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Background Supratentorial RELA fusion (ST-RELA) ependymomas (EPNs) are resistant tumors without an approved chemotherapeutic treatment. Unfortunately, the molecular mechanisms that lead to chemoresistance traits of ST-RELA remain elusive. The aim of this study was to assess RELA fusion-dependent signaling modules, specifically the role of the Hedgehog (Hh) pathway as a novel targetable vulnerability in ST-RELA. Methods Gene expression was analyzed in EPN from patient cohorts, by microarray, RNA-seq, qRT-PCR, and scRNA-seq. Inhibitors against Smoothened (SMO) (Sonidegib) and Aurora kinase A (AURKA) (Alisertib) were evaluated. Protein expression, primary cilia formation, and drug effects were assessed by immunoblot, immunofluorescence, and immunohistochemistry. Results Hh components were selectively overexpressed in EPNs induced by the RELA fusion. Single-cell analysis showed that the Hh signature was primarily confined to undifferentiated, stem-like cell subpopulations. Sonidegib exhibited potent growth-inhibitory effects on ST-RELA cells, suggesting a key role in active Hh signaling; importantly, the effect of Sonidegib was reversed by primary cilia loss. We, thus, tested the effect of AURKA inhibition by Alisertib, to induce cilia stabilization/reassembly. Strikingly, Alisertib rescued ciliogenesis and synergized with Sonidegib in killing ST-RELA cells. Using a xenograft model, we show that cilia loss is a mechanism for acquiring resistance to the inhibitory effect of Sonidegib. However, Alisertib fails to rescue cilia and highlights the need for other strategies to promote cilia reassembly, for treating ST-RELA tumors. Conclusion Our study reveals a crucial role for the Hh pathway in ST-RELA tumor growth, and suggests that rescue of primary cilia represents a vulnerability of the ST-RELA EPNs.

Medulloblastoma is a high-risk embryonal brain tumor arising in the cerebellum. Genomic profiling has revealed a striking molecular heterogeneity between medulloblastoma patients, yet treatment regimens are mostly uniform. Many children with medulloblastoma die from their disease and surviving patients often face severe long-term side effects, highlighting an urgent need for more effective treatment options. We and others have recently identified pronounced intra-tumoral heterogeneity and defined cellular hierarchies within medulloblastoma tumors. The functional role of these cellular hierarchies remains unknown. We now hypothesize the existence of an inter-cellular communication network that is maintained by receptor/ligand interactions. To test our hypothesis, we use our medulloblastoma single-cell RNA sequencing dataset of 25 patients, as well as bulk RNA sequencing, DNA methylation array, and genome sequencing data across molecular subtypes. Single-cell RNA sequencing data are analyzed to dissect cell compartments characterized by high expression of potentially oncogenic receptors and their respective ligands. Consequently, cell type-specific roles in auto- or paracrine signal transduction within the cellular community are explored. We further investigate downstream oncogenic signaling pathways by approximating transcription factor activity and explore genetic and epigenetic activation mechanisms by matched genome sequencing and DNA methylation profiling, respectively. Our findings will be applied to deconvolute bulk RNA sequencing data, thus identifying therapeutically relevant signaling networks in larger cohorts of medulloblastoma patients. Eventually, candidate targets will be validated on patient-derived cell models and xenografts by overexpression and inhibition studies. Together, here we aim at identifying tumor-driving receptor/ligand interactions in medulloblastoma, with the goal to define targets susceptible to precision oncology approaches.

