Mutant IDH1 activates MAPK signaling. (A) Row-scaled unsupervised hierarchical clustering of cells transduced with HoxA9 and CTL, IDH1wt, or IDH1mut. Three cell lines per group were generated by independent transductions of bone marrow cells and were transplanted in 1 lethally irradiated mouse each. The cells were harvested from bone marrow 4 weeks after transplantation and were sorted for GFP expression. Gene expression profiling using RNA from sorted cells was performed on Affymetrix Mouse 430.2 arrays. After robust multi-array average normalization, unsupervised hierarchical clustering was performed on variance filtered genes based on a log2 interquartile range of 1.6. (B) Graphical representation of enriched categories of gene ontology gene sets for the indicated cell comparisons based on gene set enrichment analysis from gene chip arrays. (C) Enrichment plot for the gene set “positive regulation of MAPK signaling” comparing IDH1mut and IDH1wt cells. NES, normalized enrichment score. (D) In vitro cytotoxicity assays using inhibitors of signaling pathways in HoxA9 cells transduced with CTL, IDH1wt, or IDH1mut (mean ± SEM of 3-5 independent experiments performed in duplicate). (E) In vitro cytotoxicity assay using AZD6244, an inhibitor of MEK1/2 in HoxA9 cells transduced with CTL, IDH1wt, or IDH1mut (mean ± SEM of 3 independent experiments performed in duplicate). (F) In vitro cytotoxicity assay using GSK1120212, an inhibitor of MEK1/2 in HoxA9 cells transduced with CTL, IDH1wt, or IDH1mut (mean ± SEM of 3 independent experiments performed in duplicate). (G) Representative western blot of in vitro–cultured cells for the indicated cell lines using antibodies against ERK, phospho-ERK, and β-actin as a loading control. (H) Ratio of phospho-p44 ERK/total-p44 ERK and phospho-p42 ERK/total-p42 ERK in HoxA9 CTL, HoxA9 IDH1wt, and HoxA9 IDH1mut cells (mean ± SEM of 3 independent experiments). (I) Representative western blot of in vitro–cultured cells for the indicated cell lines using antibodies against phospho-Elk1, total Elk1, phospho-p90RSK (Ser380), phospho-CREB, and β-actin as loading control. (J) Representative western blot of in vitro–cultured cells for the indicated cell lines using antibodies against activated RAS, total RAS, and β-actin, as loading control. (K) Hydrogen peroxide levels in cultured cells of the indicated cells that were incubated with Amplex red reaction mixture for 16 hours (mean ± SEM of 4 independent experiments measured in triplicate). *P < .05; ns, not significant.
