bluuebunny/crossref_metadata_2025 huggingface dataset:
NCOG-33. GLIOBLASTOMA AND PARKINSONISM, IS DEPLETED DOPAMINE A PROTECTIVE SURVIVAL FACTOR? A CASE SERIES OF 3 GBM PATIENTS
Authors: Carrillo Jose , Wagle Naveed , Sharma Akanksha , Nguyen Minhdan , Truong Judy , Mauritson Emmaline , Singh Iryna , Juarez Tiffany , Kesari Santosh
Published: November 2023

Glioblastoma multiforme (GBM) is the most common malignant brain tumor, and carries a grave prognosis with a median survival of 15 months. The pathophysiologic hallmark of Parkinson’s disease is neuronal loss of dopamine producing cells in the substantia nigra. Dopamine has been shown to modify cell-cycle status in neural progenitor cells. Dopamine agonism is adequate to increase progenitor proliferation in the subventricular zone (SVZ). Dopamine receptor antagonists have antiproliferative effects. A few clinical trials use selective dopamine receptor D2 (DRD2) inhibitors. Parkinsonism can have organic etiologies like stroke or neoplasm, while idiopathic Parkinsonism is known as Parkinson’s disease. While uncommon to have dual diagnoses, we identified three patients with both GBM and Parkinsonism, all were IDH wildtype, 2 patients were MGMT methylated, mean age was 67 years. Our 3 patients have a combined median PFS of 23.4 months, mean PFS of 25.4 months, which compares favorably to historical control median PFS of 6.9 months. Our 3 patients with dual diagnosis have a median OS of 47.3 months, and mean OS of 43.7 months, which compares favorably to historical median OS of 14.6 months. One might expect a patient with a neurodegenerative disease like Parkinson’s disease and a highly malignant tumor like GBM to have an extremely poor prognosis, yet our patient cohort reveals the opposite. The improved overall survival in GBM patients with Parkinsonism supports further clinical research into selective dopamine receptor inhibitors.
EXTH-01. STEM CELL THERAPY FOR LEPTOMENINGEAL DISEASE
Authors: Kitamura Yohei , Kanaya Nobuhiko , Moleirinho Susana , Tamura Ryota , Ueda Ryo , Sasaki Hikaru , Toda Masahiro , Shah Khalid
Published: November 2023

Leptomeningeal disease (LMD), known as carcinomatous meningitis, occurs in approximately 5% of all cancer patients. LMD presents an extremely poor prognosis, with an average life expectancy of 4-6 weeks. Although, whole brain/spinal cord irradiation and intrathecal injection of anticancer drugs are sometimes performed, the effects are limited, so new therapeutic strategies for LMD are urgently needed. We have reported that treatment using engineered stem cells (SCs) may be effective treatment option against LMD of breast cancer, lung cancer, and malignant melanoma, which are types of cancer that tends to metastasize to the central nervous system. Specifically, we reported that engineered allogeneic SCs simultaneously targeting epidermal growth factor receptor (EGFR) and death receptor (DR) has efficacy for LMD of triple negative breast cancer. Also, we reported that twin SCs releasing oncolytic herpes virus, granulocyte-macrophage colony-stimulating factor (GM-CSF), and single-chain variable region fragments (scFv) against PD-1 has efficacy in LMD of malignant melanoma. The greatest advantage of SC based therapeutic strategy for LMD is summarized by these facts: (1) LMD is a condition in which cancer cells spread within a single closed space through cerebrospinal fluid, and the SCs characteristic of homing to tumor cells has an advantage; (2) Repetitive intrathecal injection of drugs for patients is an invasive procedure, but SCs can continuously secrete/release therapeutic agents into the spinal cavity after a single injection. We believe that stem cell therapy has the potential to become a new therapeutic option for LMD in the future.
EXTH-02. A TERT-SPECIFIC TCR-LIKE ANTIBODY CAR T CELL APPROACH FACILITATES KILLING OF GLIOBLASTOMA
Authors: Zhu Eric , Gladney Susan , Ritchey Julie , Livingstone Alexandra , O'Neal Julie , Johanns Tanner
Published: November 2023

Glioblastoma (GBM) is a difficult form of brain cancer to treat as GBM cells utilize various strategies to evade host immune responses. Prior studies have shown autologous cytotoxic T cells (CTL) modified to express chimeric antigen receptors (CAR) facilitating host CTL responses against cancers. This study investigates the ability of T-cell receptor (TCR)-like antibody clones against TERT to facilitate specific CAR T cell mediated killing of GBM. Lentiviral vectors were created for five TCR-like antibody CAR constructs recognizing an HLA-A*02:01-restricted TERT-derived antigen. Nur77-GFP Jurkat reporter cell lines and 3 healthy donor-derived CTLs transduced with these vectors were used in flow-based and in vitro killing assays to assess specificity and functionality. K562 cells that did, and did not, express HLA-A*02:01 were targets in the flow-based assay. Flow cytometry captured activation of transduced Jurkat cells via GFP and CD69 upregulation. Three luciferase-transduced GBM cell lines were targets in the cytotoxic killing assay and visualized via bioluminescence imaging (BLI). Increased GFP and CD69 expression was seen on Jurkat cells transduced with clones 3A12 and 3H2 only when co-cultured with TERT antigen and A*02 positive K562 suggesting specific recognition of the TERT:HLA complex. Furthermore, healthy donor-derived CTL transduced with 3A12 and 3H2 CAR constructs demonstrated in vitro killing of tumor cells by BLI. Interestingly, the 3H2 construct only showed cytotoxicity in the presence of exogenous TERT antigen. However, when the heavy and light chains of the 3H2 clone were flipped in the lentiviral construct (3H2F), specificity to the TERT:HLA complex was preserved but the CAR T cells now demonstrated cytotoxicity of GBM without needing exogeneous peptide. TCR-like antibody clones 3A12 and 3H2F specifically recognize an HLA-A*02:01-restricted TERT-derived antigen, facilitating effective in vitro cytotoxicity of GBM cells. Ongoing experiments will evaluate the in vivo activity of these TERT-specific CAR constructs and assess potential off-target toxicity.
EXTH-04. CYTOSTATIC HYPOTHERMIA AGAINST GLIOBLASTOMA AND THE DEVELOPMENT OF A NEURAL IMPLANT FOR RODENTS AND SWINE
Authors: Enam Syed Faaiz , Chen Reed , Barwari Mahabad , Dobariya Aksharkumar , Patel Ankur , Patel Toral , Bellamkonda Ravi
Published: November 2023

