Search

Diffuse midline gliomas (DMG) are highly invasive brain tumors with rare survival beyond two years past diagnosis. The mechanism behind tumor invasion is currently not well understood. Previous reports demonstrate upregulation of the protein ID1 with H3K27M and ACVR1 mutations in DMG, but this has not been confirmed in human tumors or therapeutically targeted. Whole exome, RNA, and ChIP-sequencing were performed on the ID1 locus in DMG tissue. Scratch-assay migration and transwell invasion assays of cultured cells were performed following shRNA-mediated ID1-knockdown. In vitro and in vivo genetic and pharmacologic [cannabidiol (CBD)] inhibition of ID1 on DMG tumor growth was assessed. Additional in vitro experiments were performed to determine a potential mechanism of action for CBD-mediated effects. Self-reported CBD dosing information was collected from DMG patients. We found that increased ID1 expression in human DMG and in utero electroporation (IUE) murine tumors is associated with H3K27M mutation and brainstem location. ChIP-sequencing indicates a similar epigenetically active state at ID1 regulatory regions in human H3K27M-DMG tumors and prenatal pontine cells. Higher ID1-expressing astrocyte-like DMG cells share a transcriptional program with oligo/astrocyte-precursor cells (OAPCs) from the developing human brain and demonstrate upregulation of the migration regulatory protein SPARCL1. Genetic and pharmacologic (CBD) suppression of ID1 decreases tumor cell migration, tumor growth, and to a lesser extent invasion in both murine IUE and multiple patient-derived in vivo DMG models, improving mouse survival. ID1 knockdown significantly decreases the effect of CBD on migration, tumor growth, and invasion. CBD increases reactive oxygen species production, which also affects DMG cell proliferation in a non-ID1 mediated manner. Overall, we find that H3K27M-mediated reactivation of ID1 in DMG results in a SPARCL1+ migratory transcriptional program that is therapeutically targetable with CBD.

As the field of neuro-oncology makes headway in uncovering the key oncogenic drivers in pediatric glioma, the role of precision diagnostics and therapies continues to rapidly evolve with important implications for the standard of care for clinical management of these patients. Four studies at major academic centers were published in the last year outlining the clinically integrated molecular profiling and targeting of pediatric brain tumors; all 4 demonstrated the feasibility and utility of incorporating sequencing into the care of children with brain tumors, in particular for children and young adults with glioma. Based on synthesis of the data from these studies and others, we provide consensus recommendations for the integration of precision diagnostics and therapeutics into the practice of pediatric neuro-oncology. Our primary consensus recommendation is that next-generation sequencing should be routinely included in the workup of most pediatric gliomas.

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor with rare survival beyond two years. This poor prognosis is largely due to the tumor's highly infiltrative and invasive nature. Nearly 80% of DMGs harbor K27M mutation in the genes encoding histone H3.1 (H3F3A) or H3.3 (HISTIH3B), often with concurrent ACVR1 mutation. Inhibitor of DNA-binding (ID) proteins are key transcriptional regulators of genes involved in lineage commitment and are associated with invasiveness and poor clinical outcomes in multiple human cancers. Introduction of H3K27M and ACVR1 mutations increase ID1 expression in cultured astrocytes, but this has not been confirmed in human tumors or targeted therapeutically. We developed an in-utero electroporation (IUE) murine H3K27M-driven tumor model, which demonstrates increased ID1 expression in H3K27M- and ACVR1-mutated tumor cells. Exome and transcriptome sequencing analysis of multi-focal DMG tumors (n=52) and normal brain tissue revealed that increased ID1 expression is associated with H3K27M/ACVR1-mutation and brainstem location, and correlates with poor survival in patients. ChIP-sequencing for H3K27ac and H3K27me3 in multiple DMG tumors (n=5) revealed that the ID1 gene is epigenetically active, which matches the epigenetic state of murine prenatal hindbrain cells. Higher ID1-expressing astrocyte-like DIPG cells share a similar transcriptional program with ID1+/SPARCL1+ positive oligo/astrocyte-precursor (OAPC) cells from the developing human brain and demonstrate upregulation of gene sets involved in regulation of cell migration. Both genetic and pharmacologic [cannabidiol (CBD)] suppression of ID1 result in decreased DIPG cell invasion/migration in vitro and invasion/tumor growth in multiple in vivo models. Mechanistically, CBD reduces proliferation through production of reactive oxygen species. Further, DIPG patients treated off-trial with CBD (n=15) displayed reduced ID1 tumor expression and improved overall survival. In summary, ID1 is upregulated in DIPG through K27M-mediated epigenetic reactivation of a developmental OAPC-like transcriptional state, and ID1-driven invasiveness of DIPG is therapeutically targetable with CBD.

