Search

Includes the legends for Suppl. Table S1-S5.

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

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"

PURPOSE: Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) regulates cell growth in response to nutritional status. Central to the mTORC1 function is the Rag-GTPase heterodimer. One component of the Rag heterodimer is RagC (Ras-related GTP-binding protein C), which is encoded by the RRAGC gene. METHODS: Genetic testing via trio exome sequencing was applied to identify the underlying disease cause in 3 infants with dilated cardiomyopathy, hepatopathy, and brain abnormalities, including pachygyria, polymicrogyria, and septo-optic dysplasia. Studies in patient-derived skin fibroblasts and in a HEK293 cell model were performed to investigate the cellular consequences. RESULTS: We identified 3 de novo missense variants in RRAGC (NM_022157.4: c.269C>A, p.(Thr90Asn), c.353C>T, p.(Pro118Leu), and c.343T>C, p.(Trp115Arg)), which were previously reported as occurring somatically in follicular lymphoma. Studies of patient-derived fibroblasts carrying the p.(Thr90Asn) variant revealed increased cell size, as well as dysregulation of mTOR-related p70S6K (ribosomal protein S6 kinase 1) and transcription factor EB signaling. Moreover, subcellular localization of mTOR was decoupled from metabolic state. We confirmed the key findings for all RRAGC variants described in this study in a HEK293 cell model. CONCLUSION: The above results are in line with a constitutive overactivation of the mTORC1 pathway. Our study establishes de novo missense variants in RRAGC as cause of an early-onset mTORopathy with unfavorable prognosis.

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.

BackgroundWhile major advances have been made in improving the quality of life and survival of children with most forms of medulloblastoma (MB), those with MYC-driven tumors (Grp3-MB) still suffer significant morbidity and mortality. There is an urgent need to explore multimodal therapeutic regimens which are effective and safe for children. Large-scale studies have revealed abnormal cancer epigenomes caused by mutations and structural alterations of chromatin modifiers, aberrant DNA methylation, and histone modification signatures. Therefore, targeting epigenetic modifiers for cancer treatment has gained increasing interest, and inhibitors for various epigenetic modulators have been intensively studied in clinical trials. Here, we report a cross-entity, epigenetic drug screen to evaluate therapeutic vulnerabilities in MYC amplified MB, which sensitizes them to macrophage-mediated phagocytosis by targeting the CD47-signal regulatory protein α (SIRPα) innate checkpoint pathway.MethodsWe performed a primary screen including 78 epigenetic inhibitors and a secondary screen including 20 histone deacetylase inhibitors (HDACi) to compare response profiles in atypical teratoid/rhabdoid tumor (AT/RT, n=11), MB (n=14), and glioblastoma (n=14). This unbiased approach revealed the preferential activity of HDACi in MYC-driven MB. Importantly, the class I selective HDACi, CI-994, showed significant cell viability reduction mediated by induction of apoptosis in MYC-driven MB, with little-to-no activity in non-MYC-driven MB, AT/RT, and glioblastoma in vitro. We tested the combinatorial effect of targeting class I HDACs and the CD47-SIRPa phagocytosis checkpoint pathway using in vitro phagocytosis assays and in vivo orthotopic xenograft models.ResultsCI-994 displayed antitumoral effects at the primary site and the metastatic compartment in two orthotopic mouse models of MYC-driven MB. Furthermore, RNA sequencing revealed nuclear factor-kB (NF-κB) pathway induction as a response to CI-994 treatment, followed by transglutaminase 2 (TGM2) expression, which enhanced inflammatory cytokine secretion. We further show interferon-γ release and cell surface expression of engulfment (‘eat-me’) signals (such as calreticulin). Finally, combining CI-994 treatment with an anti-CD47 mAb targeting the CD47-SIRPα phagocytosis checkpoint enhanced in vitro phagocytosis and survival in tumor-bearing mice.ConclusionTogether, these findings suggest a dynamic relationship between MYC amplification and innate immune suppression in MYC amplified MB and support further investigation of phagocytosis modulation as a strategy to enhance cancer immunotherapy responses.