Survival rates for patients with glioblastoma (GBM) remain discouraging due to the near-impossibility of eradicating all tumor cells, resulting in frequent recurrence. However, studies have shown that recurrence is predominantly localized (80% of cases) and that survival outcomes are improved with supra-total resection. Consequently, non-destructive local therapies hold promise for preventing GBM recurrence. We have demonstrated that non-freezing, 'cytostatic' hypothermia is a unique and effective approach against GBM. Unlike ablative cryogenic hypothermia, which kills both neoplastic and healthy infiltrated tissue, this method safely and effectively inhibits GBM growth. Through extensive investigations, we have previously identified a cytostatic window of 20–25°C and examined its impact on multiple pathways. By implementing hypothermia, we successfully inhibited GBM growth, doubling median survival in two rodent models. Notably, all rats subjected to cytostatic hypothermia survived the study period. To further advance towards translation, we conducted computer simulations to demonstrate the feasibility of a scaled and implantable system. Subsequently, we designed and fabricated a multiprobe array capable of uniformly extracting heat from a small volume of brain tissue in swine. This heat would then be transferred to the dorsal flank through an innovative artificial internal circulation system (AICS). The system underwent iterative testing in vitro and validation in vivo using live, anesthetized swine. Unlike targeted therapeutics that often show promise in preclinical models but fail in clinical trials, cytostatic hypothermia capitalizes on fundamental physics that broadly influence biology. It has the potential to reduce brain tumor recurrence, limit tumor evolution, and simultaneously offer time to patients as other cytotoxic therapies are developed.
EXTH-08. IMMUNOTHERAPEUTIC TARGETING OF FIBROBLAST ACTIVATION PROTEIN (FAP) IN TREATMENT REFRACTORY GLIOBLASTOMA USING NOVEL CAR-T CELL THERAPY
Authors: Tatari Nazanin , Hedad Hayget Mohamed Seid , Zingg Andreas , Bartoszek Ewelina M , Tschan Viviane J , Schenker Timo , Ritz Marie-Françoise , Miletic Petar , Venugopal Chitra , Etter Manina M , Singh Sheila K , Läubli Heinz , Hutter Gregor
Published: November 2023

Glioblastoma (GBM), the most common malignant primary adult brain tumor, is characterized by extensive cellular and genetic heterogeneity. The standard of care therapy and clinical trials have not been successful in improving patients’ survival which underscores an urgent need for developing new effective therapies. In silico and in vitro analysis have revealed high level of Fibroblast Activation Protein (FAP) expression in GBM tissue by multiple cell populations including endothelial cells, fibroblasts, stromal and tumor cells, but not normal brain tissue, making FAP a safe and suitable therapeutic target. As chimeric antigen receptor (CAR) expressing T cells have shown promising results against multiple cancers, we aimed to target FAP in GBM by FAP specific CAR-T cells generated using a novel GMP-ready FAP-CAR construct produced based on a virus-free CAR gene transfer using advanced sleeping beauty transposon technology and pre-clinically test their anti-tumor efficacy. Our data revealed that co-culturing CAR-T cells with FAP+ GBM cells (U87) resulted in increased surface expression of T-cell activation markers. Moreover, FAP CAR-T mediated cytotoxicity against FAP+ GBM cells was also observed in the co-culture of GBM and CAR-T cells. For further validation, the effect of FAP CAR-T cells will be tested on patient-derived GBM organoids. The in vivo efficacy of FAP CAR-T cells is currently under investigation and we anticipate that the treatment of GBM tumor-bearing mice with FAP CAR-Ts results in extended survival and tumor burden reduction. These rigorously obtained data show high level of efficacy for FAP-specific CAR-T cells against GBM suggesting that clinical development of this therapeutic modality can provide a novel therapeutic strategy for GBM patients. Therefore, upon successful completion of the preclinical study, we plan to conduct a phase I dose-escalation trial of autologous FAP-CAR T cells for patients with recurrent Glioblastoma.
EXTH-14. MRNA-LOADED EXOSOMES FOR TARGETED GLIOBLASTOMA IMMUNOTHERAPY
Authors: Dong Shiyan , Antony Abin , Wang Yifan , Kim Betty , Jiang Wen
Published: November 2023

Recent successes of mRNA therapeutics against pathogenic infections have increased interest in their investigations for other human diseases including cancer. However, precisely delivering the genetic cargo to cells and tissues of interest remains challenging. Extracellular vesicles such as exosomes have gained increased interest recently as potential nucleic acid and drug carriers given their favorable immunogenic profile, long circulatory half-life, and ability to cross biological barriers such as blood-brain barrier. Here, we show an adaptive strategy that enables the docking of different targeting ligands onto the surface of mRNA-loaded exosomes. This was achieved using a microfluidic electroporation approach where a combination of nano- and milli-second pulse electroporations produced a large quantity of IFN-γ mRNA-loaded exosomes with CD64 overexpressed on their surface. The CD64 molecule serves as an adaptor to dock targeting ligands, such as anti-CD71 and anti-programmed cell death-ligand 1 (PD-L1) antibodies. The resulting immunogenic exosomes (imExo) selectively targeted glioblastoma cells and generated potent antitumor activities in vivo, including against tumors intrinsically resistant to immunotherapy. Together, these results provide a new adaptive approach to engineering mRNA-loaded exosomes with targeting functionality and pave the way for their adoption in cancer immunotherapy applications.
EXTH-17. TARGETING CTPS1 AND NUCLEOTIDE BIOSYNTHESIS PATHWAYS AS A NOVEL THERAPEUTIC APPROACH IN MYC-AMPLIFIED MEDULLOBLASTOMA
Authors: Hathaway Matthew , Jagana Hawa , Meechan Michael , Rajendran Ashmitha , Slusher Barbara , Evans II Myron
Published: November 2023

