Diffuse Intrinsic Pontine Glioma Research Articles

Laminin-associated integrins mediate Diffuse Intrinsic Pontine Glioma infiltration and therapy response within a neural assembloid model

Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive and fatal pediatric brain cancer. One pre-requisite for tumor cells to infiltrate is adhesion to extracellular matrix (ECM) components. However, it remains largely unknown which ECM proteins are critical in enabling DIPG adhesion and migration and which integrin receptors mediate these processes. Here, we identify laminin as a key ECM protein that supports robust DIPG cell adhesion and migration. To study DIPG infiltration, we developed a DIPG-neural assembloid model, which is composed of a DIPG spheroid fused to a human induced pluripotent stem cell-derived neural organoid. Using this assembloid model, we demonstrate that knockdown of laminin-associated integrins significantly impedes DIPG infiltration. Moreover, laminin-associated integrin knockdown improves DIPG response to radiation and HDAC inhibitor treatment within the DIPG-neural assembloids. These findings reveal the critical role of laminin-associated integrins in mediating DIPG progression and drug response. The results also provide evidence that disrupting integrin receptors may offer a novel therapeutic strategy to enhance DIPG treatment outcomes. Finally, these results establish DIPG-neural assembloid models as a powerful tool to study DIPG disease progression and enable drug discovery.

Management of patients with diffuse intrinsic pontine glioma in Australia and New Zealand: Australian and New Zealand Children's Haematology/Oncology Group position statement.

The main mission of the Australian and New Zealand Children's Haematology and Oncology Group (ANZCHOG) is to develop and facilitate local access to the world's leading evidence-based clinical trials for all paediatric cancers, including brain tumours, as soon as practically possible. Diffuse intrinsic pontine gliomas (DIPGs) - a subset of a larger group of tumours now termed diffuse midline glioma, H3K27-altered (DMG) - are paediatric brain cancers with less than 10% survival at two years. In the absence of any proven curative therapies, significant recent advancements have been made in pre-clinical and clinical research, leading many to seek integration of novel therapies early into standard practice. Despite these innovative therapeutic approaches, DIPG remains an incurable disease for which novel surgical, imaging, diagnostic, radiation and systemic therapy approaches are needed. All patients with DIPG should be discussed in multidisciplinary neuro-oncology meetings (including pathologists, neuroradiologists, radiation oncologists, neurosurgeons, medical oncologists) at diagnosis and at relapse or progression. Radiation therapy to the involved field remains the local and international standard of care treatment. Proton therapy does not yield a superior survival outcome compared with photon therapy and patients should undergo radiation therapy with the available modality (photon or proton) at their treatment centre. Patients may receive concurrent chemotherapy or radiation-sensitising agents as part of a clinical trial. Biopsy should be offered to facilitate consideration of experimental therapies and eligibility for clinical trial participation. After radiation therapy, each patient should be managed individually with either observation or considered for enrolment on a clinical trial, if eligible, after full discussion with the family. Re-irradiation can be considered for progressive disease. Every child diagnosed with DIPG should be offered enrolment on a clinical trial where available. Access to investigational drugs without biological rationale outside the clinical trial setting is not supported. In case of potentially actionable target identification with molecular profiling and absence of a suitable clinical trial, rational targeted therapies can be considered through compassionate access programs.

The role of focused ultrasound for pediatric brain tumors: current insights and future implications on treatment strategies.

Focused ultrasound (FUS) is an innovative and emerging technology for the treatment of adult and pediatric brain tumors and illustrates the intersection of various specialized fields, including neurosurgery, neuro-oncology, radiation oncology, and biomedical engineering. The authors provide a comprehensive overview of the application and implications of FUS in treating pediatric brain tumors, with a special focus on pediatric low-grade gliomas (pLGGs) and the evolving landscape of this technology and its clinical utility. The fundamental principles of FUS include its ability to induce thermal ablation or enhance drug delivery through transient blood-brain barrier (BBB) disruption, emphasizing the adaptability of high-intensity focused ultrasound (HIFU) and low-intensity focused ultrasound (LIFU) applications. Several ongoing clinical trials explore the potential of FUS in offering alternative therapeutic strategies for pathologies where conventional treatments fall short, specifically centrally-located benign CNS tumors and diffuse intrinsic pontine glioma (DIPG). A case illustration involving the use of HIFU for pilocytic astrocytoma is presented. Discussions regarding future applications of FUS for the treatment of gliomas include improved drug delivery, immunomodulation, radiosensitization, and other technological advancements.

