bluuebunny/crossref_metadata_2025 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

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

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 < 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

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

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

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

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

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< 0.0001) and conferred a long-term survival in 50% of mice with heterogeneous CT2AvIII:CT2A parental tumors in vivo (p< 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

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 > 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

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.
EXTH-63. COMBINED INHIBITION OF TOP1 AND PARP: A NOVEL THERAPEUTIC STRATEGY FOR GBM WITH PTEN DEFICIENCY
Authors: Kim Olga , Butler Madison , Robey Robert , Chari Raj , Sergi Zach , Zhang Meili , Pang Ying , Yu Guangyang , Zhang Wei , Song Hua , Davis Dionne , Wang Herui , Merchant Mythili , Ranjan Alice , Gilbert Mark , Gottesman Michael , Pommier Yves , Wu Jing
Published: November 2022

BACKGROUND

Glioblastoma (GBM) is the most aggressive and lethal type of brain tumor. Activation of PI3K/mTOR pathway along with the loss of its primary negative regulator, phosphatase and tensin homolog (PTEN), occurs in nearly 50% of GBM patients. As PTEN is known to promote DNA damage repair deficiency, here we investigated whether PTEN deficiency presents a vulnerability to a simultaneous induction of DNA damage and suppression of repair mechanisms by combining topoisomerase I (TOP1) and PARP inhibitors.

METHODS

We used patient-derived GBM cells and stem-like cells to determine response to LMP400 (Indotecan), a novel non-camptothecin TOP1 inhibitor, and the PARP inhibitors Olaparib or Niraparib. Treatment efficacy was also determined using cell viability, cell cycle, DNA damage, repair, and apoptosis assays in a pair of isogenic PTEN-null and PTEN-WT glioma cell lines derived from a genetically engineered mouse GBM model. RNAseq analysis was performed to identify treatment-induced dysregulated pathways.

RESULTS

PTEN-deficient cells are highly sensitive to LMP400 and PTEN rescue lessens sensitivity to the treatment. Combining LMP400 with PARP inhibitors, Olaparib or Niraparib, leads to synergistic cytotoxicity. LMP400/Niraparib combination induces G2/M cell cycle arrest, DNA damage, suppression of homologous recombination (HR)-related proteins and activation of caspase 3/7 activity significantly more in PTEN-null cells compared to isogenic PTEN-WT cells. Gene set enrichment analysis revealed suppression of cell cycle and DNA damage repair as well as activation of cell death pathways. Finally, CRISPR-Cas9 KO screening suggests that LMP400 is not likely to be a substrate for ABC transporters, suggesting the brain penetration and supporting the use in brain tumor patients.

CONCLUSION

Combined inhibition of TOP1 and PARP induces synergistic antiglioma effects selectively in PTEN-null glioblastoma cells, providing a strong scientific premise for a clinical trial of combined treatment with LMP400 and Niraparib in a subset of GBM with PTEN deficiency.
EXTH-65. SYNERGISTIC ONCOLYTIC EFFECTS OF TEMOZOLOMIDE AND ONCOMAGNETIC THERAPIES IN GLIOBLASTOMA
Authors: Pandey Arvind , Hambarde Shashank , Baskin David , Helekar Santosh
Published: November 2022

The noninvasive Oncomagnetic device developed in our laboratory selectively kills glioblastoma (GBM) and other cancer cells while sparing normal developing neurons and astrocytes in vitro. We have reported that brief 2 – 6 h of daily Oncomagnetic brain stimulation causes >30% reduction of contrast-enhanced tumor (CET) volume in an end-stage recurrent GBM patient after a ~30-day treatment. We have evidence that the spinning oscillating magnetic fields (sOMF) generated by this device disrupts electron transport in the mitochondrial respiratory chain, at least in part by inhibiting the activity of Complex II succinate dehydrogenase, leading to a marked increase in reactive oxygen species (ROS) and consequent cancer cell death. Here, we further studied the cellular effects of sOMF treatment alone and in combination with Temozolomide (TMZ) on GBM cell lines with other methods and investigated the response to Oncomagnetic monotherapy (OMT) in a mouse GBM model. We treated LN229 and U87 GBM cells with sOMF with and without a sub-therapeutic dose of TMZ. To study the anticancer effect of sOMF in vivo, we developed syngeneic GBM tumors in BALB/c mice by stereotactic intracranial injection of GL261WT cells. Our results show that cytotoxic effects of sOMF or TMZ alone, as measured by the MTT assay and reduced cell survival, are significantly potentiated by combining the two treatments. OMT of tumor-bearing mice using a whole-body sOMF stimulation method causes substantial retardation of tumor growth and reduction of the CET volume in 9.4 T MRI scans. To study the molecular pathways involved in the treatment response we are mapping alterations in various key biomarkers in treated tumor tissues and comparing them with those in untreated controls. These experiments will unravel the molecular mechanism of action underlying the response to OMT in GBM and help develop a sound rationale for an effective therapeutic protocol.
EXTH-70. IMMUNOTHERAPY REVERSES GLIOMA-DRIVEN DYSFUNCTION OF IMMUNE SYSTEM HOMEOSTASIS
Authors: Dean Bayli , Wildes Tyler , Dean Joseph , Shin David , Francis Connor , Sebastian Mathew , Font Laura Falceto , Moore Ginger , Wummer Brandon , Flores Catherine
Published: November 2022

