Mutant Diffuse Midline Glioma Research Articles

DIPG-55. DISSECTING THE ECOSYSTEM OF PEDIATRIC DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Pediatric gliomas are heterogeneous tumors encompassing high grade (pHGG) and low grade (pLGG) subtypes. For tumors not amenable to surgery, in particular high grade H3K27 mutant diffuse midline glioma (DMG), no curative options exist. DMG is characterized by 1) extensive heterogeneity of tumor cells that can exist in different interchangeable cellular states mimicking normal development and 2) extensive heterogeneity of the tumor microenvironment (TME). A better understanding of the DMG ecosystem is needed for therapeutic target identification. METHODS Publicly available single cell RNA seq data of DMG (n=17, Jessa et al. Nat. Genet. 2022), high grade pediatric glioblastoma (GBM, n=16, scPCA) and low grade tumors (ganglioglioma, n=5; pilocytic astrocytoma, n=14, scPCA) were analyzed to define key molecular characteristics and differences between these pediatric brain tumors. Doublet cells were removed from each dataset and tumor and TME cells were discriminated based on marker gene expression and inferred CNVs. RESULTS In DMG, glioma cellular states and TME heterogeneity differed depending on the tumor location. DMG arising in the pons harbored a bigger fraction of astrocyte-like glioma cells whereas oligodendrocyte-like cells were more abundant in thalamic tumors. In line with this observation, Liana cellular communication analysis inferred more intratumoral (autocrine) signaling involving oligodendrocyte-like glioma cells in thalamic tumors (e.g. NCAM1-PTPRZ1, NLGN4X-NRXN1, NTN1-DSCAM) than in pons tumors. Notably, the TME of thalamic tumors contained a bigger fraction of immunotolerant macrophages expressing CD163 or VSIG4. In contrast, pons tumors harbored a unique cluster of VEGFA expressing astrocytes, not found in GBM or pLGG. Liana analysis inferred COPA/APP-CD74 interaction between VEGFA+ astrocytes and CD163/VSIG4+ macrophages and microglia, potentially contributing to an immunotolerant TME in pons DMG. CONCLUSIONS In DMG, patterns of tumor and TME composition associated with tumor location were uncovered. Furthermore, unique TME celltypes could contribute to the particularly aggressive nature of these tumors.

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.

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.

ONC201 (Dordaviprone) in Recurrent H3 K27M-Mutant Diffuse Midline Glioma.

Histone 3 (H3) K27M-mutant diffuse midline glioma (DMG) has a dismal prognosis with no established effective therapy beyond radiation. This integrated analysis evaluated single-agent ONC201 (dordaviprone), a first-in-class imipridone, in recurrent H3 K27M-mutant DMG. Fifty patients (pediatric, n = 4; adult, n = 46) with recurrent H3 K27M-mutant DMG who received oral ONC201 monotherapy in four clinical trials or one expanded access protocol were included. Eligible patients had measurable disease by Response Assessment in Neuro-Oncology (RANO) high-grade glioma (HGG) criteria and performance score (PS) ≥60 and were ≥90 days from radiation; pontine and spinal tumors were ineligible. The primary end point was overall response rate (ORR) by RANO-HGG criteria. Secondary end points included duration of response (DOR), time to response (TTR), corticosteroid response, PS response, and ORR by RANO low-grade glioma (LGG) criteria. Radiographic end points were assessed by dual-reader, blinded independent central review. The ORR (RANO-HGG) was 20.0% (95% CI, 10.0 to 33.7). The median TTR was 8.3 months (range, 1.9-15.9); the median DOR was 11.2 months (95% CI, 3.8 to not reached). The ORR by combined RANO-HGG/LGG criteria was 30.0% (95% CI, 17.9 to 44.6). A ≥50% corticosteroid dose reduction occurred in 7 of 15 evaluable patients (46.7% [95% CI, 21.3 to 73.4]); PS improvement occurred in 6 of 34 evaluable patients (20.6% [95% CI, 8.7 to 37.9]). Grade 3 treatment-related treatment-emergent adverse events (TR-TEAEs) occurred in 20.0% of patients; the most common was fatigue (n = 5; 10%); no grade 4 TR-TEAEs, deaths, or discontinuations occurred. ONC201 monotherapy was well tolerated and exhibited durable and clinically meaningful efficacy in recurrent H3 K27M-mutant DMG.

