H3 K27M-mutant Diffuse Midline Glioma Research Articles

Integrated analyses reveal two molecularly and clinically distinct subtypes of H3 K27M-mutant diffuse midline gliomas with prognostic significance

H3 K27M-altered diffuse midline gliomas (DMGs) are highly malignant tumours that arise in the midline structures of the CNS. Most DMGs carry an H3 K27M-mutation in one of the genes encoding for histone H3. Recent studies suggested that epigenetic subgroups of DMGs can be distinguished based on alterations in the MAPK-signalling pathway, tumour localisation, mutant H3-gene, or overall survival (OS). However, as these parameters were studied individually, it is unclear how they collectively influence survival. Hence, we analysed dependencies between different parameters, to define novel epigenetic, clinically meaningful subgroups of DMGs. We collected a multifaceted cohort of 149 H3 K27M-mutant DMGs, also incorporating data of published cases. DMGs were included in the study if they could be clearly allocated to the spinal cord (n = 31; one patient with an additional sellar tumour), medulla (n = 20), pons (n = 64) or thalamus (n = 33), irrespective of further known characteristics. We then performed global genome-wide DNA methylation profiling and, for a subset, DNA sequencing and survival analyses. Unsupervised hierarchical clustering of DNA methylation data indicated two clusters of DMGs, i.e. subtypes DMG-A and DMG-B. These subtypes differed in mutational spectrum, tumour localisation, age at diagnosis and overall survival. DMG-A was enriched for DMGs with MAPK-mutations, medullary localisation and adult age. 13% of DMG-A had a methylated MGMT promoter. Contrarily, DMG-B was enriched for cases with TP53-mutations, PDGFRA-amplifications, pontine localisation and paediatric patients. In univariate analyses, the features enriched in DMG-B were associated with a poorer survival. However, all significant parameters tested were dependent on the cluster attribution, which had the largest effect on survival: DMG-A had a significantly better survival compared to DMG-B (p < 0.001). Hence, the subtype attribution based on two methylation clusters can be used to predict survival as it integrates different molecular and clinical parameters.

H3K27M diffuse midline glioma is homologous recombination defective and sensitized to radiotherapy and NK cell-mediated antitumor immunity by PARP inhibition.

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. 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. 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. 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. 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. 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.

Sequential intravenous and intracerebroventricular GD2-CAR T-cell therapy for H3K27M-mutated diffuse midline gliomas.

H3K27M-mutant diffuse midline gliomas (DMGs) express high levels of the GD2 disialoganglioside and chimeric antigen receptor modified T-cells targeting GD2 (GD2-CART) eradicate DMGs in preclinical models. Arm A of the Phase I trial NCT04196413 administered one IV dose of autologous GD2-CART to patients with H3K27M-mutant pontine (DIPG) or spinal (sDMG) diffuse midline glioma at two dose levels (DL1=1e6/kg; DL2=3e6/kg) following lymphodepleting (LD) chemotherapy. Patients with clinical or imaging benefit were eligible for subsequent intracerebroventricular (ICV) GD2-CART infusions (10-30e6 GD2-CART). Primary objectives were manufacturing feasibility, tolerability, and identification of a maximally tolerated dose of IV GD2-CART. Secondary objectives included preliminary assessments of benefit. Thirteen patients enrolled and 11 received IV GD2-CART on study [n=3 DL1(3 DIPG); n=8 DL2(6 DIPG/2 sDMG). GD2-CART manufacturing was successful for all patients. No dose-limiting toxicities (DLTs) occurred on DL1, but three patients experienced DLT on DL2 due to grade 4 cytokine release syndrome (CRS). Nine patients received ICV infusions, which were not associated with DLTs. All patients exhibited tumor inflammation-associated neurotoxicity (TIAN). Four patients demonstrated major volumetric tumor reductions (52%, 54%, 91% and 100%). One patient exhibited a complete response ongoing for >30 months since enrollment. Eight patients demonstrated neurological benefit based upon a protocol-directed Clinical Improvement Score. Sequential IV followed by ICV GD2-CART induced tumor regressions and neurological improvements in patients with DIPG and sDMG. DL1 was established as the maximally tolerated IV GD2-CART dose. Neurotoxicity was safely managed with intensive monitoring and close adherence to a management algorithm.

