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The recent trial Pediatric Neuro-Oncology Consortium 003 (PNOC003) utilized a molecular tumor board to recommend personalized treatment regimens based on tumor sequencing results in children with DIPG. We separately developed the Central Nervous System Targeted Agent Prediction (CNS-TAP) tool, which numerically scores targeted anticancer agents using preclinical, clinical, and patient-specific data. We hypothesized that highly scored agents from CNS-TAP would overlap with the PNOC003 tumor board's recommendations. For each of the 28 participants, actionable genetic alterations were derived from PNOC003 genomic reports and input to CNS-TAP to identify the highest scoring agents. These agents were then compared with PNOC003 recommendations, with a resultant concordance percentage calculated. Overall, 38% of the total agents recommended by the tumor board were also selected by CNS-TAP, with higher concordance (63%) in a subanalysis including only targeted anticancer agents. Furthermore, nearly all patients (93%) had at least 1 drug chosen by both methods. We demonstrate overlap between agents recommended by CNS-TAP and PNOC003 tumor board, though this does not appear to improve survival. We do observe some discordance, highlighting strengths and limitations of each method. We propose that a combination of expert opinion and data-driven tools may improve targeted treatment recommendations for children with DIPG., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Tissue-agnostic, molecularly targeted therapies are becoming increasingly common in cancer treatment. The molecular drivers of some classes and subclasses of tumors are rapidly being uncovered in an era of deep tumor sequencing occurring at the time of diagnosis. When and how targeted therapies should fit within up-front cytotoxic chemotherapy and radiation paradigms is yet to be determined, because many of them have been studied in single-arm studies in patients with relapsed or refractory cancer. Infant high-grade gliomas (HGGs) are biologically and clinically distinct from older child and adult HGGs, and are divided into 3 molecular subgroups. Group 1 infant HGGs are driven by receptor tyrosine kinase fusions, most commonly harboring an ALK, ROS1, NTRK, or MET fusion. Both larotrectinib and entrectinib are tropomyosin receptor kinase inhibitors with tissue-agnostic approvals for the treatment of patients with solid tumors harboring an NTRK fusion. This report discusses an 11-month-old female who presented with infantile spasms, found to have an unresectable, NTRK fusion-positive infant HGG. Larotrectinib was prescribed when the NTRK fusion was identified at diagnosis, and without additional intervention to date, the patient has continued with stable disease for >3 years. The only adverse event experienced was grade 1 aspartate transaminase and alanine transaminase elevations. The patient has a normal neurologic examination, is developing age-appropriately in all domains (gross motor, fine motor, cognitive, language, and social-emotional). She is no longer on antiseizure medications. To our knowledge, this is the first report of a patient with an infantile HGG receiving targeted therapy as first-line treatment with prolonged stable disease. A prospective study of larotrectinib in patients with newly diagnosed infant HGG is ongoing, and will hopefully help answer questions about durability of response, the need for additional therapies, and long-term toxicities seen with TRK inhibitors.

Background: Radiotherapy (RT) is the primary treatment for diffuse midline glioma (DMG), a lethal pediatric malignancy defined by histone H3 lysine 27-to-methionine (H3K27M) mutation. Based on the loss of H3K27 trimethylation producing broad epigenomic alterations, we hypothesized that H3K27M causes a functional double-strand break (DSB) repair defect that could be leveraged therapeutically with PARP inhibitor and RT for selective radiosensitization and antitumor immune responses., Methods: H3K27M isogenic DMG cells and orthotopic brainstem DMG tumors in immune deficient and syngeneic, immune competent mice were used to evaluate the efficacy and mechanisms of PARP1/2 inhibition by olaparib or PARP1 inhibition by AZD9574 with concurrent RT., Results: H3K27M mutation caused an HRR defect characterized by impaired RT-induced K63-linked polyubiquitination of histone H1 and inhibition of HRR protein recruitment. H3K27M DMG cells were selectively radiosensitized by olaparib in comparison to isogenic controls, and this effect translated to efficacy in H3K27M orthotopic brainstem tumors. Olaparib and RT induced an innate immune response and induction of NK cell (NKG2D) activating ligands leading to increased NK cell-mediated lysis of DMG tumor cells. In immunocompetent syngeneic orthotopic DMG tumors, either olaparib or AZD9574 in combination with RT enhanced intratumoral NK cell infiltration and activity in association with NK cell-mediated therapeutic responses and favorable activity of AZD9574., Conclusions: The HRR deficiency in H3K27M DMG can be therapeutically leveraged with PARP inhibitors to radiosensitize and induce an NK cell-mediated antitumor immune response selectively in H3K27M DMG, supporting the clinical investigation of best-in-class PARP inhibitors with RT in DMG patients., Key Points: H3K27M DMG are HRR defective and selectively radiosensitized by PARP inhibitor.PARP inhibitor with RT enhances NKG2D ligand expression and NK cell-mediated lysis.NK cells are required for the therapeutic efficacy of PARP inhibitor and RT., Importance of the Study: Radiotherapy is the cornerstone of H3K27M-mutant diffuse midline glioma treatment, but almost all patients succumb to tumor recurrence with poor overall survival, underscoring the need for RT-based precision combination therapy. Here, we reveal HRR deficiency as an H3K27M-mediated vulnerability and identify a novel mechanism linking impaired RT-induced histone H1 polyubiquitination and the subsequent RNF168/BRCA1/RAD51 recruitment in H3K27M DMG. This model is supported by selective radiosensitization of H3K27M DMG by PARP inhibitor. Notably, the combination treatment results in NKG2D ligand expression that confers susceptibility to NK cell killing in H3K27M DMG. We also show that the novel brain penetrant, PARP1-selective inhibitor AZD9574 compares favorably to olaparib when combined with RT, prolonging survival in a syngeneic orthotopic model of H3K27M DMG. This study highlights the ability of PARP1 inhibition to radiosensitize and induce an NK cell-mediated antitumor immunity in H3K27M DMG and supports future clinical investigation.