BACKGROUND

Glioma-induced immune disfunction has been described in a limited number of studies, and here we further demonstrate that gliomas also interrupt the cellular differentiation programming and outcomes of hematopoietic stem cells (HSC) in the bone marrow. HSCs from glioma-bearing mice are re-programmed and driven towards expansion of myeloid lineage precursors myeloid-derived suppressor cells (MDSC) in secondary lymphoid organs. However, we found this is reversed by immunotherapy. Adoptive cellular therapy (ACT) has been demonstrably efficacious in multiple preclinical models of CNS malignancies, and here we describe how glioma-induced disfunction is reversed by this immunotherapeutic platform.

METHODS

The impact of orthotopic KR158B glioma on HSCs was evaluated in an unbiased fashion using single cell RNAseq of lineage- cells and phenotypically using flow cytometry. Mature myeloid cell frequencies and function were also evaluated using flow cytometry. Finally, ACT containing total body irradiation, tumor-specific dendritic cells, tumor-reactive T cells, and HSCs isolated from glioma-bearing or non-tumor-bearing mice was used to evaluate cell fate differentiation and survival.

RESULTS

We observed differential gene expression of HSCs in tumor-bearing versus healthy mice coupled with an expansion of myeloid lineage subsets in glioma-bearing mice. Interestingly, MDSCs from glioma-bearing mice demonstrated hyper suppressive capacity as compared to MDSCs from non-tumor-bearing hosts. Interestingly, treatment with ACT overcame the suppressive properties of glioma-bearing HSCs. When HSCs from tumor-bearing mice were transferred with ACT, we observed significant survival benefit and long-term cures in orthotopic glioma models compared with mice treated with ACT using non-glioma-bearing HSPCs.

CONCLUSIONS

These findings demonstrate the suppressive disposition in HSCs from glioma-bearing hosts can be redirected using ACT for significant survival benefit. Collectively, these data demonstrate gliomas impact the hematopoietic compartment at the progenitor level to promote expansion of suppressive myeloid cells and their progenitors. However, ACT can overcome this bias and provide lasting anti-tumor effects.
EXTH-75. INTEGRATED STRESS RESPONSE ACTIVATORS SYNERGISTICALLY INDUCE APOPTOSIS AND SUPPRESS PROLIFERATION IN DIFFUSE INTRINSIC PONTINE GLIOMA
Authors: Novak Orlandi , Arnold Antje , Eberhart Charles , Raabe Eric
Published: November 2022

