoxia-responsive elements (HRE)-luciferase reporter assay. Intriguingly, DIPG but not glioma cells overexpressing a mutant ACVR1 construct significantly elevated hypoxia signaling at hypoxia. BMP ligand stimulation resulted in a dose-dependent increase of hypoxia signaling in DIPG cells. By querying clinical gene expression data, we found elevated hypoxia signature scores in pediatric gliomas with mutant ACVR1 as compared to those with wildtype ACVR1. Finally, ACVR1 mutations frequently co-occur with Histone 3.3 and 3.1 mutations, and the hypoxia signature score was significantly higher than baseline only in tumors also harboring these Histone 3.3 or 3.1 mutations. Together, these data support the notion that hypoxia signaling is elevated downstream of ACVR1 specifically in pediatric gliomas and prompts for evaluation of hypoxia signaling as potential therapeutical target in DIPG.

Panobinostat penetrates the blood-brain barrier and achieves effective brain concentrations in a murine model.

Panobinostat, an orally bioavailable pan-HDAC inhibitor, has demonstrated potent activity in multiple malignancies, including pediatric brain tumors such as DIPG, with increased activity against H3K27M mutant cell lines. Given limited evidence regarding the CNS penetration of panobinostat, we sought to characterize its BBB penetration in a murine model. Panobinostat 15mg/kg was administered IV to 12 CD-1 female mice. At specified time points, mice were euthanized, blood samples were collected, and brains were removed. LC-MS was performed to quantify panobinostat concentrations. Cmax and AUC were estimated and correlated with previously published pharmacokinetic analyses and reports of IC-50 values in DIPG cell lines. Mean panobinostat plasma concentrations (ng/mL) were 27.3 ± 2.5 at 1h, 7.56 ± 1.8 at 2h, 1.48 ± 0.56 at 4h, and 2.33 ± 1.18 at 7h. Mean panobinostat brain concentrations (ng/g) were 60.5 ± 6.1 at 1h, 42.9 ± 5.4 at 2h, 33.2 ± 6.1 at 4h, and 28.1 ± 4.3 at 7h. Brain-to-plasma ratio at 1h was 2.22 and the brain to plasma AUC ratio was 2.63. Based on the published human pharmacokinetic data, the anticipated Cmax in humans is expected to be significantly higher than the IC-50 identified in DIPG models. It is expected that panobinostat would be effective in CNS tumors where the IC-50 is in the low nanomolar range. Thus, our data demonstrate panobinostat crosses the BBB and achieves concentrations above the IC-50 for DIPG and other brain tumors and should be explored further for clinical efficacy.