Abstract Cyclin-dependent kinases (CDKs) have been an attractive target for cancer research because these kinases are predominantly active during cell division. AG-012986 is a small molecule pan-CDK inhibitor that has in vitro and in vivo antitumor properties. This compound was stopped during development due in part to rapid bone-marrow independent white blood cell toxicity in non-human primate and rodent species, and the potential for acute and delayed immunosuppression in humans. Because peripheral leukocytes are largely nonproliferating cells, it was hypothesized that the effects of AG-012986 was due to an off-target mechanism and not driven by the intended pharmacology. Using primary human peripheral blood mononuclear cells (PBMCs) and T-cells, we show that AG-012986 has a toxicity profile with greater specificity and more rapid cell death compared to the prototypical ATP-competitive kinase inhibitor, staurosporine. The cell toxicity mechanism was caspase-dependent and contained the hallmarks of apoptosis as indicated by cells staining double positive for propidium iodide and annexin-V. PBMCs or T-cells treated with AG-012986 and acutely stimulated either pharmacologically or through the T-cell receptor (TCR), exhibited decreased cellular toxicity compared to non-stimulated control cells. Interestingly under activating conditions with the pan-CDK compound, T-cells did not undergo significant cell division, as measured by BrdU incorporation, and were also rescued from apoptosis. This indicates that the pharmacology of AG-012986 was functioning as expected but that the toxicity due to its off-target effect had now been decoupled through cellular activation. Investigating the downstream signaling network showed that phosphorylation of p38 MAP kinase was impaired in the presence of AG-012986. Thus we hypothesize that AG-012986 may target the upstream kinase MKK3 and/or MKK6 for inhibition under basal conditions, leading to cell death in T cells and perhaps other white blood cells. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A10.