bluuebunny/crossref_metadata_2025_split huggingface dataset:
HGG-23. IN VITRO AND IN VIVO PRECLINICAL DRUG SCREENING OF PROMISING THERAPEUTICS FOR DIFFUSE MIDLINE GLIOMA (DMG)
Authors: Cosentino Chiara Cianciolo , Laternser Sandra , Przystal Justyna M , Yadavilli Sridevi , Zhang Jie , Müller Timothy , Kritzer Bettina , Müller Sabine , Nazarian Javad 
Published: June 2021

Abstract

Introduction
Diffuse midline gliomas (DMGs) are amongst the most unforgiving pediatric brain tumors, characterized by an intrinsic resistance to therapy. Despite major advances in understanding of tumor biology, the prognosis remains exceedingly poor, and treatment options are limited. New therapeutics are being evaluated at a fast rate by different laboratories. In order to prioritize effective drug candidates for DMG treatment, we comprehensively characterized a panel of promising therapeutic agents in in vitro and in different vivo systems.

Methods
We determined the sensitivity of primary DMG cell lines to a panel of small molecule inhibitors targeting known DMG targets and pathways. Dose response curves were generated for more than 20 different compounds and possible synergistic effects were investigated by SynergieFinder. In an effort to highlight potential toxicities and associated mechanisms at a large scale, we performed a preclinical toxicity evaluation in zebrafish larvae, with a slightly modified version of the official Fish Embryo Acute Toxicity (FET) test. Drug toxicity was tested by continuous exposure of zebrafish larvae to increasing concentrations of the different compounds. Survival curves, morphological analyses and behavioral tests were performed at a maximum tolerated dose (MTD). To confirm the findings obtained in zebrafish, we further performed in vivo studies in mice for promising candidates.

Results
Among the tested drugs in vitro we found 10 drugs showing promising dose- dependent reduction in cell viability with IC50 in nM to µM range. These were further evaluated for toxicity in zebrafish. The zebrafish larvae toxicities observations strongly correlated with the findings in murine in vivo studies, reinforcing the importance of zebrafish as an accurate investigative toxicology model to assess acute toxicity of molecules in preclinical studies.

Conclusions
By testing a wide range of drugs, targeting different pathways on DMG cells and in different in vivo systems we identified promising drug candidates for clinical management of children diagnosed with DMG.
HGG-27. HNRNPA1 SPLICED VARIANT SENSITIZATION EFFECT DISCLOSED IN GLIOMA CELLS
Authors: Yadav Ajay 
Published: June 2021

Abstract
Glioblastoma is aggressive brain tumor. Glioma heterogeneity builds in hypoxic condition due to its intrinsic high apoptosis rate cause to develop a high selection clonal pressure. HnRNPA1 plays a key role in developing glycolytic tumor, shows its high expression exclusively in hypoxic glioma cells. Recently we observed one more spliced variant of hnRNPA1, encoding higher isoform, exclusively abundant in resistant glioma cell lines. Widely around the scientific community HnRNPA1 splice factor family protein was found distinctly regulating resistant glioma phenotype. To support our hypothesis, methodology we perform includes various apoptosis assays to critically understand hnRNPA1 spliced variant dependent pathway in Temozolomide resistant U87 glioma cells. Proteomic based apoptotic array and angiogenic array enable us to visualize selective knock down of hnRNPA1 has dominant role in promoting apoptotic cascade. Additionally, flow cytometry base annexin V-PI staining technique to understand early and late apoptosis was measured in selective hnRNPA1 spliced variant knockdown cells in presence or absence of PI3 kinase inhibitor wortmannin (5 micro molar). Results showed hnRNPA1 higher isoform knock down promotes more apoptosis compare to lower isoform. Interestingly, overexpression of HnRNPA1 higher isoform or lower isoform alone doesn’t promote apoptosis, however is prominently higher apoptosis in Bortezomib treated U87 glioma cells. These both isoforms are presently majorly in gliomas, but somehow for long was not recognized. Conclusion is to explore more related novel finding or therapeutic strategy to target higher isoform of hnRNPA1, using invivo mouse xenograft model.
HGG-31. HISTONE H3.3 G34R/V MUTATIONS STIMULATE PEDIATRIC HIGH-GRADE GLIOMA FORMATION THROUGH THE INDUCTION OF CHROMOSOMAL INSTABILITY
Authors: Grigore Florina , Robinson James , Langfald Alyssa , Hinchcliffe Edward , Day Charles 
Published: June 2021

