Glioblastoma Model Research Articles

NIR-II Photoacoustic Imaging-Guided Chemo-Photothermal Therapy Using PA1094T Combined with Anti-CD47 Antibody: Activating Pyroptosis against Orthotopic Glioblastoma.

Treating glioblastoma (GBM) with single-agent chemotherapy is often ineffective due to inefficient drug delivery and the immunosuppressive tumor microenvironment, which leads to drug resistance. Strategies that activate programmed cell death mechanisms and repolarized tumor-associated macrophages toward an antitumoral M1-like phenotype can help reverse the immunosuppressive tumor microenvironment. In this study, a novel approach using NIR-II (1000-1700nm) photoacoustic imaging (PAI)-guided chemo-photothermal therapy is presented. NIR-II imaging, with its superior tissue penetration and reduced background noise, enables precise tumor targeting. A targeted nano prodrug is developed using poly (lactic-co-glycolic acid) nanoparticles loaded with A1094 dye and temozolomide (TMZ), coupled with an anti-CD47 antibody. This system employs synergistic chemo-photothermal therapy activated by NIR-II light, inducing apoptosis, pyroptosis, and T-cell activation. PAI provides rapid, point-of-care GBM diagnosis, and highlighted the effective targeting of the PA1094T nanoplatform. In a recurrent GBM model, the combination of PA1094T and anti-CD47 antibody significantly enhances cancer cell phagocytosis and effectively remodels the immunosuppressive microenvironment, resulting in better therapeutic outcomes compared to conventional therapies. These results indicate that this NIR-II PAI-guided drug cocktail therapy is a promising strategy for treating GBM, potentially addressing drug resistance and improving treatment efficacy through enhanced targeting and immunomodulation.

Reciprocal links between methionine metabolism, DNA repair and therapy resistance in glioblastoma.

Glioblastoma (GBM) is uniformly lethal due to profound treatment resistance. Altered cellular metabolism is a key mediator of GBM treatment resistance. Uptake of the essential sulfur-containing amino acid methionine is drastically elevated in GBMs compared to normal cells, however, it is not known how this methionine is utilized or whether it relates to GBM treatment resistance. Here, we find that radiation acutely increases the levels of methionine-related metabolites in a variety of treatment-resistant GBM models. Stable isotope tracing studies further revealed that radiation acutely activates methionine to S-adenosyl methionine (SAM) conversion through an active signaling event mediated by the kinases of the DNA damage response. In vivo tumor SAM synthesis increases after radiation, while normal brain SAM production remains unchanged, indicating a tumor- specific metabolic alteration to radiation. Pharmacological and dietary strategies to block methionine to SAM conversion slowed DNA damage response and increased cell death following radiation in vitro. Mechanistically, these effects are due to depletion of DNA repair proteins and are reversed by SAM supplementation. These effects are selective to GBMs lacking the methionine salvage enzyme methylthioadenosine phosphorylase. Pharmacological inhibition of SAM synthesis hindered tumor growth in flank and orthotopic in vivo GBM models when combined with radiation. By contrast, methionine depletion does not reduce tumor SAM levels and fails to radiosensitize intracranial models, indicating depleting SAM, as opposed to simply lowering methionine, is critical for hindering tumor growth in intracranial models of GBM. These results highlight a new signaling link between DNA damage and SAM synthesis and define the metabolic fates of methionine in GBM in vivo . Inhibiting radiation-induced SAM synthesis slows DNA repair and augments radiation efficacy in GBM. Using MAT2A inhibitors to deplete SAM may selectively overcome treatment resistance in GBMs with defective methionine salvage while sparing normal brain.

Real-time volumetric imaging of cells and molecules in deep tissues with Takoyaki ultrasound.

Acoustic contrast agents and reporter genes play a critical role in allowing ultrasound to visualize blood flow, map molecules and track cellular function in opaque living organisms. However, existing ultrasound methods to image acoustic contrast agents predominantly focus on 2D planar imaging, while the biological phenomena of interest unfurl in three dimensions. Here, we introduce a method for efficient, dynamic imaging of contrast agents and reporter genes in 3D using multiplexed matrix array transducers. Our "Takoyaki" pulse sequence uses the simultaneous scanning of multiple focal points to excite contrast agents with sufficient acoustic pressure for nonlinear imaging while efficiently covering 3D space. Through in vitro experiments, we first show that the Takoyaki sequence produces highly sensitive volume images of gas vesicle contrast agents and compare its performance with alternative imaging schemes. We then establish its utility in cellular imaging in vivo by visualizing acoustic reporter gene-expressing tumors in a mouse model of glioblastoma. Finally, we demonstrate real-time volumetric imaging by tracking the dynamics of fluid motion in brain ventricles after intraventricular contrast injection. Takoyaki imaging enables a more comprehensive understanding of biological processes by providing spatiotemporal information in 3D within the constraints of accessible multiplexed matrix array systems.