MYC-amplified medulloblastoma (MB) tumors are the deadliest of all subgroups with a < 50% 5-year overall survival rate. High risk patients receive large doses of chemo- and radio- therapy (CT/RT) with most survivors experiencing debilitating neurological and cognitive sequelae making the identification of novel targets of the utmost importance. Recent whole-genome CRISPR screening identified the enzyme CTPS1 and other components of the de novo pyrimidine metabolism cascade as a possible cancer-specific vulnerability in MYC-amplified MB. Our study tested targeted inhibitors of this pathway to identify rational combinations for translation to pre-clinical models. A stable of treatment-naïve, patient-derived models of MYC-amplified MB were used and treated with various de novo pyrimidine synthesis inhibitors alone and in novel combinations. Analysis of proliferation, cell survival, and downstream signaling were performed both in vitro and in vivo. Inhibition of CTPS1 led to a significant decrease in cell proliferation and alterations in cell cycle with minimal increases in cell death, suggest a cytostatic mechanism. Previous work has shown de novo pathway inhibition results in reduction of the dNTP pool, leading to activation of the DNA damage response (DDR), seizing the cell cycle. Interestingly, the DDR inhibitor prexasertib (CHKi) robustly targets MYC-amplified MB tumors in pre-clinical models. Combining CHKi and CTPS1 inhibitors yields significant tumor cytotoxicity both in vitro and in vivo. We further demonstrated robust synergy of CHKi with other clinically relevant de novo pyrimidine metabolism inhibitors. The results of this investigation confirm CTPS1 and the de novo pyrimidine metabolism pathway as targets in MYC-amplified MB and identifies a novel rational combination for treatment in patients.
EXTH-18. DEVELOPMENT OF A TRANSGENIC MOUSE MODEL ENABLING COMPARATIVE ANALYSIS OF CHIMERIC ANTIGEN RECEPTOR (CAR)-EXPRESSING IMMUNE CELL POPULATIONS FOR CANCER IMMUNOTHERAPY
Authors: Hatae Ryusuke , Watchmaker Payal , Yamamichi Akane , Kyewalabye Keith , Okada Kaori , Phyu Su , Goretsky Yitzhar , Chen Tiffany , Chuntova Pavlina , Terabe Masaki , Okada Hideho
Published: November 2023

We reported the creation of C57BL/6J-background transgenic (Tg) mice carrying the anti-epidermal growth factor receptor (EGFR)vIII-CAR with CD3z-CD28-4-1BB downstream of a Lox-Stop-Lox cassette in the Rosa26 locus. Breeding these mice with CD4-Cre Tg allows rigorous and reproducible evaluation of CAR-T cell therapy in syngeneic models (Chuntova et al. Neuro-Onc 2022). As the extension of the approach, we crossed the EGFRvIII-CAR Tg mice with mice carrying Vav-Cre, which allowed the expression of anti-EGFRvIII CAR in all hematopoietic cells. In particular, we evaluated CAR-T, CAR-NKT, CAR-NK cells, and CAR-macrophages. CAR-NK cells displayed a robust antigen-specific cytolytic activity against EGFRvIII-expressing SB28 cells, comparable to CAR-T cells in vitro and mice bearing intracerebral EGFRvIII-expressing SB28 gliomas. Remarkably, CAR-NKT cells produced higher levels of inflammatory cytokines when co-cultured with EGFRvIII-expressing SB28 cells, such as IFN-g, Perforin, IL4, and IL17. On the other hand, despite their in vitro cytotoxic effects against SB28 cells, CAR-NK cells failed to mediate a therapeutic response in mice bearing intracerebral glioma. Future studies will be directed at improving the in vivo persistence of CAR-NK cells using engineered cytokines, such as IL-2 superkine. In addition, we extensively investigated CAR-macrophages in vitro and in vivo. Unfortunately, we did not observe significant CAR-specific anti-tumor effects. Consequently, we modified the intracellular domain of CAR from CD3z-CD28-4-1BB to CD3z alone. This alteration led to increased TNF-α production through CAR signaling. This transgenic mouse model offers rigorous and reproducible evaluations of CAR-expressing hematopoietic cells. Furthermore, the model provides a platform for developing combination therapies, such as CAR-T plus CAR-NKT or CAR-T plus CAR-NK cells, with potential implications for enhanced cancer treatment strategies.
EXTH-25. IN VIVO TWO-PHOTON TUMOR IMAGING IN AN ORTHOTOPIC MEDULLOBLASTOMA MOUSE MODEL
Authors: Teske Nico , Kutlu Nayad N , Herold Julika J , Dengler Laura , Eberle Constantin , Nikolaishvili Elene , Karschnia Philipp , Blobner Jens , Müller Katharina , Langer Sigrid , Buschinger Vincenz , Warmuth Linda , Busch Dirk H , Buchholz Veit , Thon Niklas , Tonn Joerg-Christian , Feuchtinger Tobias , von Baumgarten Louisa
Published: November 2023

BACKGROUND

Medulloblastoma represents the most common primary brain malignancy in pediatric patients and is associated with neuro-cognitive impairment and frequent relapses after aggressive multimodal therapy. Adoptive immunotherapies such as CAR T-cell therapy are currently being investigated, showing promising results in preclinical models but lacking efficacy in first-in-human trials. Refined in vivo models to investigate reasons for treatment failures are therefore urgently needed.

METHODS

We developed a xenogeneic orthotopic medulloblastoma (MB) model in mice by combining a chronic cerebellar window with repetitive intravital two-photon laser scanning microscopy. Red tdTomato-fluorescent DAOY (SHH MB) tumor cells expressing B7-H3 were implanted intracranially into the cerebellum of immunodeficient mice (n = 7), and tumor cell formation was followed by in vivo-microscopy. Intravenous fluorescein isothiocyanate (FITC)-dextran was used for intravascular plasma staining. B7-H3-directed CAR T-cells were injected into the adjacent brain parenchyma.

