AK2 suppression in NSD2-overexpressing MM cells results in DNA replication stress by depleting dNTPs. (A) Volcano plots from metabolomic profiling showing metabolites altered by AK2 suppression in NTKO and TKO cells. (B) Venn diagrams showing metabolites upregulated and downregulated by AK2 depletion NTKO and TKO cells. (C) Pathway analysis of metabolites significantly altered by AK2 suppression in NTKO but not TKO cells. (D) Heatmap of the relative abundance of ribonucleotides and deoxyribonucleotides in AK2-depleted NTKO and TKO cells plotted from the metabolomics data. (E-F) Quantification of dATP and dGTP levels in control and AK2-depleted NSD2-high and -low MM cells by RT-based dNTP assays. All metabolite measurements were performed 72 hours after transduction with the indicated shRNAs in 3 biological replicates. (G) Time-course analysis of replication stress and apoptosis markers after IPTG induction of shAK2 in KMS11 and LP1 MM cell lines. Immunoblots for phosphorylated CHK1 and RPA were quantified by Image J and the signal was normalized to total CHK1 or RPA proteins, respectively. (H-I) In vitro competitive growth assays showing time-dependent depletion of GFP-positive cells after transduction with LentiCRISPRv2-GFP-sgAK2 in the presence and absence of 10× Embryomax nucleoside mix (rNs; 300 μM) or deoxynucleoside pools (dNs; 250 μM). Experiments were performed in 2 or 3 biological replicates as indicated by the individual replicate symbols. (J) Schematic representation of DNA replication fork stalling due to depletion of dNTP pools resulting from impaired RNR activity. (K) Immunoblot analysis of H2AX phosphorylation and PARP cleavage 72 hours after IPTG-induced AK2 depletion in KMS11 and LP1 cells in the presence and absence of 10 μM of the ATM kinase inhibitor (ATMi) KU55933. ∗P< .05, ∗∗P<.01.