Glucosylceramide Synthase Inhibitors Induce Ceramide Accumulation and Sensitize H3K27 Mutant Diffuse Midline Glioma to Irradiation.

H3K27M mutant (mut) diffuse midline glioma (DMG) is a lethal cancer with no effective cure. The glycosphingolipids (GSL) metabolism is altered in these tumors and could be exploited to develop new therapies. We tested the effect of the glucosylceramide synthase inhibitors (GSI) miglustat and eliglustat on cell proliferation, alone or in combination with temozolomide or ionizing radiation. Miglustat was included in the therapy protocol of two pediatric patients. The effect of H3.3K27 trimethylation on GSL composition was analyzed in ependymoma. GSI reduced the expression of the ganglioside GD2 in a concentration and time-dependent manner and increased the expression of ceramide, ceramide 1-phosphate, sphingosine, and sphingomyelin but not of sphingosine 1-phosphate. Miglustat significantly increased the efficacy of irradiation. Treatment with miglustat according to dose recommendations for patients with Niemann-Pick disease was well tolerated with manageable toxicities. One patient showed a mixed response. In ependymoma, a high concentration of GD2 was found only in the presence of the loss of H3.3K27 trimethylation. In conclusion, treatment with miglustat and, in general, targeting GSL metabolism may offer a new therapeutic opportunity and can be administered in close proximity to radiation therapy. Alterations in H3K27 could be useful to identify patients with a deregulated GSL metabolism.

Abstract 2820: Defining the role of purine metabolism in radiation resistance in H3K27M–mutant diffuse midline glioma

Abstract Diffuse midline gliomas (DMG) are an aggressive and treatment resistant form of brain cancer in urgent need of new and effective treatment strategies. Radiation therapy (RT) is the standard of care for DMGs, but these tumors often recur locally within the high-dose radiation field. Therefore, it is imperative to discover methods of overcoming RT resistance. DMGs are often characterized by the presence of driving mutation in the tail domain of histone H3 that converts the 27th residue from a lysine to a methionine (termed H3K27M). Using liquid chromatography/mass spectrometry “snapshot” metabolomics, we determined that human H3K27M-expressing cells (referred to as K27M cells going forward) possess altered metabolic profiles distinct from their H3-wildtype (H3WT) counterparts characterized by enrichment in metabolites associated with purine metabolism. RT-treated K27M isogenic-paired cells displayed K27M-specific increases in the abundance of both hypoxanthine and guanine and decreased abundance of guanosine, which can be degraded into guanine from mature guanylate species (GMP/GDP/GTP). Hypoxanthine and guanine are the substrates for HGPRT (encoded by HPRT1), the rate-limiting enzyme in purine salvage. Using publicly available data, pediatric high-grade gliomas (pHGG) expressing K27M were found to have decreased HPRT1 transcript compared to H3WT tumors, and we found decreased HGPRT protein expression in K27M cell lines compared to their isogenic H3WT counterparts. The decreased abundance of guanosine, increased abundance of both purine salvage metabolites following RT, and decreased expression of HGPRT suggest impaired guanylate purine salvage in K27M cells that is exacerbated by RT. We hypothesized that K27M cells are deficient in purine salvage and may rely on de novo purine synthesis (DNPS) for purine production following RT. Consistent with this hypothesis, we found that a clinically available, blood-brain penetrant inhibitor of DNPS increased RT-mediated killing of K27M neurospheres in vitro. Combination RT+DNPS inhibition (DNPSi) increased survival (38d post-implantation) over RT alone (31.5d post-implantation) in Rag1-KO mice bearing orthotopically implanted K27M tumors, but did not cure tumors. To understand potential mechanisms of treatment resistance, we interrogated metabolic pathway utilization with stable isotope tracing. Using 2,8-deuterium-hypoxanthine to interrogate purine salvage and 15N-glutamine to interrogate de novo purine synthesis, we found an RT-mediated increase in purine salvage that could mediate resistance to DNPSi inhibition. Future experiments will determine whether inhibition of purine salvage has efficacy for those tumors that recur or progress following treatment with RT and DNPSi inhibition. Citation Format: Erik R. Peterson, Peter Sajjakulnukit, Andrew Scott, Caleb Heaslip, Costas Lyssiotis, Maria G. Castro, Daniel R. Wahl. Defining the role of purine metabolism in radiation resistance in H3K27M–mutant diffuse midline glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2820.

