Homocysteine is recycled via MTR and not BHMTs in the absence of methionine. (A-D) Normalized relative cell counts of AML cell lines K562, MOLM13, and HL60, as well as 1 primary AML sample grown with and without methionine and in the absence of methionine but increasing concentrations of homocysteine (Hcy) (n = 3) (AML#9). (E) Western blot for H3K36me3, MAT2A, and MTR protein levels in lysates from MOLM13 and K562 cells grown for 22 and 44 hours in the presence and absence of methionine and without methionine but supplemented with 1 mM Hcy. β-Actin and unmodified histone H3 were used as loading controls. M = marker (F) Ratio of C4:C5 ¹³C-labeled methionine in cell lines grown with universally labeled ¹³C methionine tracer. Data were generated by NMR spectroscopy (n = 2-3) biological replicates measured at 1 time point). (G) Western blot for MTR in K562 scrambled guide, BHMT KO, BHMT2 KO, and MTR KO cells. (H) Relative cell counts of K562 scrambled guide, BHMT KO, BHMT2 KO, and MTR KO cells grown with and without methionine and without methionine but with 1 mM Hcy. (I) Relative reverse transcription−polymerase chain reaction determined expression of BHMT2 in K562 scrambled guide, BHMT KO, BHMT2 KO, and MTR KO cells. (J) Relative cell counts of K562 scrambled guide, BHMT KO, BHMT2 KO, and MTR KO cells grown with and without methionine and without methionine but with 1 mM Hcy. (K) Transcriptome data of MTR expression in primary AML and healthy hematopoietic populations (BloodSpot.eu dataset). Probeset – 226969at. (L) Viable cell number/mL of DAPI⁻ K562 cells grown in the presence or absence of 25 or 50 μM SAHH inhibitor (3-DZA) for 48 hours, and within the last 24 hours with and without methionine and with and without 1 mM Hcy. N = 2 experiments in K562 cells, representative of 2 independent experiments in HL60 cells. (M) Percent viable DAPI⁻ K562 cells grown in the presence or absence of 25 or 50 μM SAHH inhibitor (3-DZA) for 48 hours, and within the last 24 hours with and without methionine and with and without 1 mM Hcy. N = 2 experiments in K562 cells, representative of 2 independent experiments in HL60 cells. (N) Western blot for H3K36me3 and MAT2A levels in K562 cells grown in the presence or absence of 25 or 50 μM SAHH inhibitor (3-DZA) for 72 hours, and within the last 24 hours with and without methionine and with and without 1 mM Hcy. β-Actin and unmodified histone H3 were used as loading controls. All experiments: error bars represent mean ± SEM. Statistical analysis: ordinary 1-way ANOVA or Student t test, ∗P < .05, ∗∗P < .01, ∗∗∗∗P < .0001.