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Suppl. Figure S1 shows "Initial MAPKi screen using metabolic activity as readout". Suppl. Figure S2 shows "Western blot validation of MAPK pathway alterations in HEK293T cells". Suppl. Figure S3 shows "Dose-response curves of MAPKi combination treatment in DKFZ-BT66 pDIPZ-CMV cells". Suppl. Figure S4 shows "Dose response curves of MAPKi combination treatment in BT 40 pDIPZ CMV cells". Suppl. Figure S5 shows "Isobolograms of MAPKi combination treatment in DKFZ-BT66 pDIPZ-CMV and BT-40 pDIPZ-CMV cells"

Includes the legends for Suppl. Table S1-S5.

Suppl. Table S1 - A summary of the human cohort used for DNA methylation-profiling_DRG3003

Supplementary Figures and methods

Suppl. Table S4 shows "MAPKi combinations chosen based on low estimated IC50s and pharmaceutical company in consideration of future clinical studies".

Molecular groups of supratentorial ependymomas comprise tumors with ZFTA–RELA or YAP1-involving fusions and fusion-negative subependymoma. However, occasionally supratentorial ependymomas cannot be readily assigned to any of these groups due to lack of detection of a typical fusion and/or ambiguous DNA methylation–based classification. An unbiased approach with a cohort of unprecedented size revealed distinct methylation clusters composed of tumors with ependymal but also various other histologic features containing alternative translocations that shared ZFTA as a partner gene. Somatic overexpression of ZFTA-associated fusion genes in the developing cerebral cortex is capable of inducing tumor formation in vivo, and cross-species comparative analyses identified GLI2 as a key downstream regulator of tumorigenesis in all tumors. Targeting GLI2 with arsenic trioxide caused extended survival of tumor-bearing animals, indicating a potential therapeutic vulnerability in ZFTA fusion–positive tumors.Significance:ZFTA–RELA fusions are a hallmark feature of supratentorial ependymoma. We find that ZFTA acts as a partner for alternative transcriptional activators in oncogenic fusions of supratentorial tumors with various histologic characteristics. Establishing representative mouse models, we identify potential therapeutic targets shared by ZFTA fusion–positive tumors, such as GLI2.This article is highlighted in the In This Issue feature, p. 2113

IPA upstream regulators

Supplementary Tables 1-11

Supplemental Table Legend

Supplementary Figures 1-5

Medulloblastoma is among the most common malignant brain tumors in children. Recent studies have identified at least four subgroups of the disease that differ in terms of molecular characteristics and patient outcomes. Despite this heterogeneity, most patients with medulloblastoma receive similar therapies, including surgery, radiation, and intensive chemotherapy. Although these treatments prolong survival, many patients still die from the disease and survivors suffer severe long-term side effects from therapy. We hypothesize that each patient with medulloblastoma is sensitive to different therapies and that tailoring therapy based on the molecular and cellular characteristics of patients' tumors will improve outcomes. To test this, we assembled a panel of orthotopic patient-derived xenografts (PDX) and subjected them to DNA sequencing, gene expression profiling, and high-throughput drug screening. Analysis of DNA sequencing revealed that most medulloblastomas do not have actionable mutations that point to effective therapies. In contrast, gene expression and drug response data provided valuable information about potential therapies for every tumor. For example, drug screening demonstrated that actinomycin D, which is used for treatment of sarcoma but rarely for medulloblastoma, was active against PDXs representing Group 3 medulloblastoma, the most aggressive form of the disease. Functional analysis of tumor cells was successfully used in a clinical setting to identify more treatment options than sequencing alone. These studies suggest that it should be possible to move away from a one-size-fits-all approach and begin to treat each patient with therapies that are effective against their specific tumor.Significance:These findings show that high-throughput drug screening identifies therapies for medulloblastoma that cannot be predicted by genomic or transcriptomic analysis.

Figure S1: Markers of OIS in murine PA model Figure S2: Viability of DKFZ-BT66 cells under rIL1B treatment Figure S3: SASP factor protein levels in primary PAs Figure S4: Treatments of astrocytes with senolytic agents and combinations treatments of ABT-737 in DKFZ-BT66 cells

Chromothripsis is a form of genomic instability characterized by the occurrence of tens to hundreds of clustered DNA double-strand breaks in a one-off catastrophic event. Rearrangements associated with chromothripsis are detectable in numerous tumor entities and linked with poor prognosis in some of these, such as Sonic Hedgehog medulloblastoma, neuroblastoma and osteosarcoma. Hence, there is a need for therapeutic strategies eliminating tumor cells with chromothripsis. Defects in DNA double-strand break repair, and in particular homologous recombination repair, have been linked with chromothripsis. Targeting DNA repair deficiencies by synthetic lethality approaches, we performed a synergy screen using drug libraries (n = 375 compounds, 15 models) combined with either a PARP inhibitor or cisplatin. This revealed a synergistic interaction between the HDAC inhibitor romidepsin and PARP inhibition. Functional assays, transcriptome analyses and in vivo validation in patient-derived xenograft mouse models confirmed the efficacy of the combinatorial treatment.

Glioneuronal tumors are a heterogenous group of CNS neoplasms that can be challenging to accurately diagnose. Molecular methods are highly useful in classifying these tumors—distinguishing precise classes from their histological mimics and identifying previously unrecognized types of tumors. Using an unsupervised visualization approach of DNA methylation data, we identified a novel group of tumors (n = 20) that formed a cluster separate from all established CNS tumor types. Molecular analyses revealed ATRX alterations (in 16/16 cases by DNA sequencing and/or immunohistochemistry) as well as potentially targetable gene fusions involving receptor tyrosine-kinases (RTK; mostly NTRK1-3) in all of these tumors (16/16; 100%). In addition, copy number profiling showed homozygous deletions of CDKN2A/B in 55% of cases. Histological and immunohistochemical investigations revealed glioneuronal tumors with isomorphic, round and often condensed nuclei, perinuclear clearing, high mitotic activity and microvascular proliferation. Tumors were mainly located supratentorially (84%) and occurred in patients with a median age of 19 years. Survival data were limited (n = 18) but point towards a more aggressive biology as compared to other glioneuronal tumors (median progression-free survival 12.5 months). Given their molecular characteristics in addition to anaplastic features, we suggest the term glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA) to describe these tumors. In summary, our findings highlight a novel type of glioneuronal tumor driven by different RTK fusions accompanied by recurrent alterations in ATRX and homozygous deletions of CDKN2A/B. Targeted approaches such as NTRK inhibition might represent a therapeutic option for patients suffering from these tumors.

