Glioma Stem Cells Research Articles

IMMU-69. THERAPEUTIC WINDOW ENABLING ERADICATION OF RESIDUAL GLIOMA STEM CELLS BY INTRACRANIAL NK CELL THERAPY FOLLOWING TEMOZOLOMIDE-INDUCED TUMOUR DEPOPULATION – A POSSIBLE ROLE OF CYTOTOXIC EXTRACELLULAR VESICLES

Abstract Glioblastoma (GBM) is an incurable brain cancer, where dismal outcomes result from disease recurrence driven by tumour-initiating glioma stem cells (GSCs). GSCs survive and expand in the brain after surgery, radiation and temozolomide (TMZ) amidst weak immune and natural killer (NK) cell surveillance. Efficient NK cell-mediated killing of GBM cells occurs at high effector to target ratios precluding effective eradication of large tumour remnants, as enforced by clinical trials with autologous NK cells in an adjuvant setting. Here we explore in a human GSC xenograft model whether tumor depopulation using high dose Temozolomide (TMZ) could create a window of curative opportunity for endogenous or exogenous NK cells. We observed that while subcutaneous (sc) xenografts of patient derived GSCs are infiltrated by endogenous functional (NCR1+) NK cells in SCID mice, the corresponding intracranial (ic) tumours remained NK cell depleted. Notably, while TMZ caused near complete regression of intracranial GSC xenografts this was followed by inevitable recurrence of drug-resistant lesions. To explore whether direct delivery of NK cells into depopulated intracranial tumours would change the lethal course of the disease, mice were injected with GSCs intracranially, and upon tumour formation were treated with a sequence of TMZ (systemically), at 2, 7, 14, and 21 days later with irradiated NK92MI cells. Remarkably, this combined therapy completely obliterated recurrent disease at 2 and 7 days but not beyond 14 days. To assess whether NK92MI cells could be replaced by their derived extracellular vesicles (NK-EVs), the latter were injected i.c. post TMZ in GSC xenograft bearing mice. A single injection of NK-EVs resulted in tumour eradication in some but not all mice. Thus, chemotherapy-dependent tumour depopulation may create a unique window of opportunity for curative NK-mediated immunotherapy in GBM.

EXTH-83. CHARACTERIZATION OF SPLICING ABERRATIONS INDUCED BY PRMT5 INHIBITION IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) are lethal tumors with limited treatment options, with a high unmet need for novel therapies. PRMT5, an arginine methyltransferase that regulates several histone and non-histone targets, is overexpressed in cancers including GBM and is a promising therapeutic target given its role in promoting oncogenic processes. Here, we characterized the effects of pharmacological inhibition of PRMT5 by the brain-penetrant, orally bioavailable inhibitors, PRT811 and PRT808 on treatment-induced splicing aberrations using a panel of patient-derived glioma stem cells (GSC) and organotypic human glioma slice cultures. Changes in protein expression upon PRMT5 inhibition were assessed using reverse phase protein array (RPPA) analysis. Additionally, splicing aberrations due to PRMT5 inhibition in were tested in several GSCs using RNA-seq. We observed a potent reduction of symmetrical dimethylation of arginine (SDMA protein marks by both PRMT5 inhibitors across various GSC lines and ex-vivo brain tumor tissue slices. Notably, GSCs with both mutant and wild-type p53 displayed similar sensitivity to PRMT5 inhibition, independent of MTAP status. Moreover, PRMT5 inhibition elicited reduced cell proliferation, altered cell survival pathways, and apoptosis. RNA-seq analysis revealed that splicing alterations affecting numerous oncogenes and regulatory factors, encompassing 42 cancer driver genes, 80 splicing factors, and 42 transcriptional factors across these GSC lines, with specific splicing events observed in nuclear and cytoplasmic subcellular compartments. Additional analysis is ongoing to determine which of the aberrant transcripts are translated to yield tumor specific neoantigens that can be potential targets for tumor specific immune strategies. These results underscore the efficacy of pharmacological PRMT5 inhibition in genetically and epigenetically diverse GSCs, and highlight its potential for developing novel immune and non-immune therapeutic strategies against GBM.

