Midline Glioma Research Articles

Abstract 5460: Discovery and validation of effective combination therapies targeting cell state-specific master regulator vulnerabilities by network-based protein activity inference in diffuse midline glioma

Abstract Diffuse Midline Glioma (DMG) are fatal pediatric brain tumors with no effective systemic therapies. We leveraged network-based methodologies to dissect tumor heterogeneity, discover Master Regulators (MR) representing pharmacologically accessible vulnerabilities of distinct DMG cell states, and validated candidate MR-reversing drugs predicted by the NYS CLIA-certified OncoTarget and OncoTreat algorithms (Cancer Discov 2023) in vivo. We first interrogated single cell DMG regulatory networks generated by ARACNe (Nat Genet 2005) with publicly available gene expression signatures from 3,039 tumor cells across 6 patients using VIPER (Nat Genet 2016) to infer single cell regulatory protein activity. Clustering of cells by protein activity defined 7 patient-independent cell states with distinct MR profiles reflecting known glial lineage markers (OPC-like-S1, OPC-like-S2, OC-like-S1, OC-like-S2, Cycling, AC-like, and AC/OPC-like). We then generated drug-induced differential protein activity from RNAseq profiles following perturbation with 372 oncology drugs in two DMG cell lines that together recapitulate the MR signatures of the cell states and used this to identify drugs that invert tumor MR activity profiles using OncoTreat. Candidate drugs predicted by OncoTarget (inhibitors of individual MRs) and OncoTreat were distinct across the cell states, and we selected five drugs targeting the OPC/cycling-like cells (Trametinib, Dinaciclib, Avapritinib, Mocetinostat, and Etoposide), and four drugs targeting the AC-like cells (Ruxolitinib, Venetoclax, Napabucasin, Larotrectonib) for further validation as these states comprised most tumor cells across patients. We generated single-cell RNAseq for 95,687 cells after 5 days of treatment with either vehicle control (n = 4) or candidate drug (n = 2-3/drug) in subcutaneous SU-DIPG-17 mouse models. We show this model recapitulates cell states seen in patients and confirm reduction in tumor growth and significant depletion of either OPC/cycling-like cells or AC-like cells in line with our drug predictions for 8/9 candidate drugs. We then treated a syngeneic (DIPG4423) orthotopic DMG model with each drug and demonstrate significant differences in survival with Avapritinib, Dinaciclib, and Trametinib. Notably, the combination of drugs targeting OPC/cycling-like and AC-like cells (i.e. Trametinib+Ruxolitinib and Avapritinib+Venetoclax) showed significantly lower tumor volumes after 2 weeks of treatment as compared to vehicles or each drug alone, and significant survival differences for some combinations. This work provides a precision medicine platform to nominate much-needed novel drug combinations addressing DMG tumor heterogeneity for further study to improve outcomes in this devastating disease. Citation Format: Ester Calvo Fernandez, Lorenzo Tomassoni, Xu Zhang, Aleksandar Obradovic, Pasquale Laise, Aaron T. Griffin, Lukas Vlahos, Junqiang Wang, Hanna E. Minns, Diana V. Morales, Christian Simmons, Matthew Gallito, Hong-Jian Wei, Zhiguo Zhang, Robyn Gartrell, Luca Szalontay, Stergios Zacharoulis, Cheng-Chia Wu, Andrea Califano, Jovana Pavisic. Discovery and validation of effective combination therapies targeting cell state-specific master regulator vulnerabilities by network-based protein activity inference in diffuse midline glioma [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 5460.

Abstract 1126: Extracellular vesicle mediated radioresistance in H3K27M-diffuse midline glioma

Abstract H3K27M-Diffuse Midline Gliomas (DMGs) are a subset of uncurable malignant pediatric gliomas and the deadliest form of brain tumors in children worldwide. Radiation therapy, the current standard of care, is initially effective but all tumors become resistant within 4-6 months. H3K27M-DMGs are heterogenous, comprised of an average of 6 genetically distinct subclones. Some subclones harbor mutations in genes previously linked to radiation resistance, such as TP53. We have found subclones are also functionally distinct, with some clones harboring intrinsic radiation resistance. Extracellular signaling between clones is linked with therapy resistance and tumor progression in other tumors, and we hypothesized that radioresistant H3K27M-DMG release small extracellular vesicles (sEVs) that impact the radiosensitivity of neighboring tumor cells. We found that all patient-derived H3K27M-DMG samples examined (n=6) produced sEVs (< 220nm), indicating this is a common feature in this tumor type. We isolated sEVs from a radioresistant (RR) patient-derived H3K27M-DMG cell line and used them to treat radiosensitive (RS) DMG cells. Using flow cytometry to profile sEV uptake dynamics, we found that RR-sEVs are internalized by RS cells within 2 hours of exposure and are retained at 18 hours (p<0.001, compared to control). This uptake can be significantly abrogated by proteinase K treatment of the sEVs, (p<0.001), indicating that uptake involves sEV surface proteins. We then applied a biosensor approach to assess single-cell phenotypic changes in RS DMG after RR-sEV treatment on radiation-induced cell death, DNA damage, and cell senescence, three dynamic processes. Using a genetically encoded death indicator (GEDI), we found that RR-sEVs significantly enhanced the survival of RS cells after acute radiation exposure (p<0.001). This was due to enhanced DNA damage repair as measured by 53BP1 foci (p<0.01). Small RNA sequencing of sEV cargo demonstrated that RR-sEVs express multiple miRNAs that are involved in oncogenic signaling such as the miR-7 family, miR-1246, and miR-1290 at several log fold higher than RS-sEVs. Overall, this data reveals the first functional role for sEVs in the context of radiation induced tumor transformation in DMG. We plan to further investigate the primary route of sEV uptake and define the mechanism through which sEV cargo alters radiosensitivity. This study provides new insight into the impact of heterogeneity and sEVs in mediating radioresistance in H3K27M-DMG and may lead to the development of novel radiosensitizers, which are critically needed for all patients. Citation Format: Viral Oza, Shilpa Sampathi, Yelena Chernyavskava, Kenan Flores, Jessica Blackburn. Extracellular vesicle mediated radioresistance in H3K27M-diffuse midline glioma [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 1126.