Abstract
The histone H3.3 G34R/V mutations are known drivers of high-grade pediatric glioma (pHGG). However, the mechanism(s) for H3.3 G34R/V induced tumor formation are unclear. Chk1 phosphorylates H3.3 S31 at the pericentromere during early mitosis, suggesting a novel mitotic function. We observed that H3.3 G34 mutant pHGG cells have reduced mitotic H3.3 S31 phosphorylation compare to WT H3.3 cell lines. The H3.3 G34R mutation reduced Chk1 phosphorylation at S31 by &gt;90% in an in vitro kinase assay. Overexpression of either H3.3 G34R or non-phosphorylatable S31A in H3.3 WT, diploid cells led to a significant increase in chromosome mis-segregations. Likewise, H3.3 G34 mutant pHGG cells have significantly elevated rates of mis-segregation as compare to H3.3 WT pHGG cells. During normal cell division, phospho-S31 is lost in late anaphase. However, when chromosome missegregation occurs, phospho-S31 spreads and stimulates p53 accumulation in G1 – thus suppressing aneuploid cell proliferation. Here we show that cells expressing mutant G34 fail to arrest following mis-segregation, despite having WT p53. These studies demonstrate that the H3.3 G34R/V mutations are sufficient to transform normal, diploid cells into proliferative, chromosomally instable cells. To determine if this process contributes to tumorigenesis, we used the RCAS/TVA mouse model to overexpress H3.3 WT, G34R, or S31A in the glial precursor cells of mice pups. Over 100 days, S31A and G34R mice had drastically reduced survival (averaging 77, 81, and 100 days for S31A, G34R, and WT mice). Furthermore, most G34R and S31A mice developed HGG, while H3.3 WT mice remained tumor-free and did not develop high-grade tumors. Our work strongly indicates that a major factor in H3.3 G34R pHGG formation is the induction of chromosomal instability – which occurs directly through the suppression of H3.3 S31 phosphorylation.
HGG-32. ONC201 AND ONC206 TARGET TUMOR CELL METABOLISM IN PEDIATRIC DIFFUSE MIDLINE GLIOMA PRECLINICAL MODELS
Authors: Przystal Justyna M , Cosentino Chiara Cianciolo , Yadavilli Sridevi , Zhang Jie , Laternser Sandra , Bonner Erin R , Biery Matt , Vitanza Nicholas A , Koschmann Carl , Cain Jason , Waszak Sebastian M , Mueller Sabine , Nazarian Javad 
Published: June 2021

Abstract
Diffuse midline gliomas (DMGs) remain incurable cancers and new treatments are urgently needed. One promising new therapeutic avenue for these cancers is targeting of metabolic vulnerabilities including a heightened dependence on mitochondrial metabolism. We and others have shown that the oral, brain-penetrant imipridone drugs ONC201 and ONC206 target mitochondrial metabolism in cancer cells. In particular, ONC201 and ONC206 hyper-activate the mitochondrial protease ClpP, impair mitochondrial oxidative phosphorylation (OXPHOS), activate the integrated stress response (ISR) signaling pathway, and induce apoptosis in DMG preclinical models. We validated ClpP as a key target of ONC206 by showing that CRISPR/Cas9-mediated CLPP knockout significantly decreased ONC206 sensitivity in DMG cells. We further showed that imipridone-mediated ClpP activation resulted in significant degradation of the chaperone protein ClpX. Moreover, ONC201 and ONC206 treatment inhibited mitochondrial respiration, decreased mitochondrial membrane potential and triggered extensive mitochondrial structural damage, including disintegration of mitochondrial cristae. Time-course RNA sequencing of five DMG cell lines treated with ONC201 and ONC206, alone or in combination, revealed robust ATF4 and CHOP upregulation, indicating potent activation of ISR signaling. Notably, ATF4/CHOP upregulation was strongest in ONC201/6 combination-treated cells, indicating synergy between the two drugs. We further explored drug combinations by testing ONC201 together with ONC206, Panobinostat, JQ1, and Osimertinib to identify synergistic combination treatments. The strongest synergistic effect was found over a broad IC50 range for ONC201 and ONC206. Finally, we showed that ONC201 and ONC206 significantly prolonged survival of mice bearing brainstem DIPG xenografts. Ongoing studies include assessment of the in vivo efficacy of ONC201 and ONC206 across different CNS tumor models, as well as investigation and validation of clinically relevant biomarkers of response to treatment. In summary, our preclinical data strongly support the utility of the mitochondrial targeting agents ONC201 and ONC206 for the treatment of DMG and other malignant brain tumors.
HGG-33. EXPLOITING METABOLIC DEFECTS WITH NAMPT INHIBITORS IN DIPG
Authors: Muraleedharan Ranjithmenon , Heer Collin , Sundam Ranjini , Brenner Charles 
Published: June 2021