CAR T-cell therapy: a potential treatment strategy for pediatric midline gliomas.

Pediatric brain tumors are the primary cause of death in children with cancer. Diffuse midline glioma (DMG) and diffuse intrinsic pontine glioma (DIPG) are frequently unresectable due to their difficult access location, and 5-year survival remains less than 20%. Despite significant advances in tumor biology and genetics, treatment options remain limited and ineffective. Immunotherapy using T cells with a chimeric antigen receptor (CAR) that has been genetically engineered is quickly emerging as a new treatment option for these patients. High levels of expression were detected for both disialoganglioside (GD2) and B7-H3 in pediatric DMG/DIPG. Numerous studies have been conducted in recent years employing various generations of GD2-CAR T cells. The two most prevalent adverse effects found with this therapy are cytokine release syndrome, which varies in severity from mild constitutional symptoms to a high-grade disease associated with potentially fatal multi-organ failure, and neurotoxicity, known as CAR T-cell-related encephalopathy syndrome. During the acute phase of anticancer action, peri-tumoral neuro-inflammation might cause deadly hydrocephalus. The initial results of clinical trials show that the outcomes are not highly encouraging as B cell malignancies and myelomas. In vivo research on CAR T-cell therapy for DIPG has yielded encouraging results, but in human trials, the early results have shown potentially fatal side effects and very modest, but fleeting improvements. Solid tumors present a hindrance to CAR T-cell therapy because of the antigenic dilemma and the strong immune-suppressing tumor microenvironment.

DNA Methylation Profiles Are Stable in H3 K27M-Mutant Diffuse Midline Glioma Neurosphere Cell Lines.

Diffuse midline gliomas are among the deadliest human cancers and have had little progress in treatment in the last 50 years. Cell cultures of these tumors have been developed recently, but the degree to which such cultures retain the characteristics of the source tumors is unknown. DNA methylation profiling offers a powerful tool to look at genome-wide epigenetic changes that are biologically meaningful and can help assess the similarity of cultured tumor cells to their in vivo progenitors. Paraffinized diagnostic tissue from three diffuse intrinsic pontine gliomas with H3 K27M mutations was compared with subsequent passages of neurosphere cell cultures from those tumors. Each cell line was passaged 3-4 times and analyzed with DNA methylation arrays and standard algorithms that provided a comparison of diagnostic classification and cluster analysis. All samples tested maintained high classifier scores and clustered within the reference group of H3 K27M-mutant diffuse midline gliomas. There was a gain of 1q in all cell lines, with two cell lines initially manifesting the gain of 1q only during culture. In vitro cell cultures of H3 K27M-mutant gliomas maintain high degrees of similarity in DNA methylation profiles to their source tumor, confirming their fidelity even with some chromosomal changes.

Purine salvage promotes treatment resistance in H3K27M-mutant diffuse midline glioma

BackgroundDiffuse 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.MethodsWe 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.ResultsDMG-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.ConclusionsOur 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.

181 Initial Experience With Sonodynamic Therapy Using Low-Intensity Focused Ultrasound and ALA for Pediatric Patients With Diffuse Intrinsic Pontine Glioma

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) is a devastating pediatric brain tumor with poor prognosis. Aminolevulinic acid (ALA) sonodynamic therapy (SDT) is a non-invasive strategy where tissues sensitized with ALA are exposed to MR-guided focused ultrasound (MRgFUS). Preclinical studies have demonstrated significant slowing in growth of gliomas and increased survival in animal models. METHODS: A Phase1/2 study (NCT05123534, clinicaltrials.gov) in patients with newly diagnosed DIPG is ongoing. MRIs at 24hrs and 1- and 3-months post procedure are obtained to evaluate safety and efficacy. A total of 18-45 patients will be enrolled in 9 different dose-escalation cohorts. The three patients from the first cohort are described below. RESULTS: Three patients with DIPGs were treated with 5 mg/kg of ALA and 200 J of MRgFUS in the first cohort. They were aged 5-7 years and 2 were females. They had received radiotherapy 8-10 weeks prior and interval imaging showed no disease progression. The first patient received two treatments with one half of the pons treated during each visit. The other two received one treatment targeting the entire pons. They all tolerated the procedure well. One of the patients noted a transient worsening of his diplopia after the treatment. No significant edema or hemorrhage were noted on postoperative scans obtained within 24 hrs of treatment. MRIs obtained 1- and 3-months after treatment have shown significant decrease in tumor burden in 2 of the patients. CONCLUSIONS: The first-in-human experience with a new therapeutic modality for DIPG patients demonstrates safety and efficacy. Future procedures will involve ascending drug and MRgFUS energy dose combinations. The use of focused ultrasound signals an innovative approach for challenging to treat pediatric brain tumors.

