bluuebunny/crossref_metadata_2025_split huggingface dataset:
EXTH-29. RADIOSENSITIZATION BY BRD4 INHIBITION IN DIFFUSE INTRINSIC PONTINE GLIOMA
Authors: Watanabe Jun , Clutter Matt , Suri Amreena , Abe Koki , Ishi Yukitomo , Becher Oren , Hashizume Rintaro 
Published: November 2022

Abstract
Diffuse intrinsic pontine glioma (DIPG), the most frequent brainstem tumor in pediatrics, is one of the devastating childhood cancers, and virtually all patients die within two years after diagnosis. Since these tumors occur in the brainstem which is vital area, there are no surgical options for providing relief to patients, and conventional chemotherapy. Radiation therapy (RT) remains the only effective treatment. Radiosensitization is a mean to improve the therapeutic balance between efficacy and toxicity of RT, but not used in clinically. Here, we found BRD4 inhibitor as a radiosensitizer from drug screening. DIPG shows increased H3 K27 acetylation (H3K27ac), which binds to BET bromodomain protein 4 (BRD4) and they are strongly associated with active transcription. We tested two BRD4 inhibiter (BRD4i : AZD5153 and JQ-1) and genetic BRD4 depletion enhances radiation-induced DNA damage, making it a potential radiosensitizer in the treatment of DIPG. We evaluated the effects of BRD4i on genes expression using RNA sequence, inhibited significantly DNA-repair proteins such as BRCA1 and RAD51. CUT-RUN qPCR showed decreased H3K27ac at BRCA1 and RAD51. Combination with BRD4i and RT inhibited cell viability significantly in clonogenic survival assay, apoptosis assay, and showed cell cycle arrest. Radiation-induced DNA double-strand break (DSB) repair was prolonged high level of rH2AHX and 53BP1 with BRD4i because of inhibited DNA-repair. In vivo studies revealed increased survival of animals treated with combination therapy of RT and BRD4i in compared to either monotherapy. Together, these results highlight BRD4i as a potential radiosensitizer and provide a rationale for developing combination therapy with radiation in the treatment of DIPG.
EXTH-36. MRGFUS-DELIVERED FLUORESCENT EGFR/EGFRVIII ANTIBODY ENABLES THERANOSTIC IMAGING OF PEDIATRIC HIGH-GRADE GLIOMA AND PREDICTS RESPONSE TO TARGETED THERAPY
Authors: Zhou Quan , Aryal Muna , Leonel Johana Vega , Santoso Michelle , Zlitni Aimen , Vogel Hannes , Cayrol Romain , Li Gordon , Moseley Michael 
Published: November 2022

Abstract

BACKGROUND
Pediatric high-grade glioma (pHGG) consists of the deadliest childhood brain cancers refractory to current therapies. Fluorescent antibodies against epidermal growth factor receptor (EGFR) have demonstrated clinical promise in image-guided surgery of adult gliomas. However, their potential theranostic use for pHGG remains unexamined. This study evaluated an EGFR/EGFRvIII-targeted imaging and treatment strategy as an alternative therapeutic approach for pHGG using tracer amount of fluorescent antibody delivered through blood-brain barrier modulation.

METHODS
Immunohistochemical stainings of EGFR, EGFRvIII and claudin-5 were performed on pHGG patient tissue (n = 66). In vitro competition assay assessed binding affinity of panitumumab-IRDye800 for EGFR/EGFRvIII on pHGG cells whose viability was measured upon panitumumab treatment. Mice with EGFR/EGFRvIII-positive orthotopic pHGG patient-derived xenografts received 1 mg/kg panitumumab-IRDye800 20 hours after transcranial magnetic resonance-guided focused ultrasound (MRgFUS) sonication (2 min at 0.25 MPa). Near-infrared fluorescence imaging measured tumor-to-background ratio and TUNEL assay determined apoptotic rate in resected tumor. Survival was documented for 60 days in pHGG-bearing mice receiving 1 mg/kg panitumumab weekly with or without MRgFUS.

