Extended Data Fig. 8: 2-HG disrupts interaction between Mdm2 and mutant p53, and correlation of ME2 expression with cellular 2-HG abundance and survival of cancer patients.
From: Malic enzyme 2 maintains protein stability of mutant p53 through 2-hydroxyglutarate
a-b and d-g, Purified Flag-Mdm2 proteins were incubated with HA-p53G266E (a, d, f) or HA-p53R175H (b, e, g) proteins bound on HA-beads in the presence of increasing amounts of 2-HG, α-KG or pyruvate for 1 hour. Immunoprecipitates were analyzed by western blot using indicated antibodies. c and h, Purified Flag-Mdm2 proteins were incubated with HA-tagged p53R248W (c) or p53 (h) proteins bound on HA-beads in the presence of increasing amounts of 2-HG for 1 hour. Immunoprecipitates were analyzed by western blot using indicated antibodies. i, Absolute amounts of 2-HG in SF188 cells transfected with Ctrl or ME2 siRNA. The dissociation constant Kd of p53G266E for 2-HG is indicated. j and k, Absolute amounts of 2-HG in MDA-MB-231 cells (j) and U251MG cells (k) transfected with Ctrl or ME2 siRNA for 48 hours. Western blot confirms ME2 knockdown efficiency and shows expression of p53. l, Western blot of SF188 cells treated with increasing amounts of Ocytl-2-HG for 24 hours. m, Expression of ME2 in normal brain tissues and gliomas of different stages was determined by immunohistochemistry (proteinatlas.org). Magnified regions of ME2 staining are provided. n, Kaplan–Meier survival curves of patients with glioma based on ME2 expression. Data were generated from French dataset via ‘R2: Genomics Analysis and Visualization Platform. In the cohort (n = 273), a z-score threshold of 2.0 was used to identify tumours with high levels of ME2 expression; this included 6.97% (n = 19) of tumours. o, Related to Fig. 5b. Kaplan-Meier survival curves of patients with liver cancer based on ME2 expression and IDH1/2 mutations. In this TCGA liver hepatocellular carcinoma cohort (TCGA LIHC, n = 371), IDH1/2 mutations were observed in 3% of patient tumors (n = 11), and TP53 mutations in 30% samples (n = 107). The top 1/4 and bottom 1/4 of samples were selected based on the expression levels of ME2 to test the correlation between ME2 levels and overall survival in patients with or without IDH1/2 alterations. Data in i, j, k are means ± S.D, unpaired two-tailed Student’s t-tests.