Helical tomotherapy craniospinal irradiation in primary brain tumours: Toxicities and outcomes in a peadiatric and adult population

ObjectiveAs craniospinal irradiation (CSI) is delivered more frequently by helical tomotherapy (HT) with few reports about late effects, we analysed all patients treated in our centre over an 11-year period. Methods and materialsOur study included all patients that underwent CSI by HT, between September 2009 and January 2020, in the Department of Radiation Oncology of the Toulouse Cancer Institute. Acute radiotherapy toxicities were reported and medium- to long-term outcomes analysed. ResultsAmong the 79 patients included, 70.9 % were younger than 18 years at diagnosis, the median age was 13 (range: 1–52) at the time of radiation therapy, 67.1 % of patients had medulloblastoma. Half of them (49.4 %) had a metastatic disease at diagnosis. The median dose of CSI was 36 Gy (range, 18–36). Seventy-seven patients received a radiation boost to the original location of the primary tumour (97.5 %), 32 patients also received a boost to their metastatic sites (40.5 %). Median follow-up was 55.5 months (95 %CI = [41.2; 71.8]). The 3-year event-free survival rate was 66.3 % (95 %CI = [54.2; 75.9]). Most patients presented with acute haematological toxicities during CSI (85.9 %), predominantly severe thrombocytopenia (39.7 %). Among the 64 patients assessed for medium- and long-term outcomes, 52 survived and 47 were alive and disease-free at the latest follow-up visit on record. There were 3.8 % secondary tumours: two meningiomas and one diffuse intrinsic pontine glioma. Adult and paediatric patients respectively presented with secondary cataract (4.3 % vs 22.0 %), persistent hearing disorders (26.1 % vs 29.3 %), pulmonary or cardiac late effects (4.3 % vs 2.4 %), hormonal pituitary gland deficiencies (30.0 % vs 56.8 %) and psycho-cognitive disorders (56.5 % vs 53.7 %). ConclusionCSI dispensed by HT, did not result in any additional acute or late toxicities when compared to 3D-CSI. There was no increase in the secondary tumour rate compared to that reported in the literature

A Phase 1 Study of Intravenous EGFR-ErbituxEDVsMIT in Children with Solid or CNS Tumours Expressing Epidermal Growth Factor Receptor.

Recurrent or refractory solid and central nervous system (CNS) tumours in paediatric patients have limited treatment options and carry a poor prognosis. The EnGeneIC Dream Vector (EDV) is a novel nanocell designed to deliver cytotoxic medication directly to the tumour. The epidermal growth factor receptor is expressed in several CNS and solid tumours and is the target for bispecific antibodies attached to the EDV. To assess the safety and tolerability of EGFR-Erbitux receptor EnGeneIC Dream Vector with mitoxantrone (EEDVsMit) in children with recurrent / refractory solid or CNS tumours expressing EGFR. Patients aged 2-21 years with relapsed or refractory CNS and solid tumours, or radiologically diagnosed diffuse intrinsic pontine glioma (DIPG), were treated in this phase I open-label study of single agent EEDVsMit. Thirty-seven patients' tumours were screened for EGFR expression. EEDVsMit was administered twice weekly in the first cycle and weekly thereafter. Standard dose escalation with a rolling 6 design was employed. Dosing commenced at 5 × 108 EEDVsMit per dose and escalated to 5×109 EEDVsMit per dose. EGFR expression was detected in 12 (32%) of the paediatric tumours tested. Nine patients were enrolled and treated on the trial, including three patients with diffuse midline glioma. Overall, EEDVsMit was well tolerated, with no dose-limiting toxicities observed. The most common drug-related adverse events were grade 1-2 fever, nausea and vomiting, rash, lymphopaenia, and mildly deranged liver function tests. All patients had disease progression, including one patient who achieved a mixed response as the best response. EGFR-Erbitux receptor targeted EnGeneIC Dream Vector with mitoxantrone can be safely delivered in paediatric patients aged 2-21 years with solid or CNS tumours harbouring EGFR expression. The discovery of EGFR expression in a high proportion of paediatric gliomas means that EGFR may be useful as a target for other treatment strategies. Targeted therapeutic-loaded EDVs may be worth exploring further for their role in stimulating an anti-tumour immune response. NCT02687386.