Diffuse Midline Glioma, encompassing Diffuse Intrinsic Pontine Gliomas (DIPGs), are the most aggressive pediatric brain tumors. Their meager survival has not changed despite the combination of radiotherapy with targeted therapies emphasizing the urgent need for effective treatments. TIM-3 (HAVCR2) is a member of the T-cell immunoglobulin and mucin domain protein family. It is expressed on multiple immune cell types, including T cells, NK cells, myeloid populations, and microglia, regulating adaptive and innate immunity. In silico assessment of TIM-3 expression in DIPG datasets showed a robust expression of this gene. Single-cell sequencing analyses of DIPG biopsies uncover TIM-3 expression, especially in microglia. In vivo efficacy studies showed that treatment with AbTIM-3 significantly increased overall survival in two DIPG immunocompetent orthotopic models, led to long-term survivors (50%), and showed immune memory. TIM-3 treatment led to a significant increase in the tumor microenvironment of microglia, granulocytes, NK, and CD8+ cells and higher levels of IFNγ, GrzB and TNFα corresponding with an NK and T-cell activate phenotypes. Interestingly, there was a decrease in the Treg population which causes an increase in the pro-inflammatory CD8/Treg ratio. CD4, CD8 or NK cell depletion leads to a significant but not a total loss of treatment efficacy. CD4+ and CD8+ cells were aumented in treated draining lymph nodes and expressed higher amounts of pro-inflamattory cytokines than control-mice. Population analysis and depletion experiments demonstrated the relevance of NK, CD4, CD8 and myeloid populations in the response to anti-TIM-3 therapy. Interestingly, the depletion of the different immune populations combined or using immunodeficient Rag2 mice, did not completely abrogate the treatment efficacy. These results suggest the concurrence of an additional mechanism of action that together with the immune response leads to a robust anti-DIPG effect. In conclusion, these data demonstrate that TIM-3 is a potential target for the treatment of DIPG.

Pineoblastoma (PB) is a rare and aggressive childhood brain tumor with highly variable age and treatment-associated outcomes. Our recent bulk tumor analyses of DNA methylation and mutational landscapes uncovered four discrete PB molecular subgroups (PB-miRNA1, PB-miRNA2, PB-MYC/FOXR2, and PB-RB), providing a major advance in our understanding of biological and clinical heterogeneity. However, developmental origins of PB subgroup heterogeneity and mechanisms governing how specific genetic alterations promote malignancy remain unknown. To resolve the cellular origins of PB, we assembled a large single-nucleus RNA-sequencing cohort (n=32) of primary PB tumors, including representatives from each subgroup. Transcriptomic analysis identified subgroup-specific gene expression programs driving intra-tumoral heterogeneity. In addition, we discovered substantial differences in the expression of miRNA biogenesis genes between the PB-miRNA1 and PB-miRNA2 subgroups, providing mechanistic support for their distinct subgroup identities despite overlapping driver events. The MYC/FOXR2 subgroup was characterized by over-expression of the FOXR2 proto-oncogene in bulk RNA-seq, which we validated in single-nuclei and identified co-expressed downstream target genes. To map PB subgroups to their putative developmental beginnings, we created a single-cell transcriptional atlas of the murine pineal gland across 11 developmental stages (E11-P21). Trajectory inference within the developing pineal gland revealed a differentiation continuum of early, mid, and mature alpha-/beta pinealocytes. Cross-species correlation and deconvolution identified significant associations between multiple PB subgroups and specific differentiation states of the pinealocyte lineage, suggestive of developmental origins. Characterization of pinealocyte development informed generation of biologically faithful disease models, including a novel genetically engineered mouse model of the PB-RB subgroup. PB-Rb1 mouse tumors were histologically and molecularly validated for their fidelity to human tumor counterparts, exhibiting up-regulation of key pinealocyte lineage markers that are diagnostic in patients. Finally, high-throughput drug screening identified several promising pharmacological candidates that may attenuate consequences of Rb1 deficiency in affected children.