Adenoviral delivery of the CIITA transgene induces T-cell-mediated killing in glioblastoma organoids.

The immunosuppressive nature of the tumor microenvironment poses a significant challenge to effective immunotherapies against glioblastoma (GB). Boosting the immune response is critical for successful therapy. Here, we adopted a cancer gene therapy approach to induce T-cell-mediated killing of the tumor through increased activation of the immune system. Patient-based three-dimensional (3D) GB models were infected with a replication-deficient adenovirus (AdV) armed with the class II major histocompatibility complex (MHC-II) transactivator (CIITA) gene (Ad-CIITA). Successful induction of surface MHC-II was achieved in infected GB cell lines and primary human GB organoids. Infection with an AdV carrying a mutant form of CIITA with a single amino acid substitution resulted in cytoplasmic accumulation of CIITA without subsequent MHC-II expression. Co-culture of infected tumor cells with either peripheral blood mononuclear cells (PBMCs) or isolated T-cells led to dramatic breakdown of GB organoids. Intriguingly, both wild-type and mutant Ad-CIITA, but not unarmed AdV, triggered immune-mediated tumor cell death in the co-culture system, suggesting an at least partially MHC-II-independent process. We further show that the observed cancer cell killing requires the presence of either CD8+ or CD4+ T-cells and direct contact between GB and immune cells. We did not, however, detect evidence of activation of canonical T-cell-mediated cell death pathways. Although the precise mechanism remains to be determined, these findings highlight the potential of AdV-mediated CIITA delivery to enhance T-cell-mediated immunity against GB.

Sertraline/chloroquine combination therapy to target hypoxic and immunosuppressive serine/glycine synthesis-dependent glioblastomas

The serine/glycine (ser/gly) synthesis pathway branches from glycolysis and is hyperactivated in approximately 30% of cancers. In ~13% of glioblastoma cases, we observed frequent amplifications and rare mutations in the gene encoding the enzyme PSPH, which catalyzes the last step in the synthesis of serine. This urged us to unveil the relevance of PSPH genetic alterations and subsequent ser/gly metabolism deregulation in the pathogenesis of glioblastoma. Primary glioblastoma cells overexpressing PSPH and PSPHV116I showed an increased clonogenic capacity, cell proliferation, and migration, supported by elevated nucleotide synthesis and utilization of reductive NAD(P). We previously identified sertraline as an inhibitor of ser/gly synthesis and explored its efficacy at suboptimal dosages in combination with the clinically pretested chloroquine to target ser/glyhigh glioblastoma models. Interestingly, ser/glyhigh glioblastomas, including PSPHamp and PSPHV116I, displayed selective synergistic inhibition of proliferation in response to combination therapy. PSPH knockdown severely affected ser/glyhigh glioblastoma clonogenicity and proliferation, while simultaneously increasing its sensitivity to chloroquine treatment. Metabolite landscaping revealed that sertraline/chloroquine combination treatment blocks NADH and ATP generation and restricts nucleotide synthesis, thereby inhibiting glioblastoma proliferation. Our previous studies highlight ser/glyhigh cancer cell modulation of its microenvironment at the level of immune suppression. To this end, high PSPH expression predicts poor immune checkpoint therapy responses in glioblastoma patients. Interestingly, we show that PSPH amplifications in glioblastoma facilitate the expression of immune suppressor galectin-1, which can be inhibited by sertraline treatment. Collectively, we revealed that ser/glyhigh glioblastomas are characterized by enhanced clonogenicity, migration, and suppression of the immune system, which could be tackled using combined sertraline/chloroquine treatment, revealing novel therapeutic opportunities for this subgroup of GBM patients.

Tumoroid-On-a-Plate (ToP): Physiologically Relevant Cancer Model Generation and Therapeutic Screening.

