{
  "abstract": "Background Improving clinical responses to immunotherapy is limited by the source of novel, actionable neoantigens exclusively presented on patient cancer cells. A recent breakthrough in cancer immunotherapy is the identification of non-canonical tumor-specific antigens derived from the dark genome. Tumor antigens derived from the dark genome may evade central tolerance and can be shared across cancer types making them attractive for both vaccine and adoptive T-cell therapies. The immunoproteasome is a highly specialized proteasome variant that degrades intracellular and viral proteins to generate peptides which are transported into the endoplasmic reticulum where they bind with MHC-class I molecules for presentation to cytotoxic T-cells. Molecular glues (MGs) target undruggable proteins to create a ternary complex and stabilize protein-protein interactions.Methods We performed a high-throughput screen using a library of 9,600 HDAC-related compounds to identify novel molecular entities that increased immunoproteasome activity. Cell-based assays were performed using multiple myeloma (MM), cutaneous lymphoma and melanoma cells and the lead compound (Compound A). Patient tumor cells were treated with Compound A, lysates prepared, immunoprecipitated using a pan-MHC class I antibody, bound peptides acid-eluted and released peptides quantitated and sequenced by mass spectroscopy (MS). Cell-based, biochemical and biophysical studies characterized the effect of Compound A on the association of accessory proteins with immunoproteasomes.Results Compound A increased immunoproteasome activity in cancer cells up to 3-fold. Treatment of patient tumor cells with Compound A also increased the presentation of pan-MHC bound peptides, tumor-specific neoantigens and human endogenous retroviral (HERV) peptides up to 100-fold relative to untreated cells. Global proteomic integral stability assays revealed that Compound A binds the proteasome structural subunit PSMA1 and promotes association of the proteasome activator PA28 (encoded by PSME1/PSME2) with immunoproteasomes. Silencing of PSMA1, PSME1, or PSME2 as well as treatment with immunoproteasome-specific suicide inhibitors abolished the effects of Compound A. Biophysical studies revealed that Compound A promoted stable association of PA28 with immunoproteasomes. Treatment of patient tumor cells with Compound A significantly increased the cytotoxic effect of allogeneic and autologous CD8+ T-cells.Conclusions The immunoproteasome shapes the MHC-class I antigenic repertoire on tumor cells derived from the dark genome. MGs that target immunoproteasomes promote the identification of immunogenic neoepitopes recognized by CD8 + T cells [1, 2]. Identification of commonly shared and private neoantigens provides a promising avenue to develop broadly applicable immunotherapies and facilitates the development of therapies that can be exploited to expand the scope of TCR-engineered T-cells, T-cell engagers and cancer vaccines.",
  "authors": [
    {
      "affiliations": [
        "University Hospitals, Shaker Heights, OH, USA"
      ],
      "name": "James J Driscoll"
    },
    {
      "affiliations": [
        "University Hospitals of Cleveland, Cleveland, OH, USA"
      ],
      "name": "James J Ignatz-Hoover"
    }
  ],
  "title": "1162 Exploring the dark proteome using molecular glues that activate immunoproteasomes to reveal actionable HLA-restricted tumor-specific peptides",
  "uid": "7260f487-7e89-5894-942d-b70c1ef652bc"
}