Diffuse hemispheric gliomas, H3G34R/V-mutant (DHG-H3G34), are lethal brain tumors lacking targeted therapies. They originate from interneuronal precursors; however, leveraging this origin for therapeutic insights remains unexplored. Here, we delineate a cellular hierarchy along the interneuron lineage development continuum, revealing that DHG-H3G34 mirror spatial patterns of progenitor streams surrounding interneuron nests, as seen during human brain development. Integrating these findings with genome-wide CRISPR-Cas9 screens identifies genes upregulated in interneuron lineage progenitors as major dependencies. Among these, CDK6 emerges as a targetable vulnerability: DHG-H3G34 tumor cells show enhanced sensitivity to CDK4/6 inhibitors and a CDK6-specific degrader, promoting a shift toward more mature interneuron-like states, reducing tumor growth, and prolonging xenograft survival. Notably, a patient with progressive DHG-H3G34 treated with a CDK4/6 inhibitor achieved 17 months of stable disease. This study underscores interneuronal progenitor-like states, organized in characteristic niches, as a distinct vulnerability in DHG-H3G34, highlighting CDK6 as a promising clinically actionable target., Competing Interests: Declaration of interests M.G.F. is a consultant for Twentyeight-Seven Therapeutics and Blueprint Medicines. M.M. is an SAB member for Cygnal Therapeutics. K.L.L. is founder and equity holder of Travera Inc. and receives consulting fees from BMS, Integragen, and Rarecyte, and research support from Lilly, BMS, and Amgen. D.H. has acted as an advisor for Novartis in relation to ribociclib., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Pediatric high-grade gliomas (pHGGs), including diffuse midline gliomas (DMGs), are aggressive pediatric tumors with one of the poorest prognoses. Delta-24-RGD and ONC201 have shown promising efficacy as single agents for these tumors. However, the combination of both agents has not been evaluated., Methods: The production of functional viruses was assessed by immunoblotting and replication assays. The antitumor effect was evaluated in a panel of human and murine pHGG and DMG cell lines. RNAseq, the seahorse stress test, mitochondrial DNA content, and γH2A.X immunofluorescence were used to perform mechanistic studies. Mouse models of both diseases were used to assess the efficacy of the combination in vivo. The tumor immune microenvironment was evaluated using flow cytometry, RNAseq, and multiplexed immunofluorescence staining., Results: The Delta-24-RGD/ONC201 combination did not affect the virus replication capability in human pHGG and DMG models in vitro. Cytotoxicity analysis showed that the combination treatment was either synergistic or additive. Mechanistically, the combination treatment increased nuclear DNA damage and maintained the metabolic perturbation and mitochondrial damage caused by each agent alone. Delta-24-RGD/ONC201 cotreatment extended the overall survival of mice implanted with human and murine pHGG and DMG cells, independent of H3 mutation status and location. Finally, combination treatment in murine DMG models revealed a reshaping of the tumor microenvironment to a proinflammatory phenotype., Conclusions: The Delta-24-RGD/ONC201 combination improved the efficacy compared to each agent alone in in vitro and in vivo models by potentiating nuclear DNA damage and in turn improving the antitumor (immune) response to each agent alone., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Diffuse midline gliomas (DMGs) are lethal primary brain tumors in children. The imipridones ONC201 and ONC206 induce mitochondrial dysfunction and have emerged as promising therapies for DMG patients. However, efficacy as monotherapy is limited, identifying a need for strategies that enhance response. Another hurdle is the lack of biomarkers that report on drug-target engagement at an early timepoint after treatment onset. Here, using 1 H-magnetic resonance spectroscopy, which is a non-invasive method of quantifying metabolite pool sizes, we show that accumulation of ψ-aminobutyric acid (GABA) is an early metabolic biomarker that can be detected within a week of ONC206 treatment, when anatomical alterations are absent, in mice bearing orthotopic xenografts. Mechanistically, imipridones activate the mitochondrial protease ClpP and upregulate the stress-responsive transcription factor ATF4. ATF4, in turn, upregulates glutamate decarboxylase, which synthesizes GABA, and downregulates ABAT , which degrades GABA, leading to GABA accumulation in DMG cells and tumors. Functionally, GABA secreted by imipridone-treated cells acts in an autocrine manner via the GABAB receptor to induce expression of superoxide dismutase (SOD1), which mitigates imipridone-induced oxidative stress and, thereby, curbs apoptosis. Importantly, blocking autocrine GABA signaling using the clinical stage GABAB receptor antagonist SGS-742 exacerbates oxidative stress and synergistically induces apoptosis in combination with imipridones in DMG cells and orthotopic tumor xenografts. Collectively, we identify GABA as a unique metabolic adaptation to imipridones that can be leveraged for non-invasive assessment of drug-target engagement and therapy. Clinical translation of our studies has the potential to enable precision metabolic therapy and imaging for DMG patients., One Sentence Summary: Imipridones induce GABA accumulation in diffuse midline gliomas, an effect that can be leveraged for therapy and non-invasive imaging.

Background: This study evaluated the safety and pharmacokinetics (PK) of oral ONC201 administered twice-weekly on consecutive days (D1D2) in pediatric patients with newly diagnosed DIPG and/or recurrent/refractory H3 K27M glioma., Methods: This phase 1 dose-escalation and expansion study included pediatric patients with H3 K27M-mutant glioma and/or DIPG following ≥1 line of therapy (NCT03416530). ONC201 was administered D1D2 at 3 dose levels (DLs; -1, 1, and 2). The actual administered dose within DLs was dependent on weight. Safety was assessed in all DLs; PK analysis was conducted in DL2. Patients receiving once-weekly ONC201 (D1) served as a PK comparator., Results: Twelve patients received D1D2 ONC201 (DL1, n = 3; DL1, n = 3; DL2, n = 6); no dose-limiting toxicities or grade ≥3 treatment-related adverse events occurred. PK analyses at DL2 (D1-250 mg, n = 3; D1-625 mg, n = 3; D1D2-250 mg, n = 2; D1D2-625 mg, n = 2) demonstrated variability in Cmax, AUC0-24, and AUC0-48, with comparable exposures across weight groups. No accumulation occurred with D1D2 dosing; the majority of ONC201 cleared before administration of the second dose. Cmax was variable between groups but did not appear to increase with D1D2 dosing. AUC0-48 was greater with D1D2 than once-weekly., Conclusions: ONC201 given D1D2 was well tolerated at all DLs and associated with greater AUC0-48., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Diffuse midline glioma (DMG) with H3K27M mutation is an aggressive and difficult to treat pediatric brain tumor. Recurrent gain of function mutations in H3.3 (H3.3A) and H3.1 (H3C2) at the 27th lysine to methionine (H3K27M) are seen in over 2/3 of DMGs, and are associated with a worse prognosis. Due to the anatomical location of DMG, traditional biopsy carries risk for neurologic injury as it requires penetration of vital midline structures. Further, radiographic (MRI) monitoring of DMG often shows nonspecific changes, which makes therapeutic monitoring difficult. This indicates a critical need for more minimally invasive methods, such as liquid biopsy, to understand, diagnose, and monitor H3K27M DMG. Here, we review the use of all modalities to date to detect biomarkers of H3K27M in cerebrospinal fluid (CSF), blood, and urine, and compare their effectiveness