bluuebunny/crossref_metadata_2025_split huggingface dataset:
EXTH-68. ONCOMAGNETIC TREATMENT SELECTIVELY KILLS GLIOMA CANCER CELLS BY INDUCING OXIDATIVE STRESS AND DNA DAMAGE
Authors: Hambarde Shashank , Pandey Arvind , Baskin David , Helekar Santosh 
Published: November 2022

Abstract
A new noninvasive therapeutic device developed in our laboratory called the Oncomagnetic device provides a novel approach to glioblastoma (GBM) treatment. It involves repeated stimulation with oscillating magnetic fields (sOMF) produced by spinning permanent magnets. It is technologically and mechanistically distinct from the Optune® device approved by the FDA for the treatment of newly diagnosed and recurrent GBM. Use of this device in one end-stage GBM patient reversed the progression of his recurrent tumor causing &gt;30% reduction in its contrast-enhanced volume within 4 weeks of treatment. Mice with implanted mouse glioma cells in their brains also showed marked reduction in tumor size, increased survival (p&lt; 0.05, n = 10) and higher DNA damage (g-H2AX foci) after sOMF treatment with a whole-body stimulation method developed by us. Normal mice exposed to sOMF for 4 months had no adverse effects on the brain and other organs. In-vitro, sOMF markedly increased reactive oxygen species (ROS) levels in cancer cells leading to the selective death of these cells, while sparing normal neurons and astrocytes. Detection of g-H2AX and 53BP1 foci showed that sOMF caused significant DNA damage in GBM cells and diffuse intrinsic pontine glioma (DIPG) cells but not in normal astroglial SVGp12 cells. Furthermore, sOMF exposure for just 2 h resulted in &gt;40% loss of surviving GBM and DIPG cell colonies detected by clonogenic cell survival assay, similar to that produced by 2 Gy radiation dose. This loss was rescued by the antioxidant Trolox. These results indicate that sOMF stimulation has high anticancer potency comparable to low dose radiation therapy at the cellular level with an underlying mechanism of action that is substantially different from that proposed for Optune® TTF.
EXTH-76. PRMT5 INHIBITION SENSITIZES GLIOBLASTOMA NEUROSPHERES TO TEMOZOLOMIDE
Authors: Rivas Sarah , Rivera-Caraballo Kimberly , Murphy Sara , Otani Yoshihiro , Shelbourn Allison , Ampie Leo , Maric Dragan , Walbridge Stuart , Shah Ashish , Yan Yuanqing , Yoo Ji young , Heiss John , Kaur Balveen , Banasavadi-Siddegowda Yeshavanath 
Published: November 2022

Abstract

INTRODUCTION
The median survival of Glioblastoma (GBM) patients is less than two years with the standard of care of maximal surgical resection, radiation, and temozolomide (TMZ) chemotherapy. Protein Arginine Methyltransferase 5 (PRMT5), which regulates cellular functions by symmetrically di-methylating arginine residues, is overexpressed in GBM. Inhibiting PRMT5 induces apoptosis in differentiated and senescence in stem-like GBM tumor cells. We inhibited PRMT5 in GBM neurospheres to determine if PRMT5 inhibition would enhance TMZ’s antitumor effect.

METHODS
We depleted PRMT5 activity, in vitro, using target-specific siRNA or LLY-283 and combined these with TMZ treatment. We evaluated the antitumor effect of this combination using cell viability assay, cell cycle analysis, apoptosis assay, and western blot.

RESULTS
TMZ reduced the viability of GBMNS with PRMT5 knockdown significantly more than the viability of PRMT5 intact GBMNS. The combination of TMZ and PRMT5 knockdown elevated the expression of cleaved caspase 3 and caspase3/7 indicating that PRMT5 knockdown enhanced the apoptotic effects of TMZ. Cell cycle analysis showed that depleting PRMT5 abrogated TMZ-induced G2/M cell cycle arrest. Further, treatment of PRMT5-depleted GBMNS with TMZ increased ɣ-H2AX expression compared PRMT5 intact GBMNS treated with TMZ, suggesting that PRMT5 depletion enhanced TMZ-induced DNA damage. PRMT5 knockdown also inhibited the symmetric di-methylation of RUVBL1 that is required for homologous recombination repair of TMZ treatment-related DNA damage.