Abstract 3173: Impact of hypoxia on the integrated stress response activated by imipridones ONC201 and ONC206 in pediatric diffuse midline glioma cells

Abstract Diffuse midline gliomas (DMGs) are highly aggressive, high-grade gliomas which typically arise in children and young adults. With currently approved treatments the median survival from time of diagnosis for pediatric DMG is 8-10 months with only 10% of children living to 2 years post diagnosis. Despite being only 15% of the cases it makes up 40% of the pediatric brain cancer deaths making it the leading cause of death for all pediatric glioma cases. Two recent clinical studies, NCT03416530 and NCT03134131, have shown the clinical efficacy of Dordaviprone (ONC201/TIC10) for the treatment of DMG; increasing median survival to 22 months in patients following radiation treatment prior to recurrence. Imipridone ONC206, a chemical derivative of ONC201, is under clinical development for treatment of pediatric and adult patients with primary brain tumors (NCT04732065 and NCT04541082). To further improve treatment, we must continue to study how the tumor microenvironment impacts the efficacy of imipridones. One crucial aspect of all brain cancers is hypoxia, so the IC50s of the SU-DIGP-25, SU-DIPG-XIII and SU-DIPGIV pediatric DMG cell lines treated with ONC201 or ONC206 were measured using the CellTiter-Glo assay under conditions of hypoxia and normoxia. Imipridones mediate apoptosis through the upregulation of the TRAIL death receptor DR5 and the activation of the integrated stress response (ISR), so western blots were used to show changes in the ISR protein expression in ONC201 treated DMG cells at multiple different degrees of hypoxia. To understand how hypoxia inducible factors (HIFs) play a role in resistance and susceptibility to ONC201 or ONC206, HIF-1a, HIF-2a, and HIF-3a were knocked down showing altered ISR protein expression after treating with the imipridones under hypoxic and normoxic conditions. Citation Format: Tyler J. Roady, Nolan Stubbs, Josephine Chen, Yutong Xia, Ashley Sanchez Sevilla Uruchurtu, Xiaobing Tian, Lanlan Zhou, Wafik S. El-Deiry. Impact of hypoxia on the integrated stress response activated by imipridones ONC201 and ONC206 in pediatric diffuse midline glioma cells [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 3173.

Abstract 155: Clinico-radiological and histomolecular analyses identify of a new subtype of diffuse midline glioma, H3 K27 and BRAF/FGFR1 co-altered

Abstract Diffuse midline gliomas (DMG) H3 K27-altered are incurable grade 4 gliomas and represent a major challenge in neuro-oncology. This tumor type is now classified in four subtypes by the 2021 edition of the WHO Classification of the Central Nervous System (CNS) tumors. However, the H3.3-K27M subgroup still appears clinically and molecularly heterogenous. Recent publications reported that rare patients presenting a co-occurrence of H3.3K27M with BRAF or FGFR1 alterations tended to have a better prognosis. To better study the role of these co-driver alterations, we assembled a large pediatric and adult cohort of 29 tumors H3K27-altered with co-occurring activating mutation in BRAF or FGFR1 as well as 31 previous cases from the literature. We performed a comprehensive histological, radiological, genomic, transcriptomic and DNA methylation analysis. Interestingly, unsupervised t-distributed Stochastic Neighbor Embedding (tSNE) analysis of DNA methylation profiles regrouped BRAFV600E and all but one FGFR1MUT DMG in a unique methylation cluster, distinct from the other DMG subgroups and also from ganglioglioma (GG) or high-grade astrocytoma with piloid features (HGAP). This new DMG subtype harbors atypical radiological and histopathological profiles with calcification and/or a solid tumor component both for BRAFV600E and FGFR1MUT cases. Contrary to other DMG, these tumors occur more frequently in the thalamus (70% for BRAFV600E and 58% for FGFR1MUT) and patients have a longer overall survival with a median above three years. The analyses of a H3.3-K27M BRAFV600E tumor at diagnosis and corresponding in vitro cellular model showed that mutation in H3-3A was the first event in the oncogenesis, indicating a distinct oncogenesis from classical DMG despite an identical founder H3K27 alteration, and possible subclonal evolution.In conclusion, DMG, H3 K27 and BRAF/FGFR1 co-altered represent a new subtype of DMG with distinct genotype-phenotype characteristics, which deserve further attention with respect to trial interpretation and patient management. Our work also lays the foundations for therapeutic development highly-needed against this deadly disease. Citation Format: Lucie Auffret, Yassine Ajlil, Arnault Tauziede-Espariat, Thomas Kergrohen, Chloé Puiseux, Laurent Riffaud, Pascale Blouin, Anne-Isabelle Bertozzi, Pierre Leblond, Klas Blomgren, Sebastien Froelich, Alberto Picca, Mehdi Touat, Marc Sanson, Kevin Beccaria, Thomas Blauwblomme, Volodia Dangouloff-Ros, Nathalie Boddaert, Pascale Varlet, Marie-Anne Debily, Jacques Grill, David Castel. Clinico-radiological and histomolecular analyses identify of a new subtype of diffuse midline glioma, H3 K27 and BRAF/FGFR1 co-altered [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 155.

Abstract 525: A comprehensive approach: Copper chelation therapy modulates epigenetic, kinase signaling and metabolic pathways in diffuse midline gliomas (DMG)

Abstract Diffuse Midline Gliomas (DMG) are incurable pediatric brain tumors driven by histone mutation H3K27M, globally reducing H3K27 trimethylation altering the epigenome and deregulating gene expression. These mutations often partner with and are strongly integrated into kinase signaling and metabolic alterations, driving aggressive tumor growth with many mechanisms which can compensate for one another and therefore evade traditional single agent therapies. Copper over-accumulation has been linked to epigenetic, kinase and metabolic dysregulation in various cancers and neurological disorders. Copper chelators are clinically approved for use in pediatric Wilson’s Disease patients, improve neurological symptoms and are under clinical investigation for several cancers. We propose copper chelators may simultaneously target these mechanisms in DMG. We performed transcriptomic interrogation of patient datasets, in vitro investigations in a panel of DMG cell lines and an in vivo investigation using an orthotopic xenograft model of DIPG (SU-DIPGVI-Luc). Copper chelator tetraethylenepentamine-pentachloride (TEPA) was used to treat DMG cells. Transcriptomic (RNA-Seq), proteomic, phospho-proteomic and metabolomic (Mass Spectrometry) investigations, western blots and immunofluorescent microscopy determined efficacy and mechanism. In vivo investigation consisted of survival study with concurrent luminescence imaging, histology, and copper assay. We demonstrate copper chaperone transcript expression is increased in DMG patient samples compared to non-DMG samples. In vitro, apoptosis and growth assays demonstrated H3K27M DMG were more sensitive to TEPA than H3-WT DMG. In H3K27M DMG, TEPA downregulated ATP, GTP and SAM metabolism which corresponded with downregulated kinase signaling and altered H3K27 trimethylation and DNA methylation patterns around cell cycle genes. Affected genes included those regulating centrosome segregation and ATP synthesis. Further transcriptomic and proteomic investigations highlighted TEPA downregulated G2-M phase kinase pathways, with immunofluorescence demonstrating TEPA reduced expression of mitotic marker p-H3S10 and epigenetic methylators EZH2 and DNMT1. In vivo, TEPA reduced brain copper accumulation, eliminated tumors and increased survival in an orthotopic patient-derived xenograft DMG model. Collectively, our data indicates TEPA induced multi-modal targeting of M-phase cell cycle progression through epigenetic, kinase signaling and metabolic mechanisms. Efficacy was observed both in vitro and in vivo, providing a promising basis for future investigation of copper chelators in DMG. We propose copper-chelation is a viable therapeutic strategy for DMG patients warranting clinical investigation. Citation Format: Filip Michniewicz, Riccardo Cazzoli, Jessica Bell, Federica Saletta, Ensieh Poursani, Jourdin R. Rouaen, Tyler Shai-Hee, Vu Pham, Toni Rose Jue, Matthew Biery, Liesl Bramberger, Terry Lim, Aaminah Khan, Federico M. Giorgi, Daniele Mercatelli, Chelsea Mayoh, Giuseppe Cirillo, Alexander Macmillan, Renee Whan, Christopher T. Barlow, Maria Tsoli, Nicholas Vitanza, Scott Berry, Pouya Faridi, David Ziegler, Orazio Vittorio. A comprehensive approach: Copper chelation therapy modulates epigenetic, kinase signaling and metabolic pathways in diffuse midline gliomas (DMG) [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 525.