Brain tumors are the leading cause of disease-related death in childhood and strong efforts are required to develop innovative and efficient therapeutic strategies for patients with high-risk disease. Key critical factor of pediatric brain tumors is their molecular heterogeneity; one of the aspects that contributes to such heterogeneity is the intrinsic capacity of the tumor cells to organize, shape and exploit the surrounding brain tumor microenvironment (TME) to sustain tumor growth and malignant progression. TME was proved to play a crucial role in several malignancies, but in pediatric brain malignancies this has not been fully elucidated yet. Here, we aimed at characterizing the TME cell populations and their contributions in ependymoma and medulloblastoma, two of the most common pediatric brain tumor entities. Single-cell transcriptome analysis (n=65 ependymomas; n=39 medulloblastomas) of publicly available tumor datasets, as well as newly generated data of primary tumors and matching patient-derived tumor xenografts (PDX), showed an extensive heterogeneity of TME cell types with distinctive expression signatures. Amongst the identified TME populations, analysis revealed pro-inflammatory and proliferating myeloid cells, tumor-infiltrating lymphocytes, active regulatory T cells and vascular progenitor cells. Comparative analysis between primary and PDX tumors showed that tumor cells stimulated the host microenvironment, which in turn exhibited tumor-associated stromal signatures. Applying a deconvolution method, using our single cell data as reference, on a bulk tumor cohort including PFA ependymomas with different degree of immune infiltration, we observed an enrichment of polarized macrophages and microglia cells in tumor with high infiltration. Next, we identified TME markers involved in tumor-supportive functions, such as immune suppression (TIGIT, FOXP3, BCL2, CD19, ICAM2), pro-inflammatory stimuli (CCL3, CCL4, IL1B, GPNMB), extracellular matrix remodeling (COL4A1) and angiogenic processes (ANGPT2). TME markers with emerging role in ependymoma and medulloblastoma can be considered as possible targets for tailored and more effective anti-tumor therapeutic strategies.

Embryonal tumor with multilayered rosettes (ETMR) is a malignant brain tumor that typically occurs in children under the age of three. Most patients die within two years of diagnosis, and more effective, targeted therapies are urgently needed. To better characterize the oncogenic mechanisms of key driver alterations and identify novel therapeutic targets, we studied the cellular heterogeneity of ETMR using single-cell RNA sequencing. Analyses conducted on >3,000 high-quality cells collected from ten primary and relapse specimens revealed a common cellular hierarchy across all tumors: A highly proliferative neural stem cell-like population (SOX2+) gives rise to intermediate progenitors (ASCL1+) and more differentiated neuron-like cells (STMN2/4+). These malignant populations closely match histological patterns of ETMR (i.e. rosettes, neuropil), as observed by immunofluorescence microscopy. Comparison to single-cell datasets from human embryos indicates resemblance to cell populations of the developing brain, but also reveals key ETMR-specific differences, including expression of the chromosome 19 miRNA cluster (C19MC, the presumed genetic driver of most ETMRs), which is restricted to the stem cell-like population. We next investigated if targeting C19MC is a viable strategy to disrupt the cellular hierarchy of ETMR. Silencing with antisense oligonucleotides shows pronounced reduction of cell line growth for a specific subset of the 46 members of C19MC. These miRNAs share seed sequences with evolutionary conserved miRNAs that have been shown to regulate pluripotency and self-renewal of embryonic stem cells. We hypothesize that select C19MC members play similar roles in ETMR and represent bona fide targets for therapeutic targeting using antisense technology.

Malignant brain tumors are the leading cause of childhood death in Germany, with Diffuse Midline Glioma (DMG) being the most lethal of all paediatric brain tumors. Current treatment strategies are limited to irradiation which prolongs survival only by a few months. Preclinical studies have identified effective drug candidates, but translation into the clinic remains a major obstacle. It is known that interactions between tumor cells and components of the TME (tumor microenvironment), such as cell to cell contacts between malignant and non-malignant cells or secreted factors, can increase therapy resistance and progression of brain tumors. However, these important factors are not present in most conventional cell culture models for drug testing. Consequently, there is a need for more realistic DMG models to improve the relevance and translational potential of current drug screening. Therefore, the goal of this study was to develop a new DMG model for drug testing, consisting of induced pluripotent stem cell (iPSC) derived human brain cells and patient derived DMG cells to better mimic the complex tumor microenvironment. We co-cultured three-dimensional cerebral organoids with DMG tumor spheres resulting in the formation of DMG-Brain-Organoids (DBO). Preliminary results show that co-culture induces distinct tumor cell subpopulations corresponding to those detected in DMG tumors by single cell RNA sequencing (Filbin et al., 2018). These subpopulations mainly differ in their proliferative capacity and their differential response to clinical interventions may be critical for therapeutic success. DBOs subjected to drug treatments (single or combination) were sectioned and individual therapy effects on tumor cell subpopulations and proliferative capacity were monitored using multiplexed immunofluorescence imaging. By observing drug effects in a realistic setup, we hope to improve the predictive power of our preclinical drug screens and to find new combination therapies for DMG.

