bluuebunny/crossref_metadata_2025 huggingface dataset:
BIOM-03. IMMUNO-ONCOLOGIC PROFILING OF PEDIATRIC BRAIN TUMORS REVEALS MAJOR CLINICAL SIGNIFICANCE OF THE TUMOR IMMUNE MICROENVIRONMENT
Authors: Levine Adrian , Nobre Liana , Das Anirban , Milos Scott , Bianchi Vanessa , Johnson Monique , Fernandez Nicholas , Stengs Lucie , Ku Michelle , Rana Mansuba , Fedoráková Ivana , Bennett Julie , Ramaswamy Vijay , Siddaway Robert , Tabori Uri , Hawkins Cynthia
Published: June 2024

With the success of immunotherapy in cancer, understanding the tumor immune microenvironment (TIME) has become increasingly important; however in pediatric brain tumors this remains poorly characterized. Accordingly, we developed a clinical immune-oncology gene expression assay, including the 18-gene tumor inflammation signature (TIS) as a marker of overall immune activation, and validated this with immunohistochemistry for key cell-type markers. We used this assay, in conjunction with public data from the Pediatric Brain Tumor Atlas (PBTA), to profile a diverse range of 1382 samples in total with detailed clinical and molecular annotation. Overall among the main types of pediatric brain tumors, low-grade gliomas (LGG) had the highest inflammation levels (regardless of genetic driver alteration) and medulloblastomas the lowest. In LGG we identified three distinct patterns of immune activation with prognostic significance in BRAF V600E-mutant tumors, specifically that higher inflammation predicted worse outcomes. In high-grade gliomas (HGG), we observed immune activation and T-cell infiltrates in tumors that have historically been considered exclusively immune cold. There were genomic correlates of inflammation levels, with BRAF-V600E-mutant HGG having the highest inflammation levels and suggesting these may be candidates for combination MEK inhibition and immunotherapy. In mismatch repair deficient HGG, we found that high TIS was a significant predictor of response to immune checkpoint inhibition, and demonstrated the potential for multimodal biomarkers (TIS plus tumor mutation burden) to improve treatment stratification. Importantly, while overall patterns of immune activation were observed for histologically and genetically defined tumor types, there was significant variability within each entity, indicating that the TIME must be evaluated as an independent feature from diagnosis. In sum, in addition to the histology and molecular profile, this work underscores the importance of reporting on the TIME as an essential axis of cancer diagnosis in the era of personalized medicine.
CNSC-05. THE GABAERGIC INTERNEURONAL AND NEURAL PROGENITOR LINEAGE OF THE DIFFUSE HEMISPHERIC GLIOMA, H3 G34-MUTANT, SHOW CALCIUM EVENTS IN THE FORM OF INTERCELLULAR WAVES AND SPONTANEOUS ELECTRICAL ACTIVITY
Authors: Cruzeiro Gustavo Alencastro Veiga , Cascio Costanza Lo , Neyazi Sina , Junior Carlos Alberto Oliveira Biagi , Haase Rebecca , Nascimento Andrezza , Frederico Stephen Charles , Sarnow Katharina , Venkataramani Varun , Liu Ilon , Huang Kun , Tang Xin , Segal Rosalind , Monje Michelle , Filbin Mariella
Published: June 2024

BACKGROUND

High-grade gliomas are one of the deadliest pediatric brain cancers. A diffuse subtype of hemispheric high-grade gliomas in adolescents and young adults is assigned as DHGH3G34-mutant. Notably, DHGH3G34-mutant gliomas distinctively contain cancer cell types resembling progenitors and GABAergic interneurons (eIN-like). Normal GABAergic interneurons influence neural network dynamics in the brain by communicating with other cells through the transmission of calcium and electrical signals. Our goal is to investigate the spatial organization of eIN-like glioma cells and evaluate whether they form networks and exert electrical activity relevant to tumor progression.

METHODS

Calcium Imaging: We used the Genetically encoded calcium indicator GCAMP7s with hDCX tdTomato promoter reporter construct. Voltage Imaging: We used the voltage indicator ASAP3 for detecting changes in membrane potential. Light microscopy: The whole fixed DHGH3G34-mutant PDX brain was mounted and imaged using the Zeiss Lightsheet 7 Microscope.

RESULTS

Using single-cell RNA sequencing and immunocytochemistry on DHGH3G34-mutant primary samples, we found that eIN-like cells express DCX (doublecortin) and form structures observed in ganglionic eminences. Calcium imaging revealed that DHGH3G34-mutant cells exhibit intercellular calcium waves. To track eIN-like cells, we developed a tdTomato fluorescent reporter for the DCX gene promoter activity (hDCX). Combining hDCX with GCAMP7s, we conducted calcium imaging and observed rhythmic calcium events in eIN-like cells at a frequency of 62.5mHz. Additionally, voltage imaging demonstrate that DHGH3G34-mutant cells show spontaneous action potential bursts. Furthermore, using light sheet microscopy on DHGH3G34-mutant PDX models, we observed glioma cells spreading into the contralateral hemisphere of the brain parenchyma.

CONCLUSION

DHGH3G34-mutant tumors represent highly invasive gliomas distinguished by the presence of progenitors and cells of the GABAergic interneuron lineage, exhibiting calcium waves and spontaneous action potential bursts. We are currently delving into the biological implications of these distinctive traits to gain insights into novel therapeutic strategies for the unique biology of DHGH3G34-mutant gliomas.
CP-06. PEDIATRIC CRANIOPHARYNGIOMA: SINGLE CENTER RETROSPECTIVE STUDY OF SURVIVAL, ENDOCRINOLOGIC, AND VISION OUTCOMES IN 90 PATIENTS
Authors: Ronsley Rebecca , Tam Lydia T , Do Olivia , Ermoian Ralph P , Leary Sarah E S , Werny David , Leonard Skyler , Ferreria Manuel , Ojemann Jeffrey , Ellenbogen Richard G , Lee Amy , Ruzevick Jacob
Published: June 2024

BACKGROUND

Craniopharyngioma is histologically benign. However, due to its location, morbidity is high and may be impacted by therapeutic approach.

