Glioma Cell Lines Research Articles

Chemical Information Network and Molecular Docking Inspire the Novel Indole Discovery Against Glioma from Tabernaemontana corymbosa.

The alkaloidingredients were considered to be responsible forthe diverse pharmacological activitiesof the medicinalplant Tabernaemontana corymbosa(Roxb. ex Wall.). In the current finding, the systematic phytochemical investigation onT.corymbosahave been achieved. One new indole alkaloidtabercorympyline A(1) along with sevenknown indoles (2-8) wereisolated from T.corymobsa. Their structures were elucidated by means of spectroscopic techniquesand quantum chemical calculations. Tabercorympyline A(1) possessedthe indoleskeleton with rare N-containing nine membered ring.Chemical informationnetwork was used to comprehensively discover the cluesfor the glioma therapeutic leadsfromT.corymbosaalkaloids (TA). Inspired by chemical information networkanalysis, all the isolated compoundshave been furthervalidated theiranti glioma activities in glioma cell line U251.Interestingly, the compounds2, 3,5, and6exhibited significantinhibitory effects on gliomacellsin vitro.Molecular docking was ultimately used to indicatepossible binding performance and mechanism between active compounds(2-3) andthe core targets.This study sequentially assembled chemical informationand networkanalysis, phytochemistry, molecular docking, and in vitroactivity validation to comprehensively explore the effective compounds, related targets, and potential mechanisms of TA therapy for glioma.

MicroRNA-429 overexpression overcomes imatinib resistance of glioma cells by negatively regulating lysophosphatidic acid receptor 1

ABSTRACT Background Glioma is one of the most aggressive and lethal malignancies in central nervous system. It has been reported that miR-429 is declined in glioma and functions as a tumor suppressor. Nonetheless, the potential role of miR-429 in drug resistance of glioma is still ambiguous. Methods Stable imatinib-resistant lines U251-AR and T98G-AR were established using glioma cell lines U251 and T98G. Cell apoptosis and cycle were analyzed by flow cytometry, and CCK-8 assay was utilized to measure cell viability. Protein and RNA levels were tested with western blot and RT-qPCR. The predicted binding site was confirmed by dual luciferase reporter assay. Results Imatinib-resistant U251-AR and T98G-AR cells presented lower level of miR-429 and higher level of LPAR1. MiR-429 overexpression obviously promoted imatinib sensitivity in glioma cells, indicated by the reduced IC50 value, facilitated cell apoptosis and cell cycle arrest at G0/G1 phase, and downregulated multidrug resistance-related proteins. LPAR1 was verified as a direct target of miR-429 and its expression was negatively regulated by miR-429. Additionally, overexpression of LPAR1 restrained the biological function of miR-429 on imatinib chemoresistance. Conclusion MiR-429 partly sensitized glioma cells to imatinib via downregulation LPAR1, which might provide an approach to overcome imatinib chemoresistance during glioma treatment.

Inulae Flos is a potential herbal medicine to treat glioma: a study based on gene expression profile analysis, network pharmacology and molecular docking

Inulae Flos is a promising herbal medicine; however, its underlying mechanism in treating glioma remains unclear. This study aimed to elucidate the potential active ingredients and mechanisms of Inulae Flos in glioma treatment. Differentially expressed genes (DEGs) associated with gliomas were identified by analyzing the GSE186057 dataset. The bioactive components of Inulae Flos were searched in the Traditional Chinese Medicine Systems Pharmacology database, and related targets were obtained from the Swiss Target Prediction database. A protein-protein interaction (PPI) network was constructed using the STRING database. Next, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) of these common targets were analyzed using the DAVID database. Finally, AutoDock Vina was used for molecular docking analysis. The tumor-suppressive properties of the crude extract of Inulae Flos (XFH) and britanin were then evaluated with in vitro and in vivo assays. Nineteen active ingredients were obtained from Inulae Flos, and 20 DEGs were identified as the targets of Inulae Flos in glioma treatment. Britanin, luteolin and 1-O-Acetylbritannilactone were identified as the key ingredients. PIK3R1, CDK2, CCNB1, MAPK1, MAPK8, CCNA2, and AR were considered to be the hub target genes of Inulae Flos in glioma treatment. Good binding affinity was observed between the key components and hub targets. In addition, in vitro studies showed that XFH and britanin, a key component of Inulae Flos, could inhibit the growth, migration and invasion glioma cell lines U87 and U138, and repress the tumorigenesis of U138 cells in nude mice. Inulae Flos is promising for glioma treatment, and it exerts its anti-tumor activities via multiple components, targets and pathways.