Employing three-dimensional (3D) in vitro models, including tumor organoids and spheroids, stands pivotal in enhancing cancer therapy. These models bridge the gap between two-dimensional (2D) cell cultures and complex in vivo environments and offer versatile tools for comprehensive studies into cancer progression, drug responses, and tailored therapies. This study introduces the Tumoroid-on-a-Plate (ToP) device, an innovative ope-surface microfluidic platform designed to create predictive 3D models of solid tumors. The ToP device combines tumor mass, stromal cells, and extracellular matrix (ECM) components, to closely replicate the microenvironment of glioblastoma (GBM) and pancreatic adenocarcinoma (PDAC). Using the advanced ToP model and testing various GBM ECM compositions such as collagen and Rreelin within the model, we can assess how specific elements affect GBM invasiveness. The ToP in vitro model also enables screening chemotherapeutics such as temozolomide and iron-chelators in a single and binary treatment setting on the complex ECM-embedded tumoroids to evaluate their toxicity on GBM and PDAC models viability and apoptosis. Furthermore, co-culturing PDAC tumoroids with human-derived fibroblasts reveals the pro-invasive influence of stromal elements on tumor growth and drug response. This research underscores the value of advanced 3D models like ToP in advancing the understanding of cancer complexity and therapy responses.

CSIG-03. MT-125 INHIBITS NON-MUSCLE MYOSIN IIA AND IIB, SYNERGIZES WITH ONCOGENIC KINASE INHIBITORS, AND PROLONGS SURVIVAL IN GLIOBLASTOMA

Abstract We have shown that members of the non-muscle myosin II (NMII) family of molecular motors are non-redundant drivers of both invasion and proliferation in glioblastoma (GBM), consistent with the canonical roles NMII isoforms play in cell motility and cytokinesis. However, we have also found that NMII also has a non-canonical role by regulating oncogenic signaling pathways relevant to GBM pathogenesis. This unexpected finding has led us to identify and characterize MT-125, a highly specific small molecule inhibitor of the two predominant NMII isoforms in GBM (NMIIA and NMIIB), and we have studied its effects in pre-clinical GBM models. MT-125 has high brain penetrance and retention; an excellent safety profile in rodents and canines with no changes in metabolic or hematologic indices after prolonged administration; is well-tolerated with daily dosing for at least 85 consecutive days; blocks GBM invasion and cytokinesis, consistent with the canonical roles of NMII; and prolongs median survival by 35% as a single agent in murine GBM models. MT-125 increases signaling along both the PDGFR- and MAPK-driven pathways through a mechanism that involves the upregulation of reactive oxygen species, leading to oncogene addiction to these pathways. As a consequence, MT-125 is synthetically lethal when combined with FDA-approved, CNS-permeant inhibitors of PDGFR and mTOR in vitro. Combining MT-125 with sunitinib, a PDGFR inhibitor, or paxalisib, a combined PI3 Kinase/mTOR inhibitor, in vivo doubles median survival in a highly aggressive murine orthotopic GBM model. Furthermore, combining MT-125 with sunitinib produces tumor free remissions of >100 days--2.5-fold greater than median survival of either drug alone—in approximately 40% of mice. Our results provide a powerful rationale for developing NMII targeting strategies to treat cancer and demonstrate that MT-125 has strong clinical potential for the treatment of GBM.

EXTH-12. POTENTIATING THE EFFICACY OF IMMUNE CHECK-POINT INHIBITORS IN GLIOBLASTOMA BY INHIBITION OF CXCL12

Abstract BACKGROUND The tumor immune microenvironment (TIME) in glioblastoma (GBM) is rich in CXCL12, a chemokine known for stimulating angiogenesis. CXCL12 also controls immune cell trafficking and promotes polarization to an immunosuppressive phenotype. We postulated that inhibiting CXCL12 will modulate the immunosuppressive TIME in GBM, thereby augmenting the efficacy of immunotherapy agents like immune checkpoint inhibitors (ICIs). We examined the immunomodulatory and therapeutic efficacy of CXCL12 inhibition, using a small molecule NOX-A12, with and without ICIs in pre-clinical murine GBM models. METHODS B6 immunocompetent mice bearing intracranial (i.c.) or subcutaneous (s.c.) SB28 tumors received either vehicle, NOX-A12, anti-PD1 and anti-CTLA4 (dual ICI) or NOX-A12 + dual ICI (combination). Tumor growth was measured by IVIS imaging and immune cells in brain were analyzed using high dimensional flow cytometry. Survival was analyzed using Kaplan-Meier curves and log rank test. RESULTS Combination treatment resulted in tumor regression and long-term survival in 40% of s.c. tumor bearing mice compared to 10% treated with dual ICI only. Three of 4 mice rechallenged with contralateral s.c. tumor remained tumor free while all naive mice reached endpoint. TIME from treated s.c. tumors revealed increase in CD4 and effector memory CD8 T cells by combination treatment compared to dual ICI. In i.c. GBM tumors, while no survival benefit or growth inhibition was seen, combination treatment induced an early increase in effector memory CD8 T cells and PD-L1+ B cells, and a later increase in MHC-II+ microglia compared to dual ICI. CONCLUSION Inhibition of CXCL12 enabled the increase of effector cytotoxic T cells by ICI, improving survival in the s.c. GBM model, but not in i.c. GBM model. This might be due to differences in CXCL12 effect between extra and intra-CNS tumor or due to robust immune response causing excessive brain edema and death. These will be further explored in ongoing and future studies.