in detection, diagnosis, and monitoring treatment response. We provide specific detail of recent efforts to monitor CSF and plasma H3K27M cell-free DNA in patients undergoing therapy with the imipridone ONC201. Lastly, we discuss the future of therapeutic monitoring of H3K27M-DMG, including biomarkers such as mitochondrial DNA, mutant and modified histones, and novel sequencing-based approaches for improved detection methods., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Background Diffuse midline glioma (DMG) is a devastating pediatric brain tumor unresponsive to hundreds of clinical trials. Approximately 80% of DMGs harbor H3K27M oncohistones, which reprogram the epigenome to increase the metabolic profile of the tumor cells. Methods We have previously shown preclinical efficacy of targeting both oxidative phosphorylation and glycolysis through treatment with ONC201, which activates the mitochondrial protease ClpP, and paxalisib, which inhibits PI3K/mTOR, respectively. Results ONC201 and paxalisib combination treatment aimed at inducing metabolic distress led to the design of the first DMG-specific platform trial PNOC022 (NCT05009992). Conclusions Here, we expand on the PNOC022 rationale and discuss various considerations, including liquid biome, microbiome, and genomic biomarkers, quality-of-life endpoints, and novel imaging modalities, such that we offer direction on future clinical trials in DMG., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Background: Constitutional mismatch repair deficiency (CMMRD) syndrome is a rare and aggressive cancer predisposition syndrome. Because a scarcity of data on this condition contributes to management challenges and poor outcomes, we aimed to describe the clinical spectrum, cancer biology, and impact of genetics on patient survival in CMMRD., Methods: In this cohort study, we collected cross-sectional and longitudinal data on all patients with CMMRD, with no age limits, registered with the International Replication Repair Deficiency Consortium (IRRDC) across more than 50 countries. Clinical data were extracted from the IRRDC database, medical records, and physician-completed case record forms. The primary objective was to describe the clinical features, cancer spectrum, and biology of the condition. Secondary objectives included estimations of cancer incidence and of the impact of the specific mismatch-repair gene and genotype on cancer onset and survival, including after cancer surveillance and immunotherapy interventions., Findings: We analysed data from 201 patients (103 males, 98 females) enrolled between June 5, 2007 and Sept 9, 2022. Median age at diagnosis of CMMRD or a related cancer was 8·9 years (IQR 5·9-12·6), and median follow-up from diagnosis was 7·2 years (3·6-14·8). Endogamy among minorities and closed communities contributed to high homozygosity within countries with low consanguinity. Frequent dermatological manifestations (117 [93%] of 126 patients with complete data) led to a clinical overlap with neurofibromatosis type 1 (35 [28%] of 126). 339 cancers were reported in 194 (97%) of 201 patients. The cumulative cancer incidence by age 18 years was 90% (95% CI 80-99). Median time between cancer diagnoses for patients with more than one cancer was 1·9 years (IQR 0·8-3·9). Neoplasms developed in 15 organs and included early-onset adult cancers. CNS tumours were the most frequent (173 [51%] cancers), followed by gastrointestinal (75 [22%]), haematological (61 [18%]), and other cancer types (30 [9%]). Patients with CNS tumours had the poorest overall survival rates (39% [95% CI 30-52] at 10 years from diagnosis; log-rank p<0·0001 across four cancer types), followed by those with haematological cancers (67% [55-82]), gastrointestinal cancers (89% [81-97]), and other solid tumours (96% [88-100]). All cancers showed high mutation and microsatellite indel burdens, and pathognomonic mutational signatures. MLH1 or MSH2 variants caused earlier cancer onset than PMS2 or MSH6 variants, and inferior survival (overall survival at age 15 years 63% [95% CI 55-73] for PMS2, 49% [35-68] for MSH6, 19% [6-66] for MLH1, and 0% for MSH2; p<0·0001). Frameshift or truncating variants within the same gene caused earlier cancers and inferior outcomes compared with missense variants (p<0·0001). The greater deleterious effects of MLH1 and MSH2 variants as compared with PMS2 and MSH6 variants persisted despite overall improvements in survival after surveillance or immune checkpoint inhibitor interventions., Interpretation: The very high cancer burden and unique genomic landscape of CMMRD highlight the benefit of comprehensive assays in timely diagnosis and precision approaches toward surveillance and immunotherapy. These data will guide the clinical management of children and patients who survive into adulthood with CMMRD., Funding: The Canadian Institutes for Health Research, Stand Up to Cancer, Children's Oncology Group National Cancer Institute Community Oncology Research Program, Canadian Cancer Society, Brain Canada, The V Foundation for Cancer Research, BioCanRx, Harry and Agnieszka Hall, Meagan's Walk, BRAINchild Canada, The LivWise Foundation, St Baldrick Foundation, Hold'em for Life, and Garron Family Cancer Center., Competing Interests: Declaration of interests ALa reports payment from Alexion, support from Servier and stock from Gilead, outside of the submitted work. AV is co-lead role of the Consortium for Childhood Cancer Predisposition, outside of the submitted work. BH reports payment and stock from Invitae, outside of the submitted work. BC reports participation as data safety and monitoring board member in ReRad Study, participation in the chapter advisory board for Make a Wish Canada Nova Scotia, and participation in the medical advisory committee for Make a Wish Canada, outside of the submitted work. CCP reports grants from St Baldrick's Foundation, outside of the submitted work. DSZ reports consulting fees for Bayer, AstraZeneca, Accendatech, Novartis, Day One, FivePhusion, Alexion, Amgen, and Norgine, outside of the submitted work. DTB reports grants from MSD and Novocure, consulting fees from Nanocarry Therapeutics and Servier, and payment from Servier, outside of the submitted work. EO reports payment and support from Alexion for educational event, outside of the submitted work. EB reports grants from Roche and board participation for Novartis, Alexion and Gilead, outside of the submitted work. FC reports grants from Hall Hunter Foundation (UK), outside of the submitted work. HBF reports payments from Illumina and Sanofi Genzyme, support from Illumina, participation as scientific advisory committee for Sanofi Genzyme, International Gaucher Alliance and Igentify, stock from Igentify, and receipt of materials from Illumina, outside of the submitted work. IW reports grants from Chimerix and payment from COG Partners, outside of the submitted work. IS reports grants from Fondation Saint-Luc and FNRS-CDR, outside of the submitted work. JK reports other financial interests at Servier and PRA Health Sciences, outside of the submitted work. JRG-S reports participation on the board of the Philippine Society of Pediatric Oncology and Philippine Board of Pediatric Oncology, and stock in Macrogenics, Moderna, Mirati Therapeutics, CRISPR Therapeutics, Repligen, Quidelortho, and Shockwave Medical, outside of the submitted work. KJB reports consulting fees from US WorldMeds, Springworks Therapeutics, Alexion, and YmAbs, and payment from Alexion, outside of the submitted work. MS reports grants and support from the Swedish Childhood Cancer Fund, participation as a data safety and monitoring board member for clinical trial NCT05230758, and participation in the Swedish Pediatric CNS tumour group, outside of the submitted work. MAC reports