bluuebunny/crossref_metadata_2025 huggingface dataset:
EXTH-14. INHIBITION OF THE ANGIOTENSIN II TYPE 2 RECEPTOR AT2R IS A NOVEL THERAPEUTIC STRATEGY FOR GLIOBLASTOMA
Authors: Perryman Richard , Renziehausen Alexander , Shaye Hamidreza , Kostagianni Androniki , Tsiailanis Antonis , Thorne Tom , Chatziathanasiadou Maria , Sivolapenko Gregory , El Mubarak Mohamed Ahmed , Han Gye Won , Zarzycka Barbara , Katritch Vsevolod , Lebon Guillaume , Nigro Cristiana Lo , Lattanzio Laura , Morse Sophie , Choi James , O’Neill Kevin , Kanaki Zoe , Klinakis Apostolos , Crook Tim , Cherezov Vadim , Tzakos Andreas , Syed Nelofer
Published: November 2022

Glioblastoma (GBM) is a primary malignant brain tumor with poor clinical outcomes. Standard of care consists of surgical debulking followed by radiation and temozolomide, but the tumor invariably recurs, and median survival is only ~18 months. Repurposing drugs used for the treatment of other diseases is a promising avenue to identify novel treatments for this highly aggressive form of cancer. One such class of compounds is angiotensin II (AngII) receptor blockers, commonly used to control blood pressure. We show that ~80% of primary human GBM express the angiotensin II type 2 receptor (AT2R). In the presence of AngII, inhibition of AT2R using either PD123319 or EMA401 significantly inhibits GBM proliferation. This effect was lost in GBM cells with CRISPR/Cas9 mediated knockdown of AT2R. EMA401 inhibited invasion, angiogenesis, reduced GBM spheroid growth and induced apoptosis through caspase 3/7 activation. Furthermore, EMA401 induced changes in a number of growth regulatory pathways including apoptosis, DNA replication and focal adhesion. The crystal structure of AT2R bound to EMA401 revealed the receptor to be in an active-like conformation with helix-VIII blocking G protein or β-arrestin recruitment. We demonstrate that the architecture and interaction of EMA401 with AT2R differs drastically from complexes of AT2R with other compounds. Conjugation of EMA401 to angiopep-2 enhanced its blood brain barrier passage and reduced tumor volume in an orthotopic xenograft model of GBM. Targeting AT2R is a novel therapeutic strategy to treat GBM that should be explored in patients.
EXTH-27. TRANSLATABLE CCL3 THERAPY TO PROMOTE DC VACCINE MIGRATION AND EFFICACY AGAINST GLIOMA
Authors: Hotchkiss Kelly , Halabi Dahlia , Batich Kristen , Cook Sarah , Norberg Pam , Archer Gary , Sampson John
Published: November 2022

INTRODUCTION

Prior evidence from our studies have identified chemokine ligand 3 (CCL3) as a key modulator to increase DC vaccine migration to vaccine draining lymph nodes (VDLNs) which is associated with improved long-term survival clinically.

METHODS

The effect of CCL3 treatment on DC vaccine migration ex vivo and in vivo (n=5), antigen-specific T cell responses (n=5), and efficacy against orthotopic GL261-OVA and SMA560 tumors (n=10) was studied in C57Bl/6 and VMdK mice. CCL3 treatment of mouse and human DC vaccines ex vivo was also tested in transwell assays. DCs were electroporated with OVA-mRNA or pulsed with ODC1 neoantigen peptide. DC migration and T cell responses were quantified by flow cytometry. Median overall survival (mOS) was measure in days (d) post-intracranial implantation.

RESULTS

Ex-vivo treatment of DC vaccines with 20ng/mL CCL3 (18 and 1hr pre-injection) increased transwell (mouse and human) migration and to VDLN. In vivo systemic CCL3 treatment also resulted in a dose-dependent increase in migration of DC vaccine to VDLN (10µg, 20µg, 50µg). CCL3 added to DC vaccination generated more tumor antigen-specific CD8+IFNγ+ T cells at 7-days. CCL3 with tumor antigen-DC treatment also resulted in significantly greater survival compared to OVA-DC alone (GL261-OVA: mOS DCvac: 19.5d, CCL3+DCvac 37d, p=0.0174; SMA560: mOS: DCvac: 25d, CCL3+DCvac: 48d, p=0.002).

CONCLUSIONS

These data combined with previous success of our DC vaccine clinical trials reflect the potency of CCL3 to enhance DC vaccine-specific migration, immune responses, and survival. CCL3 is a novel and safe adjuvant to overcome prior limitations in DC vaccine therapy and may be translatable to increase heterogeneous tumor antigen presentation following vaccine-targeted tumor killing. CCL3 used as a drug could increase vaccine migration without potential systemic toxicity in patients with glioblastoma.
EXTH-33. PRIMING OF SYNNOTCH CAR T CELLS VIA CNS-SPECIFIC ANTIGEN ALLOWS SPATIAL AND TEMPORAL REGULATION OF CAR EXPRESSION, EFFECTIVE HOMING AND PERSISTENCE OF T CELLS IN THE CNS
Authors: Watchmaker Payal , Simic Milos , Diebold David , Pineo-Cavanaugh Psalm , Deucker Jason , Yu Wei , Lim Wendell , Okada Hideho
Published: November 2022