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.

Ependymoma represents a heterogeneous disease affecting the entire neuraxis. Extensive molecular profiling efforts have identified molecular ependymoma subgroups based on DNA methylation. However, the intratumoral heterogeneity and developmental origins of these groups are only partially understood, and effective treatments are still lacking for about 50% of patients with high-risk tumors. We interrogated the cellular architecture of ependymoma using single cell/nucleus RNA-sequencing to analyze 24 tumor specimens across major molecular subgroups and anatomic locations. We additionally analyzed ten patient-derived ependymoma cell models and two patient-derived xenografts (PDXs). Interestingly, we identified an analogous cellular hierarchy across all ependymoma groups, originating from undifferentiated neural stem cell-like populations towards different degrees of impaired differentiation states comprising neuronal precursor-like, astro-glial-like, and ependymal-like tumor cells. While prognostically favorable ependymoma groups predominantly harbored differentiated cell populations, aggressive groups were enriched for undifferentiated subpopulations. Projection of transcriptomic signatures onto an independent bulk RNA-seq cohort stratified patient survival even within known molecular groups, thus refining the prognostic power of DNA methylation-based profiling. Furthermore, we identified novel potentially druggable targets including IGF- and FGF-signaling within poorly prognostic transcriptional programs. Ependymoma-derived cell models/PDXs widely recapitulated the transcriptional programs identified within fresh tumors and are leveraged to validate identified target genes in functional follow-up analyses. Taken together, our analyses reveal a developmental hierarchy and transcriptomic context underlying the biologically and clinically distinct behavior of ependymoma groups. The newly characterized cellular states and underlying regulatory networks could serve as basis for future therapeutic target identification and reveal biomarkers for clinical trials.

Patients with diffuse midline glioma (DMG) harboring H3 K27M mutation rarely survive longer than two years and have no proven therapies following first-line radiation. ONC201, a bitopic DRD2 antagonist and allosteric ClpP agonist, has shown encouraging efficacy in early phase studies in H3 K27M-mutant DMG. In order to define response rates in H3 K27M DMG patients and to clarify the genomic, anatomic and molecular predictors of response, we performed an integrated pre-clinical and clinical analysis of ONC201 treatment. ONC201 was effective in intra-uterine electroporation (IUE)-generated H3 K27M-mutant murine glioma models with excellent CNS penetration and survival benefit. Patients with H3 K27M-mutant DMG treated with ONC201 on active clinical trials (n=50, 27 thalamic, 23 brainstem) showed an overall survival (OS) of 28.1 (range: 5.9–105) months from diagnosis (enrollment by 4/29/19, data cut-off 12/28/19), compared to historical median OS of 12 months. Median OS for non-recurrent patients has not been reached (n=16, median follow-up: 16.8 from diagnosis). For non-recurrent thalamic patients (n=8), median PFS is 20.1 (range: 9.3–27.6) months from diagnosis (median time on drug: 14.5 months). Best response for thalamic patients by RANO: 1 CR, 5 PR, 7 SD, 8 PD, 6 not reported. Decreased H3 K27M cell-free tumor DNA in plasma and CSF at 6 months correlated with long-term response. Baseline tumor gene expression profiling in patients treated with ONC201 (n=14) identified EGFR and the cortical developmental transcription factor FOXG1 as the strongest biomarkers of radiographic response to ONC201. Analysis of 541 ONC201-treated human cancer cell lines from DepMap, provided evidence for an EGFR-dependent ONC201 resistance mechanism. Analysis of 38 glioma cell lines further supports FOXG1 as a glioma-specific predictive biomarker of ONC201 response. The unprecedented survival results and radiographic responses to ONC201 in H3K27M DMG make a compelling case for later phase and combinatorial studies.