DDEL-17. INTRA-TUMORAL CONVECTION ENHANCED DELIVERY OF DEXAMETHASONE REDUCES INFLAMMATORY SIGNATURES AND EXTENDS SURVIVAL IN GBM MODEL

Abstract Dexamethasone is the most widely utilized drug for glioblastoma (GBM). However, systemic delivery of dexamethasone in GBM patients is associated with significant side effects and increasing doses with worse survival. Convection-enhanced delivery (CED) can deliver high doses of immunotherapy directly into the tumor microenvironment (TME) while avoiding systemic toxicity. Here, we report that high doses of dexamethasone can be delivered locally by CED without side effects and increase survival in a GBM model along with reduced inflammatory myeloid signatures. We investigated CED of dexamethasone treatment on survival(n=40), adverse side effects, and the immunological TME through peripheral analyses, liquid chromatography–mass spectrometry, and histology in a preclinical syngeneic mouse model. Additionally, we assessed the transcriptional responses of human GBM slices and stem-cell-derived human microglia after steroid treatment through bulk and single-cell RNA sequencing. We found that 7-day treatment with CED of dexamethasone produced a significant survival advantage in glioma-bearing mice compared to non-treated control(p=0.03). Systemic dexamethasone achieved low levels of drug in the TME and caused dysregulated blood glucose, blood counts, and peripheral organ weights, while high CED doses avoided these side effects(p< 0.05 each). Steroid treatment of acute GBM slices and lipopolysaccharide-activated microglia in-vitro both reversed inflammatory myeloid signatures associated with poor survival and recurrence on RNA sequencing analyses. We demonstrate the preclinical efficacy of delivering high local doses of dexamethasone in a GBM model through CED and observed prolonged survival along with reduced adverse inflammatory myeloid signatures. No side effects were demonstrated after chronic CED treatment of dexamethasone while systemic delivery achieved poor tumor penetration and caused known hematologic and metabolic side effects supporting the potential to optimize the clinical use of this therapy. CED of dexamethasone provides us a new treatment paradigm to locally control inflammatory signatures in the glioma TME in a controlled fashion.

EXTH-72. INTEGRATED MULTIOMIC ANALYSIS OF ISOGENIC EGFRVIII MODELS OF GBM REVEALS EXPLOITABLE THERAPEUTIC VULNERABILITIES

Abstract Glioblastoma (GBM) is the most common primary malignant brain tumor with an abysmal 15-month median survival. Therefore, novel therapeutic interventions are urgently needed. EGFR is a receptor tyrosine kinase that is mutated in over 50% of GBM and is a logical target for precision oncology approaches. While aberrant EGFR signaling has been successfully targeted in other cancers, early attempts to target EGFR in GBM clinical trials have not been successful. Since these early trials, several studies have revealed that GBM EGFR biology is unique and cannot be generalized from other EGFR-driven neoplasms. To better understand EGFR biology in a GBM-specific context, we have characterized the GBM transcriptome with RNAseq, the epigenome with CUT&RUN, and the kinase proteome with Multiplexed inhibitor beads with Mass Spectrometry (MIB-MS), collectively referred to as ‘multiomics’, after modeling EGFR resistance. An isogenic mouse astrocyte (mAc) model of GBM was genetically engineered to overexpress EGFRvIII (CEv3), the most common EGFR mutation in GBM. Expression of EGFRvIII induces a unique multiomic profile that mediates many hallmark cancer phenotypes including proliferation and stemness. Chronic EGFR resistance was modeled both in vitro and in vivo through continuous exposure to erlotinib or gefitinib, EGFR tyrosine kinase inhibitors (TKI). Additionally, acute EGFR resistance was modeled using a single exposure to EGFR TKI afatinib or neratinib in vitro over a 48-hour time course. Preliminary multiomic characterization of these data has revealed several targets that can be further investigated for therapeutic exploitation. Further investigation into these targets using orthotopic allografts with CEv3 cells shows that combinatorial therapy with neratinib and abemacilib, a CDK inhibitor, significantly (p < 0.001, n= 20 mice per group) extends survival (56 days) compared to neratinib alone (31.5 days). Future integrated multiomic analysis aims to elucidate the synergistic relationship between neratinib and abemacilib.

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.