One of the key challenges in the development of CAR-T therapy is the absence of target antigens that are tumor-specific and have homogenous expression. To safely target glioblastoma-associated antigens (GAAs) in the tumor without attacking normal tissue expressing the same GAAs outside of the brain, we adapted a novel synthetic Notch (synNotch) receptor system and established a “prime and kill” sequential two-receptor CAR circuit. We used glioblastoma- (GBM) specific neoantigen EGFRvIII as a priming signal for synNotch receptor and have reported robust antitumor response in the mice bearing intracerebral PDX tumor with heterogenous EGFRvIII expression. However, less than 20% of adult GBM cases express EGFRvIII. Furthermore, EGFRvIII expression can diminish over time even after its detection in the primary GBM, and the EGFRvIII-synNotch primed CAR T cells may deplete EGFRvIII-expressing GBM cells via their cytotoxic effects, thereby losing the priming signal. To overcome these inherent challenges of the EGFRvIII-priming strategy, we used CNS tissue-specific antigens as priming signal to drive localized expression of the CAR against EphA2 and IL-13Rα2 and bypassed the need for tumor-specific antigen. We have found BCAN (also known as Brevican) as the most promising priming antigen among several CNS-specific cell surface proteins that we evaluated. When mice bearing intracerebral GBM6 PDX received a single IV infusion of T cells engineered with the α-BCANsynNotch- > α-EphA2/IL-13Rα2 CAR (B-SYNC) circuit, all mice (10/10) demonstrated complete regression of the tumor. Furthermore, these B-SYNC T cells were significantly more efficacious than constitutively expressed EphA2/IL-13Rα2 CAR T cells, associated with superior persistence and less exhausted phenotype. Thus, the B-SYNC approach represents a conceptual novelty of CNS tissue-specific CAR activation. This also enhances the translational significance of synNotch-CAR T cells by widely extending the eligibility to EGFRvIII-negative glioma patients. We will develop and conduct a phase I study to evaluate our hypothesis.
EXTH-37. TARGETING THE IDH1R132H MUTATION IN GLIOMAS BY CRISPR-CAS PRECISION BASE EDITING
Authors: Weber Remi , Vasella Flavio , Klimko Artsiom , Silginer Manuela , Lamfers Martine L M , Regli Luca , Schwank Gerald , Weller Michael
Published: November 2022

Gliomas are the most frequent malignant primary brain tumors and continue to lack curative therapies. To aid the development of novel therapeutic approaches, a better understanding of glioma-specific molecular patterns is essential. We therefore investigate the role of the canonical point mutation in the isocitrate dehydrogenase 1 gene (IDH1R132H). Its early occurrence in oncogenesis and ubiquitous expression suggests a causative driver function; however, current literature is inconclusive on the impact of IDH1R132H, requiring innovative approaches to further elucidate the functional consequences and therapeutic potential of IDH1R132H.

METHODS

Clustered, regularly interspaced, short palindromic repeat (CRISPR)-Cas precision base editing systems enable the alteration of a specific base pair without suffering from off-target editing associated with previous, double strand brake-inducing systems and thus making them an ideal tool to revert the point mutation in IDH1R132H gliomas.

RESULTS

By selecting a CRISPR-Cas precision base editing system, the IDH1R132H site was edited with editing efficiencies of up to 50%. Primary patient derived cell lines and organoids were corrected in their IDH1R132H locus. Phenotypical changes, such as a change in IDH1R132H protein levels, 2-hydroxyglutarate concentration as well as proliferation rates were observed upon the reversal of the point mutation. Furthermore, the precision base editing system was packaged into a dual-AAV-vector split intein system and showed successful in vitro gene editing. In conclusion, this precise genetic intervention provides a methodology to create accurate patient derived models to analyze the impact of IDH1R132H on glioma biology and provides a framework for in vivo gene therapy.
EXTH-38. NEOADJUVANT PD1 BLOCKADE ALTERS THE TUMOR IMMUNE MICROENVIRONMENT AFTER SURGERY IN A PRECLINICAL MODEL OF RECURRENT GLIOBLASTOMA
Authors: Hoang-Minh Lan , Klippel Kelena , Doonan Bently , Von Roemeling Christina , Trivedi Vrunda , Yang Changlin , Sriharan Akshayaa , Francis Connor , Shin David , Karachi Aida , Galochkina Zhanna , Sarkisian Matthew
Published: November 2022

SIGNIFICANCE

Promising clinical trials for glioblastoma are evaluating the efficacy of neoadjuvant immunotherapy in the context of recurrent tumor surgery.

OBJECTIVE

We investigated the effects of neoadjuvant PD1 blockade on the glioma tumor microenvironment after surgery in a clinically relevant murine model of recurrent tumor.

RESULTS

Using a mouse resection model of KR158 glioblastoma that we established, we show that neoadjuvant PD1 inhibition and surgery enhance mouse survival and enhance the recruitment of CD8+ and CD4+ T cells at recurrent tumor sites following bulk tumor resection when compared with surgery followed by adjuvant immunotherapy. Transcriptome and spatial genomic analyses reveal alterations in immune exhaustion and activation pathway signaling genes after neoadjuvant anti-PD1 treatment when compared with adjuvant anti-PD1-treatment or surgery alone.

CONCLUSIONS

These studies provide insights into the effects of neoadjuvant PD1 blockade on the tumor microenvironment after surgery and uncover additional treatment targets that could be used in combination with this neoadjuvant therapy.
EXTH-39. BIOMARKER DRIVEN TARGETING OF RECURRENT IDH MUTANT GLIOMA WITH ABEMACICLIB
Authors: Nasser Ali , Melamed Lisa , Nagashima Hiroaki , Wakimoto Hiroaki , Cahill Daniel , Miller Julie
Published: November 2022

Despite initial responsiveness to standard treatments like radiation and chemotherapy, IDH mutant gliomas inevitably recur, become more clinically aggressive, and lead to untimely death. The aggressive change in clinical outcome is driven by insensitivity to conventional methods of treatment, necessitating an improved therapeutic strategy at this stage of the disease. Greater than 20% of recurrent IDH mutant gliomas exhibit homozygous loss of CDKN2A, leading to the loss of the endogenous CDK4/6 inhibitor p16 and aberrant signaling through the CDK-RB pathway. We hypothesized that CDKN2A loss leads to enhanced sensitivity to CDK4/6 inhibitors which have been approved for use in a variety of other cancer types. We examined the sensitivity of a panel of patient-derived IDH mutant gliomas with either endogenous CDKN2A loss or CRISPR-mediated gene deletion to treatment with CDK4/6 inhibitors. We observed a correlation between CDKN2A loss and enhanced cytotoxicity following abemaciclib treatment in these glioma models. We have characterized the cell cycle profile following abemaciclib treatment by flow cytometry and western blotting in both sensitive and resistant lines. Further, abemaciclib treatment resulted in significant slowing of tumor growth rate in a subcutaneous patient-derived IDH mutant glioma model. Survival studies in an intracranial model are ongoing. These preclinical results provide foundational data to support a biomarker-driven clinical trial of CDK4/6 inhibitors in patients with recurrent IDH mutant glioma.
EXTH-45. TARGETING GLIOBLASTOMA BY NOVEL INTEGRIN-SPECIFIC CAR T CELLS
Authors: Villars Danielle , Haensch Lena , Silginer Manuela , Weiss Tobias , Weller Michael , Roth Patrick
Published: November 2022

