Project summary Diffuse large B cell lymphoma (DLBCL), a non-Hodgkin’s lymphoma, is the most common hematologic malignancy in the United States, and occurs in both adults and children. Approximately 40% of adult DLBCL patients relapse or are refractory to current frontline therapy. This highlights an urgent need for new therapeutic options in DLBCL. Many DLBCL cell lines are highly sensitive to compounds that cause replicative stress and/or inhibit the DNA damage response (DDR). Thus, we chose to develop a novel nucleoside analog as a pre-clinical DLBCL therapeutic since this compound class typically induces replicative stress and are highly active in hematological cancers. Nucleoside analogs are the most widely successful drug-class and have been approved for treating hematological cancers, among other indications. Using phenotypical chemical biology combined with a high-throughput multiplexed 930 cancer cell line screen, we identified 4’-ethynyl-deoxycytidine (EdC) as a highly effective and unique pre-clinical therapeutic for DLBCL. EdC exhibits nanomolar potency in DLBCL (GCB and ABC subtypes) harboring alterations in epigenetic factors (KMT2D, EZH2), BCL2 and/or MYC and regresses DLBCL tumors in mice within 3-5 days without weight loss or toxicity. In contrast to traditional nucleoside analogs, EdC uniquely acts as a monophosphate and induces replicative stress by inhibiting de novo dCTP synthesis. To further characterize and develop EdC as a unique and highly promising pre-clinical therapeutic for DLBCL, we plan to develop the following Aims: 1. Elucidate the mechanism of action of EdC; 2. Characterize biomarkers for DLBCL sensitivity to EdC; 3. Investigate the efficacy of EdC on DLBCL in vivo. This proposal is expected to further elucidate the mechanisms, biomarkers, and in vivo efficacy of EdC as a new pre-clinical candidate for DLBCL.