METHODS

A retrospective study of patients treated for craniopharyngioma at Seattle Children’s Hospital from 1996-2023 was performed. Surgical approach, extent of resection, subsequent surgery, radiation and medical therapies, and serial surveillance magnetic resonance imaging for progression were reviewed. Five-year overall survival (OS) and progression free survival (PFS) were calculated and morbidity including endocrine and vision dysfunction was described.

RESULTS

90 patients were included of which 53(58.9%) were male. Median age at diagnosis was 8.70 (range 1.39-25.17) years. Surgical approach included endoscopic (N=24), craniotomy (N=64), and combined (N=2). Extent of resection was biopsy N=2, subtotal resection N=7, near total resection N=38, gross total resection N=30. Pathology was adamantinomatous subtype in all cases and 68 had a documented CTNNB1 alteration and 3 had a BRAF alteration in those with molecular analysis. 5-year OS was 96.1% (SE 0.13) and 5-year PFS was 51.9% (SE0.21), with median follow-up of 97 months. Transcranial compared to endoscopic endonasal approach was associated with increased risk of progression (62% vs 30%, OR 1.23). 34 patients were treated with radiation upfront (N=19) or at progression (N=15). 55% of patients at diagnosis and an additional 30% in follow up had pituitary hormone dysfunction. Hypothalamic obesity was documented in 31% of patients and varied by surgical approach. At presentation, ophthalmologic abnormalities included optic nerve edema (18%) and optic nerve pallor (24%) and 30% had abnormal visual acuity. Following initial surgery, 50% had improvement in visual acuity, 35% stable and 15% had worsened visual acuity.

CONCLUSIONS

Upfront surgical strategy may impact risk of disease recurrence; however, its relationship to long term morbidity is not clear. Further work to delineate therapeutic strategies and their relationship to morbidity in this patient population is needed.
CP-09. HTDS ON NOVEL CELL LINES IDENTIFIED BETA-CATENIN INHIBITOR TEGATRABETAN AND PROTEASOME INHIBITOR BORTEZOMIB AS TWO NOVEL POTENTIAL AGENTS AGAINST ADAMANTINOMATOUS CRANIOPHARYNGIOMA
Authors: Cocito Carolina , Padilla Christopher C , Pisapia David J , Hertz Charli Ann J , Dahmane Nadia , Greenfield Jeffrey P
Published: June 2024

BACKGROUND

Adamantinomatous craniopharyngiomas (ACPs) are rare intracranial suprasellar tumors, primarily affecting pediatric population. ACPs are histologically benign but their location makes them highly debilitating and clinically challenging with no standard of care available. Mutations in exon 3 of the CTNNB1 gene, codifying for β-catenin, are pathognomonic hallmark of ACPs. Although several cell lines and patient-derived models have been sporadically described, no reproducible pre-clinical models are available for these tumors.

METHODS

Eight ACP patients underwent gross total resections and we were able to generate novel cell lines from four of them. The lines, named CBTP-77, CBTP-97, CBTP-101 and CBTP-119, grow consistently and have been passaged more than 15 times. Immunocytochemistry and RNA sequencing were performed, highlighting inter- and intra-line heterogeneity, typical of ACP tumors. To identify new therapeutic targets, High Throughput Drug Screening (HTDS) was performed on two lines.

RESULTS

β-catenin inhibitor tegatrabetan, FGFR inhibitor erdafitinib, and the proteasome inhibitor bortezomib resulted as top effective agents. In vitro validation of the three agents confirmed strong cytotoxicity and on target activity shown by downregulation of the WNT and PI3K/AKT/mTOR pathways. The limited CNS permeability of these agents, makes them great candidates for local intra-cystic delivery, as described for other therapeutic options for ACP.

CONCLUSIONS

Our study represents the first large scale screening for new therapeutic agents for ACP, highlighting the importance of establishing consistent cell lines and preclinical models for these rare and devastating tumors.
CP-10. DOES PRE-IRRADIATION GROSS TUMOR VOLUME PREDICT THE RISK OF PROGRESSION AFTER RADIATION THERAPY IN PEDIATRIC PATIENTS WITH ADAMANTINOMATOUS CRANIOPHARYNGIOMA?
Authors: Brooks Tamar , Gao Dexiang , Dorris Katie , Mirsky David M , Staulcup Susan , Prince Eric , Moskalenko Marina , Ignowski Elizabeth , Wandrey Narine , Fakhoury Kareem , Hankinson Todd C , Milgrom Sarah A
Published: June 2024

OBJECTIVE

In a cohort of patients who were treated with resection and adjuvant radiotherapy (RT) for adamantinomatous craniopharyngioma (ACP), we explored whether gross tumor volume (GTV) at the initiation of RT was associated with the risk of progressive disease (PD) following treatment.

METHODS

Pediatric and adolescent patients who received surgery and RT for ACP at a single institution from 1998-2021 were identified. Univariable Cox regression analyses (UVA) were performed to assess the association between pre-RT GTV and PD after RT. Multivariable analyses (MVA) were used to control for potential confounders. Two different endpoints were used to define PD. The first definition was based on radiographic tumor growth and the second definition was the requirement for an additional tumor-directed intervention following the completion of RT.

RESULTS

48 patients were eligible for inclusion. The median age at diagnosis was 7.9 years (range: 2.1-17.4). All patients were treated with surgery and RT with median dose of 52.2 Gy (range: 45-55.8 Gy) and median GTV of 9.86 cm3 (range 0.7-117.7 cm3). Upon follow-up, 8 patients experienced PD based on both definitions. Five-year EFS was 85.4% (95% CI: 74.1 -98.3%). On both UVA and MVA, GTV was significantly associated with a small increased likelihood of PD (HR 1.02, 95% CI 1.00 - 1.04, p=0.022). However, after exclusion of a single outlier with GTV of 117.7 cm3 prior to RT, a second analysis identified no significant association between GTV and PD (UVA: HR 1.03, p=0.4; MVA: HR 1.06, p=0.2).

