Project Summary: The small molecule drug JTE-607 was discovered 20 years ago that inhibits the production of pro-inflammatory cytokines. Animal studies showed that JTE-607 can be used to treat multiple inflammation diseases, including septic shock, acute injury, and endotoxemia, and it has progressed through healthy human volunteer clinical studies. More recently it was discovered that this compound also displays anti-tumor activities. It is particularly potent against acute myeloid leukemia (AML) and Ewing’s sarcoma. Despite these promising pharmacological studies, the mechanism of action of JTE-607 remained unknown until recently. Two studies published recently revealed that JTE-607 is a prodrug that is converted to Compound 2 (Cmp2) in cells, which specifically binds to the CPSF73 protein, an essential mRNA 3’ end processing factor. The 3’ ends of almost all eukaryotic mRNAs are formed in two catalytic steps, an endonucleolytic cleavage and the addition of a string of adenosines (polyadenylation). CPSF73 is the endonuclease responsible for the cleavage step. It has been proposed that JTE-607 kills cancer cells by inducing mRNA 3’ processing and transcription termination defects, which, in turn, cause genome instability and cell death. However, JTE-607 has not been directly tested in human mRNA 3’ processing and it is unclear why JTE-607 only targets specific cancer types. We aim to identify the RNA sequences and the protein “sensors” that determine the drug sensitivity.These studies will reveal the in-depth mechanism of action of a novel anti-inflammation and anti-cancer compound. Characterization of the JTE-607-resistant mRNA 3’ processing may identify novel target(s) for blocking mRNA 3’ processing in future drug development. From the perspective of basic science, JTE-607 provides a unique tool for dissecting the mechanism of mRNA 3’ processing and the discovery of multiple modes of mRNA 3’ processing will have implications for our fundamental understanding of eukaryotic gene expression.