Abstract
Diffuse intrinsic pontine glioma (DIPG) are universally lethal pediatric brain tumors with limited treatment options. We recently performed synthetic lethal drug screen with a panel of DNA repair and metabolic inhibitors in vitro, in patient-derived DIPG cells and isogenic cell lines engineered to contain key DIPG-associated mutations. Nearly 80% of DIPGs harbor a recurrent H3K27M mutation in H3.3 (H3F3A) or H3.1 (HIST1H3B) histones. This has prompted us to consider H3K27M mutation-induced exploitable defects for a therapeutic gain. This screen identified synthetic lethal interactions between H3K27M mutations and the nicotinamide phosphoribosyl transferase (NAMPT) inhibitor, FK866. The association between H3K27M mutations and NAMPTi sensitivity was validated in follow-up studies using isogenic WT and H3K27M-mutant expressing pairs of human immortalized astrocytes and neural progenitor cells (NPCs). In addition, we tested the effects of FK866 in a panel of unique DIPG patient-derived tumor neurospheres in short-term viability assays. We found that FK866 induced depletion of NAD and exhibited toxicity towards H3K27M mutant DIPG cell lines with an IC50 of ~2.5 nM. These findings were consistent across three structurally unique NAMPT inhibitors. Finally, we found that inducible expression of mutant H3K27M results in a gradual, 50% decrease in cellular NAD levels over the course of five days, suggesting that H3K27M mutations may induce an inherent NAD metabolic defect that is exploited by NAMPTi’s. We now seek to understand the mechanistic basis for the observed metabolic defect and NAMPTi sensitivity associated with mutant H3K27M. In addition, we aim to identify specific NAMPTi and DNA damaging agent combinations which maximally exploit this H3K27M-associated NAD metabolic defects.
HGG-34. A DNA DAMAGE REPAIR SIGNATURE IN PRIMARY AND RECURRENT PEDIATRIC HIGH-GRADE GLIOMAS: PROGNOSTIC AND THERAPEUTIC VALUE
Authors: Entz-Werle Natacha , Poidevin Laetitia , Nazarov Petr , Lhermitte Benoit , Chenard Marie Pierre , Poch Olivier , Dontenwill Monique , Van Dyck Eric 
Published: June 2021

Abstract
Pediatric high-grade gliomas (pHGGs), including diffuse intrinsic pontine gliomas (DIPGs), despite their low incidence, are the leading cause of mortality in pediatric neuro-oncology. Frequently, pHGGs, harboring mainly histone mutations, are or become resistant to standard therapies like irradiation or chemotherapies. Recent insights showed in adult HGG the predominant role of DNA damage repair (DDR) as a way of prognostic classification. Given the recent evidence that transcription conflicts like in histone-mutated gliomas can induce replication stress and be linked to DDR abnormalities, this study is aiming to establish a DDR signature able to classify specifically pHGGs and to cluster among them poor responders to radiation. Transcriptomic analyses were performed to discriminate seven pHGGs comparatively to a cohort of 10 pilocytic astrocytomas with specific DDR deregulations. The specific transcriptomic signature obtained from this differential gene expression analysis was compared to the aHGG signature already established. To strengthen, refine and finalize the DDR signature able to classify and cluster the pHGGs, we explored both signatures and their common genes in already published transcriptomic analyses of DIPGs and sus-tentorial pHGGs. To check DDR protein expressions correlated to loss of trimethylation as well as histone and TP53 mutations, an immunohistochemical assessment of several DDR markers was performed on a collection of 21 pHGG diagnostic samples and 9 paired relapses. To validate the DDR functional inhibition, we used 3 patient-derived cell lines bearing H3.3K27M mutations. A finalized signature of 28 genes involved in DNA repair and cell cycle machineries was used to cluster in two groups the pHGG cohorts. The differential protein expression of PARP1, XRCC1, p53 and stem cell markers was linked significantly to the more resistant pHGGs and the rapid progressions after radiotherapy. Those DDR makers might be used as theranostic and therapeutic targets, which were screened in PDCLs with promising results.
HGG-37. UPFRONT TARGETED THERAPY FOR THE TREATMENT OF BRAFV600E-MUTANT PEDIATRIC HIGH-GRADE GLIOMA – A MULTI-INSTITUTIONAL EXPERIENCE
Authors: Rosenberg Tom , Yeo Kee Kiat , Joshirao Mrinal , Michaiel George , Tran Hung , Dahiya Sonika , Kachurak Kara , Friedman Gregory , Huang Michael , Wright Karen , Aguilera Dolly , MacDonald Tobey , Chi Susan , Karajannis Matthias 
Published: June 2021

Abstract

Introduction
Sustained responses to molecular targeted therapy with BRAF with or without MEK inhibitors have been reported in patients with recurrent BRAFV600E-mutant pediatric high-grade gliomas (pHGG). The role of upfront targeted therapy in this population, however, has not yet been established.