Brain metastases (BMs) are a devastating complication of advanced cancers associated with poor prognosis. Contrarily to the current improvement in systemic therapies, BMs are still incurable and one of the main causes of death in cancer patients. We analyzed BMs from thirty patients with various primary tumor origins by RNA sequencing and identified the upregulation of UBE2C, a gene involved in the correct transition from metaphase to anaphase. Using an independent cohort of patients with BMs, we demonstrated that high protein expression of UBE2C was associated with worse survival. UBE2C-driven cancer cells promoted migration and invasion in vitro and induced an aggressive phenotype and decreased survival in mouse orthotopic xenografts. PI3K/mTOR inhibition effectively blocked cancer cell signaling and prevented the development of leptomeningeal metastases. Therefore, we have identified UBE2C as a molecular marker of worse outcome in BMs patients and pre-clinically validated an effective therapy against UBE2C-driven brain metastatic disease.

Medulloblastoma is the most common malignant brain cancer in children. Since previous studies have mainly focused on alterations in the coding genome, our understanding of the contribution of long noncoding RNAs (lncRNAs) to medulloblastoma biology is just emerging. Using patient-derived data, we show that the promoter of lncRNA TP73-AS1 is hypomethylated and that the transcript is highly expressed in the SHH subgroup. Furthermore, high expression of TP73-AS1 is correlated with poor outcome in patients with TP53 wild-type SHH tumors. Silencing TP73-AS1 in medulloblastoma tumor cells induced apoptosis, while proliferation and migration were inhibited in culture. In vivo, silencing TP73-AS1 in medulloblastoma tumor cells resulted in reduced tumor growth, reduced proliferation of tumor cells, increased apoptosis and led to prolonged survival of tumor-bearing mice. Together, our study suggests that the lncRNA TP73-AS1 is a prognostic marker and therapeutic target in medulloblastoma tumors and serves as a proof of concept that lncRNAs are important factors in the disease.

The utility of patient-derived tumor cell lines as experimental models for glioblastoma has been challenged by limited representation of the in vivo tumor biology and low clinical translatability. Here, we report on longitudinal epigenetic and transcriptional profiling of seven glioblastoma spheroid cell line models cultured over an extended period. Molecular profiles were associated with drug response data obtained for 231 clinically used drugs. We show that the glioblastoma spheroid models remained molecularly stable and displayed reproducible drug responses over prolonged culture times of 30 in vitro passages. Integration of gene expression and drug response data identified predictive gene signatures linked to sensitivity to specific drugs, indicating the potential of gene expression-based prediction of glioblastoma therapy response. Our data thus empowers glioblastoma spheroid disease modeling as a useful preclinical assay that may uncover novel therapeutic vulnerabilities and associated molecular alterations.

Medulloblastoma is the most common malignant brain tumor in childhood and comprises four distinct molecular subgroups with further layers of intertumoral heterogeneity. Amplification of the oncogene MYC drives tumorigenesis and constitutes a hallmark feature underlying Group 3 biology. Employing our in-house drug screening pipeline, we evaluated a library of epigenetic inhibitors (n=78) in various brain tumor cell lines followed by a secondary HDACi library (n=20) screen, we identified the clinically established, class I selective HDACi CI-994 as the compound with the most preferential antitumoral effect in MYC-driven medulloblastoma. We confirmed that the inhibitor response was in part MYC-dependent as our lentiviral-based MYC-overexpression model showed higher sensitivity towards CI-994 treatment as compared to the isogenic control with low endogenous MYC expression. CI-994 showed significant antitumoral effects at the primary site and at the metastatic compartment in two orthotopic mouse models of MYC-driven medulloblastoma. RNA sequencing profiling of tumor cells treated with CI-994 at IC50 revealed an up-regulation of multiple innate inflammatory pathways like NFκB, TLR4, Interferon-gamma, and TGFbeta. Flow cytometry analysis revealed an increased surface expression of MHC-I. We combined CI-994 with an anti-body against the innate checkpoint CD47 which acts as a “don’t eat me” signal previously shown by us to have significant anti-tumor activity against MYC-driven MB. Combining CI-994 with anti-CD47 shows a significant increase in macrophage-mediated phagocytosis of tumor cells and a significant increase in the survival of tumor-bearing mice.