PATH-03. FOXR2-OVEREXPRESSING CENTRAL NERVOUS SYSTEM (CNS) TUMORS EXHIBIT SUBSTANTIAL DIVERSITY ACROSS HISTOLOGICAL, MOLECULAR, AND CLINICAL DIMENSIONS

Abstract BACKGROUND FOXR2 activation is regarded as the pathognomonic molecular signature of CNS neuroblastoma. However, a comprehensive understanding of the landscape for CNS tumors exhibiting FOXR2 activation is lacking. METHODS Integrated histopathologic, molecular, and clinical data analysis of 36 FOXR2-overexpressing CNS tumors identified through screening of an institutional dataset. RESULTS FOXR2-overexpressing CNS tumors showed a broad anatomic distribution throughout the neuraxis. Of the 36 tumors, 17 (47.2%) were gliomas and 17 (47.2%) were embryonal tumors. The gliomas included 16 high-grade gliomas (eight histone H3 K27M-mutant diffuse midline gliomas, seven radiation-associated tumors, and one with MN1::PATZ1 fusion) and one pilocytic astrocytoma. The embryonal tumors included ten CNS neuroblastomas, six pineoblastomas, and one medulloblastoma. Two were difficult-to-classify high-grade neuroepithelial tumors. The gliomas and embryonal tumors demonstrated comparable FOXR2 expression based on normalized RNA-sequencing data. FOXR2-overexpressing gliomas, but not embryonal tumors, displayed diverse concomitant genetic alterations. The most common mechanisms of FOXR2 activation involved structural alterations leading to proximity effects, frequently incorporating non-canonical non-coding exons, followed by activation of the exon -3 promoter. Tumor location, histology, and concomitant alterations correlated with patient outcomes. While FOXR2-overexpressing gliomas were aggressive with a 5-year overall survival of 13.3%, FOXR2-overexpressing embryonal tumors showed a diverse prognosis. FOXR2-activated CNS neuroblastoma showed a 5-year overall survival of 100%. Pineoblastomas with FOXR2 activation in the PB-FOXR2 DNA methylation group showed a 3-year survival of 0% despite multimodal therapy. The one medulloblastoma patient in the cohort is alive at three years post-diagnosis. CONCLUSION FOXR2-overexpressing CNS tumors manifest significant histological, molecular, and clinical diversity and can occur as post-radiation tumors. Glioma histology and concomitant genetic alterations correlate with treatment resistance and inferior prognosis, while FOXR2-overexpressing embryonal tumors show more heterogeneous outcomes. Integrating histologic and molecular diagnostic approaches along with therapeutic interventions and clinical outcomes is imperative for accurate prognostication and optimal therapeutic decision-making.

LGG-17. NATURAL HISTORY AND MANAGEMENT OF INCIDENTALLY DISCOVERED BRAIN LESIONS SUSPECT FOR NEOPLASM IN CHILDREN

Abstract BACKGROUND The rise in neuroimaging has resulted in an increase of incidentally discovered brain lesions suspect for a neoplasm (‘incidentaloma’) in children. With the experience in our national hospital, we aim to diversify and broaden the knowledge on the management and natural history of pediatric incidentaloma. METHODS A retrospective analysis of all incidentaloma patients in the Princess Maxima Center for Pediatric Oncology from June 2018 until March 2023. RESULTS Our research population consists of 23 patients (10M/13F) with an incidentaloma. The lesions were mostly located in the cerebellum (21.7%), thalamus and ventricle (both 17%) and cerebral hemisphere (13%) The average age at diagnosis was 7.9 years (range 0-15 years) and the mean duration of follow-up was 4.6 years (range 0-11 years). The initial management was clinical and radiological follow-up in seventeen (74%), resection in five (22%) and biopsy in one patient (4%). Pathological examination performed in the six neurosurgical patients revealed low grade gliomas (LGGs) in three patients, medulloblastoma in one and plexus papilloma in one patient respectively. The pathological analysis of the patient with biopsy only was indecisive. One patient became symptomatic with clear tumor growth on imaging after 6.8 years of follow-up. Biopsy showed a H3K27M mutant diffuse midline glioma. She died 8.1 years after initial diagnosis. In 11 other patients radiographic changes (mostly tumor progression) were observed with a median of 23 months after diagnosis (range 3-70 months). Two of these underwent complete resection of an LGG. CONCLUSIONS For most incidentalomas, clinical and radiological follow-up is sufficient. As late disease progression can occur as seen in our patient who became symptomatic after 7 years, this follow-up should be long-term. Future studies should focus on identifying (radiological) prognostic factors for tumor progression, so follow-up of incidentaloma can be more personalized.

