biostudies-literatureCalbert MLAlex's Lemonade Stand Foundation for Childhood CancerFrench Infrastructure for Integrated Structural BiologyNational Cancer InstituteNCI NIH HHS683-699https://www.ebi.ac.uk/biostudies/studies/S-EPMC11286238biostudies-literatureUnknown23(5)Anticancer nucleosides are effective against solid tumors and hematologic malignancies, but typically are prone to nucleoside metabolism resistance mechanisms. Using a nucleoside-specific multiplexed high-throughput screening approach, we discovered 4'-ethynyl-2'-deoxycytidine (EdC) as a third-generation anticancer nucleoside prodrug with preferential activity against diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). EdC requires deoxycytidine kinase (DCK) phosphorylation for its activity and induces replication fork arrest and accumulation of cells in S-phase, indicating it acts as a chain terminator. A 2.1Å cocrystal structure of DCK bound to EdC and UDP reveals how the rigid 4'-alkyne of EdC fits within the active site of DCK. Remarkably, EdC was resistant toMolecular cancer therapeutics4'-Ethynyl-2'-Deoxycytidine (EdC) Preferentially Targets Lymphoma and Leukemia Subtypes by Inducing Replicative Stress.PMC11286238K22 CA251649R01 CA279364P30 CA010815P30 CA0560362022 A AwardK22CA251649R01CA279364ANR-10-INSB-05-01R01 CA214799R01 CA236853R01CA236853Childers WEBetzi SRees MGJohnson NGordon JKent TGrana XToma MMRonan MMPomerantz RTWang YRoth JAMitra RBoghossian ASGorman NSaez-Ayala MTyagi MHurtz CEischen CMChandramouly GAdams CMMorelli XCalbert MLAtkins JGoldman ARSkorski TAyyadevara VSSAKrais JJfalse4'-Ethynyl-2'-Deoxycytidine (EdC) Preferentially Targets Lymphoma and Leukemia Subtypes by Inducing Replicative Stress.Anticancer nucleosides are effective against solid tumors and hematologic malignancies, but typically are prone to nucleoside metabolism resistance mechanisms. Using a nucleoside-specific multiplexed high-throughput screening approach, we discovered 4'-ethynyl-2'-deoxycytidine (EdC) as a third-generation anticancer nucleoside prodrug with preferential activity against diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). EdC requires deoxycytidine kinase (DCK) phosphorylation for its activity and induces replication fork arrest and accumulation of cells in S-phase, indicating it acts as a chain terminator. A 2.1Å cocrystal structure of DCK bound to EdC and UDP reveals how the rigid 4'-alkyne of EdC fits within the active site of DCK. Remarkably, EdC was resistant to2024-01-01T00:00:00Z2024 May2025-04-04T00:29:09.698Z2025-04-04T00:29:09.698ZS-EPMC112862383806471210.1158/1535-7163.MCT-23-048710.1158/1535-7163.mct-23-0487