The dissemination of cancer cells to the brain parenchyma (brain metastases - BMs) and to the leptomeninges (leptomeningeal dissemination – LD) are a late-stage complication of systemic cancers, with a poor survival. Despite advances in radiation and chemotherapy, including intrathecal administration of anticancer agents, these forms of advanced cancer are incurable. Therefore, there is an unmet clinical need for novel effective therapies that target both parenchymal and leptomeningeal disease. We analysed the transcriptomic profile of BMs from patients with diverse primary tumors, treated at CHULN, to identify genetic drivers of cancer cell dissemination to the brain and potential novel targets for therapy. The most differentially expressed gene codifies a ubiquitin conjugating enzyme (UCE). UCE levels were evaluated in tissue microarrays of BMs from an independent cohort of patients and correlated with clinical data. UCE functional role was assessed in vitro and in vivo using modulated lung and breast cancer cell lines. A high-throughput drug screening was performed to find UCE-targeting compounds. High protein levels of the UCE were associated with decreased survival in patients with BMs, independently of the primary tumor origin. High levels of UCE led to increased migration and invasion abilities in cancer cell lines in vitro, with no effect in proliferation. In vivo, high levels of UCE increased leptomeningeal dissemination and decreased survival in orthotopic models of breast cancer BMs. Leptomeningeal disease promoted by UCE was prevented by oral administration of inhibitor A, identified in our high-throughput drug screening. In conclusion, we have identified UCE as a prognostic marker in patients with BMs from different primary tumors. In orthotopic mouse models of the disease, UCE led to a worse survival and promoted leptomeningeal dissemination. Strikingly, this aggressive disease phenotype was prevented by oral therapy with inhibitor A.

Introduction Resistance to chemotherapy is a common cause of treatment failure in cancer patients and a major problem facing current cancer research. Targeted modulation of oncogenic signaling pathways may be used to systematically characterize drug resistance mechanisms across tumor entities and may help to identify new therapeutic strategies. Since the transcription factor MYC is aberrantly activated in many cancers including pediatric malignant brain tumors, like medulloblastoma (MB), our study focused on MYC-related drug resistance. Methods and Results We performed high-throughput drug screening using our in-house semi-automated platform and identified the HDAC inhibitor Entinostat as a drug that shows promising effects in MYC-driven MB. Investigating genome-wide dCas9-based transcriptional activation screening, potential drug response modulators, mainly TGFB1/Erk/MKNK1 signaling including neural EGFL like 2 (NELL2), were discovered. For further validation, we stably overexpressed NELL2 in MYC-driven MB cells and treated overexpressing cells and the corresponding control cells with Entinostat. Using PI staining, cell cycle status was tracked. Entinostat treatment led to modest induction of cell death in MYC-driven MB control cells but only slightly increased cell death rate in MYC-driven MB cells with NELL2-overexpression. Conclusion We report that the combination of genetic and pharmacological approaches is a powerful approach to study drug resistance. Our data suggest that activation of the TGFB1/Erk/MKNK1 signaling pathway desensitizes MYC-driven MB cells to Entinostat. Synergistic targeting of TGFB1/Erk/MKNK1 signaling and MYC could therefore provide a novel therapeutic option in this aggressive MB subtype.

Background: Glioblastoma is the most common and most lethal primary brain cancer. CBF1 (also known as Recombination signal Binding Protein for immunoglobulin kappa J, RBPJ) is the cardinal transcriptional regulator of the Notch signalling network and has been shown to promote cancer stem-like cells (CSCs) in glioblastoma. Recent studies suggest that some of the malignant properties of CSCs are mediated through the activation of pro-invasive programme of epithelial-to-mesenchymal transition (EMT). Little is known whether CBF1 is involved in the EMT-like phenotype of glioma cells. Methods: In a collection of GBM neurosphere lines, we genetically inhibited CBF1 and investigated the consequences on EMT-related properties, including in vitro invasiveness by Boyden chambers assay, chemoresistance using a clinical drug library screen and glycolytic metabolism assessing live-cell extracellular acidification rate. We also compared CBF1 expression in cells exposed to low and high oxygen tension. In silico analysis in large-scale Western and Eastern patient cohorts investigated the clinical prognostic value of CBF1 expression in low- and high-grade glioma as well as medulloblastoma. Results: Mean CBF1 expression is significantly increased in isocitrate dehydrogenase 1 (IDH1) R132H mutant glioblastoma and serves as prognostic marker for prolonged overall survival in brain tumours, particularly after therapy with temozolomide. Hypoxic regions of glioblastoma have higher CBF1 activation and exposure to low oxygen can induce its expression in glioma cells in vitro. CBF1 inhibition blocks EMT activators such as zinc finger E-box-binding homeobox 1 (ZEB1) and significantly reduces cellular invasion and resistance to clinically approved anticancer drugs. Moreover, we indicate that CBF1 inhibition can impede cellular glycolysis. Conclusions: Mean CBF1 activation in bulk tumour samples serves as a clinical predictive biomarker in brain cancers but its intratumoral and intertumoral expression is highly heterogeneous. Microenvironmental changes such as hypoxia can stimulate the activation of CBF1 in glioblastoma. CBF1 blockade can suppress glioblastoma invasion in vitro in particular in cells undergone EMT such as those found in the hypoxic niche. Targeting CBF1 can be an effective anti-EMT therapy to impede invasive properties and chemosensitivity in those cells.