Abstract 1437: Generating primary mouse models of diffuse midline glioma through a combination of the RCAS/tv-a retrovirus system and CRISPR/Cas9 gene editing

Abstract Diffuse midline gliomas (DMGs) are deadly brain tumors that arise in the brainstem of children and young adults, resulting in a median survival of less than two years. Genetically engineered mouse models (GEMMs) are critical to studying tumorigenesis and tumor-immune interactions in DMG, which may inform the design of new urgently-needed treatment approaches. One approach to generate DMG GEMMs uses RCAS/tv-a, an avian retrovirus gene delivery system to express transgenes in specific brain cell lineages in the mouse brain. However, this approach often requires resource-intensive breeding to generate mice with the desired genetic alterations in their germline. As a result, we aimed to establish a new approach that combines this system with CRISPR/Cas9 gene editing technology. This methodology has been described for model adult supratentorial gliomas in mice but has not been described for pediatric or brainstem gliomas. To express Cas9 and the avian retrovirus receptor TVA in mouse neural stem cells, we bred mice of genotype Nestin-TVA; Nestin-Cre; LoxP-STOP-LoxP-Cas9-EGFP. RCAS retrovirus plasmids were generated containing various guide RNAs (gRNAs) targeting DMG-relevant genes for disruption by Cas9, including Trp53, Cdkn2a, Atm, Ppm1d, Pten, and a non-targeting control (Cntl). For each gRNA, two plasmids were generated, one with a platelet-derived growth factor beta ligand (PDGFB) oncogene driver and one with a blue fluorescent protein (BFP) marker. In a pilot experiment, avian fibroblast cells transduced with RCAS-Trp53_gRNA_BFP and RCAS-Cntl_gRNA-PDGFB were injected into postnatal day 4 mouse brainstems of the aforementioned genotype and observed for tumor formation. High grade tumor formation was observed. We also observed high grade tumors in mice of genotype Nestin-TVA; LoxP-STOP-LoxP-Cas9-EGFP when injected with an additional RCAS-Cre construct, indicating that Cre can be expressed from the mouse germline or delivered via a retrovirus payload to drive tumorigenesis. Immunohistochemistry confirmed tumor features that model human DMG, expression of Cas9 and PDGFB, and loss of p53. Thus, RCAS/tv-a and CRISPR/Cas9 can be combined to model pediatric glioma tumorigenesis in the mouse brainstem. This approach to mouse modeling will streamline future GEMM DMG research by allowing new genes of interest to be rapidly perturbed by simply generating RCAS retrovirus constructs. The workflow may also enable sophisticated experiments such as pooled in vivo genetic experiments that screen gRNAs targeting various tumor suppressor genes or therapeutic targets. Citation Format: Sophie R. Wu, Harrison Q. Liu, Joshua Tolliver, Spencer M. Maingi, Vennesa Valentine, Nerissa T. Williams, Zachary J. Reitman. Generating primary mouse models of diffuse midline glioma through a combination of the RCAS/tv-a retrovirus system and CRISPR/Cas9 gene editing [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 1437.

Abstract 3636: Myeloid cell rebound associated with GD2-CAR-T cell therapy in solid tumor patients

Abstract GD2 is a validated cancer immunotherapy target with highly restricted normal tissue expression. Promising activity of GD2-CAR-T cell therapy in diffuse midline glioma and neuroblastoma has promoted interest in this therapy. We initiated three phase 1 clinical trials of escalating doses of intravenously administered autologous GD2-CAR-T cells in patients with advanced GD2-positive solid tumors. Emerging evidence suggests that CAR-T cells may increase circulating myeloid derived suppressor cells (MDSCs) and that this event may be mediated and/or triggered by CAR-T cell-derived cytokines and chemokines. These findings may help to explain the limited persistence of CAR-T cell therapy in solid tumor patients. Hence, we sought evidence of MDSC generation and secretion of relevant cytokines and chemokines in our clinical trial patients. The CARPETS trial (www.anzctr.org.au: ACTRN12620000622909) recently closed to recruitment after enrolling 12 patients (7 melanoma, 4 colorectal cancer, and 1 sarcoma). No significant adverse events were attributable to CAR-T cell therapy. We opened two other GD2-CAR-T cell therapy trials in 2023: LEVI’S-CATCH (ACTRN12622000675729) in patients with diffuse midline glioma at Sydney Children’s Hospital and KARPOS (ACTRN12622001514796) in adult patients with recurrent glioblastoma at Royal Adelaide Hospital. To date, 3 patients have enrolled to LEVI’S-CATCH with the first patient clearing the dose limiting (DLT) evaluation period. Three patients completed enrolment to the first dose cohort of KARPOS without DLTs. In all CARPETS patients, CAR-T cell expansion, which was measured by by DNA quantitative PCR and flow cytometry, peaked during the 6-week post-CAR-T infusion DLT evaluation period. In 6 CARPETS patients for whom peripheral blood samples were available, we found by flow cytometry, newly formed or increased size of pre-existing subpopulations of non-classical monocytes and/or MDSCs of mononuclear and/or polymorphonuclear class, which peaked at or after the time of peak CAR-T cell expansion. Typically, serum levels of inflammatory cytokines and chemokines such as TNF, IL-6, IL-8, CCL2, GM-CSF, and CXCL10, were detected only after the CAR-T cell infusion with variable patterns of the timing and concentration of analyte observed among the 12 patients. At the time of writing, blood samples from the first KARPOS patient showed CAR-T cell expansion and the subsequent increases in circulating myeloid derived suppressor cells and inflammatory cytokines. We expect that full data sets for these parameters will be presented for current and future enrolled patients to both studies. To improve CAR-T cell persistence in solid tumor patients, these data indicate the need for new therapeutic strategies to mitigate effects of CAR-T cell induced myeloid cells with antitumor activity. Citation Format: Michael P. Brown, Tessa Gargett, Nga T. Truong, Orazio Vittorio, David S. Ziegler. Myeloid cell rebound associated with GD2-CAR-T cell therapy in solid tumor patients [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 3636.