The landscape of tumor cell states and spatial organization in H3-K27M mutant diffuse midline glioma across age and location

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.

EPCO-21. THE SPATIAL ORGANIZATION OF H3-K27M MUTANT DIFFUSE MIDLINE GLIOMA

Abstract Histone 3 lysine27-to-methionine mutant diffuse midline gliomas (H3-K27M DMGs) are among the most lethal brain tumors. Their putative cellular hierarchy has been shown to be driven by self-renewing stem-like cells arrested in an oligodendrocyte precursor-like (OPC-like) state, of which few cells are able to differentiate towards more mature astrocyte (AC)-like and oligodendrocyte (OC)-like cells. However, the spatial organization underlying this tumor cell architecture and its microenvironmental interactions in intact H3-K27M DMG tissues remain unknown. Here, we profiled the single cell transcriptomes of 45 patient H3-K27M DMGs and derived cell population-specific marker gene combinations to characterize the single cell spatial organization of 16 tumors using targeted in situ sequencing. We thereby resolved different malignant and non-malignant cell populations including cycling, OPC-like, AC-like, OC-like, mesenchymal tumor cells, and non-malignant oligodendrocytes, astrocytes, neurons, myeloid cells, T cells, and vascular cells directly in situ. Global neighborhood analyses indicate a higher tendency of cycling OPC-like cells, vascular cells, and neurons to localize within a more restricted homogeneous compartment, whereas AC-like cells, non-malignant astrocytes and myeloid cells tend to intermingle with different cell populations in a more diffuse manner. Among malignant cells, we observed cycling OPC-like and OC-like cells to co-localize within a niche-like structure that is surrounded by more differentiated AC-like cells. We further validated this stem-like niche at the protein level using multiplexed immunofluorescence via the CODEX system. Finally, we characterized relationships between malignant and non-malignant cells, consistently identifying preferred neighborhoods of mesenchymal tumor cells with vascular and myeloid cells. Together, this study resolves the spatial architecture of H3-K27M DMG malignant and non-malignant cells at single cell resolution and identifies a local niche of the oligodendroglial lineage containing the OPC-like cancer stem-like cells, thus providing novel insights into the cancer stem-like compartment in H3-K27M DMGs and suggesting potential avenues for its perturbation.

MODL-11. NOVEL IMMUNOTHERAPY MODEL IN AGGRESSIVE PEDIATRIC H3K27M MUTANT GLIOMAS WITH INDUCIBLE TGFBR2 AND LAG3 EXPRESSION

Abstract INTRODUCTION Gliomas are considered immunologically “cold” tumors that mask themselves from the immune system. Immunotherapy, a breakthrough for cancer treatment, remains inefficient for gliomas. H3K27M mutant diffuse midline glioma has poor survival. Using our innovative mosaic analysis with dual recombinases technology (Cell 2019), we further created a novel murine model of H3K27M glioma with an inducible expression of LAG3 and TGFBR2 to make the tumor recognizable by the immune system. This allows studying molecular mechanisms of “perfect” immunotherapy in pediatric gliomas with histone H3.3 mutations. METHODS To make a novel murine model, H3K27M plasmids with doxycycline-inducible expression for TGFBR2 and LAG3 were designed, sequenced, and replicated. For in vitro validation, murine neuronal stem cells were nucleofected with these plasmids, grown and sorted. Doxycycline was added to the experimental group in vitro to induce expression. The plasmids were then electroporated into neonatal MGMT mice (n=21) of both genders. Control mice were used on a standard diet (n=9) and experimental mice (n=12) were given doxycycline in food to induce TGFBR2 and LAG3 expression in vivo. Mice were followed up for survival. RESULTS Plasmid sequencing validation demonstrated their high purity. In vitro nucleofected neuronal stem cells showed significant increase in TGFBR2 and LAG3 expression by immunofluorescent staining after addition of doxycycline. The mouse survival study showed a significant benefit with treatment group surviving 46% longer (135 ± 41.9 days vs. 92.6 ± 32.7 days in the control group, p=0.030). Conclusion and Future Directions: We have successfully modified H3K27M glioma to have inducible expression of TGFBR2 and LAG3 to simulate “perfect” immunotherapy. The survival study showed a significant survival benefit. The development of organoid transplants for more uniform testing and next-generation sequencing is ongoing. This can lead to the emergence of novel immune therapies and uncover novel mechanisms of tumor response to treatment.