RATIONALE

Innovative treatment strategies are urgently needed to improve the prognosis of glioblastoma patients. Chimeric antigen receptor (CAR) T cell therapy has shown great success in hematological malignancies. However, it remains to be shown that CAR T cells are also active against solid tumors. αv integrins are overexpressed in gliomas and have already been used as therapeutic targets for small molecule inhibitors and antibodies. While these approaches were well tolerated, they did not exert meaningful clinical activity in glioblastoma patients. We aimed at exploiting the presence of αv integrins as targets for anti-glioma CAR T cell therapy.

METHODS

CAR T cells targeting specific integrin heterodimers (αvβ3, αvβ5 or αvβ8) were generated and their anti-glioma activity was determined in co-culture assays with different glioma cells. The efficacy of CAR T cells to control tumor growth in vivo was assessed in clinically relevant orthotopic mouse glioma models.

RESULTS

All newly generated integrin-targeting CAR T cells exerted strong anti-tumor activity in vitro. The highest lytic activity against glioma cells was observed for αvβ5 and αvβ8 integrin-specific CAR T cells. CAR T cells generated from patient-derived immune cells displayed strong lytic activity when tested against autologous tumors cells. Glioma cells with a CRISPR/Cas9-mediated knockout of the target antigen were resistant to CAR T cell-mediated cytotoxicity demonstrating their specificity. The in vivo activity of integrin-specific CAR T cells was tested in 2 orthotopic glioma models. Intratumoral treatment of glioma-bearing mice with αvβ5 or αvβ8 CAR T cells significantly prolonged their survival and cured a substantial fraction of these animals.

CONCLUSION

αvβ5- and αvβ8-specific CAR T cells exert strong and target-specific anti-glioma activity in vitro and prolong the survival of glioma-bearing mice. These data form the basis for a clinical evaluation of integrin-specific CAR T cells against glioblastoma.
EXTH-50. IDENTIFYING TRIM11 AS A POTENTIAL THERAPEUTIC TARGET FOR MALIGNANT GLIOMAS
Authors: Di Kaijun , Abrams Daniela , Yadav Pratik , Das Bhaskar , Bota Daniela
Published: November 2022

TRIM11 (tripartite motif-containing protein 11) belongs to the TRIM/RBCC (the RING B-box coiled-coil) family of E3 ubiquitin ligases. Members of this family have been implicated in development, neurodegenerative diseases, cellular response to viral infection and cancer. Glioblastoma (GBM) is an aggressive infiltrative brain tumor with poor prognosis. Our previous work demonstrated that TRIM11 is over-expressed in high-grade gliomas and promotes proliferation, invasion, migration and tumor growth, suggesting TRIM11 is a target for malignant glioma treatment. Here we reported the effect of TRIM11 on GBM progression in vivo using immunocompromised mice intracranially implanted with GBM xenografts over-expressing TRIM11. The control group mice survived longer than the mice bearing TRIM11 over-expressing xenografts (P< 0.5), suggesting TRIM11 enhances tumor progression in vivo. The oncogenic effect of TRIM11 may be related to its influence on apoptosis pathway as a robust induction of poly (ADP-ribose) polymerase (PARP) was observed in TRIM11 over-expressing GBM cells. PARP could repair DNA damage caused by UV light, radiation, and certain anticancer drugs, etc. Blocking PARP may prevent cancer cells from repairing their damaged DNA, causing them to die. Temozolomide (TMZ) is a standard-of-care chemotherapeutic agent for GBM through alkylating/methylating DNA, leading to DNA damage and death of tumor cells. TRIM11 over-expressing GBM cells were found more resistant to TMZ and showed a higher survival rate compared to parental cells, indicating that TRIM11 might be a therapeutic target for GBM. Based on homology modeling we designed and synthesized small library of TRIM11 inhibitors, and identified one compound (BT# 592) significantly inhibited the growth of GBM cells in a dose-dependent manner, implicating that TRIM11 inhibitors may serve as novel agents for malignant glioma treatment. We are in the process to develop more active compounds using SAR studies to identify hit-to-lead compounds as potential therapeutics for GBM.
EXTH-52. HARNESSING A B CELL THERAPY TO PROMOTE ANTI-GLIOBLASTOMA HUMORAL RESPONSE
Authors: Castro Brandyn , Fischietti Mariafausta , Zolp Andrew , Zhao Junfei , Hou David , Nguyen Linh , Wan Hanxiao , Zhang Peng , Han Yu , Lopez-Rosas Aurora , Platanias Leonidas , Stupp Roger , Miska Jason , Lesniak Maciej , Lee-Chang Catalina
Published: November 2022

