Revalidating DNA Damage Inducible 1 Homolog II (DDI2) as a Novel Target for the Treatment of Cancer

Abstract

Cancer cells are under proteotoxic stress and are more sensitive to the perturbation of protein quality control pathways than normal cells. The proteasome is a critical component of the protein quality control machinery. Proteasome inhibitors are approved for treating multiple myeloma, a cancer of plasma cells that produce large amounts of immunoglobulins leading to exceptionally high levels of proteotoxic stress. They have not demonstrated clinical efficacy in other cancers. Recovery of proteasome activity after bolus treatment may contribute to intrinsic and acquired resistance to proteasome inhibitors. Upon treatment with sub-toxic doses of proteasome inhibitors, transcription factor NFE2L1 is activated by a novel protease DDI2 and upregulates the proteasome genes. However, it is not known whether this transcriptional upregulation is responsible for activity recovery. We used RNAi and CRISPR KO cells in this dissertation to answer this question. Although we confirmed that DDI2 processes NFE2L1, DDI2 was not required to recover proteasome activity. The recovery depended on the translation of proteasomal mRNA, and we hypothesize that nascent proteasome subunits assemble more efficiently in the presence of proteasome inhibitors. Based on the CRISPR KO screen and transcriptome analysis, we also report transcriptional downregulation of immunoproteasome subunits and decreased immunoproteasome activity at the basal level. Finally, we describe the design and initial steps of synthesizing an activity-based probe of DDI2, a protease that cannot be assayed with traditional peptide substrates.