Abstract
Abstract Chordoid gliomas (ChG) are a rare low-grade brain tumor, believed to be derived from tanycytes. An analysis by the team identified a novel mutation present in all ChGs: PRKCA p.D463H. This mutation involves a D463H substitution at the kinase domain of the Protein kinase C alpha (PKCα), it is not found in any other cancer, and represents the hallmark of ChG. The aim of the project is to identify novel and biologically relevant PKCαD463H signaling pathways, which will demonstrate the involvement and the role of this mutated kinase in cellular functions implicated in the development of ChGs. The D463H mutation affects a critical residue of the kinase domain of PKCα, suggesting that such a change modifies substrate affinity. Following the purification of PKCαD463H, we have shown its inactivation via in vitro kinase assays and peptide array. In cellulo fret-based activity reporter assays have shown that PKCαD463H has a dominant negative effect over PKCαWT. Through co-immunoprecipitation we have shown that the mutant protein can bind the WT form and also colocalizes in the cell. Our results also show that the mutation affects the tertiary structure of the protein, resulting in a more open, unstable protein compared to the WT. Phosphoproteomic analysis of HEK cells overexpressing PKC⍺D463H show a decrease of phosphorylation specifically on proteins involved with cell-cell adhesion, including functionally relevant phosphosites. This is in line with our Co-IP mass spectrometry data which shows a decrease in interaction of PKC⍺D463H with proteins involved in cell junctions. The cell of origin, tanycytes, are highly specialized cells that contain important cell-cell junctions and therefore their perturbation could be a potential route of tumorigenesis. By understanding these changes integrated with our snRNAseq and bulk RNAseq of ChGs, and exploration in cellular models, we hope to elucidate the mechanism by which the mutation leads to the development of Chordoid Glioma. Citation Format: Charlotte Bellamy, Hannah Tovell, Florent Dingli, Damarys Loew, Stephane Liva, Selina Schwaighofer, Eduard Stefan, Alexandra Newton, Marc Sanson, Franck Bielle. Functional characterisation of a novel mutation in PRKCA, a major driver of chordoid gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4370.
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