CONCLUSION
Overall, PRMT5 inhibition sensitized GBMNS to TMZ and enhanced TMZ-related DNA damage and cytotoxicity. These findings support further development of this potential therapeutic combination.
EXTH-77. TARGETING CD73 IN GBM SENSITIZES TUMORS TO ONCOLYTIC VIRUS THERAPY
Authors: Murphy Sara , Mullarkey Matthew P , Sahu Upasana , Hong Bangxing , Lewis Cole , Diaz Miguel , Wenzel Pamela , Kamal Abu Hena Mostafa , Putluri Nagireddy , Park Junhyoung , Kaipparettu Benny , Kaur Balveen 
Published: November 2022

Abstract
Glioblastoma Multiforme (GBM) is the most aggressive malignant primary brain tumor and has abysmal 5-year overall survival. With the approval of oHSV Imlygic by FDA for metastatic melanoma and more recently, conditional approval of G47Δ, marketed by Daiichi Sankyo for GBM treatment in Japan, oncolytic viral therapy has emerged as a promising biological approach to treat solid tumors. Our laboratory has previously shown that oHSV expressing long isoform of PTEN (PTENα), HSV-P10, has faster kinetics of virus replication associated with enhanced killing of the glioma cells compared to control HSV. Additionally, HSVP-10 is known to increase mitochondrial membrane potential and cellular ATP production while stimulating anti-tumor immune responses in vivo. RNA sequencing of primary GBM cells infected with HSV-P10 shows altered metabolic pathways relative to control HSV infected cells. 13C metabolic flux analysis in uninfected and control or HSV-P10 infected primary GBM cells reveals that while HSV-P10 infection shuttles most of the glutamine towards citrate by reductive carboxylation of α-ketoglutarate resulting in faster replication of HSV-P10, simultaneously it increased glucose utilization and shuttling towards TCA cycle. This corroborates with enhanced mitochondrial activity leading to increased oxidative phosphorylation and increased cellular and extracellular ATP (eATP). eATP binds to purinergic receptors on tumor and immune cells and boosts anti-tumor immunity. However, the use of extracellular ATP in cancer therapy is limited owing to its short half-life and rapid hydrolysis by ectoenzymes CD39 and CD73 into immune-suppressing adenosine. CD73 carries out the conversion of AMP to adenosine, making it a key regulator of this pathway. Using mice with CD73 knocked out globally (CD73KO), we show that combination of CD73 inhibition with HSV-P10 imparts significant survival benefit compared to WT mice treated with HSV-P10. Our findings will further the understanding of oHSV therapy and the role of the ATP/adenosine in the tumor microenvironment.
EXTH-78. CRISPR/CAS9 KNOCKOUT SCREENS UNRAVEL EPIGENETIC REGULATORS AS DRUGGABLE DEPENDENCIES FOR SONIC HEDGEHOG MEDULLOBLASTOMA
Authors: Tsiami Foteini , Piccioni Federica , Root David , Bandopadhayhay Pratiti , Segal Rosalind , Tabatabai Ghazaleh , Merk Daniel 
Published: November 2022