Abstract 7555: Selective inhibition of the CoREST complex as a novel therapeutic target of atypical teratoid rhabdoid tumors

Abstract Atypical teratoid/rhabdoid tumors (AT/RT) are deadly infantile brain tumors, primarily arising from a single recurring biallelic loss-of-function mutation in SMARCB1 which is a member of the SWI-SNF chromatin remodeling complex. The partially functional SWI-SNF complex (without SMARCB1) is able to inhibit EZH2 at most locations of the genome except at gene promoter regions where EZH2 co-localizes with the REST complex. This results in hypermethylation and subsequent repression of genes coding for neuronal differentiation and tumor suppressors. CoREST is an important transcriptional repressor and consists of LSD1 and HDAC1/2 bound together by the scaffolding protein RCOR. Increased activity of EZH2 and the CoREST complex may help AT/RT cells maintain a stem cell-like state, driving its aggressive growth and therapy resistance. We hypothesize that functional inhibition of the CoREST complex by targeting LSD1 and HDAC1/2 might help restore the epigenetic balance lost in AT/RT cells.We selectively inhibited the CoREST complex in AT/RT cells (BT37, CHLA05 and CHLA06) by using a bi-functional LSD1 and HDAC1/2 inhibitor, Corin, which has previously been shown to be effective against melanoma and diffuse midline glioma. Administration of corin to AT/RT cells significantly increased H3K4me1, H3K9Ac and H3K27Ac histone marks (p < 0.001, Western blotting). Corin inhibited AT/RT cell growth and proliferation (MUSE Viacount p<0.05, BrdU immunofluorescence compared to control), and increased apoptosis (cPARP Western blotting, CC3 immunofluorescence, MUSE Annexin V assay, p<0.05 compared to control). Corin appears to be specific in targeting AT/RT as it did not affect brain organoids derived from iPSCs (cPARP Western blotting). Corin induced neuronal differentiation in AT/RT as seen by the increased expression of Beta-3-tubulin, MAP2 and Synaptophysin (Immunofluorescence). Enrichment analysis of differentially expressed genes after whole mRNA sequencing of AT/RT cells treated with corin for 24 h revealed upregulation of pathways related to neuronal differentiation and axonal development and downregulation of pathways related to proliferation and cell cycle (pFDR < 0.05, FC>2). In mice bearing orthotopic CHLA06 tumors, intratumoral delivery of a single dose of corin for 24 h increased H3K27me3 and H3K27ac, demonstrating that corin engaged its target in vivo, reduced growth and induced apoptosis (Ki67, cPARP). Survival studies show a trend toward single agent activity against the very aggressive CHLA06 orthotopic xenograft (increased in median survival from 14 to 35 days compared to control). These studies demonstrate that targeting the CoREST complex disrupts AT/RT epigenetic abnormalities, reversing their stem cell-like state, slowing tumor cell growth, and inducing apoptosis, that may translate into the clinical setting to help improve AT/RT’s dismal survival. Citation Format: Anupa Geethadevi, Marianne Collard, Tyler Findlay, Yiming Deng, Robert Fisher, Nikhil Vaidya, Charles Eberhart, Philip A. Cole, Rhoda Alani, Jeffrey Rubens, Eric Raabe. Selective inhibition of the CoREST complex as a novel therapeutic target of atypical teratoid rhabdoid tumors [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 7555.

Abstract 2858: Phenotypic screens identify genetic regulators of nanoparticle delivery to pediatric brain tumors

Abstract Pediatric central nervous system tumors are the leading cause of cancer death in children. Diffuse midline glioma (DMG) is a universally fatal pediatric brain tumor and despite many clinical trials over the past decades, less than 10% of patients are long-term survivors. The clinical standard of care is radiotherapy, however it only provides limited symptom relief. There is a clear need to develop creative treatment strategies for patients with DMG. Nanomedicine has the potential to address this challenge, allowing the encapsulation of difficult to deliver therapeutic cargoes, and selectively targeting them to tumors while crossing innate biological barriers. However, a deep understanding of disease-specific nano-bio interactions is crucial to leverage the full potential of these therapies. To functionally validate the role of candidate biomarkers in nanoparticle delivery to DMGs, we performed pooled genetic perturbation screens using a flow based phenotypic output. We developed a lentiviral all-in-one CRISPR/Cas9 knock-out library paired with fluorescence-activated cell sorting to probe the functional impact of 700 genes on the delivery of nanoparticles to DMG. Layer-by-layer electrostatic assembly was used to generate liposomal nanoparticles with a polymer surface coating of hyaluronic acid. Two patient-derived neurosphere models of DMG were transduced with the pooled all-in-one CRISPR/Cas9 library. Following antibiotic selection, cells were incubated with hyaluronic acid or polymeric nanoparticles for 4 or 24 hours and sorted into populations based on nanoparticle affinity. A dynamic cell-sorting strategy was optimized to separate cells infected with inert DNA barcodes based on their degree of nanoparticle-association. DNA was isolated from sorted cells and subjected to next generation sequencing. The enrichment of each single guide was determined relative to matched controls. We found strong agreement between biologic replicates, and determined which genes, when knocked down, shifted nanoparticle uptake into DMG cells. Our pooled phenotypic screen revealed strong biologic regulators of nanoparticle uptake. These can be categorized as formulation dependent and independent, as well as time-dependent. We identified several negative regulators of nanoparticle uptake including members of the mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) pathways. The top target genes are currently being validated in DMG and human neural stem cell models. In ongoing work, we are leveraging genetic and chemical inhibition to sensitize DMG cells to nanoparticle therapeutics. We plan to leverage our screening technique to develop novel combination therapy approaches that could be implemented across multiple administration routes (intravenous, intraventricular, or intratumoral) to enhance the use of nanomedicine for DMG and other pediatric brain tumors. Citation Format: Marissa Coppola, Julianna Kenny-Serrano, John G. Doench, Paula Hammond, Pratiti Bandopadhayay, Jessica W. Tsai, Joelle Straehla. Phenotypic screens identify genetic regulators of nanoparticle delivery to pediatric brain tumors [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 2858.