CONCLUSIONS

For most patients with ACP, the GTV at the initiation of RT is not associated with the risk of PD. This finding suggests that aggressive tumor debulking for the purpose of improving the efficacy of RT may not be necessary. For giant ACPs, however, novel strategies may be necessary for tumor control.
CP-11. BODY MASS INDEX INCREASE FOLLOWING CHILDHOOD CRANIOPHARYNGIOMA SURGERY IS MOST SEVERE IN THE FIRST THREE MONTHS; TIME TO ACT!
Authors: Hulsmann Sanne C , Hoving Eelco W , de Vos-Kerkhof Evelien , Janssens Geert O , Bakker Boudewijn , van Santen Hanneke M
Published: June 2024

BACKGROUND

Centralization of care for childhood craniopharyngioma (cCP) in the Netherlands (June 2018), including the involvement of a multidisciplinary team, has resulted in a decreased prevalence of hypothalamic obesity. Unfortunately, in certain patients, increasing body mass index standard deviation scores (BMI SDS) are still encountered after tumor resection. The aim of this analysis is to identify timing of BMI change, incidence/prevalence and predictive factors for BMI changes enabling early preventive strategies.

METHODS

A retrospective cohort study was performed of all newly diagnosed cCP patients since centralization of care in the Netherlands (June 2018). Data on anthropometrics, radiology, tumor treatment (surgery, radiotherapy, interferon and tocilizumab), hypothalamic-pituitary function, medication and visual function, were collected. Uni- and multivariate analyses were done with the ‘mean’ change in BMI SDS at 3, 6 and 12 months after resection as primary endpoint. Body mass index trends were assessed as secondary outcome.

RESULTS

Of 34 patients, median age 8.93 (range 0.79-17.36), mean ∆BMI SDS was most pronounced at 3 months (+1.09 SDS (SD 1.17), p<0.01), followed by a further mean increase of +0.25 SDS from 3 to 6 months (SD 0.50, p=0.01). In 17 patients, BMI SDS increased with ≥1.0 SDS in the first 3 months after resection (50%). A Muller grade 2 at diagnosis was associated with ∆BMI SDS at 3 months. Children with the highest BMI at diagnosis had the highest BMI at three months, but not the highest ∆BMI SDS.

CONCLUSION

Changes in BMI SDS ≥1.0 are observed in half of cCP patients following surgical resection despite expert multidisciplinary care, with most pronounced BMI changes during the first 3 months. These observations are a plea for more early interventions to prevent (morbid) hypothalamic obesity.
CP-12. PHASE 0 AND FEASIBILITY SINGLE-INSTITUTION CLINICAL TRIAL OF INTRAVENOUS TOCILIZUMAB FOR ADAMANTINOMATOUS CRANIOPHARYNGIOMA
Authors: Dorris Kathleen , Mettetal Ashley , Hemenway Molly , Winzent-Oonk Shelby , Dahl Nathan , Levy Jean Mulcahy , Prince Eric , McWilliams Jennifer , Jordan Kimberly R , Mirsky David , Hoffman Lindsey , Hankinson Todd
Published: June 2024

BACKGROUND

Adamantinomatous craniopharyngioma (ACP) is a devastating skull-base tumor believed to derive from epithelial remnants of the primordial craniopharyngeal duct (Rathke’s pouch), which gives rise to the anterior pituitary gland. ACP lacks medical antitumor therapies. Current standard therapy with surgery and radiation is associated with poor quality of life. Clinical and preclinical data indicate that IL-6 blockade may contribute to ACP tumor control.

METHODS

Children aged 2–21 years with newly diagnosed or previously treated ACP with measurable disease are eligible for the Phase 0/feasibility single-institution clinical trial (NCT03970226) of intravenous (IV) tocilizumab at Children’s Hospital Colorado (Aurora, CO, USA). The phase 0 stratum is completed and involved a single IV tocilizumab dose prior to a standard-of-care surgical resection. The feasibility portion of the trial involves IV tocilizumab every two weeks for up to 13 28-day cycles. Tocilizumab is administered at the established weight-based pediatric dosage of 8 mg/kg for patients who weigh ≥30kg or 12 mg/kg for patients who weigh <30kg.

RESULTS

Six patients (4 female; median age—10 years, range—6-15 years; 4 who had progressive disease after radiation prior to enrollment) have been treated on the feasibility stratum. The subjects have received a median of 6 cycles (range 0.5-13 cycles). The best response to date is minor response. One dose reduction for myelosuppression has been required. One patient discontinued therapy due to anaphylaxis related to tocilizumab on the first dose.

CONCLUSION

Systemic delivery of tocilizumab at the established pediatric dosage is promising for treatment of ACP based on preclinical work and its demonstrated penetration into cystic and solid portions of ACP tumors. The therapy to date has been well tolerated overall. Further study is underway through a CONNECT consortium international Phase II trial (NCT05233397) to explore clinical efficacy.
DIPG-06. FUNCTIONAL MULTI-OMIC PROFILING OF THE LANDSCAPE OF DIFFUSE MIDLINE GLIOMAS TO IDENTIFY THERAPEUTIC VULNERABILITIES
Authors: Findlay Izac J , Staudt Dilana , Duchatel Ryan J , Jackson Evangeline R , Kearny Padraic , Persson Mika L , McEwen Holly , Smith Nathan D , Vitanza Nicholas A , Cain Jason E , Waszak Sebastian M , Hansen Mitchell , Alvaro Frank , Dun Matthew D
Published: June 2024

BACKGROUND

Diffuse midline glioma (DMG) is a devastating pediatric high-grade glioma (pHGG) located along the midline structures of the brain and spine. Palliative radiotherapy is the only approved treatment outside of clinical trials, with patients given 9–11-months survival post-diagnosis. Over the last 10-years, it has become common for patients to be prescribed precision therapies targeting genetic alterations, however largely, DMG harbor genetic alterations that are untargetable.

METHODS

To improve DMG patient outcomes, we are performing multi-omic analysis on 210 DMG, normal controls and other pHGG samples to functionally characterize the genomic landscape of these tumors and understand the protein-controlled functional consequences of recurring somatic alterations.