Simultaneous Integrated Boost Technique for Pediatric High-Grade Gliomas: A Single-Institution Experience

<h3>Purpose/Objective(s)</h3> The prognosis for pediatric patients with high grade glioma (HGG) remains dismal despite aggressive multidisciplinary treatment, and there is marked variability regarding the optimal radiation approach. We report outcomes from a retrospective review of pediatric patients with HGG treated using a simultaneous integrated boost (SIB) technique at a single institution. <h3>Materials/Methods</h3> The medical records of patients ages 0–22y with biopsy-proven non-DIPG localized HGG treated with radiotherapy (RT) from January 2006 to May 2021 were retrospectively reviewed on an IRB approved protocol. Endpoints were Kaplan-Meier estimates of overall survival (OS) and progression free survival (PFS), and patterns of failure. <h3>Results</h3> There were 32 patients in the cohort; 24 patients had glioblastoma without further molecular characterization, 2 had H3 K27M mutant diffuse midline gliomas, 2 had G34R mutant glioma, 2 had glioblastoma with IDH1 mutation, 1 had high-grade astrocytoma, and 1 had high-grade glial neoplasm. Median age at diagnosis was 14.9 (1.9–21.5) years. Prior to RT, 6 patients obtained both spinal MRIs and lumbar punctures, 5 obtained spinal MRIs, and 1 had a lumbar puncture; none had evidence of distant metastases. All patients were treated with intensity modulated radiotherapy (IMRT) using SIB with two dose levels. The most common (n=25) dose to the high dose volume was 60 Gy (range, 40–60 Gy); the most common (n=24) dose to the low dose volume was 50 Gy (range, 30–55 Gy). Treatment was most commonly (n=26) delivered in 30 fractions (range, 15-33). One patient did not complete planned RT due to tumor hemorrhage. Median follow up for all patients was 17 (0–164.4) months. For patients who completed planned RT, median PFS was 20.1 (1–140) months from RT start. Median OS was 25.9 months with one- and two-year survival rates of 80% and 56%, respectively. Of 19 patients with known recurrence, 8 recurred locally, 7 recurred in the distant brain, and 4 developed leptomeningeal dissemination. Three patients were alive at 10 years after initial diagnosis; none of these patients were known to have tumors with IDH mutations. <h3>Conclusion</h3> This study explores outcomes in a cohort of pediatric HGG patients treated with SIB technique, with encouraging initial results. Future efforts will be necessary to identify additional molecular and treatment factors that impact outcome.

The landscape of tumor cell states and spatial organization in H3-K27M mutant diffuse midline glioma across age and location

The landscape of tumor cell states and spatial organization in H3-K27M mutant diffuse midline glioma across age and location

JS04.6.A The landscape of tumor cell states and spatial organization in H3-K27M mutant diffuse midline glioma across age and location

Abstract Background Histone 3 lysine27-to-methionine mutations (H3-K27M) frequently occur in childhood diffuse midline gliomas (DMGs) of the pons, thalamus and spinal cord, presumed to be driven by the specific spatiotemporal context of these midline locations during postnatal development. While most common in the pons and at mid-childhood ages, the same oncohistone mutation is recurrently detected in adult DMGs and throughout different midline regions. The potential heterogeneity of tumors at different ages and in different anatomical locations of the midline are vastly understudied. Material and Methods Through dissecting the transcriptomic, epigenomic and spatial architectures of a comprehensive cohort of patient H3-K27M DMGs - spanning the age range from 2-68 years and locations from spinal cord to thalamus - at single cell resolution, we delineate how age- and location-dependent contexts shape glioma cell-intrinsic and -extrinsic features in light of the shared driver mutation. Results We identify that oligodendrocyte precursor (OPC)-like cells constitute the stem-like compartment in H3-K27M DMGs across all clinico-anatomical groups, however, depending on location, display varying levels of maturity resembling less differentiated pre-OPCs or more mature OPCs further differentiated along the oligodendroglial lineage. We further demonstrate increased mesenchymal cell states in adult tumors, which we link to age-related differences in glioma-associated immune cell compartments. We for the first time resolve the spatial organization of H3-K27M DMG cell types in intact patient tissues, identifying a local niche of the oligodendroglial lineage. Conclusion Our study provides a powerful resource for rational modeling and therapeutic frameworks taking into account determinants of age and location in this lethal glioma group.