TMOD-03.BRAFV600E MUTATION INDUCES TWIST1 UPREGULATION ASSOCIATED WITH MIGRATION AND CEREBRAL FLUID DISSEMINATION IN GLIOBLASTOMA

Abstract INTRODUCTION BRAFV600E-mutant glioblastoma (GBM) with epithelioid features frequently show cerebrospinal fluid dissemination (CSFD), which results in poor prognoses. BRAF mutation is worth investigating since BRAF or downstream MEK inhibitors show dramatic response for BRAFV600E-mutant GBM including CSFD. This suggests BRAF mutation could play a role for inducing the clinical manifestation of CSFD found in newly diagnosed and recurrent BRAFV600E-mutant GBM. Because, BRAFV600E-mutant GBM often recur with CSFD, elucidating the mechanism by which BRAFV600E-mutant GBM exhibits CSFD is important to improve prognosis. (Objective) To elucidate the molecular mechanism of CSFD involved in BRAFV600E-mutant GBM. METHODS We introduced the heterozygous BRAFV600E mutation in human induced pluripotent stem cells (iPSC) in the context of GBM genetic alterations, CDKN2A/2B, and PTEN deletion, TERT promoter mutation and with EGFRvIII overexpression. We implanted these iPSC-derived neural progenitor cells into immunodeficient mouse brains to establish a BRAFV600E-mutant GBM model. At the same time, we created a model with the same genetic background without BRAFV600E. We compared their gene expression, pathology, and migration patterns to identify BRAFV600E specific characteristics. RESULTS RNA sequencing revealed BRAFV600E-mutant tumors upregulated migration, and downregulated cell-to-cell adhesion genes. TWIST1 was one of the most upregulated genes, and was validated by qPCR. Concomitant with TWIST1 expression we detected downregulation of E-cadherin. The BRAFV600E GBM model histologically showed a cluster-like invasion phenotype, while non-BRAF tumors showed homogenous invasion with typical GBM pathological findings. Immunohistochemical staining showed relatively high TWIST1 expression in cluster-like tumor cells of BRAFV600E tumors. Upon re-engraftment of primary tumor-derived cells, secondary BRAFV600E tumors presented leptomeningeal dissemination. TWIST1 knockdown resulted in a decrease in the migratory ability of our BRAFV600E model in vitro, and is under investigation in vivo. CONCLUSION BRAV600E mutation induces TWIST1 upregulation, and might facilitate migration and CSFD. This work nominates TWIST1 and its regulated genes as targets for BRAFV600E-mutant GBM with CSFD.

TMET-28. RECIPROCAL LINKS BETWEEN METHIONINE METABOLISM, DNA REPAIR AND THERAPY RESISTANCE IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is uniformly lethal due to profound treatment resistance. Altered cellular metabolism is a key mediator of GBM treatment resistance. Uptake of the essential sulfur-containing amino acid methionine is drastically elevated in GBMs compared to normal cells, however, it is not known how this methionine is utilized or whether it relates to GBM treatment resistance.Here, we find that radiation acutely increases the levels of methionine-related metabolites in a variety of treatment-resistant GBM models. Stable isotope tracing studies further revealed that radiation acutely activates methionine to S-adenosyl methionine (SAM) conversion through an active signaling event mediated by the kinases of the DNA damage response. In vivo tumor SAM synthesis increases after radiation, while normal brain SAM production remains unchanged, indicating a tumor-specific metabolic alteration to radiation. Pharmacological and dietary strategies to block methionine to SAM conversion slowed DNA damage response and increased cell death following radiation in vitro. Mechanistically, these effects are due to depletion of DNA repair proteins and are reversed by SAM supplementation. These effects are selective to GBMs lacking the methionine salvage enzyme methylthioadenosine phosphorylase. Pharmacological inhibition of SAM synthesis hindered tumor growth in flank and orthotopic in vivo GBM models when combined with radiation. By contrast, methionine depletion does not reduce tumor SAM levels and fails to radiosensitize intracranial models, indicating depleting SAM, as opposed to simply lowering methionine, is critical for hindering tumor growth in intracranial models of GBM. These results highlight a new signaling link between DNA damage and SAM synthesis and define the metabolic fates of methionine in GBM in vivo. Inhibiting radiation-induced SAM synthesis slows DNA repair and augments radiation efficacy in GBM. Using MAT2A inhibitors to deplete SAM may selectively overcome treatment resistance in GBMs with defective methionine salvage while sparing normal brain.

