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Supplementary Table from Epigenome Programming by H3.3K27M Mutation Creates a Dependence of Pediatric Glioma on SMARCA4

Patients with diffuse midline gliomas that are H3K27 altered (DMG) display a dismal prognosis. However, the molecular mechanisms underlying DMG tumorigenesis remain poorly defined. Here we show that SMARCA4, the catalytic subunit of the mammalian SWI/SNF chromatin remodeling complex, is essential for the proliferation, migration, and invasion of DMG cells and tumor growth in patient-derived DMG xenograft models. SMARCA4 colocalizes with SOX10 at gene regulatory elements to control the expression of genes involved in cell growth and the extracellular matrix (ECM). Moreover, SMARCA4 chromatin binding is reduced upon depletion of SOX10 or H3.3K27M, a mutation occurring in about 60% DMG tumors. Furthermore, the SMARCA4 occupancy at enhancers marked by both SOX10 and H3K27 acetylation is reduced the most upon depleting the H3.3K27M mutation. Taken together, our results support a model in which epigenome reprogramming by H3.3K27M creates a dependence on SMARCA4-mediated chromatin remodeling to drive gene expression and the pathogenesis of H3.3K27M DMG. Significance: DMG is a deadly pediatric glioma currently without effective treatments. We discovered that the chromatin remodeler SMARCA4 is essential for the proliferation of DMG with H3K27M mutation in vitro and in vivo, identifying a potentially novel therapeutic approach to this disease. See related commentary by Beytagh and Weiss, p. 2730. See related article by Panditharatna et al., p. 2880. This article is highlighted in the In This Issue feature, p. 2711

Diffuse Midline Glioma (DMG), H3K27M altered, confers a dismal survival of 9-15 months and has a non-inflammatory tumor immune microenvironment (TIME). Radiation therapy (RT) is the mainstay treatment for DMG and has been shown in other cancers to recruit an immune component. However, the effect of RT on the DMG TIME has not been explored. In a syngeneic murine model of pontine DMG (PDGFB+, H3.3K27M, p53−/−), mice were treated with single fraction 15Gy RT or sham control, four mice per group. We performed single cell sequencing after CD45 isolation to evaluate the TIME 4 days post RT and compare to untreated tumor (sham control). Unsupervised clustering of 14,848 CD45+ cells revealed 16 immune cell subsets, most abundantly microglia at 75% of cells, with four subtypes representing a spectrum of homeostatic to activated. Microglia from RT are more concentrated in the activated subtypes with an upregulation of interferon response (i.e. Isg15, Ifit3) compared to untreated tumor with an increase in several interferon pathways using REACTOME. Consistent with RT response, RT treated tumors have increase in cell cycle regulatory genes such as Cdkn1a, across all clusters. In non-resident myeloid cells, compared to untreated tumor, RT is associated with a robust upregulation of interferon response genes in both macrophages (Isg15 Fold Change (FC) 2.30; Ifit1 FC 1.64; Ifit3 FC 2.02; Cxcl10 FC 2.29) and dendritic cells (Isg15 FC 2.67; Ifit1 FC 1.72; Ifit3 FC 2.06; Cxcl10 FC 1.50). We also find differential expression of immune checkpoints in RT-treated versus untreated tumor with decreased expression of Lag3, Tim3 (Havcr2), and Csf1R and increased expression of Cd47, Sirpa and Gitr (Tnfrsf18) post RT. In summary, RT stimulates a pro-inflammatory TIME response and alters immune checkpoints in DMG, highlighting the potential for combining RT and immunotherapy in these tumors.