// 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

Medulloblastoma is the most common malignant brain tumor in childhood and comprises four distinct molecular subgroups with further layers of intertumoral heterogeneity. Amplification of the oncogene MYC drives tumorigenesis and constitutes a hallmark feature underlying Group 3 biology. Metastatic dissemination is frequently observed at diagnosis or recurrence and confers a dismal prognosis. Employing our in-house drug screening pipeline, we evaluated a library of epigenetic inhibitors (n=78) in various brain tumor cell lines including glioblastoma (n=14), medulloblastoma (n=14) and atypical teratoid/rhabdoid tumors (n=11). By this cross-entity approach we revealed preferential activity of histone deacetylase inhibitors (HDACi) in MYC-driven medulloblastoma. In a secondary HDACi (n=20) screen, we identified the clinically established, class I selective HDACi CI-994 as the compound with the most preferential antitumoral effect in MYC-driven medulloblastoma. We confirmed that the inhibitor response was in part MYC-dependent as our lentiviral based MYC-overexpression model showed higher sensitivity towards CI-994 treatment as compared to the isogenic control with low endogenous MYC expression. Moreover, we observed significantly reduced MYC mRNA and protein expression levels, decreased cell viability and induction of apoptosis following CI-994 treatment. Notably, CI-994 showed significant antitumoral effects at the primary site and the metastatic compartment in two orthotopic mouse models of MYC-driven medulloblastoma. Additionally, a screen for synergistic drug interaction with a clinical inhibitor library of approved chemotherapeutics and clinical phase III/IV agents (n=199) revealed a favorable interaction of the NFκB inhibitor bardoxolone methyl with CI-994. In line with our synergy data, RNA sequencing profiling confirmed the functional relevance of NFκB pathway induction upon CI-994 treatment. In all, our pharmacogenomic approach suggests that the combination with NFκB inhibitors has the potential to increase the anitumoral activity of HDACi and the pronounced activity in MYC amplified medulloblastoma, where therapeutic options are limited, is particular noteworthy and warrants further clinical investigation.

Most medulloblastoma protocols worldwide include vincristine during radiation and chemotherapy. A significant dose-limiting toxicity is peripheral neuropathy; however, there is a paucity of data to support the view that omission of vincristine does not impact survival. Herein we report two adolescent patients with Group 4 and SHH medulloblastoma, where vinblastine successfully replaced vincristine with resolution of their peripheral neuropathy. We furthermore show vinblastine is highly active in vitro and demonstrates equivalent antitumoral activity compared to vincristine. Substitution of vincristine with vinblastine in future studies should be considered for all patients with medulloblastoma, particularly those with hereditary neuropathy, severe vincristine toxicity, and adults.