STEM-01. SHARED ASTROCYTE-LIKE GLIOMA STEM CELLS DRIVE HETEROGENEITY AND IMMUNE DYNAMICS IN ADULT-TYPE DIFFUSE GLIOMAS

Abstract Adult-type diffuse gliomas present significant treatment challenges due to their complex cellular composition and evolving nature. Understanding this heterogeneity is critical for the development of effective, targeted therapies. We conducted single-cell and single-nucleus RNA sequencing on a unique cohort of 66 tumours (single surgery) and 54 paired samples (two surgeries). This comprehensive dataset of approximately 900,000 cells allows us to dissect conserved cellular landscapes and dynamic evolution across diverse glioma subtypes. Using a novel phylogeny-based approach, we identified an astrocyte-like glioma stem cell (GSC) as the root of diverse glioma lineages. This discovery transforms our understanding of glioma development, pinpointing the GSC as a potential therapeutic target. The GSC’s similarity to healthy adult neural stem cells provides clues as to the origin of glioma heterogeneity and the shared basis of intratumoural diversity. Our analysis uncovered a shared cellular architecture across astrocytomas, oligodendrogliomas, and glioblastomas (GBMs). This architecture encompasses seven recurring malignant cell states: neuro-, oligo-, neuro-oligo-lineage, cycling (G1/S, G2/M), hypoxia-associated, and a quiescent astrocyte-like GSC. Importantly, these cell state proportions vary significantly between glioma subtypes. Additionally, we demonstrate that GBMs display substantially higher T-cell and myeloid cell infiltration than IDH-mutant gliomas. Counterintuitively, this correlates with a poorer prognosis, implying profound T-cell dysfunction and intricate myeloid cell-tumour interactions. Longitudinal analysis further highlights the dynamic nature of glioma cell populations and the immune landscape. Subtype-specific T-cell and myeloid gene expression profiles were identified, including potential targets such as PARD6G (GBM) and CXCL13 (IDH-mutant-astrocytoma), laying the groundwork for tailored immunotherapies and prognostic biomarkers. This study presents a comprehensive model of glioma heterogeneity and evolution. The model reveals a shared astrocyte-like GSC that fuels cellular diversity, coupled with distinct, dynamic immune landscapes in different glioma subtypes. These findings hold extraordinary potential for developing transformative treatment strategies. Future research functionally validating the GSC and its impact on tumour dynamics will be essential for translating these insights into improved patient outcomes.

STEM-07. UNVEILING THE ROLE OF HYPOXIA AND TAM-SECRETED TGF-Β1 IN PROMOTING GLIOMA STEM CELL MAINTENANCE VIA YAP/TAZ ACTIVATION IN GLIOBLASTOMA PERI-NECROTIC NICHE

Abstract Glioblastoma (GBM) is a highly malignant brain tumor with rapid growth and poor outcomes. In the tumor microenvironment (TME), necrosis triggers significant reshaping and rapid progression, marked by tumor-associated macrophages (TAMs) and glioma stem cells (GSCs) accumulation in peri-necrotic zones. Analysis of Ivy-GAP and single-cell RNA-Seq data revealed consistent Hippo pathway activation in the hypoxic peri-necrotic zone, despite canonical stem-cell transcription factors such as SOX2, OLIG2, and FOXM1 not being upregulated. We hypothesize that Hippo transcription activation may promote GSC stemness via direct effects of hypoxia and/or TAM-secreted cytokines. Exposing patient-derived GBM neurospheres to 1% hypoxia increased nuclear YAP1 and TAZ expression, along with CYR61, a Hippo pathway readout, compared to normoxia at 48 hours. Single-cell RNA-Seq identified potential hypoxia-regulated genes upstream of YAP/TAZ, including Zyxin, upregulated in 1% hypoxia-treated cultures, possibly facilitating nuclear YAP/TAZ transport. Culturing GBM cells with primary macrophage-conditioned media activated Hippo transcription, with TGF-β1 identified as a hypoxia-specific driver of malignant cell gene expression with single-cell RNA-Seq analysis using NichenetR. Treating GBM cells with TGF-β1 increased CYR61 expression, signifying Hippo transcription activation. Furthermore, we showed that hypoxia and TGF-β1 synergistically maintained GSCs with extreme limiting dilution assays. In the immunocompetent RCAS/tv-a mouse model, Verteporfin, a YAP/TAZ inhibitor, significantly reduced tumor size and prolonged mouse lifespan. TAZ knockdown similarly extended lifespan and reduced tumor growth, with flow cytometry showing a decrease in CD133/CD44 positive GSC populations in knockdown mice. These findings suggest the conclusion that hypoxia and TAM-secreted TGF-β1 directly stimulate YAP/TAZ activation and potentially promote GSC stemness in the GBM peri-necrotic niche.