IMMU-18. ENHANCING GD2.CAR T CELL THERAPY FOR DIFFUSE MIDLINE GLIOMA WITH LSD1 AND HDAC INHIBITORS

Abstract BACKGROUND Chimeric antigen receptor T cells against the glycolipid GD2 (GD2.CARTs) have shown promise against Diffuse Midline Glioma (DMG) in clinical trials but are not yet curative. Previous studies in neuroblastoma demonstrate that histone deacetylase inhibitor (HDACi) treatment can increase GD2 on tumor cells and improve targeting by GD2.CARTs. Our lab previously found that simultaneous HDACi and lysine-demethylase 1 inhibitor (LSD1i) treatment in DMG cells causes synergistic alterations in gene expression and reduces DMG growth. Here, we find that treating GD2-low DMG cell lines with combinations of LSD1i and HDACi improves DMG response to GD2.CARTs. METHODS Surface expression of GD2 before and after LSD1i/HDACi treatment were analyzed using immunofluorescence microscopy and flow cytometry. The cytotoxicity of GD2.CARTs against DMG cells was determined from co-culture experiments and in patient-derived orthotopic xenograft models using flow cytometry and bioluminescent imaging. RESULTS Since low target antigen expression is one mechanism by which tumor cells can escape CAR T cell therapy, we profiled GD2 expression on 9 different H3K27M-mutant DMG cell lines. This analysis shows that only 5 of the 9 cell lines exhibit high GD2 expression while the remaining 4 lines have low GD2 expression. When treating these cell lines with GD2.CARTs, we find that low GD2 expression on DMG cells correlates with tumor cell escape and resistance to GD2.CARTs. Treating GD2-low DMG cells with LSD1i+HDACi increases GD2 expression and enhances tumor cell targeting by GD2.CARTs in subsequent co-culture assays. Further studies in mice bearing GD2-low DMG tumors were initially GD2.CART-insensitive but showed reduced tumor growth when pre-treated with LSD1i+HDACi before intravenous GD2.CART infusion. CONCLUSIONS These studies suggest that variable GD2 expression is a major driver of DMG resistance to GD2.CARTs and that LSD1i+HDACi pre-treatment may improve DMG patient responses to GD2.CART therapy by increasing target antigen expression.

DIPG-47. SEQUENTIAL INTRAVENOUS AND INTRACEREBROVENTRICULAR GD2-CAR T-CELL THERAPY FOR H3K27M-MUTATED DIFFUSE MIDLINE GLIOMAS

Abstract BACKGROUND H3K27M-mutant diffuse midline gliomas (DMGs) express uniformly high levels of the GD2 disialoganglioside. In preclinical models, chimeric antigen receptor modified T-cells (CAR T-cells) targeting GD2 robustly regressed orthotopically xenografted DMGs. METHODS This Phase I trial (NCT04196413) administered one IV dose of autologous T-cells transduced with a GD2-CAR retroviral vector to patients with H3K27M-mutant pontine (DIPG) or spinal (sDMG) diffuse midline gliomas at two dose levels (DL1=1e6 GD2-CAR T-cells/kg; DL2=3e6 GD2-CAR T-cells/kg) following standard lymphodepleting (LD) chemotherapy. Patients with clinical or imaging benefit following IV infusion were eligible for subsequent intracerebroventricular (ICV) GD2-CAR T-cell infusions (10-30e6 GD2-CAR T-cells). Primary objectives were to determine feasibility of manufacturing, assess tolerability of IV GD2-CAR T-cells in patients with DIPG and sDMG, and identify a maximally tolerated dose of IV GD2-CAR T-cells following lymphodepleting chemotherapy. Secondary objectives included preliminary assessments of benefit. Here we report the final results of Arm A. RESULTS Thirteen patients enrolled and 11 received one IV GD2-CAR T infusion on study [n=3 DL1(3 DIPG); n=8 DL2(6 DIPG/2 sDMG). GD2-CAR T-cells were successfully manufactured for each patient. After IV infusion, no dose-limiting toxicities (DLTs) occurred on DL1, but three patients experienced DLT on DL2 due to grade 4 cytokine release syndrome (CRS). Nine patients received ICV infusions, which were not associated with DLTs. All patients exhibited tumor inflammation-associated neurotoxicity (TIAN). Four patients demonstrated major volumetric reductions of 52%, 54%, 91% and 100%. One patient exhibited a complete response durable for &amp;gt;30 months since therapy began. Eight patients demonstrated neurological benefit based upon a protocol-directed Clinical Improvement Score. CONCLUSIONS Sequential IV followed by ICV GD2-CAR T-cells infusions induced tumor regressions and neurological improvements. DL1 was established as the maximally tolerated IV GD2-CAR T-cell dose. Neurotoxicity was safely managed with intensive monitoring and close adherence to a management algorithm.