CNSC-59. EPIGENETIC POTENTIATION OF ANTI-TUMOR IMMUNE RESPONSES USING VIRAL MIMICRY IN GLIOMAS

Abstract Clinical trials using immune checkpoint inhibition(ICI) have traditionally failed in glioblastoma (GBM). Viral mimicry, which augments anti-tumor immune responses and sensitizes response to immunotherapy in other cancers, involves the epigenetic activation of endogenous retroelements (REs). REs are silenced via the HUSH complex and H3K9me3. This process is mediated by ZNF638. We aimed to elucidate the role of viral mimicry in enhancing ICI through epigenetic reprogramming of the HUSH complex. We demonstrated that RE expression among 48 superfamilies inversely correlated with ZNF638 expression in gliomas, based on data from 71 newly-diagnosed GBMs. Using transcriptional deconvolution, we showed ZNF638 negatively correlates with innate antiviral immune response signatures(TLR3)(RTLR3=-0.300,pTLR3=0.00006). We validated these in-silico results in pure glioma cell lines, patient-derived GBM neurospheres, and syngeneic murine models. ZNF638 knockdown induced intracellular RE-mediated dsRNA signaling cascades via RIG-I and TLR3. This knockdown significantly increased PD-L1 expression(p<0.001), reduced H3K9 trimethylation, and enhanced cytoplasmic dsRNA accumulation in glioma cell lines. In patient-derived GBM neurospheres, ZNF638 knockdown upregulated immune and antiviral programs. Additionally, knockdown resulted in upregulation of several endogenous repeat elements (Alu, LTR). Single-cell RNA sequencing showed ZNF638 clustering in neural progenitor-like and oligodendrocyte-like cells, with increased retroelement expression in low ZNF638 cells. Tumors with lower ZNF638 expression showed increased CD8+ populations. Bulk RNA sequencing deconvolution revealed that ZNF638 is linked to reduced CD8 and DC infiltration in gliomas (RCD8=-0.202,RDC=-0.198;pCD8=8.79E-06,pDC=1.34E-05). In immunocompetent mice, ZNF638 KO and PD-L1 inhibition significantly improved survival (mOS >50 days, p<0.0001) and reduced tumor volume (p<0.001). Analysis of cohorts from multiple cancer types treated with anti-PD1 therapy demonstrated that ZNF638 expression predicts therapeutic response and overall survival. Our findings suggest that epigenetic reprogramming through HUSH inhibition may potentiate immunotherapy for GBM.

DDDR-28. SPHINGOSINE AND N,N-DIMETHYLSPHINGOSINE MODULATE CHOLESTEROL HOMEOSTASIS IN IDH1MUT GLIOMAS, CAUSING PRO-APOPTOTIC MEMBRANE DEGRADATION

Abstract BACKGROUND Previous work in our team revealed a therapeutic potential for metabolic reprogramming along the sphingolipid pathway within IDH1mut gliomas. The dosing of various IDH1mut glioma subtypes with sphingosine and sphingosine kinase inhibitor (SphKi), N,N-dimethylsphingosine (NDMS) led to the enrichment of pro-apoptotic ceramides and sphingosines while suspending the proliferation of the IDH1mut neurospheres. However, the mechanism of drug action (MOA) was not well understood. We postulated that suppressing the production of S1P production with SphKi treatment combined with the global accumulation of sphingosine and certain ceramides triggers apoptotic membrane stress in highly susceptible IDH1mut gliomas. METHODS We cultured and treated IDH1mut (TS603, BT142 & NCH1681) and IDH1wt (GSC923) glioma cell lines with a combination of NDMS and C17-sphingosine. We used a comprehensive multi-omics approach involving LC-MS lipidomic, RNA sequencing, immunoblotting, flow cytometry, and fluorescent microscopy. RESULTS Following combination treatment, a global increase in sphingosines, ceramides, and their derivatives was accompanied by a decline in cholesterol levels. The transcriptomic expression was elevated for NR1H3, MYLIP, ABCA1, and ABCG1, which regulate the trafficking and biosynthesis of membrane-associated cholesterol. In contrast, total gene expression for catalytic enzymes involved in cholesterol (and isoprenoid) biosynthesis was negatively impacted. In addition, early onset changes included an elevation in the expression of ROCK (a marker for apoptotic membrane blebbing). Moreover, fluorescent microscopy revealed that sphingosine and cholesterol tend to become associated within the cellular membranes. This interaction then saturates the plasma membrane (PM), causing apparent vesiculation, permeation, and disruption of membrane integrity. CONCLUSION The MOA involved concomitant attenuation of cholesterol metabolism and influx in response to membrane saturation with sphingosine-associated cholesterol. As a result, PM stress was incurred, leading to PM disintegration in a blebbing fashion. Our study revealed how reprogramming sphingolipid metabolism increases the susceptibility of IDHmut gliomas to sphingosine-associated cholesterol-induced blebbing and resulting cell death.

Towards Aptamer-Targeted Drug Delivery to Brain Tumors: The Synthesis of Ramified Conjugates of an EGFR-Specific Aptamer with MMAE on a Cathepsin B-Cleavable Linker