RESULTS

Genomic sequencing has been performed on 40/210 pHGG samples, identifying 290 unique mutations and several recurrent chromosomal alterations. Additionally, proteomic/phosphoproteomic profiling identified both unique and similar features of post-translational landscapes across genotypes. Examining H3.3K27M DMG cell-lines, SU-DIPG-XIII and UON-VIBE5, genomic sequencing identified 11 and 6 mutations respectively, however, only UON-VIBE5 possessed a genomically-predicted target, a PDGFRA-D842V mutation, potentially targetable by Avapritinib. Conversely, investigation of proteomes/phosphoproteomes revealed several potential therapeutic vulnerabilities. SU-DIPG-XIII possessed high enrichment of HDAC/BRD and CDK pathways and UON-VIBE5, ATM-centric pathways. In vitro proliferation assays of Avapritinib treated UON-VIBE5 failed to elicit a response at clinically relevant doses, while all proteomically-predicted therapies (HDAC – Fimepinostat, BRD – JQ1, CDK – Ribociclib and ATM – WSD-0628) successfully influenced tumor cell growth whilst leaving microglia controls cells unaffected. Avapritinib and WSD-0628 (CNS-active ATM inhibitor) drug predictions were further validated in the RA055 DMG model, which possesses similar genomic and proteomic features to UON-VIBE5. Avapritinib alone failed to influence tumor growth, while WSD-0628 notably reduced cell proliferation. The combination of these drugs, however, synergistically inhibited RA055 proliferation.

CONCLUSIONS

These findings thus provide preliminary evidence supporting the need to take a multi-omic approach in treating DMGs.
DIPG-10. SUBCLONAL POPULATIONS OF DIFFUSE MIDLINE GLIOMA CELLS PROMOTE TUMORIGENESIS THROUGH AP-1 TRANSCRIPTION FACTOR DYSREGULATION
Authors: Kessler Ketty , Grabovska Yura , Carvalho Diana , Burford Anna , Temelso Sara , Tari Haider , Molinari Valeria , Crampsie Shauna , Yu Lu , Choudhary Jyoti , Proszek Paula , Hubank Mike , Mackay Alan , Jones Chris
Published: June 2024

BACKGROUND

Paediatric-type diffuse high-grade glioma (PDHGG) are brain tumours occurring largely in children, classified into distinct subgroups based upon their location and defining molecular alterations, with very poor clinical outcomes. The presence of a wide diversity of genotypically- and phenotypically-distinct subclones within individual tumours provides a substantial barrier to developing effective treatments.

METHODS

To understand their role, and how this may be exploited therapeutically, we established 218 subclones in vitro from 8 patient-derived PDHGG different cell lines, and characterised them using single-cell and bulk multi-omic approaches linked to high-content phenotypic screening.

RESULTS

Highly migratory subpopulations of diffuse midline glioma (DMG) cells were isolated, whereby RNAseq and proteomics identified a specific metaprogram associated with migration, enriched in AP-1 target genes, and common across a series of DMG patient-derived cells. Data integration of scRNAseq/scATACseq showed an opening of the chromatin at loci such as the early development transcription factors FOS and JUN (AP-1 targets) associated with their overexpression in highly migratory cells. Co-culturing these subclones with less motile patient-matched counterparts conferred an enhanced migratory capacity in these cells, so we sought to abrogate this via high-throughput screening of over 900 compounds aimed at selectively targeting DMG subclones grown in co-culture. Inter-clonal interactions were disrupted by a diverse set of compound classes including previously identified secreted protein inhibitors (MMP2/9i). Moreover, hits were seen with numerous neuroactive drugs inhibiting a series of neural receptors, ion channels, and transporters shown to be highly expressed in subpopulations of DMG cells, and linked to a common mechanism of action via AP-1.

CONCLUSIONS

Clinically available neuroactive drugs may therefore represent a novel therapeutic approach in targeting highly migratory cells within DMG, which undergo chromatin remodelling to confer a pro-tumorigenic programme via upregulation of AP-1 targets.
DIPG-18. MULTIMODALITY VISUALIZATION OF A PRECLINICAL MODEL OF DIFFUSE MIDLINE GLIOMA DEMONSTRATES CLEAR TREATMENT EFFECT AFTER RNA NANOPARTICLE THERAPY
Authors: Moor Rachel , Weidert Frances , von Roemeling Christina , Robles Jonathan Chardon , Merritt Matthew , Rushin Anna , Sharma Guarav , McGuiness James , Thomas Nagheme , DeVries Anna , Qdaisat Sadeem , Zhang Dingpeng , Rahman Maryam , Ogando-Rivas Elizabeth , Castillo Paul , Ghiaseddin Ashley , Carrera-Justiz Sheila , Mitchell Duane , Mendez-Gomez Hector , Silver Natalie , Ligon John , Sayour Elias
Published: June 2024

BACKGROUND

Diffuse Midline Glioma (DMG) is an inoperable pediatric brain tumor with insufficient treatment options. Our group has developed a novel treatment modality for this disease and others, using mRNA vaccines consisting of tumor derived antigens in a unique lipid-nanoparticle (NP).

METHODS

We have treated mice, canines, and human patients with primary brain tumors to define local immunotherapuetic treatment response. We treated our neonatally implanted DMG model with NPs generated from total-tumor mRNA beginning day 31 and used MRI, MR spectroscopy, suprerspectral imaging, and immunofluorescence to visualize and quantify treatment effects.

RESULTS

In canine glioma patients enrolled in clinical trial, we visualized radiographically enlarging tumors of animals that ultimately became long-term survivors, supporting the concept of a process by which lesion size increases in the absence of true disease progression. In our clinical trials investigating NPs as a treatment for human and canine glioma, we found features of both reactive gliosis and traditional pseudoprogression (immune infiltration), suggesting a new radiographic diagnosis, thus named paraprogression. In mice bearing DMG, NP therapy led to long-term survivors, despite the development of hydrocephalus. We were able to visualize treatment response during the course of NP therapy using MRI.

CONCLUSIONS

These results are rapidly translatable to aid clinical decision making in patients with brain tumors treated with immunotherapy. Future studies will aim to mitigate effect of paraprogression without compromising benefit of NP therapy.
DIPG-22. A NOVEL ROLE FOR ACVR1/ALK2 IN REGULATING CHOLESTEROL BIOSYNTHESIS PROVIDES A NEW COMBINATORIAL THERAPEUTIC APPROACH FOR PATIENTS WITH DMG
Authors: Rogers Rebecca , Grabovaska Yura , Mackay Alan , Carvalho Diana , Dobinson Claire , Pereira Rita , Sejdiu Drenusha , Burford Anna , Molinari Valeria , Ruddle Ruth , Raynaud Florence , Chew Nicole , Tsui Vanessa , Sun Claire , Firestein Ron , Jones Chris
Published: June 2024

BACKGROUND

The discovery of somatic mutations in ACVR1, which encodes the serine/threonine kinase ALK2, in 20-25% of diffuse midline glioma H3K27-altered (DMG-H3K27) patients has led to the development of numerous chemotypes of ALK2 inhibitor (ALK2i). However, in models of ACVR1-mutant DMG-H3K27 the efficacy of single-agent ALK2i’s has been modest and new combinatorial treatment approaches are desperately needed.