Abstract 7578: BRD4 protein degradation for p53 mutant diffuse intrinsic pontine glioma (DIPG) via various delivery approaches for ARV-825 by using camel milk exosomes

Abstract DIPGs (diffuse intrinsic pontine gliomas) are a particularly aggressive and malignant type of pediatric brain tumor. Given the tumor's location, surgery has a restricted role, and it is insensitive to most chemotherapies. Although fractionated radiation is the standard of therapy for DIPG, the cancer invariably worsens in 6-12 months. TP53 tumor suppressor protein mutations are found in 70-80% of DIPG tumors. These TP53 mutations have been linked to radiation resistance in DIPG patients. Our goal was to find new pharmacological compounds that would improve the radiation sensitivity of p53 mutant DIPG cell lines and to determine their molecular mechanism of action. Numerous bromodomain-containing protein 4 (BRD4) inhibitors have entered clinical trials in recent years, yielding promising outcomes in tumor treatment since BRD4 expression in DIPG is substantially higher than in neighboring normal brain tissue. So consequently, BRD4 is a target for DIPG treatment. ARV-825 is an emerging PROTAC (Proteolysis Targeting Chimera) compound which can cause rapid and persistent BRD4 protein degradation. However, delivering ARV-825 to pons is a major challenge owing to poor oral bioavailability of PROTACS. In this study, we used camel milk derived exosomes (CME) for delivery of ARV-825 to pons by both nasal and oral routes. To begin with, goat milk exosomes were also considered but CME was chosen because of its innate cytotoxicity against DIPG cell lines. CME were isolated using ultracentrifugation and nanoparticle tracking analysis and revealed a size distribution of 112.5 ± 2.8 nm, zeta potential of -26.38 ± 0.12 mV. CME (1*1011) were cytotoxic to both SF8628 (with a H3.3 K27M mutation) and SF188 (grade IV pediatric glioblastoma with wild type H3.3) cell lines and had 51% and 44% cell viability respectively. ARV-825 showed IC50 of 546.58 ± 22.14 nM and 608.94 ± 30.04 nM in SF8628 cells and SF188 cells respectively. ARV-825’s IC50 was lowered when ARV-825 loaded CME were used. ARV-825 was loaded using sonication and entrapment efficiency was found to be 42.96 ± 2.14 % by HPLC analysis. CME were labelled with Vybrant DiO (CME-DIO) and instilled into each nostril of Sprague Dawley rats daily. Brain sections were viewed with fluorescent microscope and CME-DIO were seen in olfactory region after 3 days and in the pons region after 5 days demonstrating the ability of CME to reach the target site. In-vitro permeability studies using MDCK cells showed approximately 9µg/mL (60%), 15µg/mL (80%) and 16µg/mL (100%) of ARV-825 permeation in 2, 4 and 24 hours respectively when loaded into CME. The results of the present study suggest the possibility of using CME as a viable delivery strategy for ARV-825 against DIPG. Further in vitro and in vivo studies are currently undergoing to validate the results and understand kinetics and the molecular mechanisms of ARV-825 loaded CME. Citation Format: Aakash Nathani, Mounika Aare, Li Sun, Yan Li, Mandip Singh. BRD4 protein degradation for p53 mutant diffuse intrinsic pontine glioma (DIPG) via various delivery approaches for ARV-825 by using camel milk exosomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7578.