DIPG has elevated baseline activation of the integrated stress response (ISR), an evolutionarily conserved system that enables cells to tolerate various forms of stress. Activation of the ISR is indicated by increased expression of activating transcription factor 4 (ATF4). Intermediate levels of ATF4 protect cells from stress, while persistent high levels result in cell death. The imipridone drug ONC201 increases mitochondrial stress and ATF4 expression by binding to and activating the mitochondrial protease ClpP. Sal003 inhibits dephosphorylation of ATF4 upstream regulator, eIF2α. The retinoic acid derivative fenretinide induces ATF4, increases reactive oxygen species, and has clinical activity in pediatric patients with neuroblastoma. Because DIPG has a high baseline level of ATF4, we hypothesized that the ISR activators Sal003, ONC201, and fenretinide would synergize to kill DIPG. After determining the IC50 of Sal003, fenretinide, and ONC201, we treated patient-derived DIPG cell lines with low micromolar doses. The combination of Sal003 and ONC201 significantly increased apoptosis as measured by CC3 immunofluorescence in comparison to DMSO and single treatment (p< 0.0001, ANOVA). Western blots for cleaved PARP expression detected induction of apoptosis in DIPG treated with both Sal003 and ONC201 over DMSO and monotherapy treated cells. The combination also increased ATF4 expression. Since Sal003 is not clinically available, we treated DIPG cells with ONC201 and fenretinide. Synergy testing in JHH-DIPG1 showed an overall ZIP synergy score of 14 with inhibitory concentrations of both drugs in the nanomolar range. CC3 immunofluorescence indicated elevated apoptosis in the combination vs. DMSO (p< 0.0001, ANOVA). Western blots showed increased cleaved PARP, ATF4, p-eIF2α, and CHOP expression in DIPG treated with ONC201 and fenretinide. We are currently testing the efficacy of this combination in brain organoids and orthotopic DIPG xenografts. Our results suggest the combination of ONC201 with fenretinide could potentially serve as a therapy for DIPG.
EXTH-82. THERAPEUTIC TARGETING OF EZH2 AND BET BRD4 IN PEDIATRIC RHABDOID TUMORS
Authors: Ishi Yukitomo , Zhang Yongzhan , Zhang Ali , Sasaki Takahiro , Piunti Andrea , Suri Amreena , Watanabe Jun , Abe Kouki , Natsumeda Manabu , Zou Lihua , Shilatifard Ali , Hashizume Rintaro
Published: November 2022

BACKGROUND

Aberrant activity of the H3K27 modifiers EZH2 and BRD4 is an important oncogenic driver for atypical teratoid/rhabdoid tumor (AT/RT), and each is potentially a possible therapeutic target for treating AT/RT. Thus, targeting distinct histone modifier activities would be an effective approach for treating AT/RT.

METHODS

The effects of EZH2 and BRD4 inhibition on histone modification, cell proliferation, and cell invasion were analyzed by immunoblotting, MTS assay, colony formation assay, and cell invasion assay. RNA- and chromatin immunoprecipitation-sequencing were used to determine transcriptional and epigenetic changes in AT/RT cells treated with EZH2 and BRD4 inhibitors. We treated mice bearing human AT/RT xenografts with EZH2 and BRD4 inhibitors. Intracranial tumor growth was monitored by bioluminescence imaging, and the therapeutic response was evaluated by animal survival.

RESULTS

AT/RT cells showed elevated levels of H3K27 trimethylation (H3K27me3) and H3K27 acetylation (H3K27ac), with expression of EZH2 and BRD4, and lack of SMARCB1 proteins. Targeted inhibition of EZH2 and BRD4 activities reduced cell proliferation and invasiveness of AT/RT in association with decreasing H3K27me3 and H3K27ac. Differential genomic occupancy of H3K27me3 and H3K27ac regulated specific gene expression in response to EZH2 and BRD4 inhibitions. Genes associated with DNA-repair pathway was downregulated upon EZH2 and BRD4 inhibition, and the sensitivity to cisplatin was elevated upon combination of EZH2 and BRD4 inhibitors in vitro. A combination of EZH2 and BRD4 inhibition increased the therapeutic benefit in vitro and in vivo, outperforming either monotherapy.

CONCLUSION

Histone H3K27me3 and H3K27ac were elevated in AT/RT cells and distributed in distinct chromatin regions to regulate specific gene expression and to promote AT/RT growth. Targeting EZH2 and BRD4 activity is therefore a potential combination therapy for AT/RT.
EXTH-90. HDAC INHIBITOR, CAY10603, IS A POTENT RADIOSENSITIZER IN MENINGIOMAS
Authors: Hanemann Clemens Oliver , Na Juri
Published: November 2022