Brain tumors are comprised of cells with heterogeneous genetic and transcriptional states, resulting in substantial phenotypic diversity. This diversity is particularly evident in embryonal tumor with multilayered rosettes (ETMR), which shows a striking bi-phasic pattern for which it is named. A better understanding of its underlying molecular makeup is urgently needed to develop more effective therapeutic strategies that eliminate all malignant cell types underlying ETMR initiation, maintenance, progression, and relapse. Furthermore, the cellular origin of ETMR is currently poorly understood. We used plate-based single-cell RNA sequencing to assess the intratumoral heterogeneity in 6 fresh and 4 snap-frozen surgical biopsies, following a workflow that we have previously established to study pediatric high grade gliomas, medulloblastomas, and ependymomas. Computational analyses conducted on >4,000 single cells identified cellular hierarchies ranging from a proliferative, undifferentiated cell population to more differentiated, predominantly neural-like progeny in all samples. Patient-derived cell line and xenograft models partially recapitulated this hierarchy. We further integrated transcriptional programs identified in single cells with available datasets of the developing normal brain, as well as with programs identified in other pediatric brain tumor entities, to inform both putative cellular origins and ETMR-specific oncogenic pathways. These timely results provide unparalleled insights into the molecular underpinnings of the phenotypic heterogeneity observed in ETMR. Analyses aimed at further integrating malignant cell type abundances with genetic alterations and clinical annotations, and therapeutical targeting of malignant cell populations using in-vitro models are currently ongoing.

INTRODUCTION DMG-ACT (DMG- multi-arm Adaptive and Combinatorial Trial) aims to implement a highly innovative clinical trial design of combinatorial arms for patients with diffuse midline gliomas (DMGs) at all disease stages that is adaptive to pre-clinical data generated in eight collaborating institutions. The goals of the team are to: i) rapidly identify and validate promising drugs for clinical use, and ii) predict biomarkers for promising drugs. METHODS In vitro (n=15) and in vivo (n=8) models of DMGs across seven institutions were used to assess single and combination treatments with ONC201, ONC206, marizomib, panobinostat, Val-083, and TAK228. In vivo pharmacokinetic assays using clinically relevant dosing of ONC201, ONC206, and panobinostat were performed. Predictive biomarkers for ONC201 and ONC206 were identified using extensive molecular assays including CRISPR, RNAseq, ELISA, FACS, and IHC. RESULTS Inhibitory concentrations (IC50) were established and validated across participating sites. In vivo validation of single and combination drug assays confirmed drug efficacy as increased survival for: ONC201 (p=0.01), ONC206 (p=0.01), ONC201+ONC206 (p=0.02), and ONC201+panobinostat (p=0.01). Marizomib showed toxicity in murine/zebrafish PDXs models. Murine pharmacokinetic analysis showed peak brain levels of ONC201 and ONC206 above pre-clinical IC50. Molecular testing and analyses of existing drug screen across 537 cancer cell lines validated mitochondrial stress and ATF4 as the main targets induced by ONC201/6. CONCLUSION Thorough preclinical testing in a multi-site laboratory setting is feasible and identified ONC201 in combination with ONC206 as promising therapeutics for DMGs. Preclinical and correlative-clinical studies are ongoing.