Medulloblastoma (MB) is the most common malignant brain tumor in children and is frequently metastatic at diagnosis. Treatment with surgery, radiation and multi-agent chemotherapy may leave survivors of these brain tumors with long-term deficits as a consequence. One of the four consensus molecular subgroups of MB is the MYC-driven group 3 MB, which is the most malignant type and has a poor prognosis under current therapy. Thus, it is important to discover more effective targeted therapeutic approaches. We conducted a high-throughput drug screening to identify novel compounds showing efficiency in group 3 MB using both clinically established inhibitors (n=196) and clinically-applicable compounds (n=464). More than 20 compounds demonstrated a significantly higher anti-tumoral effect in MYChigh (n=7) compared to MYClow (n=4) MB cell models. Among these compounds, Navitoclax and Clofarabine showed the strongest effect in inducing cell cycle arrest and apoptosis in MYChigh MB models. Furthermore, we show that Navitoclax, an orally bioavailable and blood-brain barrier passing anti-cancer drug, inhibits specifically Bcl-xL proteins. In line, we found a significant correlation between BCL-xL and MYC mRNA levels in 763 primary MB patient samples (Data source: “R2 https://hgserver1.amc.nl”). In addition, Navitoclax and Clofarabine have been tested in cells obtained from MB patient-derived-xenografts, which confirmed their specific efficacy in MYChigh versus MYClow MB. In summary, our approach has identified promising new drugs that significantly reduce cell viability in MYChigh compared to MYClow MB cell models. Our findings point to novel therapeutic vulnerabilities for MB that need to be further validated in vitro and in vivo.

Subgroups of ependymoma, especially RELA fusion-positive and posterior fossa type A tumors, are associated with poor prognosis. Curative therapeutic strategies have not yet been identified. We set up a high-throughput drug screening (HTS) pipeline to evaluate clinically established compounds (n=196) in primary ependymoma cultures (n=12). As culturing ependymoma is challenging, assay miniaturization to 1536-well microplates emerged as a key feature to process HTS despite smallest cell numbers. DNA methylation profiling showed that entity and subgroup affiliation from primary diagnosis was maintained in primary cultures, as assessed through molecular neuropathology 2.0 based classification (MNP 2.0, Capper, D. et al., Nature, 2018). A comparison of HTS data of ependymoma and other pediatric brain tumor models (n=48) revealed a remarkable chemoresistance in vitro. However, we identified Neratinib, an irreversible ERBB2 inhibitor, as the most prominent candidate which was preferentially active in a subset of the investigated ependymoma cultures (n=5). Combinatory treatment with Copanlisib, a PI3K inhibitor, was able to overcome resistance to single agent treatment using Neratinib in established cell lines of ependymoma (n=3) and 2/4 primary cultures for which combinatory treatment could be tested. Finally, we validated efficacy of Neratinib combined with Copanlisib in mice bearing ependymoma xenografts which revealed significantly reduced tumor size compared to vehicle-treated animals. In summary, our study demonstrates that HTS may reveal targeted therapies for pediatric brain tumors. Specifically, we found a synergistic interaction of Neratinib and Copanlisib for treatment of ependymoma, thereby providing a novel therapeutic approach in an otherwise largely chemoresistant entity.

Pilocytic astrocytomas (PAs) and other pediatric low-grade gliomas (pLGGs) exhibit aberrant activation of the MAPK signaling pathway caused by genetic alterations, most commonly KIAA1549:BRAF fusions, BRAF V600E and NF1 mutations. In such a single-pathway disease, novel drugs targeting the MAPK pathway (MAPKi) are prime candidates for treatment. We developed an assay suitable for pre-clinical testing of MAPKi in pLGGs, aiming at the identification of novel MAPK pathway suppressing synergistic drug combinations. We generated a reporter plasmid (pDIPZ) expressing destabilized firefly luciferase driven by a MAPK-responsive ELK-1-binding element, packaged in a lentiviral vector system. We stably transfected pediatric glioma cell lines with a BRAF fusion (DKFZ-BT66) and a BRAFV600E mutation (BT-40) background, respectively. Measurement of MAPK pathway activity was performed using the luciferase reporter. pERK protein levels were detected for validation. We performed a screen of a MAPKi library and calculated Combination Indices of selected combinations. The MAPKi library screen revealed MEK inhibitors as the class inhibiting the pathway with the lowest IC50s, followed by ERK and second generation RAF inhibitors. Synergistic effects in both BRAF-fusion and BRAFV600E mutation backgrounds were observed following combination treatments with different MAPKi classes (RAFi/MEKi, > RAFi/ERKi > MEKi/ERKi). We have generated a novel reporter assay for medium- to high-throughput pre-clinical drug testing of MAPKi in pLGG cell lines. MEK, ERK and next-generation RAF inhibitors were confirmed as potential treatment approaches for KIAA1549:BRAF and BRAFV600E mutated pLGGs. Synergistic suppression of MAPK pathway activity upon combination treatments was revealed using our assay in addition.