Developing and evaluating potential radiosensitisers to enhance therapeutic efficacy are urgently needed due to the lack of efficient chemotherapy for meningioma. Histone deacetylase (HDACs) expression is generally increased in many cancer types and regulate the expression of numerous proteins involved in tumorigenesis. Targeting HDAC using HDAC inhibitor (HDACi) represent promising radiosensitisers that affect various biological processes, such as cell survival, apoptosis, and DNA repair. Recently, one of the HDAC inhibitor, AR42 monotherapy trials for patients with neurofibromatosis type2 started Nov 2021. However, we were not able to detect the actual HDAC downregulation after applying AR42 (0.5-2 μM) in immortalised meningioma. Hence, we investigated whether pretreatment with the hydroxamate-based HDAC6 inhibitor, Cay10603, impacts HDAC expression, radiation-induced DNA double-strand break (DSB) induction, cell survival, and cell cycle arrest in both immortalised and human meningioma cell lines. Cay10603 at nanomolar level (100 nM) was treated prior to high energy x-ray irradiation (2 Gy) by a medical linear accelerator (LINAC). We found that surviving rate was synergistically decreased after combination treatment of Cay10603 and radiation. Combination therapy induced DNA damage with activation of histone gH2AX and increased G2/M arrest compared to drug or radiation alone. To focus on the mechanisms of action of HDAC6 inhibition followed by radiation, we further investigated nuclear localisation of beta-catenin levels. The results showed the beta-catenin in the nucleus was suppressed after combination therapy. Our findings demonstrate a potential therapeutic strategy of Cay10603 to improve the radiosensitization for meningiomas.
EXTH-92. TARGETING PRIMARY CENTRAL NERVOUS SYSTEM B CELL LYMPHOMA IGH CLONOTYPES USING NOVEL RNA-NPS
Authors: Ogando-Rivas Elizabeth , Von Roemeling Christina , Castillo Paul , Liu Ruixuan , Mendez-Gomez Hector , Thomas Nagheme , Weidert Frances , Chardon-Robles Jonathan , Qdaisat Sadeem , Cascio Matthew , Ligon John , Huang Jianping , Mitchell Duane , Sayour Elias
Published: November 2022

BACKGROUND

Recurrent PCNSBLs represent a therapeutic challenge. Up to 60% of PCNSBL patients relapse to later face survival rates as low as 22%. Unfortunately, tumor heterogeneity and off-target effects have limited the success of immunotherapy against PCNSBL.

METHODS

We propose a novel immunotherapy to overcome PCNSBL heterogeneity and off-target effects in an exquisitely tumor specific manner using nanoparticle vaccination, capable of delivering personalized tumor derived mRNAs, that induces systemic orchestration of innate and adaptive immunity. We target tumor antigens derived from the B cell receptor (i.e., heavy chain immunoglobulin - IgH) clonotypes. IgH clonotypes are hypervariable gene rearrangements clonally generated by B cells. Tumor IgH clonotypes are unique for each malignant B cell clone and hence attractive immune targets, not shared by normal B cell clones avoiding undesirable off-target effects.

RESULTS

RNA-NPs can reprogram tumor microenvironment while activating the innate immunity via IFN type I (i.e., IFNα) and priming of hypervariable region clonotype specific T cell responses in naïve mice. We determined the rearranged IgH sequences (predominant clone 99% and nine additional clones with frequencies < 1%) of clinically relevant inbred murine PCNSBL models (BAL17 and A20) by PCR. The number of identified clonotypes confirmed the IgH variability observed in human B cell hematological malignancies. In preliminary experiments targeting lymphoma derived single clonotypes with RNA-NPs, we showed the feasibility of priming in-vivo T cells specific against hypervariable regions after 3 weekly i.v. RNA-NPs (median IFNγ: 58 pg/ml; range: 50-70 pg/ml vs controls < 30 pg/ml; p=0.008). Targeting of clonotype RNA-NPs was associated with decreased tumor growth (p=0.04). Interestingly, we have observed tumor reactive lymphangiogenesis that communicates with regional skull bone marrow observed in 3D microscopy that might direct future routes of RNA-NP administration.

CONCLUSION

Our RNA-NP systemic vaccination platform can induce PCNSBL clonotype specific T cell responses, sparing normal tissues.
EXTH-93. THE IRAK-4 INHIBITOR EMAVUSERTIB (CA-4948) FOR THE TREATMENT OF PRIMARY CNS LYMPHOMA
Authors: von Roemeling Christina , Doonan Bently , Klippel Kelena , Hoang-Minh Lan , Ogando-Rivas Elizabeth , Martinez Elizabeth , Castillo Paul , Iqbal Madiha , Tun Han , von Roemeling Reinhard , Mitchell Duane
Published: November 2022

BACKGROUND

PCNSL is an aggressive brain tumor accounting for 3% of all CNS malignancies and is associated with poor prognosis. Standard of care treatment includes induction with high-dose methotrexate based chemoimmunotherapy followed by consolidation with autologous stem cell transplant or whole brain radiation. However, more than half of PCNSL patients cannot tolerate this intensive therapeutic intervention. Novel treatments are urgently needed. Toll-like receptor signaling pathway via MyD88/IRAK-4 signalosome is constitutively active in PCNSL secondary to MYD88 L265P mutation and represents an excellent therapeutic target. Emavusertib (CA-4948) is an oral first-in-class small molecule inhibitor of IRAK-4 that demonstrates clinical activity in patients with systemic Non-Hodgkin’s Lymphoma, however it has not been evaluated in the CNS space. HYPOTHESIS: Emavusertib has anti-tumor activity against preclinical PCNSL.