Glioblastoma (GBM) continues to retain its dismal prognosis despite numerous new therapeutic modalities to target various aspects of the tumor. There is a need to identify new, and even personalized, targetable GBM specific antigens. Our B-cell-based vaccine (BVax) is generated by isolating immune experienced B cells, identified by 4-1BBL, from murine secondary lymphoid organs or patient blood. We have shown this subset of B cells to have anti-tumoral potential in GBM. These cells are strengthened with BAFF, CD40, and IFNg stimulation to form BVax and then activated in vitro to form plasmablasts. Immunoprecipitation-mass spectrometry is performed using BVax-derived antibodies and tumor lysate from the paired patient specimen. We have identified unique antigens bound by BVax-derived antibodies. Given the significant therapeutic benefit we have seen using BVax-derived antibodies in preclinical murine models, we hypothesize that targeting these unique antigens would have significant therapeutic benefit in GBM patients. We focused on antigens that showed a survival benefit with lower expression using CGGA database analysis. We then confirm the presence and prevalence of the antigens within tumor cells in the patient’s tissue using immunohistochemistry, identifying some antigens in up to 15% of tumor cells. Using our brain tumor bank repository, we screened other GBM patient’s tissue for the presence of these markers to determine the broader applicability of targeting each antigen. We have identified proteins key in extracellular matrix formation which promote tumor growth and progression in cancer, including the ability to induce epithelial-mesenchymal transition. Simultaneously we have developed a computational algorithm to predict which antigens BVax-derived antibodies will bind using single cell RNA sequencing data from GBM patient-derived tumor and BVax, which may have broader applicability to a larger number of patients. Both techniques serve as exciting platforms to identify new therapeutic targets in GBM.
EXTH-56. TARGETING GBM WITH AN ONCOLYTIC PICORNAVIRUS SVV-001 ALONE AND IN COMBINATION WITH FRACTIONATED RADIATION IN A NOVEL PANEL OF ORTHOTOPIC PDX MODELS
Authors: Zhang Huiyuan , Du Yuchen , Qi Lin , Xiao Sophie , Braun Frank K , Kogiso Mari , Abdallah Aalaa , Baxter Patricia A , Su Jack M , Brat Daniel , Hallenbeck Paul L , Teo Wan-Yee , Patel Akash , Li Xiao-Nan
Published: November 2022

BACKGROUND

Large panels of clinically relevant animal models are needed for the development of new therapies for GBM. SVV-001, a replication-competent oncolytic virus that can pass through the blood brain barrier, represents an attractive novel approach. Material and

METHODS

Surgical tissues from 23 patients were implanted into the brains of NOD/SCID mice (1x105 cells/mouse) to develop patient-derived orthotopic xenograft (PDOX) models. Analysis of histology, gene expression (RNAseq), tumor growth was performed and compared with the patient tumors during subtransplantations (up to 5 passages). Anti-tumor activities of SVV-001 were examined in vitro in primary monolayer cultures, 3D neurospheres and purified glioma stem cells derived from 13 PDOX models; and its therapeutic efficacy validated in vivo in PDOX models through single i.v. injection (1x1011 viral particle) alone and in combination with fractionated (2 Gy/day x 5 days) radiation. Changes of animal survival times were analyzed together with viral infection, cell proliferation and DNA damage.

RESULTS

PDOX formation was confirmed in 17/23 (73.9%) GBMs. They replicated key histopathological features and diffuse invasion of the patient tumors. RNAseq identified the differentially expressed genes and subclassified PDOX models into proneural, classic and mesenchymal groups. SVV-001 killed primary monolayer cultures (4/13 models) and neurosphere (7/13 models) and putative glioma stem cells (CD133+ and/or CD15+ cells) in vitro, infected PDOX tumors without harming normal brain cells in vivo and significantly prolonged survival times in 2/2 models. Combination with radiation showed a trend in further prolonging animal survival times through enhanced DNA damages by SVV-001.

CONCLUSION

A panel of 17 clinically relevant and molecularly annotated PDOX modes of GBM is developed. The strong anti-tumor activities of SVV-001 in vitro and in vivo in GBM models provided experimental rational that support the initiation of clinical trials in GBM patients.
EXTH-62. EXAMINATION OF THE TUMOR MICROENVIRONMENT OF GLIOMAS TREATED WITH VIROIMMUNOTHERAPY REVEALS THE PRODUCTION OF ONCOMETABOLITES LEADING TO IMPROVED THERAPEUTIC STRATEGIES
Authors: Nguyen Teresa , Shin Dong Ho , Kim Debora , Singh Sanjay K , Jiang Hong , Fan Xuejun , Yi Yanhua , Gumin Joy , Lang Frederick , Alvarez-Breckenridge Christopher , Alonso Marta M , Zhai Lijie , Ladomersky Erik , Lauing Kristen , Wainwright Derek , Fueyo Juan , Gomez-Manzano Candelaria
Published: November 2022

Oncolytic viruses are considered part of immunotherapy and have shown promise in preclinical experiments and clinical trials in a small fraction of patients. To improve the response rate, it is necessary to reshape the tumor microenvironment that shields the tumor from the immune system of the patient. We engineered Delta-24-RGDOX (DNX-2440), an oncolytic adenovirus to express the T-cell activator OX40L, which triggered, in murine glioblastoma models, a dramatic reshaping of the tumor microenvironment dominated by changes in immune processes in an RNA-seq analysis of ingenuity pathways. Paradoxically, network analyses also revealed that Delta-24-RGDOX induced robust activation of the cytokine-driven immunosuppressive IDO-Kynurenine-AhR circuitry, indicating a potential mechanism of resistance of gliomas to oncolytic virotherapy. To reverse this immunosuppression, we combined Delta-24-RGDOX with clinically relevant IDO inhibitors to treat glioma-bearing mice. The combined therapy decreased the activation of the IDO network without affecting viral activity. Flow cytometry assays revealed a decrease in the presence of the immunosuppressive cell populations, MDSCs and Tregs, which presence was higher in Delta-24-RGDOX-treated tumors. These data were further confirmed in gene set enrichment analyses. Functional co-culture studies showed that the combined therapy activated splenocytes against tumor antigens and that this activation was reversed by Kynurenine. Importantly, the combination treatment eradicated the tumors in a CD4-dependent manner and significantly prolonged the survival of glioma-bearing mice. Altogether, these studies indicate that the combination treatment promotes an adaptive immune response while decreasing the immunosuppression caused by the virus-induced IDO activation. Furthermore, our data identified the striking role of immunosuppressive pathways in the resistance of gliomas to oncolytic virotherapy. Specifically, the activity of the tumor microenvironment IDO circuitry was responsible, at least partially, for the remodeling of local immunosuppression after tumor infection. Combining molecular and immune-related therapies may improve outcomes in human gliomas treated with virotherapy.
EXTH-64. IPSC-DERIVED NK CELLS EXHIBIT POTENT IN VITRO AND IN VIVO TUMORCIDAL ACTIVITY AGAINST PATIENT-DERIVED GLIOBLASTOMA STEM CELLS (GSCS)
Authors: Ning Jianfang , Davis Zachary , Cichocki Frank , Wang Hongbo , Tuininga Katie , Bjordahl Ryan , Lee Tom , Miller Jeffrey , Chen Clark
Published: November 2022