RESULTS

Chronic cranial window implantation was well tolerated and allowed repetitive visualization of the mouse cerebellum. After intracranial tumor cell implantation, continuous tumor growth could be evaluated with epifluorescence as well as 2-photon laser scanning microscopy. Tumor formation was identified as early as day 5 in every mouse and could continuously be followed up to 35 days until mice succumbed due to tumor burden. Window quality and fluorescence intensity remained high until the end of the experiments. Studies on GFP-expressing CAR T-cell injection directed against B7-H3 are currently ongoing.

CONCLUSIONS

We herein establish a robust orthotopic medulloblastoma mouse model that allows repetitive in vivo tracking of fluorescence-expressing tumor cells, CAR T-cells and blood vessels over weeks. Such models may be used to study medulloblastoma tumor growth under the influence of different immunotherapies and reveal interactions between tumor cells and CAR T-cell therapies on a single-cell level.
EXTH-29. INTERNEURONS THAT BITE, A CELLULAR THERAPY FOR HIGH GRADE GLIOMA
Authors: Brosius Stephanie , Manley William , Anderson Stewart , De Raedt Thomas
Published: November 2023

Glioblastoma has a poor prognosis with limited therapeutic options. Cellular therapies, like CAR-T therapy, have unfortunately shown limited efficacy for brain tumors, due to the lack of unique tumor antigens, the downregulation of targeted antigens, and the immune suppressive tumor microenvironment (TME). To circumvent these brain tumor specific obstacles, we developed a cellular immunotherapy delivery system, derived from post-mitotic Migratory Cortical Inhibitory Interneuron Precursors (MCIPs), that induces a cytotoxic tumor response in glioblastoma independent of unique tumor antigens. During fetal brain development, MCIPs are chemoattracted to migrate long distances from their subcortical origins into the cerebral cortex. The MCIP chemoattractant CXCL12 activates CXCR4 to induce this migration. Intriguingly, CXCL12 is secreted by glioblastoma, suggesting they chemoattract MCIPs. Excitingly, our in vitro and in vivo data indeed show that MCIPs robustly migrate to the majority of glioblastoma cell lines evaluated. Crucially, this migration is independent of unique tumor antigens and the TME and provides us with the opportunity to use MCIPs as a delivery vector for cytotoxic agents. In one approach, we successfully eliminated EGFR expressing glioblastoma in vivo by modifying MCIPs to secrete EGFR Bispecific T-cell Engagers (BiTE). BiTE engagement with a tumor cell results in T-cell activation and elimination of the engaged tumor cell. Importantly, the T-cells have no innate recognition of the tumor cell, this is provided by the BiTE. Local EGFR BiTE secretion has limited systemic toxicity, thereby making EGFR a viable non-unique target. MCIPs can be equipped with a range of agents that eliminate glioblastoma. As a therapy, we envisage injecting modified MCIPs, differentiated from patient or donor “off the shelf” induced pluripotent stem cells, into the margin of the surgical cavity to “clean-up” tumor cells remaining post-surgery. An interneuron clinical trial for epilepsy (NCT05135091) is ongoing, highlighting the translational potential of our MCIP cellular therapy.
EXTH-45. DEVELOPMENT OF COMBINATION THERAPIES OF KRASG12C INHIBITOR ADAGRASIB IN PRECLINICAL MODELS OF BRAIN METASTASIS
Authors: Torrini Consuelo , Migliarese Christian , Summers Elizabeth , Demir Fatma Turna , Ijad Nazanin , Nayyar Naema , Yamazawa Erika , De Sauvage Magali , Brastianos Priscilla , Wakimoto Hiroaki
Published: November 2023

BACKGROUND

The highly selective KRASG12C inhibitor adagrasib (MRTX849) is FDA-approved for KRASG12C+ advanced non-small cell lung cancer (NSCLC). We recently provided preclinical evidence of MRTX849 brain penetration and activity, allowing MRTX849 inclusion in a national genomically-guided multicenter clinical trial (NCT03994796) for patients with brain metastasis (BM). However, multiple genetic alterations such as CDKN2A/B deletions, a common driver of NSCLC BM, can limit MRTX849 efficacy. Furthermore, although MRTX849 can sensitize extracranial tumors to immune checkpoint inhibitors (ICI), its intracranial immune benefit is unknown. Our objective was to improve MRTX849 monotherapy, evaluating combination therapies with CDK4/6 inhibitor abemaciclib or anti-PD-1 in preclinical models of BM.

METHODS

We tested in vitro sensitivity of MRTX849 and abemaciclib, using KRASG12C+, CDKN2A-deleted human lung cancer cell lines SW1573 and H2122. The efficacy of a 5-week treatment of MRTX849 and abemaciclib was evaluated in an intracranial NSCLC BM model in nude mice. Additionally, we utilized a syngeneic murine KrasG12C colorectal cancer BM model (CT26G12C) to test different MRTX849 dosages and assess its therapeutic effects in combination with anti-PD-1 antibody.

RESULTS

MRTX849 and abemaciclib demonstrated mild synergy in reducing cell viability in vitro. Combination treatment of MRTX849 and abemaciclib significantly improved both survival and intracranial tumor control relative to controls in SW1573 NSCLC BM model. In a syngeneic colorectal BM model, we identified benefit of a 2-week MRTX849 single treatment at 100 mg/kg (BID) in controlling brain tumor growth. In a dual subcutaneous intracranial BM model with CT26G12C, MRTX849 alone and combination therapy mediated complete regression of extracranial tumors. Although monotherapies reduced intracranial tumor growth, MRTX849 combination with anti-PD-1 resulted in a greater benefit. Both MRTX849 monotherapy and the combination therapy extended animal survival.