METHODS

Using multiparameter immunohistochemistry (IHC) and proteomics analysis, IRAK-1, IRAK-4, and NF-κB pathway activation were examined in patient PCNSL tissues as compared to normal controls. Plasma, cerebrospinal fluid, and brain tissue were assessed via UPLC-MS/MS for emavusertib drug concentration following single oral high dose in a murine model. Survival responses in preclinical PCNSL models were measured in response emavusertib treatment, along with in vivo tumor proliferation and downstream biomarker expression by multi-parameter IHC.

RESULTS

Our data confirm elevated IRAK-1, IRAK-4, and NF-κB signaling in patient PCNSL. We show that emavusertib can achieve therapeutically relevant concentrations in the brain and CSF. Emavusertib impairs tumor cell proliferation in vitro and in vivo, and shows dose-dependent single-agent activity in preclinical PCNSL models. We confirm decreased ERK1/2, MAPK and NF-κB activation, indicative of downstream IRAK-4 inhibition.

CONCLUSIONS

Our data supports that emavusertib demonstrates anti-tumor activity in the CNS space, warranting further preclinical and clinical evaluation of this agent for the treatment of PCNSL.
EXTH-94. LOCALIZED INTRATUMORAL TREATMENT WITH ADOPTIVE T CELL THERAPY FOLLOWED BY ONCOLYTIC ADENOVIRUS DELTA-24-RGDOX INDUCES SUSTAINABLE REGRESSION OF DISSEMINATED SOLID TUMORS THROUGH ANTIGEN SPREAD
Authors: Jiang Hong , Shin Dong Ho , Yi Yanhua , Fan Xuejun , Gumin Joy , Gillard Andrew , Alonso Marta M , Lang Frederick , Gomez-Manzano Candelaria , Fueyo Juan
Published: November 2022

Tumor-targeting biological therapies, including adoptive T cell therapy (ACT) and oncolytic viruses, are being tested clinically to address the challenges in treating advanced solid tumors with heterogeneity and immune suppressive microenvironment (TME). Since complementary properties of these two approaches have been observed, we hypothesize that localized intratumoral treatment with combination of ACT and Delta-24-RGDOX (third generation oncolytic adenovirus developed by our group) induces not only potent therapeutic effect in treated tumor but also abscopal benefit to eliminate distant disseminated tumors. Using a B16-C57BL/6 melanoma model with disseminated tumors in C57BL/6 mice, we found gp100-specific CD8+ T cells injected into the first subcutaneous (s.c.) tumor showed tropism for distant s.c. and intracranial tumors, tumor draining lymph nodes, compared to spleen, blood and normal brain hemisphere. In a B16-OVA-C57BL/6 s.c./s.c. tumor model, Delta-24-RGDOX injections following gp100-specific T cells in the first tumor increased total cytotoxic T cell presence within both tumors, including gp100- and OVA- specific T cells. Further analysis revealed that Delta-24-RGDOX reduced the exhaustion of OVA-specific T cells while that of gp100-specific T cells kept at a low level. Moreover, Delta-24-RGDOX augmented the activity of the splenocytes against the tumor cells and other tumor-associated antigens (OVA, TRP2) than gp100. Consequentially, the combination of OVA-specific T cells and Delta-24-RGDOX was more potent to inhibit the injected and untreated distant tumor growth and caused improved survival rate than either of the agent alone (p < 0.05). Importantly, we observed relapse of the regressed tumors in the T cell treatment group, but not in the combination group. The survivors developed immune memory against rechallenging with B16-OVA cells. Collectively, our data demonstrate that localized treatment with intratumoral ACT followed by Delta-24-RGDOX induces global immune activation and antigen spread to expand antitumor T cell repertoire, leading to efficacious systemic anti-tumor immunity and sustainable tumor regression.
EXTH-96. BIOPROCESSING OF SURGICAL PEDIATRIC BRAIN TUMOR SPECIMENS FOR GENOME-GUIDED PERSONALIZED DRUG TESTING
Authors: Nasajpour Emon , Lyle Geoff , Lancero Hope , Garcia Cesar , Learned Katrina , Gibson Eden , Tran Caitlynn , Schouten Troy , Vogel Hannes , Mahaney Kelly , Prolo Laura , Vaske Olena , Grant Gerald , Petritsch Claudia
Published: November 2022