INTRODUCTION

Natural Killer (NK) cells play pivotal roles in cancer immune surveillance and harbor potent anti-tumor activities. We have engineered an induced pluripotent stem cells (iPSCs) derived, NK cell (termed FT538) that express 1) a high-affinity non-cleavable CD16 Fc receptor to facilitate antibody engagement, 2) a membrane-bound IL-15/IL-15 receptor fusion to enhance NK activity and persistence and 3) a knock-out of CD38 to enhance NK metabolic fitness. FT538 is produced in a GMP-compliant manner as an off-the-shelf immunotherapy. Clinical experiences with FT538 have shown evidence of durable anti-tumor immunity after single administration in select lymphoma patients.

METHODS

We performed pre-clinical studies to characterize the tumoricidal activity of FT573 against glioblastomas.

RESULTS

Without specific antibody to engage CD16, FT538 exhibited potent in vitro activity against ~80% of a panel of isocitrate dehydrogenase wild-type patient-derived xenograft (PDX) glioblastoma lines, including MGG8. When directly injected into the murine brain, 50% of the FT538 persisted for > 2 weeks without addition of exogenous cytokine. Moreover, the mice exhibited no evidence of neurologic compromise, and brains harvested after injection show no signs of FT538 activation. In vivo intratumoral injection of FT538 into an orthotopically established MGG8 glioblastoma caused a ~20 fold increase in granzyme B release by FT538, with a comparable increase of cleaved-caspase 3 in the tumor. In independent experiments, > 90% of mice orthotopically implanted MGG8 survived beyond 100 days after intra-tumoral FT538 while all mice treated with mock injection died by 60 days. For glioblastoma PDX refractory to FT538, such as MGG4, tumoricidal activity of FT583 can be induced in vitro and in vivo by the addition of a tri-specific engager targeting B7H3, a surface protein highly expressed in glioblastomas.

CONCLUSION

Our results demonstrate great promise of FT538 as a glioblastoma therapy, with plans for translation into a first-in-human clinical trial.
EXTH-66. BIOENERGETIC SWITCH AND BIOLOGIC RESPONSE WITH SLC25A11 INHIBITION TARGETING MALATE-ASPARTATE SHUTTLE (MAS) ON GLIOBLASTOMA TUMORSPHERE
Authors: Cho Hejoung , Yoo Jihwan , Choi Ran Joo , Park Junseong , Yoon Seon-Jin , Oh Yoojung , Moon Ju Hyung , Kim Eui Hyun , Chang Jong Hee , Kang Seok-Gu
Published: November 2022

BACKGROUND: Cancer cells depended on cytosolic nicotinamide adenine dinucleotide (NADH) transported into mitochondria via the malate-aspartate shuttle (MAS) for ATP production. KN612 (N-phenylmaleimide), a MAS inhibitor, is known to interfere with cancer growth by reducing ATP production, supported by several studies except on glioblastoma (GBM). Therefore, this study was designed to elucidate whether MAS could be an aimable target in GBM. METHODS: We compared expression levels of MAS conforming enzymes between normal and GBM samples. Gene expression profiles were analyzed using RNA-sequencing. Mitochondrial activity was measured by oxygen consumption (OCR), tetramethylrhodamine-ethylester (TMRE) staining, and liquid chromatograph-tandem mass spectrometer (LC-MS/MS). Also, biological functions were measured by cell viability, ATP levels, NADH levels, stemness, and invasiveness. In vivo efficacies were confirmed using a mouse orthotopic xenograft model. RESULTS: An analysis of the microarray database revealed that expression levels of several MAS enzymes including OGC (SLC25A11) were elevated in GBM. Through RNA sequencing, it was confirmed that KN612 accurately targeted SLC25A11 and decreased its expression. KN612-treated cells showed decreased viability, ATP production, and NADH levels compared with control cells. Under the same conditions, a significant decrease in stemness, invasion, and MMP was confirmed. In addition, KN612 confirmed remarkable therapeutic responses in a mouse orthotopic xenograft model. CONCLUSION: Our results show that KN612 effectively inhibits cancer cells both at the cellular level and at the in vivo level. This shows that targeting MAS could be a potential treatment option in addition to the currently limited standard GBM therapy.
EXTH-71. CHEMOTACTIC RECOMBINANT ADENO-ASSOCIATED VIRUS (RAAV) VIROTHERAPY IMPROVES LYMPHOCYTE RECRUITMENT IN GLIOBLASTOMA (GBM)
Authors: von Roemeling Christina , Klippel Kelena , Trivedi Vrunda , Yegorov Oleg , Yang Changlin , Hoang-Minh Lan , Doonan Bently , Ran Yong , Golde Todd , Mitchell Duane
Published: November 2022

BACKGROUND

Although immunotherapy holds promise for producing durable outcomes in GBM, a major obstacle is the limited ability of cytotoxic T lymphocytes (CTLs) to home to the tumor. CTLs are recruited via long-range signaling mediated by the diffusion of chemokines present in inflammatory environments. During glioma formation, tumors manufacture immune-suppressive chemokines and cytokines that co-opt resident cells, resulting in the preferential recruitment of immune suppressor cells from the periphery. We explored a novel strategy to selectively reprogram the tumor microenvironment (TME) using focal delivery of rAAV encoding a powerful call-and-receive chemokine for CTLs: CXCL9. HYPOTHESIS: rAAV transduction of the GBM TME to express CXCL9 (rAAV6-CXCL9) will enhance CTL tumor infiltration and improve responsiveness to immunotherapy.