RESULTS
61% and 24% pHGG patient tissue respectively expressed EGFR and EGFRvIII (17% expressing both). Heterogeneous claudin-5 expression indicated partial blood-brain barrier permeability. Panitumumab-IRDye800 bound to EGFR/EGFRvIII-positive pHGG with high affinity (IC50: 4.2 nM). EGFR blocking enhanced therapeutic response of panitumumab by 48 ± 13%. Following MRgFUS in pHGG-bearing mice, 19 ± 6.3% injected dose of intratumoral panitumumab-IRDye800 uptake resulted in 2.1 ± 0.4 tumor contrast and 3.9 ± 1.1% apoptotic cell death. Weekly MRgFUS with 1 mg/kg panitumumab prolonged median survival (27.5 vs. 17.5 days, P &lt; 0.0001, n = 10).

CONCLUSION
Tracer dose of panitumumab-IRDye800 improved tumor contrast and predicted antitumor activity via EGFR/EGFRvIII dual targeting following MRgFUS, potentially benefiting 68% of pHGG population. Reduced therapeutic dose of panitumumab by EGFRvIII-focused targeting may improve treatment safety and efficacy against EGFRvIII-stratified gliomas.
EXTH-41. COMBINED PHARMACOLOGICAL AND GENETIC SCREENING TO IDENTIFY DEPENDENCIES AND COMBINATIONS IN ACVR1-MUTANT DIFFUSE MIDLINE GLIOMA
Authors: Rogers Rebecca , Carvalho Diana , Grabovska Yura , Fernandez Elisabet , Izquierdo Elisa , Mackay Alan , Jones Chris 
Published: November 2022

Abstract
Somatic mutations in ACVR1, which encodes the serine/threonine kinase ALK2, are found in 20-25% of DMG-H3K27 patients. Treatment of ACVR1-mutant patient-derived models with multiple chemotypes of ALK2 inhibitors (ALK2i) results in reduced cell viability in vitro and extended survival in orthotopic xenografts in vivo but, as single agents, these inhibitors were unable to achieve a complete anti-tumour response. Recently we reported that combinatorial treatment of ACVR1-mutant DIPG cells with vandetanib (RTK inhibitor) and everolimus (mTOR/ABC transporter inhibitor) was synergistic both in vitro and in vivo and was shown to be a feasible combination to trial clinically in this setting. To identify specific dependencies in ACVR1-mutant cells which may be translatable with novel synergistic drug combinations alongside ALK2i, we have implemented both candidate and unbiased drug and genetic screening approaches. Using a panel of 13 patient-derived ACVR1-mutant models (and 6 wild-type controls), we identified synergy between multiple chemotypes of ALK2i (M4K2009/LDN-214117) and PI3K/mTOR (AZD8055/everolimus) and MEK inhibitors (trametinib), reflecting the common co-segregation of PIK3CA/PIK3R1 alterations in these tumours. Whole-genome CRISPR/Cas9 screening of ACVR1-mutant SU-DIPG-IV cells in combination with two ALK2i (M4K2009/LDN-193189), confirmed a specific MTOR genetic dependency, as well as for the protein phosphatase regulatory subunit PPP2R1A, known to play a role in MAPK pathway activation. Additional hits include the serine/threonine kinase PKMYT1, a negative regulator of the G2/M checkpoint via a functionally redundant phosphorylation of CDK1/CCNB1 alongside WEE1; confirmatory drug assays with the WEE1 inhibitor AZD1775 resulted in a synergistic interaction with ALK2i in ACVR1-mutant cells. Hits were integrated with DepMap using ‘gene effect’ scores (Chronos) enabling filtering of common essential genes. Preliminary pathway enrichment analysis (MAGeCKFlute) identified ALK2i-specific vulnerabilities involving TGFB1/SMAD signalling and histone deacetylation. These data highlight functionally rational and novel combinatorial possibilities for children with ACVR1-mutant DMG, with systematic preclinical assessment required for prioritisation for the clinic.
EXTH-44. PERSONALIZED IMMUNOTHERAPY VACCINES FOR GLIOBLASTOMA AND THEIR TREATMENT EFFICACY
Authors: Trivedi Vrunda , Yang Changlin , Fenton Graeme , Yegorov Oleg , Von Roemeling Christina , Klippel Kelena , Ogando-Rivas Elizabeth , Castillo Paul , Hoang-Minh Lan , Dyson Kyle , Moore Ginger , Mitchell Duane 
Published: November 2022

