Abstract Immunotherapy has emerged as a powerful tool for myeloma-directed cytotoxicity with the unique potential to induce immune memory and reduce the risk of relapse. However, evasion of immune destruction is a cancer hallmark that thwarts the efficacy of immunotherapy. Certain patients do not develop sustained responses and many patients that do benefit eventually develop drug resistance through defective antigen presentation. Immunoproteasomes are a highly specialized proteasomes that play a crucial role in the generation of MHC class I antigen (Ag) presentation. Immunoproteasomes degrade intracellular and viral proteins to generate peptides which are transported into the ER and associate with MHC-class I molecules for presentation to T-cells. The proteasome activator PA28α/ß is a heteroheptameric complex that interacts with immunoproteasomes and modulates Ag processing. The immunoproteasome exhibits distinct catalytic activities compared to constitutive (standard) proteasomes. This difference is due to the substitution of the 3 constitutive catalytic subunits: β1 (peptidylglutamyl-like), β2 (trypsin-like), and β5 (chymotrypsin-like) compared with 3 immune-specific subunits β1i (chymotrypsin-like), β2i (trypsin-like), and β5i (chymotrypsin-like). These immune subunits alter the proteasome's catalytic activity with a shift toward cleavage after hydrophobic and aromatic residues, thus impacting the types of peptides it generates and influencing the immune repertoire of (Neo)Ags presented to T-cells. Molecular glues (MGs) are highly attractive small molecules that target currently undruggable proteins to stabilize or induce protein-protein interactions (PPIs) by binding to two protein surfaces, creating a ternary complex that leads to beneficial downstream events. Here, we hypothesized that MGs could function to stabilize the PA28α/ß-immunoproteasome complex leading to the downstream activation of Ag presentation to modulate the immunopeptidomic landscape and promote T-cell-mediated tumor lysis. Ags bound to MHC class I molecules and presented on the cell surface constitute the targets of cytotoxic T-lymphocytes. Methods to identify and govern the generation of these Ags are critical to the development of more effective forms of immunotherapy, specifically TCR-engineered T-cells and cancer vaccines, for which the goal is the generation of effective adaptive immune responses accompanied by long-lasting immune memory to preclude disease relapse. Methods: We performed a HTS to identify novel molecules that increased immunoproteasome activity. Cell-based assays were performed using MM, cutaneous lymphoma and melanoma cells and the lead compound (Compound A). CD138+ tumor cells were obtained from MM patient bone marrow and treated with Compound A (1 uM, 48h), lysates prepared, immunoprecipitated using a pan-MHC class I antibody, bound peptides acid-eluted and released peptides then quantitated and sequenced by mass spectroscopy (MSBioWorks). Cell-based, biochemical and biophysical studies further characterized the effect of Compound A on the association of PA28α/ß with immunoproteasomes. Results. Treatment of multiple cancer cell types with Compound A increased immunoproteasome activity up to 3-fold. Treatment of MM patient tumor cells with Compound A ex vivo also increased the presentation of pan-MHC bound peptides, tumor-specific neoantigens and human endogenous retroviral (HERV) peptides 100-fold relative to the presentation of these peptide forms detected on untreated MM cells. Treatment of MM patient tumor cells ex vivo with Compound A also increased the relative proportion of MHC-bound peptides that were 8-9 amino acids in length. The optimal length of antigens bound in the MHC peptide cleft generally is 8-9 aa residues. Moreover, treatment with Compound A increased the relative frequency of hydrophobic and aromatic amino acids detected at the C-terminus, consistent with a dominant role of immunoproteasomes in the generating MHC class I Ags. Discussion: We demonstrate that MGs that promote PA28α/ß association with immunoproteasomes and not only enhance the degradation of proteins with hydrophobic C-termini but also generate immunopeptides with a similar enhanced frequency of hydrophobic C-termini. The results support the development of a platform to pharmacologically modulate the presentation of commonly shared and private NeoAgs and expand the scope of TCR-engineered T-cells, T-cell engagers and cancer vaccines.
James J. Ignatz-Hoover (Mon,) studied this question.