Diffuse intrinsic pontine glioma (DIPG) is a lethal childhood brain tumor. Radiotherapy remains the standard of care, but tumors recur due to radioresistance. Tumor hypoxia contributes to radioresistance, and evidence of oxidative metabolism and hypoxia-associated transcriptomic programs suggests that hypoxia may be relevant in DIPG. We therefore investigated the FDA-approved mitochondrial inhibitor atovaquone as a strategy to target oxidative metabolism and enhance radiation response in DIPG. Methods: Patient-derived DIPG cell lines were used to evaluate atovaquone by extracellular flux analysis, hypoxia and reactive oxygen species assays, clonogenic survival assays, metabolomics, and RNA sequencing. To improve brain exposure, an amorphous solid dispersion (ASD) atovaquone formulation was evaluated and tested in an orthotopic DIPG model. Results: In patient-derived DIPG cultures, atovaquone suppressed mitochondrial respiration, reduced hypoxia-associated readouts, decreased HIF-1α expression in 3D models, and enhanced radiation response. At higher concentrations, atovaquone also increased oxidative stress and enhanced the radiosensitivity of DIPG monolayers. Transcriptomics analysis revealed disruption of cell-cycle and mitotic pathways, supporting additional treatment-associated effects beyond hypoxia reduction alone. Commercial and ASD formulations showed comparable in vitro activity. In vivo, ASD atovaquone combined with radiation prolonged survival in an orthotopic DIPG model. Conclusions: Targeting mitochondrial metabolism enhances radiosensitivity in DIPG and supports mitochondrial metabolism as a potential therapeutic weakness in this disease. Its effects are associated with reduced hypoxia-related signaling and broader metabolic and transcriptional changes.

MYCN functions as a developmental oncogene, but its role in pediatric high-grade gliomas (pHGGs) remains unclear. In co-operation with Trp53 and Pten loss, MYCN initiates tumorigenesis and establishes an origin for MYCN-driven pHGGs. This transformation creates a vulnerability to PI3K and mTOR inhibition. However, prolonged treatment drives adaptive resistance through MYCN protein rebound, mediated by the attenuation of IGFBP5 and the induction of insulin-like growth factor 2. Although insulin pathway feedback has been implicated in resistance to PI3K targeted therapies, MYCN emerges as the central node of this adaptive program. Resistance can be overcame by sustained MYCN suppression using PI3K and mTOR inhibitors, combined with insulin-like growth factor 1 receptor and insulin receptor inhibitors or dietary intervention. A degradation-resistant MYCN isoform abolishes this response, establishing MYCN as both an initiating oncogene and a resistance driver and revealing a mechanistically defined therapeutic vulnerability.

Abstract Background Diffuse midline glioma (DMG), including tumors originating in the brainstem known as diffuse intrinsic pontine glioma (DIPG), are childhood and adolescent brain tumors that remain uniformly fatal despite extensive genomic and epigenomic characterization. Their unique anatomy, genetic heterogeneity, cell states, immunosuppressive microenvironment, and variable drug penetration within the midline structures create a disease that is biologically complex and difficult to treat with current therapeutic strategies. Methods Spatial multi-omics technologies have emerged as powerful tools for addressing these challenges, particularly given the limited amount of tumor tissue available from biopsy. These methods require minimal material while preserving anatomical context, enabling simultaneous profiling of thousands of transcripts, proteins, and metabolites within a single tissue section. Such approaches can profile tumor cell states, immune interactions, and pharmacological gradients within their native tissue architecture. Results Building on discussions from the 2025 ChadTough Defeat DIPG Research Workshop, we outline a practical roadmap for implementing spatial transcriptomics, spatial proteomics, imaging-based mass spectrometry, and related spatial approaches across the DMG disease course, from diagnostic biopsy through autopsy. Key considerations are discussed, including tissue triage, spatial platform selection, quality control standards, analytical workflows, and data governance frameworks needed to support collaborative analysis. Conclusions Spatial multi-omics offers an opportunity to link tumor biology, microenvironmental features, and therapeutic exposure at cellular or near-cellular resolution. Establishing shared standards and coordinated international frameworks will be essential to translate these technologies from discovery research into clinically meaningful applications for patients with DMG and DIPG. Importance of the study For lethal tumors such as DMG, including DIPG, genetic heterogeneity, anatomical location, diverse malignant cell states, and a profoundly immunosuppressive tumor microenvironment means that no single therapy is likely to achieve durable survival. Understanding how these factors interact within the spatial architecture of the tumor is therefore essential for developing more effective therapeutic strategies. Spatial multi-omics technologies enable high-resolution mapping of tumor biology directly within intact tissue, allowing researchers to identify cellular states, immune interactions, and regional differences in drug exposure that are not captured by bulk molecular profiling approaches. These approaches are particularly valuable in DMG, where biopsy tissue is scarce. By outlining practical considerations and a coordinated framework for implementing spatial multi-omics throughout the DMG disease course, this review highlights how these technologies can accelerate biological discovery and support the development of rational combination therapies for patients with these devastating tumors.