Abstract 2854: Delineating mediators of oncogenesis in FOXR2-expressing cancers

Abstract Background: Pediatric brain tumors are associated with the highest rates of morbidity and mortality of all childhood cancers. Diffuse midline gliomas (DMGs) are universally fatal pediatric tumors with a median survival of less than one year. DMGs are thought to arise from stalled developmental programs in the context of histone mutations (H3 K27M) which are thought to be a critical initiating event. However, these histone mutations alone are insufficient for gliomagenesis and cooperate with other alterations, including mutations in the TP53 pathway (TP53, PPM1D), growth factor receptor pathway activation (PDGFRA, EGFR, PIK3CA), and MYC amplifications (MYC, MYC-N) to induce tumor formation. There is a high degree of genetic heterogeneity amongst DMGs, and other alterations are also critical to DMG pathogenesis. This work is focused on Forkhead Box R2 (FOXR2), a forkhead family TF that we have found to be aberrantly expressed in many cancers, including DMGs. Expression of FOXR2 is sufficient to enhance glioma formation, and we have shown that regulation of FOXR2 occurs through a previously unrecognized epigenetic mechanism. Expression of FOXR2 is regulated by novel promoters, and we have demonstrated that these promoters are necessary for FOXR2 expression in several cancer lineages using CRISPR interference technology, including DMGs, melanoma, neuroblastoma, and non-small cell lung cancers. Methods: We applied an integrative approach using transcriptomics, epigenetics, proteomics, in vitro cancer models, and in vivo mouse models to systematically evaluate how FOXR2 mediates gliomagenesis. Results: We have found that FOXR2 is activated across multiple cancer lineages, including DMGs. FOXR2 is required for proliferation in vitro, and it is sufficient to enhance gliomagenesis using an in utero electroporation in vivo mouse model. FOXR2 enhances MYC protein stability, even in the presence of cycloheximide. However, FOXR2 also exerts its oncogenic effects through MYC independent functions. FOXR2 is highly enriched at E26-transformation (ETS) motifs and specifically activates ETS transcriptional gene sets. To determine protein-protein interactions of FOXR2, we have performed immunoprecipitation and mass spectrometry to identify FOXR2 protein interactors. Moreover, we have performed both proteomic and phospho-proteomic analyses of FOXR2-expressing human neural stem cells. These proteomic studies have allowed us to identify proteins and phospho-sites that are highly enriched in FOXR2-expressing cells, and that could be potential therapeutic targets in FOXR2-expressing cancers. Conclusion: Taken together, this study elucidates how FOXR2 protein interactors mediate oncogenesis in FOXR2-expressing diffuse midline gliomas. These studies proposed here have broad applicability across a range of cancers as FOXR2 is aberrantly expressed in 8% of all cancers. Citation Format: Jessica W. Tsai, Marissa Coppola, Phonepasong Arounleut, Jared B. Collins, Patrick Hart, Hasmik Keshishian, David T. Jones, Pratiti Bandopadhayay, Timothy N. Phoenix. Delineating mediators of oncogenesis in FOXR2-expressing cancers [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 2854.

Abstract 5110: Exploring the effects of Indirubins in pediatric diffuse high grade glioma models

Abstract Diffuse midline glioma (DMG) is an invasive pediatric brain tumor which grows in the pons of the brainstem. The current course of treatment is focal radiation however, DMG continues to have a very poor prognosis with a median survival of less than 10 months. A major barrier for chemotherapeutic treatment of DMG is the blood brain barrier (BBB) which tightly controls access of molecules to the brain. This limits the efficacy of drugs to treat brain cancer and other diseases of the central nervous system. Previous studies from the Lawler lab and others have shown that the indirubin derivative, 6-bromoindirubin-3′-acetoxime(BIO-acetoxime/BIA), has anti-invasive properties and can enhance survival in glioblastoma xenograft models. Here, we investigated the effects of BIA on pediatric glioma models using a range of relevant assays. In an in vitro collagen migration assay, BIA was observed to significantly slow cell migration at a concentration of 1µM in DIPG 36 cells, over 72 hours. Furthermore, we have also shown that BIA modulates the tumor vasculature, which could limit tumor growth, and improves drug delivery to tumors by targeting tight junctions in tumor associated endothelium. The effect on BBB drug penetration was assessed in a multicellular 3D in vitro BBB model and effects of BIA on endothelial barrier functions was determined using trans-endothelial resistance assays (TEER). BIA treatment increased dextran uptake into 3D BBB models, and also lowered endothelial cell permeability in vitro via a reduction in the expression of tight junction proteins as shown by staining and a decrease in TEER values. In order to assess potential synergistic interactions, a drug screen was performed on a panel of distinct pediatric glioma cell lines (DIPG 36 and KNS42). This identified several FDA approved drugs that combine well with BIA. From this screen, BIA was shown to synergize with DNA damaging chemotherapy to enhance toxicity and in combination with cisplatin promote DNA damage in pediatric glioma cell lines. Other drugs identified in the screen are currently under investigation and details will be reported. Thus, our hypothesis is that BIA may provide an effective approach for further development as a DMG therapeutic through targeting invasion and enhancing drug potency and delivery via modulation of endothelial cell tight junctions. Interrogating the mechanisms involved and developing drug formulations informed by the drug screen for in vivo studies to determine the translational potential of this approach is our focus for future studies. Citation Format: Jasmine S. Clark, Jorge Jimenez-Macias, Philippa Vaughn-Beaucaire, Shengliang Zhang, Wafik El-Deiry, Rishi Lulla, Sean Lawler. Exploring the effects of Indirubins in pediatric diffuse high grade glioma models [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 5110.