CONCLUSIONS

MRTX849 combination therapies with abemaciclib or anti-PD-1 demonstrate intracranial activity, inhibiting BM progression and prolonging survival, in preclinical models of KRASG12C+ BM.
EXTH-50. ACUTE AND LONG-TERM RESPONSES TO QUISINOSTAT TREATMENT IN PDX MODELS OF GLIOBLASTOMA
Authors: Orndorff Keely , McNamara James , LoCascio Costanza , White Connor , Stavnichuk Mariya , Shaik Kamal , Margaryan Tigran , Tovmasyan Artak , Sanai Nader , Mehta Shwetal
Published: November 2023

BACKGROUND

Histone deacetylase inhibitors (HDACi) promote reactive oxygen species production, result in DNA damage, and trigger cell fate changes. Quisinostat is a Class I-II selective HDAC inhibitor with high specificity for HDAC1. Here, we evaluated acute and long-term quisinostat treated glioblastoma (GBM) PDX tissues and glioma stem cells for increase in reactive oxygen species and cell fate change.

METHODS

GBM PDX models (n = 6) and multiple PDX-derived glioma stem cells were treated with quisinostat, radiation, or a combination of quisinostat and radiation. Tissue and cells were processed via LC-MS/MS, RNA-seq, qRT-PCR, immunoblotting, and immunocytochemistry assays to examine increase in cellular reactive oxygen species, oxidative stress biomarkers, and markers of stemness and differentiation.

RESULTS

RNA sequencing of tumor tissue revealed that chronic co-treatment with quisinostat and radiation led to increased cellular expression of oxidative stress- and neuronal-related genes compared to control group. Immunoblotting confirmed upregulation of neuronal signaling proteins in the quisinostat treated tumor tissue in vivo. qRT-PCR and LC-MS/MS analysis demonstrated an increase in oxidative stress biomarkers following acute quisinostat treatment in vitro.

CONCLUSIONS

Treatment with quisinostat increased the expression of genes involved in promoting oxidative stress and neuronal signaling in vivo. LC-MS/MS, qRT-PCR, immunoblotting and immunocytochemistry assays confirmed increase in oxidative stress and neuronal markers upon acute treatment in vitro. Cancer cells often express higher levels of reactive oxygen species compared to normal tissue, and oxidative stress is linked to greater survival and proliferation of cancer cells. However, there is much interest in increasing oxidative stress in glioma cells to induce selective cell death in cancer cells. Ongoing studies are aimed at evaluating the significance of these cellular and molecular changes resulting from quisinostat treatment and whether the quisinostat-induced oxidative stress and cell fate changes can be exploited for future combination therapy for GBM.
EXTH-52. LONG-ACTING RECOMBINANT INTERLEUKIN-7, NT-I7, IMPROVES SURVIVAL FOLLOWING ONCOLYTIC ZIKA VIRUS TREATMENT IN THE SB28 IMMUNOSUPPRESSIVE AND TREATMENT-RESISTANT MURINE GLIOMA MODEL
Authors: Li Yuping , Hu Tong , Kesarwani Ashwani , Costa Amanda De Andrade , Illindala Ritvik , Wolfarth Alexandra , Ferrando-Martinez Sara , Yang Se Hwan , Lee Byung Ha , Diamond Michael S , Chheda Milan
Published: November 2023

Glioblastoma (GBM) does not respond to immunotherapy for several reasons, including limited cancer-specific neoantigens and poor T-cell infiltration. The SB28 murine glioma model recapitulates these aspects of human GBM. Zika virus (ZIKV) treatment of GL261 and CT2A-bearing mice resulted in significant survival improvement through multiple mechanisms, including direct tumor killing and induction of a local CD8+ T-cell anti-tumor response, but ZIKV is less effective against SB28 tumors. Previously, we published that a long-acting human IL-7, rhIL-7-hyFc (NT-I7, efineptakin alfa; NeoImmuneTech Inc.), increases T-cell abundance and improves tumor infiltration. Therefore, we hypothesize that combining NT-I7 with oncolytic ZIKV therapy will enhance the anti-tumor response and improve survival in mice bearing SB28 syngeneic tumors. 1×103 SB28 tumor cells were intracranially implanted in C57BL/6J mice. Mice were treated with NT-I7 on post tumor implantation day 7 and 10, followed by intratumoral ZIKV on day 14. Tumor burden was assessed using bioluminescence imaging; mice were monitored for survival and bled weekly to assess the systemic T-cell response to NT-I7. Subcutaneous injection of NT-I7 significantly boosted systemic T-cell abundance compared to control (5,623 cells/uL vs. 136 cells/uL, p < 0.05), particularly cytotoxic CD8+ T-cells (4,776 cells/uL vs. 47 cells/uL, p < 0.05). This increased abundance of CD8+ T-cells peaked at 7 days after initial treatment and persisted for 21 days. NT-I7 monotherapy did not confer any survival benefit. However, the combination of NT-I7 and ZIKV treatment resulted in significant improvement in survival compared to NT-I7 or ZIKV treatment alone (p < 0.05). Treatment with NT-I7 primes the immune system by expanding the circulating CD8+ T-cell population by 100-fold. Timing the oncolytic ZIKV injection with the peak in peripheral CD8+ T-cells greatly improved survival in the immunotherapy resistant SB28 glioma model. These data suggest a role for NT-I7 as an adjunct to various T-cell mediated treatment strategies for GBM.
EXTH-57. THE IGF-2 SIGNALING PATHWAY IS A PROMISING MENINGIOMA TREATMENT TARGET
Authors: Uhlmann Erik , Rabinovsky Rosalia , Mackel Charles , Deforzh Evgeny , Vega Rafael , Varma Hemant , Krichevsky Anna
Published: November 2023