Molecular groups of supratentorial ependymomas comprise tumors with ZFTA–RELA or YAP1-involving fusions and fusion-negative subependymoma. However, occasionally supratentorial ependymomas cannot be readily assigned to any of these groups due to lack of detection of a typical fusion and/or ambiguous DNA methylation–based classification. An unbiased approach with a cohort of unprecedented size revealed distinct methylation clusters composed of tumors with ependymal but also various other histologic features containing alternative translocations that shared ZFTA as a partner gene. Somatic overexpression of ZFTA-associated fusion genes in the developing cerebral cortex is capable of inducing tumor formation in vivo, and cross-species comparative analyses identified GLI2 as a key downstream regulator of tumorigenesis in all tumors. Targeting GLI2 with arsenic trioxide caused extended survival of tumor-bearing animals, indicating a potential therapeutic vulnerability in ZFTA fusion–positive tumors. Significance: ZFTA–RELA fusions are a hallmark feature of supratentorial ependymoma. We find that ZFTA acts as a partner for alternative transcriptional activators in oncogenic fusions of supratentorial tumors with various histologic characteristics. Establishing representative mouse models, we identify potential therapeutic targets shared by ZFTA fusion–positive tumors, such as GLI2. This article is highlighted in the In This Issue feature, p. 2113

Precision oncology requires diagnostic accuracy and robust detection of actionable alterations. The Pediatric Targeted Therapy (PTT) 2.0 program aims at improving diagnostic accuracy by addition of molecular analyses to the existing histological diagnosis and detection of actionable alterations for relapsed paediatric oncology patients, in cases with limited availability of tumour material.Paediatric patients diagnosed with relapse or progression of a central nervous system tumour (n = 178), a sarcoma (n = 41) or another solid tumour (n = 44) were included. DNA methylation array, targeted gene panel sequencing on tumour and blood (130 genes), RNA sequencing in selected cases and a pathway-specific immunohistochemistry (IHC) panel were performed using limited formalin-fixed paraffin embedded tissue from any disease episode available. The clinical impact of reported findings was assessed by a serial questionnaire-based follow-up.Integrated molecular diagnostics resulted in refined or changed diagnosis in 117/263 (44%) tumours. Actionable targets were detected in 155/263 (59%) cases. Constitutional DNA variants with clinical relevance were identified in 16/240 (7%) of patients, half of which were previously unknown. Clinical follow-up showed that 26/263 (10%) of patients received mechanism-of-action based treatment matched to the molecular findings.Next-generation diagnostics adds robust and relevant information on diagnosis, actionable alterations and cancer predisposition syndromes even when tissue from the current disease episode is limited.

PURPOSE: The prognosis for SHH-medulloblastoma (MB) patients with Li-Fraumeni syndrome (LFS) is poor. Due to lack of comprehensive data for these patients, it is challenging to establish effective therapeutic recommendations. We here describe the largest retrospective cohort of pediatric LFS SHH-MB patients to date and their clinical outcomes. PATIENTS AND METHODS: N=31 patients with LFS SHH-MB were included in this retrospective multicenter study. TP53 variant type, clinical parameters including treatment modalities, event-free survival (EFS) and overall survival (OS), as well as recurrence patterns and incidence of secondary neoplasms, were evaluated. RESULTS: All LFS-MBs were classified as SHH subgroup, in 30/31 cases based on DNA methylation analysis. The majority of constitutional TP53 variants (72%) represented missense variants, and all except two truncating variants were located within the DNA-binding domain. 54% were large cell anaplastic, 69% gross totally resected and 81% had M0 status. The 2-(y)ear and 5-(y)ear EFS were 26% and 8,8%, respectively, and 2y- and 5y-OS 40% and 12%. Patients who received post-operative radiotherapy (RT) followed by chemotherapy (CT) showed significantly better outcomes (2y-EFS:43%) compared to patients who received CT before RT (30%) (p

Introduction A hallmark of pediatric low-grade glioma (pLGG) is aberrant signaling of the mitogen activated protein kinase (MAPK) pathway. Hence, inhibition of MAPK signaling using small molecule inhibitors such as MEK inhibitors (MEKi) may be a promising strategy. Methods In this multi-center retrospective centrally reviewed study, we analyzed 18 patients treated with the MEKi trametinib for progressive pLGG as an individual treatment decision between 2015 and 2019. We have investigated radiological response as per central radiology review, molecular classification and investigator observed toxicity. Results We observed 6 partial responses (PR), 2 minor responses (MR), and 10 stable diseases (SD) as best overall responses. Disease control rate (DCR) was 100% under therapy. Responses were observed in KIAA1549:BRAF- as well as neurofibromatosis type 1 (NF1)-driven tumors. Median treatment time was 12.5 months (range: 2 to 27 months). Progressive disease was observed in three patients after cessation of trametinib treatment within a median time of 3 (2–4) months. Therapy related adverse events occurred in 16/18 patients (89%). Eight of 18 patients (44%) experienced severe adverse events (CTCAE III and/or IV; most commonly skin rash and paronychia) requiring dose reduction in 6/18 patients (33%), and discontinuation of treatment in 2/18 patients (11%). Conclusions Trametinib was an active and feasible treatment for progressive pLGG leading to disease control in all patients. However, treatment related toxicity interfered with treatment in individual patients, and disease control after MEKi withdrawal was not sustained in a fraction of patients. Our data support in-class efficacy of MEKi in pLGGs and necessity for upfront randomized testing of trametinib against current standard chemotherapy regimens.

Background The sensitivity of myelocytomatosis oncogene (MYC) amplified medulloblastoma to class I histone deacetylase (HDAC) inhibition has been shown previously; however, understanding the underlying molecular mechanism is crucial for selection of effective HDAC inhibitors for clinical use. The aim of this study was to investigate the direct molecular interaction of MYC and class I HDAC2, and the impact of class I HDAC inhibition on MYC function. Methods Co-immunoprecipitation and mass spectrometry were used to determine the co-localization of MYC and HDAC2. Chromatin immunoprecipitation (ChIP) sequencing and gene expression profiling were used to analyze the co-localization of MYC and HDAC2 on DNA and the impact on transcriptional activity in primary tumors and a MYC amplified cell line treated with the class I HDAC inhibitor entinostat. The effect on MYC was investigated by quantitative real-time PCR, western blot, and immunofluorescence. Results HDAC2 is a cofactor of MYC in MYC amplified medulloblastoma. The MYC-HDAC2 complex is bound to genes defining the MYC-dependent transcriptional profile. Class I HDAC inhibition leads to stabilization and reduced DNA binding of MYC protein, inducing a downregulation of MYC activated genes (MAGs) and upregulation of MYC repressed genes (MRGs). MAGs and MRGs are characterized by opposing biological functions and by distinct enhancer-box distribution. Conclusions Our data elucidate the molecular interaction of MYC and HDAC2 and support a model in which inhibition of class I HDACs directly targets MYC’s transactivating and transrepressing functions.