BACKGROUND Diffuse intrinsic pontine gliomas (DIPGs) are lethal pediatric brain tumors with no curative therapies. Inhibitor of DNA binding (ID) proteins are key regulators of gene differentiation during embryogenesis. Previous work has shown that H3F3A and ACVR1 mutations increase ID1 expression in cultured astrocytes, but this has not been validated in human DIPG, nor has the regulation and targetability of ID1 been explored in DIPG. RESULTS Analysis of post-mortem tissue and multiple human datasets showed ID1 to be elevated in DIPG, and to correlate with reduced survival. In a multi-focal autopsy of a DIPG case, we also found ID1 expression to be heterogeneous and to correlate with tumor invasion. Chromatin immunoprecipitation qPCR (ChIP-qPCR) revealed elevated H3K27ac and low H3K27me3 at ID1 regulatory regions (enhancers/promoters) in DIPG tissue compared to normal brain, regardless of H3 or ACVR1 mutation status. Analysis of publicly-available ISH and ChIP-sequencing data of developing murine brains revealed H3K27ac at ID1 enhancers to be elevated in the prenatal hindbrain compared to prenatal forebrain and midbrain, and all postnatal brain regions. ID1 shRNA-mediated knockdown of primary human H3K27M DIPG cells (DIPG007) significantly reduced invasion and migration. We also treated DIPG007 cells with cannabidiol (CBD) and found reduced viability at clinically relevant dosing (IC50=2.4 uM) with dose-dependent reduction in ID1 protein. CONCLUSIONS These findings indicate that a multifactorial (genetic and regionally-based) epigenetic upregulation of ID1 drives DIPG invasiveness and is targetable with CBD. ID1 knockdown and CBD treatment experiments in murine models of DIPG are ongoing.

Diffuse intrinsic pontine gliomas (DIPGs) are lethal brain tumors with no effective therapies other than radiation. Inhibitor of DNA binding (ID) proteins, key regulators of lineage commitment during embryogenesis, are implicated in tumorigenesis in multiple human cancers. Prior work showed that recurrent H3F3A and ACVR1 mutations increase ID1 expression in cultured astrocytes. However, this has not been validated in human DIPG. The regulation and targetability of ID1 in DIPG has not been explored either. Exome and transcriptome sequencing analysis of multi-focal DIPG tumors and normal brain tissue from autopsy (n=52) revealed that ID1 expression is significantly elevated in DIPG tissues. Higher ID1 expression correlates with reduced survival in DIPG patients and increased regional invasion in multi-focal autopsy samples. Analyses of developing mouse brain RNA/ChiP-Seq data revealed high ID1 expression and H3K27ac promoter binding in prenatal hind brain compared to all other prenatal and postnatal brain regions. ChIP-qPCR for H3K27ac and H3K27me3 revealed that ID1 gene regulatory regions are epigenetically poised for upregulation in DIPG tissues compared to normal brain, regardless of H3/ACVR1 mutational status. These data support that the developing pons is regionally poised for ID1 activation. Genetic (shRNA) ID1 knockdown in primary human H3.3K27M-DIPG cells (DIPG007) resulted in significantly reduced invasion and migration in vitro. Additionally, DIPG-ID1-KO cells showed improved sensitivity to radiation therapy. Phospho-kinase array analysis of DIPG cells revealed that Akt and WNK1 activity were significantly downregulated upon ID1 knockdown, which was previously shown in lung tumors. Treatment of DIPG007 cells with cannabidiol (CBD) reduced ID1 expression levels and viability/proliferation of DIPG cells in vitro. ID1 knockdown and CBD treatment studies in vivo are ongoing. In summary, our findings indicate that multifactorial (genetic and regional) epigenetic upregulation of ID1 drives DIPG invasiveness and targeting ID1 using CBD may be a potential strategy for the treatment of DIPGs.