METHODS

We performed unbiased combinatorial genetic and drug screens in patient-derived ACVR1-mutant DMG-H3K27 cells in vitro, using multiple chemotypes of ALK2i. We also generated in vitro transcriptomic, proteomic and metabolomic profiles of three ACVR1-mutant DMG-H3K27 models following ALK2i treatment.

RESULTS

The top ALK2i sensitising CRISPR hits (vehicle vs. ALK2i) were analysed using gene set enrichment analysis (GSEA) which revealed specific genetic dependencies in cholesterol synthesis/regulation following both M4K2009 and TP-0184 treatment. The top CRISPR hits included multiple key nodes of cholesterol biosynthesis such as HMGCR (HMG-CoA>mevalonate), LSS (squalene-2-2-epoxide>lanosterol) and DHCR24 (lanosterol>cholesterol). These hits were pharmacologically mirrored in the top hits identified from the drug screen, including drugs that either directly or indirectly target cholesterol synthesis. Integration of the multi-omic data revealed a raft of changes associated with cholesterol biosynthesis/efflux. The expression of HMGCR/HMGCS1 (cholesterol synthesis) and LXR/ABCA1 (cholesterol export) were down and upregulated, respectively, with metabolomic data showing a reduction in cholesterol and it’s derivative 4-cholesten-3-one and an increase in desmosterol, an immediate precursor to cholesterol synthesis. Highly synergistic interactions were observed between multiple ALK2i and a panel of pharmacological agents targeting the cholesterol synthesis/efflux pathway, including routinely used drugs such as simvastatin and lovastatin, clinically well-tolerated and with good CNS penetrance in immuno-compromised and immune-competent mouse models, in whom in vivo combination studies are ongoing.

CONCLUSIONS

These data provide evidence for a previously unappreciated link between ALK2 signalling and metabolism, and identify readily translatable combination partners for ALK2i in the clinic.
DIPG-24. COMBINED EPIGENETIC THERAPY WITH FACT AND BET INHIBITORS REMODELS CHROMATIN AND DISRUPTS ONCOGENIC TRANSCRIPTION IN DIFFUSE MIDLINE GLIOMA
Authors: Holliday Holly , Khan Aaminah , Jayatilleke Nisitha , Mayoh Chelsea , Ross Samuel E , Guo Xinyi , Sanguino Yolanda Colino , Gopalakrishnan Anjana , Ehteda Anahid , Tsoli Maria , Ziegler David S
Published: June 2024

BACKGROUND

Aberrant epigenetic regulation is a hallmark of Diffuse Midline Glioma (DMG), an incurable pediatric brain tumor. The H3K27M driver histone mutation leads to transcriptional dysregulation, indicating that targeting the epigenome and transcription may be a key therapeutic strategy against this aggressive cancer. One such target is the Facilitates Chromatin Transcription (FACT) histone chaperone. FACT is targeted by the curaxin compound CBL0137 which is currently in Phase I/II Children’s Oncology Group clinical trial as a single agent for pediatric cancer patients (NCT04870944).

METHODS

We applied a multi-omics approach by integrating epigenetic profiling with transcriptomics (CUT&RUN, ATAC-seq and RNA-seq) to interrogate the role of FACT in DMG. We then employed in vitro cytotoxicity assays and in vivo orthotopic patient-derived xenograft (PDX) pre-clinical models to test a novel, mechanistic-anchored epigenetic combination therapy.

RESULTS

We found FACT to be enriched at developmental gene promoters, coinciding with regions of open chromatin and binding motifs of core DMG regulatory transcription factors (OLIG2, TCF12, and SNAI1). FACT interacted and co-localized with the Bromodomain and Extra-Terminal Domain (BET) protein BRD4 at active promoters and enhancers, suggesting functional cooperation between FACT and BRD4 in DMG. Investigating the combination of CBL0137 with two BET inhibitors (JQ1 and trotabresib) revealed potent cytotoxicity in vitro, particularly against H3K27M-mutant cells. Furthermore, this approach was recapitulated in vivo, significantly extending the survival of three independent orthotopic PDX models of DMG. Mechanistically, CBL0137 decreased chromatin accessibility, synergising with BET inhibition to disrupt transcription, leading to transcriptional silencing of several key oncogenes (MYC, PDGFRA and MDM4) as well as causing alterations to the splicing landscape.

CONCLUSIONS

This study highlights the therapeutic promise of simultaneously targeting FACT and BRD4 in DMG, and provides the rationale for a combination clinical trial, currently in development.
DIPG-26. H3.3K27M ENHANCES SENSITIVITY TO ATR INHIBITION IN PEDIATRIC GLIOMA
Authors: Wang Heng , Kaplan Jakub P , Chowdhury Dipanjan , Pal Sharmistha , Haas-Kogan Daphne
Published: June 2024

BACKGROUND

Diffuse midline glioma (DMG) is an aggressive, uniformly fatal childhood brain tumor. Radiation remains the standard of care but provides limited survival benefit. The most prevalent driver mutation in DMG is lysine to methionine mutation at position 27 (K27M) of histone H3.3, resulting in global loss of H3K27 trimethylation and remodeled chromatin, thus promoting DMG tumorigenesis. We have previously shown that inhibition of de novo pyrimidine synthesis induces replication stress that enhances DMG dependency on ATR. In this study, we sought to explore the contribution of H3K27M to basal replication stress in DMGs and vulnerability to ATR inhibition.

METHODS

To investigate ATR sensitivity of DMG, we employed ART0380, a potent and selective ATR inhibitor currently in Phase 2 clinical trials in adults. Using isogenic models and a panel of patient tumor-derived DMG cell lines we perform viability assays, immunoblotting, microscopy and flow cytometry-based assays to determine various endpoints/measures of ATR inhibition in DMGs.