Pilocytic astrocytomas as well as other pediatric low-grade gliomas (pLGG) exhibit genetic events leading to aberrant activation of the MAPK pathway. The most common alterations are KIAA1549:BRAF fusions and BRAFV600E and NF1 mutations. Novel drugs targeting the MAPK pathway (MAPKi) are prime candidates for the treatment of these single-pathway diseases. We aimed to develop an assay suitable for preclinical testing of MAPKi in pLGGs with the goal to identify novel MAPK pathway–suppressing synergistic drug combinations. A reporter plasmid (pDIPZ) with a MAPK-responsive ELK-1–binding element driving the expression of destabilized firefly luciferase was generated and packaged using a lentiviral vector system. Pediatric glioma cell lines with a BRAF fusion (DKFZ-BT66) and a BRAFV600E mutation (BT-40) background, respectively, were stably transfected. Modulation of the MAPK pathway activity by MAPKi was measured using the luciferase reporter and validated by detection of phosphorylated protein levels. A screening of a MAPKi library was performed, and synergy of selected combinations was calculated. Screening of a MAPKi library revealed MEK inhibitors as the class inhibiting the pathway with the lowest IC50s, followed by ERK and next-generation RAF inhibitors. Combination treatments with different MAPKi classes showed synergistic effects in BRAF fusion as well as BRAFV600E mutation backgrounds. Here, we report a novel reporter assay for medium- to high-throughput preclinical drug testing in pLGG cell lines. The assay confirmed MEK, ERK, and next-generation RAF inhibitors as potential treatment approaches for KIAA1549:BRAF and BRAFV600E-mutated pLGGs. In addition, the assay revealed that combination treatments synergistically suppressed MAPK pathway activity.

Large-scale molecular profiling studies in recent years have shown that central nervous system (CNS) tumors display a much greater heterogeneity in terms of molecularly distinct entities, cellular origins and genetic drivers than anticipated from histological assessment. DNA methylation profiling has emerged as a useful tool for robust tumor classification, providing new insights into these heterogeneous molecular classes. This is particularly true for rare CNS tumors with a broad morphological spectrum, which are not possible to assign as separate entities based on histological similarity alone. Here, we describe a molecularly distinct subset of predominantly pediatric CNS neoplasms (n = 60) that harbor PATZ1 fusions. The original histological diagnoses of these tumors covered a wide spectrum of tumor types and malignancy grades. While the single most common diagnosis was glioblastoma (GBM), clinical data of the PATZ1-fused tumors showed a better prognosis than typical GBM, despite frequent relapses. RNA sequencing revealed recurrent MN1:PATZ1 or EWSR1:PATZ1 fusions related to (often extensive) copy number variations on chromosome 22, where PATZ1 and the two fusion partners are located. These fusions have individually been reported in a number of glial/glioneuronal tumors, as well as extracranial sarcomas. We show here that they are more common than previously acknowledged, and together define a biologically distinct CNS tumor type with high expression of neural development markers such as PAX2, GATA2 and IGF2. Drug screening performed on the MN1:PATZ1 fusion-bearing KS-1 brain tumor cell line revealed preliminary candidates for further study. In summary, PATZ1 fusions define a molecular class of histologically polyphenotypic neuroepithelial tumors, which show an intermediate prognosis under current treatment regimens.

Long-term complications such as radiation-induced second malignancies occur in a subset of patients following radiation-therapy, particularly relevant in pediatric patients due to the long follow-up period in case of survival. Radiation-induced gliomas (RIGs) have been reported in patients after treatment with cranial irradiation for various primary malignancies such as acute lymphoblastic leukemia (ALL) and medulloblastoma (MB). We perform comprehensive (epi-) genetic and expression profiling of RIGs arising after cranial irradiation for MB (n = 23) and ALL (n = 9). Our study reveals a unifying molecular signature for the majority of RIGs, with recurrent PDGFRA amplification and loss of CDKN2A/B and an absence of somatic hotspot mutations in genes encoding histone 3 variants or IDH1/2, uncovering diagnostic markers and potentially actionable targets., Radiation-induced gliomas (RIGs) have been reported in patients after treatment with cranial irradiation for various primary malignancies but their origin are still unclear. Here, the authors define the genomic, epigenetic and transcriptional landscape of 32 RIGs cases.

Molecular markers have become pivotal in brain tumor diagnostics. Mutational analyses by targeted next-generation sequencing of DNA and array-based DNA methylation assessment with copy number analyses are increasingly being used in routine diagnostics. However, the broad variety of gene fusions occurring in brain tumors is marginally covered by these technologies and often only assessed by targeted assays. Here, we assessed the feasibility and clinical value of investigating gene fusions in formalin-fixed paraffin-embedded (FFPE) tumor tissues by next-generation mRNA sequencing in a routine diagnostic setting. After establishment and optimization of a workflow applicable in a routine setting, prospective diagnostic application in a neuropathology department for 26 months yielded relevant fusions in 66 out of 101 (65%) analyzed cases. In 43 (43%) cases, the fusions were of decisive diagnostic relevance and in 40 (40%) cases the fusion genes rendered a druggable target. A major strength of this approach was its ability to detect fusions beyond the canonical alterations for a given entity, and the unbiased search for any fusion event in cases with uncertain diagnosis and, thus, uncertain spectrum of expected fusions. This included both rare variants of established fusions which had evaded prior targeted analyses as well as the detection of previously unreported fusion events. While the impact of fusion detection on diagnostics is highly relevant, it is especially the detection of "druggable" fusions which will most likely provide direct benefit to the patients. The wider application of this approach for unbiased fusion identification therefore promises to be a major advance in identifying alterations with immediate impact on patient care.

