BACKGROUND:Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric brain tumor affecting over 300 children annually in the United States. Chimeric antigen receptor (CAR) T cells are a targeted immune effector cell therapy with substantial clinical benefit against hematologic cancers. Against central nervous system (CNS) tumors, CAR T cells targeting B7-H3, a protein highly expressed on DIPG, have rapidly advanced from preclinical studies to clinical trials. BrainChild-03 (NCT04185038), a phase 1 trial of repeatedly delivered intracerebroventricular (ICV) B7-H3-targeting CAR T cells (B7-H3 CAR T cells), demonstrated tolerability and potential efficacy for children and young adults with DIPG. However, clinical benefits were not uniformly seen, and multi-agent treatment strategies may be required against such an aggressive disease. Here, we combined B7-H3 CAR T cells with ONC206, an imipridone molecule also under clinical investigation. METHODS:We tested B7-H3 CAR T cells combined with ONC206 across multiple DIPG cell cultures and orthotopic xenograft mouse models. RESULTS:B7-H3 CAR T cell monotherapy induced robust cytotoxicity while ONC206 treatment resulted in significant mitochondrial dysfunction against DIPG cells. The combination of low effector-to-target ratios of B7-H3 CAR T cells and IC50 concentrations of ONC206 led to significantly enhanced cytotoxicity in vitro (P < .003) and increased IL-2, IL-29, VEGF-A, and Granzyme B levels. In vivo combinatorial studies of ONC206 and a single ICV dose of B7-H3 CAR T cells extended survival in DIPG xenograft mouse models. CONCLUSIONS:B7-H3 CAR T cells combined with ONC206 are a feasible and efficacious multi-agent approach against multiple DIPG models.

Fewer than 10 % of children with diffuse midline glioma (DMG) survive 2 years from diagnosis. Radiation therapy remains the cornerstone of treatment and there are no medicinal products with regulatory approval. Although the biology of DMG is better characterized, this has not yet translated into effective treatments. H3K27-alterations initiate the disease but additional drivers are required for malignant growth. Hence, there is an urgent unmet need to develop new multi-modality therapeutic strategies, including alternative methods of drug delivery. ONC201 (DRD2 antagonist and mitochondrial ClpP agonist) is the most widely evaluated investigational drug. Encouraging early data is emerging for CAR T-cells and oncolytic viruses. GD2, B7-H3 and PI3K signalling are ubiquitous targets across all subtypes and therapeutics directed to these targets would potentially benefit the largest number of children. PI3K, ACVR1, MAPK and PDGFRA pathways should be targeted in rational biological combinations. Drug discovery is a very high priority. New specific and potent epigenetic modifiers (PROTACS e.g. SMARCA4 degraders), with blood-brain penetrance are needed. Cancer neuroscience therapeutics are in early development. Overall survival is the preferred regulatory endpoint. However, the evaluation of this can be influenced by the use of re-irradiation at the time of progression. An efficient clinical trial design fit for regulatory purposes for the evaluation of new therapeutics would aid industry and facilitate more efficient therapy development. Challenges in conducting clinical trials such as the need for comparator data and defining endpoints, could be addressed through an international, first-in-child, randomised, complex innovative design trial. To achieve progress: i) drug discovery; ii) new multi-modality, efficient, collaborative, pre-clinical approaches, possibly including artificial intelligence and, iii) efficient clinical trial designs fit for regulatory purposes are required.