Diffuse midline gliomas (DMG) are pediatric tumors with dismal prognosis. When these tumors emerge in the brainstem, there exists no feasible method of surgical resection or systemic intervention, making ionizing radiation the sole therapeutic avenue to date. However, radiotherapy (RT) provides only marginal survival benefit as the topographically diffuse and highly infiltrative tumors spread in areas in which the blood-brain barrier (BBB) is relatively intact. Focused ultrasound (FUS) with intravenous microbubbles provides a compelling solution, transiently and non-invasively opening the BBB to allow drug delivery across the cerebrovasculature. Nonetheless, it remains unclear whether FUS can be safely administered at the brainstem in patients receiving RT. Therefore, the goal of this study was to assess the safety and feasibility of FUS administered concurrent with and adjuvant to a clinical hypofractionated radiation scheme for brainstem DMG. Non-tumor bearing B6 albino mice were randomly assorted into control, RT, FUS, and RT+FUS groups. Mice designated RT+FUS received 39Gy/13fx (hypofractionated RT scheme) to the brainstem with two sessions of FUS approximately 1 week apart. A single-element, spherical-segment FUS transducer driven by a function generator through a power amplifier was used with concomitant microbubble injection to sonicate the brainstem. Magnetic resonance imaging (MRI) was used to confirm BBB opening and cardiopulmonary measures were recorded throughout sonication. Vitals were assessed daily, and all treatment animals underwent Kondziela inverted screen testing and sequential weight lifting to assess brainstem-related strength and motor coordination deficits. In both FUS and RT+FUS mice, MRI confirmed brainstem BBB opening and subsequent closure within 96 hours. Mouse weights were stable, with slight drops (mean=5.5%) following FUS that resolved within three days. No attenuation in cardiorespiratory, strength, and motor coordination measurements was observed from FUS. FUS is a safe and feasible technique for brainstem BBB opening concurrent with and adjuvant to clinical hypofractionated RT.

Diffuse Midline Glioma – H3K27M mutant (DMG), is a fatal and inoperable pediatric brain tumor with limited treatment options as radiation provides only temporary reprieve, leaving the median survival between 9-15 months. Conventional dose-rate radiation (2Gray/minute, CONV) has been shown in other cancers to recruit an immune component, however, this has not been studied in DMG. Ultra-high dose-rate radiation given at 90 Gray/second (FLASH), is a novel technique associated with decreased toxicity and effective tumor control. Using a syngeneic model of brainstem DMG, we performed single-cell RNA sequencing on CD45+ immune cells isolated from tumors irradiated with 15Gray using FLASH or CONV, and compared to unirradiated tumor and normal brainstem. Isolation of 33,308 immune cells revealed 17 unique clusters, most abundant of which was microglia (73.8%), present in four distinct subtypes representing a spectrum from homeostatic to activated. In the most activated microglia, both FLASH and CONV showed an enrichment in type 1 interferon (IFN1) pathway scores compared to untreated tumors (p< 0.001 and p< 0.001, respectively). The most differential response was found in macrophages (MAC) and dendritic cells (DC) with a robust enrichment of IFN1 pathway scores for CONV compared to FLASH (p< 0.001, MAC and p< 0.001 DC). FLASH showed an increase in anti-inflammatory MAC markers such as Mrc1, Cd163, and Maf and an enrichment of myeloid-derived suppressor cell (MDSC) signature in monocytes, not seen in CONV (p< 0.001). Finally, we correlated our data with publicly available single-cell data taken from the cerebrospinal fluid of DMG patients treated with anti-GD2 CAR T Cell therapy and found similar inflammatory markers characteristic of our unirradiated murine tumors. In summary, our work is the first to study immune alterations comparing different dose-rates of radiation with single-cell resolution in DMG, highlighting the potential for combining radiation and immunotherapy in these tumors.