CSIG-31. ANNEXIN A7 ALTERNATIVE SPLICING IN GLIOMA STEM CELLS FACILITATES THE DEGRADATION OF EGFR BY PHOSPHORYLATING TYROSINE RESIDUES EXCLUSIVE TO PROTEIN ISOFORM 1

Abstract The majority of GBMs express aberrant receptor tyrosine kinase (RTK) activity, which are canonically endocytosed and degraded. This process is disrupted in GBM to promote receptor recycling and resulting in hyperactive RTK signaling. Exon six of Annexin A7 (ANXA7) can be alternatively spliced to yield two distinct protein isoforms through its inclusion (I1) or exclusion (I2). We investigated ANXA7 isoform differences for epidermal growth factor receptor (EGFR) trafficking fates in PDX glioma stem cells (GSCs). We demonstrated that ANXA7 exon skipping supports the impaired endosomal trafficking of RTKs to favor recycling in GBM. JX14 GSCs were made to overexpress ANXA7-I1 with lentivirus to conduct immunofluorescence experiments measuring EGFR-ANXA7 colocalization (Pearson) with markers of the endocytic pathway for the early endosomes (EEA1), recycling endosomes (Rab4/Rab11), and lysosomes (LAMP1). EV and I1 cells were also incubated with siRNA targeting both ANXA7 isoforms to determine the impact of ANXA7 loss on endosomal EGFR trafficking. Interestingly, we found drastic differences in endosomal EGFR trafficking in I1 expressing cells. EGFR colocalized with EEA1 in both EV and I1 cells (R2=0.01336, P>0.6275). In EV cells, EGFR was recycled via fast/slow recycling endosomes demonstrated by Rab4/Rab11 colocalization while I1 did not (R2=0.9267, P<0.0001). Furthermore, I1 cells and not EV preferentially degraded EGFR as seen by LAMP1 colocalization (R2=0.9398, P<0.0001). On the other hand, ANXA7 siRNA KD in I1 cells impaired EGFR sorting via a 40% increase (P<0.0010) in total EGFR protein and a 40% reduction (P<0.0001) in colocalization with EEA1 to prefer recycling again while EV cells were unaffected. I1 overexpression significantly reduced EGFR signaling through degradation, which is driven by the phosphorylation of I1-exclusive tyrosine residues in phosphomimetic (p<0.01) and non-phosphorylatable protein mutants. I1-induced reduction in EGFR signaling suggests a new therapeutic vulnerability for GSCs by potentially reducing tumorigenicity or improving the efficacy of EGFR inhibition.

TMOD-14. ESTABLISHING A CLINICALLY RELEVANT MOUSE MODEL TO STUDY NECROSIS AS A PRIMARY VARIABLE IN GLIOBLASTOMA

Abstract All glioblastoma (GBM) molecular subsets share the common trait of accelerated progression following necrosis, which cannot be adequately explained by cellular proliferation arising from accumulated genetic alterations. We suggest that necrosis is much more than a passive phenomenon related to rapid growth but rather is a driving force causing tumor microenvironment (TME) restructuring and biologic progression. yet mechanisms remain poorly understood due to a lack of animal models to study necrosis as a primary variable. In GBM, the most malignant primary brain tumor, vascular pathology and central necrosis precede rapid, radial expansion. To reveal spatio-temporal changes directly following vascular damage in the brain, we developed methodology to induce clinically relevant thrombosis vaso-occlusion within GBMs in immunocompetent RCAS/tv-a mouse model to study TME restructuring by intravital microscopy and demonstrate its impact on glioma progression in real-time. We found that following Rose Bengal photothrombosis, GBMs rapidly expanded radially, with glioma migration away from central necrosis and tumor-associated macrophages (TAMs) and glioma stem cells (GSCs) increased dramatically in the peri-necrotic zones. Through the single cell tracking analysis, we found that TAMs displaying increased displacement toward necrosis and greater overall migration distances. Collectively, this model introduces necrosis as the primary variable and captures glioma growth dynamics and TME restructuring in a manner that will facilitate the development of therapies that antagonize these mechanisms to improve outcomes.

ANGI-03. GLIOMA STEM CELLS INDUCE VASECTASIA – A NON-ANGIOGENIC BRAIN TUMOUR NEOVASCULARIZATION PROCESS DRIVEN BY INTERCELLULAR EGFR TRANSFER THROUGH EXTRACELLULAR VESICLES