Rapid tumor DNA analysis of cerebrospinal fluid accelerates treatment of central nervous system lymphoma

AbstractDelays and risks associated with neurosurgical biopsies preclude timely diagnosis and treatment of central nervous system (CNS) lymphoma and other CNS neoplasms. We prospectively integrated targeted rapid genotyping of cerebrospinal fluid (CSF) into the evaluation of 70 patients with CNS lesions of unknown cause. Participants underwent genotyping of CSF-derived DNA using a quantitative polymerase chain reaction–based approach for parallel detection of single-nucleotide variants in the MYD88, TERT promoter, IDH1, IDH2, BRAF, and H3F3A genes within 80 minutes of sample acquisition. Canonical mutations were detected in 42% of patients with neoplasms, including cases of primary and secondary CNS lymphoma, glioblastoma, IDH-mutant brainstem glioma, and H3K27M-mutant diffuse midline glioma. Genotyping results eliminated the need for surgical biopsies in 7 of 33 cases (21.2%) of newly diagnosed neoplasms, resulting in significantly accelerated initiation of disease-directed treatment (median, 3 vs 12 days; P = .027). This assay was then implemented in a Clinical Laboratory Improvement Amendments environment, with 2-day median turnaround for diagnosis of CNS lymphoma from 66 patients across 4 clinical sites. Our study prospectively demonstrates that targeted rapid CSF genotyping influences oncologic management for suspected CNS tumors.

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.

Demonstrated efficacy and mechanisms of sensitivity of ONC201: H3K27M-mutant diffuse midline glioma in the spotlight.

Demonstrated efficacy and mechanisms of sensitivity of ONC201: H3K27M-mutant diffuse midline glioma in the spotlight.

Abstract A001: Silencing of EYA4 regulates stemness in H3K27M-mutant diffuse midline glioma

Abstract A001: Silencing of EYA4 regulates stemness in H3K27M-mutant diffuse midline glioma

Single-cell RNA sequencing of anaplastic ependymoma and H3K27M-mutant diffuse midline glioma.

Anaplastic ependymoma and H3K27M-mutant diffuse midline glioma are two common subtypes of brain tumors with poor long-term prognosis. The present study analyzed and compared the differences in cell types between two tumors by single-cell RNA sequencing (scRNA-seq) technology. ScRNA-seq was performed to profile cells from cancer tissue from anaplastic ependymoma patient and H3K27M-mutant diffuse midline glioma patient. Cell clustering, marker gene identification, cell type annotation, copy number variation analysis and function analysis of differentially expressed genes were then performed. A total of 11,219 cells were obtained from anaplastic ependymoma and H3K27M mutant diffuse midline glioma, and these cells categorized into 12 distinct clusters. Each cell cluster could be characterized with specific cell markers to indicate cellular heterogeneity. Five cell types were annotated in each sample, including astrocyte, oligodendrocytes, microglial cell, neural progenitor cell and immune cell. The cluster types and proportion of cell types were not consistent between the two brain tumors. Functional analyses suggest that these cell clusters are involved in tumor-associated pathways, with slight differences in the cells of origin between the two tumors. In addition, cell communication analysis showed that the NRG3-ERBB4 pair is a key Ligand-receptor pair for anaplastic ependymoma, while in H3K27M-mutant diffuse midline glioma it is the PTN-PTPRZ1 pair that establishes contact with other cells. There was intratumor heterogeneity in anaplastic ependymoma and H3K27M mutant diffuse midline glioma, and that the subtype differences may be due to differences in the origin of the cells.