Diffuse Midline Glioma with H3K27M mutation (DMG) is an aggressive unresectable central nervous system tumor of the brainstem, midline thalamus, or spine. Prognosis is poor and systemic agents have been ineffective partially due to limited permeability of the blood brain barrier (BBB). Non-invasive low intensity focused ultrasound (FUS) can be used for BBB opening (BBBO). Our preclinical studies showed safety and feasibility of targeting and BBBO in the brainstem; however, to our knowledge, FUS-guided BBBO for thalamic DMG has yet to be reported. RNA-seq was performed on mouse syngeneic DMG cells 4423 (PDGFB+, H3.3K27M, p53−/−) and results recapitulated molecular programs seen in human tumors. 4423 cells were injected into the thalamus of male B6 (Cg)-Tyrc-2J/J mice at 1.6mm lateral and 1.8mm posterior to the bregma at a depth of 3.2mm. MRI at 14-21 days post injection confirmed thalamic tumor growth. Histological analysis with Hematoxylin and Eosin stain was consistent with thalamic DMG. Tumor implantation rate was 85% and median survival was 38 days post injection. To test safety and feasibility of BBBO for thalamic tumors, a 1.5 MHz FUS transducer was used with concurrent microbubble injection. BBBO assessed by contrast-enhanced MRI. FUS achieved BBBO targeting the thalamic tumor without increased morbidity or mortality and BBB closure was observed on day 3 post-sonication. Next we tested the tolerance of drug delivery with panobinostat post-FUS at a dose of 20mg/kg weekly. The results are preliminary at this time; however, animals tolerated the combination therapy without morbidity or mortality. Preclinical models are crucial to improve the development of new therapeutic strategies. Establishing this syngeneic thalamic DMG murine model provides the opportunity to test FUS as a non-invasive drug delivery technology for thalamic DMG compared to brainstem DMG, and to re-visit therapeutic agents previously considered ineffective due to limited penetration of the BBB.

Diffuse midline glioma (DMG) is a pediatric tumor with dismal prognosis. Systemic therapeutic strategies have been unsuccessful to date and radiotherapy (RT) remains the standard of care. A central impediment to systemic therapy is the blood-brain barrier (BBB), which precludes drug delivery to the tumor site. Focused ultrasound (FUS) with intravenous microbubbles can transiently and non-invasively circumvent the BBB to enhance drug delivery. Nevertheless, it remains unclear whether FUS is safe at the brainstem in combination with clinical doses of RT. In this study, we hypothesized that FUS-mediated BBB-opening (BBBO) is safe and feasible with 39 Gy RT. To establish a safety timeline, we administered FUS to the brainstem of nontumor bearing mice concurrent with or adjuvant to radiation; then, we validated our findings in a syngeneic orthotopic xenograft DMG model which received repeated sonication concurrent with RT. Male B6 (Cg)-Tyrc-2J/J albino mice received intracranial injection of 4423 mouse DMG cells (PDGFB+, H3.3K27M, p53−/−) at a location posterior and lateral to the lambda. A clinical RT dose of 39 Gy in 13 fractions was delivered to the brainstem with the Small Animal Radiation Research Platform (SARRP) or the XRAD-320 irradiator. FUS was administered with a 0.5 MHz transducer, and both BBBO and tumor volume were monitored with MRI. FUS-mediated BBBO in nontumor bearing mice receiving RT did not affect cardiorespiratory rate, motor function, and tissue integrity. Moreover, tumor bearing mice tolerated repeated brainstem BBBO concurrent with RT. 39 Gy over 13 fractions offered local control, although disease progression occurred in all animals approximately 3-4 weeks post-RT. Ultimately, repeated FUS-mediated BBB opening concurrent with RT is safe and feasible. In our brainstem DMG model, relapse occurs, making it ideal for future tests of combinatorial RT and FUS-mediated drug delivery.