BACKGROUND For midline tumors, surgical biopsy risks neurological injury. Non-invasive methods for diagnosis and surveillance are greatly needed. Tumors release DNA into cerebrospinal fluid (CSF-ctDNA), allowing for potential detection and serial monitoring of tumor-associated genetic mutations by CSF sampling. Current detection platforms are limited by their requirement for assay development for each mutation (digital droplet PCR), or cost and timeliness (Illumina sequencing). We hypothesized that direct, electronic analysis of CSF-ctDNA with a novel, hand-held platform (Oxford Nanopore MinION) could provide real-time, ultra-deep sequencing of patient-specific alterations in CSF-ctDNA. METHODS We established multiple clinical trials for pediatric high-grade glioma with required multi-time point (0, 2, and 6 month) correlate lumbar puncture (LP) at time of MRI, with accrual ongoing. We performed amplicon-based PCR on CSF-ctDNA for recurrent mutations and sequenced patient samples (tumor tissue n=8, tumor CSF n=60) and normal controls (tissue n=5, CSF n=24) using NanoPore technology. Variant allele fractions (VAF) were determined via MinKNOW, Guppy, MiniMap2, and Integrated Genome Browser. RESULTS Sensitivity was 79% and specificity 100% by NanoPore. Time from LP to results was 12 hours. A 17-year-old female presented with a biopsy-proven grade IV thalamic glioma with clonal mutations in H3F3A K27M, PIK3CA E545G, TP53 R158G, and TP53 R248Q. After failing standard treatment, she was enrolled in the ONC201 clinical trial and underwent serial LPs. MRI showed stable tumor at 2 months and 40% decrease at 6 months of treatment. H3K27M VAF increased from baseline at 2 months, but decreased to 1% at 6 months of treatment, results that were confirmed by ddPCR. PIK3CA E545G, TP53 R158G, and TP53 R248Q demonstrated the same decrease in VAF, with p-value of < 0.0001. CONCLUSIONS We demonstrate a rapid, reliable method to detect tumor mutations in CSF, and further show molecular remission of H3K27M glioma by CSF sampling.

Primary brain tumors and CNS metastases shed circulating tumor DNA (ctDNA) into the CSF, which can be assessed for tumor-associated mutations. Thus far, there have been no extensive studies using droplet digital PCR (ddPCR) to detect and quantify ctDNA in the CSF of pediatric high-grade brain tumor patients. There are also gaps in our knowledge, including the potential dependence of ctDNA amount on location of sample collection and whether ctDNA can be used to quantify tumor growth and treatment response. To address these questions, we developed a novel H3F3A K27M ddPCR assay and applied it to four pediatric patients with H3F3A K27M-mutant DIPG and GBM. We found that ddPCR was able to detect the K27M mutation in patient CSF and that the closest relation emerged between mutant K27M copies per ng of total DNA (henceforth K27M copies) and contrast-enhancing tumor area on MRI. Multi-focal CSF sampling at autopsy of a DIPG patient exhibited differences in K27M copies by proximity to the tumor. To better understand changes in K27M copies in response to both growth and treatment of DIPG, we developed an in vitro system comprised of astrocytes (NHAs) co-cultured with luciferase-expressing human DIPG cell line DIPG007 as a means to simulate ctDNA release into the CSF. We found that DIPG007 cells released ctDNA into culture media in proportion to their proliferation, even when the media was changed frequently to approximate the constant production and resorption of CSF. Irradiation with 8 Gy resulted in a spike in mutant ctDNA 72–120 hours post-radiotherapy before decreasing. In summary, our study suggests that H3F3A K27M copies in the CSF of children with high-grade brain tumors have a linear relation with contrast-enhancing tumor area and that ddPCR can be used to follow treatment response including ctDNA release shortly after effective therapies.

