Abstract
Introduction: Large population studies have shown that elevated vitamin B12 serum levels are associated with increased all-cause mortality and myeloid cell malignant transformation, and our lab has recently shown that serum B12 levels are elevated in individuals with clonal hematopoiesis (CH). B12 is an essential dietary nutrient and a key regulator of one-carbon metabolism, where it acts as a cofactor of methionine synthase (MS) for the recycling of methionine from homocysteine. Regulation of the methionine cycle by B12 maintains the production of s-adenosylmethionine (SAM), the methyl donor used in all DNA, RNA, and protein methylation reactions in the cell. Dysregulation of DNA methylation plays a driving role in AML, and given the influence of B12 and 1C metabolism on DNA methylation maintenance, B12 may influence the epigenetic state of AML cells. B12 also feeds Met and odd-chain fatty acids into the tricarboxylic acid (TCA) cycle by acting as a cofactor for Methylmalonyl-CoA Mutase (MCM), generating the oncometabolite succinate, a known inhibitor of DNA methylation regulator tet methylcytosine dioxygenase 2 (TET2). Therefore, we hypothesized that high B12 levels may promote malignant transformation of the myeloid lineage by altering DNA methylation maintenance and metabolic activity in pre-malignant HSPCs and sustain DNA hypermethylation patterns associated with worse survival.
Methods: Using human myelomonocytic leukemia cell lines, (MOLM-13 and THP-1), we sought to determine the effect of altering B12 levels on the metabolic, epigenetic and transcriptional profile of AML. Cells were grown in RPMI media formulated with dialyzed fetal bovine serum to remove exogenous sources of B12, then supplemented with CNCbl at the normal amount of B12 typical of this media (5ng/L) and low (no supplementation) or high (2000-fold higher) concentrations compared to the normal amount of B12 in RPMI. After 4 months of culture in altered B12 media, we performed liquid-chromatography and high-resolution mass spectrometry (LC-MS) and seahorse assays, RNA sequencing, and reduced representative bisulfite sequencing (RRBS) and 5-hydroxymethylcytosine DNA-IP sequencing to assess metabolic, transcriptomic, and DNA methylation/hydroxymethylation changes associated with B12 supplementation. To test the impact of elevated B12 on leukemic cells in vivo, we transduced Tet2-deficient murine cKit+ cells with Aml1-Eto9a (Ae9a), transplanted them 1:10 (25k tumor cells:250k CD45.1+ support bone marrow) into lethally irradiated mice placed on diets ranging from deficient, normal, high B12, high Met, and combined high B12&Met. We also tested the impact of B12 supplementation on a primary mouse model of AML with both Tet2-deficiency and Flt3-ITD mutations (Tet2+/-Flt3ITD/+). Mice between 3-6 month of age were placed on 4 diets (normal, high B12, high Met, and combined high B12&Met) and monitored for leukemic burden and survival. To assess the cell-intrinsic impact of B12 on leukemic cells, we generated Ae9a or Mll-Af9 leukemic cells using cKit+ cells isolated from B12 receptor (Cd320)WT or KO mice and performed CFU assays and in vivo survival studies.
Results: Leukemic cells cultured in the presence of high B12 exhibit elevated levels of 1C metabolites, such as Met and SAM, and elevated succinate levels, indicating an increased supply of succinyl CoA entering the TCA and an increase in the activity of both the two B12-dependent enzymes MS and MCM, respectively. These metabolic perturbations correlated with DNA hypermethylation and reduced hydroxymethylation, which is known to drive myeloid malignancy and confer a poor prognosis in AML. RNAseq evaluation revealed that B12 treatment drives transcriptional changes linked to altered metabolism and increased innate inflammatory signaling, which is associated with worse survival in AML patients. We determined in vivo that increasing B12 serum levels through dietary supplementation of B12 and Met rapidly accelerates leukemia formation and progression in both transplantation and primary mouse models. Genetic deletion of CD320 in Ae9a or Mll models decreased self-renewal in vitro and increased survival in vivo, even in the presence of high B12&Met supplementation.
Conclusions: Our studies show that alterations in B12 levels can impact the epigenome, transcriptome and metabolome of AML cells and provide genetic evidence that targeting B12 metabolism has therapeutic potential for the treatment of AML.