B7-H3 CAR T cell treatment in combination with ONC206 aims to assess benefit and significant extension of survival in orthotopic DIPG mouse models. Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric tumor arising in the pons and affecting over 300 children each year in the United States. Chimeric antigen receptor (CAR) T cells, a targeted immunotherapy showing remarkable results in treating hematologic cancers, have recently been translated to children with DIPG. B7-H3 is a protein expressed in nearly all DIPG but present at only low levels in normal brain tissue. Seattle Children’s first-in-human phase 1 clinical trial of repeated intracranial anti-B7-H3 CAR T cells (BrainChild-03 trial, NCT04185038) found the highest planned dose (100 million CAR T cells) to be tolerable. We also demonstrated a survival of 20.1 months for treated patients, but while 3 patients are alive beyond 42 months, response to therapy was not universal. To uncover a beneficial combinatorial strategy, we tested B7-H3 CAR T cells with ONC206, a small molecule imipridone targeting ClpP and DRD2, being evaluated in the PNOC023 clinical trial (NCT04732065) for pediatric patients with CNS tumors. The combinatorial strategy was performed against multiple DIPG models (i.e., PBT-22FH, PBT-24FH, PBT-27FH, PBT-29FH, and SU-DIPG-XIII) before being validated in orthotopic xenograft DIPG mouse models. B7-H3 CAR T cells (1 million) were intracranially delivered at day 7, whereas ONC206 (50 mg/kg) was administered twice a week via oral gavage. We initially assessed an ONC206 IC50 at approximately 250 nM and demonstrated a pro-apoptotic effect in various DIPG models. Next, we showed that ONC206 induces mitochondrial dysfunction, which limits tumor cell respiration and activates cell death pathways. After a 120h treatment with ONC206, we observed upregulation of Caspase-3, Caspase-7, ATF4, CHOP, ClpP, and DR5, whereas levels of B7-H3 and DRD2 remained constant.Given that B7-H3 CAR T cells are highly effective as single agents in preclinical settings, we used lower Effector:Tumor (E:T) ratios and low concentrations of ONC206, revealing a significant additive effect in the models studied (p-value < 0.003). Interestingly, the phenotype of CAR T cells was unaffected by ONC206, and multiplex cytokine assays not only showed unaltered levels of TNF-α and IFN-γ, but also detected increased levels of IL2, IL29, VEGFA, IP10, and Granzyme B post-treatment. Preclinical studies confirmed an additive benefit in the aggressive and metastatic SU-DIPG-XIII model, significantly extending the survival of mice up to 94 days (p-value < 0.005), compared to mice treated solely with B7-H3 CAR T cells (median survival = 25 days). B7-H3 CAR T cells and ONC206 may have a combinatorial benefit. Therefore, future studies will continue to validate these findings, optimize dosing strategies, and assess immunocompetent DIPG models. Andrea Timpanaro, Edward Song, Matthew C. Biery, Ryma Toumi, Davina Lau, Leo Elena-Sanchez, Michael Meechan, Ashmitha Rajendran, Jason Wendler, Joshua Gustafson, Lily Winter, Carl Koschmann, Myron Evans, Siobhan Pattwell, Shannon Oda, Jessica Foster, Matthew D. Dun, Michael C. Jensen, Nicholas A. Vitanza. Preclinical efficacy of combinatorial B7-H3 CAR T cell and ONC206 treatment in DIPG/DMG [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6440.

Background. Despite its clinical promise in non-solid tumor, immunotherapy is yet to show significant clinical efficacy for brain tumors including pediatric diffuse midline glioma (DMG). This indicated the need to fully explore DMG immune tumor microenvironment (TME). Method. Whole brains (49 DMGs, 20 non-DMG, 10 non-malignant) from 79 pediatric patients were used to establish a tissue microarray (918 cores) representing primary, metastatic, and adjacent healthy sites. CellDIVE MxIF multiplex assay was used to probe for 33 immune and cell type markers. RNA sequencing (n=62 patients) defined additional immune signatures. Findings were validated using patient plasma and DMG PDX models. Our annotated single-cell atlas was used to train a spatial AI model to predict antigens from H&E staining. Findings. We found enrichment of M1-activated microglia in primary versus adjacent healthy tissue. PD1 positive cells were significantly (p<0.01) higher in tumor compared to adjacent controls. This was validated by mRNA profiling, further indicating two distinct groups with top 35 significant (p<0.05) genes revealing synaptic signature in the metastatic cohort. We stratified the patient cohort by treatment. Imipridone cohort (n=5) showed decreased progenitor (Nestin+, Vimentin+, and SOX2+) and increased macrophages/microglia infiltration. Increased T and B cells was validated in patient plasma following imipridone therapy. Combination therapy of imipridone and immunotherapy (n=7) resulted in increased myeloid (Iba1, CD68, CD163) and lymphoid (CD3, CD8) cells. Enhanced immune engagement was validated in DMG PDX models. Machine learning resulted in a spatial AI model capable of predicting 22 antigens using H&E slides. Interpretations. DMG tumors maintain a cold immune microenvironment, which is nevertheless dynamic and responsive to therapy, indicating the need to explore combination therapies. AI-assisted antigen detection is suitable for rapid interpretation of clinical biospecimens. ### Competing Interest Statement The authors have declared no competing interest.

