Purpose: To assess, in dogs with naturally occurring non-Hodgkin's lymphoma, pharmacokinetics, safety, and activity of GS-9219, a prodrug of the nucleotide analogue 9-(2-phosphonylmethoxyethyl) guanine (PMEG), which delivers PMEG and its phosphorylated metabolites to lymphoid cells with preferential cytotoxicity in cells with a high proliferation index such as lymphoid malignancies. Experimental Design: To generate proof-of-concept, a phase I/II trial was conducted in pet dogs (n = 38) with naturally occurring non-Hodgkin's lymphoma using different dose schedules of GS-9219. A subset of dogs was further evaluated with 3′-deoxy-3′-18 F-fluorothymidine positron emission tomography/computed tomography imaging before and after treatment. Results: The prodrug had a short plasma half-life but yielded high and prolonged intracellular levels of the cytotoxic metabolite PMEG diphosphate in peripheral blood mononuclear cells in the absence of detectable plasma PMEG. Dose-limiting toxicities were generally manageable and reversible and included dermatopathy, neutropenia, and gastrointestinal signs. Antitumor responses were observed in 79% of dogs and occurred in previously untreated dogs and dogs with chemotherapy-refractory non-Hodgkin's lymphoma. The median remission durations observed compare favorably with other monotherapies in dogs with non-Hodgkin's lymphoma. High 3′-deoxy-3′-18 F-fluorothymidine uptake noted in lymphoid tissues before treatment decreased significantly after treatment (P = 0.016). Conclusions: GS-9219 was generally well tolerated and showed significant activity against spontaneous non-Hodgkin's lymphoma as modeled in pet dogs and, as such, supports clinical evaluation in humans.Non-Hodgkin's lymphoma is the fifth leading cause of cancer deaths and the second fastest-growing form of cancer in the United States. In the latest compilation of Surveillance, Epidemiology and End Results reports, the incidence and death rate for non-Hodgkin's lymphoma continue to increase despite an overall decrease in overall cancer incidence (1). Therefore, there is still a major unmet medical need in non-Hodgkin's lymphoma patients for novel agents with improved efficacy compared with existing treatment modalities, especially in patients who have failed frontline therapy.The acyclic nucleotide phosphonate 9-(2-phosphonylmethoxyethyl) guanine (PMEG) forms an active phosphorylated metabolite, PMEG diphosphate, in cells and causes cytotoxicity in dividing cells due to potent inhibition of the nuclear DNA polymerases α, δ, and ε by causing DNA chain termination, resulting in inhibition of DNA synthesis (2). In rodent