CSIG-18. YAP/TAZ TARGET GENES MAY DETERMINE THE SELF-RENEWAL AND SURVIVAL OF GLIOBLASTOMA STEM CELLS

Abstract Glioblastoma, a neoplasm arising from neuro-glial stem/progenitor cells, is the most aggressive and lethal form of brain cancer, with a median survival of 14 months. Despite intensive treatments like surgery, radiation, and chemotherapy, recurrence is inevitable, underscoring the urgent need for new treatment strategies. Progress is limited by an incomplete understanding of the biological mechanisms that drive glioblastoma formation and progression, hindering the development of effective therapies. Key molecular pathways, particularly the receptor tyrosine kinases, such as EGFR, and their effector pathways play a critical role in the initiation and maintenance of glioblastoma. Our lab’s research using patient-derived glioblastoma neurospheres and mouse models has shown that the transcriptional co-activators YAP and TAZ are overexpressed downstream of EGFR activity in EGFR-amplified or mutant glioblastoma tumors. YAP/TAZ proteins interact with the TEAD family DNA-binding proteins to promote expression of transcriptional target genes, including SOX2, C-MYC, and EGFR itself in glioblastoma, creating a positive feedback loop that enhances tumor stem cell renewal, proliferation, and survival. Furthermore, our research has demonstrated that inhibiting YAP/TAZ activity with the drug Verteporfin induces apoptosis and promotes growth arrest in patient-derived EGFR-amplified/mutant glioblastoma models. Our findings highlight YAP/TAZ-TEAD as key mediators of glioblastoma stem cell (GSC) self-renewal and survival. Ongoing experiments aim to identify tumor cell-specific YAP/TAZ-TEAD transcriptional target genes predictive of response to YAP/TAZ inhibition in GSCs within EGFR-amplified glioblastomas. Our research seeks to uncover the epigenetic basis of YAP/TAZ dependencies and to determine which genes and cellular processes are critical for tumor stem cell survival, tumor progression, and sensitivity to YAP/TAZ inhibition. These target genes could serve as biomarkers for predicting therapeutic response to YAP/TAZ inhibitors in glioblastoma.

EXTH-51. ENHANCING GLIOBLASTOMA TREATMENT: ALLOGENEIC CAR-T CELLS OVERCOMES FUNCTIONAL DEFICITS OF PATIENT-DERIVED AUTOLOGOUS PRODUCTS

Abstract Glioblastoma (GBM) is the most common primary brain malignancy in adults, with a dismal prognosis despite an intensive standard of care. Recently, chimeric antigen receptor T-cell (CAR-T) therapy has shown promising outcomes in treating liquid malignancies. However, clinical trials targeting various tumor antigens in GBM failed to show durable clinical benefit. Though this may stem from various tumor-intrinsic immune evasion strategies typical of GBM, there has been little work investigating whether the problem lies in the quality of the CAR-T products treatment itself. Currently, CAR-T cells for clinical studies are produced in an autologous setting, where T-cells are extracted from patients, engineered ex-vivo, and then re-infused. However, peripheral T-cells taken from GBM patients have shown qualitative and functional deficits, which may contribute to suboptimal treatment outcomes. Thus, we aimed to explore whether CAR-Ts generated from GBM patients had any functional deficits in comparison to healthy donors, utilizing our previously validated CD133 CAR-T. In this study, we show pre-treatment exhaustion, poor tumor control, and reduced survival advantage in autologous, patient-derived CD133-targeting CAR-T cell products using an orthotopic xenograft model of human GBM. To address the functional and logistical considerations of autologous therapy, we also sought to generate an “off-the-shelf” allogeneic CD133 CAR-T. Using CRISPR gene editing technology, we generated TCR-knockout CAR-T cells with comparable pre-clinical efficacy to our healthy donor derived autologous models. Ultimately, this work highlights the need to reevaluate autologous CAR-T therapy for GBM and consider allogeneic approaches as biologically-informed therapeutic alternatives.