METHODS

Proteomic arrays were used to identify CTL chemokines absent in human glioma. 3D immunohistochemistry and flow cytometry was used to evaluate geospatial rAAV transgene expression and lymphocyte trafficking in vitro and in vivo. Survival studies of rAAV6-CXCL9 alone and in combination with anti-PD-1 checkpoint blockade was performed in preclinical models of GBM. Tumor immunogenicity following treatment was evaluated by scRNAseq and proteomic analysis.

RESULTS

We identify rAAV6 as a reliable capsid for transducing murine tumor-reactive astrocytes. Transgene expression following a single intra-tumor injection is focal, stable, and results in high levels of CXCL9 production. CXCL9 is effective at promoting tumor T-cell chemotaxis in vitro and in vivo. rAAV6-CXCL9 treatment sensitizes GBM to PD-1 blockade, improving survival in two distinct syngeneic preclinical models- an effect that is largely dependent on CD8 T-cells.

CONCLUSIONS

We combine the excellent safety profile of rAAV with focal delivery, enhancing exposure within the tumor compartment and mitigating systemic toxicities seen with conventional therapies. rAAV therapy was successful at stimulating the intended biological response to our encoded transgene (CXCL9): increased lymphocyte recruitment. This strategy has far-reaching potential across other immunotherapy platforms.
EXTH-72. TTFIELDS ENHANCE THE ANTINEOPLASTIC ACTIVITY OF THE DRUG-REPURPOSING APPROACH CUSP9V3 IN GLIOBLASTOMA IN VITRO
Authors: Cao Qiyu , Hajosch Annika , Kast Richard , Hlavac Michal , Westhoff Mike-Andrew , Siegelin Markus , Wirtz Christian Rainer , Halatsch Marc-Eric , Karpel-Massler Georg
Published: November 2022

OBJECTIVE

Drug repurposing represents a promising strategy to safely accelerate the clinical use of therapeutics with antineoplastic activity. In this study, we examined whether Tumor Treating Fields (TTFields) enhance the biological effects of CUSP9v3, a treatment strategy including nine repurposed drugs, in an in vitro setting of glioblastoma.

METHODS

We performed MTT-assays to examine effects of the combination treatment on the viability of different glioblastoma cells. Tumor spheroids were used as a model to examine effects of the combination treatment in a 3-dimensional setting. Staining with annexin V/propidium iodide or MitoTrackerTM followed by flow cytometry was done to assess pro-apoptotic effects. Specific protein expression of caspases and members of the Bcl-2 family of proteins was determined by Western blot analyses.

RESULTS

TTFields had at least additive inhibitory effects on the cell viability of established (U251), primary cultured (PC38, PC40, PC128) and stem-like (SC38, SC40) glioblastoma cells when combined with CUSP9v3. In addition, flow cytometric analyses revealed that a simultaneous treatment with TTFields and CUSP9v3 significantly increased the fraction of annexin V-positive (apoptotic) glioblastoma cells. Moreover, the fraction of cells with a reduced mitochondrial outer membrane potential was significantly higher following a simultaneous treatment with TTFields and CUSP9v3. On the molecular level, these observations were associated with enhanced cleavage of effector caspase 3 and a reduced expression of the anti-apoptotic Bcl-2 family proteins Bcl-2 and Mcl-1.

CONCLUSION

These data suggest that TTFields enhance the susceptibility of glioblastoma cells towards CUSP9v3, potentially allowing significant dose reduction and decreased toxicity. This observation seems to rely at least in part on a caspase-dependent cell death mechanism. TTFields are widely used for the treatment of glioblastoma patients and CUSP9v3 was recently shown to have a favorable safety profile in a phase Ib/IIa trial (NCT02770378) which facilitates transition of a combined approach to the clinical setting.
EXTH-73. A NOVEL ANTI-GALECTIN-3 MONOCLONAL ANTIBODY THERAPEUTIC DIMINISHES MIGRATION AND SLOWS TUMOR GROWTH TO PROLONG SURVIVAL IN GLIOBLASTOMA
Authors: Barrette Anne Marie , Deshpande Krutika , Cadiente Marvin , Wang Xi , Sun Dongxu
Published: November 2022

Glioblastoma (GBM) is the most aggressive, invasive, and malignant primary brain cancer with a standard of care that hasn’t been improved in over 15 years. Heterogenous tumor cell populations that evade resection and develop resistance to chemoradiation 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 and microglia/macrophages, is a potent modulator of cell proliferation, migration, angiogenesis, T-cell inhibition, and M2 macrophage polarization. Increased Galectin-3 expression levels correlate to lower survival in glioma patients, and these levels further increase following exposure to temozolomide or radiation alone or in combination, thus more greatly emphasizing the need to target Galectin-3 in GBM patients following initial standard-of-care treatment. Here, we characterize the therapeutic effects of a monoclonal anti-Galectin-3 antibody (Gal3 Ab) we have developed for its ability to inhibit GBM tumorigenic and metastatic potential in several in vitro assays and in vivo heterotopic and orthotopic mouse models. We find that Galectin-3 directly binds to EGFR, the most commonly amplified gene locus in GBM affecting roughly 40% of patients, and our Gal3 Ab can block this binding. Human GBM cells treated with Gal3 Ab show a significant decrease in migration and invasion and reduction in EGFR activation in vitro. In vivo, the Gal3 Ab additionally decreases tumor burden and provides a survival benefit in combination with temozolomide compared to the current standard-of-care treatment alone. Our studies indicate the potential for our novel anti-Galectin-3 antibody to further benefit GBM patients receiving chemoradiation treatment by diminishing tumor cell migration and slowing tumor growth.
EXTH-81. ITGA5 IS A NOVEL IMMUNOTHERAPEUTIC TARGET AGAINST TREATMENT REFRACTORY MEDULLOBLASTOMA
Authors: Bakhshinyan David , Suk Yujin , Kuhlman Laura , Adile Ashley , Ignatchenko Vladimir , Gwynne William , Custers Stefan , Macklin Andrew , Venugopal Chitra , Kislinger Thomas , Singh Sheila
Published: November 2022

