Mutant IDH1 impedes heme synthesis by reducing succinyl-CoA production. (A) Immunoblot to detect ALAS1, ALAS2, and ferrochelatase (FECH) in Ter-119⁺ and Ter-119^– nucleated cells from BM and spleen of control and IDH1-KI mice (n = 3). (B) Relative mRNA levels of ALAS1, ALAS2, HMBS, and FECH in Ter-119⁺ nucleated cells from BM and spleen of control and IDH1-KI mice. Data are the mean ± SEM (n = 4). β-actin, endogenous control. (C) Relative levels of glycine, succinyl-CoA, and 5-ALA in Ter-119⁺ nucleated cells from BM and spleen of control and IDH1-mutant mice as determined by HPLC/LC-MS. Data are the mean ± SEM (n = 6). (D) Diagram outlining the Krebs cycle and its intersection with heme metabolism. (E) Top, microscopic views of representative May-Grünwald-Giemsa–stained mutant IDH1-expressing K562 cells that were treated with glycine, citrate, α-KG, succinate, succinyl-CoA, 5-ALA, or BAY1436032 (mutant IDH1 inhibitor). Scale bar, 50 μm. Bottom, corresponding cell pellets for the cultures in the top panels. (F) Flow cytometric determination of expression of the erythroid marker CD235a by mutant IDH1-expressing K562 cells that were treated with glycine, citrate, α-KG, succinate, succinyl-CoA, 5-ALA, or BAY1436032. Results are representative of 3 trials. (G) Quantitation of heme content in the cells in panel F. Data are the mean ± SEM (n = 5). (H) Immunoblot to detect hemoglobin proteins in mutant IDH1-expressing K562 cells that were treated with succinyl-CoA, 5-ALA, or BAY1436032 (n = 3). *P < .05; **P < .01; ***P < .001. ACO, aconitase; CS, citrate synthase; FH, fumarate hydratase; HMBS, hydroxymethylbilane synthase; MDH, malate dehydrogenase; PBS, phosphate-buffered saline; SDH, succinate dehydrogenase.