Purpose: Pilocytic astrocytoma is the most common childhood brain tumor, characterized by constitutive MAPK activation. MAPK signaling induces oncogene-induced senescence (OIS), which may cause unpredictable growth behavior of pilocytic astrocytomas. The senescence-associated secretory phenotype (SASP) has been shown to regulate OIS, but its role in pilocytic astrocytoma remains unknown. Experimental Design: The patient-derived pilocytic astrocytoma cell culture model, DKFZ-BT66, was used to demonstrate presence of the SASP and analyze its impact on OIS in pilocytic astrocytoma. The model allows for doxycycline-inducible switching between proliferation and OIS. Both states were studied using gene expression profiling (GEP), Western blot, ELISA, and cell viability testing. Primary pilocytic astrocytoma tumors were analyzed by GEP and multiplex assay. Results: SASP factors were upregulated in primary human and murine pilocytic astrocytoma and during OIS in DKFZ-BT66 cells. Conditioned medium induced growth arrest of proliferating pilocytic astrocytoma cells. The SASP factors IL1B and IL6 were upregulated in primary pilocytic astrocytoma, and both pathways were regulated during OIS in DKFZ-BT66. Stimulation with rIL1B but not rIL6 reduced growth of DKFZ-BT66 cells and induced the SASP. Anti-inflammatory treatment with dexamethasone induced regrowth of senescent cells and inhibited the SASP. Senescent DKFZ-BT66 cells responded to senolytic BCL2 inhibitors. High IL1B and SASP expression in pilocytic astrocytoma tumors was associated with favorable progression-free survival. Conclusions: We provide evidence for the SASP regulating OIS in pediatric pilocytic astrocytoma, with IL1B as a relevant mediator. SASP expression could enable prediction of progression in patients with pilocytic astrocytoma. Further investigation of the SASP driving the unpredictable growth of pilocytic astrocytomas, and its possible therapeutic application, is warranted.

BACKGROUND: The Pediatric Targeted Therapy (PTT) 2.0 program was established to enable precision oncology for relapsed pediatric oncology patients by performing a set of molecular analyses on tumor samples to improve diagnostic accuracy and to detect actionable alterations. METHODS: International pediatric oncology patients with relapse or progression after standard of care treatment independent of histological diagnosis were included. Required material was an FFPE sample from any disease episode available to perform a DNA methylation array, customized targeted gene panel sequencing (130 genes), RNA Sanger sequencing in selected cases, and a pathway-specific immunohistochemistry (IHC) panel. For sequencing a blood sample was used as paired constitutional DNA, allowing for detection of potential cancer predisposition syndromes. All molecular results were discussed in an interdisciplinary tumor board and reported back to the submitting centers. The clinical impact of reported findings was assessed by a serial questionnaire-based two year follow-up. RESULTS: n=266 patients were registered, the molecular workup was successfully performed for n=263 (99%) patients. The most frequent diagnostic category was central nervous system tumors (n=172/263, 65%). Integrated molecular diagnostics suggested a refined or changed diagnosis in n=95/172 (55%) brain tumors. Actionable targets were detected in n=106/172 (61%) cases. In n=11/155 (7.1%) of brain tumor patients pathogenic or likely pathogenic constitutional DNA variants with clinical relevance were identified, n=5 (45%) of which were previously unknown. Clinical follow-up of revealed that n=11/131 (12%) of brain tumor patients received mechanism-of-action based treatment matched to the molecular findings in PTT2.0. CONCLUSIONS: Molecular diagnostics adds robust and clinically relevant information on diagnosis, actionable alterations, and cancer predisposition syndromes in CNS and other pediatric tumors even when tissue from the current disease episode is limited.

The ROS proto-oncogene 1 (ROS1) gene is rearranged in various cancers. The translated fusion protein presents an attractive therapeutic target, since specific inhibitors have been approved for several tumor types. In glioma, ROS1 fusions are frequent within infantile hemispheric glioma, and single case reports on occurrences in other glioma types exist. However, a comprehensive analysis spanning the full width of glioma types and subtypes is lacking. We here assessed the spectrum and distribution of ROS1 fusions by screening >20,000 glioma cases for typical chromosomal alterations, with subsequent RNA-sequencing for confirmation of candidate cases. ROS1 fusions were identified in 16 cases, from low grade pilocytic astrocytoma WHO grade 1 to glioblastoma, IDH wildtype WHO grade 4. Thus, despite being enriched in some tumor types, ROS1 fusions are not pathognomonic for specific glioma types and may consitute a relevant target in a variety of cases.

Chromothripsis is a form of genomic instability characterized by the occurrence of tens to hundreds of clustered DNA double-strand breaks in a one-off catastrophic event. Rearrangements associated with chromothripsis are detectable in numerous tumor entities and linked with poor prognosis in some of these, such as Sonic Hedgehog medulloblastoma, neuroblastoma and osteosarcoma. Hence, there is a need for therapeutic strategies eliminating tumor cells with chromothripsis. Defects in DNA double-strand break repair, and in particular homologous recombination repair, have been linked with chromothripsis. Targeting DNA repair deficiencies by synthetic lethality approaches, we performed a synergy screen using drug libraries (n = 375 compounds, 15 models) combined with either a PARP inhibitor or cisplatin. This revealed a synergistic interaction between the HDAC inhibitor romidepsin and PARP inhibition. Functional assays, transcriptome analyses, and in vivo validation in patient-derived xenograft mouse models confirmed the efficacy of the combinatorial treatment.

Sarcomas are malignant soft tissue and bone tumours affecting adults, adolescents and children. They represent a morphologically heterogeneous class of tumours and some entities lack defining histopathological features. Therefore, the diagnosis of sarcomas is burdened with a high inter-observer variability and misclassification rate. Here, we demonstrate classification of soft tissue and bone tumours using a machine learning classifier algorithm based on array-generated DNA methylation data. This sarcoma classifier is trained using a dataset of 1077 methylation profiles from comprehensively pre-characterized cases comprising 62 tumour methylation classes constituting a broad range of soft tissue and bone sarcoma subtypes across the entire age spectrum. The performance is validated in a cohort of 428 sarcomatous tumours, of which 322 cases were classified by the sarcoma classifier. Our results demonstrate the potential of the DNA methylation-based sarcoma classification for research and future diagnostic applications., This work was funded by Deutsche Krebshilfe grant 70112499, the NCT Heidelberg and an Illumina Medical Research Grant. Part of this work was funded by the National Institute of Health Research (to S.B. and Z.J.) and to UCLH Biomedical research centre (BRC399/NS/RB/101410). Human tissues were obtained from University College London NHS Foundation Trust as part of the UK Brain Archive Information Network (BRAIN UK, Ref: 18/004) which is funded by the Medical Research Council and Brain Tumour Research UK. The methylation profiling at NYU is supported by a grant from the Friedberg Charitable Foundation (to M.Sn.). M.Mi. would like to thank the Luxembourg National Research Fond (FNR) for the support (FNR PEARL P16/BM/11192868 grant). Open Access funding enabled and organized by Projekt DEAL.