Abstract
Sonic hedgehog medulloblastoma (SHH-MB) is a malignant, highly heterogeneous brain tumor entity, accounting for 30% of all MBs in the pediatric population and is characterized by aberrant activation of the canonical SHH signaling pathway. Although current therapies targeting Smoothened (Smo) have proven a promising treatment approach for SHH-MB patients, pre-existing or acquired resistance impedes its clinical efficacy. Therefore, novel targeted approaches that overcome mechanisms of resistance are urgently needed. Here, we performed a genome-wide CRISPR/Cas9 knockout screen in a murine and a human SHH-MB cell line, SMB21 and DAOY, respectively, in order to decipher tumor-specific genetic essentialities. Our data demonstrate that SMB21 cells highly depend on key mediators of the SHH pathway, such as Gli2) for their proliferation, as opposed to DAOY cells, suggesting that the latter does not represent a faithful model of SHH-MB. Among other dependencies for SHH-MB are members of the epigenetic machinery such as Dnmt1) and Smarca5). Pharmacologically, we show that DNMT1 inhibition is efficacious at clinically relevant concentrations against Smo inhibitor- sensitive, as well as resistant SHH-MB cell lines. By performing RNA sequencing of SMB21 cells, we identified early and late alterations in global gene expression induced by DNMT1 inhibition, including decreased expression of positive regulators of SHH signaling. An additional knockout drug screen in SMB21 cells unraveled synthetic lethal interactors for DNMT1 inhibitors, as validated in vitro) by drug combination treatments. Further global DNA methylation profiling in SMB cells will help to define the molecular basis of sensitivity to DNMT1 inhibitors in SHH-MB. Last but not least, genetic ablation of epigenetic regulators and combinatorial treatments using DNMT1 inhibition will be investigated in in vivo) established mouse models of SHH-MB. Summarizing, our data indicate the potential of inhibiting epigenetic regulators as novel therapeutic avenues in SHH-MB sensitive, as well as resistant to Smo inhibition.
EXTH-85. TARGETING CLAUDIN 6 WITH CAR T CELL THERAPY FOR ATYPICAL TERATOID/RHABDOID TUMOR
Authors: Madsen Peter , Stern Allison , Griffin Crystal , Oehm Petra , Rengstl Benjamin , Flemming Carina , Resnick Adam C , Storm Philip B , Foster Jessica 
Published: November 2022

Abstract
Atypical teratoid/rhabdoid tumor (ATRT) is an aggressive brain tumor that predominantly affects young children and has an average 5-year survival under 50%. Novel, targeted therapies are desperately needed. Claudin 6 (CLDN6) is a tight junction protein present during development and expressed in up to 70% of ATRT specimens but not in normal tissue, making it a promising immunotherapeutic target. CLDN6-targeted chimeric antigen receptor (CAR) T cells in combination with a CAR T cell–amplifying mRNA vaccine have demonstrated antitumor activity against other CLDN6-expressing cancers in pre-clinical and phase I adult trial (NCT04503278; Haanen J et al AACR, 2022). To assess the effectiveness of CLDN6-targeted CAR T cells against ATRT, we utilized a second-generation mRNA CAR with a 4-1BB costimulatory domain and single-chain variable fragment against CLDN6 (Reinhard et al, 2020). Patient-derived ATRT specimens were assessed by RNAseq for CLDN6 expression (mean FPKM= 11.4) and by immunohistochemistry (positive staining in 53% of specimens). Tumor-derived cell lines were validated for CLDN6 expression by flow cytometry. Co-culture of CLDN6-directed mRNA CAR T cells with ATRT cell line 7316-2187 resulted in tumor-specific cytotoxicity compared to CD19-directed control CAR T cells (92% versus 15% at 10:1, p&lt; 0.0001; 86% versus 0% at 5:1, p&lt; 0.0001). Similar results were seen with ATRT cell line 7316-2141 (75% versus 7% at 10:1, p&lt; 0.0001; 53% versus 0% at 5:1, p&lt; 0.0001). Both CLDN6- and CD19-directed CAR T cells showed no cytotoxicity against CLDN6-negative cell line 7316-4149. Patient-derived xenograft models were also created through intracranial injection of multiple ATRT patient cell lines, and ongoing work will evaluate locoregional administration of CLDN6-directed CAR T cells in orthotopic xenograft models to test in vivo efficacy. This work highlights the potential for targeting CLDN6 via CAR T cell therapy in patients with ATRT as a novel therapeutic strategy for these devastating tumors.
EXTH-87. DECITABINE POTENTIATES MENINGIOMA IMMUNOTHERAPY TARGETING NY-ESO-1
Authors: Sun Matthew , Orpilla Joey , Contreras Erick , Imbroane Marisa , Treger Janet , Wang Anthony , Ko MyungJun , Vitte Jeremie , Giovannini Marco , Liau Linda M , Prins Robert , Everson Richard G 
Published: November 2022

Abstract

INTRODUCTION
No immunotherapy exists for meningiomas. NY-ESO-1 is a potential target because in meningiomas it’s the most frequently expressed Cancer-Testis-antigen. Decitabine is a hypomethylating agent known to upregulate CT-antigens. We tested the effects of decitabine on NY-ESO-1 expression in primary and immortalized meningioma cultures, and its impact on the efficacy of NY-ESO-1 T-cell-receptor-transduced T-cells(TCRs), to understand its potential role in meningiomas immunotherapy.