Pilocytic astrocytoma (PA) is the most common pediatric brain tumor. Aberrant MAPK signaling, typically mediated by BRAF alterations, drives PA formation. While five-year overall survival rates exceed 95%, incompletely resected tumors recur frequently despite treatment. Therefore, we used proteogenomics to discern the biological heterogeneity of PA to improve classification of this tumor entity and identify novel therapeutic targets. Our proteogenomics approach utilizes RNA sequencing and LC/MS-based proteomic profiling and Similarity Fusion Network (SNF) analysis reveals the biological heterogeneity of PA. Integrative genomics dissects aberrant pathway activation in biological subgroups. Lastly, we utilize a drug screening pipeline to evaluate selective therapeutic activity of conventional anti-cancer and phase III/IV clinical trial drugs in PA culture models. PAs segregate into three groups with distinct clinical and molecular features. Age and tumor location are significantly associated with the SNF groups. BRAF fusions were predominantly observed in Groups 1 and 2, while Group 3 PA largely harbored other alterations leading to MAPK activation. Pathway enrichment analyses reveals genesets involved in primary ciliogenesis in Group 3, while immune response signatures, many SYK-related, are associated with Group 1. Confirming this analysis, the SYK inhibitor R788 was specifically active in Group 1, and less active in other brain tumors models (n=27). In summary, our proteogenomic approach reveals important biological heterogeneity with novel therapeutic targets emerging in PA. These biological insights may improve biological classification and reveal novel therapeutic targets specifically useful for non-resectable tumors with high risk of progressive disease.

Medulloblastoma is the most common malignant pediatric brain tumor and comprises at least four distinct biological subgroups. MYC-driven tumorigenesis constitutes a hallmark feature underlying Group 3 biology and metastatic dissemination at diagnosis or recurrence constitutes a major clinical problem in this highly aggressive subgroup. Employing our institutional drug screening platform, we evaluated an in-house library of over 200 histone deacetylase inhibitors (HDACi) in various brain tumor cell lines and patient-derived primary cultures including glioblastoma (n=8), medulloblastoma (n=10) and atypical teratoid/rhabdoid tumors (n=11). Thereby, we identified CI-994, a clinically established class I specific HDAC inhibitor, which selectively inhibited proliferation of MYC-driven medulloblastoma in our primary and secondary screen. We confirmed the MYC-dependent response in medulloblastoma cell lines with CRISPR/CAS9-based MYC overexpression compared to their isogenic controls with low MYC expression. Notably, inhibitor treatment resulted in significantly reduced MYC mRNA and protein expression levels, decreased cell viability and induction of apoptosis. Additionally, a screen for synergism with a clinical inhibitor library (clinical phase III/IV and approved chemotherapeutics) revealed favorable interaction with NFκB inhibition. Furthermore, integrated proteogenomics using RNA sequencing and proteomic profiling corroborated NFκB pathway activation upon CI-994 treatment. Finally, we demonstrated a significantly prolonged survival, a decrease in tumor growth and spinal metastasis in two orthotopic xenograft mouse models of MYC-driven medulloblastoma. In conclusion, our results suggest a MYC-dependent response to class I HDAC inhibition in medulloblastoma and provide compelling rationale for further development of a novel, potentially highly effective therapeutic strategy against the primary site and, importantly, the metastatic compartment.