Abstract 5108: Immunomodulatory and anti-tumor effect of radiation and induced telomere damage to treat pediatric high-grade gliomas

Abstract Pediatric high-grade gliomas (pHGGs), including diffuse intrinsic pontine glioma (DIPG) are among the most treatment refractory cancers in children. Despite multimodal therapy, prognosis is dismal with survival of <1 year for DIPG and a 5-year overall survival <10% for other pHGGs. Radiotherapy remains the only standard of care for DIPG that extends the survival by few months. In recent years, immunotherapy is emerging as a potential alternative treatment option. However, several factors have hindered the use of immunotherapy for pHGGs including “cold” immune environment with lack of immune cells infiltration, low tumor mutational burden, and lack of neo-antigen expression that prevents tumor recognition by the immune system. Therefore, novel therapies that activate the immune system while evading the tumor immunosuppression are highly needed. Here, we aim to demonstrate the immunomodulatory anti-tumor effects of THIO (6-thio-2’deoxyguanosine), a nucleoside analog and a telomerase-dependent telomere targeting agent, in DIPG mouse models. Our preclinical in vitro and in vivo data have demonstrated the efficacy of THIO in inducing telomeric damage, G2/M cell cycle arrest, and apoptosis in high-risk medulloblastoma and DIPG. Importantly, we have demonstrated that THIO crosses the blood-brain barrier and specifically targets tumor cells in an orthotopic mouse model of DIPG. THIO was shown to induce anti-tumor immunity through micronuclei formation leading to cGAS-STING pathway mediated activation of the innate and adaptive immune response in colon, lung, and hepatocellular carcinoma tumor models. Our preliminary data demonstrate the THIO-dependent induction of micronuclei formation co-localizing with cGAS and leading to STAT1 activation in DIPG cell lines. We have also shown that THIO synergistically sensitizes DIPG cells to ionizing radiation (IR), significantly decreasing cell proliferation. We are currently testing the combination of THIO+IR in an orthotopic mouse model for DIPG. We will present results on the potential of THIO and IR treatments to stimulate anti-tumor immunity through the activation of STING pathway in a syngeneic mouse model of DIPG. These preclinical studies will support the potential use of THIO and IR to treat children with high-risk pediatric brain tumors. Citation Format: Umaru Banlanjo, Shiva Senthil Kumar, Sergei Gryaznov, Rachid Drissi. Immunomodulatory and anti-tumor effect of radiation and induced telomere damage to treat pediatric high-grade gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5108.

Abstract 508: Determining the nuclear and cytoplasmic function of BMI-1 in diffuse intrinsic pontine glioma during M phase

Abstract Diffuse Intrinsic Pontine Glioma (DIPG) represents one of the most aggressive malignancies of the central nervous system, predominantly affecting children. DIPG is a particularly poor prognosis brain tumor with a median overall survival of less than 1 year. Hence, there is an urgent need to develop novel therapies that not only improve outcome but mitigate long-term complications in children with DIPG. The protein BMI-1, a critical component of the Polycomb Repressive Complex 1 (PRC1), is a proto-oncogene implicated in development, stemness of normal and malignant cells, and self-renewal. BMI-1 has emerged as a potential therapeutic target in DIPG; however, its role in DIPG tumorigenesis and progression remains elusive. We have previously shown that PTC596, a potent BMI-1 modulator, induces chromosome scattering, M phase cell-cycle arrest, BMI-1 phosphorylation and translocation to the cytoplasm leading to the inhibition of its PRC1 canonical function and cell death. These findings imply the induction of the spindle assembly checkpoint (SAC), thereby preventing mitotic exit followed by BMI-1 phosphorylation, probably at multiple sites, suggesting a survival role of BMI-1 in M phase in DIPG. The objective of the present study is to delineate the molecular pathways governing BMI-1 function during M phase and to establish the temporal sequence of these biochemical events related to BMI-1. Our data demonstrated that following PTC596 treatment, BMI-1 is actively transported from the nucleus to the cytoplasm. Furthermore, we observed colocalization of BMI-1 with COX4 in the mitochondria during M phase. Moreover, using Alphafold prediction model, BMI-1 could interact with CRM1 transporter. These findings suggest a potential PRC1-independent role of BMI-1 during early M phase required for DIPG proliferation. We are currently investigating potential pathways involved in BMI-1 nuclear-cytosol translocation through bioinformatic analyses, protein-protein interaction simulations, and pharmacological inhibitor assays. Collectively, these studies aim to elucidate the molecular mechanisms underlying BMI-1 functions in DIPG, thereby potentially paving the way for the development of targeted therapeutic strategies related to M phase progression. Citation Format: Hao-Han Pang, Banlanjo Umaru, Shiva Senthil Kumar, Rachid Drissi. Determining the nuclear and cytoplasmic function of BMI-1 in diffuse intrinsic pontine glioma during M phase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 508.