DIPG-29. Highly multiplexed digital spatial profiling of the tumor immune environment of pediatric and adultH3 K27M-mutant diffuse midline gliomas

Abstract BACKGROUND: H3 K27M-mutant diffuse midline gliomas (DMGs) are highly aggressive malignancies of the central nervous system that affect both pediatric and adult populations. The immune layout and genetic changes within the tumor microenvironment associated with these high-grade malignancies are thought to play an integral role in the phenotypic differences in tumor presentation and clinical course between both populations. Comparative immune landscape between pediatric and adult DMGs is not known. METHODS: The NanoString GeoMxTM Digital Spatial Profiler platform was used to determine the immune marker and genetic layout in a cohort of both pediatric and adult H3 K27M-mutant DMG tissue samples. Three fluorescently labeled antibodies targeting immune cells (CD45), epithelial cells (PanCK), tumor cells (H3 K27M) and a nucleic acid stain (SYTO-13) were used to identify and separate out the various components within the tumor tissues from selected regions of interest. The resultant information was then pooled into libraries that were run through the Illumina sequencing system to assess gene expression and proteomics for both cohorts of samples. RESULTS: Our data revealed that there were immune and genetic marker changes that were seen within both populations across multiple regions of interest within the tumor tissues. A number of immune cell types and protein markers exhibited significant difference between the pediatric and adult tissue cohorts. CONCLUSION: The digital spatial analysis of pediatric and adult H3 K27M-mutant DMGs show different immune and genetic profiles which are proposed to contribute to the clinical differences between both populations. These observed differences may play a role in future treatments for H3 K27M-mutant DMGs and further research is needed to explore this.

DIPG-43. Glucosylceramide synthase inhibitors induce ceramide accumulation and sensitise H3K27 Mutant Diffuse midline Glioma to irradiation

Abstract BACKGROUND AND AIMS: Glycosphingolipids (GSL) are amphipathic lipids particularly abundant in the brain where their amount and expression patterns change drastically during the embryonic to postnatal stages and during tumorigenesis. The biosynthesis of GSL begins with the formation of glucosylceramide from ceramide, a step catalysed by the glucosylceramide synthase (UGCG). UGCG can be inhibited by eliglustat, which is used for treating children with Gaucher‘s disease. We have previously shown that the GSL composition is deregulated in H3K27M mutant diffuse midline glioma (H3K27M mut) and that eliglustat inhibits cell proliferation. Here we analysed the mechanism of action of eliglustat in H3K27M mut and its effect on irradiation. METHODS: The concentration of different components of the sphingolipid metabolism (ceramide, ceramide-1-Phosphate (CIP), sphingomyelin, Sphingosine and Sphingosine-1-Phosphate (S1P)) was assessed by mass spectrometry in the H3K27M mut cell line SF8628, before and after treatment with eliglustat. The combination of eliglustat with ionizing radiation was analysed by clonogenic assay. RESULTS: The treatment of H3K27M mut cells with eliglustat resulted in a significant increase in the concentration of ceramide, Sphingosine, C1P, but not S1P. The increase was concentration and time dependent and was not observed after longer incubation. Eliglustat treatment reduced the colony formation ability after irradiation. CONCLUSIONS: Ceramide is a known mediator of apoptosis involved in the molecular mechanisms underlying cellular response to irradiation. Increased endogenous ceramide levels, induced by blocking the synthesis of GSL, may sensitize H3K27M mut cells to irradiation. However, ceramide can be converted in C1P, a potent inhibitor of apoptosis and inducer of cell survival. Thus, the time and concentration dependent shift to ceramide and C1P requires further investigation in order to achieve an appropriate balance between the levels of these two metabolites and identify the optimal therapeutic window for combination with irradiation and potentially chemotherapy

Magnetic Resonance Imaging Characteristics of Molecular Subgroups in Pediatric H3\xa0K27M Mutant Diffuse Midline Glioma