METHODS
We established primary meningiomas cultures from fresh tumor specimens and quantified NY-ESO-1 expression using Western blot and qRT-PCR. Cultures were pre-treated with 1uM of decitabine for at least 48 hours before assays. In vitro cytolysis was measured using the xCelligence Real-Time Cell Analyzer System in meningioma co-cultures with MHC-Class-I(HLA-A*0201)–restricted NY-ESO-1(157 – 165)-specific TCRs.

RESULTS
Nine primary and three immortalized meningioma cultures(Grade I – III) were evaluated for baseline NY-ESO-1 expression. High expression was defined as &gt; 100-fold overexpression compared to glioma(U251) cultures. Decitabine treatment significantly increased NY-ESO-1 expression in 6 primary and 2 immortalized(SF1335, grade 1; IOMM-LEE, grade 3) cultures with low baseline expression(median increase over 100 fold; p &gt; 0.001); whereas it slightly decreased NY-ESO-1 expression in 3 primary and 1 immortalized culture(CH157, WHO grade 3) with high baseline expression(median decrease 20%;p&lt; 0.001). Decitabine pre-treatment of meningioma before co-culture with NY-ESO-1 TCRs at a ratio of 1:1, increased tumor cytolysis for SF1335, from 20% to 40%, and for IOMM-LEE, from 0% to 85%, measured at 10 hours. NY-ESO-1 expression increased 5000-fold vs 10-fold for IOMM-LEE vs SF1335, respectively. Decitabine treatment also increased MHC-Class-I expression for IOMM-LEE but not SF1335. Decitabine pre-treatment did not change cytolysis of CH157 at 65%(10 hrs).

CONCLUSIONS
Decitabine upregulates NY-ESO-1 expression in meningiomas with low baseline expression, and it increases cytolytic effects of NY-ESO-1 TCRs proportional to NY-ESO-1 and MHC-Class-I molecule upregulation. Decitabine may be a clinically feasible adjunct to meningioma immunotherapy.
EXTH-95. UNCOVERING THERAPEUTIC VULNERABILITIES IN MISMATCH REPAIR-DEFICIENT GLIOMAS
Authors: Pal Sangita , Boynton Adam , Johnston Ryan , Currimjee Naomi , Qian Kenin , Touat Mehdi , Bellamy Charlotte , Ho Patricia , Root David , Persky Nicole , Goodale Amy , Berstler Jim , Miller Lisa , Guletsky Alex , Cortes-Cros Marta , Ligon Keith , Beroukhim Rameen , Bandopadhayay Pratiti 
Published: November 2022

Abstract
Approximately 25% of recurrent gliomas exhibit hypermutation, which is most often acquired in chemotherapy sensitive gliomas post-treatment with standard of care alkylating agent temozolomide, Intriguingly, nearly all of these recurrent hypermutant tumors harbor deficiencies in mismatch repair (MMR) genes. Unlike other MMR-deficient cancers, hypermutant gliomas do not exhibit detectable microsatellite instability (MSI) at the population level, and do not share similar dependencies. Thus, strategies to therapeutically target MMR-deficient and hypermutant gliomas are urgently needed, and likely to impact many patients. To evaluate vulnerabilities associated with MMR-deficient gliomas, we have generated isogenic MMR-deficient models by systematic ablation of core MMR genes MSH2, MSH6, MLH1, and PMS2, in multiple patient-derived glioma cell lines using a robust all-in-one CRISPR-Cas9 system. We characterized these isogenic MMR-deficient glioma lines in comparison to respective MMR-proficient control line by performing differential gene expression analysis and subsequent gene set enrichment analyses. Our analyses reveal in an unbiased fashion an enrichment of several hallmark gene sets including cell cycle control and DNA repair in the MMR-deficient cell lines. We performed a CRISPR-Cas9 knockout screen in these MMR deficient and hypermutant models and identified candidate genes involved in DNA repair, cell cycle, RNA metabolism and other pathways as preferential dependencies in the MMR-deficient glioma cells. We also screened multiple MMR-deficient isogenic models against the high-throughput drug repurposing (REPO) chemical library and our preliminary results reveal a number of compounds from a variety of drug classes that selectively kill the MMR-deficient glioma cells, indicating that the loss of MMR confers differential dependencies to these small molecule inhibitors. The candidate drugs and genes identified from these high-throughput assays are currently being subjected to further validation, and they possibly will help us identify novel therapeutic strategies or improve existing therapeutic strategies to target recurrent MMR-deficient gliomas.
EXTH-97. BONE MARROW-DERIVED ENGINEERED MYELOID CELLS ENHANCE THE TRAFFICKING AND ACTIVATION OF CYTOTOXIC T CELLS IN THE GLIOMA MICROENVIRONMENT
Authors: Canella Alessandro , Nazzaro Matthew , Schmitt Claire , Rajendran Sakthi , Haffey Abigail , Nigita Giovanni , Thomas Diana , Rajappa Prajwal 
Published: November 2022