Meningioma is the most common primary central nervous system tumor often causing serious complications while there is no effective medical treatment. We developed a tumor-derived primary culture model featuring tumor cells with retained PDG2S lineage marker as well as non-arachnoid cells mimicking the tumor cell microenvironment. We used this model to test the relevance of miRNA differentially expressed in meningioma tumor samples identified by small RNA sequencing. We found that in all meningiomas studied, there was activation of the IGF2/miR-483 locus with high expression of both miR-483-5p and IGF2. Inhibition of miR-483-5p reduced the growth of cultured meningioma cells, whereas a miR-483 mimic increased cell proliferation. Anti-IGF2 neutralizing antibodies reduced meningioma cell proliferation. Small molecule tyrosine kinase inhibitor blockade of the IGF2 receptor (IGF1R) resulted in rapid loss of viability of cultured meningioma tumor-derived cells. Autocrine IGF2 feed-back appears to be obligatory for meningioma tumor cell survival and growth. Inhibition of this circuit with IGF1R small molecule tyrosine kinase inhibitors or receptor-binding antibodies as well as anti-IGF2 neutralizing antibodies may have therapeutic potential for meningioma.
EXTH-62. COMBINATIONAL TREATMENT OF G-QUADRUPLEX STABILIZER CX-5461 AND IONIZING RADIATION POTENTIATES SELECTIVE LETHALITY IN PRECLINICAL ATRX-DEFICIENT GLIOMA MODELS
Authors: Dharmaiah Sharvari , Malgulwar Prit Benny , Johnson William E , Chen Brandon A , Farooqi Ahsan S , Huse Jason
Published: November 2023

Mutations in α-thalassaemia/mental retardation X-linked (ATRX) are a critical molecular marker for fatal high-grade glioma (HGG). Standard of care has remained stagnant in the last 30 years, with no therapies approved specifically for ATRX-deficient glioma. ATRX mutations give rise to abnormal DNA secondary G-quadruplex (G4) structures at GC-rich regions of the genome, altering genome-wide accessibility of chromatin and enhancing DNA damage. We aim to use G4 stabilizers, a class of novel small molecule compounds that selectively bind to and stabilize G4s, to enhance DNA damage and induce cell death in ATRX-deficient glioma. Using patient-derived glioma stem cells (GSCs) and the G4 stabilizing compound CX-5461 (Senhwa Biosciences), we evaluated the efficacy of G4 stabilization as a single agent and in combination with ionizing radiation (IR). As IR is standard of care and DNA-damaging, it may hold strong potential to synergize with CX-5461. Here, we report that ATRX-deficient GSCs demonstrate dose-sensitive lethality to CX-5461, compared to controls. Mechanistic studies reveal that CX-5461 activates p53-independent apoptosis, exhibits G2/M arrest in ATRX-deficient GSCs, and upregulates G4s (BG4), replication stress (ATR, RPA32) and DNA damage (ATM, γH2AX) pathways specifically in ATRX-deficient GSCs. These functional findings are corroborated by ATRX-deficient and -intact GSC flank xenograft mouse models, which were separated into the following treatment groups: 1) vehicle, 2) CX-5461 alone, 3) IR alone, 4) CX-5461 and IR (n = 20/group). Excitingly, we demonstrate that combinational treatment leads to profound tumor growth delay exclusively in ATRX-deficient flank tumors compared to vehicle (p = 0.0004), CX-5461 alone (p = 0.0001), or IR alone (p = 0.0002) groups. Multiplex immunohistochemistry of CX-5461 alone and the combinational treatment tumors shows enhanced induction of G4s, replication stress, and DNA damage, recapitulating in vitro findings. Taken together, our work defines mechanisms of action and efficacy for a novel therapeutic strategy for pre-clinical ATRX-deficient HGG, with strong implications for clinical translation.
EXTH-70. UTILIZING ONCOLYTIC VIROTHERAPY AS AN APPROACH TO ENHANCE NEOANTIGEN SPECIFIC IMMUNE RESPONSES IN GLIOBLASTOMA
Authors: Piranlioglu Raziye , Ahn Ryujin , Islam Anan , Nowicki Michal , Ling Alexander , Soni Himanshu , Ahuja Ved V , Goins William F , Irvine Darrell J , White Forest M , Nakashima Hiroshi , Antonio Chiocca E
Published: November 2023

Despite the success in treating many solid cancers, immunotherapy has not shown significant efficacy in patients with glioblastoma (GBM). The lack of tumor-infiltrating T cells recognizing tumor antigens displayed with major histocompability complex I (MHC I) is one of the reasons for GBM’s evasion from immunotherapy. Our preclinical GBM models and an ongoing clinical trial with oncolytic herpes simplex virus-1 (oHSV) at our institution (NCT03152318) demonstrated that oHSV treatment leads to an increase influx of CD8+ and CD4+ T cells into tumor. However, it remained unclear whether these T cells are specific to the tumor antigens (neoantigens) or reactive to only viral antigens. To determine whether infiltrating CD8 T cells target tumor antigens, we first performed whole-exome and RNA sequencing of CT2A, a preclinical model of GBM impervious to immunotherapies for identification of neoantigens. We screened immunogenicity of identified neoantigens by ELISPOT assay. TILs from the oHSV-treated (but not from control group) reacted to two of the neoantigens that were identified with our pipeline and this activation was similar in magnitude to the reaction to the immunodominant HSV antigen. FACS analyses of TILs seven days after virotherapy showed that oHSV-treated tumor had higher level of TILs than vehicle control tumors, a finding that mirrored what we observed in our phase 1 clinical trial with oHSV. High dose of oHSV treatment not just induces the infiltration of viral specific T cells, but infiltration of T cells targeting neoantigens that were not presented without OV infection. Phosphoproteomic analysis of bulk tumor revealed activation of key inflammatory pathways, including that of T cells receptor signaling upon virus treatment. We evaluated the efficacy of therapeutic neoantigen vaccination in combination with oncolytic intratumoral virotherapy. Survival analysis showed that combination therapy not just delays the tumor progression but also provides survival advantage compared to monotherapies.
EXTH-79. A NOVEL ANTI-GALECTIN-3 ANTIBODY THERAPEUTIC SYNERGIZES WITH TEMOZOLOMIDE IN PRECLINICAL MODELS TO DIMINISH TUMOR GROWTH, DECREASE MIGRATION, AND PROLONG SURVIVAL
Authors: Barrette Anne Marie , Deshpande Krutika , McDonald Bailey , Cadiente Marvin , Wang Xi , Sun Dongxu
Published: November 2023