Medulloblastoma (MB) is the most common type of malignant pediatric brain cancer. Current standard of care (SOC) involves maximal safe resection and neuraxis radiotherapy and chemotherapy in individuals older than 3 years. To date, these cytotoxic SOC combined with craniospinal irradiation led to devastating neurocognitive and developmental deficits impacting quality of life for pediatric patients. The biological heterogeneity of MB is highlighted by the existence of four distinct molecular subgroups (WNT, SHH, Group 3, and Group 4). Group 3 and Group 4 have the poorest patient outcomes because of their aggressive, metastatic nature, and so often remain treatment refractory to SOC. Group 3 has a poor prognosis due to its high incidence of leptomeningeal spread and an overall survival rate of less than 50%. The cytotoxic nature and lack of response in specific subtypes to SOC underscores the urgent need for developing and translating novel treatment options including immunotherapies. In our earlier work, we have developed a therapy-adapted patient derived xenograft (PDX) model of the Group 3 MB as the tumor cells undergoes therapy in vitro and in vivo. N-glycocapture surfaceome profiling of the MB cells through this PDX model identified Integrin α5 (ITGA5) as one of the most differentially expressed targets found at recurrence when compared to engraftment and untreated timepoints. Through shRNA knockdown and small molecule inhibition, we identify ITGA5 expression marks a MB cell subpopulation with increased self-renewal ability both in vitro and in vivo. Access to recurrent MB (rMB) post-therapy allowed us to investigate the changes in the surfaceome of MB cells using proteomics profiling to identify promising rMB-specific targets for rational development of novel immunotherapies.
EXTH-83. DEVELOPMENTALLY REGULATED ANTIGENS FOR IMMUNOLOGIC TARGETING OF EMBRYONAL PEDIATRIC BRAIN CANCERS
Authors: Sebastian Mathew , Pham Christina , Wummer Brandon , Woodworth Delaney , Reid Alexandra , Abraham Rebecca , Gil Jorge , Drake Jeffrey , Mitchell Duane , Flores Catherine
Published: November 2022

BACKGROUND

Mutated neoantigens have shown promise as targets for cancer immunotherapy but embryonal pediatric brain tumors with notoriously low mutational burden have more limited opportunities for neoantigen directed therapies. Evidence suggests that embryonal brain tumors such as medulloblastoma (MB) and brain stem gliomas (BSG) arise from the aberrant reactivation of fetal developmental programming. We explored the hypothesis that neonatal mouse cerebellum and brainstem express developmentally regulated proteins that could serve as potent tumor rejection antigens for a preclinical model of Group 3 MB (NSC) and H3.3K27M positive BSG (K2), respectively. We thus generated tumor-reactive T cells by using P5 cerebellum and P4 brain stem RNA as sources of antigens. P5-cerebellum-specific T cells (cDevAg-T cells) and P4-brain stem-specific T cells (bsDevAg-T cells) demonstrated effector function against respective MB and BSG tumor cells with exquisite specificity.

METHODS

RNA was isolated from P5 cerebellum and P4 brain stem and pulsed into bone marrow-derived dendritic cells. These were then used to in vitro activate splenocytes from previously immunized mice, generating either cDevAg-T cells or bsDevAg-T cells. In vitro functionality assays against tumor targets were conducted to determine reactivity and specificity. DevAg-T cells were used in adoptive cellular therapy in orthotopic models to determine therapeutic efficacy of DevAg-specific T cells in vivo. Results and

CONCLUSIONS

DevAg-T cells produce high levels of Th1-type cytokines that recognize distinct subtypes of MB and BSG, show no cross-reactivity with normal brain. Adoptive cellular therapy employing DevAg-specific T cells demonstrate a significant survival benefit in orthotopic models of established Group 3 NSC MB and H3.3K27M mutation positive BSG. Our studies demonstrate that RNA encoding non-mutated and organ-specific developmental antigens can serve as novel tumor rejection antigens for pediatric brain cancers.
EXTH-88. DOCETAXEL TARGETS AGGRESSIVE METHYLATION PROFILES AND SERVES AS A RADIOSENSITIZER IN HIGH-RISK MENINGIOMAS
Authors: Youngblood Mark , Tran Anh , Wang Wenxia , An Shejuan , Scholtens Denise , Zhang Lyndsee , O’Shea Kaitlyn , Pokorny Jenny , Magill Stephen , Sachdev Sean , Lukas Rimas , Ahmed Atique , Unruh Dusten , Walshon Jordain , McCortney Kathleen , Wang Yufen , Baran Aneta , Sahm Felix , Aldape Kenneth , Chandler James , Heimberger Amy , James David , Horbinski Craig
Published: November 2022