Abstract 1736: Elucidating the oncogenic mechanisms in diffuse midline glioma: Comparative analysis of h3k27m mutation and k27 methylation loss on gene expression profiles via RNA sequencing

Abstract Background: Diffuse Midline Glioma (DMG), a severe pediatric brain tumor, is characterized by its aggressive nature and poor prognosis. Common in brain midline structures like the thalamus and brainstem, DMGs often harbor H3K27M mutations, leading to reduced histone trimethylation at lysine 27 and loss of serine 31 phosphorylation. Building on these findings, our laboratory has undertaken a novel approach by engineering a knockout of the Polycomb Repressive Complex 2 (PRC2) in selected cell lines. This was achieved using CRISPR-Cas technology to target the EZH1 and EZH2 methyltransferases (PRC2KO), alongside the introduction or reversal of histone H3.3 mutations. The goal of this study is to investigate the effects of K27 Methylation loss and H3.3 mutations on gene expression in DMG to better understand DMG pathogenesis and potential therapeutic avenues. Methods: RNA and ATAC sequencing were performed on DMG cell lines including H3.3WT, H3.3 S31A, H3.3K27M, and PRC2KO. Utilizing 150 bp paired-end reads, 25 to 45 million unique reads were mapped to the human genome (hg38) using STAR. Differential gene expression was analyzed using DESeq2 in R, complemented by ATAC-sequencing to explore potential epigenetic changes. Results: The analysis identified 2,564; 2,946; 4,618; and 5,490 differentially expressed genes (DEGs) in the K27M, S31A, K27M-PRC2KO, and WT-PRC2KO cell lines, respectively, when compared to WT. A four-way Venn diagram analysis identified unique and shared gene cohorts among the different mutations. H3K27M cells showed 414 downregulated and 385 upregulated genes, mainly affecting cell migration. In contrast, PRC2KO cells, characterized by a complete loss of histone methylation, showed extensive changes with 1,302 genes downregulated and 1,779 upregulated, primarily impacting cell cycle and developmental processes. The S31A cells displayed a unique profile with 548 genes uniquely downregulated and 547 genes upregulated. The lack of enrichment in specific pathways for these genes suggests their involvement in a broader range of affected pathways. A significant discovery was the identification of a set of genes common to both S31 and K27M cells, but absent in PRC2 knockouts. This cohort, consisting of 350 downregulated and 389 upregulated genes, may represent a molecular link between serine phosphorylation and the K27M mutation. Additionally, ATAC-sequencing analysis was conducted to determine if the expression changes were a direct result of epigenetic dysregulation or an indirect effect. Conclusion: This study delineates the intricate gene regulation in DMG resulting from H3K27M mutations beyond loss of loss of K27 Methylation. It highlights unique and overlapping pathways influenced by, providing valuable insights into the molecular basis of DMG and guiding future therapeutic strategies. Citation Format: Suraj Bhattarai, Faruck L. Hakkim, Charles Day, Florina Grigore, Alyssa Langfald, Edward Hinchcliffe, James Robinson. Elucidating the oncogenic mechanisms in diffuse midline glioma: Comparative analysis of h3k27m mutation and k27 methylation loss on gene expression profiles via RNA sequencing [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 1736.

Abstract 3331: Preclinical combination of ONC206 with radiotherapy and temozolomide in a GBM mouse orthotopic model

Abstract Glioblastoma multiforme (GBM) is the most common form of primary malignant brain tumor in adults. It is also the most aggressive and lethal. Standard of care therapy comprises maximal safe surgical resection followed by adjuvant alkylating agent temozolomide (TMZ) and radiotherapy (RT). There have only been five drugs and one device ever approved by the FDA for the treatment of GBM. The five-year survival rate for GBM patients has shown no notable improvement in the last three decades. We previously reported that the first-in-class imipridone small-molecule Dordaviprone (ONC201) decreases protein chaperone ClpX to unleash mitochondrial protease ClpP activity, integrated stress response and cell death. Preclinical combination of ONC201 (100 mg/kg weekly) with radiotherapy and Temozolomide in a GBM mouse orthotopic model results in reduced tumor burden and prolonged survival. The second generation imipridone ONC206 also activates integrated stress response and decreases ClpX in GBM cells but ONC206 is approximately 20 times more potent than its parent imipridone ONC201. ONC206 0.08 uM in combination with 12.5 uM TMZ and 2 Gy RT reduced the cell viability of GBM cells significantly compared to all single treatments and double treatments. During a short-term treatment of a GBM mouse orthotopic model, ONC206 (50 mg/kg weekly, half of ONC201 doses) synergizes with TMZ and RT to induce more tumor cell apoptosis and inhibits more tumor cell proliferation compared to other groups with single or double treatments. There are two ongoing clinical trials with ONC206: single weekly or multiple-day weekly dose regimens of single-agent, oral ONC206 in patients with recurrent, primary central nervous system (CNS) neoplasms (NCT04541082) and ONC206 alone or in combination with radiation therapy in treating patients with diffuse midline gliomas that is newly diagnosed or has come back (recurrent) or other recurrent primary malignant CNS tumors (NCT04732065). Future studies evaluating the role of immune function, mitochondrial metabolism, dopamine receptors, the ISR and TRAIL pathway in the synergistic effect would support further development of the triple combination regimen of ONC206, TMZ and radiation therapy for GBM clinical trial. Citation Format: Lanlan Zhou, Leiqing Zhang, Jun Zhang, Shengliang Zhang, Wafik S. El-Deiry. Preclinical combination of ONC206 with radiotherapy and temozolomide in a GBM mouse orthotopic model [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 3331.

Abstract 5470: ONC206 inhibits tumour growth and is a potential novel therapeutic strategy for refractory medulloblastoma

Abstract Medulloblastoma is the most common malignant pediatric brain tumor with a for certain molecular subgroups very low overall survival and therefore an urgent need for novel treatment approaches. Dordaviprone (ONC201) and its chemical derivative with nanomolar potency ONC206 are inhibitors of the dopamine receptor 2 (DRD2) and more importantly induce apoptosis of cancer cells by activation of the mitochondrial caseinolytic protease P (ClpP). ONC201 has shown objective responses in patients with diffuse midline gliomas and is currently being evaluated in the Phase 3 ACTION study. ONC206 is currently in Phase I clinical trials for pediatric and adult patients with primary brain tumors. In this study, we evaluated the preclinical therapeutic effects of ONC206 in medulloblastoma in vitro and in vivo and investigated the molecular mode of action for this imipridone. We found evidence for high expression of ClpP at both the RNA and protein level in medulloblastoma tumours, compared to very low expression in normal brain and normal cerebellum. In addition, we saw a pronounced reduction in cell viability of human Group 3 and Group 4 and murine medulloblastoma cells treated with ONC206 with extremely low IC-50s (range: 14nM - 2μM).After treatment with ONC206, we observed a significant downregulation of mitochondrial electron transport chain complexes I and III and mitochondrial hyperpolarization, as well as an induction of the ATF4 pathway, thereby implying that ONC206 induces an integrated stress response and mitochondrial damage. In order to test the efficacy of ONC206 in vivo, we used murine models of SHH-driven and Group 3 medulloblastoma as well as Group 3 patient-derived xenografts (PDXs). ONC206 led to a significant prolongation of survival in both murine models, with the SHH mice demonstrating a remarkable survival benefit. Mice from both Group 3 PDXs also responded to ONC206, with 25% of ONC206 treated animals exhibiting long-term tumor-free survival. Our results highlight ONC206 as a novel attractive therapeutic option for patients with relapsed medulloblastoma and importantly pave the way for a clinical trial testing the efficacy of ONC206 in the treatment of these patients. Citation Format: Theophilos Tzaridis, Jingbo Liu, Varun Prabhu, Robert Wechsler-Reya, Tobey MacDonald. ONC206 inhibits tumour growth and is a potential novel therapeutic strategy for refractory medulloblastoma [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 5470.