PurposeRecent research identified histone H3 K27M mutations to be associated with a dismal prognosis in pediatric diffuse midline glioma (pDMG); however, data on detailed MRI characteristics with respect to H3 K27 mutation status and molecular subgroups (H3.1 and H3.3 K27M mutations) are limited.MethodsStandardized magnetic resonance imaging (MRI) parameters and epidemiologic data of 68 pDMG patients (age <18 years) were retrospectively reviewed and compared in a) H3 K27M mutant versus H3 K27 wildtype (WT) tumors and b) H3.1 versus H3.3 K27M mutant tumors.ResultsIntracranial gliomas (n = 58) showed heterogeneous phenotypes with isointense to hyperintense signal in T2-weighted images and frequent contrast enhancement. Hemorrhage and necrosis may be present. Comparing H3 K27M mutant to WT tumors, there were significant differences in the following parameters: i) tumor localization (p = 0.001), ii) T2 signal intensity (p = 0.021), and iii) T1 signal homogeneity (p = 0.02). No significant imaging differences were found in any parameter between H3.1 and H3.3 K27M mutant tumors; however, H3.1 mutant tumors occurred at a younger age (p = 0.004). Considering spinal gliomas (n = 10) there were no significant imaging differences between the analyzed molecular groups.ConclusionWith this study, we are the first to provide detailed MR imaging data on H3 K27M mutant pDMG with respect to molecular subgroup status in a large patient cohort. Our findings may support diagnosis and future targeted therapeutic trials of pDMG within the framework of the radiogenomics concept.

Abstract 635: Response to novel imipridone combination therapies targeting H3K27M mutant diffuse midline glioma (DMG)

Abstract ONC201, a first-in-class, small molecule, imipridone therapy, is known to induce apoptosis via upregulation of TNF-related apoptosis inducing ligand (TRAIL) and its proapoptotic death receptor (DR5), independent of p53. Current trials have been exploring ONC201 as a monotherapy or in conjunction with radiotherapy, in treatment of patients with newly diagnosed and refractory diffuse intrinsic pontine gliomas (DIPG), a disease which remains incurable at present. We sought to identify synergy between ONC201 or second generation impridones (ONC206 and ONC212), and other chemotherapeutics with known promising pre-clinical activity against DMGs. Seven patient derived H3K27M mutant DIPG cell lines (SU-DIPG-IV, SU-DIPG-13, SU-DIPG-25, SUDIPG-27, SU-DIPG-29, SU-DIPG-36, SF8628) were grown in culture and exposed to ONC201, ONC206, or ONC212 either as monotherapy or in combination with other FDA approved chemotherapeutics (histone de-acetylase inhibitors [HDACi], marizomib, etoposide, and temozolomide). Dose dependent response to imipridone therapy was noted in all cell lines, with mean half maximal inhibitory concentration (IC50) of 1.46 µM in ONC201, 0.11 µM in ONC206, and 0.03 µM in ONC212. Synergy was identified via combination studies, with combination indices of &amp;lt;1 indicating some degree of synergy, and &amp;lt;0.5 indicating very strong synergy. We identified strong synergy between ONC201 and HDAC inhibitors: Panobinostat (CI 0.01) and Romidepsin (CI 0.08), with lesser synergy detected with Entinostat (CI 0.59). Romidepsin similarly showed robust synergy with ONC206 (CI 0.1), as did proteasome inhibitor Marizomib (CI 0.1). Combination of ONC212 and Panobinostat demonstrated induction of apoptosis as measured by the induction of poly (ADP-ribose) polymerase (PARP) cleavage. Our results suggest an increased sensitivity of H3K27M DIPG cell lines to second generation imipridones as compared to ONC201. Additionally, we have shown promising synergism between imipridones with Panobinostat or Romidepsin, which should be considered for clinical translation. Citation Format: Robyn Borsuk, Lanlan Zhou, Yiqun Zhang, Varun V. Prabhu, Joshua E. Allen, Nikos Tapinos, Rishi Lulla, Wafik S. El-Deiry. Response to novel imipridone combination therapies targeting H3K27M mutant diffuse midline glioma (DMG) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 635.