financial support from SUNY Upstate Department of Pediatrics and board participation for Paige's Butterfly Run, outside of the submitted work. MO reports payment from Aptitude Health and participation on a data safety monitoring board or advisory board for Ultragenyx and Abeona, outside of the submitted works. MZ reports payment and support from and board participation for AstraZeneca. NW reports grants from CANSEARCH Foundation, Childhood Cancer Research Switzerland, and the Foundation for Children and Adolescents with Cancer; payment, support, and advisory board participation for Swedish Orphan Biovitrum; and board participation for Childhood Cancer Switzerland, outside of the submitted work. NH reports grants from the National Institutes of Health (NIH) and board participation for Incyte and Pfizer, outside of the submitted work. PCN reports grants from the Canadian Institutes for Health Research (CIHR) Foundation, US Department of Defense, and Garron Family Cancer Centre, outside of the submitted work. RP reports participation in the Collaborative Group of the Americas on Inherited Gastrointestinal Cancer, outside of the submitted work. RT reports consulting fees from Fennec Pharmaceuticals and Day One Biopharmaceuticals and payment from Fennec Pharmaceuticals, outside of the submitted work. SS reports payments from Sanofi Pharmaceuticals and Mylan Pharmaceutical, and board participation for Sanofi Pharmaceuticals, outside of the submitted work. SB reports consulting fees from Hoffmann-La Roche, YmAbs, MAP Biopharma and SERB SAS, and payment from Zschimmer & Schwarz Mohsdorf, outside of the submitted work. UI reports board participation in Pakistan Society of Pediatric Oncology, outside of the submitted work., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPGs), are a fatal form of brain cancer. These tumors often carry a driver mutation on histone H3 converting lysine 27 to methionine (H3K27M). DMG-H3K27M are characterized by altered metabolism and resistance to standard of care radiation (RT) but how the H3K27M mediates the metabolic response to radiation and consequent treatment resistance is uncertain., Methods: We performed metabolomics on irradiated and untreated H3K27M isogenic DMG cell lines and observed an H3K27M-specific enrichment for purine synthesis pathways. We profiled the expression of purine synthesis enzymes in publicly available patient data and our models, quantified purine synthesis using stable isotope tracing, and characterized the in vitro and in vivo response to de novo and salvage purine synthesis inhibition in combination with RT., Results: DMG-H3K27M cells activate purine metabolism in an H3K27M-specific fashion. In the absence of genotoxic treatment, H3K27M-expressing cells have higher relative activity of de novo synthesis and apparent lower activity of purine salvage demonstrated via stable isotope tracing of key metabolites in purine synthesis and by lower expression of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), the rate-limiting enzyme of purine salvage into IMP and GMP. Inhibition of de novo guanylate synthesis radiosensitized DMG-H3K27M cells in vitro and in vivo. Irradiated H3K27M cells upregulated HGPRT expression and hypoxanthine-derived guanylate salvage but maintained high levels of guanine-derived salvage. Exogenous guanine supplementation decreased radiosensitization in cells treated with combination RT and de novo purine synthesis inhibition. Silencing HGPRT combined with RT markedly suppressed DMG-H3K27M tumor growth in vivo., Conclusions: Our results indicate that DMG-H3K27M cells rely on highly active purine synthesis, both from the de novo and salvage synthesis pathways. However, highly active salvage of free purine bases into mature guanylates can bypass inhibition of the de novo synthetic pathway. We conclude that inhibiting purine salvage may be a promising strategy to overcome treatment resistance in DMG-H3K27M tumors., (© 2024. The Author(s).)

Background: Diffuse intrinsic pontine gliomas (DIPG/DMG) are devastating pediatric brain tumors with extraordinarily limited treatment options and uniformly fatal prognosis. Histone H3K27M mutation is a common recurrent alteration in DIPG and disrupts epigenetic regulation. We hypothesize that genome-wide H3K27M-induced epigenetic dysregulation makes tumors vulnerable to epigenetic targeting., Methods: We performed a screen of compounds targeting epigenetic enzymes to identify potential inhibitors for the growth of patient-derived DIPG cells. We further carried out transcriptomic and genomic landscape profiling including RNA-seq and CUT&RUN-seq as well as shRNA-mediated knockdown to assess the effects of chaetocin and SUV39H1, a target of chaetocin, on DIPG growth., Results: High-throughput small-molecule screening identified an epigenetic compound chaetocin as a potent blocker of DIPG cell growth. Chaetocin treatment selectively decreased proliferation and increased apoptosis of DIPG cells and significantly extended survival in DIPG xenograft models, while restoring H3K27me3 levels. Moreover, the loss of H3K9 methyltransferase SUV39H1 inhibited DIPG cell growth. Transcriptomic and epigenomic profiling indicated that SUV39H1 loss or inhibition led to the downregulation of stemness and oncogenic networks including growth factor receptor signaling and stemness-related programs; however, D2 dopamine receptor (DRD2) signaling adaptively underwent compensatory upregulation conferring resistance. Consistently, a combination of chaetocin treatment with a DRD2 antagonist ONC201 synergistically increased the antitumor efficacy., Conclusions: Our studies reveal a therapeutic vulnerability of DIPG cells through targeting the SUV39H1-H3K9me3 pathway and compensatory signaling loops for treating this devastating disease. Combining SUV39H1-targeting chaetocin with other agents such as ONC201 may offer a new strategy for effective DIPG treatment., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Immune checkpoint inhibition (ICI) is effective for replication-repair-deficient, high-grade gliomas (RRD-HGG). The clinical/biological impact of immune-directed approaches after failing ICI monotherapy is unknown. We performed an international study on 75 patients treated with anti-PD-1; 20 are progression free (median follow-up, 3.7 years). After second progression/recurrence (n = 55), continuing ICI-based salvage prolonged survival to 11.6 months (n = 38; P < 0.001), particularly for those with extreme mutation burden (P = 0.03). Delayed, sustained responses were observed, associated with changes in mutational spectra and the immune microenvironment. Response to reirradiation was explained by an absence of deleterious postradiation indel signatures (ID8). CTLA4 expression increased over time, and subsequent CTLA4 inhibition resulted in response/stable disease in 75%. RAS-MAPK-pathway inhibition led to the reinvigoration of peripheral immune and radiologic responses. Local (flare) and systemic immune adverse events were frequent (biallelic mismatch-repair deficiency > Lynch syndrome). We provide a mechanistic rationale for the sustained benefit in RRD-HGG from immune-directed/synergistic salvage therapies. Future approaches need to be tailored to patient and tumor biology., Significance: Hypermutant RRD-HGG are susceptible to checkpoint inhibitors beyond initial progression, leading to improved survival when reirradiation and synergistic immune/targeted agents are added. This is driven by their unique biological and immune properties, which evolve over time. Future research should focus on combinatorial regimens that increase patient survival while limiting immune toxicity. This article is featured in Selected Articles from This Issue, p. 201., (©2023 American Association for Cancer Research.)