RESULTS

Our data show that H3K27M histone oncoproteins enhance dependency on ATR and ATR inhibition synergizes with radiation to induce DMG cell death. After ART0380 treatment, H3K27M oncoproteins result in greater induction in apoptosis and accumulation of DNA damage (as measured by g-H2AX) compared with their histone H3 wild-type isogenic counterparts. Inhibition of ATR by ART0380 is confirmed by effects on phosphorylation of bona fide ATR targets-CHK1 and RPA. To gain mechanistic insights into how H3K27M modulates dependency on ATR, we have evaluated accumulation of chromatin-bound RPA, R-loops, and frequency of transcription and replication collisions (TRC) in isogenic cells.

CONCLUSIONS

Our data indicate a heightened sensitivity of H3K27M mutated DMG to ATR inhibition thus identifying a therapeutic window for DMG treatment with ATR inhibitor as monotherapy or in combination with radiotherapy.
DIPG-32. NEOANTIGEN HEAT SHOCK PROTEIN VACCINE, RHSC-DIPGVAX, IS ASSOCIATED WITH INCREASED VACCINE-INDUCED B CELLS AND BCR/TCR REPERTOIRE DIVERSITY IN PEDIATRIC PATIENTS WITH DIFFUSE MIDLINE GLIOMAS
Authors: Boland Lauren K , Wang Si , Fourati Slim , DeLay Suzi , Chia Tzu-Yi , Billingham Leah , Katz Joshua , Wei Chao , Geng Yuheng , Sipila Patrick , Narendran Aru , Chi Susan , Sato Mariko , Lee-Chang Caty , Miska Jason M , Plant-Fox Ashley S
Published: June 2024

BACKGROUND

Diffuse midline gliomas (DMG/DIPG) remain a devastating class of tumors with limited treatment options. Cancer vaccines educate the immune system to tumor neoantigens to facilitate immune-mediated tumor destruction. DMGs have high-frequency, co-occurring neoantigens and are therefore a relevant target for activation of anti-tumor immunity via vaccine. To this end, we have initiated a clinical trial (NCT04943848) to determine if a neoantigen peptide-based vaccine with recombinant human heat-shock constitutive protein 70 and QS-21 adjuvant can promote anti-tumor immunity against DMG/DIPG. NCT04943848 is ongoing and now enrolling subsequent arms with rHSC-DIPGVax plus checkpoint blockade.

METHODS

Whole blood was collected before the first vaccine cycle (C1D1). Vaccine doses were administered every 2 weeks and blood was collected prior to the third or fifth vaccine cycle (C3D1 & C5D1). Peripheral blood mononuclear cells (PBMCs) were isolated and stored at -160°C. PBMCs from each patient, pre- and post-vaccine, were stained with human T-cell, myeloid, and B-cell antibody panels before spectral flow cytometry, single-cell RNA sequencing, and genomic DNA isolation for T- and B-cell receptor (TCR/BCR) sequencing. Spectral flow cytometry data was analyzed via FlowJo v10. TCR/BCR sequencing was processed via MiXCR and Immunarch.

RESULTS

Following vaccination, there were higher percentages of two mature CD20+CD19+IgM+ B cell populations, including a 4-1BBL+CD86+ population previously shown to induce robust CD8+ T-cell activation in adults with glioblastoma. Interestingly, given the recent discovery of TIM3’s importance in DIPG, we also identified TIM3-expressing CD4+ and CD8+ T cell populations. Finally, TCR/BCR sequencing demonstrated increased receptor repertoire diversity after vaccination, which is indicative of the vaccine shaping adaptive immunity.

CONCLUSIONS

Peripheral immune populations and TCR/BCR sequences change during vaccination with rHSC-DIPGVax. Ongoing analyses will determine the specificity of the patient immune response to vaccine antigens and correlate changes in immune populations to clinical course.
DIPG-38. VAL-083 IN COMBINATION WITH AZD1775 FOR THE TREATMENT OF DIFFUSE MIDLINE GLIOMA
Authors: Subramaniam Bavani , Roy Debasish , Woldegerima Ayda , Przystal Justyna , Zhang Jie , Laternser Sandra , Kritzer Bettina , Yadavilli Sridevi , Mueller Sabine , Packer Roger , Nazarian Javad
Published: June 2024

BACKGROUND

Diffuse midline gliomas (DMGs) are malignant pediatric brain tumors characterized by H3K27M mutation and global genome hypomethylation. This results in enhanced transcription of MGMT, a DNA-repair enzyme, rendering alkylating chemotherapeutics ineffective. Therefore, we investigated the efficacy of a functionally different DNA damaging agent, Val-083, that induces double strand breaks and triggers S/G2 phase cell cycle arrest for homologous DNA repair. We further evaluated its combined efficacy with Wee1 (G2/M checkpoint) inhibitor, AZD1775, to override cell cycle arrest and propel cells with DNA damage into premature mitosis and apoptosis.

METHODS

DMG cells were treated with increasing concentration of Val-083 and/or AZD1775 and their viability was evaluated using CellTiter-Glo luminescent assay. Next, the drugs’ mechanisms of action were investigated by immunoblotting for biomarkers involved in DNA damage response and cell cycle arrest. Flow cytometry was used to investigate cell cycle profile and apoptosis. Additionally, in vivo tumor growth inhibition and survival benefit were evaluated using zebrafish and mouse xenograft DMG models.

RESULTS

The IC50 values of Val-083 range between 0.69 and 4.38 µM while the IC50 values of AZD1775 range between 0.22 to 0.98 µM. Combination of both drugs showed high areas of synergy on multiple DMG lines, DNA double strand breaks and significant increase in cell population at the S (p<0.01) and G2/M (p<0.05) phases. Importantly, on zebrafish xenograft tumor model, combination treatment significantly (p<0.001) inhibited tumor growth in comparison to control and single treatment groups. Furthermore, the combined treatment demonstrated significant (p<0.01) survival benefit (median survival (MS) = 62 days) on tumor-bearing nude mice models, in comparison to vehicle (MS= 44 days), Val-083 (MS= 54.5 days) and AZD1775 (MS= 47 days) treatment groups.