Abstract Tumour neovascularization in glioblastoma (GBM) is abundant, abnormal and poorly understood, and it likely involves multiple co-existing mechnanisms with distinct modes of regulation. Since glioma stem cells (GSCs) engage in bidirectional interactions with the vasculature we dissected the resulting vascular responses elicited by either proneural (PN), or mesenchymal (MES) GSC subtypes established from patients isolates and able to drive progression of orthotopic intracranial xenografts in mice. We report here that while PN-GSCs elicited mostly angiogenesis-type vascular capillary growth processes, their MES-GSC counterparts elicited non-angiogenic growth of enlarged vessels (vasectesia), paradoxically associated with reduced blood vessel density. Vasectasia was dependent on the expression of oncogenic EGFR/EGFRvIII by MES-GSCs, and on the related kinase/signalling activity, as EGFR/EGFRvIII gene disruption and Dacomitinib treatment suppressed formation of large blood vessels in vivo and resulted in a preponderance of a small calliber (angiogenic) vasculature throughout xenografts. Notably, the analysis of spatial transcriptomes and single cell RNA sequencing revealed distinct molecular traits of endothelial cells asociated with vasectasia and angiogesis, the former enriched in Birc5, but depleted for angiogenic tip cell markers (Vegfr2, Apln). Mechnaistically, vasectasia was linked to the intercellular transfer of EGFR from MES-GSCs to endothelial cells resulting in ectopic activation of EGFR signalling in the endothelial cell bacground, both in vitro and in a model of in vivo blood vessel recruitment into implanted matrigel pellets. Simultaneous pharmacological blockade of both, angiogenesis (anti-VEGR2) and vasectasia (Dacomitinib) lead to a marked increase in survival of mice with aggressive GBM lesions initiated by MES-GSCs. We suggest that, vasectasia driven by intercellular transfer of oncogenic EGFR may represent a new, subtype-specific mechanism of GBM neovascularization, and a plausible target for agents aiming to modify vascular tumour microenvironment.

STEM-03. TARGETING F-ACTIN DYNAMICS OF GLIOBLASTOMAS TO OVERCOME THERAPY RESISTANCE: IMPACTS ON DNA REPAIR AND GLIOMA STEM CELL PHENOTYPE

Abstract Glioblastoma (GBM) is a highly aggressive brain tumor with high recurrence and resistance to therapies. The F-actin cytoskeleton in GBM is related to cell invasion and the glioma stem cell (GSC) phenotype, affecting self-renewal and migration. Our research identifies a novel role for the F-actin pathway in DNA repair, impacting genomic stability and contributing to therapy resistance. Understanding F-actin’s role is crucial for developing targeted strategies to improve GBM treatment outcomes. This study examines the effects of pharmacological F-actin depolymerization on reversing resistance to ionizing radiation (IR) and temozolomide (TMZ) in GBM spheroids. We established resistant sublines of GBM U87-MG cells through IR and TMZ cycles, observing significant resistance increases via MTT assays. Also, resistant cells had enhanced spheroid formation capacity, with higher size and proliferation. Alterations in DNA repair and F-actin pathway were observed, with intensified and disorganized F-actin polymerization and efficient repair in resistant cells. Elevated expression of stemness markers (Nestin, CD133, and CD44) indicated a GSC-like phenotype in resistant cells, corroborated by increased tumorigenicity and de novo spheroid formation. These markers were further enhanced by DNA damage. Treating cells with actin polymerization inhibitors reduced resistance to TMZ and IR, partly due to impaired DNA repair capacity. Additionally, F-actin disassembly affected stemness marker expression, reinforcing the link between cytoskeletal dynamics and the GSC phenotype. Our findings suggest that F-actin dynamic is crucial for resistance in GBM. Targeting F-actin could resensitize recurrent GBM tumours by diminishing DNA repair capabilities and affecting the GSC-like phenotype, offering new therapeutic avenues.

DDDR-30. ENHANCED CHEMO AND RADIO-SENSITIZATION OF GLIOMAS BY HSP90 INHIBITION WITH JIN-001, A NOVEL BLOOD-BRAIN BARRIER PENETRANT HSP90 INHIBITOR

Abstract BACKGROUND HSP90, an abundant molecular chaperone, plays a pivotal role as a key regulator in the growth and survival of malignant cells and has been a therapeutic target in various cancers. We have previously reported a strong role for HSP90 inhibitors to disable DNA damage repair mechanisms and induce cell death. Here, we determined the effects of JIN-001 mediated HSP90 inhibition on alkylator and radiation induced DNA damage. Method: Using a panel of patient-derived glioma stem cells (GSCs) we determined the synergy scores for cell viability of JIN-001 (0.1µM to 0.5µM) in combination with radiation (RT) and Temozolomide (TMZ). We also examined cell cycle dynamics and cell death of the combination using flow cytometry (PI and PI/Annexin method) and cell viability assays. Additionally, organotypic human glioma slice cultures treated with JIN-001 and chemoradiation were analyzed for changes in gene and protein expression as well as cell viability in situ. Ongoing in-vivo studies are being conducted to further elucidate drug combination efficacy in mouse models. RESULTS JIN-001 sensitized the cells to chemoradiation leading to cell cycle arrest and cell death which was higher in the combination compared to single agent. Significant changes in expression of downstream molecules of homologous recombination pathway was noted (ATM, pATM, CHK1, pCHK1, ATR, pATR) in the cell lines and confirmed in human glioma slice culture. Based on the synergy score from four cell lines, an effective dose for combination of JIN-001+RT+TMZ was identified; Results of ongoing ex-vivo human glioma gene expression changes and in-vivo mouse studies will be presented. CONCLUSION Through a comprehensive analysis, we demonstrated that HSP90 inhibitor JIN-001 sensitizes glioma cells to radio-chemotherapy including in human glioma tissue. Our study underscores the potential of combining JIN-001 with standard therapies in glioma treatment in future clinical trials for patients with glioblastoma.