Targeting oncogenic fusion-genes in pediatric high-grade gliomas (pHGG) with entrectinib has emerged as a highly promising therapeutic approach. Despite ongoing clinical studies, to date, no reports on the treatment of cerebrospinal fluid (CSF) disseminated fusion-positive pHGG exist. Moreover, clinically important information of combination with other treatment modalities such as intrathecal therapy, radiotherapy and other targeted agents is missing. We report on our clinical experience of entrectinib therapy in two CSF disseminated ROS1/NTRK-fusion-positive pHGG cases. Combination of entrectinib with radiotherapy or intrathecal chemotherapy appears to be safe and has the potential to act synergistically with entrectinib treatment. In addition, we demonstrate CSF penetrance of entrectinib for the first time in patient samples suggesting target engagement even upon CSF dissemination. Moreover, in vitro analyses of two novel cell models derived from one case with NTRK-fusion revealed that combination therapy with either a MEK (trametinib) or a CDK4/6 (abemaciclib) inhibitor synergistically enhances entrectinib anticancer effects. In summary, our comprehensive study, including clinical experience, CSF penetrance and in vitro data on entrectinib therapy of NTRK/ROS1-fusion-positive pHGG, provides essential clinical and preclinical insights into the multimodal treatment of these highly aggressive tumors. Our data suggest that combined inhibition of NTRK/ROS1 and other therapeutic vulnerabilities enhances the antitumor effect, which should be followed-up in further preclinical and clinical studies.

Introduction Precise diagnoses and robust detection of actionable alterations is required for individualized treatments. By using extended molecular diagnostics, the Pediatric Targeted Therapy (PTT) 2.0 program aims at the improvement of diagnostic accuracy and detection of actionable alterations for pediatric high-risk patients. The impact of these analyses on clinical management is reported. Methods Pediatric patients with relapsed or progressive tumors after standard of care treatment were included, independent of histological diagnosis. Formalin fixed paraffin embedded material and a blood sample for germline correction were requested. DNA methylation array, targeted gene panel sequencing (130 genes), RNA and Sanger sequencing in selected cases, and immunohistochemistry (IHC) of selected markers (pERK, pAKT, pS6, PD-L1) were performed. A questionnaire-based follow-up was used to determine the clinical impact of the analysis. Results We enrolled n=263 patients from February 2017 to February 2019. Complete molecular analysis was possible for n=260 cases (99%). The most common entities were brain tumors (n=172/260, 65%). In brain tumors, DNA methylation array alone allowed robust diagnostic classification (score of >=0.9) in n=104/172 cases (60%). Actionable targets as detected by copy number calculation, gene panel sequencing, RNA sequencing and IHC were found in n=94/172 (55%) brain tumor cases. The most common actionable targets in brain tumors were MAPK (pERK, BRAF fusions, BRAF V600E), mTOR (pS6), PI3K (pAKT), CDK4/6 (CDKN2A/B loss), and immune checkpoints (PD-L1). Pathogenic germline alterations with clinical relevance were identified in n=12/172 brain tumor cases (6.9%) and were confirmed by Sanger sequencing, 5/12 (41%) of which were previously unknown. Clinical follow-up of subsequent treatment and outcome are ongoing. Conclusion The combination of next-generation diagnostics such as methylation arrays and targeted sequencing in addition to selected IHC markers added robust information with regard to diagnosis and actionable alterations. The impact on clinical decision-making and on outcome is currently being evaluated.

Background Medulloblastoma (MB) is a highly aggressive brain tumour in children. Patients with Group 3 MB harbouring a MYC-amplification (subtype II) show a particularly poor outcome despite high-intensity multimodal therapy. We and others have previously shown that MYC amplified Group 3 MB cells are highly susceptible towards treatment with class I histone deacetylase (HDAC) inhibitors such as entinostat. However, in clinical trials HDACi as a monotherapy show only modest efficacy in solid tumours. We propose to increase the efficacy of class I HDACi by drug combinations. Methods To identify synergistic drug combinations (entinostat + X) for the treatment of MYC amplified MB we performed a drug screen with a library of n=75 clinically available compounds as single agents and in combination with entinostat in n=3 MYC amplified vs. n=1 MYC-non amplified cell lines. Synergistic behaviour of the six most promising drug combinations was validated by metabolic activity assays, cell count experiments and gene expression profiling. Synergy was assessed by the Loewe additivity model using a combination of ray design and checkerboard matrix. Results The drug screen revealed n=20/75 drugs that were particularly effective (drug sensitivity score ≥10) in combination with entinostat treatment in all three MYC amplified cell lines. Synergy assessment of the top n=6 drugs confirmed strong synergistic activity with entinostat for n=2 drugs (navitoclax, irinotecan). The BCL-2 family inhibitor navitoclax showed the most robust synergy with entinostat in subsequent validation experiments. Conclusion Several drugs either clinically available or currently in clinical trials, including the BCL-2/Xl/w inhibitor navitoclax, show promising effects in a combination therapy with entinostat for the treatment of MYC amplified Group 3 MB.

Aims KIAA1549-BRAF fusions occur in certain brain tumours and provide druggable targets due to a constitutive activation of the MAP-kinase pathway. We introduce workflows for calling the KIAA1549-BRAF fusion from DNA methylation-array-derived copy number as well as DNA panel sequencing data. Methods Copy number profiles were analysed by automated screening and visual verification of a tandem duplication on chromosome 7q34, indicative of the KIAA1549-BRAF fusion. Pilocytic astrocytomas of the ICGC cohort with known fusion status were used for validation. KIAA1549-BRAF fusions were called from DNA panel sequencing data using the fusion callers Manta, Arriba with modified filtering criteria and deFuse. We screened DNA methylation and panel sequencing data of 7790 specimens from brain tumour and sarcoma entities. Results We identified the fusion in 337 brain tumours with both DNA methylation and panel sequencing data. Among these, we detected the fusion from copy number data in 84% and from DNA panel sequencing data in more than 90% using Arriba with modified filters. While in 74% the KIAA1549-BRAF fusion was detected from both methylation-array-derived copy number and panel sequencing data, in 9% it was detected from copy number data only and in 16% from panel data only. The fusion was almost exclusively found in pilocytic astrocytomas, diffuse leptomeningeal glioneuronal tumours and high-grade astrocytomas with piloid features. Conclusions The KIAA1549-BRAF fusion can be reliably detected from either DNA methylation array or DNA panel data. The use of both methods is recommended for the most sensitive detection of this diagnostically and therapeutically important marker.