Diffuse midline glioma (DMG), including tumors diagnosed in the brainstem (diffuse intrinsic pontine glioma; DIPG), are uniformly fatal brain tumors that lack effective treatment. Analysis of CRISPR/Cas9 loss-of-function gene deletion screens identified PIK3CA and MTOR as targetable molecular dependencies across patient derived models of DIPG, highlighting the therapeutic potential of the blood-brain barrier-penetrant PI3K/Akt/mTOR inhibitor, paxalisib. At the human-equivalent maximum tolerated dose, mice treated with paxalisib experienced systemic glucose feedback and increased insulin levels commensurate with patients using PI3K inhibitors. To exploit genetic dependence and overcome resistance while maintaining compliance and therapeutic benefit, we combined paxalisib with the antihyperglycemic drug metformin. Metformin restored glucose homeostasis and decreased phosphorylation of the insulin receptor in vivo, a common mechanism of PI3K-inhibitor resistance, extending survival of orthotopic models. DIPG models treated with paxalisib increased calcium-activated PKC signaling. The brain penetrant PKC inhibitor enzastaurin, in combination with paxalisib, synergistically extended the survival of multiple orthotopic patient-derived and immunocompetent syngeneic allograft models; benefits potentiated in combination with metformin and standard-of-care radiotherapy. Therapeutic adaptation was assessed using spatial transcriptomics and ATAC-Seq, identifying changes in myelination and tumor immune microenvironment crosstalk. Collectively, this study has identified what we believe to be a clinically relevant DIPG therapeutic combinational strategy.

Recent clinical trials for H3K27-altered diffuse midline gliomas (DMGs) have shown much promise. We present a consensus roadmap and identify three major barriers: (1) refinement of experimental models to include immune and brain-specific components; (2) collaboration among researchers, clinicians, and industry to integrate patient-derived data through sharing, transparency, and regulatory considerations; and (3) streamlining clinical efforts including biopsy, CNS-drug delivery, endpoint determination, and response monitoring. We highlight the importance of comprehensive collaboration to advance the understanding, diagnostics, and therapeutics for DMGs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Therapeutic resistance remains a major obstacle to successful clinical management of diffuse intrinsic pontine glioma (DIPG), a high-grade pediatric tumor of the brain stem. In nearly all patients, available therapies fail to prevent progression. Innovative combinatorial therapies that penetrate the blood-brain barrier and lead to long-term control of tumor growth are desperately needed. We identified mechanisms of resistance to radiotherapy, the standard of care for DIPG. On the basis of these findings, we rationally designed a brain-penetrant small molecule, MTX-241F, that is a highly selective inhibitor of EGFR and PI3 kinase family members, including the DNA repair protein DNA-PK. Preliminary studies demonstrated that micromolar levels of this inhibitor can be achieved in murine brain tissue and that MTX-241F exhibits promising single-agent efficacy and radiosensitizing activity in patient-derived DIPG neurospheres. Its physiochemical properties include high exposure in the brain, indicating excellent brain penetrance. Because radiotherapy results in double-strand breaks that are repaired by homologous recombination (HR) and non-homologous DNA end joining (NHEJ), we have tested the combination of MTX-241F with an inhibitor of Ataxia Telangiectasia Mutated to achieve blockade of HR and NHEJ, respectively, with or without radiotherapy. When HR blockers were combined with MTX-241F and radiotherapy, synthetic lethality was observed, providing impetus to explore this combination in clinically relevant models of DIPG. Our data provide proof-of-concept evidence to support advanced development of MTX-241F for the treatment of DIPG. Future studies will be designed to inform rapid clinical translation to ultimately impact patients diagnosed with this devastating disease., (©2023 The Authors; Published by the American Association for Cancer Research.)

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain stem tumor and the leading cause of pediatric cancer-related death. To date, these tumors remain incurable, underscoring the need for efficacious therapies. In this study, we demonstrate that the immune checkpoint TIM-3 (HAVCR2) is highly expressed in both tumor cells and microenvironmental cells, mainly microglia and macrophages, in DIPG. We show that inhibition of TIM-3 in syngeneic models of DIPG prolongs survival and produces long-term survivors free of disease that harbor immune memory. This antitumor effect is driven by the direct effect of TIM-3 inhibition in tumor cells, the coordinated action of several immune cell populations, and the secretion of chemokines/cytokines that create a proinflammatory tumor microenvironment favoring a potent antitumor immune response. This work uncovers TIM-3 as a bona fide target in DIPG and supports its clinical translation., Competing Interests: Declaration of interests A.C.A. is a member of the SAB for Tizona Therapeutics, Trishula Therapeutics, Compass Therapeutics, Zumutor Biologics, ImmuneOncia, and Excepgen, which have interests in cancer immunotherapy. A.C.A. is a paid consultant for iTeos Therapeutics and Larkspur Biosciences. A.C.A.’s interests were reviewed and managed by the Brigham and Women’s Hospital. The rest of authors do not have potential conflicts of interest to disclose., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Patients with H3K27M-mutant diffuse midline glioma (DMG) have no proven effective therapies. ONC201 has recently demonstrated efficacy in these patients, but the mechanism behind this finding remains unknown. We assessed clinical outcomes, tumor sequencing, and tissue/cerebrospinal fluid (CSF) correlate samples from patients treated in two completed multisite clinical studies. Patients treated with ONC201 following initial radiation but prior to recurrence demonstrated a median overall survival of 21.7 months, whereas those treated after recurrence had a median overall survival of 9.3 months. Radiographic response was associated with increased expression of key tricarboxylic acid cycle-related genes in baseline tumor sequencing. ONC201 treatment increased 2-hydroxyglutarate levels in cultured H3K27M-DMG cells and patient CSF samples. This corresponded with increases in repressive H3K27me3 in vitro and in human tumors accompanied by epigenetic downregulation of cell cycle regulation and neuroglial differentiation genes. Overall, ONC201 demonstrates efficacy in H3K27M-DMG by disrupting integrated metabolic and epigenetic pathways and reversing pathognomonic H3K27me3 reduction., Significance: The clinical, radiographic, and molecular analyses included in this study demonstrate the efficacy of ONC201 in H3K27M-mutant DMG and support ONC201 as the first monotherapy to improve outcomes in H3K27M-mutant DMG beyond radiation. Mechanistically, ONC201 disrupts integrated metabolic and epigenetic pathways and reverses pathognomonic H3K27me3 reduction. This article is featured in Selected Articles from This Issue, p. 2293., (©2023 The Authors; Published by the American Association for Cancer Research.)