CNSC-49. ELUCIDATING THERAPEUTIC POTENTIAL OF USP1 INHIBITION IN DIFFUSE MIDLINE GLIOMAS

Abstract Diffuse midline gliomas (DMGs) are aggressive childhood brain tumors with the H3K27M mutation in the H3F3A or H3C2 gene. Despite treatment advances, radiotherapy is the only viable option, but 99% of patients die within 1.5-2 years. Therefore, there is an urgent need to identify potential novel therapeutic targets for the treatment of DMGs. Ubiquitin-specific protease 1 (USP1) is a key deubiquitinating enzyme involved in genome integrity, cell cycle regulation, and cell homeostasis. USP1 is overexpressed in various cancers, including gliomas, is linked to poor prognosis. It promotes glioma stem cell (GSC) self-renewal and radio-resistance by regulating ID1 and CHEK1. Our recent findings show that ID1 is crucial for DMG invasion and migration. Inhibiting USP1 suppresses malignant growth, enhances radiation sensitivity, and improves chemotherapy response, highlighting its potential for combined therapies. However, USP1’s role in DMGs and its feasibility as a therapeutic target remain poorly understood. In this study, we investigated USP1 in DMGs for the first time. RNA sequencing revealed high USP1 expression in DMG patient samples and cell lines, directly linked to the H3K27M mutation. Correcting this mutation reduced USP1 expression in DMG cell lines. Pharmacologic inhibition of USP1 with the specific inhibitor SJB3-019A significantly reduced DMG cell proliferation, triggered apoptosis, and altered cell cycle profile. SJB3-019A-mediated USP1 inhibition decreases migration and invasion in multiple patient-derived DMG cell lines. Additional in vitro experiments revealed that USP1 inhibition leads to a significant decrease in the expression of ID1 and pAKT, suggesting the ID1/AKT pathway as a potential mechanism underlying the observed effects. H3K27M-mediated activation of USP1 in DMG is a promising therapeutic target, with inhibition by SJB3-019A reducing tumor aggressiveness by inhibiting invasion and migration. We will next assess SJB3-019A’s efficacy in vivo using genetically engineered isogenic mouse models (GEMM) with DNp53/PDGFRA/H3.3K27M (PPK) alterations and evaluate USP1 inhibition combined with radiation therapy as a potential radiosensitizer in DMGs.

TMIC-46. TRANSCRIPTIONAL HETEROGENEITY AND MECHANISTIC PATHWAYS OF RECURRENT GLIOBLASTOMA: INSIGHTS FROM A PRE-CLINICAL RECURRENT TUMOR MODEL

Abstract Despite aggressive therapy combining surgical resection, radiation and chemotherapy, glioblastoma (GBM) almost always recurs. Currently, there is no standard treatment for recurrent GBM, and patient inclusion in clinical trials is based on the genetic and molecular profile of the primary tumor even though the effects of chemoradiation on the transcriptional subtype in matched primary and recurrent GBM remain largely understudied. Paralleling the GLASS consortium, we sought to perform a longitudinal assessment of GBM recurrence after therapy based on molecular subtype with the hypothesis that recurrent tumors exhibit a distinct transcriptional landscape different from treatment-naive tumors. To address this, we utilized primary tumor-derived glioma stem cells (GSCs) representing classical and proneural subtypes. We then undertook in vivo intracranial implantation studies and performed standard concomitant treatment that mimics the Stupp protocol. Upon bulk RNAseq data analyses of untreated (n=21) and recurrent tumors (n=17) we identified that while the molecular subtype of recurrent tumors does not change post therapy, their transcriptional profile is more heterogenous compared to untreated tumors for both subtypes. Hierarchical cluster analyses revealed distinct sub-clusters of recurrent tumors. Common mechanisms of recurrence include upregulation of angiogenesis pathways and PI3k/Akt/mTOR signaling pathways. Interestingly, recurrent tumors exhibit upregulation of genes involved in cellular metabolism and membrane transport/ potential. The source of heterogeneity in recurrent tumors in both subtypes appears to be due to the diversity in expression of gene sets involved in hallmark of cancer pathways such as cell cycle, Notch and Wnt signaling pathways and heterogeneity in oncogenic signatures and cell states resembling fetal or neural cells. Further research into the recurrent tumor microenvironment by spatial multi-omics approaches using this pre-clinical recurrent GBM model will elucidate the linkage between cellular as well as molecular heterogeneity and enable identification of targets that can help drive therapeutic decisions for recurrent GBM patients.