Background/Objectives: Targeted delivery of chemotherapeutic agents is a well-established approach to cancer therapy. Antibody–drug conjugates (ADCs) typically carry toxic payloads attached to a tumor-associated antigen-targeting IgG antibody via an enzyme-cleavable linker that releases the drug inside the cell. Aptamers are a promising alternative to antibodies in terms of antigen targeting; however, their polynucleotide nature and smaller size result in a completely different PK/PD profile compared to an IgG. This may prove advantageous: owing to their lower molecular weight, aptamer-drug conjugates may achieve better penetration of solid tumors compared to ADCs. Methods: On the way to therapeutic aptamer–drug conjugates, we aimed to develop a versatile and modular approach for the assembly of aptamer–enzymatically cleavable payload conjugates of various drug–aptamer ratios. We chose the epidermal growth factor receptor (EGFR), a transmembrane protein often overexpressed in brain tumors, as the target antigen. We used the 46 mer EGFR-targeting DNA sequence GR-20, monomethylauristatin E (MMAE) on the cathepsin-cleavable ValCit-p-aminobenzylcarbamate linker as the payload, and pentaerythritol-based tetraazide as the branching point for the straightforward synthesis of aptamer–drug conjugates by means of a stepwise Cu-catalyzed azide–alkyne cycloaddition (CuAAC) click reaction. Results: Branched aptamer conjugates of 1:3, 2:2, and 3:1 stoichiometry were synthesized and showed higher cytotoxic activity compared to a 1:1 conjugate, particularly on several glioma cell lines. Conclusions: This approach is convenient and potentially applicable to any aptamer sequence, as well as other payloads and cleavable linkers, thus paving the way for future development of aptamer–drug therapeutics by easily providing a range of branched conjugates for in vitro and in vivo testing.

IMMU-55. TUMOR-WIDE RNA SPLICING ABERRATIONS GENERATE IMMUNOGENIC PUBLIC NEOANTIGENS IN GLIOBLASTOMAS AND OTHER CANCER TYPES

Abstract BACKGROUND Immunotherapy in gliomas is limited by tumor heterogeneity and low mutational burden. Aberrant RNA-splicing (neojunctions) offers a new source for targets, and our neoantigen discovery platform (SNIPP) characterizes a novel class of clonally-expressed splicing-derived neoantigens that elicit a CD8+ T-cell-mediated tumor killing response. METHODS SNIPP identified public neojunctions expressed in TCGA RNA-seq (positive sample rate (PSR) > 10%) and not in GTEx normal tissue RNA-seq data (PSR < 1%) across 12 cancer types. To characterize intratumorally-conserved neojunctions, we performed maximally-distanced multi-site biopsies (n=535) within glioma patients (n=56) and obtained RNA-seq data from each intratumoral site. Two independent algorithms then predicted peptide processing likelihood and HLA-binding affinity of ASN candidates. Neoantigen-specific TCR sequences characterized from subsequent PBMC in vitro sensitization and 10x V(D)J scRNA-seq were transduced into CD8+ T-cells for immunogenicity and cytotoxicity assays against glioma cell lines. RESULTS Our pipeline identified 789 public neojunctions, with 32 neojunctions concurrently identified in transcriptomic and proteomic glioma data and predicted to be presented by HLA-A*02:01 with high confidence. IVS and subsequent 10x VDJ scRNA-seq identified TCR clonotypes reactive against neojunctions in RPL22 (n=7) and GNAS (n=1), the latter being highly intratumorally-conserved (detected in > 90% of spatially-mapped biopsies across 17/56 patients (26.78%)). TCR-transduced T-cells demonstrated recognition and immunogenic activation against endogenously processed and presented neoantigens in multiple GBM PDX cell lines, and co-culturing these T-cells against both glioblastoma and melanoma cell lines demonstrated tumor-specific cytotoxicity. Furthermore, IDH1-mutant oligodendroglioma samples demonstrated significantly elevated expression of neojunctions over IDH1-mutant astrocytoma and IDH1wt subtypes. Differential gene expression (DESeq2) identified decreased expression of splicing factors due to oligodendroglioma-specific co-deletion of Chromosomes 1p/19q. siRNA knockdown of these splicing factors (e.g. SF3A3, SNRPD2) in IDH1wt glioma cells resulted in significantly increased expression of corresponding neojunctions. CONCLUSION Our unique SNIPP neoantigen discovery platform identified novel public tumor-wide splice-derived neoantigens and reactive TCRs, and its pan-cancer application characterized candidates that are targetable across multiple diseases.

IMMU-39. CAR T CELLS TARGETING ICAM-1 SHOW A SURVIVAL BENEFIT IN BOTH SYNGENEIC AND XENOGRAFT GLIOMA MOUSE MODELS

Abstract BACKGROUND Chimeric antigen receptor (CAR) T-cell therapy, while effective against hematological malignancies, has faced significant hurdles in its application to solid tumors. Challenges in CAR T-cell therapy for glioblastoma include the immunosuppressive tumor microenvironment, the limited infiltration of CAR T-cells into the tumor, heterogeneous target expression and antigen escape. In this study, we focused on targeting Intracellular adhesion molecule-1 (ICAM-1), a protein expressed on both glioma cells and tumor-associated cells like macrophages and endothelial cells. METHODS Glioblastoma and normal brain tissue microarray sections (TMA) were immunohistochemically analyzed for ICAM-1 expression. Second-generation human and murine ICAM-1-targeting CAR T cells were generated by lentiviral or retroviral transduction of T cells from healthy human donors or murine splenocytes. Their efficacy was evaluated in co- culture assays with human and murine glioma and endothelial cell lines. Syngeneic and xenograft orthotopic glioma mouse models were used to assess the in vivo activity of CAR T cells. Ex vivo analysis of these tumors included examination of changes in the tumor microenvironment using high-dimensional flow cytometry. RESULTS Immunohistochemical staining of TMA revealed a significant upregulation of ICAM-1 in human glioblastoma tissue compared to normal brain tissue. Single-cell RNA sequencing data from glioblastoma specimens showed a high level of ICAM-1 expression in tumor- associated macrophages. Human and murine ICAM-1-targeting CAR T cells displayed strong lysis of ICAM-1-expressing glioma and endothelial cells in vitro. Intratumoral application of CAR T cells resulted in a survival benefit in both syngeneic and xenograft glioma mouse models. Ex vivo analysis of the tumor microenvironment revealed treatment-induced changes from our CAR T cell therapy and indicates potential for further enhancements. CONCLUSION Our study demonstrates that ICAM-1-targeting CAR T cells improve survival in human and murine glioma mouse models. Flow cytometry of the tumor microenvironment reveals therapy-induced changes, for future improvements of the CAR T cell therapy.