BACKGROUND: Histone deacetylases (HDACs) are crucial regulators of epigenetic and posttranslational modifications and therefore represent a promising therapeutic target in cancer cells that harbor distinct epigenomes from normal cells. To exploit the therapeutic potential of HDAC inhibitors (HDACi) we synthesized a unique in-house library of more than 200 inhibitors. For the evaluation of the antitumor effects of the compound library in brain tumor cell lines, we successfully established an optimal screening workflow. METHODS: The screening procedure was streamlined by automated dispensing of cell lines, reagents and inhibitors using state of the art equipment. The drugs were evaluated for their effect on tumor cell viability in a panel of cell lines derived from different brain tumor entities (8 glioblastoma, 10 medulloblastoma and 6 atypical teratoid/rhabdoid tumor cell lines) and compared to 5 normal control tissues. Corresponding dose-response profiles were generated using an optimized bioinformatics workflow. In addition to our in-house library commercially available and clinically used HDACi (e.g. Vorinostat) were included. RESULTS: The semi-automated setup enabled the miniaturization of the assay format to 384-well plates. In combination with additional modifications, a remarkable increase of the overall throughput was realized, while generating accurate and reproducible results. Based on this workflow, we created a unique and comprehensive data set and could thereby identify various HDACi acting universally across brain tumors or being specifically active in distinct tumor entities. Promising candidates are currently being further characterized, e.g. Panobinostat showed activity in the majority of brain tumor models at low nanomolar concentrations. CONCLUSION: The growing importance of HDAC inhibitors is reflected by an increasing number of HDACi in clinical trials, with four HDACi already approved by the FDA for treating lymphomas and multiple myelomas. Our study supports the finding that HDACi are valid therapeutic agents and that selected inhibitors are promising candidates for future epigenetic therapy of primary brain tumors.

Pilocytic astrocytoma (PA) is a well-circumscribed, slow growing, low-grade brain tumor predominantly affecting children and considered a single-pathway disease with activation of MAPK signaling, typically driven by BRAF alterations. While five-year overall survival rates exceed 95%, incompletely resected tumors recur frequently despite radiation and/or chemotherapy. Less favorable responses to multimodal therapies are observed in adults compared to pediatric patients. Thus, we utilized a proteogenomics approach to discern the biological heterogeneity of PA across all age-groups to identify novel therapeutic targets. Our proteogenomic approach encompasses RNA sequencing (RNAseq) and LC/MS-based proteomic profiling in a cohort of pediatric (n=55) and adult cases (n=24). Unsupervised hierarchical clustering is used to determine the biological heterogeneity of the disease. Integrative genomics, combined with downstream pathway analysis, dissects aberrant pathway activation in biological subgroups. Lastly, we utilize a drug screening pipeline encompassing conventional chemotherapeutics and targeted therapeutics (n=211) currently evaluated in phase III and IV clinical trials to validate novel molecular targets in PA culture models (n=8). Pediatric and adult PA segregate into two subgroups identified by unsupervised hierarchical clustering using RNAseq and proteomic profiling (p=0.001). Adult tumors form a single entity, while we observed additional heterogeneity in pediatric tumors segregating into two subgroups (p=0.01). Initial analyses show that adult PA demonstrate an over-representation of genes involved in translation initiation and ciliogenesis according to GeneSet Enrichment Analysis, whereas pediatric PA show a significant enrichment of pathways involved in glycolysis, signal transmission and AKT signaling. In all, our proteogenomic approach reveals important biological heterogeneity in pediatric and adult PA. Ongoing drug profiling is used to determine therapeutics with preferential activity against pediatric and adult PA cultures compared to neural stem cells and astrocytes. These biological insights may improve biological classification and reveal novel therapeutic targets specifically useful for non-resectable tumors with high risk of progressive disease.

Juvenile myelomonocytic leukemia (JMML) is a chronic, poor prognostic myeloid neoplasm of childhood that is characterized by malignant expansion of monocytic cells. Chemo- and radiotherapy are not effective in JMML, therefore allogeneic hematopoietic stem cell transplantation is the only therapy option for most affected children. Relapse is the most frequent cause of treatment failure and event-free-survival at five years is low (approximately 50%). Recent studies showed that in 90% of JMML patients the proliferation of monocytic tumor cells is driven by mutations in a confined set of genes (KRAS, NRAS, PTPN11, NF1 or CBL) that activate the RAS signalling pathway. Drugs specifically targeting this pathway are therefore attractive candidates for therapy of JMML patients. As in vitro models of JMML, we generated inducible pluripotent stem cells (iPSC) stably expressing wildtype or activating oncogenic versions of KRAS (G12D) or NRAS (G13D) as well as iPSCs with CRISPR interference mediated downregulated NF1 expression. Manipulation of KRAS, NRAS, and NF1 expression and activation of downstream signaling targets (MEK, ERK) of the Ras pathway were confirmed by RT-PCR and western blot analyses, respectively. After transduction iPSCs retained typical pluripotency markers and could be differentiated into CD34+ and CD45+ cells of the hematopoietic lineage. We then carried out a screen to test the response of these iPSC cell lines to experimental and clinical drugs targeting the Ras signaling pathway, as well as to other compounds suggested to be promising candidate drugs or drugs already in clinical trial for JMML. In our screen the model cell lines were resistant to all tested MEK-inhibitors, including Selumetinib and Trametinib. The broad receptor tyrosine kinase inhibitor Dovitinib and the DNA methyltransferase inhibitor Azacytidine elicited strong responses in all iPSC cell lines regardless of their KRAS, NRAS or NF1 state. This underlines their extensive, but non-targeted killing potential. In our screen, an experimental small molecule drug induced significantly more cell death in KRAS-G12D iPSCs (IC50 1.5 µM) than in comparable wildtype cells (IC50 3.3 µM, p Our results suggest, that iPSCs with RAS pathway activation due to stable expression of oncogenic KRAS or NRAS or downregulation of NF1 expression are valuable tools for preclinical testing and may identify promising novel lead compounds for JMML treatment. Disclosures No relevant conflicts of interest to declare.