High-grade gliomas (HGG) are among the most prevalent and fatal cancers in pediatric, adolescent, and young adult (AYA) patients. Especially understudied are older children and young adults, aged 16–39 years. Previously, we profiled primary pediatric HGGs through single-cell transcriptomics and identified the genetic, epigenetic and developmental programs that drive their malignant progression. However, the questions of how these programs compare to those in older HGG patients, what the mechanisms are by which these tumors ultimately evolve to drive recurrence and treatment resistance, and how distinct tumor cell subpopulations bidirectionally communicate with their microenvironment remain to be elucidated. Here, we use single-nucleus RNA sequencing to compare 11 paired, matched high-grade gliomas at diagnosis and recurrence and 15 additional H3K27M primary and recurrent DMG samples in pediatric and AYA patients. In all tumors, we find both undifferentiated and differentiated tumor cells recapitulating distinct glial lineages, as well as diverse microenvironmental cell populations. When longitudinally comparing this tumor architecture within matched pairs, we find substantial differences in transcriptional program expressions. Diagnostic samples include more differentiated, astrocyte-like tumor cells, while cells from recurrent samples more highly express ribosomal and heat-shock protein genes, suggesting tumor progression- and treatment-related shifts. Ongoing sequencing and analysis will allow for unprecedented insight into the evolutionary dynamics of pediatric and AYA high-grade gliomas as well as delineate differences in the biology of DMGs occurring in different age groups. This multi-institutional project was funded by the National Institute of Health.

Children diagnosed with diffuse intrinsic pontine glioma (DIPG), a type of high grade glioma in the brainstem, currently have a dismal 5-year overall survival of only 2%. The majority of DIPG patients harbor a K27M mutation in histone 3.3 encoding genes (H3.3K27M). To understand if the aberrant epigenetic landscape induced by H3.3K27M provides an opportunity for novel targeted therapies, we conducted the first CRISPR/Cas9 screen using a focused library of 1,350 epigenetic regulatory and cancer related genes in six H3.3K27M DIPG patient-derived primary neurosphere cell lines. We identified gene dependencies in chromatin regulators, polycomb repressive complexes 1 and 2 (PRC1 and PRC2), histone demethylases, acetyltransferases and deacetylators as novel tumor cell dependencies in DIPG. We hypothesized that targeting dysregulated functions of chromatin regulators by genetically deleting and chemically targeting these epigenetically induced vulnerabilities, we could ameliorate, or even reverse the downstream oncogenic effects of the aberrant epigenetic landscape of DIPG. In our secondary CRISPR nanoscreen, we first used six single guide RNAs (sgRNA) to knockout each gene using CRISPR/Cas9 ribonucleoprotein nucleofections, followed by use of three best sgRNAs combined with homology directed repair templates. Compared to lentiviral delivery, nucleofection is a rapid method, with reduced off-target toxicity, suitable for single gene knockouts in DIPG neurospheres. Secondary CRISPR validations confirmed dependencies in BMI1, CBX4, KDM1A, EZH2, EED, SUZ12, HDAC2, and EP300. Next, we conducted a chemical screen using 20 inhibitors and degraders to target the aberrant activity of HDAC, KDM1A, P300/CBP, PRC1 and PRC2. We identified eight chemical compounds that were effective in H3.3K27M DIPG neurosphere cell lines at low drug concentrations. Among these, an inhibitor and degrader targeting P300/CBP activity indicates a novel strategy of epigenetic therapy in DIPG. Through our combinatorial testing, we will identify a synergistic combination of epigenetic therapy for treating children diagnosed with H3.3K27M DIPG.

Diffuse midline gliomas are characterized by a lysine27-to-methionine mutation in histone H3 (H3K27M-glioma) and represent a highly aggressive molecular entity of high-grade gliomas. These mutations have primarily been described in children, but are also increasingly recognized in adults. In pediatric H3K27M-gliomas, the transcriptional architecture and cellular composition have been studied at the single-cell level. Data suggest that these tumors arise through malignant transformation of a glial progenitor during a specific neurodevelopmental time window. On the other hand, little is known about the architecture and cellular context of adult H3K27M-gliomas, and it remains to be elucidated whether they are driven by the same or by distinct oncogenic programs as their pediatric counterparts. Here, we utilize single-cell transcriptomics to characterize the transcriptional landscape of five H3K27M-gliomas from adult patients aged 22 to 56 years (median 33 years). We describe the specific cellular and microenvironmental architecture of the adult tumors, which comprises distinct populations of cancer and normal cells. We contrast our findings to 12 location-matched pediatric H3K27M-tumors from patients aged 2.5 to 15 years (median 8 years) to identify features related to tumorigenesis and developmental context in light of the shared hallmark H3K27M mutation. Our preliminary data indicate shared gene expression programs between adult and pediatric tumors. However, we find significant differences in the composition of the immune compartment as well as less pronounced differentiation programs in the adult tumors. Our findings provide an unprecedented insight into the composition of adult H3K27M-gliomas and advance our understanding of this molecular tumor class.