TMET-32. THE ROLE OF HOXC10 IN DRIVING GLIOBLASTOMA AGGRESSIVENESS

Abstract BACKGROUND Glioblastoma (GBM) is an aggressive malignancy, with a tumor doubling time of just 49.6 days. Despite maximal safe surgical resection, radiation, and temozolomide, median survival is only 14.6 months. Homeobox gene, homeobox C10 (HOXC10), a transcription factor of the HOX10 family, is pivotal in neuronal development, upregulated in several cancers, including glioma, and correlates with poor survival. METHODS RNA sequencing analysis was done on our mouse glioma syngeneic cell lines. HOXC10 expression analysis was done on human and mouse GBM cell lines and tissues by immunoblotting and immunohistochemistry, respectively. HOXC10 and p16-/-silencing were done using 4 unique 29mer shRNA constructs in the GFP vector, respectively. Gene Ontology predicted HOXC10 functions. HOXC10 and pyrroline-5-carboxylic acid reductase 1 (PYCR 1), a proline biosynthetic enzyme co-expression was studied using immunofluorescence analysis. RESULTS HOXC10 is highly expressed in human and mouse GBM tissues. HOXC10 was upregulated only in the high-grade/aggressive cell line [EGFRvIII; p16-/- & GFAP Cre] and demonstrated increased expression with p16 deletion in the mouse glioma cell line. Compellingly, TCGA analysis of GBM correlated the negatively prognostic p16 homozygous deletion (HD) with HOXC10 upregulation. Gene Ontology supports the role of HOXC10 for proline biosynthesis and metabolism. Immunofluorescence on mouse GBM sections revealed co-expression of HOXC10 and PYCR1. HOXC10 silencing significantly decreased GBM cell line proliferation, induced apoptosis, and decreased PYCR1 expression in vitro. CONCLUSIONS HOXC10 expression is increased in GBM and with p16 HD. HOXC10 silencing significantly decreased GBM cell line proliferation and decreased PYCR1 expression. HOXC10-mediated proline biosynthesis may present a novel target to improve GBM survival.

TMET-29. TARGETING IDH1-MUTATED OLIGODENDROGLIOMA WITH ACID CERAMIDASE INHIBITORS

Abstract BACKGROUND Currently, an estimated 14,950 people lives with oligodendroglioma in the United States. Oligodendroglioma is genetically defined as a tumor harboring isocitrate dehydrogenase 1 or 2 mutations (IDH1mut/IDH2mut). Previously, we reported that in IDH1mut gliomas, D-2HG, the product of IDH1 mutant enzyme produces an increase in monounsaturated fatty acid levels that are incorporated into ceramides, tilting the S1P-to-ceramide rheostat toward apoptosis. Herein, we exploited this imbalance to further induce and IDHmut-specific glioma cell death. METHODS TCGA and CCGA data were used to explore the association between acid ceramidase (AC) gene expression and patient survival. CCK8 viability assays were carried out to measure the sensitivity of glioma cell lines to acid ceramidase inhibitors. Western blot and liquid chromatography-mass spectrometry analyses were performed for studying the mechanism of action of acid ceramidase inhibitors. The in vivo efficacy of the SABRAC drug, an irreversible inhibitor of AC, was evaluated in an orthotopic oligodendroglioma xenograft mice model using TS603 patient-derived oligodendroglioma cells. RESULTS. We report for the first time that the inhibition of acid ceramidase (AC) induces apoptosis and increases the survival of mice with IDH1mut oligodendroglioma. We demonstrated an IDH1mut-specific cytotoxicity of SABRAC, in patient-derived oligodendroglioma cells. Exploring the mechanism of action of this drug, we found that SABRAC activates both extrinsic and intrinsic apoptosis in an ER stress-independent manner, pointing to a direct action of AC-related ceramides in mitochondria permeability. The activation of apoptosis detected under SABRAC treatment was associated with up to a 30-fold increase in some ceramide levels and its derivatives from the salvage pathway. CONCLUSIONS We propose AC as a novel therapeutic target in oligodendroglioma with IDH1mut and we are conducting additional preclinical testing.

