Immunoproteasome activation expands the MHC class I immunopeptidome, unmasks neoantigens and enhances T-cell antimyeloma activity.

Proteasomes generate antigenic peptides that are presented on the tumor surface to cytotoxic T-lymphocytes (CTLs). Immunoproteasomes are highly-specialized proteasome variants that are expressed at higher levels in antigen-presenting cells and contain replacements of the three constitutive proteasome catalytic subunits to generate peptides with a hydrophobic C-terminus that fit within the groove of MHC class I (MHC-I) molecules. A hallmark of cancer is the ability to evade immunosurveillance by disrupting the antigen presentation machinery and downregulating MHC-I antigen presentation. High-throughput screening was performed to identify Compound A, a novel molecule that selectively increased immunoproteasome activity and expanded the number and diversity of MHC-I-bound peptides presented on multiple myeloma (MM) cells. Compound A increased the presentation of individual MHC-I-bound peptides >100-fold and unmasked tumor-specific neoantigens on myeloma cells. Global proteomic integral stability assays determined that Compound A binds the proteasome structural subunit PSMA1 and promotes association of the proteasome activator PA28α/β (PSME1/PSME2) with immunoproteasomes. CRISPR/Cas9 silencing of PSMA1, PSME1, or PSME2 as well as treatment with immunoproteasome-specific suicide inhibitors abolished the effects of Compound A on antigen presentation. Treatment of MM cell lines and patient bone marrow-derived CD138+ cells with Compound A increased the antimyeloma activity of allogenic and autologous T-cells. Compound A was well-tolerated in vivo and co-treatment with allogeneic T-cells reduced the growth of myeloma xenotransplants in NSG mice. Taken together, our results demonstrate the paradigm-shifting impact of immunoproteasome activators to diversify the antigenic landscape, expand the immunopeptidome, potentiate T-cell-directed therapy, and reveal actionable neoantigens for personalized T-cell immunotherapy.