Abstract

BACKGROUND
Glioblastoma multiforme (GBM) remains a disease with debilitating survival outcomes. Cancer immunogenomics represents a complementary approach to the application of genomics in developing novel immunotherapies for brain malignancies. Using a cancer immunogenomics approach that we developed, called the Open Reading Frame Antigen Network (O.R.A.N.), we identified the immunogenic neoantigens and tumor-associated antigens (TAAs) including cancer-testis and developmental antigens, that are aberrantly overexpressed in murine models of GBM. The aim of this study is to evaluate the immunologic effects and safety of immunotherapy vaccines targeting neoantigens and TAAs in preclinical models of GBM.

METHODS
RNAseq and WES were performed for murine glioblastoma tumors- KR158-Luc and GL261, and the results were fed to the O.R.A.N pipeline for antigen prediction. A tumor antigen-specific RNA library was created for each tumor using a gene enrichment strategy and validated by RNAseq. Tumor-bearing animals were treated with adoptively transferred ex vivo expanded lymphocytes and dendritic cell vaccines loaded with the tumor antigen-specific RNA. Tumor volume, and thus progress, was determined using in vivo luciferase imaging technique and the survival outcomes were noted. We also evaluated the efficacy of the tumor vaccines in combination with checkpoint blockade therapy by treating tumor-bearing animals with dendritic cell vaccines loaded with the tumor antigen-specific RNA combined with an anti-PD-1 antibody.

RESULTS
The dendritic cell vaccines loaded with tumor antigen-specific RNA were significantly effective in slowing the progression of murine GBM tumors in combination with both the adoptive cellular therapy as well as checkpoint blockade therapy. Additionally, we identified antigen-specific T cells targeting several of our predicted antigens and an increase in tumor-infiltrating lymphocytes and memory T cells in the treated animals.

CONCLUSION
We developed a dendritic cell-based vaccination approach targeting neoantigens and TAAs identified as being tumor-specific and evaluated the efficacy of immunotherapy vaccines in preclinical models of GBM.
EXTH-46. RNA LIPID PARTICLES INDUCE BI-DIRECTIONAL IMMUNITY AGAINST DIFFUSE MIDLINE GLIOMA
Authors: McGuiness James , Weidert Frances , Zhang Dingpeng , Grippin Adam , Karachi Aida , Qdaisat Sadeem , Thomas Nagheme , Castillo Paul , Huang Jianping , Ligon John , Silver Natalie , Rahman Maryam , Hwang Eugene , Mitchell Duane , Mendez-Gomez Hector , Sayour Elias 
Published: November 2022

Abstract

BACKGROUND
Diffuse Intrinsic Pontine Glioma (DIPG) is uniformly fatal. Upon diagnosis, DIPG cannot be safely debulked and systemic therapies have unproven benefit. Immunotherapy can overcome these obstacles, but objective responses are rare; and cancer antigens, even when tumor specific (i.e. H3K27M), are usually poorly immunogenic.

OBJECTIVE
We sought to develop a murine model of diffuse midline glioma (DMG) that better recapitulates human disease. In these models, we tested different iterations of mRNA loaded nanoparticle (NP) vaccines for their therapeutic effect. APPROACH: We implanted murine gliomas (expressing H3K27M mutation) midline into developing neonatal brains. We manufactured different mRNA constructs encoding for foreign proteins (e.g. GFP), H3K27M or wildtype H3K27 and evaluated immunogenicity and anti-tumor efficacy. These mRNA vaccines were layered into lipid particles (RNA-NP) and administered intravenously when animals became acutely symptomatic (similar to when patients are diagnosed).