Glioblastoma (GBM) is the most aggressive, invasive, and malignant primary brain cancer whose standard of care for nearly 20 years has been surgical resection followed by radiotherapy and temozolomide (TMZ) treatment, resulting in a median survival of 15 months following diagnosis. Heterogenous tumor cell populations that evade resection and develop chemoradiation resistance highlight the need for novel therapies that can target both the migratory and proliferative nature of GBM cells. Galectin-3, a carbohydrate-binding protein overexpressed in the GBM parenchyma and stromal astrocytes, is a potent modulator of cell proliferation, migration, angiogenesis, T-cell inhibition, and M2 macrophage polarization, and is seen to increase following TMZ and radiation treatment. Heightened Galectin-3 expression levels correlate to lower survival in glioma patients, thus greatly underscoring the need for targeting Galectin-3 in conjunction with initial standard-of-care treatment. Here, we characterize the therapeutic effects of a brain-penetrant monoclonal anti-Galectin-3 antibody (Gal3 Ab) we have developed for its ability to inhibit GBM growth and migration in several in vitro assays and in vivo heterotopic and orthotopic mouse models. Our Gal3 Ab significantly decreases cell viability, neurosphere migration, and cell invasion in vitro in the presence of full length or truncated Gal3, whose expression and secretion in GBM cells is enhanced following short and long-term TMZ stimulation. We find that Galectin-3 directly binds to EGFR, amplified in ~40% of GBM patients, as well as its most commonly mutated form, EGFRvIII, and our Gal3 Ab can block these bindings, decrease EGFR activation, and alter EGFR localization. In vivo, our Gal3 Ab decreases tumor burden and improves survival in combination with temozolomide, compared to standard-of-care treatment alone in multiple cell-line models. Our pre-clinical studies highlight the potential for our novel Gal3 antibody to further improve TMZ efficacy and benefit GBM patients by diminishing tumor cell migration and slowing tumor growth.
EXTH-81. RE-EXPRESSION OF CXCL9 FOLLOWING ADENO-ASSOCIATED VIRUS (AAV) TRANSGENE DELIVERY TRANSFORMS THE IMMUNOLOGICAL LANDSCAPE OF GLIOBLASTOMA, SENSITIZING TUMORS TO PD-1 IMMUNE CHECKPOINT BLOCKADE (ICB)
Authors: von Roemeling Christina , Yegorov Oleg , Yang Changlin , Russell Rylynn , Carpenter Savannah , Trivedi Vrunda , Bhatia Alisha , Doonan Bently , Golde Todd , Mitchell Duane
Published: November 2023

BACKGROUND

To date, few glioblastoma (GBM) patients have demonstrated clinical benefit from treatment with anti-PD-1 ICB. GBM is notoriously devoid of lymphocytes, signifying that a possible obstacle for effective ICB response may be the limited ability of cytotoxic T-lymphocytes (CTLs) to home to these tumors. CTLs are recruited via long-range signaling mediated by the diffusion of chemokines present in inflammatory environments. Following a comprehensive chemokine screen of clinical tumor specimens, we identified that CXC motif ligand 9 (CXCL9) is notably absent in GBM. To test if GBM reconstitution with CXCL9 could transform these intractable tumors into ICB-responsive, we leveraged AAV gene therapy to generate durable production of this potent lymphotactic signal.

HYPOTHESIS

AAV mediated re-expression of CXCL9 will improve lymphotaxis in GBM, sensitizing these tumors to PD-1 ICB.

METHODS

3-dimensional whole brain fluorescent imaging was performed to characterize the distribution, tropism, and durability of AAV transgene expression in preclinical GBM models. Single-cell RNA sequencing (scRNAseq) and multiparameter flow cytometry was utilized to immunologically profile tumors in response to single and combination treatment. Survival studies with and without lymphocyte depletion were performed to evaluate immunogenic responsiveness of therapy.

RESULTS

Intra-tumor delivery of AAV6 yields stable transduction of tumor-reactive astrocytes, producing a localized, peri-tumoral pattern of transgene signal distribution. Combination therapy increases tumor infiltration by lymphocytes. scRNAseq analyses reveal widespread innate and adaptive immune activation in response to each CXCL9 and PD-1 ICB treatment. Infiltrating CD8 T-lymphocytes demonstrate inflammatory signatures associated with increased effector function, including antigen presentation, cytotoxicity, and TCR signaling. Combination treatment considerably improved overall survival in two distinct GBM models, with durable responses dependent on CD8 T-lymphocyte infiltration.

CONCLUSIONS

This work suggests direct conditioning of the tumor microenvironment by AAV-CXCL9 could potentially overcome a key resistance factor in GBM, sensitizing tumors to immune mediated therapies such as PD-1 ICB.
EXTH-82. THE ROLE OF PTEN AND EPIGENETIC KINOME PROGRAMMING IN EGFR TKI RESPONSE IN GLIOBLASTOMA
Authors: Ziebro Julia , Shelton Abigail , Smithberger Erin , Lin Benjamin , Skinner Kasey , Stamper Allie , Bash Ryan , Furnari Frank , Miller Christopher
Published: November 2023