TMET-02. CLINICAL EFFICACY OF ONC201 IN H3K27M-MUTANT DIFFUSE MIDLINE GLIOMAS IS DRIVEN BY DISRUPTION OF INTEGRATED METABOLIC AND EPIGENETIC PATHWAYS

Abstract H3K27M-mutant diffuse midline glioma (DMG) patients have no proven effective therapies beyond radiation. ONC201, a mitochondrial protease ClpP agonist, has recently demonstrated efficacy in these patients, but the mechanism behind this remains unknown. We assessed clinical outcomes, tumor sequencing, and tissue samples from patients treated in two completed multi-site clinical studies (n=71). Patients treated with ONC201 monotherapy following initial radiation but prior to recurrence (n=35) demonstrated a median overall survival of 21.7 months and a median progression-free survival of 12.2 months. Radiographic response was associated with increased expression of key tricarboxylic acid cycle-related genes in baseline tumor sequencing. Similar survival benefits were observed using in utero electroporation and orthotopic mouse models of H3K27M-DMG (median 107 versus 77 days [p=0.02] and median 97 versus 141 days [p=0.004], respectively). Integrated transcriptomic and metabolomic analysis of ONC201-treated H3K27M-DMG cells revealed downregulation of TCA cycle, glycolysis, and pyruvate metabolism genes, most notably oxoglutarate dehydrogenase (OGDH), and concomitant changes in levels of associated metabolites including ɑ-ketoglutarate (ɑ-KG). Interestingly, L-2-hydroxyglutarate (L-2HG), a known inhibitor of the Jumonji C domain family of histone lysine demethylases, was also increased. This corresponded with increases in repressive H3K27me3 in vitro and in human tumors accompanied by epigenetic downregulation of cell cycle regulation and neuro-glial differentiation genes. Genetic knockdown of OGDH recapitulated ONC201-mediated increases in H3K27me3. In contrast, genetic knockdown of ClpP or lactate dehydrogenase A or overexpression of L-2-hydroxyglutarate dehydrogenase (L-2HGDH), but not D-2HGDH, abrogated ONC201-mediated increases in H3K27me3, demonstrating that ONC201 induces the production of L-2HG in H3K27M-DMG cells leading to increased H3K27me3. Overall, our data demonstrates the efficacy of ONC201 in H3K27M-mutant DMG and supports ONC201 as the first monotherapy to improve outcomes in patients with H3K27M-mutant DMG for whom few therapeutic options currently exist. Mechanistically, ONC201 disrupts integrated metabolic and epigenetic pathways and reverses pathognomonic H3K27me3 reduction.

P10.11.A CHARACTERISTICS OF DIFFUSE MIDLINE GLIOMA WITH H3 K27M MUTATION IN ADULTS

Abstract BACKGROUND H3 K27M-mutant diffuse midline gliomas (DMG) occur primarily in children but can also be encountered in adults. Adult H3 K27M-mutant DMG is a still challenging disease with no effective medical therapies. MATERIAL AND METHODS We retrospectively analyzed the characteristics and prognostic factors of 49 adult patients with intracranial midline gliomas treated in Kobe University Hospital from 2006 to 2022. RESULTS The median age at diagnosis in midline gliomas was 50.8 years (range: 17-83 y). All tumors were located in thalamus n=25, brainstem n=10, cerebellum n=14. Isocitrate dehydrogenase 1 (IDH1) and H3 K27M mutation was recognized in 6 and 16 patients, respectively, which was mutually exclusive. In all midline gliomas, the identification of H3 K27M mutation was significantly a poor prognosis factor (median 18.5 months vs 34.2 months, p=0.041). Among IDH1 wild-type midline gliomas, however, H3 K27M-mutant DMG had no worse overall survival (median 18.8 months vs 22.2 months, p=0.805). CONCLUSION H3 K27M-mutant DMG is considered as a distinct WHO Grade 4 regardless of histological features, but the biological properties of H3 K27M mutation is not yet defined in IDH1 wild-type midline gliomas in adults.

Histone H3 K27M-mediated regulation of cancer cell stemness and differentiation in diffuse midline glioma

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.

Clinical Efficacy of ONC201 in H3K27M-Mutant Diffuse Midline Gliomas Is Driven by Disruption of Integrated Metabolic and Epigenetic Pathways.

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.

A comprehensive genomic study of 390 H3F3A-mutant pediatric and adult diffuse high-grade gliomas, CNS WHO grade 4.