CONCLUSION

Val-083 in combination with AZD1775 demonstrate promising efficacy in DMG preclinical models, providing a strong rationale for positioning these arms for clinical testing.
DIPG-43. CHARACTERIZATION OF THE BLOOD BRAIN BARRIER IN DIPG MURINE MODELS AND EFFECT OF ULTRASOUND ON ITS SPECIFICITIES AND PERMEABILITY TO ASIDNA™
Authors: BIARD adam , LE Ludivine DRET , Marie DUTREIX , Alexandre CARPENTIER , Manon LANCIEN , Alexandre PELLISSIER , Marie-Anne Debily , David CASTEL , Jacques GRILL , Kevin BECCARIA
Published: June 2024

BACKGROUND

The poor prognosis of pediatric diffuse midline gliomas, including diffuse intrinsic pontine gliomas (DIPGs), is partly due to the blood-brain barrier (BBB). The BBB can be opened by ultrasound (US), but few studies have focused on its characteristics in DIPGs, especially when submitted to US. We aimed to characterize the BBB in DIPGs and the influence of US on its characteristics, and to evaluate the effect of US-induced BBB opening on the kinetics of AsiDNA™ in DIPG murine models.

METHODS

US-induced BBB opening was performed with the SonoCloud® device and controlled with Evans blue dye. Permeability of DIPGs’ BBB was assessed with fluorescent dextrans. A histological evaluation of BBB markers was carried out by immunohistochemistry and immunofluorescence, including endothelial markers (CD34, GLUT1, PLAVP), tight junctions proteins (claudin 5, Zona occludens 1), and the efflux transporter ABCB1. An in vivo and ex vivo kinetic study of AsiDNA™ was performed with fluorescence.

RESULTS

We could repeatedly open the BBB with low intensity US (acoustic pressure 0.4 Mpa). In vivo, 3 kDa and 70 kDa dextrans delivery to DIPG tissue was increased by US-induced opening of the BBB, while 500 kDa dextrans did not cross the BBB spontaneously or after US. ABCB1 gene expression was variable at baseline in different DIPGs but was downregulated after US-induced BBB opening. The spontaneous passage of AsiDNA™ through the BBB was limited but could be increased after US-induced BBB opening.

CONCLUSIONS

We have shown that the BBB was specific in DIPG murine models, particularly in the expression of ABCB1. This BBB can be modulated and permeabilized by US, a technique that could allow for an improved delivery of AsiDNA. This study is a preliminary step before the introduction of a such technique and treatment in future clinical trials in children and adolescents with DMGs.
DIPG-46. TOTAL THERAPY FOR DIFFUSE MIDLINE GLIOMAS: A SEQUENTIAL, INTENSIVE, MULTI-AGENT COMBINATION PLATFORM IS A POTENT TREATMENT STRATEGY
Authors: Khan Aaminah , Upton Dannielle , Gopalakrishnan Anjana , Chen Yongjuan , Tsoli Maria , Ziegler David S
Published: June 2024

BACKGROUND

Diffuse Midline Glioma (DMG) has a dire prognosis, with a mortality rate nearing 100%. In the past, children diagnosed with Acute Lymphoblastic Leukemia (ALL) faced a similar prognosis until multi-agent chemotherapy revolutionized their survival rates. We hypothesized that implementing a comprehensive treatment strategy for DMG could yield similar efficacy. This study aimed to test the safety and efficacy of sequential, intensive, multi-agent combinations targeting epigenetic/chromatin modification (CBL0137-Panobinostat, CP), polyamine metabolism (DFMO-AMXT1501, DA), and the NF-κB pathway (ACT001) in preclinical DMG models.

METHODS/RESULTS

Maximum tolerated dose studies, which integrated comprehensive biochemical and hematological analyses with clinical data, tested different treatment durations and drug sequences. Tolerable doses and drug sequences were assessed in the aggressive patient-derived SU-DIPGVI orthotopic model for activity. In total six different drug combination sequences were tested for impact on survival. One particular sequence – DA/ACT001/CP – was significantly more effective compared with both vehicle and other regimens. This specific sequential regimen resulted in a 50% tumour regression rate with surviving animals maintaining tumor-free status > 1 year, compared with 81-day survival in controls. This 5-agent treatment sequence (DA/ACT001/CP) was next tested in the HSJD-DIPG007 orthotopic model with a significant extension of survival (119 days) compared with single or two-agent treatments (vehicle-59 days, DA-86 days, ACT001-66 days, CP-62 days, DA/ACT001-93 days). Further enhancement of this treatment regimen through addition of radiotherapy significantly extended survival to 130 days, with one-third of the cohort showing sustained tumor regression and remaining tumor-free (by Xenogen imaging) for > 220 days.

CONCLUSIONS

There are no effective therapies for DMG. Single and combination regimens in vivo often extend survival by days or weeks but do not generally eliminate tumors. Our “Total Therapy” strategy, inspired by successful ALL therapies, suggests that multi-agent sequential combination therapy can induce profound responses and unprecedented long-term survival in DMG animal models.
DIPG-55. DISSECTING THE ECOSYSTEM OF PEDIATRIC DIFFUSE MIDLINE GLIOMA
Authors: Morscio Julie , Jacobs Sandra , De Smet Frederik
Published: June 2024

BACKGROUND

Pediatric gliomas are heterogeneous tumors encompassing high grade (pHGG) and low grade (pLGG) subtypes. For tumors not amenable to surgery, in particular high grade H3K27 mutant diffuse midline glioma (DMG), no curative options exist. DMG is characterized by 1) extensive heterogeneity of tumor cells that can exist in different interchangeable cellular states mimicking normal development and 2) extensive heterogeneity of the tumor microenvironment (TME). A better understanding of the DMG ecosystem is needed for therapeutic target identification.

METHODS

Publicly available single cell RNA seq data of DMG (n=17, Jessa et al. Nat. Genet. 2022), high grade pediatric glioblastoma (GBM, n=16, scPCA) and low grade tumors (ganglioglioma, n=5; pilocytic astrocytoma, n=14, scPCA) were analyzed to define key molecular characteristics and differences between these pediatric brain tumors. Doublet cells were removed from each dataset and tumor and TME cells were discriminated based on marker gene expression and inferred CNVs.