DNA methylation profiling has emerged as a transformative prognostic tool in meningiomas, identifying distinct epigenetic subgroups that share unique clinical and biological features. Hypothesizing that methylation patterns may also reflect underlying pathogenic mechanisms, we investigated if this modality could be informative for the selection of efficacious chemotherapies in meningiomas, focusing on FDA-approved compounds that are well-tolerated in patients. Using a methylation dataset of 210 tumors, we identified 981 genes in which adjacent CpG sites were predictive of progression-free survival. Pathway enrichment of these genes nominated Docetaxel as a candidate therapeutic; a well-established taxane that is routinely used for breast, prostate, and lung cancers. Using 15 primary patient-derived cultures and 2 cell lines, we found that Docetaxel could inhibit meningioma growth at concentrations that were favorable relative to other cancers. This drug triggered apoptosis in meningiomas of all tumor grades and methylation classes, with IC50 values ranging from 0.8nm to 4.4nm in vitro. Combining Docetaxel with radiation therapy resulted in synergistic cytotoxic effects on meningioma cultures, associated with elevated markers of cell death and dsDNA breaks. We confirmed our results via orthotopic meningioma mouse models of the cell lines CH157 and IOMM-Lee, which exhibited dose-dependent improvements in mortality and tumor volume in response to Docetaxel and radiation. Our results leverage DNA methylation patterns to repurpose FDA-approved medications in the treatment of meningiomas, and suggest a clinical trial of Docetaxel with radiotherapy may be efficacious in higher-grade lesions.
EXTH-91. GALECTIN-3 INHIBITOR PAIRED WITH FOCAL LOW-INTENSITY NONINVASIVE TDCS CAN ACHIEVE MAXIMAL THERAPEUTIC BENEFIT IN CLINICALLY RELEVANT MENINGIOMA MOUSE MODELS
Authors: Das Arabinda , McDonald Daniel G , Yazdani Milad , Stephenson Connor , Mehrotra Shikhar , Welsh Cynthia A , Infinger Libby Kosnik , Vandergrift William A , Varma Abhay K , Patel Sunil J , Giglio Pierre , Zukas Alicia , Cachia David , Lindhorst Scott M
Published: November 2022

Atypical and anaplastic meningiomas (WHO Grade 2 and WHO Grade 3 respectively) represent a subgroup of meningiomas that tend to have higher rates of recurrence with associated’s higher morbidity and mortality than the more common WHO Grade 1 meningiomas. Our laboratory data demonstrated that galectin-3 (Gal-3), a multifunctional β-galactoside-binding protein, is highly expressed in these meningioma subtypes as compared to normal brain tissue. However, the root of the profoundly immunosuppressive tumor microenvironment in meningioma cells is not fully understood. To address this, we used a Gal-3 inhibitor TD 139 paired with low-intensity non-invasive transcranial direct current stimulation (tDCS) to suppress tumor growth and enhance the immune response in the preclinical model. Treatment of tDCS (50 mV/mm for 30 min) followed by 1 mM of TD139 induced cell death in anaplastic meningioma in vitro human triple co-culture model but has no effect on mouse cortical neurons. Furthermore, we demonstrated that inhibiting of Gal-3 expression significantly decreased the protein levels of urokinase-type plasminogen activator receptor as well as phosphorylation of Akt and upregulates Bax expression. Treatment with TD139 (1mg/kg per day for 14 days) with tDCS [350 μA (1 x 30 min/day) for 7 times (within 14 Days) showed a significantly (∼60%) decrease in MGS2 atypical meningioma tumor growth in orthotopic allograft model (at Day 41). The percent survival of tumor-bearing mice treated with TD139 and tDCS was significantly higher than an untreated tumor or single treatment. Following TD139 plus tDCS treatment, our results demonstrated significant meningioma cell death via a decrease in NDRG4 (Meningioma marker) and were associated with downregulation of Ki-67 in treated tumors. Based on our substantial preliminary data, we believe that this proposed translational work has the potential to be an effective combinational modality form of treatment for atypical and malignant meningiomas.
QLTI-07. IDENTIFYING RISK FACTORS FOR PROLONGED TRANSFUSION-DEPENDENT TEMOZOLOMIDE-INDUCED THROMBOCYTOPAENIA IN PATIENTS RECEIVING CONCURRENT CHEMO-RADIOTHERAPY FOR GLIOBLASTOMA (GBM)
Authors: Ismail Nashwah , Patel Karan , Parikh Tapan , Colaco Rovel , McBain Catherine
Published: November 2022

BACKGROUND

Temozolomide-induced thrombocytopaenia is a well-recognised clinical issue with significant inter-patient variation. However, knowledge of the extent of the problem and its potential risk factors remains limited. Our aim was to determine the incidence of patients requiring platelet transfusion and ascertain associated risk factors using real-world data.

METHODS

Data from patients with GBM receiving concurrent temozolomide-chemo-radiotherapy (CRT) from 01/01/18-31/12/2019 retrospectively reviewed. Patients receiving platelet transfusion identified (Local policy: transfusion for platelet count ≤ 30x109/L). Logistic regression and chi-squared tests used to compare variables in patients requiring platelet transfusion and those not.

RESULTS

111 patients with GBM received TMZ-CRT (60Gy/30#; 75mg/m2 temozolomide). 12/111 patients received platelet transfusion: 10 during concurrent-CRT and 2 during adjuvant-treatment. Numbers of transfusions: 1-2: 7 patients; 3-4: 2 patients; ≥ 5 transfusions: 3 patients. Median time from platelet count ≥ G3 to ≥ 100 in 11/12 patients: 38 days (range 6-72). 1 did not recover, patient died 84 days following start of TMZ-CRT with thrombocytopaenia. Comparison of the transfused vs the non-transfused cohort shows: transfused: 50% female; non-transfused: 36% female. Mean BSA: transfused: 1.73, SD0.21; non-transfused:1.96, SD0.22 (OR 0.01; 95% CI:0.0003 – 0.16). Mean age: transfused: 57, SD15.8; non-transfused: 58, SD10.7 . Mean baseline platelet count: transfused: 329, SD185; non-transfused: 319, SD117. Mean PTV: transfused: 400, SD115; non-transfused: 414, SD123. There were no statistically significant associations with demographics, age, blood parameters or planning target volume.

CONCLUSION

This study demonstrates a subset of patients who experience severe and prolonged thrombocytopaenia. Risk factors elucidated are female sex and lower BSA. Large-scale prospective trials evaluating a panel of predictors including concomitant medications, patient MGMT variants and temozolomide-metabolite concentrations would be valuable. Precise stratification of this vulnerable subgroup may permit consideration of temozolomide dose-capping in the future. Careful informed consent with all patients and caution with intensifying treatment under trials is vital presently.