IMMU-63. MESSENGER RNA CHALLENGE PREDICTS CANCER IMMUNOGENICITY AND RESPONSE TO CHECKPOINT INHIBITORS

Abstract BACKGROUND Checkpoint inhibition has emerged as a promising cancer therapeutic approach. Checkpoint inhibitors (CIs) function by overcoming the adaptive T cell exhaustion induced by solid tumor microenvironment including brain tumors, particularly in the neoadjuvant setting. Unfortunately, not every patient who receives CIs will unlock effective antitumor immunity. Objectives: Microsatellite instability and high mutational burden are considered key criteria to predict response to CIs. However, only a minority of patients will fulfill these criteria suggesting the need to develop innovative technologies to identify CI responsive patients. METHODS To determine whether glioma tumors will respond to CIs, we have developed a novel multilamellar mRNA-lipid particle tool to predict response ex vivo. Messenger RNA is complexed into multilamellar lipid particles to form aggregates (RNA-LPAs) to induce pathogen recognition receptor signaling in tumor cells to define an inflammatory profile as an indirect predictor to CI response. RESULTS In naïve mice, we found that multilamellar RNA-LPAs induce activation of Type I IFN system as supported by decreased secretion of IFN-α in MyD88 KO mice compared with wild type mice. We next selected murine glioma cell lines with known response profile to CIs (responders – GL261 and SMA560 vs non-responders – KR158b and CT2A). Prior to evaluation of cytokine profiling, successful uptake of RNA-LPAs was achieved in all cell lines. In 2D cell lines cultures and their corresponding 3D tumor spheroids, we demonstrated that treatment of CI responsive murine glioma models showed a differential cytokine signature (increased secretion of IFN-β/IL-6/CCL4 versus non-responsive models). CONCLUSION We identified a unique inflammatory signature after ex vivo RNA-LPAs challenge that correlates with checkpoint blockade activity. These findings allow for creation of a diagnostic assay to quickly assess tumor immunogenicity, allowing for informed immunotherapeutic treatment.

DNAR-16. TARGETING APOBEC CYTIDINE DEAMINASES TO ENHANCE RADIOSENSITIVITY IN GLIOMA

Abstract Glioma is the most common and aggressive primary adult brain tumor. Radiation therapy (RT) is a critical part of glioma treatment, but radioresistance severely limits its therapeutic efficacy. To better understand how radioresistance develops, we analyzed 212 paired primary and recurrent gliomas from the Glioma Longitudinal Analysis Consortium. We found a significant increase in mutational signatures associated with the activity of the Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) family of cytidine deaminases in RT-treated recurrent gliomas (p<0.001, paired Wilcoxon signed-rank test). Further analysis of post-treatment mutations showed that RT increased APOBEC mutational signatures independent of other factors (p<0.001, multivariable log-linear regression), suggesting that APOBEC proteins become activated by radiation. However, their role in promoting tumor evolution and treatment resistance is currently unknown. Further analyses of extended signature contexts and expression data nominated APOBEC3B (A3B) and APOBEC3G (A3G) as candidates for creating these mutational signatures. To investigate their roles in RT response, we knocked out (KO) A3B and A3G individually in patient-derived glioma cell lines before RT. Compared to Control cells, irradiated A3B-KO and A3G-KO cells acquired significantly fewer APOBEC mutational signatures by whole genome sequencing (p<0.05, Kruskal–Wallis test) and exhibited enhanced radiosensitivity in vitro. Interestingly, the KO cells displayed a significant decrease in mutational signatures associated with Non-Homologous End-Joining (NHEJ), one of the main pathways for repairing the RT-induced DNA damage (p<0.05, Kruskal–Wallis test), suggesting that APOBEC deletion impaired DNA repair. This was further supported by persistent gH2AX DNA damage foci 48h post-RT and reduced autophosphorylation of DNA-dependent protein kinase (DNA-PK), a vital component of the NHEJ pathway, particularly in A3G KO cells. Overall, our results identify APOBEC proteins as potential targets for radiosensitizing gliomas and highlight the clinical utility of using mutational signatures to predict and monitor response to treatment.

