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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.

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.

(R)-(−)-Mellein 1 and its (S)-(+)-antipode 2 are prepared in six steps and ca. 30% overall yield form (R)- and (S)-propylene oxide, 3 and ent- 3 , respectively.

A new synthetic approach to enantiopure pyranonaphthoquinones is described. (S)-Mellein 10, prepared in 6 steps from (S)-propylene oxide 16, is converted stereospecifically to the (1R,3S)-dimethylpyran 15. The pyran 15 is then converted to the benzoquinone 14, which undergoes regiospecific Diels-Alder reactions with a variety of oxygenated butadienes to give pyranonaphthoquinones including ventiloquinones E, G, L, eleutherin and ent-deoxyquinone A.