Malignant brain tumors, known as H3K27-altered diffuse midline glioma (DMG) and H3G34-mutant diffuse hemispheric glioma (DHG), can affect individuals of all ages and are classified as CNS WHO grade 4. We comprehensively characterized 390 H3F3A-mutant diffuse gliomas (201 females, 189 males) arising in pediatric patients (under 20years old) and adults (20years and older) evaluated by the CGP program at Foundation Medicine between 2013 and 2020. We assessed information from pathology reports, histopathology review, and clinical data. The cohort included 304 H3K27M-mutant DMG (156 females, 148 males) and 86 H3G34-mutant DHG (45 females, 41 males). Median patient age was 20years (1-74years). The frequency of H3K27M-mutant DMG was similar in both pediatric and adult patients in our cohort-48.6% of the patients were over 20years old, 31.5% over 30, and 18% over 40 at initial diagnosis. FGFR1 hotspot point mutations (N546K and K656E) were exclusively identified in H3K27M-mutant DMG tumors (64/304, 21%; p = 0.0001); these tend tooccur in older patients (median age: 32.5years) and mainly arose in the diencephalon. H3K27M-mutant DMG had higher rates of mutations in NF1 (31.0 vs 8.1%; p = 0.0001) and PIK3CA/PIK3R1 (27.9% vs 15.1%; p = 0.016) compared to H3G34-mutant DHG. However, H3G34-mutant DHG had higher rates of targetable alterations in cell-cycle pathway genes (CDK4 and CDK6 amplification; CDKN2A/B deletion) (27.0 vs 9.0%). Potentially targetable PDGFRA alterations were identified in ~ 20% of both H3G34-mutant DHG and H3K27M-mutant DMG. Overall, in the present study H3K27M-mutant DMG occurred at similar rates in both adult and patient patients. Through our analysis, we were able to identify molecular features characteristic of DMG and DHG. By identifying the recurrent co-mutations including actionable FGFR1 point mutations found in nearly one-third of H3K27M-mutant DMG in young adults, our findings can inform clinical translational studies, patient diagnosis, and clinical trial design.

A validated prognostic nomogram for patients with H3 K27M-mutant diffuse midline glioma

H3 K27M-mutant diffuse midline glioma (H3 K27M-mt DMG) is a rare, highly invasive tumor with a poor prognosis. The prognostic factors of H3 K27M-mt DMG have not been fully identified, and there is no clinical prediction model for it. This study aimed to develop and validate a prognostic model for predicting the probability of survival in patients with H3 K27M-mt DMG. Patients diagnosed with H3 K27M-mt DMG in the West China Hospital from January 2016 to August 2021 were included. Cox proportional hazard regression was used for survival assessment, with adjustment for known prognostic factors. The final model was established using the patient data of our center as the training cohort and data from other centers for external independent verification. One hundred and five patients were ultimately included in the training cohort, and 43 cases from another institution were used as the validation cohort. The factors influencing survival probability in the prediction model included age, preoperative KPS score, radiotherapy and Ki-67 expression level. The adjusted consistency indices of the Cox regression model in internal bootstrap validation at 6, 12, and 18 months were 0.776, 0.766, and 0.764, respectively. The calibration chart showed high consistency between the predicted and observed results. The discrimination in external verification was 0.785, and the calibration curve showed good calibration ability. We identified the risk factors that affect the prognosis of H3 K27M-mt DMG patients and then established and validated a diagnostic model for predicting the survival probability of these patients.

Differences in survival prognosticators between children and adults with H3K27M-mutant diffuse midline glioma.

H3K27M-mutant diffuse midline glioma (DMG) is a rare and aggressive central nervous system tumor. The biological behavior, clinicopathological characteristics, and prognostic factors of DMG have not yet been completely uncovered, especially in adult patients. This study aims to investigate the clinicopathological characteristics and identify prognostic factors of H3K27M-mutant DMG in pediatric and adult patients, respectively. A total of 171 patients with H3K27M-mutant DMG were included in the study. The clinicopathological characteristics of the patients were analyzed and stratified based on age. The Cox proportional hazard model was used to determine the independent prognostic factors in pediatric and adult subgroups. The median overall survival (OS) for the entire cohort was 9.0 months. Significant differences were found in some clinicopathological characteristics between children and adults. The median OS was also significantly different between the pediatric and adult subgroups, with 7.1 months for children and 12.3 months for adults (p < 0.001). In the overall population, the multivariate analysis identified adult patients, single lesion, concurrent chemoradiotherapy/radiotherapy, and intact ATRX expression as independent favorable prognostic factors. In the age-stratified subgroups, the prognostic factors varied between children and adults, with intact ATRX expression and single lesion being independent favorable prognostic factors in adults, while infratentorial localization was significantly associated with worse prognosis in children. The differences in clinicopathological features and prognostic factors between pediatric and adult patients with H3K27M-mutant DMG suggest the need for further clinical and molecular stratification based on age.