Methods
We performed a retrospective, multi-institutional record review of patients with BRAFV600E-mutant pHGGs, treated with off-label BRAF and/or MEK inhibitors as part of their initial adjuvant therapy.

Results
Seventeen patients were identified (median age at diagnosis, 8.8 years, range 1.8–20.2). Histologic diagnoses included HGG/glioblastoma (n=10), anaplastic ganglioglioma (n=3), high-grade neuroepithelial tumor (n=2), diffuse midline glioma (n=1) and anaplastic astroblastoma (n=1). Ten patients underwent biopsy (n=8) or subtotal resection (n=2), while near-total or complete resection was accomplished in seven. Concomitant genetic alterations including CDKN2A/B loss, H3K27M and TERT promotor mutations were found in eight tumors. Thirteen patients received focal radiation therapy (RT) and one received craniospinal irradiation prior to targeted therapy. Adjuvant targeted therapy was initiated shortly after diagnosis or completion of RT. Five patients received BRAF-inhibitor monotherapy (dabrafenib or vemurafenib). Twelve patients received combination therapy with the addition of a MEK inhibitor (trametinib). For patients with measurable disease, best responses per COG criteria were CR (n=3), PR (n=7), SD (n=3) and PD (n=1). With median follow-up of 29 months (range 8–78), two-year PFS and OS for the cohort were 74.7% and 81.1%, respectively. Ten (59%) patients remain free of disease recurrence or progression. Grade 3 or higher toxicities were reported in four patients (neutropenia, skin toxicity/photosensitivity, fatigue and weight loss), leading to therapy discontinuation in two.

Conclusions
Upfront targeted therapy for patients with BRAFV600E mutant pHGG appears tolerable and effective, with a durable disease control rate that is superior to historical data. This promising paradigm is currently being evaluated prospectively in the COG ACNS1723 clinical trial.
HGG-38. DE NOVO PYRIMIDINE SYNTHESIS INHIBITION INDUCES REPLICATION CATASTROPHE MEDIATED CELL DEATH IN DIFFUSE MIDLINE GLIOMA
Authors: Pal Sharmistha , Kaplan Jakub , Stopka Sylwia , Regan Michael , Kann Benjamin , Agar Nathalie , Stiles Charles , Cooney Tabitha , Mueller Sabine , Chowdhury Dipanjan , Haas-Kogan Daphne 
Published: June 2021

Abstract
Diffuse midline gliomas (DMG) are aggressive and lethal pediatric brain tumors that cannot be cured by conventional therapeutic modalities. Using a genome wide CRISPR screen we identified the de novo pyrimidine biosynthesis pathway as a metaboilic vulnerability in DMGs. BAY2402234 is a small molecule inhibitor of DHODH -a rate liminting enzyme in the de novo pyrimidine biosynthesis pathway. BAY2402234 induces cell death in DMG cells at low nanomolar concentrations while sparing adult glioblastoma cells and normal astrocytes. Further investigations revealed drammatic reduction in cellular UMP pools, the precursor for all pyrimidine nucleotides, after DHODH inhibition, specifically in DMG cells. Cytotoxicity of DHODH inhibition in DMG cells is rescued by exogenous uridine, supporting UMP depletion as the mechanism underlying DMG cell death and also showing that cell death is an “on target” response to BAY2402234. Cell death induced by BAY2402234 is a consequence of replication fork stalling as evident by accumulation of chromatin-bound RPA foci and g-H2AX. Stalled replication forks eventually collapse, resulting in replication catastrophy and apoptosis. Cytotoxic effects of DHODH inhibition are further exacerbated by inhibition of the intra-S checkpoint protein, ATR. Combined treatment of DMG cells with DHODH and ATR inhibitors resulted in enhanced accumulation of chromatin-bound RPA, g-H2AX, replication fork collapse and apoptosis. Importantly, in vivo studies verify that both BAY2402234 (DHODHi), and BAY1895344 (ATRi), cross the blood-brain barrier, accumulate in the brain at therapeutically relevant concentrations, and induce DNA damage in intracranial DMG xenografts in mice. Taken together, our studies have identified DHODH inhibition as a DMG-specific vulnerability resulting in cell death; the mechanism of DHODHi-induced cell death led us to identify combined inhibition of DHODH and ATR as a synergistic therapy against DMG tumors.
IMMU-14. COMPUTATIONAL DECONVOLUTION OF TUMOR-INFILTRATING IMMUNE COMPONENTS IN PEDIATRIC NERVOUS SYSTEM TUMORS
Authors: Beck Pengbo , Nabbi Arash , Sill Martin , Sudhaman Sumedha , Kool Marcel , Resnick Adam C , Jones David T W , Pfister Stefan M , Pugh Trevor J , Jäger Natalie 
Published: June 2021