Abstract 6516: Single-cell protein activity inference reveals lymphocytic immune infiltration in diffuse intrinsic pontine glioma induced by focused ultrasound

Abstract Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor, located in the brainstem, and traditionally thought to be immune-excluded with no significant infiltration by cytotoxic T-cells, and therefore not amenable to immunotherapies. Focused ultrasound technology (FUS) for drug delivery is being investigated for DIPG in both preclinical and clinical trial settings. In this study, we sought to study the effects of FUS and radiation therapy (RT) on immune cell infiltration in DIPG tumors to assess their ability to alter the immune microenvironment and potentially enhance combination immunotherapies. Specifically, we generated single-cell RNAseq profiles for 27,780 cells from syngeneic orthotopic DIPG tumors (KPAP cell line containing H3K27M mutation, ATRX and P53 loss, and PDGFRA overexpression) after 14 days of tumor growth across the following treatment arms: vehicle control, FUS, and RT (n=2 each group). FUS was applied by 4-point sonication around the tumor with a pressure of 0.6 Mpa at days 8 and 11 post tumor implantation (PTI). RT was administered in fractions of 3 Gy daily from days 8 to 11 PTI (12Gy total). We leveraged a set of algorithms for network-based inference of regulatory protein activity to infer a set of context-matched gene regulatory networks from which protein activity can be inferred by expression of downstream targets. By this approach, we identified distinct clusters of immune cells segregating by cell type as inferred by singleR and activity of known lineage markers. Strikingly, we found that both RT and FUS significantly depleted a TGFb-expressing population of immune-suppressive macrophages as compared to vehicle controls. Importantly, FUS only additionally increased T-cell infiltration into the tumor micro-environment over three-fold as compared to vehicle controls, which was not observed in response to RT. Further protein validation is underway. This has significant implications for therapeutic combinations of FUS-mediated drug delivery with immune checkpoint inhibitors in DIPG, such that in addition to drug delivery, FUS-mediated immune modulatory effects may provide more proinflammatory response to enhance immunotherapy. Citation Format: Erin White, Hong-Jian Wei, Ester Calvo Fernández, Hanna Minns, Jovana Pavisic, Robyn Gartrell, Cheng-Chia Wu, Andrea Califano, Aleksandar Obradovic. Single-cell protein activity inference reveals lymphocytic immune infiltration in diffuse intrinsic pontine glioma induced by focused ultrasound [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6516.

Discovery of Conformationally Constrained ALK2 Inhibitors.

Despite decades of research on new diffuse intrinsic pontine glioma (DIPG) treatments, little or no progress has been made on improving patient outcomes. In this work, we explored novel scaffold modifications of M4K2009, a 3,5-diphenylpyridine ALK2 inhibitor previously reported by our group. Here we disclose the design, synthesis, and evaluation of a first-in-class set of 5- to 7-membered ether-linked and 7-membered amine-linked constrained inhibitors of ALK2. This rigidification strategy led us to the discovery of the ether-linked inhibitors M4K2308 and M4K2281 and the amine-linked inhibitors M4K2304 and M4K2306, each with superior potency against ALK2. Notably, M4K2304 and M4K2306 exhibit exceptional selectivity for ALK2 over ALK5, surpassing the reference compound. Preliminary studies on their in vivo pharmacokinetics, including blood-brain barrier penetration, revealed that these constrained scaffolds have favorable exposure and do open a novel chemical space for further optimization and future evaluation in orthotopic models of DIPG.