RESULTS
Surprisingly, all mRNA constructs tested were sufficient to elicit anti-tumor efficacy against DMGs. This was true for both antigen specific (H3K27M) and non-antigen specific constructs (GFP, H3K27 wildtype). While H3K27M encoding RNA-NPs were superior to H3K27 wildtype encoding RNA-NPs (p=0.057), the adaptive immune effects appear marginal relative to the innate immune responses generated by all RNA-NP constructs. These innate responses are characterized by induction of type I interferon response from plasmacytoid DCs. Interestingly, these effects could not be recapitulated in murine models of adult type glioblastoma (KR158b) which required antigen specificity for induction of anti-tumor efficacy.

CONCLUSION
These data suggest that pediatric gliomas may be particularly sensitive to innate immunity. We have optimized development of a target H3K27M mRNA construct that balances innate and adaptive bi-directional immune induction. We are completing FDA-IND enabling data to support translation of H3K27M loaded RNA-NPs into human clinical trials.
EXTH-47. PRECLINICAL EFFICACY OF LP-184, A TUMOR SITE ACTIVATED SYNTHETICALLY LETHAL THERAPEUTIC, IN GLIOBLASTOMA
Authors: Lal Bachchu , Kulkarni Aditya , McDermott Joseph , Lopez-Bertoni Hernando , Bhatia Kishor , Sharma Panna , Laterra John 
Published: November 2022

Abstract
Temozolomide (TMZ) is currently the most effective standard-of-care chemotherapy based on its ability to prolong survival of patients with newly diagnosed MGMT-methylated GBM. Blood-brain barrier (BBB) permeable agents effective against TMZ-resistant GBMs (i.e. recurrent GBM, MGMT unmethylated GBM) are desperately needed. Lantern Pharma is advancing LP-184, an acylfulvene prodrug activated by the enzyme PTGR1, in GBM. In an MGMT expressing TMZ resistant GBM PDX model, LP-184 was found to be ~5000X more potent than TMZ. DNA damage induced by acylfulvenes is primarily repaired via transcription-coupled nucleotide excision repair (TC-NER) mediated by ERCC complexes and gene set variation analyses associate LP-184 cell sensitivity with low TC-NER. ERCC3 is a key NER factor that is proteolytically degraded in response to the FDA approved BBB permeable diuretic, spironolactone (SP). After verifying that spironolactone induces rapid ERCC3 degradation by up to 95% in GBM cells, we validated that SP-induced ERCC3 loss led to substantial increases in LP-184 response in vitro and in subcutaneous tumor xenografts. Co-treatment of GBM cells with LP-184 and 5 or 25 µM SP resulted in a 3-6 fold decrease in LP-184 IC50s in M1123, Mayo39 and U87 cultures in vitro. Mice bearing pre-established subcutaneous U87 xenografts were treated with SP alone, LP-184 alone or their combination. Tumors recurred after initial regression in 5/5 animals treated with LP-184 alone whereas tumors showed durable complete regression without recurrence in 4/5 animals treated with LP-184 + SP. LP-184 is a promising chemotherapeutic with potential clinical translation in GBM patients. Our results indicate that LP-184 is MGMT-agnostic and effective in TMZ-resistant pre-clinical GBM models. Our findings also identify ERCC3-dependent TC-NER activity as a determinant of LP-184 synthetic lethality predicting that LP-184’s therapeutic potential will be significantly enhanced in patients with intrinsic or spironolactone-induced NER deficient tumors.
EXTH-53. IL-12 ARMORED CAR T CELL THERAPY FOR HETEROGENEOUS GLIOBLASTOMA
Authors: Shen Steven , Cook Sarah , Suryadevara Carter , Hotchkiss Kelly , Snyder David , Sanchez-Perez Luis , Sampson John 
Published: November 2022