Background:High-grade gliomas (HGGs), including diffuse midline glioma (DMG), represent the most aggressive and deadly pediatric brain cancers. Despite recent advances in understanding their molecular underpinnings, these tumors remain universally fatal. A hallmark feature of pediatric HGGs is the frequent presence of mutations and amplifications in components of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling pathway. These alterations drive unchecked tumor growth, confer resistance to standard therapies, and contribute to the dismal survival outcomes observed in affected children. Main Body:While the PI3K/mTOR axis has been recognized as a critical dependency in DMG and other pediatric HGGs, clinical translation of pathway inhibitors has been limited by several major barriers. Most notably, the blood-brain barrier (BBB) restricts the delivery of conventional PI3K and mTOR inhibitors, many of which lack sufficient central nervous system (CNS) penetration. Furthermore, even when delivered to the tumor site, these agents often encounter rapid adaptive resistance through activation of compensatory pathways, reducing their therapeutic benefit. Treatment-related toxicities, including hyperglycemia, rash, and mucositis, further limit tolerability and patient adherence.Emerging brain-penetrant PI3K/mTOR inhibitors represent a new generation of targeted therapies with the potential to overcome these pharmacological limitations. However, increasing drug exposure does not necessarily equate to improved outcomes, particularly when used in combination with immunotherapies or other targeted agents. Achieving optimal therapeutic efficacy while minimizing systemic toxicity remains a central challenge, requiring careful consideration of drug dosing, timing, and combination strategies tailored to each individual patient. Conclusion:This review explores the current landscape of PI3K/mTOR targeting in DMG, highlighting both the therapeutic promise and inherent challenges. We discuss known resistance mechanisms, the need for better CNS-optimized compounds, and the importance of individualized treatment strategies. Finally, we propose a roadmap for future research, emphasizing rational drug combinations, refined patient stratification, and the development of next-generation therapies aimed at improving outcomes for children with these devastating malignancies.

GSEA of genes associated with positive or negative correlation with radiographic response to ONC201 treatment.

GSEA analysis of genes with significantly lower chromatin accessibility at promoters and enhancers and reduced gene expression in DIPG007 cells treated with ONC201 versus vehicle.

