Higher B12 serum levels increase clonal fitness in a CH mutant murine model. (A) B12 acts as a cofactor for 2 mammalian enzymes, MS and MCM. MS is the central regulator of one-carbon metabolism that coordinates folate with Met recycling after its processive conversion to SAM and Hcy. Met, upon its breakdown to PP-CoA and, subsequently, MM-CoA, is then converted to Succ-CoA by MCM for entry into the TCA cycle. (B) BM reconstitution assays were performed using CD45.2+Sf3b1+/+ or Sf3b1+/K700E donor cells mixed 1:5 with congenic WT CD45.1+ support BM (equivalent to 10% variant allele frequency). Upon confirmation of engraftment (4 weeks after transplant), altered B12 and Met dietary supplementation was initiated compared to a normal diet until 8 months after transplant (7 months of dietary intervention). (C) Serum B12 (pg/ml) measured by enzyme-linked immunosorbent assay in WT mice after 4 months of supplementation with high B12 and/or Met diets compared to normal chow. (D-F) Frequency of CD45.2+Sf3b1+/+ or Sf3b1+/K700E cells from mice fed high B12 and Met compared to a normal diet measured by flow cytometry in total cells (D) and CD11b+ cells (E) of peripheral blood, spleen, and BM at month 2 or month 8 after transplant; frequency of CD45.2+Sf3b1+/+ or Sf3b1+/K700E cells in BM Lin–, LK, LSK, hematopoietic stem cells (CD150+CD48–), and myeloid-primed multipotent progenitor cells (CD150–CD48+) at month 8 after transplant (F). (G-H) Fold change in competitiveness of CD45.2+Sf3b1+/+ or Sf3b1+/K700E cells at month 8 after transplant in CD11b+ cells (G) and BM hematopoietic stem and progenitor cells (H). Data shown are the mean and range of cell frequencies and fold changes. Student t test; ∗P < .05; ∗∗P < .01. BM, bone marrow; Hcy, homocysteine; Lin–, lineage negative; LK, ckit+Sca1–; LSK, Lin–cKit+Sca1+; Met, methionine; MM-CoA, methylmalonyl coenzyme A; MS, methionine synthase; PP-CoA, propionyl coenzyme A; SAM, s-adenosylmethionine; Succ-CoA, succinyl coenzyme A; TCA, Tricarboxylic acid cycle; WT, wild-type.