Pilocytic astrocytoma (PA) is the most common pediatric brain tumor. Aberrant MAPK signaling, typically mediated by BRAF alterations, drives PA tumorigenesis. While five-year overall survival rates exceed 95%, tumor recurrence constitutes a major clinical challenge in incompletely resected tumors despite chemotherapeutic or radiation based therapeutic approaches. Therefore, we used proteogenomics to discern the biological heterogeneity of PA to improve classification of this tumor entity and identify novel therapeutic targets. Our proteogenomics approach integrates RNA sequencing, DNA methylation profiling and LC/MS-based proteomic profiling data from a cohort of 75 primary PA samples. Similarity Fusion Network (SNF) analysis was conducted to discern the biological heterogeneity of PA. Integrative genomics was used to identify aberrant pathway activation in these biological subgroups. Lastly, we utilized a drug screening pipeline to evaluate selective therapeutic activity of conventional anti-cancer and phase III/IV clinical trial drugs in PA culture models. Pilocytic astrocytomas segregate into two groups with distinct clinical and molecular features. Age is significantly associated with the SNF groups. BRAF fusions were predominantly observed in Group 1, while Group 2 PAs largely harbored other alterations leading to MAPK activation. Importantly, we could confirm these two distinct biological subgroups in two independent, non-overlapping cohorts. Pathway enrichment analyses reveal genesets involved in ion channel activity, neurotransmitter signaling and cellular respiration, whereas largely SYK-related immune response signatures are associated with Group 1. Confirming this analysis, the SYK inhibitor R788 was specifically active in Group 1 PAs, and less active in other brain tumor models (n=13).

Ependymoma is the third most common brain tumor in children. Subsets of ependymomas, in particular RELA fusion-positive supratentorial ependymomas and posterior fossa type A ependymomas, are associated with frequent recurrence and dismal outcome. Neurosurgical resection and radiotherapy are the main therapeutic options, while response to conventional chemotherapy is limited. We aimed to establish a high-throughput drug screening (HTS) pipeline for comprehensive individualized testing of patient-derived primary cultures to identify novel therapeutic targets for ependymomas. Ependymoma primary cultures (n=6) were established following tumor resection and underwent HTS in 1536-well plates to allow large-scale drug screening despite limited cell numbers. We generated dose-response data with 9 dilution steps (dose range 32.5nM to 25µM) for a clinical inhibitor library (n=196) comprising established chemotherapeutic agents and novel anti-cancer compounds currently in phase III and IV studies. The Infinium MethylationEPIC Array was used to characterize primary tumors and matched primary cultures genomically and epigenetically. DNA methylation and copy number profiles revealed that short-term primary cultures faithfully recapitulate the genomic and epigenetic landscape of the corresponding primary tumors. Comparison of drug response data from ependymomas, other pediatric brain tumors (n=25) and controls (i.e. non-neoplastic neural progenitor cells) confirmed extraordinary chemoresistance of ependymomas but also revealed promising selectively active drugs, such as neratinib, an irreversible ERBB2 inhibitor. Thus, our data demonstrate the feasibility of HTS in primary cultures derived from pediatric brain tumors and provide preclinical evidence suggesting inhibition of ERBB2 as a promising therapeutic option in a subset of otherwise highly chemoresistant ependymomas.