Glioblastoma (GBM) is the most common brain tumor and standard therapy only extends survival from 12 to 15 months. The receptor tyrosine kinase EGFR is altered in ~60% of GBM, resulting in aberrant activation of downstream pathways, including PI3K, that potentiate tumorigenesis. Though EGFR targeting has been successful in other cancers, clinical trials with EGFR tyrosine kinase inhibitors (TKI) have failed in GBM, in part due to resistance. Trial results suggested that the most common oncogenic EGFR variant, EGFRvIII (vIII), and negative regulator of PI3K signaling, PTEN, were biomarkers of response. To dissect the role of PTEN in GBM tumorigenesis and EGFR TKI response, we utilized vIII-expressing mouse astrocytes with and without functional Pten. Pten loss potentiated tumorigenesis in vitro and in vivo, as proliferation and stemness were significantly increased, and mouse survival was decreased upon implantation as orthoptic allografts. Both RNA-seq and proteomic analysis using multiplex inhibitor beads with mass spectrometry (MIB-MS) showed significantly altered kinome profiles. Pten deleted cells were 5-fold more sensitive to the EGFR TKI neratinib, a drug that induced Pten-dependent modulation of the kinase transcriptome over 48h. This acute transcriptional adaptation implies alteration of the epigenetic landscape. We hypothesized that disruption of enhancer dynamics via BET bromodomain inhibition would improve EGFR TKI durability. We found that cells expressing Pten were 10-fold more sensitive to multiple BET inhibitors, including JQ1, birabresib, and molibresib, and these inhibitors synergized with neratinib to reduce proliferation only in the Pten wild type model. Ongoing work is focused on defining the role of PTEN in maintenance of differential transcriptional and epigenetic programs. We will explore potential mechanistic roles of both the enzymatic and non-enzymatic functions of Pten and characterize its effect on hallmark cancer phenotypes such as proliferation, stemness/self-renewal, and tumorigenesis in the presence and absence of EGFR ± BET inhibitors.
EXTH-83. BIRC3 AS A PREDICTOR OF OUTCOME AND A NEW THERAPEUTIC TARGET IN TMZ RESISTANT GLIOBLASTOMA TUMORS
Authors: Morelli Mariangela , Franceschi Sara , Lessi Francesca , Aretini Paolo , Pastore Aldo , Corradi Elena , Marranci Andrea , Gambacciani Carlo , Pieri Francesco , Grimod Gianluca , Montemurro Nicola , Giacomarra Manuel , Menicagli Michele , Ferri Gianmarco , Pasqualetti Francesco , Sanson Marc , Picca Alberto , Stefano Anna Luisa Di , Santonocito Orazio Santo , Mazzanti Chiara Maria
Published: November 2023

Glioblastoma (GBM) is an incurable malignant brain tumor, with a highly unfavorable prognosis. Apart from MGMT promoter methylation and TMZ response, molecular biomarkers associated with therapeutic response or survival prognosis are still lacking in GBM. Previously, using NAD(P)H-fluorescence lifetime imaging, a new drug screening precision medicine ex-vivo approach, we perturbed patients-derived vital GBM tumor explants (GB-EXPs) by TMZ treatment and obtained a stratification in TMZ responder (Res) and non-responder (Non-Resp) tumors, identifying several genes differentially expressed between the two groups. To validate the approach we enlarged our glioblastoma case study. Transcriptome analysis was performed to characterize TMZ Res and Non-Resp cases. In-silico and functional cellular investigations were carried on to explore the biomarker's efficiency. The majority of the genes found dysregulated in the previous study, showed the same directional regulation of expression and we confirmed, in TMZ-resistant samples the statistically significant upregulation of BIRC3, a strong inhibitor of apoptosis. The TCGA dataset provided an opportunity to fully assess the BIRC3 mRNA expression complementarity with the MGMT status as a predictor of outcome. BIRC3 was validated as a prognostic factor of survival also in large French and Italian glioblastoma cohorts. Most importantly, AZD5582 drug, which is a specific antagonist of BIRC3, combined with TMZ treatment, is able to reverse the TMZ resistance, in glioblastoma cell lines, by restoring apoptosis, and in GB-EXPs that were resistant to TMZ treatment. BIRC3 represents a potential biomarker of prognosis and a predictor of TMZ treatment response in GBM and complements MGMT status as an outcome predictor. The level of BIRC3 expression could stratify patients who will benefit from AZD5582 or its combination with TMZ. Our study provides a potentially new treatment approach and valuable leads to determine whether functional precision medicine combined with BIRC3 expression assessment become a standard tool in glioblastoma clinical oncology.
QLTI-01. HIGH-GRADE ASTROCYTOMA WITH PILOID FEATURES RESPONSE TO TREATMENT WITH CARBOPLATIN AND VINCRISTINE
Authors: Cielo Brandon
Published: November 2023

High-grade astrocytoma with piloid features (HGAP) is a brain tumor that requires distinct DNA methylation profiling for diagnosis. The current literature on HGAP response to treatment is limited. This case details the response to treatment of a young female patient initially with a pilocytic astrocytoma that at progression transformed into HGAP, both diagnoses confirmed with methylation testing. At 12 years old, the patient initially presented with unremitting headaches and syncopal episodes. Imaging revealed a non-enhancing non-cystic lesion in the roof of the fourth ventricle measuring 5 mm in the greatest dimension. She was followed by serial surveillance until undergoing surgical resection four years later. Histologically the tumor was grade I pilocytic astrocytoma containing a KIAA1549-BRAF fusion; the diagnosis was subsequently confirmed with methylation array testing. The patient transitioned to serial surveillance until she was lost to follow-up. At 27 years old, she presented with progressive headaches, gait instability, and agraphia. MRI revealed a posterior fossa cerebellar mass and surgical resection was performed. Histologically this was an anaplastic pilocytic astrocytoma with KIAA1549-BRAF fusion; however, DNA methylation profiling confirmed the diagnosis of HGAP. The patient was treated with postoperative radiation and demonstrated asymptomatic tumor progression in the left cerebellar hemisphere fourteen months later. She received carboplatin and vincristine with radiographic response and stability for four months until discontinuation due to tumor progression with infiltrative disease within the posterior fossa and brainstem. Her treatment was transitioned to selumetinib with mixed radiographic response but continued clinical deterioration after three months. Selumetinib was terminated and best supportive care was initiated. The patient died 216 months from initial presentation with pilocytic astrocytoma and 28 months from transformation into HGAP. To our knowledge, this is the first report of HGAP with treatment response to carboplatin and vincristine. Further research into the best treatments for HGAP is needed.