DIPG-80. CLINICAL AND RADIOGRAPHIC RESPONSE TO ONC201 IN A PEDIATRIC PATIENT WITH A THALAMIC H3K27M AND BRAFV600E MUTANT DIFFUSE MIDLINE HIGH GRADE GLIOMA

Recent improved understanding of the molecular markers of high grade glioma has shifted the approach to these aggressive CNS tumors to increasingly use molecularly guided targeted therapies. Treatment of patients with BRAFV600E mutant high grade gliomas with BRAF inhibitors has shown efficacy, however the impact of concomitant H3K27M mutation is unknown. ONC201 targets dopamine receptor D2 (DRD2), which is shown to be broadly overexpressed in the thalamus as well as multiple tumor types; its antagonism has demonstrated anti-tumor efficacy and immunomodulatory properties in preclinical studies. ONC201 has also demonstrated clinical efficacy in patients with H3K27M mutant gliomas. We present the case of a 9-year-old male with a right thalamic H3.3K27M mutant diffuse midline glioma with a concomitant BRAFV600E mutation with an ongoing partial response to ONC201 treatment. The patient was diagnosed in May 2018. He underwent biopsy, followed by standard focal proton radiation therapy (54Gy) and subsequent treatment with dasatinib, bevacizumab and everolimus over the course of five months. After continued radiographic progression on serial imaging, in April 2019 he started ONC201 375mg orally once per week through an expanded access trial. He has tolerated the medication well with grade 1 nausea and fatigue. Over the next nine months, he demonstrated clinical and radiographic improvement with modest increased use of his left side and MRIs showing progressive decrease in size of the thalamic lesion with a 70 % decrease in the target lesion (measuring 53x62mm prior to treatment, decreased to 38x26mm in January 2020).

Abstract 5407: Novel imipridone combination therapies targeting oncohistone H3K27M mutant diffuse midline glioma (DMG)

Abstract Malignant glioma including diffuse midline glioma (DMG) is the leading cause of cancer related death in children. Recent advances in sequencing have identified the oncohistone H3K27M mutation in ~80% of pediatric DMG, which represents a target for new therapeutics. ONC201 is a first-in-class small molecule which upregulates TNF-related apoptosis inducing ligand (TRAIL) and its proapoptotic receptor DR5, independent of p53, selecting for apoptosis/growth arrest of many different tumor types including H3K27M mutant DMG. While single agent ONC201 along with RT is currently being tested in clinical trials for newly diagnosed and refractory DMG, there is an urgent need to evaluate ONC201 and other imipridone analogs in combination with other agents. Patient derived H3K27M mutant DMG cell lines (SU-DIPG-IV, SU-DIPG-13, SU-DIPG-25, SU-DIPG-27, SU-DIPG-29, SU-DIPG-36, and SF8628), were grown in culture and exposed to imipridones (ONC201, ONC206 and ONC212) alone, and in combination with other chemotherapeutic agents that have demonstrated pre-clinical activity against DMG (HDAC inhibitors and etoposide). Response to treatment was determined by Cell Titer-Glo cell viability assays as well as Western Immunoblotting for evidence of apoptosis and integrated stress response (ISR) activation. All H3K27M-mutant DMG cell lines tested to date exhibit a dose-dependent response to imipridone therapy. Mean IC50 values were significantly lower with the imipridone analogs ONC206 (0.11 µM) and ONC212 (0.03 µM) as compared to ONC201 (1.46 µM) when used as a single agent. ONC206 and ONC212 were associated with robust induction of apoptosis evidenced by increased expression of cleaved PARP, and ISR by increased expression ATF4. In the SU-DIPG-IV cell line, combination studies of ONC201 and panobinostat demonstrated a synergistic effect of both agents, as calculated by CompuSyn software. Combination indices below one were observed at concentrations of 0.003 - 0.05 µM of panobinostat with ONC201 at 0.156 - 5 µM, with the best combination index of 0.22. We similarly observed synergy between either ONC201 or ONC206 with Etoposide. Additional combination testing and synergy analysis is ongoing and will be presented. Our findings suggest that H3K27M DMG cells demonstrate increased sensitivity to imipridones ONC206 and ONC212 as single agents and combinational strategies with HDAC inhibitors and etoposide, and provide insights into novel therapies for further clinical testing. Citation Format: Robyn Borsuk, Lanlan Zhou, Rishi Lulla, Wafik El-Deiry. Novel imipridone combination therapies targeting oncohistone H3K27M mutant diffuse midline glioma (DMG) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5407.