EXTH-80. A NOVEL BISAMINOQUINOLINE DERIVATIVE NANOPARTICLE (BAQ13-NP) FOR THE TREATMENT OF GLIOBLASTOMA

Abstract Aminoquinolines, such as hydroxycholoquine, are known to inhibit autophagy and slow cancer cell growth, improving survival in preclinical models of glioblastoma. Unfortunately, they failed to demonstrate benefit in clinical trials due to a lack of sufficient potency and BBB penetration. We have developed a novel bisaminoquinoline derivative packaged in a nanoparticle (BAQ13-NP) with improved potency and intratumoral accumulation after systemic administration, confirmed by distribution and toxicity studies in animals. In this study, we evaluated the impact of BAQ13-NP on tumor cell autophagy and survival using patient-derived glioma cell lines and murine models. GBM patient-derived cell lines (n=4) and murine glioma cells (GL261, CT-2A) were treated with BAQ13-NP (1-4 µM) in culture, demonstrating growth restriction to less than 50% of control at 5-7 days (p<0.01) by MTT assay. Cells treated with BAQ13-NP also demonstrated reduced autophagy with significant increases in accumulation of LC3B and p62 (p<0.01) and reduced IL-6 dependent STAT3 phosphorylation in tumor cells, T cells, and myeloid cells (p<0.01). In addition, treatment of tumor cells with BAQ13-NP reduced IL-6 production over 50% (p<0.01). Mice with orthotopic GL261 or CT2A tumors were treated with BAQ13-NP by tail vein injection (25 mg/kg q2 days) starting 7 days post implantation. Significantly increased survival was observed in BAQ13-NP treated GL261 (HR 0.75, p=0.030) and CT2A (HR 0.78, p=0.033) bearing mice compared to vehicle control treated animals. Reduced tumor size at specific intervals was also observed with BAQ13-NP treatment, corelating with improved survival. BAQ13-NP can inhibit autophagy and slow glioma tumor growth, improving survival in preclinical models. Given the excellent distribution and low toxicity of this novel agent when delivered intravenously, we believe it has great potential to improve outcomes for GBM patients. Our drug has recently received IND approval and will soon be tested in a phase I clinical trial.

TMIC-33. MICROENVIRONMENT-DRIVEN CHANGES IN GLIOBLASTOMA CELL STATE SIGNATURES EXAMINED USING REGION-SPECIFIC HUMAN BRAIN ORGANOIDS

Abstract Cell state plasticity is retained by all stem-like and differentiated cells within glioblastoma (GBM) tumors. GBM cells can exploit these state-shifting capacities to evade and resist therapeutics. Thus, there is a need to identify and target the regulators of GBM cell state transitions. To address this question, we use a fully human platform where hiSPC-derived organoids serve as a recipient environment to host cells engrafted directly from surgically resected GBMs. This allows us to precisely examine how microenvironmental variables impact the molecular signatures of GBM cells. We acutely isolate GBM cells from surgical resections, label cells with a GFP-lentivirus for 4 hours, and then engraft these tumor cells into parallelly patterned region-specific organoids that mimic dorsal forebrain, ventral forebrain, midbrain, and hindbrain identities. These regions were chosen as they capture derivatives of the three primary brain vesicles and because these regions contain unique cell types and signaling molecules. We performed paired single-cell RNA sequencing on tumor cells pre-engraftment and 14 days post-engraftment. Universal to all regional organoids, we find that GBM cells shift towards a neural progenitor-like and neuron-like signature within organoids, regardless of the cell state of the parent tumor prior to engraftment. To support our findings, we also observed that patient-derived glioma cell lines converge on a neural progenitor-like state within organoids. Thus, the dominance of this shared GBM cell state within organoids implies the existence of shared extrinsic factors in these immature (neurogenic) organoids that favor a progenitor-like or neuron-like identity across all four regions. We are now asking whether this bias towards neuronal and/or neural progenitor identity is a result of engrafting into young neurogenic organoids, or if the same phenomenon could be true in late-stage (post-gliogenic) mature organoids. We are also using this valuable platform and data to identify the extrinsic signals that GBM cells rely on to fulfill their plastic potential, adapt to the microenvironment, and resist therapeutics.

PATH-55. RAMAN-BASED MACHINE LEARNING PLATFORM REVEALS UNIQUE METABOLIC DIFFERENCES BETWEEN IDHMUT AND IDHWT GLIOMA

Abstract BACKGROUND Formalin-fixed, paraffin-embedded (FFPE) tissue slides are routinely used in cancer diagnosis, clinical decision-making, and stored in biobanks, but their utilization in Raman spectroscopy-based studies has been limited due to the background coming from embedding media. METHODS Spontaneous Raman spectroscopy was used for molecular fingerprinting of FFPE tissue from 46 patient samples with known methylation subtypes. Spectra were used to construct tumor/non-tumor, IDH1WT/IDH1mut, and methylation-subtype classifiers. Support vector machine and random forest were used to identify the most discriminatory Raman frequencies. Stimulated Raman spectroscopy was used to validate the frequencies identified. Mass spectrometry of glioma cell lines and TCGA were used to validate the biological findings. RESULTS Here we develop APOLLO (rAman-based PathOLogy of maLignant glioma) – a computational workflow that predicts different subtypes of glioma from spontaneous Raman spectra of FFPE tissue slides. Our novel APOLLO platform distinguishes tumors from nontumor tissue and identifies novel Raman peaks corresponding to DNA and proteins that are more intense in the tumor. APOLLO differentiates isocitrate dehydrogenase 1 mutant (IDH1mut) from wildtype (IDH1WT) tumors and identifies cholesterol ester levels to be highly abundant in IDHmut glioma. Moreover, APOLLO achieves high discriminative power between finer, clinically relevant glioma methylation subtypes, distinguishing between the CpG island hypermethylated phenotype (G-CIMP)-high and G-CIMP-low molecular phenotypes within the IDH1mut types. CONCLUSIONS Our results demonstrate the potential of label-free Raman spectroscopy to classify glioma subtypes from FFPE slides and to extract meaningful biological information thus opening the door for future applications on these archived tissues in other cancers.