INTRODUCTION Pediatric diffuse midline glioma (DMG) with histone H3 mutations (80%), are highly morbid tumors with poor response to therapy. We previously detected H3 mutations in circulating tumor DNA (ctDNA) from CSF derived from children with DMG and high grade glioma. Here, we describe a high-throughput, sensitive and specific approach for H3 mutation detection and quantification in plasma and CSF specimens, validated across multiple centers. METHODS DNA extracted from tissue-validated H3.3K27M specimens (4 tumor, 5 CSF, 4 plasma) and pediatric glioma cell lines (1 wild-type, 1 H3.3K27M) were used to standardize ddPCR workflows. ctDNA extracted from 500uL of CSF or plasma was pre-amplified using sequence-specific primers, then analyzed on RainDance and BioRad ddPCR systems using two sets of custom primers and fluorescent LNA probes (Assays A and B). H3 mutation allelic frequency (MAF) was determined across specimens, and validated through parallel analysis of additional matched H3.3K27M tumor tissue, CSF and plasma specimens (n=3). RESULTS ctDNA processing and detection was standardized across three institutions and two platforms with sensitive, specific and reproducible H3 mutation detection. H3K27M mutations were detected in as little as 0.3-2pg input DNA on both platforms, with comparable MAFs across instruments and assays. RainDance yielded greater overall positive droplet number detection (increased sensitivity). Differences in optimal sample input volume between platforms were circumvented by vacuum concentration or dilution to maintain equal input DNA. Assay A yielded superior specificity so was used for subsequent matched specimen analysis. Mutation detection in plasma, as in previous studies, remained challenging due to low [ctDNA], while CSF analysis yielded reliable results across assays and platforms. CONCLUSIONS We demonstrate the utility of liquid biopsy for identifying H3K27M mutations in plasma and CSF via ddPCR with low starting [DNA], representing a rapid, minimally invasive method for diagnosis and therapeutic monitoring of DMG.

Pediatric high-grade glioma (HGG) and diffuse intrinsic pontine glioma (DIPG) frequently harbor alterations in PDGFRA. The CNS penetration of PDGFRA inhibitors, such as dasatinib, is limited by the tumor-efflux protein P-glycoprotein (P-gp). We hypothesized that co-treatment with everolimus, which has been shown to block P-gp in non-tumor models, would increase CNS penetration and efficacy of dasatinib in PDGFRA-driven HGG and DIPG. Dasatinib effectively treated mouse DIPG cells generated from an intra-uterine electroporation (IUE) model (TP53, PDGFRA and H3K27M mutations), with an IC(50) of 100 nM and a dose-dependent reduction in PDGFRA and pPDGFRA by western blot. Using an in vitro P-gp inhibitor assay, we confirmed that everolimus strongly blocks P-gp activity at 1 uM (p=0.0028 vs untreated, and NS vs complete P-gp block). Treatment studies using the IUE model are ongoing. Brief treatment with everolimus resulted in sub-IC(50) dasatinib average mouse cortex (23 nM) and tumor (65 nM) concentrations by mass spectroscopy, but prolonged (>24 hours) everolimus exposure resulted in improved average cortex (288 nM) and brainstem tumor (360 nM) concentrations. Based on this promising pre-clinical data, we established a phase 2 trial employing dasatinib and everolimus in children with HGG and DIPG that contain PDGFRA alterations (NCT03352427). The first two patients (a recurrent PDGFRA-amplified HGG and a recurrent PDGFRA D842V-mutated DIPG) treated with dasatinib and everolimus after re-irradiation survived 6 months and 9 months (ongoing), respectively, after progression, which compares very favorably to historical controls. Paired CSF samples (before and after addition of everolimus) from the PDGFRA-amplified patient showed a 50% increase in CSF dasatinib level after addition of everolimus. In summary, we demonstrate that dasatinib treatment of PDGFRA-driven pediatric HGG and DIPG is improved with everolimus blockade of P-gp. This represents a novel route for improving the CNS penetration and efficacy of precision therapies for pediatric HGG.