Clinico-pathological and molecular characterization of diffuse midline gliomas: is there a prognostic significance?

H3K27M mutant diffuse midline gliomas (DMGs) are considered grade IV irrespective of histological features and have dismal prognosis. We evaluated clinico-pathologic, radiological, and molecular characteristics of DMGs across all ages. One twenty-six DMGs were identified over 10years. Immunohistochemistry was done for H3K27M, ATRX, IDH1, and p53, and Sanger sequencing performed for IDH1 and H3K27M mutation. Patient demographics and clinico-radiologic characteristics were reviewed and survival analysis performed. DMGs comprised 5.3% of all gliomas with 49.2% H3K27M mutant and 50.8% wild types. Majority (75.68%) of pediatric and 38.20% of adults were H3K27M mutant (p = 0.0001). Amongst H3K27M mutants, brainstem (46.43%) was the commonest location in pediatric and thalamus (61.76%) in adults. H3K27M mutation was mutually exclusive with IDH mutation in 93.55%, while p53, ATRX mutation were seen in 56.4% and 30.6% cases respectively. Software-based immunohistochemistry evaluation (H-scoring) showed 99.2% concordance with sequencing for H3K27M mutation. Radiologically, no significant difference in contrast enhancement was seen between mutant and wild types (p = 0.05). The difference in overall survival (OS) was not significant in mutant versus wild types, with age or location. Tumor resection independently and on correlation with H3K27M did not influence OS (p = 0.51 and p = 0.47). Adjuvant therapy impacted survival significantly in adults (p = 0.0009), however, not in pediatric cases (p = 0.06). The study highlights the differences in frequency and location of pediatric and adult DMGs. IHC (H-scoring) for H3K27M mutation is an excellent surrogate for sequencing. Prognosis remains dismal irrespective of age, location, and H3K27M status. Potential therapeutic targets need to be explored.

Pediatric Diffuse Midline Gliomas H3 K27M-Mutant and Non-Histone Mutant Midline High-Grade Gliomas in Neurofibromatosis Type 1 in Comparison With Non-Syndromic Children: A Single-Center Pilot Study.

Background: Pediatric neurofibromatosis type 1 (NF1) patients rarely develop aggressive central nervous system tumors. Among high-grade gliomas (HGGs), histone mutant diffuse midline gliomas (DMGs H3 K27M-mutant) have exceptionally been reported. The aim of this retrospectives single-center study was to compare the clinical behavior of DMGs H3 K27M-mutant and non-histone mutant midline HGGs in NF1 vs. non-syndromic children and to report imaging features of NF1 HGGs.Method: We conducted a retrospective review of cerebral DMGs H3 K27M-mutant or non-histone mutant HGGs in 18 patients with or without NF1 followed at our institution between 2010 and 2018. Differences in outcomes, notably progression-free survival (PFS) and overall survival (OS), were evaluated.Results: Two patients were identified with genetically confirmed diagnosis of NF1 and cerebral HGGs (one DMG H3 K27M-mutant and one histone wild type). Both subjects presented with midline mass lesions with imaging features of aggressive biological activity on advanced MRI or amino-acid PET. During the same time period, 16 non-NF1 patients (11 subjects with DMGs H3 K27M-mutant and 5 with non-histone mutant midline HGGs) were treated at our institution. The two patients with NF1 and HGGs presented a PFS of 3 months and an OS of 5 and 7 months. Median PFS and OS of children without NF1 were respectively 6 and 10 months in DMGs H3 K27M-mutant, and 6 and 11 months in H3 K27M wild-type tumors. Seventy-five percent of subjects with non-NF1 HGGs presented a PFS >4 months compared to 0% in NF1 patients. The 8-month OS of patients with non-NF1 HGGs was 81% compared to 0% in NF1 patients.Conclusions: Cerebral HGGs arising in midline structures rarely occur in pediatric patients with NF1 and present with extremely poor prognosis, worse than HGGs developing in non-NF1 patients, independent of the presence or absence of H3 K27M mutation. Imaging features of aggressive biological activity on advanced MRI or amino-acid PET imaging suggest prompt neuropathological and molecular investigations.