EPCO-27. CHROMATIN REMODELING REPROGRAMS THE TRANSCRIPTION NETWORK IN GLIOBLASTOMA

Abstract Fundamentally, cancer arises from genetic and epigenetic alterations that interplay to reprogram gene expression networks, leading to unrestrained proliferation. Glioblastoma (GBM) is the most prevalent and aggressive primary brain cancer, with a median survival of approximately one year and a 5-year survival rate of just 5%. The standard care for GBM patients has remained unchanged for decades, underscoring the need for a better understanding of GBM biology to develop more effective therapies. The relatively low genetic mutation burden in GBM highlights the significant role of epigenetic mechanisms in its pathogenesis. Our recent work shows that chromatin remodeling plays pivotal roles in GBM pathogenesis. The BRD8-driven EP400 chromatin remodeling complex maintains GBM by crippling p53-mediated tumor suppression in an unprecedented way through hijacking the histone variant H2AZ at p53 target loci, enforcing a repressive chromatin state that prevents p53-mediated transactivation. Targeting BRD8 in TP53WT GBM, which makes up ~71% of all GBM cases, enhances chromatin accessibility by evicting H2AZ. This restores p53-mediated transactivation of its targets, reestablishes cell cycle arrest, inhibits gliomagenesis, and prolongs survival in xenograft models of TP53WT GBM. Conversely, the NuRD chromatin remodeling complex driven by CHD5 governs the MYC-mediated oncogenic network to prevent gliomagenesis. Mechanistically, CHD5 forms a CHD3-containing but CHD4-independent NuRD complex that directly binds to the MYC promoter and super enhancers. Chd5 loss triggers the remodeling of chromatin, enhances chromatin accessibility at Myc loci, augments Myc expression, significantly impairs NSCs differentiation while promotes proliferation, transformation, and gliomagenesis in vivo. Furthermore, reactivating CHD5 in human GBM xenograft models significantly extends survival. Thus, our findings reveal previously unappreciated epigenetic mechanisms by which GBM cells reprogram both tumor-suppressive and oncogenic transcription networks to facilitate their outgrowth. This sheds new light on our understanding of GBM malignancy and provides promising therapeutic opportunities for treating patients with this devastating malignancy.

IMMU-65. TARGETING AXONAL GUIDANCE DEPENDENCIES IN GLIOBLASTOMA WITH ROBO1 CAR T CELLS

Abstract Resistance to genotoxic therapies and subsequent disease recurrence are hallmarks of aggressive cancers, including glioblastoma (GBM). Here, we uncover functional drivers of post-treatment recurrent GBM using genome scale CRISPR-Cas9 screens accompanied by integrative genomic analysis. Using patient-matched pre- and post-treatment GBM models, our findings uncover large-scale reorganization of functional dependencies at tumor recurrence and implicate protein tyrosine phosphatases 4A2 (PTP4A2) as a novel driver of tumorigenicity in recurrent GBM. Small molecule inhibition and phospho-proteomic analyses reveal a novel PTP4A-ROBO1 signaling axis that modulates tumor invasion, self-renewal and proliferation in recurrent GBM. Since a pan-PTP4A targeting small molecule suffers from poor blood-brain-barrier penetrance, we engineered a novel second generation chimeric antigen receptor (CAR) targeting human ROBO1, a cell surface receptor enriched across GBM specimens. Not only do ROBO1 CAR T cells exhibit a potent and specific anti-tumor effect in vitro, a single dose of ROBO1 CAR T cells doubles median survival in a patient-derived xenograft (PDX) model of recurrent GBM. Expansion of ROBO1 CAR T cells to other invasive brain cancers leads to tumor eradication in ~50% of mice in PDX models of pediatric medulloblastoma and adult lung-to-brain metastases. Together, we provide insights into functional remodeling of GBM at recurrence and present a multi-targetable PTP4A-ROBO1 signaling axis with potential across multiple malignant brain tumors.

EXTH-23. NEW DNA-CROSSLINKING CHEMOTHERAPY IS EFFECTIVE AGAINST POST-TEMOZOLOMIDE MISMATCH REPAIR-DEFICIENT PATIENT-DERIVED HYPERMUTANT GLIOMAS

Abstract The DNA mismatch repair (MMR) pathway triggers glioblastoma (GBM) apoptosis when base mismatches induced by the alkylating agent temozolomide (TMZ) cannot be repaired. GBMs often develop hypermutation and resistance to TMZ through acquired inactivation of DNA MMR enzymes (MSH2, MSH6, MLH1, PMS2). A newly developed therapeutic, KL-50, fluoro-ethylates DNA bases, leading to DNA inter-strand cross-links. This form of DNA damage triggers MMR-independent apoptosis. Thus, KL-50 can target MMR-deficient tumor cells. Previous work showed that KL-50 is effective against GBM cell lines that were rendered TMZ-resistant through shRNA knockdown of MMR genes (PMID 35901163). In those studies, KL-50 was most effective when used in combination with an MGMT enzyme inhibitor, or in MGMT-deficient cells. Here, we extend those findings with a preclinical trial of KL-50 in GBM patient-derived xenografts (PDX), including PDX models of post-TMZ GBM that acquired MMR mutations and hypermutation through serial TMZ exposure (PMID 31852831). Among TMZ-naïve PDX, KL-50 extended survival of engrafted mice by 24 days for GBM6 (partially MGMT-deficient), and by 38 days for GBM12 PDX (fully MGMT-deficient) compared to vehicle (p<0.0001, Log-Rank). Combining KL-50 with 4.0 Gy radiation further extended survival of GBM12 mice (9 days, p=0.02), but not GBM6 mice. With engraftments of GBM6R-m185, a post-TMZ, MMR-deficient derivative of GBM6, 14/14 (100%) of KL-50 treated mice are still alive 60 days post-engraftment compared to 0/13 (0%) of vehicle control mice (median survival 37 days, p<0.0001, experiment ongoing). Complementary in vitro studies showed that GBM6R-m185 cells are more sensitive to KL-50 than TMZ at matched concentrations of 50µM (p=0.001, one-way ANOVA) and 100µM (p=0.047). These data demonstrate the potential of KL-50 in treating post-TMZ MMR-deficient recurrent gliomas.