Pediatric high-grade glioma (pHGG), including diffuse midline glioma (DMG), are the most aggressive and fatal pediatric cancers. Mutations and amplifications within the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) pathway drive tumor growth, treatment resistance, and poor outcomes. Although PI3K and mTOR have been identified as genetic dependencies in pHGGs, translating this knowledge into effective treatment remains challenging. The blood-brain barrier (BBB) restricts the delivery of most PI3K/mTOR inhibitors and , hence, often show poor CNS penetration. Even when present in the brain, these agents frequently encounter adaptive resistance mechanisms that blunt efficacy. Side effects, including hyperglycemia, rash, and mucositis, further complicate their use and reduce compliance. Encouragingly, novel brain-penetrant PI3K/mTOR inhibitors offer new opportunities for treatment, but combining these agents with other therapies, including chemotherapy, other small molecules, and immunotherapies, requires careful balancing of toxicity and efficacy. Therefore, achieving optimal dosing for each patient remains a significant hurdle. This review examines the promise and pitfalls of targeting the PI3K/mTOR pathway in DMG, including the limitations of available therapies, mechanisms of resistance, and the critical need for improved regimen design. We propose a roadmap to guide future efforts, emphasizing rational combination strategies and better patient stratification to improve survival for children diagnosed with these devastating cancers.

Diffuse midline glioma (DMG) is a universally fatal high-grade glioma. Its immunologically cold tumor immune microenvironment (TIME) presents a major barrier to durable anti-tumor responses. Dordaviprone (ONC201) is a brain-penetrant DRD2 antagonist and ClpP agonist that disrupts mitochondrial complex II (CII) activity; currently under clinical evaluation for DMG. In melanoma, CII inhibition has been shown to enhance MHC presentation and T cell–mediated killing. Here, we investigated the immunomodulatory effects of dordaviprone in DMG mouse models, patient tumor tissues, and blood samples. Immunocompetent DMG-PPK mice (PdgfraD842V, Trp53DN, H3f3aK27M mutations) were treated with dordaviprone (125 mg/kg, twice weekly) for 1 week (acute) or 3 weeks (chronic) and compared to naïve, sham (orthotopic surgery without tumor engraftment), and vehicle-treated controls. Murine tumors were analyzed by single-cell multiome (scRNA-seq and scATAC-seq) and spatial transcriptomics. Murine blood and bone marrow (BM) and human blood samples were profiled by scRNA-seq and flow cytometry. In models, dordaviprone promoted microglial activation and macrophage infiltration within the TIME. Spatial transcriptomics identified increased expression of the MHC-I subunit B2m in tumor cells and tumor-infiltrating Cd8+ T cells following dordaviprone treatment. Flow cytometry of murine and human peripheral blood confirmed CD4+ and CD8+ lymphopenia at baseline. Dordaviprone increased circulating lymphocytes in models (log2 fold change=1.03; patient data pending), reversing DMG-induced CD3+ T cell sequestration in the BM. Immunohistochemistry confirmed elevated levels of B2M (p=0.0281) and tumor-infiltrating lymphocytes (TILs), including CD45+ (p=0.0275) and CD3+ T cells (p=0.0103) in dordaviprone-treated mice. Despite enhanced TIL recruitment, dordaviprone did not improve survival (n=10), likely due to persistent immunosuppressive myeloid activity, i.e. Havcr2 (TIM-3), Cd74, Cd274 (PD-L1). This study demonstrates that DMG tumors contribute to systemic lymphopenia, partially reversed by dordaviprone. While supporting its immunomodulatory effects, including enhanced antigen presentation and TIL recruitment, our findings highlight future combination strategies to leverage dordaviprone-induced immunoreactive TIME.