EPCO-13. HUMAN ENDOGENOUS RETROVIRUS (HERV) PLAYS A CRITICAL ROLE IN ONCOGENIC MECHANISM INIDH1-WILD TYPE GLIOBLASTOMAS

Abstract IDH1-wildtype (WT) GBMs are the most common and aggressive tumors of the central nervous system (CNS), with a median survival rate of 12-15 months, and are resistant to clinical therapies due to their heterogenous molecular phenotype. In this study, we developed a pipeline to dissect the potential role of human endogenous retroviruses (HERVs) in patient derived glioma stem cells (GSCs) and integrated our findings with published epigenomic datasets for enhancers (H3K27ac), CTCF, and chromatin toplogy (Hi-C). We found high expression of several HERV transcripts in IDH1-WT GBMs as compared to IDH1-mutant variants. Epigenomic analysis of these upregulated HERVs showed association with both enhancer marks and CTCF deposition. Annotation of HERVs to their nearest genes, followed by Gene Set Enrichment Analysis (GSEA), identified involvement of actin-filament based mechanisms and regulation of immune phenotype. Integration of these findings identified MER61 (ERVMER61-1), a potential regulator of Interleukin-6 and showed alteration in chromatin looping and enhancer occupancy. To summarize, our work points to novel mechanisms of GBM pathogenesis, through involvement of HERVs in cell migratory and immune dysfunction, warranting further investigations into these oncogenic mechanism to identify and develop novel therapeutic options for GBM.

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.

DDDR-01. TUMOR ASSOCIATED MACROPHAGES VIS-À-VIS TMZ RESISTANCE IN PTEN-DEFICIENT GLIOMA

Abstract Drug resistance is one of the most important obstacles during chemotherapy. Currently temozolomide (TMZ) is still first line chemotherapy agent for gliomas, while drug resistance is one of the main reasons for chemotherapy failure. It has been reported that tumor-associated macrophages (TAMs) could be related to drug resistance, and thus we have investigated the association between TAMs and TMZ response in gliomas. Firstly, our study have found that PTEN-deficient glioma can specifically induce a large number of M2 phenotype TAMs, which can attenuate the TMZ-mediated the killing effect on glioma cells. Further search for the possible mechanism, we have found TAMs may secrete cytokine IL-6 in PTEN-deficient gliomas, which in turn mediate TMZ resistance. We also revealed that IL-6 highly secreted by TAMs can enhance the stemness of glioma cells through activation of the JAK-STAT3 signaling pathway in PTEN-deficient glioma cells. Secondly, have found valproic acid (VPA) can promote the transformation of TAMs from M2 to M1, reduce its IL-6 secretion, and in turn enhancing sensitivity of TMZ in PTEN-deficient gliomas. Our study revealed that TMZ resistance may modulated through soluble factors released by TAMs. Conventional anti-epilepsy drug- VPA may enhance TMZ sensitivity in PTEN-deficient gliomas, and worth to further clinical investigation. Keywords: PTEN-deficient gliomas; tumor-associated macrophages; valproic acid; temozolomide resistance

EXTH-78. A NOVEL BISPECIFIC S100A4/TFR ANTIBODY TO REPROGRAM THE GLIOMA TUMOR MICROENVIRONMENT AND TARGET GLIOMA STEM CELLS

Abstract GBM (glioblastoma) is the most common and aggressive primary brain tumor in adults. While immunotherapy is a promising treatment modality for GBM, most clinical trials have thus far only provided a modest benefit to GBM patients. We propose that enhancing the efficacy of immunotherapy for GBM requires approaches that increase T cell infiltration and reprogram the myeloid cells to a more pro-inflammatory state. S100A4 is a major regulator of stemness, epithelial-mesenchymal transition, and a critical regulator of immune suppressive myeloid and T cell phenotypes in GBM. Targeting S100A4 in either glioma or stromal cells is sufficient to reprogram the tumor immune landscape and extend survival of glioma bearing mice, indicating that S100A4 is a promising immunotherapy target. We recently developed a S100A4 blocking monoclonal antibody; however, this antibody does not cross the blood brain barrier (BBB) efficiently to confer significant therapeutic benefit, limiting its efficacy in treating GBM. To overcome this challenge, we generated a bispecific S100A4-TfR antibody (BsA) that allows robust BBB penetration, and tested this in a pre-clinical syngeneic, orthotopic mouse model of GBM. Using ELISA, confocal immunofluorescent microscopy, flow cytometry, and bulk RNA sequencing, we report that systemic administration of the S100A4-TfR BsA results in increased immune cell infiltration with a preferential increase in CD4 T cell infiltration, and potent immune remodeling of the tumor microenvironment to a more pro-inflammatory state. Furthermore, the BsA is taken up by glioma cells intracellularly, which is correlated with depletion of intracellular S100A4 and decreased nuclear SOX2 localization, which would reduce stemness of glioma cells. Functionally, in vitro BsA treatment of primary glioma cell cultures shows decreased S100A4 expression, proliferation, and self-renewal. Our results suggest that S100A4-TfR BsA is a novel dual activity inhibitor to simultaneously reprogram the GBM immune landscape and reduce glioma stemness.