Abstract 1719: Targeting H3.3 serine 28 phosphorylation as a novel therapeutic strategy for diffuse midline glioma

Abstract Background: Diffuse midline glioma (DMG) is a highly aggressive pediatric brain cancer with a poor prognosis [1]. Mechanistically, DMG harbors a lysine-to-methionine substitution at position 27 of histone H3.3 (H3.3K27M), which leads to global hypomethylation and a dramatically altered epigenetic landscape [2, 3]. Aim: This study aims to explore the H3.3K27M mutation from a novel perspective by investigating its effects on H3.3 ubiquitination, with the goal of identifying effective, novel therapeutic targets. Methods: Mass spectrometry was used to identify potential E3 ligases for H3.3, while immunoprecipitation to verify this interaction. Cell viability and survival were determined by MTS and clonogenic survival assays, respectively. Nucleosome fractionation was performed to detect the distribution of wild-type (WT) or K27M mutant H3.3 histones. Results: Mass spectrometry identified UBR7, an E3 ligase, as exhibiting a stronger binding affinity with WT H3.3 than its K27M mutant counterpart. Further nucleosome fractionation assay revealed that K27M histones are more likely deposited in nucleosomes than WT H3.3. Further analysis demonstrated that a non-phosphorylation mimic mutation (serine to alanine) at amino acid 28 (S28A) restored the binding affinity of K27M mutant H3.3 with UBR7. Importantly, this mutation disrupted H3.3 phosphorylation and significantly diminished cell survival, whereas the constitutively activated phosphorylation mutation (S to glutamine (E)) on S28 (S28E) mimics K27M mutation in promoting cell proliferation and survival. Conclusion: These findings suggest that S28ph impairs UBR7-mediated K27M ubiquitination and promotes DMG cell growth, suggesting that targeting S28ph is an effective therapeutic strategy. However, the underlying mechanisms by which K27M interplays with S28ph in promoting tumorigenesis require further investigation. Citation Format: Yuqian Yan. Targeting H3.3 serine 28 phosphorylation as a novel therapeutic strategy for diffuse midline glioma [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 1719.

Abstract 2648: CD155 regulates cell growth and immune evasion in diffuse midline glioma

Abstract There is an unmet need for effective treatment strategies for diffuse midline glioma (DMG), a devastating brain tumour arising in children and young adults. While immunotherapy is emerging as a powerful approach for treatment of other cancers, clinical trials with immune checkpoint inhibitors have failed to show a survival benefit for DMG patients. In this study, we analysed the expression of known immune checkpoint molecules on the surface of human and murine DMG cells by flow cytometry and identified CD155 and B7-H3 as the most highly expressed checkpoint molecules, with minimal expression of others, including PD-L1 and PD-L2. These findings were confirmed in primary patient samples from pediatric brain tumours (gliomas, medulloblastomas and ependymomas). To test whether CD155 regulates susceptibility to CD8+ T cell killing, we cultured murine DMG cells expressing ovalbumin (OVA) with CD8+ T cells from OT-I mice, which express T cell receptors specific for OVA. shRNA mediated silencing of CD155 led to a strong increase in T cell-mediated killing. In vivo, adoptive transfer of OT-I T cells into OVA-positive DMG-bearing immuno-compromised mice resulted in delayed tumour growth, and this effect was enhanced when tumours lacked CD155. Strikingly, CD155-deficient DMG cells failed to grow at all in immuno-competent mice, and depletion of CD8 T cells allowed these tumours to grow, highlighting a role for CD8 T cells in rejection of CD155-deficient cells. In addition to its effects on susceptibility to T cells, CD155 also exerted cell-autonomous effects on tumour cells: silencing of CD155 led to induction of apoptosis of DMG cells in vitro and to delayed tumour growth in vivo, even in immune-compromised mice. These studies demonstrate that CD155 functions as an immune checkpoint and as a regulator of tumour cell survival in DMG, and suggest that targeting CD155 could be a valuable double-pronged therapeutic strategy for this devastating disease. Citation Format: Theophilos Tzaridis, Tanja Eisemann, Augusto Faria Andrade, Jennifer L. Hope, Oren Becher, Nada Jabado, Linda M. Bradley, Peter Adams, Robert Wechsler-Reya. CD155 regulates cell growth and immune evasion in diffuse midline glioma [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 2648.

Bone Morphogenic Proteins in Pediatric Diffuse Midline Gliomas: How to Make New Out of Old?

The BMP pathway is one of the major signaling pathways in embryonic development, ontogeny and homeostasis, identified many years ago by pioneers in developmental biology. Evidence of the deregulation of its activity has also emerged in many cancers, with complex and sometimes opposing effects. Recently, its role has been suspected in Diffuse Midline Gliomas (DMG), among which Diffuse Intrinsic Pontine Gliomas (DIPG) are one of the most complex challenges in pediatric oncology. Genomic sequencing has led to understanding part of their molecular etiology, with the identification of histone H3 mutations in a large proportion of patients. The epigenetic remodeling associated with these genetic alterations has also been precisely described, creating a permissive context for oncogenic transcriptional program activation. This review aims to describe the new findings about the involvement of BMP pathway activation in these tumors, placing their appearance in a developmental context. Targeting the oncogenic synergy resulting from this pathway activation in an H3K27M context could offer new therapeutic perspectives based on targeting treatment-resistant cell states.

High-Grade Astrocytoma with Piloid Features: A Dual Institutional Review of Imaging Findings of a Novel Entity.