CSIG-34. TYROSINE PHOSPHORYLATION OF NON-CANONICAL NF-ΚB P52 PROMOTES GLIOMA GROWTH AND CHEMORESISTANCE IN GBM

Abstract p52 is a nuclear factor-κB (NF-κB) transcription factor that comprises the non-canonical NF-κB pathway. The impact and mechanism of non-canonical NF-κB signaling remains understudied in glioblastoma (GBM). Subunit post-translational modification is a known mechanism of NF-κB activation, however phosphorylation of p52 has never been demonstrated. Elevated tyrosine kinase activity is a known-feature of GBM and portends worse survival. Further, p52 has been shown to upregulate factors that likely promote invasion of GBM, thereby reducing survival. Activation of p52 may serve as a link between increased kinase activity and poor survival in GBM. p52-knockout cell lines were generated using the CRISPR-Cas9 system and assessed for growth and survival differences in vitro and in syngeneic murine glioma models. Public glioma datasets and an institutional cohort were surveyed to corroborate p52-dependent survival differences in GBM. To determine whether p52 is post-translationally modified, we performed tandem mass spectrometry on p52 in GBM cells. Interacting factors were also analyzed and tested for site-specific modification of p52 via co-IP studies. We then generated PTM-null, knock-in glioma cell lines using CRISPR-CAS9 editing to demonstrate the functional significance of p52 PTMs, which were assessed using assays of relative cell growth, stemness, chemosensitivity and survival. Finally, we performed unbiased analysis of the p52 PTM-dependent transcriptome via RNA-seq. p52 loss significantly reduced glioma growth rate in vitro and improves murine survival. Retrospective analysis of GBM in patients suggests that p52 levels are negatively prognostic. Mass spectrometry identified multiple p52 PTMs with tyrosine phosphorylation sites as top hits. Identified phospho-tyrosine residues specifically interact with SRC-family kinases. PTM site editing significantly reduces glioma cell growth and stemness while enhancing chemosensitivity, reflecting broad transcriptome level changes. In sum, p52 activity enhances GBM growth and chemoresistance, which is partially modulated by tyrosine-specific phosphorylation. p52 warrants investigation as a therapeutic target in GBM, possibly in combination with kinase inhibitors and standard alkylating chemotherapy.

SURG-58. COLD ATMOSPHERIC PLASMA (CAP) CAUSES SELECTIVE TUMOR CELL DEATH VIA APOPTOSIS AND FERROPTOSIS IN MALIGNANT GLIOMA CELLS

Abstract BACKGROUND High-grade gliomas (HGGs) are primary brain tumors associated with significant morbidity and mortality, and despite advances in neuro-oncology, the prognosis remains poor. Cold atmospheric plasma, a novel experimental anti-cancer therapeutic tool, is a near-room temperature ionized gas generated at atmospheric pressure. It has many promising results in vitro and in vivo with a variety of cancers. CAP cytotoxicity appears to be selective to cancer cells without significant damage to surrounding healthy tissues. We aim to evaluate the efficacy of CAP in generating tumor-selective cytotoxicity against high-grade glioma. METHODS B16, U87, GL261, patient derived high grade glioma, human neural stem cells (LM-NSC008) and astrocytes (NHA hTERT) were treated with CAP. Proliferation and propidium iodide (PI)/annexin V flow cytometry assays were employed to quantify cytotoxicity, cell cycle phases and apoptosis. Cells were pre-treated with ferroptosis inhibitors Ferrostatin-1 and Deferoxamine (DFO) in rescue assays. Cerebral organoids were transplanted with fluorescent tumor cell lines and stained with cleaved caspase-3, ki67 and transferrin receptor. RESULTS CAP induces dose dependent cell death in all glioma cell lines tested. CAP induces an accumulation of intracellular ROS, most prominent in glioma cells. CAP-mediated cell death involves apoptosis and ferroptosis. CAP selectively leads to apoptosis in tumor implanted organoids and leads to decreased proliferation in malignant and non-malignant cells. CAP leads to a shift into G0 phase of all treated cells, malignant and non-malignant. CONCLUSIONS CAP treatment induces dose-dependent increases in ROS and apoptosis in HGG lines tested more significantly than in NHAs and hNSCs. CAP induces decreased proliferation and G0 phase cell cycle arrest in all CAP-treated cells. CAP-mediated cytotoxicity was significantly mitigated with DFO pre-treatment in HGG cells, suggesting that ferroptosis plays a critical role in the mechanism of CAP treatment in HGG. CAP led to selective glioma cell death in tumor implanted cerebral organoids.