Adjuvant Wilms’ tumour 1-specific dendritic cell immunotherapy complementing conventional therapy for paediatric patients with high-grade glioma and diffuse intrinsic pontine glioma: protocol of a monocentric phase I/II clinical trial in Belgium

IntroductionDiffuse intrinsic pontine glioma (DIPG) and paediatric high-grade glioma (pHGG) are aggressive glial tumours, for which conventional treatment modalities fall short. Dendritic cell (DC)-based immunotherapy is being investigated as a...

Just a spoonful of metformin helps the medicine go down.

Diffuse intrinsic pontine glioma (DIPG) is a devastating brain tumor with a need for novel therapies. So far, monotherapies have failed to prolong survival for these patients, and combinatorial strategies have often shown severe, dose-limiting toxicities. In this issue of the JCI, Duchatel, Jackson, and colleagues address this challenge by introducing a drug combination that mitigates side effects and overcomes resistance. After identifying the PI3K/mTOR pathway as a therapeutic vulnerability, they treated DIPG-bearing mice with paxalisib and saw responses but also observed hyperglycemia as a severe side effect. Combining paxalisib with metformin mitigated this toxicity, but also upregulated protein kinase C (PKC) signaling. To tackle this mechanism of resistance, the authors added the PKC inhibitor enzastaurin to their drug combination and showed that this triple therapy led to improved survival. This approach paves the way for improved outcomes for patients with DIPG and other brain tumors.

Bone Morphogenic Proteins in Pediatric Diffuse Midline Gliomas: How to Make New Out of Old?

The BMP pathway is one of the major signaling pathways in embryonic development, ontogeny and homeostasis, identified many years ago by pioneers in developmental biology. Evidence of the deregulation of its activity has also emerged in many cancers, with complex and sometimes opposing effects. Recently, its role has been suspected in Diffuse Midline Gliomas (DMG), among which Diffuse Intrinsic Pontine Gliomas (DIPG) are one of the most complex challenges in pediatric oncology. Genomic sequencing has led to understanding part of their molecular etiology, with the identification of histone H3 mutations in a large proportion of patients. The epigenetic remodeling associated with these genetic alterations has also been precisely described, creating a permissive context for oncogenic transcriptional program activation. This review aims to describe the new findings about the involvement of BMP pathway activation in these tumors, placing their appearance in a developmental context. Targeting the oncogenic synergy resulting from this pathway activation in an H3K27M context could offer new therapeutic perspectives based on targeting treatment-resistant cell states.

Nimotuzumab-vinorelbine combination therapy versus other regimens in the treatment of pediatric diffuse intrinsic pontine glioma.

Pediatric diffuse intrinsic pontine glioma (DIPG) is a fatal disease associated with a median survival of < 1year despite aggressive treatments. This retrospective study analyzed the treatment outcomes of patients aged < 18years who were diagnosed with DIPG between 2012 and 2022 and who received different chemotherapy regimens. After radiotherapy, patients with DIPG received nimotuzumab-vinorelbine combination or temozolomide-containing therapy. When nimotuzumab was unavailable, it was replaced by vincristine, etoposide, and carboplatin/cyclophosphamide (VECC). Temozolomide was administered as a single agent or a part of the combination chemotherapy comprising temozolomide, irinotecan, and bevacizumab. Furthermore, 1- and 3-year overall survival (OS), progression-free survival (PFS), and median OS and PFS were analyzed. The median age of 40 patients with DIPG was 97 ± 46.93 (23-213) months; the median follow-up time was 12months. One and 3-year OS were 35.0% and 7.5%, respectively. Median OS was 12months in all patients (n = 40), and it was 16, 10, and 11months in those who received first-line nimotuzumab-vinorelbine combination (n = 13), temozolomide-based (n = 14), and VECC (n = 6) chemotherapy regimens, respectively (p = 0.360). One patient who received gefitinib survived for 16months. Conversely, patients who never received radiotherapy and any antineoplastic medicamentous therapy (n = 6) had a median OS of 4months. Nimotuzumab-vinorelbine combination therapy prolonged OS by 6months compared with temozolomide-containing chemotherapy, although the difference was not statistically significant.

ACTION: a randomized phase 3 study of ONC201 (dordaviprone) in patients with newly diagnosed H3 K27M-mutant diffuse glioma.