10015 Background: The gut microbiome exerts a multifaceted influence on treatment outcomes across various cancers, yet its potential role in diffuse midline gliomas (DMGs) remains under-explored. In this report, we present the gut microbiome findings from cohort 2 in the DMG–Adaptive Combination Trial (DMG-ACT, PNOC022). Methods: PNOC022 is an open-label, multi-institutional, international clinical trial using a Bayesian drug combination platform trial design. This report focuses on the combination therapy arm involving dordaviprone and paxalisib, administered to patients (aged 2–39 years) who had completed standard-of-care radiation therapy (Cohort 2). Stool samples were collected at baseline (n = 22), cycle 1 day 1 (n = 15), cycle 7 day 1 (n = 9), and at progression (n = 4). Microbiome profiling was performed with shotgun metagenomic sequencing (NovaSeq X Plus Series, PE150). Progression-free survival (PFS) and overall survival (OS) were analyzed using the Kaplan-Meier method. Longitudinal shifts in microbial communities were evaluated using α-diversity (Shannon-index) and β-diversity (Bray-Curtis dissimilarity index). Baseline α-diversity associations with PFS and OS were examined with log-rank tests and further validated through age-adjusted Cox regression analysis. Results: Between November 2021 and October 2023, 69 biopsy-confirmed DMG patients enrolled (median age 9 years [range 3-37], n = 42 female [61%]) in cohort 2. Median OS from time of diagnosis was 15.6 months (95% CI 12.9-19.5), with a median follow-up time of 19.5 months (95% CI 17.9-23.9). Microbiome analyses were performed for 33 DMG patients (48%). Alpha-diversity and β-diversity remained stable across timepoints. Using baseline samples (n = 22) and a median α-diversity cutoff of the respective group’s values, patients were stratified into two categories (low- vs. high-diversity). Low-diversity was associated with significantly worse PFS and trended worse for OS, resulting in a 6-month PFS: 73% (95%CI 51-100) vs. 100%; p < 0.001) and 12-month OS: 46% (95%CI 24-87) vs. 78% (95%CI 55-100; p = 0.19). Validation using age adjusted Cox regression analysis confirmed a decrease in the risk of progression or death with increasing α-diversity. PFS hazard ratio (HR) was 0.2 (95% CI: 0.1–0.5; p < 0.01) and OS HR was 0.3 (95% CI: 0.1–0.7; p < 0.01). Conclusions: Baseline high alpha-diversity in the gut microbiome is significantly associated with improved PFS and trended towards improved OS in pediatric patients with DMG in cohort 2 of PNOC022. Age-adjusted survival models reinforced its prognostic value for PFS and OS. These findings highlight the potential impact the gut microbiome has on outcomes and will be explored further and warrant our ongoing investigation in PNOC022.

VST-transformed gene expression values for all samples and protein-coding genes.

Diffuse intrinsic pontine glioma (DIPG) is a fatal central nervous system (CNS) tumor that confers a median survival of 11 months. As B7-H3 is expressed on pediatric CNS tumors, we conducted BrainChild-03, a single-center, dose-escalation phase 1 clinical trial of repetitive intracerebroventricular (ICV) dosing of B7-H3-targeting chimeric antigen receptor T cells (B7-H3 CAR T cells) for children with recurrent or refractory CNS tumors and DIPG. Here we report results from Arm C, restricted to patients with DIPG. The primary objectives were to assess feasibility and tolerability, which were both met. Secondary objectives included assessments of CAR T cell distribution and survival. A total of 23 patients with DIPG enrolled, and 21 were treated with repeated doses of ICV B7-H3 CAR T cells using intra-patient dose-escalation regimens without previous lymphodepletion. Concurrent tumor-directed therapy, including re-irradiation, was not allowed while on protocol therapy. We delivered a total of 253 ICV doses and established the highest planned dose regimen, DR4, which escalated up to 10 × 107 cells per dose, as the maximally tolerated dose regimen. Common adverse events included headache, fatigue and fever. There was one dose-limiting toxicity (intratumoral hemorrhage) during DR2. For all treated patients (n = 21), the median survival from their initial CAR T cell infusion was 10.7 months and the median survival from diagnosis was 19.8 months with 3 patients still alive at 44, 45 and 52 months from diagnosis. Ultimately, this completed first-in-human trial shows that repetitive ICV dosing of B7-H3 CAR T cells in pediatric and young adult patients with DIPG is tolerable, including multiyear repeated dosing, and may have clinical efficacy that warrants further investigation on a multisite phase 2 trial. ClinicalTrials.gov registration: NCT04185038 . In the final report of a phase 1 trial evaluating intracerebroventricular B7-H3-targeting CAR T cells in children and young adults with diffuse intrinsic pontine glioma, repeated intracranial infusions were feasible and well tolerated with a median overall survival of 19.8 months and 3 patients surviving over 40 months from diagnosis.