Therapeutic resistance remains a major obstacle to preventing progression of H3K27M-altered Diffuse Midline Glioma (DMG). Resistance is driven in part by ALDH-positive cancer stem cells (CSC), with high ALDH1A3 expression observed in H3K27M-mutant DMG biopsies. We hypothesized that ALDH-mediated stemness and resistance may in part be driven by the oncohistone itself. Upon deletion of H3K27M, ALDH1A3 expression decreased dramatically and was accompanied by a gain in astrocytic marker expression and a loss of neurosphere forming potential, indicative of differentiation. Here we show that the oncohistone regulates histone acetylation through ALDH1A3 in a Wnt-dependent manner and that loss of H3K27M expression results in sensitization of DMGs to radiotherapy. The observed elevated Wnt signaling in H3K27M-altered DMG likely stems from a dramatic suppression of mRNA and protein expression of the Wnt inhibitor EYA4 driven by the oncohistone. Thus, our findings identify EYA4 as a bona fide tumor suppressor in DMG that upon suppression, results in aberrant Wnt signaling to orchestrate stemness and differentiation. Future studies will explore whether overexpression of EYA4 in DMG can impede growth and invasion. In summary, we have gained mechanistic insight into H3K27M-mediated regulation of cancer stemness and differentiation, which provides rationale for exploring new therapeutic targets for DMG., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: CAL has received consulting fees from Astellas Pharmaceuticals, Odyssey Therapeutics, and T-Knife Therapeutics, and is an inventor on patents pertaining to Kras regulated metabolic pathways, redox control pathways in pancreatic cancer, and targeting the GOT1-pathway as a therapeutic approach (US Patent No: 2015126580-A1, 05/07/2015; US Patent No: 20190136238, 05/09/2019; International Patent No: WO2013177426-A2, 04/23/2015). All other authors declare that no conflicts of interest exist., (Copyright © 2023. Published by Elsevier Inc.)

It is often challenging to distinguish cancerous from non-cancerous lesions in the brain using conventional diagnostic approaches. We introduce an analytic technique called Real-CSF (repetitive element aneuploidy sequencing in CSF) to detect cancers of the central nervous system from evaluation of DNA in the cerebrospinal fluid (CSF). Short interspersed nuclear elements (SINEs) are PCR amplified with a single primer pair, and the PCR products are evaluated by next-generation sequencing. Real-CSF assesses genome-wide copy-number alterations as well as focal amplifications of selected oncogenes. Real-CSF was applied to 280 CSF samples and correctly identified 67% of 184 cancerous and 96% of 96 non-cancerous brain lesions. CSF analysis was considerably more sensitive than standard-of-care cytology and plasma cell-free DNA analysis in the same patients. Real-CSF therefore has the capacity to be used in combination with other clinical, radiologic, and laboratory-based data to inform the diagnosis and management of patients with suspected cancers of the brain., Competing Interests: Declaration of interests B.V., K.W.K., and N.P. are founders of Thrive Earlier Detection, an Exact Sciences Company. K.W.K., N.P., and C.D. are consultants to Thrive Earlier Detection. B.V., K.W.K., N.P., and C.D. hold equity in Exact Sciences. B.V., K.W.K., and N.P. are founders of and own equity in ManaT Bio. K.W.K. and N.P. are consultants to and own equity in Haystack Oncology, Neophore, and Personal Genome Diagnostics. K.W.K., B.V., and N.P. hold equity in and are consultants to CAGE Pharma. B.V. is a consultant to and holds equity in Catalio Capital Management and may be a consultant to and hold equity in Haystack Oncology. B.V. owns equity in CAGE, Neophore, and Personal Genome Diagnostics. C. Bettegowda is a consultant to Depuy-Synthes, Bionaut Labs, Haystack Oncology, and Galectin Therapeutics. C. Bettegowda is a co-founder of OrisDx. C. Bettegowda and C.D. are co-founders of Belay Diagnostics. The companies named above, as well as other companies, have licensed previously described technologies related to the work described in this paper from Johns Hopkins University. B.V., K.W.K., N.P., C. Bettegowda, and C.D. are inventors on some of these technologies. Licenses to these technologies are or will be associated with equity or royalty payments to the inventors as well as to Johns Hopkins University. Patent applications on the work described in this paper may be filed by Johns Hopkins University. The terms of all these arrangements are being managed by Johns Hopkins University in accordance with its conflict of interest policies. M.H. is on the data safety monitoring board for Parexel and Advarra and has received an honorarium from Pfizer. K.S. received an honorarium from Springworks Therapeutics and receives research funding from Springworks Therapeutics. J.N. receives research funding from Merck, AstraZeneca, BMS, Amgen, Novartis, and Roche/Genetech and is a consulting/advisory board for Merck, AstraZeneca, BMS, Amgen, Novartis, Roche/Genetech, Takeda, Pfizer, Daiichi Sankyo, and NGM Biosciences. C.J. is a co-founder with equity interest in Egret Therapeutics and receives research support from Biohaven., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9 to 11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring PIK3CA mutations showed increased sensitivity to ONC201, whereas those harboring TP53 mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992., Significance: PI3K/Akt signaling promotes metabolic adaptation to ONC201-mediated disruption of mitochondrial energy homeostasis in diffuse intrinsic pontine glioma, highlighting the utility of a combination treatment strategy using ONC201 and the PI3K/Akt inhibitor paxalisib., (©2023 The Authors; Published by the American Association for Cancer Research.)

Diffuse intrinsic pontine glioma (DIPG) is a rare but highly lethal pediatric and adolescent tumor located in the pons of the brainstem. DIPGs harbor unique and specific pathological and molecular alterations, such as the hallmark lysine 27-to-methionine (H3K27M) mutation in histone H3, which lead to global changes in the epigenetic landscape and drive tumorigenesis. While fractionated radiotherapy, the current standard of care, improves symptoms and delays tumor progression, DIPGs inevitably recur, and despite extensive efforts chemotherapy-driven radiosensitization strategies have failed to improve survival. Advances in our understanding of the role of epigenetics in the cellular response to radiation-induced DNA damage, however, offer new opportunities to develop combinational therapeutic strategies selective for DIPGs expressing H3K27M. In this review, we provide an overview of preclinical studies that explore potential radiosensitization strategies targeting the unique epigenetic landscape of H3K27M mutant DIPG. We further discuss opportunities to selectively radiosensitize DIPG through strategic inhibition of the radiation-induced DNA damage response. Finally, we discuss the potential for using radiation to induce anti-tumor immune responses that may be potentiated in DIPG by radiosensitizing-therapeutic strategies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Malignant primary brain tumors are the most common cancer in children aged 0-14 years, and are the most common cause of death among pediatric cancer patients. Compared to other cancers, pediatric brain tumors have been difficult to diagnose and study given the high risk of intracranial biopsy penetrating through vital midline structures, where the majority of pediatric brain tumors originate (Ostrom et al., 2015). Furthermore, the vast majority of these tumors recur. With limitations in the ability to monitor using clinical and radiographic methods alone, minimally invasive methods such as liquid biopsy will be crucial to our understanding and treatment. Liquid biopsy of blood, urine, and cerebrospinal fluid (CSF) can be used to sample cfDNA, ctDNA, RNA, extracellular vesicles, and tumor-associated proteins. In the past year, four seminal papers have made significant advances in the use of liquid biopsy in pediatric brain tumor patients (Liu et al., 2021; Cantor et al., 2022; Miller et al., 2022; Pagès et al., 2022). In this review, we integrate the results of these studies and others to discuss how the newest technologies in liquid biopsy are being developed for molecular diagnosis and treatment response in pediatric brain tumors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tripathy, John, Wadden, Kong, Sharba and Koschmann.)