H3 K27M-mutant diffuse glioma primarily affects children and young adults, is associated with a poor prognosis, and no effective systemic therapy is currently available. ONC201 (dordaviprone) has previously demonstrated efficacy in patients with recurrent disease. This phase 3 trial evaluates ONC201 in patients with newly diagnosed H3 K27M-mutant glioma. ACTION (NCT05580562) is a randomized, double-blind, placebo-controlled, parallel-group, international phase 3 study of ONC201 in newly diagnosed H3 K27M-mutant diffuse glioma. Patients who have completed standard frontline radiotherapy are randomized 1:1:1 to receive placebo, once-weekly dordaviprone, or twice-weekly dordaviprone on 2 consecutive days. Primary efficacy endpoints are overall survival (OS) and progression-free survival (PFS); PFS is assessed by response assessment in neuro-oncology high-grade glioma criteria (RANO-HGG) by blind independent central review. Secondary objectives include safety, additional efficacy endpoints, clinical benefit, and quality of life. Eligible patients have histologically confirmed H3 K27M-mutant diffuse glioma, a Karnofsky/Lansky performance status ≥70, and completed first-line radiotherapy. Eligibility is not restricted by age; however, patients must be ≥10 kg at time of randomization. Patients with a primary spinal tumor, diffuse intrinsic pontine glioma, leptomeningeal disease, or cerebrospinal fluid dissemination are not eligible. ACTION is currently enrolling in multiple international sites.

Abstract PR-007: H3.3K27M diffuse midline gliomas are sensitive to SWI/SNF chromatin remodeler degradation

Abstract Diffuse Midline Gliomas (DMG) including Diffuse Intrinsic Pontine Glioma (DIPG) is a lethal pediatric brain tumor. The lysine 27 (K27)-to-methionine (M) point mutation in histone H3.1 or H3.3 (H3.1K27M or H3.3K27M, respectively) affects about 80% of patients establishing H3K27M mutation as a tumor hallmark. Mutated H3 histones inhibit the function of Polycomb repressive complex 2 resulting in global reduction of repressive H3K27me3. In addition, increased acetylation of H3K27 is observed, further contributing to an aberrant chromatin state. Considering the impact of epigenetic alterations in DIPG, we investigated the role of Switch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodelers in shaping the oncogenic chromatin state in DIPG cells. DIPG cells harboring H3.3K27M mutation were sensitive to combined SMARCA4/SMARCA2/PBRM1 degradation by the proteolysis targeting chimera (PROTAC) AU-15330 degrader. Our premise is based on the SWI/SNF complex altering chromatin states of tumor cells to regulate the tumor immune microenvironment. We hypothesized that the SWI/SNF complex in tumor cells modulates immune responses in DIPG. Cytokine profiler arrays in AU-15330 versus vehicle treated H3.3K27M cells showed a marked decrease of Intercellular Cell Adhesion Molecule-1 (ICAM-1) glycoprotein levels. In addition to its well-known role in leukocyte endothelial transmigration, ICAM-1 contributes to malignant potential of tumor cells. We validated downregulation of ICAM-1 mRNA and protein levels by AU-15330 by immunoblotting, proteomics, and transcriptomics, suggesting an epigenetic regulation of ICAM-1 gene transcription by the SWI/SNF complex. Moreover, silencing of mutant H3.3-encoding gene decreased ICAM-1 protein levels, suggesting ICAM-1 expression is promoted by H3.3K27M mutation-induced SWI/SNF chromatin remodeling in DIPG. In conclusion, our work suggests the H3.3K27M mutation renders DIPG cells dependent on chromatin remodeling by SWI/SNF complexes that can in turn alters the expression of immune system-associated components, such as ICAM-1. Our future work is geared to understanding the role of the SWI/SNF complex in regulating the immune tumor microenvironment in H3K27M DMGs. Citation Format: Mateus Mota, Stefan Sweha, Matt Pun, Siva Kumar Natarajan, Yujie Ding, Chan Chung, Debra Hawes, Fusheng Yang, Alexander Judkins, Susanta Samajdar, Xuhong Cao, Lanbo Xiao, Abhijit Parolia, Arul Chinnaiyan, Sriram Venneti. H3.3K27M diffuse midline gliomas are sensitive to SWI/SNF chromatin remodeler degradation [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr PR-007.