Abstract

Introduction
In the last decade, checkpoint inhibitor-based immunotherapy has been a groundbreaking development in the treatment of cancer. However, only a subset of patients treated with immune checkpoint inhibitors show long-lasting clinical benefit. Studies showed the tumor immune microenvironment (TME) as a particularly important factor influencing treatment response, critical for the design of other or combinatorial immunotherapy treatment strategies. Extensive research has been performed in the adult cancer field to unravel its immunogenomic aspects. However, in pediatric cancer this insight into tumor-infiltrating immune components is still lacking. This study aims to provide insight into the landscape of the immune microenvironment in pediatric primary nervous system tumors.

Methods
Bulk RNA-seq data of 936 pediatric primary solid tumors acquired from multiple international initiatives including Therapeutically Applicable Research To Generate Effective Treatments (TARGET), the International Cancer Genome Consortium (ICGC) and the Children’s Brain Tumor Tissue Consortium (CBTTC) were included in this study. We applied computational tumor immune microenvironment deconvolution, repurposed RNA-seq data to recover infiltrating T- and B-cell clonotypes and studied checkpoint gene expression across pediatric neural tumors.

Results
Among pediatric neural tumors, embryonal tumors with multilayered rosettes (ETMR) and medulloblastomas (MB) were least immune infiltrated. Neuroblastomas (NBL) had the highest T-cell infiltration among pediatric cancers, while atypical teratoid/rhabdoid tumors (ATRT) had the highest levels of CD8 T cell infiltration among pediatric CNS tumors. While tumor mutational burden (TMB) was associated with immune cell infiltration in adult lung cancers and melanomas, we found no significant associations in pediatric cancers. The majority of NBL samples expressed LAG3, but ~10% of samples had elevated levels of TIM3 gene expression, suggesting a distinct mode of immunosuppression in this subset.
IMMU-17. USE OF MRNA FOR SAFE AND EFFECTIVE GD2-DIRECTED CAR T CELLS TO TREAT DIFFUSE MIDLINE GLIOMAS
Authors: Foster Jessica , Griffin Crystal , Stern Allison , Brimley Cameron , Storm Phillip , Barrett David , Resnick Adam 
Published: June 2021

Abstract
Chimeric antigen receptor (CAR) T cells targeting the disialoganglioside GD2 have garnered interest as an effective therapeutic for treating diffuse midline glioma (DMG). However, prior studies raised significant concerns of neurotoxicity and fatality when using virally transduced CAR T cells against these midline tumors. Building upon our prior work optimizing mRNA for use in CAR T cells (Hum Gen Ther, 2019), we hypothesized transient GD2-directed mRNA CAR T cells could be successfully employed for safe and effective treatment of DMG. GD2-directed CAR T cells were created using mRNA encoding the 14G2a single chain variable fragment paired with 41BB and CD3-zeta co-stimulatory domains and transfected into human T cells. GD2-directed CAR T cells were tested against a panel of DMG cell lines and two murine xenograft models of DMG: 7316-6349 and SU-DIPG13P*. In all DMG cellular models, GD2-directed mRNA CAR T cells induced significant tumor cell death compared to CD19-directed mRNA CAR T cell controls. In vivo, mRNA CAR T cells were delivered locoregionally using an indwelling infusion catheter to allow for repeated dosing. Four intratumoral doses of 5 x 106 GD2-directed mRNA CAR T cells induced significant tumor regression measured by bioluminescence in DMG model 7316-6349 (p&lt;0.0001). In addition, GD2-directed mRNA CAR T cells prolonged survival of mice harboring the aggressive DMG model SU-DIPG13P* by 61% (mean survival 29 days versus 18 days, p&lt;0.01) following four intratumoral doses of 4 x 106 CAR T cells. No GD2-directed CAR T cell treatment-related deaths or toxicities were observed. These data highlight the utility of using mRNA to titrate CAR T cell therapy in the brain, and establish GD2-directed mRNA CAR T cells as a safe and effective method for treating DMG.