Abstract

INTRODUCTION
Gliomas are the most prevalent type of brain tumors in the adolescent and young adult population (AYA) and one of the leading causes of cancer- related death. Two-third of glioma AYA patients are affected by low-grade gliomas (LGGs), but yet there are no specific treatments. Therefore, a significant percentage of LGG patients experience tumor recurrence and malignant progression to high-grade glioma (HGG) and the immune microenvironment plays a critical role in mediating mechanisms of tumor progression and treatment resistance. Specifically, tumor associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) have been shown to restrict cytotoxic effector trafficking and activation. To that end, first-line immunotherapies have limited efficacy in improving outcomes for HGGs.

HYPOTHESIS
We investigated the preclinical efficacy of a novel immunotherapeutic approach for the treatment of LGGs in AYA mice to modulate and reprogram the immunosuppressive tumor microenvironment (TME) and enhance the trafficking of activated effector T cells.

METHODS
Immunocompetent RCAS n/tv-a LGG bearing animals were treated at day 28 with a single retroorbital dose of bone-marrow derived myeloid cells, engineered for the release of Interlekin-2 (GEMys-IL2). The treatment was well tolerated and no sign of toxicity was noted.

RESULTS
Our results demonstrated that GEMys-IL2 efficiently crossed the BBB, infiltrate the glioma microenvironment, and activate infiltrating immune cells while altering the TME underscored by an increased CD8+ T cell population. Of note, the RCAS/t-va immunocompetent LGG to HGG progression mouse model treated with a single dose of GEMys-IL2 demonstrated a transient arrest of tumor burden within 14 days post treatment, which was associated with a significant advantage in overall survival.

CONCLUSIONS
These results highlight the pre-clinical efficacy and anti-tumoral therapeutic activity of GEMYys-IL2 on CD8+ T cell trafficking and activation to the TME in low-grade glioma in vivo, thus supporting its potential as a novel cell-mediated innate immunotherapy.
EXTH-102. PLASMA AND CEREBROSPINAL FLUID (CSF) PHARMACOKINETICS (PK) OF MIRDAMETINIB IN A NON-HUMAN PRIMATE (NHP) MODEL
Authors: McCully Cynthia Lester , Shearer Todd , Gross Andrea , Langseth Abraham , Peer Cody , Killoran Kristin , Garcia Rafael Cruz , Figg William , Widemann Brigitte 
Published: November 2022

Abstract

Mirdametinib is a MEK inhibitor with reported CSF penetration in pre-clinical models. Murine studies reported ERK phosphorylation inhibition in brain tissue at 1.15 nM (0.73 ng/mL) and tumor cell lines at 0.33–0.59 nM (0.16-0.28 ng/mL). A phase II clinical trial evaluating mirdametinib for neurofibromatosis type 1-related plexiform neurofibromas reported a 42% partial response rate and a mean plasma exposure (AUC0-12h) of 443 h*ng/mL. This study determined the plasma and CSF pharmacokinetic (PK) profile of mirdametinib in a non-human primate (NHP) model where CSF penetration serves as a proxy for CNS penetration.