Abstract

INTRODUCTION
Chimeric antigen receptor (CAR) T cells specific for the glioblastoma (GBM)-specific epidermal growth factor receptor variant III (EGFRvIII; CARvIII) have successfully treated tumors homogeneously expressing EGFRvIII when combined with lymphodepletion, however this is not recapitulated clinically. We generated an “armored” CARvIII which constitutively secretes IL-12, a stimulatory cytokine that enhances T cell persistence and function, that is capable of treating orthotopic heterogeneous GBM in immune competent mice.

METHODS
C57Bl/6 mice were intracranially (IC) implanted with 5 x 105 of either homogenous (CT2AvIII) or heterogeneous (1:1 CT2AvIII and CT2A parental) tumor cells. Mice were treated with 2 x 106 CARvIII or IL-12 CARvIII seven days post tumor implant and monitored for survival. TCRα -/-, CD8-/- and C57Bl/6 mice were IC implanted with homogeneous or heterogeneous tumors and treated 7 days post implant with intracranial IL-12 CARvIII therapy to assess the role of endogenous T cells. T cells within the tumor microenvironment were characterized by flow cytometry days 8, 14, and 17 post tumor implantation.

RESULTS
IL-12 CARvIII cells were successfully generated, secreted IL-12, and were cytotoxic against EGFRvIII-expressing tumor cells in vitro. IL-12 CARvIII therapy was curative in homogenous CT2AvIII tumors (p&lt; 0.0001) and conferred a long-term survival in 50% of mice with heterogeneous CT2AvIII:CT2A parental tumors in vivo (p&lt; 0.0001). Furthermore, IL-12 CARvIII therapy successfully eradicated the homogeneous CT2AvIII tumors in TCRα -/- and CD8-/- mice but failed to produce any efficacy against the heterogeneous CT2AvIII:CT2A parental tumors (p=0.0002). Endogenous T cells in IL-12 CARvIII treated mice were found to be more migratory, and interestingly more exhausted than in CARvIII treated mice.

CONCLUSIONS
Our findings show that IL-12 CARvIII can effectively eradicate IC homogenous tumors without lymphodepletion. Surprisingly, IL12 CARvIII therapy also treated IC heterogeneous glioma. Heterogeneous tumor clearance required an endogenous CD8 T cell response.
EXTH-54. CO-CULTURE OF PATIENT-DERIVED GLIOBLASTOMA CELL LINES WITH AUTOLOGOUS PBMCS FOR THE INVESTIGATION OF ONCOLYTIC VIRUS RESPONSE
Authors: Stavrakaki Eftychia , van den Bossche Wouter B L , Balvers Rutger , Vogelezang Lisette B , Teodosio Cristina , Mustafa Dana A M , de Koning Willem , Stubbs Andrew P , Goins William F , Nakashima Hiroshi , Chiocca E Antonio , van Dongen Jacques J M , Dirven Clemens M F , Lamfers Martine L M 
Published: November 2022

Abstract

BACKGROUND
The dismal prognosis of glioblastoma (GBM) patients, with a median survival of less than 15 months despite maximal therapy urgently warrants new therapeutic approaches. Clinical trials employing oncolytic viruses (OVs) have shown encouraging results, however, in each OV clinical trial only a small subset of patients responded to treatment. As inter-tumoral heterogeneity has been the key challenge in treating GBM, we hypothesized that development of an in vitro co-culture model for assessment of viral replication and subsequent immune response might ultimately predict in vivo OV efficacy for individual GBM patients.

METHODS
20 patient-derived GBM cell cultures were tested with dose-ranges of two clinically relevant OVs (DNX2401 and rQnestin34.5 V1) to determine the EC50 values (half-maximal effective concentration). To discriminate between responders and non-responders six of these cultures were infected using MOI 10 for DNX2401 and 0.25 for rQnestin34.5V1, and co-cultured with autologous PBMCs. OV-induced changes in gene and protein expression of immune associated genes were assessed using targeted gene expression (NanoString Technology) and ELISA.

