NSD2-derived creatine depletion underlies increased dependence on AK2. (A) DNA methylation in KMS11 NTKO and TKO cells measured by whole-genome bisulfite sequencing (coverage at 19,190,528). (B) Differential abundance of metabolites related to SAM/creatine metabolism in NTKO and TKO MM cells. Relative guanidinoacetate, homocysteine, and spermidine levels were calculated from the metabolomics data. Creatine levels were determined using an enzyme-based fluorometric assay. (C) Immunoblot analysis of H2AX phosphorylation and PARP cleavage in KMS11 NTKO cells 72 hours after IPTG induction of AK2 suppression with and without creatine (50 mM) supplementation. (D) Time-dependent depletion of GFP-positive NTKO cells after transduction with LentiCRISPRv2-GFP-sgAK2 in the presence and absence of creatine (50 mM). (E) Immunoblot analysis of H2AX phosphorylation and PARP cleavage in KMS11 TKO cells 72 hours after IPTG induction of AK2 suppression with or without cyclocreatine (10 mM) treatment. (F) Time-dependent depletion of GFP-positive TKO cells after transduction with LentiCRISPRv2-GFP-sgAK2 in the presence and absence of cyclocreatine (10 mM). Quantitative experiments were performed in 2 or 3 biological replicates as indicated by the individual replicate symbols. (G) Schematic diagram showing the compensatory role of creatine in replenishing cytosolic ATP levels. dcSAM, decarboxylated SAM; SAH, S-adenosyl homocysteine; GAMT, guanidinoacetate N-methyltransferase; CK, creatine kinase. MAT, methionine adenosyltransferase. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001.