METHODS
Four NHP received mirdametinib PO q.d, 0.50 mg/kg, in serial studies as a single dose (Single-Dose), three doses (Multiple-Dose), or IV, 0.20 mg/kg, followed by paired plasma and CSF sample collections through 96 hours. Mirdametinib was quantified by LC-MS/MS. PK parameters were calculated via noncompartmental methods. Plasma protein binding was determined by rapid equilibrium dialysis.

RESULTS
Mean □ standard deviation values reported. Three subjects were evaluable for both plasma (total and unbound) and CSF drug concentrations. Plasma – AUClast: Single-Dose 500.3 □ 253.4 and IV 552.3 □ 43 h*ng/mL; AUCtau: Multiple-Dose 456.3 □ 120.6 h*ng/mL. CSF Cmax (ng/mL): Single-Dose 0.43 □ 0.18, IV 2.0 □ 1.8, Multiple-Dose 0.56 □ 0.18. CSF Penetration (%): Single-Dose 1.3 □ 0.45, IV-1.6 □ 0.95 (AUCcsf-last:AUCplasma-last); Multiple-Dose 1.3 □ 0.56 (AUCcsf-tau:AUCplasma-tau). CSF/unbound plasma ratios ranged from 2.5-3.0 Plasma free fraction (%): 0.52 □ 0.1.

CONCLUSION
Notable mirdametinib CSF penetration was demonstrated in this NHP model. A higher CSF to unbound plasma ratio suggests that the efflux of mirdametinib from the CSF was delayed. The NHP CSF Cmax approached the ERK phosphorylation inhibition concentrations previously reported. The ability of this NHP model to predict human PK parameters was demonstrated via the comparable NHP to patient plasma exposure reported in the Phase II clinical trial.
EXTH-104. ROLE OF CLPP AND MITOCHONDRIAL METABOLISM IN THE ANTI-CANCER EFFECTS OF IMIPRIDONE ONC201 AND ONC206 IN GLIOBLASTOMA AND DIPG
Authors: Foster Scott , Lee Andrew , Maranto Cristina , Allen Joshua E , Lanier Randall , Sethna Phiroze , Prabhu Varun V 
Published: November 2022

Abstract
ONC201 is the first bitopic dopamine receptor D2 (DRD2) antagonist and allosteric mitochondrial protease ClpP agonist. ONC201 is well tolerated and induces durable tumor regressions in H3 K27M-mutant glioma patients. ONC206, a derivative of ONC201 currently in Phase I trials, is also a bitopic DRD2 antagonist and ClpP agonist with enhanced nanomolar potency relative to ONC201. We evaluated in vitro efficacy, mechanism(s) of action and acquired resistance for ONC201 and ONC206 in glioblastoma (GBM) (T98G, A172, U87MG), H3.3 K27M mutant DIPG (SF8628, SF7761) and astrocytoma (H4, U118MG) cell lines. ONC206 was more potent than ONC201 in cell viability assays for all line. A mutation in ClpP has previously been associated with resistance to imipridones. Accordingly, SF8628 cells with a CRISPR-mediated ClpP knockout were resistant to both ONC201 and ONC206. Polyclonal resistant cell lines were generated through prolonged passaging in increasing concentrations of ONC201 (SF8628 and T98G cells) or ONC206 (T98G cells). Cells with acquired resistance to ONC201 (ONC201Res) or ONC206 (ONC206Res) showed cross resistance to both imipridones. Time-stability of resistance was confirmed by removing drug for 2 to 4 weeks and retesting. Whole genome sequencing revealed ClpP mutations in both ONC201Res and ONC206Res T98G cells. RNAseq analysis revealed upregulation of the integrated stress response, apoptosis pathways, and amino acid metabolism with imipridone treatment in T98G parental cells but not resistant clones. Depleting glucose in the media enhanced cell viability inhibition by imipridones, indicating mitochondrial metabolism may contribute to their mechanism of action. Our results identify ClpP and mitochondrial metabolism as key aspects for the mechanism of action of ONC201 and ON206 in DIPG and GBM cell lines.