DDDR-32. EXPLORING ANDROGENIC MODULATION IN GLIOBLASTOMA: POTENCY AND SELECTIVITY OF NOVEL NORETHINDRONE DERIVATIVES

Abstract BACKGROUND Glioblastoma (GBM) represents the most frequently diagnosed malignant CNS tumor in adults and remains incurable. Despite the identification of several oncogenic drivers contributing to tumor formation, targeted therapy with brain-penetrant inhibitors has afforded little progress in survival outcomes. Interestingly, gender has been shown to increase the incidence of GBM, with men having roughly a 50-60% increase in incidence compared to women in the US. This statistic led to research on the involvement of and subsequent inhibition of androgen synthesis and signaling in cell culture models and tumor samples. METHODS To further investigate the potential involvement of steroidal hormones in GBM proliferation, our lab synthesized a series of norethindrone (NE) derivatives with varied molecular weights, steric bulk, and lipophilicity to modulate the androgenic activity of NE. The small library was tested in several GBM cell lines, including U87, A172, and T98G cells, using cell viability assays. RESULTS One derivative, a lipophilically modified version of NE synthesized via a one-pot click reaction, was shown to have improved potency compared with parental NE (>5-fold). The lipophilic NE derivative had IC50 value of 22 uM, 15 uM, and 35 uM in the U87, A172, and T98G cells, respectively. This molecule was tested in immortalized normal human astrocyte (NHA) cells and found to have an IC50 value of 34 uM suggesting some selectivity. DISCUSSION Given this derivative’s activity in GBM models and modest selectivity, we plan to screen it in additional GBM cell lines (LN-18 and U251) and eventually in glioma stem cell models. Future research will focus on identifying key targets involved in the mechanism of action, including the impact of lipophilic addition to the steroid nucleus and its effect on androgenic activity and downstream signaling. We will investigate these effects using RPPA and co-treatments with androgen receptor antagonists and 5-alpha reductase inhibitors.

EPCO-25. TRANSPOSABLE ELEMENTS INDUCE ONCO-EXAPTATION EVENTS IN MALIGNANT ATRX-DEFICIENT GLIOMAS

Abstract Deficiency of ATRX (Alpha Thalassemia/Mental Retardation Syndrome X-linked), a core member of SWI/SNF family chromatin regulator is altered in diffuse gliomas and coexist with IDH1/2 mutations in adults and histone G34R/V alterations in pediatric populations. Loss of ATRX has been associated with global alterations in chromatin accessibility and epigenomics landscape, however, the detail mechanism behind the oncogenic transformation remains elusive. Integrating transcriptomics and in-depth positional epigenome analysis over repetitive DNA sequences in progenitor and tumors models of murine and human gliomas, we describe ATRX-deficient gliomas is fundamentally a disease of altered Transposable Elements (TE’s) and regulates neuronal differentiation and cell motility in gliomas, as validated pharmacologically and genetically. Epigenome assessment reported de-repression of LINE-1 (Long interspersed nuclear elements-1), specifically at Lamina-Associated Domains (LADs) with gain of enhancer histone marks, with association in chromatin loops/topology in ATRX-deficient glioma cells. Moreover, our single cell-RNA seq analysis focusing on TE’s and molecular docking studies in human glioma stem cells demonstrated LINE-1 regulates oncogenic alternative splicing events and neuro-developmental signatures. Expanding the analysis, we identified a subset of evolutionary young regulatory TE’s to play critical role in ATRX-deficient gliomas involved in neuronal differentiation phenotype. To summarize, our cross-species analysis establishes tangible links between ATRX deficiency and multiscale dysregulated 3D-epigenomic architecture that hijack regulates onco-exaptation programs in gliomas. We further discover that enhancer-like TE’s fine-tune the transcriptional program with important clinical implications, that could help in developing treatment options in malignant gliomas.