TMET-12. TARGETING AMINO ACID METABOLIC VULNERABILITIES IN GLIOMAS

Abstract Isocitrate dehydrogenase (IDH) wild-type gliomas and IDH mutant gliomas are similar in histologic appearance but differ in their genetic and metabolic backgrounds. This study aimed to reveal the difference in metabolites between IDH-wildtype glioma and IDH-mutant glioma, and to find the effective treatment according to the status of IDH in gliomas. Two artificial cell lines made from human astrocyte were used: NHAE6E7hTERTRas (IDH-wildtype) and NHAE6E7hTERTIDHmut (IDH-mutant). Using capillary electrophoresis- and ion chromatography–coupled mass spectrometry, the amount of asparagine was lower in NHAE6E7hTERTRas cells compared with NHAE6E7hTERTIDHmut cells, whereas the amount of glutamine, glutamate and 2-oxoglutarate in NHAE6E7hTERTIDHmut cells was lower than NHAE6E7hTERTRas cells. L-asparaginase, which converts asparagine into aspartate, was more effective in NHAE6E7hTERTRas cells than NHAE6E7hTERTIDHmut cells. L-asparaginase induced autophagy and inhibition of autophagy by 3-MA suppressed L-asparaginase-induced antitumor effect, which meant that the antitumor effect was at least partially due to the L-asparaginase-induced autophagy. Pharmacological or genetical inhibition of GLUD1 which converts glutamate to 2-oxoglutarate, suppressed proliferation of the cells by inducing ROS and apoptosis in NHAE6E7hTERTIDHmut cells. ROS inhibitor, NAC suppressed GLUD1 inhibitor-induced ROS, apoptosis, and cytotoxicity in NHAE6E7hTERTIDHmut cells, revealing that cytotoxicity by GLUD1 inhibitor was at least partially due to the inhibitor-induced ROS. The experiments using mutant IDH1 overexpressed glioma cell line showed similar sensitivity to GLUD1 inhibitor to NHAE6E7hTERTIDHmut, which suggested that the difference of sensitivity to GLUD1 inhibitor was due to the status of mutant IDH. In vivo experiments, L-asparaginase suppressed the growth of xenografted glioma cells without mutant IDH, whereas GLUD1 inhibitor inhibited the proliferation of xenografted glioma cells with overexpressed mutant IDH1. In conclusion, L-asparaginase and Glud1 inhibitor will be new therapeutic option for IDH-wildtype glioma and IDH-mutant glioma, respectively.

EXTH-56. INVESTIGATING THE EFFECTS OF INDIRUBINS IN PEDIATRIC DIFFUSE HIGH GRADE GLIOMA MODELS

Abstract Diffuse midline glioma (DMG) is a highly invasive and fatal pediatric brain tumor that arises from glial cells within the pons of the brainstem. Despite focal radiation treatment, DMG continues to have a poor prognosis, with a median survival of less than 10 months. A significant challenge in treating DMG is the blood-brain barrier (BBB), which tightly restricts access to therapeutic agents, preventing drugs from reaching the brain at effective concentrations. Previous studies from the Lawler lab 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. We investigated the effects of BIA on pediatric glioma models using various assays. In an in vitro scratch migration assay, BIA significantly slowed cell migration at a concentration of 1µM in pediatric glioma cells over 72 hours. Additionally, BIA was shown to modulate tumor vasculature and improve drug delivery to tumors by targeting tight junctions in tumor-associated endothelium. BIA treatment increased dextran uptake into 3D BBB models and lowered endothelial cell permeability in vitro by reducing the expression of tight junction proteins, as shown by staining and a decrease in TEER values. Furthermore, BIA’s anti-angiogenic effects were demonstrated through staining, showing significant alterations in angiogenesis-related protein expression. To assess potential synergistic interactions, a drug screen was performed on a panel of pediatric glioma cell lines identifying several FDA-approved drugs that combine well with BIA. Drugs identified in the screen are currently under investigation. Our hypothesis is that BIA could be an effective therapeutic agent for DMG by targeting tumor invasion, angiogenesis, and improving drug potency and delivery through modulation of endothelial cell tight junctions. Future studies will focus on elucidating the underlying mechanisms and developing drug formulations for in vivo studies to assess the translational potential of this approach.

TMIC-34. INFILTRATIVE BEHAVIOR OF PATIENT-DERIVED GLIOBLASTOMA CELL LINES IN REGION-SPECIFIC HUMAN BRAIN ORGANOIDS

Abstract Glioblastoma (GBM) is the most common malignant brain tumor in adults. GBMs are known for their highly invasive and proliferative properties and are associated with a dismal prognosis (~12-18 months post-diagnosis). Neuroanatomical disparities in the occurrence of GBMs suggest that in addition to microenvironmental impacts, tumor cells are impacted by the cellular and molecular diversity across varying regions of this nervous system. This idea has been difficult to study due to the lack of human-specific models that effectively recapitulate the biological behaviors of GBMs in region-specific microenvironments. To address these challenges, we engrafted GFP-tagged patient-derived glioma cell lines into human induced pluripotent stem cell (hiPSC)-derived organoids patterned to mimic the most forebrain (dorsal cortex) and hindbrain (spinal cord) regions of the nervous system. Immunostaining assays at 2, 7, 14, and 21 days post-engraftment compared the extent of infiltration, as measured by the distance traveled by glioma cells, in both regions. We found that glioma cells infiltrated a significantly greater distance in the dorsal cortical organoids compared to the spinal cord organoids. This suggests a more conducive environment for glioma infiltration in the forebrain vs. hindbrain at baseline. We are currently determining if these regional biases in infiltration capacity are modified with either optogenetic stimulation of the host brain organoids or in the presence of interregional interactions in an assembloid system. Thus, using this fully human-specific platform, we aim to gain insights into the infiltrative behaviors of GBM cells, which will advance our understanding of this devastating disease.