Metabolism-based targeting of MYC via MPC-SOD2 axis-mediated oxidation promotes cellular differentiation in group 3 medulloblastoma

Group 3 medulloblastoma (G3 MB) carries the worst prognosis of all MB subgroups. MYC oncoprotein is elevated in G3 MB tumors; however, the mechanisms that support MYC abundance remain unclear. Using metabolic and mechanistic profiling, we pinpoint a role for mitochondrial metabolism in regulating MYC. Complex-I inhibition decreases MYC abundance in G3 MB, attenuates the expression of MYC-downstream targets, induces differentiation, and prolongs male animal survival. Mechanistically, complex-I inhibition increases inactivating acetylation of antioxidant enzyme SOD2 at K68 and K122, triggering the accumulation of mitochondrial reactive oxygen species that promotes MYC oxidation and degradation in a mitochondrial pyruvate carrier (MPC)-dependent manner. MPC inhibition blocks the acetylation of SOD2 and oxidation of MYC, restoring MYC abundance and self-renewal capacity in G3 MB cells following complex-I inhibition. Identification of this MPC-SOD2 signaling axis reveals a role for metabolism in regulating MYC protein abundance that has clinical implications for treating G3 MB.


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Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection. Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. and visualization online platform (http://r2.amc.nl), accessible from GEO (accession number GSE85217).Patient MB tumor proteomics data was accessed from Archer et al. 2018Archer et al. (doi: 10.1016Archer et al. /j.ccell.2018. Gene sets used in GSEA analysis were accessed from the Molecular Signatures Database (mSigDB, Broad Institute) https://www.gsea-msigdb.org/gsea/msigdb/. No data are excluded.
For vitro/molecular experiments (ie. tumorsphere assays, qPCR, immunoblotting, microscopy, etc.), studies were carried out in 3 or greater independent biological replicates for each cell line with the majority also being replicated in 1-2 additional cell lines for reproducibility. All statistics were performed on independent biological replicates. All attempts at replication were successful and provide an overall reflection of each individual experiment's findings. Two independent vivo studies were performed on 10 and 12 male NOD-SCID gamma mice equally divided to randomly receive either placebo control or experimental IACS-010759 treatment. Tumors from all animals were collected and IHC analysis was performed on slides from all individual animals. IHC for all samples were processed in parallel with representative images displayed and quantification of all samples present in the main figures.
Following intracerebellar transplantation, NOD SCID gamma mice were randomly distributed into groups for IACS-010759 treatment. For all other experiments, cells/samples were randomly assigned into groups Blinding was performed during tissue preparation for IHC. Remaining studies were not blinded because the individual(s) performing the experiment/analysis were also responsible for preparing the samples. Instead, the experiments were replicated as described above and/or repeated in 1-2 additional cell lines.

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Ki67 Cell Signaling Technology 9449S (Immunohistochemistry): Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human Ki-67 protein. cell lymphoma tissue labelling c-Myc with purified ab32072 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.
SOX2 Abcam ab97959 (Immunohistochemistry): Rabbit polyclonal to SOX2. Reacts with: Mouse, Rat, Human. IHC image of SOX2 staining in Human brain glioma formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab97959, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
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