EXTH-44. ENHANCING THERAPEUTIC APPROACHES IN HIGH-GRADE GLIOMAS: SYNERGISTIC EFFECTS OF LSD1 AND KINASE INHIBITION

Abstract BACKGROUND Glioblastoma (GBM) and Diffuse Intrinsic Pontine Glioma (DIPG) are devastating high-grade gliomas (HGGs) with poor prognoses. Lysine-specific demethylase 1 (LSD1) is a promising therapeutic target and a key regulator in tumor initiation and recurrence. Crosstalk between LSD1 and other signaling pathways, such as the MAPK pathway, frequently hyperactivated in HGG, offers an opportunity to enhance the efficacy of LSD1 inhibitors. This study investigates the combined inhibition of LSD1 and kinase signaling to improve therapeutic outcomes in HGG. METHODS Transcriptional changes following LSD1 knockdown in human GBM cells were analyzed using RNA-seq and GSEA. RPPA and western blotting assessed the impact of LSD1 inhibition on kinase signaling. Pharmacological LSD1 inhibition was tested using NCD38 or bomedemstat in HGG models and normal human astrocytes (NHA). The effects of combining LSD1 inhibitors with kinase inhibitors (osimertinib, afatinib, and ulixertinib) on cell viability were evaluated. Combination treatment was also assessed in orthotopic xenograft models of GBM. RESULTS GSEA revealed LSD1 expression was enriched for gene sets involved in kinase signaling processes. Increased phosphorylated ERK1/2 levels were observed following NCD38 treatment in MDA-GSC17, as shown by RPPA and western blot analyses. The LSD1 inhibitor, NCD38, increased phospho-ERK1/2 levels (72% increase from baseline), while co-treatment with osimertinib mitigated this effect. Combined LSD1 and kinase inhibition (EGFR and ERK inhibition) showed synergistic effects in vitro in GBM and DIPG models but not in the NHA. DISCUSSION Our results demonstrate that kinase activity can be modulated by inhibiting LSD1, potentially leading to a resistant subpopulation. This study underscores the potential of combining LSD1 and kinase inhibition to enhance therapeutic efficacy in HGG, including GBM and DIPG. Future research will focus on elucidating the mechanisms by which LSD1 regulates kinase signaling, aiming to optimize combination therapies.

EXTH-20. EXPLORING EUPHOL’S THERAPEUTIC EFFECT IN ADULT AND PEDIATRIC BRAZILIAN HIGH-GRADE GLIOMAS PRE-CLINICAL MODELS

Abstract High grade gliomas (HGG) are among the most aggressive brain tumors affecting both adults and children. Adult HGG (aHGG) account for almost 25% of all brain tumors in adults, with glioblastoma (GBM) being the predominant subtype. Pediatric HGG (pHGG), though less common, accounts for 3-15% of primary brain tumors in children and exhibits distinct genetic profiles compared to aHGG. Standard treatment for HGG in both adults and pediatric patients includes maximal surgical resection followed by temozolomide (TMZ)-based chemotherapy and radiation therapy. Our group previously reported that euphol, a natural compound derived from the sap of Euphorbiaceae family plants, exerts cytotoxicity effects on glioma cells. In this study, we investigated the therapeutic potential of euphol in primary cell cultures and xenograft models derived from six Brazilian HGG patients, including three adults and three pediatrics. In vitro, euphol exposure significantly reduced cell viability reduction, clonogenic capacity, and migration, while enhancing apoptosis in all six primary cultures. Additionally, euphol sensitized glioma cells to TMZ, resulting in a synergistic cytotoxic effect. In vivo, euphol reduced tumor growth in both GBM and pHGG models, and prolonged the survival of mice in the GBM orthotopic experiment. These findings highlight the promising therapeutic potential of euphol for treating high-grade gliomas in both adult and pediatric populations.