Background Diffuse intrinsic pontine glioma (DIPG) is a devastating pediatric brain cancer with limited treatment options and poor survival. The delivery of systemic therapies in this disease is severely limited by the blood-brain barrier (BBB). Focused ultrasound combined with intravenous microbubbles (FUS+MB) can effectively open the BBB permitting the entry of drugs across the cerebrovasculature. Etoposide is a chemotherapy frequently used in pediatric oncology with well-established anti-tumor effects but limited efficacy when administered systemically in DIPG. Given that FUS+MB in DIPG is not well studied, our goal was to determine the feasibility of ultrasound-mediated BBB opening and etoposide delivery in a preclinical murine pontine glioma model. Methods A syngeneic, orthotopic model was established by stereotactic injection of PDGF-B+PTEN-/-p53-/- murine glioma cells into the pons of B6 albino mice. Mice were randomly divided into control (n=6) or FUS+MB groups (n=6). A single-element, spherical-segment FUS transducer (center frequency=1.5MHz) driven by a function generator through a power amplifier was used with concurrent microbubble injection to sonicate the tumor and its margins on post-injection day 14. Immediately after treatment, 5 mg/kg of intraperitoneal etoposide was administered to all 12 mice. All animals underwent cardiac puncture and blood sampling, followed by transcardiac perfusion and brain harvesting. Liquid chromatography-mass spectometry was performed on both serum and tumor tissue to measure etoposide levels. Results Contrast-enhanced MRI demonstrated successful BBB opening in all FUS+MB mice. Compared to control (mean=20.98ng/g), etoposide concentration in the sonicated tumor tissue (mean=164.77ng/g) was nearly eight times greater. Lastly, the mean brain tumor–to-serum ratio was more than fivefold higher in the treated mice (1.50%) compared with the control mice (0.28%) (P Conclusions FUS+MB is hereby shown successful in BBB opening and enhanced delivery of etoposide in a preclinical pontine glioma model.

Purpose/Objective(s) Diffuse intrinsic pontine glioma (DIPG) is the most lethal brain tumor in children with no effective treatment options to date. The prognosis remains poor with a median overall survival of less than 1 year. Recent in vitro drug screening studies found the histone deacetylase (HDAC) inhibitor, panobinostat, to be highly effective against DIPG. However, as panobinostat has poor blood-brain barrier (BBB) penetration, phase 1 clinical trials using systemic administration of panobinostat have been unsuccessful. Therefore, improved efficacy will require enhancement of delivery into the brain. Focused ultrasound (FUS), a non-ionizing acoustic form of radiation, has been shown to safely and non-invasively open the BBB, which can increase drug delivery. In this study, we hypothesize that FUS-mediated BBB opening (BBBO) can enhance the delivery of panobinostat for a therapeutic benefit in DIPG. Materials/Methods A syngeneic DIPG model was established by injecting mouse DIPG cells (PDGFB+, H3.3K27M, p53−/−) intracranially. A single-element, spherical-segment FUS transducer (center frequency: 0.5 MHz) driven by a function generator through a power amplifier was used with concurrent microbubble injection to perform BBBO. We then utilized magnetic resonance imaging to evaluate BBBO and tumor progression. Results We first demonstrated that FUS-mediated BBBO is safe and feasible in mice with DIPG tumors by MRI and passive cavitation detection. Our DIPG cell line was shown to be very sensitive to panobinostat in cell viability assay (IC50 = 0.012 μM). The combined treatment of FUS-mediated BBBO and panobinostat showed benefits in both local control and overall survival. Conclusion The current results demonstrate that FUS could increase the treatment efficacy of panobinostat in a DIPG animal model due to the improved targeted dose of systemic panobinostat in the DIPG tumors. We believe these data establish a strong preclinical rationale to assess the combination of FUS-mediated BBB opening with panobinostat in pediatric patients with DIPG.