Medulloblastoma is among the most common malignant brain tumors in children. Recent studies have identified at least four subgroups of the disease that differ in terms of molecular characteristics and patient outcomes. Despite this heterogeneity, most patients with medulloblastoma receive similar therapies, including surgery, radiation, and intensive chemotherapy. Although these treatments prolong survival, many patients still die from the disease and survivors suffer severe long-term side effects from therapy. We hypothesize that each patient with medulloblastoma is sensitive to different therapies and that tailoring therapy based on the molecular and cellular characteristics of patients' tumors will improve outcomes. To test this, we assembled a panel of orthotopic patient-derived xenografts (PDX) and subjected them to DNA sequencing, gene expression profiling, and high-throughput drug screening. Analysis of DNA sequencing revealed that most medulloblastomas do not have actionable mutations that point to effective therapies. In contrast, gene expression and drug response data provided valuable information about potential therapies for every tumor. For example, drug screening demonstrated that actinomycin D, which is used for treatment of sarcoma but rarely for medulloblastoma, was active against PDXs representing Group 3 medulloblastoma, the most aggressive form of the disease. Functional analysis of tumor cells was successfully used in a clinical setting to identify more treatment options than sequencing alone. These studies suggest that it should be possible to move away from a one-size-fits-all approach and begin to treat each patient with therapies that are effective against their specific tumor. Significance: These findings show that high-throughput drug screening identifies therapies for medulloblastoma that cannot be predicted by genomic or transcriptomic analysis.

Aims: DNA methylation-based central nervous system (CNS) tumour classification has identified numerous molecularly distinct tumour types, and clinically relevant subgroups among known CNS tumour entities that were previously thought to represent homogeneous diseases. Our study aimed at characterizing a novel, molecularly defined variant of glioneuronal CNS tumour. Patients and methods: DNA methylation profiling was performed using the Infinium MethylationEPIC or 450 k BeadChip arrays (Illumina) and analysed using the ‘conumee’ package in R computing environment. Additional gene panel sequencing was also performed. Tumour samples were collected at the German Cancer Research Centre (DKFZ) and provided by multinational collaborators. Histological sections were also collected and independently reviewed. Results: Genome-wide DNA methylation data from >25 000 CNS tumours were screened for clusters separated from established DNA methylation classes, revealing a novel group comprising 31 tumours, mainly found in paediatric patients. This DNA methylation-defined variant of low-grade CNS tumours with glioneuronal differentiation displays recurrent monosomy 14, nuclear clusters within a morphology that is otherwise reminiscent of oligodendroglioma and other established entities with clear cell histology, and a lack of genetic alterations commonly observed in other (paediatric) glioneuronal entities. Conclusions: DNA methylation-based tumour classification is an objective method of assessing tumour origins, which may aid in diagnosis, especially for atypical cases. With increasing sample size, methylation analysis allows for the identification of rare, putative new tumour entities, which are currently not recognized by the WHO classification. Our study revealed the existence of a DNA methylation-defined class of low-grade glioneuronal tumours with recurrent monosomy 14, oligodendroglioma-like features and nuclear clusters.

Embryonal tumours with multilayered rosettes (ETMRs) are aggressive paediatric embryonal brain tumours with a universally poor prognosis1. Here we collected 193 primary ETMRs and 23 matched relapse samples to investigate the genomic landscape of this distinct tumour type. We found that patients with tumours in which the proposed driver C19MC2–4 was not amplified frequently had germline mutations in DICER1 or other microRNA-related aberrations such as somatic amplification of miR-17-92 (also known as MIR17HG). Whole-genome sequencing revealed that tumours had an overall low recurrence of single-nucleotide variants (SNVs), but showed prevalent genomic instability caused by widespread occurrence of R-loop structures. We show that R-loop-associated chromosomal instability can be induced by the loss of DICER1 function. Comparison of primary tumours and matched relapse samples showed a strong conservation of structural variants, but low conservation of SNVs. Moreover, many newly acquired SNVs are associated with a mutational signature related to cisplatin treatment. Finally, we show that targeting R-loops with topoisomerase and PARP inhibitors might be an effective treatment strategy for this deadly disease.

Central nervous system (CNS) tumors account for the highest mortality among pediatric malignancies. Accurate diagnosis is essential for optimal clinical management. The increasing use of molecular diagnostics has opened up novel possibilities for more precise classification of CNS tumors. We here report a single-institutional collection of pediatric CNS tumor cases that underwent a refinement or a change of diagnosis after completion of molecular analysis that affected clinical decision-making including the application of molecularly informed targeted therapies. 13 pediatric CNS tumors were analyzed by conventional histology, immunohistochemistry, and molecular diagnostics including DNA methylation profiling in 12 cases, DNA sequencing in 8 cases and RNA sequencing in 3 cases. 3 tumors had a refinement of diagnosis upon molecular testing, and 6 tumors underwent a change of diagnosis. Targeted therapy was initiated in 5 cases. An underlying cancer predisposition syndrome was detected in 5 cases. Although this case series, retrospective and not population based, has its limitations, insight can be gained regarding precision of diagnosis and clinical management of the patients in selected cases. Accuracy of diagnosis was improved in the cases presented here by the addition of molecular diagnostics, impacting clinical management of affected patients, both in the first-line as well as in the follow-up setting. This additional information may support the clinical decision making in the treatment of challenging pediatric CNS tumors. Prospective testing of the clinical value of molecular diagnostics is currently underway.Die höchsten Mortalitätsraten pädiatrischer Krebserkrankungen sind durch Tumore des zentralen Nervensystems (ZNS) bedingt. Eine präzise Diagnose ist essentiell für eine optimale Behandlung. Durch die zunehmende Verwendung molekularer Diagnostik ergeben sich neue Möglichkeiten der präzisen Klassifizierung von ZNS-Tumoren. Hier berichten wir über eine Fallserie pädiatrischer ZNS-Tumore, deren Diagnose durch molekulare Diagnostik präzisiert oder geändert wurde, mit Einfluss auf die klinische Entscheidungsfindung bis hin zur Anwendung molekular informierter Therapien. Es wurden 13 pädiatrische ZNS Tumore mittels konventioneller Histologie, Immunhistochemie und molekularer Diagnostik analysiert, inklusive DNA-Methylierungsprofil in 12 Fällen, DNA-Sequenzierung in acht Fällen und RNA-Sequenzierung in 3 Fällen. Nach erfolgten molekularen Analysen wurde bei 3 Tumoren die Diagnose präzisiert und bei 6 Tumoren die Diagnose geändert. Eine gezielte Therapie wurde in 5 Fällen eingeleitet. Ein zugrundeliegendes Tumorprädispositionssyndrom wurde in 5 Fällen detektiert. Da diese Fallserie retrospektiver Natur und nicht bevölkerungsbezogen ist, ist die Aussagekraft insgesamt limitiert. Dennoch können wertvolle Einsichten bezüglich präziser Diagnosestellung und daraus folgendem klinischen Management in Einzelfällen gewonnen werden. Die Genauigkeit der Diagnose wurde in den hier vorgestellten Fällen verbessert, mit Einfluss auf das klinische Management der betroffenen Patienten bei Initialtherapie als auch in der Nachsorge. Diese zusätzliche Information kann die klinische Entscheidungsfindung in der Behandlung komplexer pädiatrischer ZNS Tumore unterstützen. Der klinische Nutzen molekularer Diagnostik wird aktuell in Studien geprüft.