Alterations in the FMS-like tyrosine kinase 3 (FLT3) gene are the most frequent driver mutations in acute myeloid leukaemia (AML), linked to a high risk of relapse in patients with internal tandem duplications (FLT3-ITD). Tyrosine kinase inhibitors (TKIs) targeting the FLT3 protein are approved for clinical use, yet resistance often emerges. This resistance is mainly seen following the acquisition of additional point mutations in the tyrosine kinase domain (TKD), resulting in a double mutant FLT3-ITD/TKD, which sustains cell signalling and survival despite the presence of FLT3 inhibitors. Here, we developed a FLT3-mutant AML model with adaptive resistance to type II TKIs, sorafenib, and quizartinib by in vitro drug selection. Through global multiomic profiling, we identified upregulation of proteins involved in reactive oxygen species (ROS) production, particularly NADPH-oxidases, driving cellular 'ROS-addiction', with resistant cells relying on ROS for survival, and genome fidelity preserved by ATM-driven DNA repair. Transcriptomic analysis of adult and paediatric AML (pAML) patients identified high ATM expression as a biomarker for shorter median overall survival in both the de novo and relapsed settings. Inhibition of ATM with clinically relevant therapy WSD-0628 effectively killed TKI- and chemotherapy-resistant AML cells in vitro and significantly extended the survival of mice with sorafenib- and quizartinib-resistant FLT3-ITD AML in vivo. We propose a new treatment strategy to improve survival of patients who develop resistance to sorafenib and quizartinib, as well as relapsed and refractory pAML, exploiting resistance mechanisms to precision therapies and cell-intrinsic features of high-risk cases, highlighting a clinically relevant salvage strategy. ### Competing Interest Statement The authors have declared no competing interest.

The enhancer of zeste inhibitory protein (EZHIP) is typically expressed during germ cell development and has been classified as a cancer-testis antigen (CTA) in various cancers. In 2020, 4% of diffuse midline gliomas (DMGs) were shown to aberrantly express EZHIP, mirroring the DMG hallmark histone H3 K27M (H3K27M) oncohistone mutation. Similar to H3K27M, EZHIP is a negative regulator of polycomb repressive complex 2 (PRC2), leading to global epigenomic remodeling. In this opinion, we explore the similarities and disparities between H3K27M- and EZHIP-DMGs with a focus on their shared functional hallmark of PRC2 inhibition, their genetic and epigenomic landscapes, plausible differences in the cell of origin, and therapeutic avenues. Upcoming research on EZHIP will help better understand its role in gliomagenesis and DMG therapy.

Diffuse midline glioma (DMG) is a universally fatal CNS tumor, with a median overall survival of <12-months. Recent studies, including our own, have identified overexpression of the mitochondrial protease ClpP in DMG patient samples. ClpP plays a pivotal role in regulating energy production. Brain-penetrant ClpP agonists, including ONC201, ONC206, and TR107, induce non-specific degradation of the mitochondrial electron transport chain. However, there is considerable variability in the response of DMG cell lines to these therapies, with only about half reaching IC50. Notably, TP53-mutant cell lines exhibit reduced sensitivity. Further analysis using quantitative proteomics of TP53-mutant DMG cell lines exhibited hallmark activation of ClpP, increased mitochondrial stress and dysfunction. Importantly, ClpP agonism increased the activity of the transcription factor NRF2, driving expression of NQO1 (p=0.0017). NQO1 is a cytoprotective enzyme, promoting redox balance and cell survival. This highlights a potential adaptive response to ClpP agonist therapy in DMG cells. Analysis of DMG patient samples through publicly available RNA sequencing data identified ubiquitously high NRF2 expression compared to other pediatric CNS tumors. This led us to hypothesize that a clinically relevant, selective NQO1 inhibitor could elicit a strong response to ClpP agonism. Therefore, we examined ARQ761, a CNS-active therapy that disrupts NQO1 leading to the formation of an unstable hydroquinone, causing oxidative DNA damage and apoptosis. When ARQ761 was used concurrently with a ClpP agonist, the combination showed additive effects, average Bliss Score of 9.91 (n=4). Interestingly, administering ARQ761 24 h after ClpP agonist exposure combined synergistically, average Bliss Score of 15.87 (n=4). This synergy was further potentiated using ARQ761 48 h after ClpP agonism, when NQO1 expression peaked, resulting in an average Bliss Score of 20.30 (n=4). This study has unveiled an intrinsic antioxidant defense mechanism in DMG, which facilitates evasion from treatments inducing oxidative stress. This defense mechanism appears particularly robust in cell lines harboring TP53-mutations, which are also known to exhibit resistance to radiotherapy.