We have developed a simple and generalizable in vivo method for brain tumor modeling, mosaic analysis by dual recombinase-mediated cassette exchange (MADR). MADR allows for stable labeling of mutant cells expressing transgenic elements from a precisely-defined chromosomal locus. We have demonstrated the power and versatility of MADR by creating novel glioma models with mixed, reporter-defined zygosity, or with “personalized” H3. 3-containing driver mutation signatures from pediatric glioma--each manipulation altering the spatiotemporal profile of resulting tumors. Further we have generated ependymoma models by employing patient-derived fusion driver mutations. Notably, each model displays divergent spatiotemporal tumor expansion profiles and cellular phenotypes. Now, we use single-cell RNA-seq to compare these models to their cells of mutation and to human datasets to elucidate the fundamental transcriptional programs and markers within and across these tumor types. This investigation will assess and credential these models against their clinical counterparts by scrutinizing the resulting datasets and validating their clinical relevance as pre-clinical models for therapeutic discovery and testing.

Pilocytic astrocytoma (PAs) are pediatric low grade gliomas that frequently harbor oncogenic KIAA1549-BRAF fusions and exhibit low rates of other somatic genetic alterations. Previous transcriptomic profiling of bulk PA tissues sought to uncover gene expression programs that distinguish PA from other tumor types and which may underlie the unique biological features of PA. However, bulk PA transcriptomic data is often confounded by gene signatures thought to be contributed by normal, non-tumor cells. Also, analysis of bulk PA tissue provides incomplete information on whether distinct cell populations exist among the tumor cells. To resolve the cell types contributing to PA transcriptional profiles and to identify gene expression programs that distinguish PA tumor cells from those of higher-grade tumors, we performed RNA sequencing of >1000 single cells (scRNA-seq) collected from six PAs. To confidently distinguish tumor cells from non-tumor cells, we sorted cells by glial progenitor marker A2B5 status and also profiled KIAA1549-BRAF fusion status for every cell. This scRNA-seq approach revealed unique tumor and non-tumor cell populations within PAs. We identified tumor cell populations expressing oligodendrocyte precursor and radial glia gene signatures. Non-tumor cells consisted of populations expressing microglia, T lymphocyte, and endothelial cell gene signatures which accounted for many of the expression patterns seen in bulk PA transcriptional data. In summary, scRNA-seq has revealed the cellular ecosystem and population structure of PA.

Human cancers are composed of cells with heterogeneous genetic and epigenetic states, resulting in substantial phenotypic diversity. Medulloblastoma (MB), a clinically challenging, malignant childhood brain tumor, is no exception. A better understanding of its cellular heterogeneity is urgently needed in order to develop more efficacious therapeutic strategies that eliminate all tumor subclones underlying MB initiation, maintenance, progression, and relapse. We used single-cell RNA sequencing to assess intratumoral heterogeneity in 20 MB samples representing all four molecular subgroups. Fresh surgical biopsies were dissociated and flow sorted into single cells prior to full-length transcriptome sequencing using the Smart-seq2 protocol. Computational analyses conducted on >6,000 single cells identified cellular hierarchies ranging from proliferative, undifferentiated cell types to more differentiated neural- and astrocyte-like progeny. Transcriptional differences in undifferentiated cell populations between MB subgroups suggested distinct cellular origins, and informed oncogenic pathways believed to promote MB pathogenesis. Identification of transcriptional programs active in single cells aided the deconvolution of published bulk RNA-seq profiles, providing a deeper understanding of the heterogeneity characteristic of Group 3/4 subtypes. Lastly, inference of copy-number variations and probable driver-gene mutations from the single-cell data provided evidence for genetic subclones in a subset of patients. Overlaying transcriptional profiles on MB genetic subclones unveiled how oncogenic programs adapted during tumor evolution. These interim results provide unparalleled insights into the cellular heterogeneity of MB subgroups. Analyses aimed at further resolving molecular substructure in Group 3/4, verifying the developmental origins of MB subgroups, and implicating defined cell populations driving tumorigenesis are ongoing.