Background Medulloblastoma (MB) is one of the most common malignant pediatric CNS tumors. Patients with Group 3 MBs harboring MYC amplification exhibit low survival rates. Surviving patients suffer from therapy-induced sequelae, which calls for new targeted therapy strategies. We and others have previously shown the sensitivity of MYC-amplified MB to class I histone deacetylase (HDAC) inhibition. After demonstrating that the MYC target gene PLK1 is significantly downregulated upon class I HDACi treatment, we hypothesized that inhibition of both HDACs and PLK1 could have synergistic effects. Methods Cell metabolic activity changes upon HDAC and PLK1 inhibitor treatment were measured in MYC-amplified and non-amplified MB cell lines, as well as in an additional MYC-inducible cell line. The interaction effect of both inhibitors was determined by computation of the combination index (CI) using the Chou-Talalay method. Results were validated assessing cell viability, cell cycle, and apoptosis induction. Transcription profile changes after combination treatment were evaluated. Results MYC-amplified MB cell lines were more sensitive than non-amplified cell lines to PLK1i treatment, showing IC50 in clinically achievable concentration ranges. Inhibition of class I HDACs and PLK1 synergistically reduced cell metabolic activity in lower concentrations in MYC-amplified compared to non-amplified MB cell lines. We also observed a significant loss of viability and cells in G1 phase, as well as induction of apoptosis after combination treatment in MYC-amplified cells. MYC target gene sets were significantly downregulated in the MYC-amplified cell line HD-MB03 after treatment with combination. We demonstrated reduction of MYC protein levels upon PLK1i treatment. In vivo evaluation of combination treatment using orthotopic Group 3 MYC-amplified MB PDX models is ongoing. Conclusion Our data suggest that MYC-amplification is a predictive marker for PLK1i treatment in MB. The combination of HDACi and PLKi could be a candidate therapy for future clinical trials for MYC-amplified group 3 MB.

// Florian Selt 1, 2 , Juliane Hohloch 1 , Thomas Hielscher 3 , Felix Sahm 4, 5 , David Capper 4, 5 , Andrey Korshunov 4, 5 Diren Usta 1 , Sebastian Brabetz 6 , Johannes Ridinger 1 , Jonas Ecker 1, 2 , Ina Oehme 1 , Jan Gronych 7, 8 , Viktoria Marquardt 9 , David Pauck 9 , Heidi Bachli 10 , Charles D. Stiles 11 , Andreas von Deimling 4, 5 , Marc Remke 9 , Martin U. Schuhmann 12 , Stefan M. Pfister 2, 6 , Tilman Brummer 13 , David T.W. Jones 6 , Olaf Witt 1, 2, * , Till Milde 1, 2, * 1 Clinical Cooperation Unit Pediatric Oncology (G340), German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Heidelberg, Germany 2 Center for Individualized Pediatric Oncology (ZIPO) and Section of Pediatric Brain Tumors, Department of Pediatric Oncology, Hematology and Immunology, University Hospital Heidelberg, Heidelberg, Germany 3 Division of Biostatistics (C060), German Cancer Research Center (DKFZ), Heidelberg, Germany 4 Department of Neuropathology, University Hospital Heidelberg, Heidelberg, Germany 5 Clinical Cooperation Unit Neuropathology (G380), German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Heidelberg, Germany 6 Division of Pediatric Neurooncology (B062), German Cancer Research Center (DKFZ), Heidelberg, Germany, and German Cancer Consortium (DKTK), Heidelberg, Germany 7 Division of Molecular Genetics (B060), German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Heidelberg, Germany 8 AbbVie Deutschland GmbH & Co. KG, Medical Immunology, Wiesbaden, Germany (current affiliation) 9 Department of Pediatric Oncology, Hematology, and Clinical Immunology, Medical Faculty, University Hospital Dusseldorf, Germany, and Department of Pediatric Neuro-Oncogenomics, German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany 10 Department of Neurosurgery, University Hospital Heidelberg, Heidelberg, Germany 11 Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA 12 Department of Neurosurgery, University Hospital Tubingen, Tubingen, Germany 13 Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University and University Medical Centre, Freiburg, Germany * These authors have contributed equally to this work Correspondence to: Florian Selt, email: f. selt@dkfz.de Keywords: pediatric low grade glioma, pilocytic astrocytoma, KIAA1549:BRAF-fusion, oncogene-induced senescence (OIS), MAPK-inhibitors Received: November 18, 2016 Accepted: November 23, 2016 Published: December 17, 2016 ABSTRACT Pilocytic astrocytoma (PA) is the most frequent pediatric brain tumor. Activation of the MAPK pathway is well established as the oncogenic driver of the disease. It is most frequently caused by KIAA1549:BRAF fusions, and leads to oncogene induced senescence (OIS). OIS is thought to be a major reason for growth arrest of PA cells in vitro and in vivo , preventing establishment of PA cultures. Hence, valid preclinical models are currently very limited, but preclinical testing of new compounds is urgently needed. We transduced the PA short-term culture DKFZ-BT66 derived from the PA of a 2-year old patient with a doxycycline-inducible system coding for Simian Vacuolating Virus 40 Large T Antigen (SV40-TAg). SV40-TAg inhibits TP53/CDKN1A and CDKN2A/RB1, two pathways critical for OIS induction and maintenance. DNA methylation array and KIAA1549:BRAF fusion analysis confirmed pilocytic astrocytoma identity of DKFZ-BT66 cells after establishment. Readouts were analyzed in proliferating as well as senescent states, including cell counts, viability, cell cycle analysis, expression of SV40-Tag, CDKN2A (p16), CDKN1A (p21), and TP53 (p53) protein, and gene-expression profiling. Selected MAPK inhibitors (MAPKi) including clinically available MEK inhibitors (MEKi) were tested in vitro . Expression of SV40-TAg enabled the cells to bypass OIS and to resume proliferation with a mean doubling time of 45h allowing for propagation and long-term culture. Withdrawal of doxycycline led to an immediate decrease of SV40-TAg expression, appearance of senescent morphology, upregulation of CDKI proteins and a subsequent G1 growth arrest in line with the re-induction of senescence. DKFZ-BT66 cells still underwent replicative senescence that was overcome by TERT expression. Testing of a set of MAPKi revealed differential responses in DKFZ-BT66. MEKi efficiently inhibited MAPK signaling at clinically achievable concentrations, while BRAF V600E- and RAF Type II inhibitors showed paradoxical activation. Taken together, we have established the first patient-derived long term expandable PA cell line expressing the KIAA1549:BRAF-fusion suitable for preclinical drug testing.