Novel treatment approaches for pediatric central nervous system (CNS) tumors are urgently needed. A lack of patient-derived tumor cells impedes progress towards developing such new therapies. We intended to overcome this challenge by establishing methods to create a biorepository of viable single cell suspensions of pediatric brain tumor surgical specimens. Quantitative and qualitative comparisons of tissue processing strategies were performed to preserve viability of heterogeneous tumor and immune cells. Novel drug targets were identified by analyzing pathways affected by RNA transcripts that are highly expressed (outliers) in a patients’ tumor; outliers for each patient were determined by RNA-seq data from individual patients’ tumors compared with a compendium of 12,747 pediatric and adult samples harmonized by the Treehouse Childhood Cancer Initiative at the UCSC Genomics Institute. The predicted anti-tumor efficacy of small molecule inhibitors of the outlier pathways was tested in cell viability assays against short-term cultured cells from matched patients. Successful tissue collection required obtaining informed consent, standard operating procedures, and sample recording using a Laboratory Inventory Management Software (LIMS). Since 2020 we have banked 67 pediatric CNS brain tumor specimens at Stanford. Amongst those, 51 cases yielded sufficient tissue for RNA-seq and cryoprotection. The most common tumor histology was low-grade glioma (LGG, 26 of 67), the majority of which were pilocytic astrocytoma (18 of 26). The second and third most common tumor types are embryonal tumors (6 medulloblastoma, 3 AT/RT) and ependymoma (4), respectively. We identified significant differences in cell viability with different preservation media. An outlier pathway previously not implicated in LGG was identified and sensitivity to a small molecule inhibitor of this outlier pathway was demonstrated. Taken together, we established feasibility for validating therapeutic vulnerabilities identified by a genome-guided approach in short-term cultures from surgical specimens. This works facilitates the rapid development of personalized CNS tumor treatment.
EXTH-98. ENHANCING T CELL TRAFFICKING OF CD3-ENGAGING IMMUNOTHERAPY TO TUMORS OF THE CENTRAL NERVOUS SYSTEM
Authors: Singh Kirit , Foster Matthew , Hotchkiss Kelly , Snyder David , Khasraw Mustafa , Sampson John
Published: November 2022

BACKGROUND

Effective immunotherapy against tumors of the central nervous system (CNS) requires that the drug the blood brain barrier (BBB) and encounter immune cells. We have previously described a mechanism which may facilitate transport of CD3-engaging therapeutics into the CNS, via carriage on activated T cells. Building on this, we sought to produce specific T cell phenotypes that rapidly enter and accumulate in the CNS.

METHODS

8–10-week-old C57/Bl6 mice (n=5-6 per group) were implanted with 30,000 CT2AvIII cells which established over 14 days. Mice received (1) CD45.1 lymphocytes activated with IL-7 and Concanavalin-A (single intravenous (IV) injection, 1 x 107 adoptive lymphocyte transfer (ALT)) or (2) CD45.1 lymphocytes activated with IL-2 and serial Con-A stimulation. Mice were sacrificed 3 hours following ALT and their brains analysed via flow cytometry. A follow-up timepoint of 48 hours was also analysed. Groups were compared using a Mann-Whitney U test.

RESULTS

Ex vivo culture in group 1 yielded a mixed population of T effector and T central memory cells whereas group 2 resulted in terminally differentiated T effector memory cells only. VLA-4, a migratory integrin involved in T cell entry into the CNS was upregulated in the IL-7 group. Mice in Group 1 demonstrated significantly enhanced entry of CD8+ effector and memory T cells into tumor bearing hemispheres 3- and 48-hours following administration compared to Group 2 (p = 0.005, p = 0.0159 respectively).

CONCLUSIONS

Varying the cytokine cocktail used for ex vivo activation and expansion of T cells results in markedly different trafficking properties and phenotype composition. Ongoing work will determine how enhanced T cell localization to tumor affects accumulation of immunotherapy via the hitchhiking mechanism. Further, we will evaluate the safety of combinatorial CD3-engaging immunotherapy and ALT in Phase I trials (NCT04903795) using a cGMP Brain Bi-specific T cell engager (BRiTE).