Background: Diffuse midline gliomas (DMG) are highly invasive brain tumors with rare survival beyond two years past diagnosis and limited understanding of the mechanism behind tumor invasion. Previous reports demonstrate upregulation of the protein ID1 with H3K27M and ACVR1 mutations in DMG, but this has not been confirmed in human tumors or therapeutically targeted., Methods: Whole exome, RNA, and ChIP-sequencing was performed on the ID1 locus in DMG tissue. Scratch-assay migration and transwell invasion assays of cultured cells were performed following shRNA-mediated ID1-knockdown. In vitro and in vivo genetic and pharmacologic [cannabidiol (CBD)] inhibition of ID1 on DMG tumor growth was assessed. Patient-reported CBD dosing information was collected., Results: Increased ID1 expression in human DMG and in utero electroporation (IUE) murine tumors is associated with H3K27M mutation and brainstem location. ChIP-sequencing indicates ID1 regulatory regions are epigenetically active in human H3K27M-DMG tumors and prenatal pontine cells. Higher ID1-expressing astrocyte-like DMG cells share a transcriptional program with oligo/astrocyte-precursor cells (OAPCs) from the developing human brain and demonstrate upregulation of the migration regulatory protein SPARCL1. Genetic and pharmacologic (CBD) suppression of ID1 decreases tumor cell invasion/migration and tumor growth in H3.3/H3.1K27M PPK-IUE and human DIPGXIIIP* in vivo models of pHGG. The effect of CBD on cell proliferation appears to be non-ID1 mediated. Finally, we collected patient-reported CBD treatment data, finding that a clinical trial to standardize dosing may be beneficial., Conclusions: H3K27M-mediated re-activation of ID1 in DMG results in a SPARCL1+ migratory transcriptional program that is therapeutically targetable with CBD., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Pediatric brain tumors are the leading cause of cancer-related death in children in the United States and contribute a disproportionate number of potential years of life lost compared to adult cancers. Moreover, survivors frequently suffer long-term side effects, including secondary cancers. The Children's Brain Tumor Network (CBTN) is a multi-institutional international clinical research consortium created to advance therapeutic development through the collection and rapid distribution of biospecimens and data via open-science research platforms for real-time access and use by the global research community. The CBTN's 32 member institutions utilize a shared regulatory governance architecture at the Children's Hospital of Philadelphia to accelerate and maximize the use of biospecimens and data. As of August 2022, CBTN has enrolled over 4700 subjects, over 1500 parents, and collected over 65,000 biospecimen aliquots for research. Additionally, over 80 preclinical models have been developed from collected tumors. Multi-omic data for over 1000 tumors and germline material are currently available with data generation for > 5000 samples underway. To our knowledge, CBTN provides the largest open-access pediatric brain tumor multi-omic dataset annotated with longitudinal clinical and outcome data, imaging, associated biospecimens, child-parent genomic pedigrees, and in vivo and in vitro preclinical models. Empowered by NIH-supported platforms such as the Kids First Data Resource and the Childhood Cancer Data Initiative, the CBTN continues to expand the resources needed for scientists to accelerate translational impact for improved outcomes and quality of life for children with brain and spinal cord tumors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. David S. Ziegler is a consultant, or on the advisory board, of Bayer, AstraZeneca, Accendatech, Novartis, Day One, FivePhusion, Amgen, Alexion, and Norgine. Angela J. Waanders is on the advisory board of Alexion and Day One., (Copyright © 2022. Published by Elsevier Inc.)

Histone 3 lysine27-to-methionine (H3-K27M) mutations most frequently occur in diffuse midline gliomas (DMGs) of the childhood pons but are also increasingly recognized in adults. Their potential heterogeneity at different ages and midline locations is vastly understudied. Here, through dissecting the single-cell transcriptomic, epigenomic and spatial architectures of a comprehensive cohort of patient H3-K27M DMGs, we delineate how age and anatomical location shape glioma cell-intrinsic and -extrinsic features in light of the shared driver mutation. We show that stem-like oligodendroglial precursor-like cells, present across all clinico-anatomical groups, display varying levels of maturation dependent on location. We reveal a previously underappreciated relationship between mesenchymal cancer cell states and age, linked to age-dependent differences in the immune microenvironment. Further, we resolve the spatial organization of H3-K27M DMG cell populations and identify a mitotic oligodendroglial-lineage niche. Collectively, our study provides a powerful framework for rational modeling and therapeutic interventions., (© 2022. The Author(s).)