DIPG is the leading cause of pediatric brain tumor-related death and remains without effective clinical treatment. Through numerous genomic studies, the H3K27M mutation has emerged as the molecular hallmark of DIPG. While this mutation causes global chromatin remodeling and a broadly oncogenic transcriptional signature, the core molecular pathways driving DIPG pathobiology remain to be defined. In this study, we characterize the active chromatin epigenomic landscape of DIPG in a large cohort of primary tumor samples and identify novel epigenetic subgroups within histone-mutant DIPG. We uncover distinct signaling pathways controlling oncogenic state for each subgroup, elucidating novel mechanisms mediating proliferation and treatment resistance. Utilizing a human iPSC developmental model introducing the histone mutation at progressive stages of neural precursor differentiation, we will describe the developmental cell state most closely resembling DIPG tumors from each genomic subgroup and identify the specific molecular pathways by which the H3K27M mutant drives transformation. Taken together, this study identifies functional molecular heterogeneity between primary DIPG tumors and characterizes novel targets for potential therapeutic development.

INTRODUCTION: Clinical genomics platforms identify targetable alterations for precision medicine in pediatric neuro-oncology, allowing both the genomic profiling of tumors to inform clinical care and facilitating the potential discovery of novel driver alterations. METHODS: We applied two clinical genomic platforms (OncoPanel and OncoCopy) to profile pediatric brain tumors in a clinical setting. OncoPanel, a multiplexed targeted exome-sequencing platform that includes 300 cancer causing genes, was used to assess single nucleotide variants and rearrangements/indels. OncoCopy, a high-resolution genome-wide array comparative genomic hybridization (aCGH) assay, was used to evaluate copy number alterations and refine rearrangement breakpoints. RESULTS: Cancer genomes of 203 pediatric brain tumors were profiled across histological subtypes including 117 analyzed by OncoPanel, 146 by OncoCopy, and 60 evaluated using both methodologies. OncoPanel revealed clinically relevant alterations in 56% of patients (44 cancer mutations and 20 rearrangements), including BRAF alterations that directed the use of targeted inhibitors. EWSRa rearrangements were observed unexpectedly in two patients; one with a Atypical Teratoid Rhabdoid Tumor with loss of INI-1 (SMARCB1), and the second in an ependymoma. Whole Genome Sequencing of the rhabdoid tumor confirmed a rearrangement involving EWSR1 and PLAGL1.CONCLUSIONS: The combined deployment of OncoPanel and OncoCopy multiplex genomic assays can identify clinically relevant genomic alterations in pediatric brain tumors and facilitate the discovery of novel driver alterations. *equal contribution

While human tumors are shaped by the genetic evolution of cancer cells, evidence also suggests that they display superimposed hierarchies reminiscent of normal development. Yet, relating the genetic and hierarchical models of cancer organization has been limited by technical challenges. Here, studying H3 K27M-mutant diffuse midline gliomas we profiled thousands of single cells from fresh tumors by RNA-seq and reconstructed the genetic and epigenetic cellular architecture of tumors in patients. We identified a subset of cancer cells that are differentiated along specialized glial programs while a distinct subset expresses neural stem/progenitor expression programs. By identifying distinct genetic clones, we show that these cellular architectures evolve during the progression of the tumors, suggesting that developmental programs and genetic influences coordinately determine tumor architecture. These results provide unprecedented insight into the cellular composition of midline brain tumors at single-cell resolution and may help harmonize the cancer stem cell and the genetic models of cancer, with critical implications for disease management.