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.

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

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.

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.

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

Upregulated and downregulated metabolites in DIPG007 cells treated with ONC201 versus vehicle.

Diffuse midline glioma (DMG), including tumors diagnosed in the brainstem (diffuse intrinsic pontine glioma - DIPG), is the primary cause of brain tumor-related death in pediatric patients. DIPG is characterized by a median survival of <12 months from diagnosis, harboring the worst 5-year survival rate of any cancer. Corticosteroids and radiation are the mainstay of therapy; however, they only provide transient relief from the devastating neurological symptoms. Numerous therapies have been investigated for DIPG, but the majority have been unsuccessful in demonstrating a survival benefit beyond radiation alone. Although many barriers hinder brain drug delivery in DIPG, one of the most significant challenges is the blood-brain barrier (BBB). Therapeutic compounds must possess specific properties to enable efficient passage across the BBB. In brain cancer, the BBB is referred to as the blood-brain tumor barrier (BBTB), where tumors disrupt the structure and function of the BBB, which may provide opportunities for drug delivery. However, the biological characteristics of the brainstem's BBB/BBTB, both under normal physiological conditions and in response to DIPG, are poorly understood, which further complicates treatment. Better characterization of the changes that occur in the BBB/BBTB of DIPG patients is essential, as this informs future treatment strategies. Many novel drug delivery technologies have been investigated to bypass or disrupt the BBB/BBTB, including convection enhanced delivery, focused ultrasound, nanoparticle-mediated delivery, and intranasal delivery, all of which are yet to be clinically established for the treatment of DIPG. Herein, we review what is known about the BBB/BBTB and discuss the current status, limitations, and advances of conventional and novel treatments to improving brain drug delivery in DIPG.

Diffuse midline gliomas (DMGs) are devastating pediatric brain tumors recognized as the leading cause of cancer-related death in children. DMGs are high-grade gliomas (HGGs) diagnosed along the brain's midline. Euchromatin is the hallmark feature of DMG, caused by global hypomethylation of H3K27 either through point mutations in histone H3 genes (H3K27M), or by overexpression of the enhancer of zeste homolog inhibitory protein. In a clinical trial for adults with progressive HGGs, a 22-year-old patient with a thalamic DMG, H3 K27-altered, showed a remarkable clinical and radiological response to dordaviprone (ONC201). This response in an H3 K27-altered HGG patient, coupled with the lack of response of patients harboring wildtype-H3 tumors, has increased the clinical interest in dordaviprone for the treatment of DMG. Additional reports of clinical benefit have emerged, but research defining mechanisms of action (MOA) fall behind dordaviprone's clinical use, with biomarkers of response unresolved. Here, we summarize dordaviprone's safety, interrogate its preclinical MOA identifying the mitochondrial protease "ClpP" as a biomarker of response, and discuss other ClpP agonists, expanding the arsenal of potential weapons in the fight against DMG. Finally, we discuss combination strategies including ClpP agonists, and their immunomodulatory effects suggestive of a role for the tumor microenvironment in DMG patient response.

Upregulated and downregulated genes in DIPG007 cells treated with ONC201 versus vehicle.