High-grade astrocytoma with piloid features (HGAP) is a recently identified brain tumor characterized by a distinct DNA methylation profile. Predominantly located in the posterior fossa of adults, HGAP is notably prevalent in individuals with neurofibromatosis type 1. We present an image-centric review of HGAP and explore the association between HGAP and neurofibromatosis type 1. Data were collected from 8 HGAP patients treated at two tertiary care institutions between January 2020 and October 2023. Demographic details, clinical records, management, and tumor molecular profiles were analyzed. Tumor characteristics, including location and imaging features on MR imaging, were reviewed. Clinical or imaging features suggestive of neurofibromatosis 1 or the presence of NF1 gene alteration were documented. The mean age at presentation was 45.5 years (male/female = 5:3). Tumors were midline, localized in the posterior fossa (n = 4), diencephalic/thalamic (n = 2), and spinal cord (n = 2). HGAP lesions were T1 hypointense, T2-hyperintense, mostly without diffusion restriction, predominantly peripheral irregular enhancement with central necrosis (n = 3) followed by mixed heterogeneous enhancement (n = 2). Two NF1 mutation carriers showed signs of neurofibromatosis type 1 before HGAP diagnosis, with one diagnosed during HGAP evaluation, strengthening the HGAP-NF1 link, particularly in patients with posterior fossa masses. All tumors were IDH1 wild-type, often with ATRX, CDKN2A/B, and NF1 gene alteration. Six patients underwent surgical resection followed by adjuvant chemoradiation. Six patients were alive, and two died during the last follow-up. Histone H3 mutations were not detected in our cohort, such as the common H3K27M typically seen in diffuse midline gliomas, linked to aggressive clinical behavior and poor prognosis. HGAP lesions may involve the brain or spine and tend to be midline or paramedian in location. Underlying neurofibromatosis type 1 diagnosis or imaging findings are important diagnostic cues.

Abstract PR-007: H3.3K27M diffuse midline gliomas are sensitive to SWI/SNF chromatin remodeler degradation

Abstract Diffuse Midline Gliomas (DMG) including Diffuse Intrinsic Pontine Glioma (DIPG) is a lethal pediatric brain tumor. The lysine 27 (K27)-to-methionine (M) point mutation in histone H3.1 or H3.3 (H3.1K27M or H3.3K27M, respectively) affects about 80% of patients establishing H3K27M mutation as a tumor hallmark. Mutated H3 histones inhibit the function of Polycomb repressive complex 2 resulting in global reduction of repressive H3K27me3. In addition, increased acetylation of H3K27 is observed, further contributing to an aberrant chromatin state. Considering the impact of epigenetic alterations in DIPG, we investigated the role of Switch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodelers in shaping the oncogenic chromatin state in DIPG cells. DIPG cells harboring H3.3K27M mutation were sensitive to combined SMARCA4/SMARCA2/PBRM1 degradation by the proteolysis targeting chimera (PROTAC) AU-15330 degrader. Our premise is based on the SWI/SNF complex altering chromatin states of tumor cells to regulate the tumor immune microenvironment. We hypothesized that the SWI/SNF complex in tumor cells modulates immune responses in DIPG. Cytokine profiler arrays in AU-15330 versus vehicle treated H3.3K27M cells showed a marked decrease of Intercellular Cell Adhesion Molecule-1 (ICAM-1) glycoprotein levels. In addition to its well-known role in leukocyte endothelial transmigration, ICAM-1 contributes to malignant potential of tumor cells. We validated downregulation of ICAM-1 mRNA and protein levels by AU-15330 by immunoblotting, proteomics, and transcriptomics, suggesting an epigenetic regulation of ICAM-1 gene transcription by the SWI/SNF complex. Moreover, silencing of mutant H3.3-encoding gene decreased ICAM-1 protein levels, suggesting ICAM-1 expression is promoted by H3.3K27M mutation-induced SWI/SNF chromatin remodeling in DIPG. In conclusion, our work suggests the H3.3K27M mutation renders DIPG cells dependent on chromatin remodeling by SWI/SNF complexes that can in turn alters the expression of immune system-associated components, such as ICAM-1. Our future work is geared to understanding the role of the SWI/SNF complex in regulating the immune tumor microenvironment in H3K27M DMGs. Citation Format: Mateus Mota, Stefan Sweha, Matt Pun, Siva Kumar Natarajan, Yujie Ding, Chan Chung, Debra Hawes, Fusheng Yang, Alexander Judkins, Susanta Samajdar, Xuhong Cao, Lanbo Xiao, Abhijit Parolia, Arul Chinnaiyan, Sriram Venneti. H3.3K27M diffuse midline gliomas are sensitive to SWI/SNF chromatin remodeler degradation [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr PR-007.

Abstract B015: RNA N6-methyladenosine (m6A) as a therapeutic target in Diffuse Midline Glioma (DMG)

Abstract Diffuse Midline Glioma (DMG) is an incurable pediatric brain tumor thought to originate from oligodendrocyte precursor cells in midline brain structures. The RNA modification N6-methyladenosine (m6A) plays an important role in RNA stability and is critical to neuronal stem-cell self-renewal and differentiation. We therefore sought to investigate m6A as a therapeutic target in DMG. Moreover, targeting the epitranscriptome has shown promise in the treatment of other cancers, and several small-molecule inhibitors of m6A writers and erasers have been recently developed. To that end, we tested the sensitivity of a panel of patient-derived DMG cell lines to FB23-2, an inhibitor of the m6A eraser FTO, and STM2457, an inhibitor of the mRNA m6A writer METTL3. In order to interrogate the therapeutic mechanisms and identify predictive biomarkers for response, we then performed RNA-seq to measure gene expression changes, and native RNA-seq to measure RNA m6A levels. We found that DMG cell lines were more sensitive to FB23-2 (IC50 ~10μM) than STM2457 (IC50 ~100μM), indicating that m6A gain rather than loss may be a potential therapeutic strategy. Furthermore, we observed variation in FB23-2 sensitivity between cell lines with RNA-sequencing identifying marked changes in the expression of cell cycle, cell stress response, and differentiation associated genes in the cell lines which responded best to FB23-2. Additionally, these analyses also identified several potential biomarkers for response to FB23-2. Finally, we generated the first m6A transcriptome maps for DMG using native RNA sequencing and demonstrated that m6A is abundant in DMG cell lines and is particularly enriched on cell cycle, cell stress response, and metabolic pathway transcripts. Overall, our work identified the FTO m6A demethylase as a potential therapeutic target in DMG, whereby its inhibition with FB23-2 increases m6A abundance, altering the stability of critical transcripts and pathways. Citation Format: Samuel E. Ross, Holly Holliday, Maria Tsoli, David S. Ziegler, Marcel E. Dinger. RNA N6-methyladenosine (m6A) as a therapeutic target in Diffuse Midline Glioma (DMG) [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B015.