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

The acetylation of DNA can modulate the expression of genes involved in the development and progression of malignancies. Histone deacetylase (HDACs) inhibitors are clinically active and well tolerated in the treatment of a wide variety of tumors. HDACs inhibitors can sensitize cell response to ionizing radiation, potentially reducing treatment doses and side effects. OBJECTIVES: To assess the potential radiosensitizer effect of abexinostat (PCI-24781), a new and potent pan-HDAC inhibitor in two pediatric glioblastoma cell-lines (SF188 and KNS42). METHODS: The effect of PCI-24781 on proliferation rates, clonogenic capacity and apoptosis were compared prior and following cell irradiation. The clonogenic assay was performed without irradiation (48h of incubation) at the doses of 0.25, 0.5, 1 and 2uM of PCI-24781. The clonogenic assay combined with irradiation was performed at 48h using the inhibitor IC30 for each cell-line in combination with irradiation doses of 0.5, 1, 2 and 4Gy. For cell proliferation assays, the intervals of 24h, 48h, 72h and 96h at the doses of 0.5, 1, 2, 4, 8 and 16uM of PCI-24781 were selected for study. Apoptosis were evaluated at 48h-cultures at the doses of 2, 4, 8 and 16uM. RESULTS: The HDAC inhibitor repressed cell proliferation, displaying a more significant effect at 48h in the dose of 2uM (p < 0.05). PCI-24781 also induced apoptosis (p < 0,05); the percentage of cell death for SF188 and KNS42 (at 16uM) was 40% and 55% respectively. Cells demonstrated massive decrease in colony formation with PCI-24781; this effect was more pronounced when drug was combined with irradiation (p < 0.001). CONCLUSION: These data demonstrate potential radiosensitizer and antiproliferative effects of PCI- 24781 on pediatric glioblastoma cell-lines. This study will now focus on key proteins responsible for the double-strand breaks repair caused by irradiation, trying to elucidate epigenetic pathways potentially involved in the therapy with PCI-24781. Financial Support: Fapesp (process no. 2013/15891-8).