DIPG-43. Glucosylceramide synthase inhibitors induce ceramide accumulation and sensitise H3K27 Mutant Diffuse midline Glioma to irradiation

BACKGROUND AND AIMS: Glycosphingolipids (GSL) are amphipathic lipids particularly abundant in the brain where their amount and expression patterns change drastically during the embryonic to postnatal stages and during tumorigenesis. The biosynthesis of GSL begins with the formation of glucosylceramide from ceramide, a step catalysed by the glucosylceramide synthase (UGCG). UGCG can be inhibited by eliglustat, which is used for treating children with Gaucher‘s disease. We have previously shown that the GSL composition is deregulated in H3K27M mutant diffuse midline glioma (H3K27M mut) and that eliglustat inhibits cell proliferation. Here we analysed the mechanism of action of eliglustat in H3K27M mut and its effect on irradiation. METHODS: The concentration of different components of the sphingolipid metabolism (ceramide, ceramide-1-Phosphate (CIP), sphingomyelin, Sphingosine and Sphingosine-1-Phosphate (S1P)) was assessed by mass spectrometry in the H3K27M mut cell line SF8628, before and after treatment with eliglustat. The combination of eliglustat with ionizing radiation was analysed by clonogenic assay. RESULTS: The treatment of H3K27M mut cells with eliglustat resulted in a significant increase in the concentration of ceramide, Sphingosine, C1P, but not S1P. The increase was concentration and time dependent and was not observed after longer incubation. Eliglustat treatment reduced the colony formation ability after irradiation. CONCLUSIONS: Ceramide is a known mediator of apoptosis involved in the molecular mechanisms underlying cellular response to irradiation. Increased endogenous ceramide levels, induced by blocking the synthesis of GSL, may sensitize H3K27M mut cells to irradiation. However, ceramide can be converted in C1P, a potent inhibitor of apoptosis and inducer of cell survival. Thus, the time and concentration dependent shift to ceramide and C1P requires further investigation in order to achieve an appropriate balance between the levels of these two metabolites and identify the optimal therapeutic window for combination with irradiation and potentially chemotherapy