RESULTS
DNX2401 EC50 values ranged from 0.35 to &gt; 600 and from 0.04 to 1.77 for rQnestin34.5V1. Induction of pro-inflammatory cytokines and chemokines differed per virus and per patient. Enhanced OV infection or oncolysis efficiency did not lead to increased levels of IFNγ production. Importantly, longer exposure to dexamethasone prior to PBMC isolation correlated with suppressed IFNγ production.

CONCLUSION
We established an autologous GBM cells/PBMCs co-culture model which reflects inter-tumoral heterogeneity in terms of cytokine induction and virus-specific changes in gene and protein expression upon OV infection. Immune activation is not directly related to degree of infection or oncolysis and can be hampered by prolonged prior dexamethasone use. These first results support our hypothesis that improved prediction of response rates in oncolytic virotherapy for GBM may require a personalized approach with an in vitro test system.
EXTH-59. IMMORTALIZATION OF MOUSE BONE MARROW-DERIVED MACROPHAGES FOR BRAIN TUMOR THERAPY DEVELOPMENT
Authors: Joseph Benson , Ann Liu , Patil Chirag , Black Keith , Sun Tao 
Published: November 2022

Abstract

BACKGROUND
Macrophages can cross blood-brain-barrier (BBB) and infiltrate brain parenchyma with malignancies such as glioma. Recently, many efforts have been made to develop macrophage-based cancer therapy, including CAR–macrophages. However, lack of proliferation in differentiated macrophages hinders their utility. We sought to ameliorate this problem by immortalizing macrophages to assist potential therapy development. Bone marrow derived macrophages (BMDMs) were harvested from C57Bl/6 mice and transduced with mouse telomerase reverse transcriptase (mTert) gene via retrovirus. Following puromycin selection, morphology, proliferation, lineage marker expression and phagocytosis were measured by microscopy, flow cytometry and IncuCyte assays.

RESULTS
Through overexpression of mTert, we have successfully immortalized mouse BMDMs, termed as iBMDMs. Morphologically, iBMDMs largely maintained features of primary BMDMs. The established iBMDMs proliferated for over 20 subcultures without obvious reduction in growth. Proliferation of iBMDMs (doubling time, dt=52h) was similar to IC-21(dt=53h), an established mouse macrophage line immortalized by SV40-T antigen, and slower than Raw264.7 (dt=23h), an immortalized mouse macrophage line driven by murine leukemia virus. Furthermore, iBMDMs can grow without macrophage colony-stimulating factor (M-CSF) (dt=54h). Macrophage lineage markers CD11B and F4/80 expression levels were higher in primary BMDMs (CD11B+=62.8±0.53%, F4/80+=54.9±0.38%, CD11B+F4/80+=52.7±0.41%, n=2 for all flow results) and iBMDMs (CD11B+=39.1±5.29%, F4/80+=26.4±7.93%, CD11B+F4/80+=24.6±5.95%) than in IC-21 (CD11B+=10.7±0.37%, F4/80+=19.4±1.88%, CD11B+F4/80+=9.13±0.37%) and Raw264.7 (CD11B+=34.3±5.32%, F4/80+=14.1±4.40%, CD11B+F4/80+=8.33±4.19%) cells lines. Lastly, we evaluated the phagocytic ability of iBMDMs through IncuCyte assays. Similar to primary BMDMs, we observed a concentration dependent increase in red fluorescence, which indicates the phagocytosis of E. coli derived bioparticles, in iBMDMs with and without M-CSF.

CONCLUSIONS
We immortalized bone marrow-derived macrophages of C57Bl/6 mouse origin with competent immune function via mTert transduction. These cells can be used in preclinical immunotherapy development for glioma treatment. Ongoing work is focused on assessing their ability to penetrate BBB and infiltrate brain tumor in a mouse glioma model.