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 recent work suggests 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, the parent grant aims to identify small molecule inhibitors of this protease that could be used to enhance the efficacy of proteasome inhibitors. In line with the goals of the parent grant, and based on recent preliminary data, this supplement aims to characterize the role of the protease function of DDI2 in proteasome inhibitor-induced proteotoxic stress response mediated by NRF1. This will facilitate the characterization of the DDI2 inhibitors that emerge from the high- throughput screen that is being implemented in the parent grant.