Background: Children ≤36 months with diffuse intrinsic pontine glioma (DIPG) have increased long-term survival (LTS, overall survival (OS) ≥24 months). Understanding distinguishing characteristics in this population is critical to improving outcomes., Methods: Patients ≤36 months at diagnosis enrolled on the International DIPG Registry (IDIPGR) with central imaging confirmation were included. Presentation, clinical course, imaging, pathology and molecular findings were analyzed., Results: Among 1183 patients in IDIPGR, 40 were eligible (median age: 29 months). Median OS was 15 months. Twelve patients (30%) were LTS, 3 (7.5%) very long-term survivors ≥5 years. Among 8 untreated patients, median OS was 2 months. Patients enrolled in the registry but excluded from our study by central radiology review or tissue diagnosis had median OS of 7 months. All but 1 LTS received radiation. Among 32 treated patients, 1-, 2-, 3-, and 5-year OS rates were 68.8%, 31.2%, 15.6% and 12.5%, respectively. LTS had longer duration of presenting symptoms (P = .018). No imaging features were predictive of outcome. Tissue and genomic data were available in 18 (45%) and 10 patients, respectively. Among 9 with known H3K27M status, 6 had a mutation., Conclusions: Children ≤36 months demonstrated significantly more LTS, with an improved median OS of 15 months; 92% of LTS received radiation. Median OS in untreated children was 2 months, compared to 17 months for treated children. LTS had longer duration of symptoms. Excluded patients demonstrated a lower OS, contradicting the hypothesis that children ≤36 months with DIPG show improved outcomes due to misdiagnosis., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pediatric high-grade gliomas (pHGGs) are the leading cause of cancer-related deaths in children in the USA. Sixteen percent of hemispheric pediatric and young adult HGGs encode Gly34Arg/Val substitutions in the histone H3.3 (H3.3-G34R/V). The mechanisms by which H3.3-G34R/V drive malignancy and therapeutic resistance in pHGGs remain unknown. Using a syngeneic, genetically engineered mouse model (GEMM) and human pHGG cells encoding H3.3-G34R, we demonstrate that this mutation led to the downregulation of DNA repair pathways. This resulted in enhanced susceptibility to DNA damage and inhibition of the DNA damage response (DDR). We demonstrate that genetic instability resulting from improper DNA repair in G34R-mutant pHGG led to the accumulation of extrachromosomal DNA, which activated the cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway, inducing the release of immune-stimulatory cytokines. We treated H3.3-G34R pHGG-bearing mice with a combination of radiotherapy (RT) and DNA damage response inhibitors (DDRi) (i.e., the blood-brain barrier-permeable PARP inhibitor pamiparib and the cell-cycle checkpoint CHK1/2 inhibitor AZD7762), and these combinations resulted in long-term survival for approximately 50% of the mice. Moreover, the addition of a STING agonist (diABZl) enhanced the therapeutic efficacy of these treatments. Long-term survivors developed immunological memory, preventing pHGG growth upon rechallenge. These results demonstrate that DDRi and STING agonists in combination with RT induced immune-mediated therapeutic efficacy in G34-mutant pHGG.

Background: ONC201, a dopamine receptor D2 (DRD2) antagonist and caseinolytic protease P (ClpP) agonist, has induced durable tumor regressions in adults with recurrent H3 K27M-mutant glioma. We report results from the first phase I pediatric clinical trial of ONC201., Methods: This open-label, multi-center clinical trial (NCT03416530) of ONC201 for pediatric H3 K27M-mutant diffuse midline glioma (DMG) or diffuse intrinsic pontine glioma (DIPG) employed a dose-escalation and dose-expansion design. The primary endpoint was the recommended phase II dose (RP2D). A standard 3 + 3 dose escalation design was implemented. The target dose was the previously established adult RP2D (625 mg), scaled by body weight. Twenty-two pediatric patients with DMG/DIPG were treated following radiation; prior lines of systemic therapy in addition to radiation were permitted providing sufficient time had elapsed prior to study treatment., Results: The RP2D of orally administered ONC201 in this pediatric population was determined to be the adult RP2D (625 mg), scaled by body weight; no dose-limiting toxicities (DLT) occurred. The most frequent treatment-emergent Grade 1-2 AEs were headache, nausea, vomiting, dizziness and increase in alanine aminotransferase. Pharmacokinetics were determined following the first dose: T 1/2 , 8.4 h; T max , 2.1 h; C max , 2.3 µg/mL; AUC 0-tlast , 16.4 hµg/mL. Median duration of treatment was 20.6 weeks (range 5.1-129). Five (22.7%) patients, all of whom initiated ONC201 following radiation and prior to recurrence, were alive at 2 years from diagnosis., Conclusions: The adult 625 mg weekly RP2D of ONC201 scaled by body weight was well tolerated. Further investigation of ONC201 for DMG/DIPG is warranted., (© The Author(s) 2022. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Background: Pediatric diffuse midline gliomas (DMGs) are incurable childhood cancers. The imipridone ONC201 has shown early clinical efficacy in a subset of DMGs. However, the anticancer mechanisms of ONC201 and its derivative ONC206 have not been fully described in DMGs., Methods: DMG models including primary human in vitro (n = 18) and in vivo (murine and zebrafish) models, and patient (n = 20) frozen and FFPE specimens were used. Drug-target engagement was evaluated using in silico ChemPLP and in vitro thermal shift assay. Drug toxicity and neurotoxicity were assessed in zebrafish models. Seahorse XF Cell Mito Stress Test, MitoSOX and TMRM assays, and electron microscopy imaging were used to assess metabolic signatures. Cell lineage differentiation and drug-altered pathways were defined using bulk and single-cell RNA-seq., Results: ONC201 and ONC206 reduce viability of DMG cells in nM concentrations and extend survival of DMG PDX models (ONC201: 117 days, P = .01; ONC206: 113 days, P = .001). ONC206 is 10X more potent than ONC201 in vitro and combination treatment was the most efficacious at prolonging survival in vivo (125 days, P = .02). Thermal shift assay confirmed that both drugs bind to ClpP, with ONC206 exhibiting a higher binding affinity as assessed by in silico ChemPLP. ClpP activation by both drugs results in impaired tumor cell metabolism, mitochondrial damage, ROS production, activation of integrative stress response (ISR), and apoptosis in vitro and in vivo. Strikingly, imipridone treatment triggered a lineage shift from a proliferative, oligodendrocyte precursor-like state to a mature, astrocyte-like state., Conclusion: Targeting mitochondrial metabolism and ISR activation effectively impairs DMG tumorigenicity. These results supported the initiation of two pediatric clinical trials (NCT05009992, NCT04732065)., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pediatric high-grade glioma (pHGG), including both diffuse midline glioma (DMG) and non-midline tumors, continues to be one of the deadliest oncologic diagnoses (both henceforth referred to as "pHGG"). Targeted therapy options aimed at key oncogenic receptor tyrosine kinase (RTK) drivers using small-molecule RTK inhibitors has been extensively studied, but the absence of proper in vivo modeling that recapitulate pHGG biology has historically been a research challenge. Thankfully, there have been many recent advances in animal modeling, including Cre-inducible transgenic models, as well as intra-uterine electroporation (IUE) models, which closely recapitulate the salient features of human pHGG tumors. Over 20% of pHGG have been found in sequencing studies to have alterations in platelet derived growth factor-alpha (PDGFRA), making growth factor modeling and inhibition via targeted tyrosine kinases a rich vein of interest. With commonly found alterations in other growth factors, including FGFR, EGFR, VEGFR as well as RET, MET, and ALK, it is necessary to model those receptors, as well. Here we review the recent advances in murine modeling and precision targeting of the most important RTKs in their clinical context. We additionally provide a review of current work in the field with several small molecule RTK inhibitors used in pre-clinical or clinical settings for treatment of pHGG., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Schwark, Messinger, Cummings, Bradin, Kawakibi, Babila, Lyons, Ji, Cartaxo, Kong, Cantor, Koschmann and Yadav.)