BackgroundDiffuse intrinsic pontine gliomas (DIPG’s) are immunologically inert tumors with a median survival of 9–15 months. Radiation therapy (RT) is the mainstay treatment for DIPG but is associated with immunodepletion of the tumor microenvironment (TME) at high dose ranges. FLASH, or ultra-fast dose rate RT, represents a novel ablative technique that may spare TME immune responses while decreasing tumor burden. Here, we present single-cell immune profiling of DIPG tumors treated with FLASH, conventional dose rate RT (CONV) or no RT (SHAM).MethodsMurine H3.3K27M mutant DIPG cells were stereotactically injected and tumor induction confirmed by magnetic resonance imaging (MRI) 15 days later. DIPG-bearing mice were randomly assigned to one of three treatment groups (n=4/group), FLASH, CONV or SHAM. A fourth group with no tumor (NML) was included as a negative biological control. A modified linear accelerator was used to deliver 15 Gy of electron RT to the brainstem at dose rates of 90 Gy/second and 2 Gy/minute, for the FLASH and CONV groups, respectively. Four days post-RT, mice brainstems were harvested, homogenized, stained for CD45 and tagged with a hashtag antibody specific to each group. CD45+ immune cells were isolated and sequenced using the 10X Genomics chromium single-cell 3’ platform. After processing and alignment of the reads using CellRanger with default parameters, the data was quality checked and filtered before hashtag demultiplexing, unsupervised clustering and downstream analysis was implemented following the Seurat R package. Differential expression evaluated based on the non-parametric Wilcoxon rank sum test. Key genes determine by an adjusted p value of < 0.05 based on bonferroni correction and |avg log2FC| > 0.8.ResultsPreliminary analysis identifies 15 clusters with distinct CD45 immune phenotypes (figure 1). Differential gene expression analysis by hashtag antibody (treatment group) reveals 14 clusters differentially expressing key genes, including 3 clusters upregulated in DIPG compared to NML, and 2 clusters upregulated in irradiated tumors compared to SHAM and NML (figure 2). Notably, analysis demonstrates an individual cluster upregulated in FLASH versus all other groups (p = 3.07E-171). Further deconvolution of specific immune phenotypes represented by each cluster is ongoing.Abstract 91 Figure 1tSNE plot based on clustering of RNA signatures, grouped by RNAAbstract 91 Figure 2tSNE plot based on clustering of RNA signatures, grouped by hashtag antibodyConclusionsOur preliminary analysis shows differential immune responses among DIPG tumors compared to NML. We also find several immune cell subsets that are unique to DIPG treated with CONV or FLASH compared to unirradiated samples. Most notably, we identify a single immune cell subset that is exclusive to FLASH alone, indicating that FLASH elicits a unique immune response in murine DIPG.

Diffuse intrinsic pontine glioma (DIPG) is the lethal high-grade brain tumor in children with no effective treatment options to date. Despite excessive clinical trials, the prognosis remains poor, with a median overall survival (mOS) of less than 1 year. Genomic studies of DIPG tissue have identified highly recurrent mutations in genes encoding histone H3 resulting in the substitution of lysine to methionine at position 27 (K27M), which is found in approximately 80% of DIPG. Recent drug screening studies identified the histone deacetylase (HDAC) inhibitors panobinostat as a highly effective drug against DIPG in vitro. However, due to the poor Blood-Brain Barrier (BBB) penetration of systemic administration, to enhance the delivery of panobinostat to improve treatment efficacy is needed. Focused ultrasound (FUS) has been shown to be able to safely and non-invasively open BBB to enhance drug delivery. Hence, in this study, we hypothesize that FUS-mediated BBBO (BBBO) can enhance the delivery of panobinostat for a therapeutic benefit in DIPG. Herein we established the syngeneic DIPG model by intracranially injecting mouse DIPG cells (PDGFB+, H3.3K27M, p53−/−) and used FUS and microbubbles to open BBB and enhance the panobinostat delivery. Magnetic resonance (MR) imaging was utilized to evaluate BBBO and tumor progression. We first demonstrated that FUS-mediated BBB-opening is safe and feasible to mice with DIPG tumors by MR imaging and passive cavitation detection. Moreover, this DIPG cell line is very sensitive to panobinostat in in vitro cytotoxicity assay. The combined treatment of FUS-mediated BBBO and panobinostat showed benefits in both local control and overall survival. The current results demonstrated FUS could increase the treatment efficacy of panobinostat to DIPG animals may be due to the increase of targeted delivery of systemic panobinostat to DIPG tumors in brainstem.