BACKGROUND:The epididymis has long been of interest owing to its role in promoting the functional maturation of the male germline. More recent evidence has also implicated the epididymis as an important sensory tissue responsible for remodeling of the sperm epigenome, both under physiological conditions and in response to diverse forms of environmental stress. Despite this knowledge, the intricacies of the molecular pathways involved in regulating the adaptation of epididymal tissue to paternal stressors remains to be fully resolved. OBJECTIVE:The overall objective of this study was to investigate the direct impact of corticosterone challenge on a tractable epididymal epithelial cell line (i.e., mECap18 cells), in terms of driving adaptation of the cellular proteome and phosphoproteome signaling networks. MATERIALS AND METHODS:The newly developed phosphoproteomic platform EasyPhos coupled with sequencing via an Orbitrap Exploris 480 mass spectrometer, was applied to survey global changes in the mECap18 cell (phospho)proteome resulting from sub-chronic (10-day) corticosterone challenge. RESULTS:The imposed corticosterone exposure regimen elicited relatively subtle modifications of the global mECap18 proteome (i.e., only 73 out of 4171 [∼1.8%] proteins displayed altered abundance). By contrast, ∼15% of the mECap18 phosphoproteome was substantially altered following corticosterone challenge. In silico analysis of the corresponding parent proteins revealed an activation of pathways linked to DNA damage repair and oxidative stress responses as well as a reciprocal inhibition of pathways associated with organismal death. Corticosterone challenge also induced the phosphorylation of several proteins linked to the biogenesis of microRNAs. Accordingly, orthogonal validation strategies confirmed an increase in DNA damage, which was ameliorated upon selective kinase inhibition, and an altered abundance profile of a subset of microRNAs in corticosterone-treated cells. CONCLUSIONS:Together, these data confirm that epididymal epithelial cells are reactive to corticosterone challenge, and that their response is tightly coupled to the opposing action of cellular kinases and phosphatases.

Protocol for NCT03416530, ONC201-014: ONC201 in Newly Diagnosed Diffuse Intrinsic Pontine Glioma and Recurrent/Refractory Pediatric H3K27M Gliomas.

Forecasted increases in the prevalence and severity of extreme weather events accompanying changes in climatic behavior pose potential risk to the reproductive capacity of humans and animals of ecological and agricultural significance. While several studies have revealed that heat stress induced by challenges such as testicular insulation can elicit a marked negative effect on the male reproductive system, and particularly the production of spermatozoa, less is known about the immediate impact on male reproductive function following subchronic whole-body exposure to elevated ambient temperature. To address this knowledge gap, we exposed unrestrained male mice to heat stress conditions that emulate a heat wave (daily cycle of 8 h at 35 °C followed by 16 h at 25 °C) for a period of 7 d. Neither the testes or epididymides of heat-exposed male mice exhibited evidence of gross histological change, and similarly, spermatozoa of exposed males retained their functionality and ability to support embryonic development. However, the embryos generated from heat-exposed spermatozoa experienced pronounced changes in gene expression linked to acceleration of early embryo development, aberrant blastocyst hatching, and increased fetal:placental weight ratio. Such changes were causally associated with an altered sperm small noncoding RNA (sncRNA) profile, such that these developmental phenotypes were recapitulated by microinjection of wild-type embryos sired by control spermatozoa with RNAs extracted from heat-exposed spermatozoa. Such data highlight that even relatively modest excursions in ambient temperature can affect male reproductive function and identify the sperm sncRNA profile as a particular point of vulnerability to this imposed environmental stress.