RESULTS

In DMG, glioma cellular states and TME heterogeneity differed depending on the tumor location. DMG arising in the pons harbored a bigger fraction of astrocyte-like glioma cells whereas oligodendrocyte-like cells were more abundant in thalamic tumors. In line with this observation, Liana cellular communication analysis inferred more intratumoral (autocrine) signaling involving oligodendrocyte-like glioma cells in thalamic tumors (e.g. NCAM1-PTPRZ1, NLGN4X-NRXN1, NTN1-DSCAM) than in pons tumors. Notably, the TME of thalamic tumors contained a bigger fraction of immunotolerant macrophages expressing CD163 or VSIG4. In contrast, pons tumors harbored a unique cluster of VEGFA expressing astrocytes, not found in GBM or pLGG. Liana analysis inferred COPA/APP-CD74 interaction between VEGFA+ astrocytes and CD163/VSIG4+ macrophages and microglia, potentially contributing to an immunotolerant TME in pons DMG.

CONCLUSIONS

In DMG, patterns of tumor and TME composition associated with tumor location were uncovered. Furthermore, unique TME celltypes could contribute to the particularly aggressive nature of these tumors.
DIPG-56. BRAINSTEM TUMOR BIOPSY: A SINGLE INSTITUTE EXPERIENCE OF SAFETY AND FEASIBILITY IN A LOW-MIDDLE INCOME COUNTRY (LMIC)
Authors: Bashir Farrah , Hussain Muzamil Hamid , Javed Gohar , Bari Ehsan , Enam Ather , Qureshi Bilal Mazhar , Sirat Shayan , Thomas Diana , Drissi Richard , Khan Sara , Fouladi Maryam , Hawkins Cynthia , Minhas Khurram , Bouffet Eric , Mushtaq Naureen
Published: June 2024

BACKGROUND

Brainstem tumors account for 10-15% of all pediatric central nervous system tumors and carry a dismal prognosis. These include a wide spectrum of histological diagnosis therefore, in most specialized centers stereotactic biopsies for brainstem tumors are considered safe and standard practice. However, this approach continues to pose a challenge in LMIC’s.

METHODS

We retrospectively reviewed data of patients presenting with brainstem tumors at Aga Khan University Hospital, Pakistan, between 2014 and 2023. In addition to histopathological analysis, whole-genome sequencing and RNA sequencing were also performed on 6 tumors.

RESULTS

We identified 39 patients with brainstem tumors; amongst these 56.4% were females. Median age at diagnosis was 9 years (IQR: 5.1-11). Most cases presented with walking difficulty (74%) and cranial nerve palsies (46%). Median duration of symptoms was 30 days (IQR: 20-60). On imaging large proportion were intrinsic pontine tumors (53.8%) followed by brainstem (30.8%) and cerebellopontine angle (15.4%). 21 patients underwent stereotactic biopsy with 86% performed after 2016. 43% were intrinsic pontine tumors. Histopathology analysis showed a wide spectrum with diffuse midline glioma in 28% of cases, pilocytic astrocytoma, anaplastic astrocytoma and ganglioglioma in 33%, 14% and 9% of cases respectively. Rare entities identified included embryonal tumor with multilayered rosettes, atypical teratoid rhabdoid tumor and schwannoma (5%). Biopsies were uneventful in 81% of cases. Complication of biopsy in 2 patients included cranial nerve palsy and subdural hemorrhage. No patient died due to complications of biopsy. Surgical and medical management was altered in 9 patients due to availability of histological diagnosis.

CONCLUSIONS

We present the first study demonstrating safety and feasibility of stereotactic biopsy for brainstem tumors in LMIC. These biopsies should be considered for atypical brainstem tumors and should be performed in well-equipped tertiary neurosurgical facilities by trained neurosurgeons in LMIC.
DIPG-62. REPURPOSING OF ANTIPSYCHOTIC TRIFLUOPERAZINE FOR INHIBITING MITOCHONDRIAL FUNCTION IN DIFFUSE MIDLINE GLIOMA
Authors: Petrovic Antonela , Wojnicki Kamil , Laternser Sandra , Kitanovic Ana , Kleele Tatjana , Grotzer Michael , Müller Sabine , Nazarian Javad
Published: June 2024

BACKGROUND

Diffuse Midline Gliomas (DMGs) present formidable challenges in neuro-oncology, prompting the exploration of innovative therapeutic strategies. Repurposing approved drugs, particularly those exhibiting multifaceted effects, holds promise for addressing these challenges efficiently. In this study, we investigate the potential of trifluoperazine (TFP), an antipsychotic medication, with a specific focus on its mitochondrial-targeted actions in DMGs.

METHODS

TFP, a phenothiazine derivative, displayed robust in vitro anticancer activity against DMGs. Our investigation explored the molecular mechanisms underlying TFP’s interaction with mitochondria, emphasizing its influence on mitochondrial function, membrane potential, and reactive oxygen species (ROS) production. TFP is well known to induce irreversible Ca2+ release from intracellular stores by directly interacting with calmodulin 2 (CaM2) at the inositol triphosphate receptor (IP3R), leading to IP3R opening. In DMGs, intracellular Ca2+ plays a pivotal role in gene expression, cell motility, differentiation, and survival. TFP disrupts calcium homeostasis by elevating intracellular Ca2+ concentration, thereby inhibiting DMG invasion and growth. DMG cell lines, exhibiting heightened CaM2 expression compared to control healthy astrocyte cells, display increased sensitivity to TFP.

RESULTS

Remarkably, mitochondrial homeostasis was seriously affected by TFP, and a loss of mitochondrial membrane potential, ROS overproduction and fragmentation of mitochondrial network was observed. This mitochondrial stress, coupled with ER stress, activated the integrated stress response. We demonstrated that TFP induces cell death triggered by a poor ATP content, observed in TFP-treated cells as a consequence of a dramatic decrease in OXPHOS metabolism due to mitochondrial stress.

CONCLUSIONS

Our findings underscore TFP’s potential as a targeted therapeutic agent against DMGs, offering a better understanding of its impact on mitochondrial and intracellular signaling pathways. As the mitochondrial landscape remains critical in DMG biology, the observed mitochondrial damage-induced oxidative stress provides valuable insights for future investigations into the clinical application of TFP in neuro-oncology.