Pilocytic astrocytomas as well as other pediatric low-grade gliomas (pLGG) exhibit genetic events leading to aberrant activation of the MAPK pathway. The most common alterations are

Brain tumors presenting with histological and molecular features not matching any established category in the WHO classification are challenging for diagnosticians and clinicians. Albeit rare, these cases can belong to distinct, as of yet not recognized entities. Based on unsupervised clustering of DNA methylation data from >30,000 tumors, we identified a group of tumors forming a cluster separate from all established entities. Histological reports revealed varying diagnoses and WHO grading. DNA and/or RNA sequencing of nine cases revealed NTRK fusions in five samples and fusions involving EGFR, FGFR1 and MET in single cases. Homozygous deletion at 9p21 encompassing CDKN2A/B were detected in 4/9 patients (44%). No other recurrent, focal copy number aberrations were observed. Considering cellular differentiation, histological features of anaplasia with high proliferative activity and the endothelial proliferation, the tumor entity was termed anaplastic neuroepithelial tumor with condensed nuclei (ANTCoN). The clinical findings provisionally support its classification as WHO grade III. The high frequency of NTRK fusions opens opportunities for targeted therapy. As a pre-clinical platform, a representative mouse model of an NTRK2-fused tumor with deletion of CDKN2A/B was generated using in utero electroporation in CD1 immunocompetent mice. A combination of the fusion plus loss-of-function of either p53 or cdkn2a resulted in tumors with a penetrance of 96% and mean latencies of 42.6 and 74.5 days, respectively. Histology of the murine tumors resembled human ANTCoN. This model will allow comparing the efficacy of Trk inhibitors in vivo to accelerate pre-clinical testing towards drug approval. In summary, the identification of this novel entity underlines the concept of the current WHO classification for brain tumors, that virtually all cases can be clearly categorized based on combined histological and molecular profiling. The detection of promising therapeutic targets in such groups, as in ANTCoN, can also provide significant opportunities for treatment.

Background Until today, adult and pediatric clinical trials investigating single-agent or combinatorial HDAC inhibitors including vorinostat in solid tumors have largely failed to demonstrate efficacy. These results may in part be explained by data from preclinical models showing significant activity only at higher concentrations compared to those achieved with current dosing regimens. In the current pediatric trial, we applied an intra-patient dose escalation design. The purpose of this trial was to determine a safe dose recommendation (SDR) of single-agent vorinostat for intra-patient dose escalation, pharmacokinetic analyses (PK), and activity evaluation in children (3–18 years) with relapsed or therapy-refractory malignancies. Results A phase I intra-patient dose (de)escalation was performed until individual maximum tolerated dose (MTD). The starting dose was 180 mg/m2/day with weekly dose escalations of 50 mg/m2 until DLT/maximum dose. After MTD determination, patients seamlessly continued in phase II with disease assessments every 3 months. PK and plasma cytokine profiles were determined. Fifty of 52 patients received treatment. n = 27/50 (54%) completed the intra-patient (de)escalation and entered phase II. An SDR of 130 mg/m2/day was determined (maximum, 580 mg/m2/day). n = 46/50 (92%) patients experienced treatment-related AEs which were mostly reversible and included thrombocytopenia, fatigue, nausea, diarrhea, anemia, and vomiting. n = 6/50 (12%) had treatment-related SAEs. No treatment-related deaths occurred. Higher dose levels resulted in higher Cmax. Five patients achieved prolonged disease control (> 12 months) and showed a higher Cmax (> 270 ng/mL) and MTDs. Best overall response (combining PR and SD, no CR observed) rate in phase II was 6/27 (22%) with a median PFS and OS of 5.3 and 22.4 months. Low levels of baseline cytokine expression were significantly correlated with favorable outcome. Conclusion An SDR of 130 mg/m2/day for individual dose escalation was determined. Higher drug exposure was associated with responses and long-term disease stabilization with manageable toxicity. Patients with low expression of plasma cytokine levels at baseline were able to tolerate higher doses of vorinostat and benefited from treatment. Baseline cytokine profile is a promising potential predictive biomarker. Trial registration ClinicalTrials.gov, NCT01422499. Registered 24 August 2011

Patients with MYC-driven Group 3 medulloblastoma (MB) show particularly poor outcome. It was previously shown that MYC-driven MBs are highly sensitive to class I histone deacetylase inhibition (HDACi). We studied the molecular effects of the class I HDACi entinostat in MYC-driven MB cells to identify potentially synergistic drug combinations, prioritizing drug clinical availability to enable clinical translation. Gene expression profiles of the MYC-amplified group 3 MB cell line HD-MB03 treated with entinostat were analyzed using bioinformatic approaches, identifying 29 altered biomechanisms. Overlay with a translational drug library of n=76 compounds resulted in 44 compounds targeting 9 biomechanisms. Filtering for publications supporting each drug′s role in MYC-driven entities, or functional interaction with HDACs, without publication of this combination in MBs, resulted in 5 compounds (olaparib, idasanutlin, ribociclib, selinexor, vinblastine). Synergism testing identified olaparib as the drug with the strongest synergism. Validation of the combination olaparib and entinostat by p.H2AX and PI staining as well as trypan blue exclusion showed increased double strand breaks (DSBs), increased cell death, loss of viability and cell numbers. Selectivity of MYC-amplified MB cells was shown by comparison to MYC-non amplified cell lines, which showed higher IC50s, and reacted with cell cycle arrest as opposed to cell death to the combination treatment. The role of HDACis in DNA damage repair was confirmed by increased DSBs when entinostat was added to the combination of olaparib with doxorubicin. Our study identified olaparib as a potential combination partner with entinostat for the treatment of MYC-driven Group 3 MB.