DDDR-36. LOOKING AROUND THE CORNER: PREDICTING SYNERGY OR FUTILITY TO COMBINATION OF ARSENIC TRIOXIDE PLUS MNK1 INHIBITORS IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most prevalent type of malignant tumor within the central nervous system. The five-year survival rate of 6.8% highlights the challenges inherent to exceptionally heterogeneous tumors that grow behind the blood brain barrier. The protracted dearth in new agents approved for GBM begs innovative strategies by which to approach clinical trials. Arsenic trioxide (ATO) is the standard of care for myelodysplasia and relapsed or refractory acute promyelocytic leukemia. ATO has an idiopathic mechanism of action, but has shown beneficial effects in other solid tumors. Across six GBM PDX models a 10-fold difference in sensitivity manifests, indicative of underlying innate sensitivity or resistance to ATO. This resistance has been previously shown to correlate with MNK1 expression. In order to sensitize PDX models innately resistant to ATO we utilized combination therapy with the MNK1 inhibitor AUM001 (AUM Biosciences, Singapore; NCT05462236). In high MNK1 expressing PDX models the combination of ATO + AUM001 induces cytotoxic synergy. Additionally, when narrowly measuring effects on the glioma stem cell population (ELDA), strong synergy was observed in models, including those failing to show such using bulk cell population viability assay. GSEA analysis revealed differential high enrichment in gene sets related to oxidative phosphorylation in the models failing to show synergy. Such criteria could act as exclusionary criteria (futility) for clinical trials by excluding such putative non-responder patients from the treatment population. Synergistic models showed enrichment in gene sets related to epithelial/mesenchymal transition, skeletal and sensory organ development, and regionalization. These signatures could serve to prioritize patient inclusion in clinical trials of ATO in combination with AUM001, enriching the fraction of responsive glioma patients. Such molecular signatures may bring precision medicine in early-stage clinical trials to advance promising new agents and combinations of agents to full FDA approval.

MicroRNA-505-5p/-3p Regulates the Proliferation, Invasion, Apoptosis, and Temozolomide Resistance in Mesenchymal Glioma Stem Cells by Targeting AUF1.

Mesenchymal glioma stem cells (MES-GSCs) are a major subtype of GSCs that reside within glioma tissues and contribute to metastasis, therapy resistance, and glioma recurrence. However, the precise molecular mechanisms governing MES-GSC functions remain elusive. Our findings revealed that expression levels of miR-505-5p/-3p are elevated in MES-GSCs compared with those in proneural (PN)-GSCs, glioma cell lines, and normal brain tissue and that miR-505-5p/-3p expression levels are decreased in differentiated MES-GSCs. We assumed that miR-505-5p/-3p would play distinctive roles in MES-GSCs and performed loss-of-function experiments targeting miR-505-5p/-3p. Knockdown of miR-505-5p/-3p increased proliferation and promoted differentiation in MES-GSCs while suppressing invasion, temozolomide resistance, and enhancing apoptosis in MES-GSCs. Mechanistically, miR-505-5p/-3p directly targets the 3' UTR of AUF1, leading to its repression in MES-GSCs. Notably, AUF1 expression levels were significantly lower in MES-GSCs compared with those in PN-GSCs, glioma cell lines, and normal brain tissues. Co-inhibition of AUF1 expression with miR-505-5p/-3p knockdown ameliorated the observed cellular phenotypes caused by miR-505-5p/-3p knockdown in MES-GSCs. These results suggest that miR-505-5p/-3p exerts MES-GSC-specific roles in regulating proliferation, differentiation, invasion, apoptosis, and temozolomide resistance by repressing AUF1 expression.

Deciphering the topological landscape of glioma using a network theory framework

Glioma stem cells have been recognized as key players in glioma recurrence and therapeutic resistance, presenting a promising target for novel treatments. However, the limited understanding of the role glioma stem cells play in the glioma hierarchy has drawn controversy and hindered research translation into therapies. Despite significant advances in our understanding of gene regulatory networks, the dynamics of these networks and their implications for glioma remain elusive. This study employs a systemic theoretical perspective to integrate experimental knowledge into a core endogenous network model for glioma, thereby elucidating its energy landscape through network dynamics computation. The model identifies two stable states corresponding to astrocytic-like and oligodendrocytic-like tumor cells, connected by a transition state with the feature of high stemness, which serves as one of the energy barriers between astrocytic-like and oligodendrocytic-like states, indicating the instability of glioma stem cells in vivo. We also obtained various stable states further supporting glioma’s multicellular origins and uncovered a group of transition states that could potentially induce tumor heterogeneity and therapeutic resistance. This research proposes that the transition states linking both glioma stable states are central to glioma heterogeneity and therapy resistance. Our approach may contribute to the advancement of glioma therapy by offering a novel perspective on the complex landscape of glioma biology.