PROJECT SUMMARY Proteasome inhibitors are currently being used in the clinic against Multiple myeloma. This approach is thought to work at least in part because cancer cells appear to rely more heavily on proteasomes than do normal cells. However, our previous studies showed that proteasome inhibition invokes an adaptive program driven by the transcription factor NRF1 which upregulates proteasome genes resulting in the recovery of proteasome activity, thus limiting the efficacy of this approach. Consistent with this notion, our preliminary data suggest that depletion of NRF1 potentiates the action of proteasome inhibition therapy in a breast cancer xenograft model. Taking advantage of the fact that a protease DDI2 is essential for NRF1 activation, here we propose to identify small molecule inhibitors of this protease that could be used to enhance the efficacy of proteasome inhibitors. Moreover, based on our preliminary data that showed depleting DDI2 in itself retards tumor growth, DDI2 inhibitors could also find use as a single-agent. Based on our pilot screen that demonstrates feasibility and consistent with the goals of PAR-20-271, this 4-year project will pursue the following specific aims. In AIM 1, we will express and purify DDI2 protein and perform high-throughput screens using a protein thermal shift (PTS) assay to identify compounds that bind DDI2. In AIM 2, we will perform hit selection, confirmation and profiling using a panel of secondary assays that includes orthogonal and counter-screen assays as well as biophysical assays. In AIM 3, we will perform hit validation, hit expansion, probe selection and profile the mechanism of action of hits, followed by cellular assays to assess DDI2 target engagement, impact on the DDI2-NRF1 axis and cancer cell apoptosis.