A Novel Immunoproteasome Activator That Expands and Amplifies the Multiple Myeloma Immunopeptidome and Promotes Cytotoxic T-Cell Activity
Authors: Driscoll James J. , Malek Ehsan , Rana Priyanka S. , Ignatz-Hoover James J 
Published: December 2024

Background: Multiple myeloma (MM) cells develop strategies to evade immune surveillance and resist current cellular and pharmacologic immunotherapies, while many other tumors present a low tumor mutation burden limiting the benefit of antigenicity. Novel strategies are required to overcome current shortcomings in order to make tumors more antigenic and to improve the success and broaden the applicability of cancer immunotherapy. Evasion of host immunity is a cancer hallmark achieved through disruption of the host antigen processing and presentation machinery (APM). The overwhelming majority of T-cell responses require proteasomal degradation of intracellular and viral proteins followed by downstream processing and presentation of oligopeptides that are complexed with major histocompatibility complex (MHC) class I molecules and presented to antigen-restricted CD8+ cytotoxic T-cells. Immunoproteasomes are highly specialized proteasome variants that degrade proteins exposed to oxidative stress and proinflammatory stimuli. Immunoproteasomes function as the source of most antigenic peptides presented on tumor cells to the immune system. Here, we hypothesized that immunoproteasome activators could increase proteasome-dependent protein-degrading activity, increase neoantigen presentation, and promote cytotoxic T-cell activity.

Results: We performed high-throughput library screening and identified novel molecules that specifically increased immunoproteasome peptide-hydrolyzing activity. Global proteomic integral stability assays determined that the lead compound (Compound A) binds proteasome structural subunit PSMA1 and to promote association of the proteasome activator PA28a/b (encoded by 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. MM cell lines and patient bone marrow CD138+ cells were treated with Compound A, total lysates prepared, immunoprecipitated with a pan-MHC class I molecule. Immunopeptides were eluted and sequenced by mass spectroscopy. Treatment of MM cells with the immunoproteasome activator Compound A increased the presentation of specific, known individual MHC class I-bound antigenic peptides by 100-fold. Strikingly, Compound A also increased the presentation of neoantigens on MMCLs and MM patient tumor cells. Compound A treatment unmasked a BCMA-derived neoantigen on tumor cells from an HLA-B018 myeloma patient and unmasked human endogenous retrovirus (HERV) neoantigens on tumor cells of an HLA-A02 patient. Ex vivo experiments demonstrated that Compound A increased the anti-myeloma activity of allogenic and autologous cytotoxic T-cells. Compound A was well-tolerated in vivo and co-treatment with allogenic 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.

Conclusions: Pharmacologic modulation of proteasome activity in tumor cells represents a cutting-edge strategy to expand and amplify the antigenic landscape as a means to increase antitumor immunity. A keystone to achieving a successful antitumor response is the presence of potent tumor neoantigens. Our studies have established a rapid, feasible, cost-effective platform to identify neoantigens on MM patient tumor cells that can then be targeted through the adoptive transfer of autologous and allogenic T-cells, TCR-restricted and chimeric antigen receptor (CAR)-engineered T-cell immunotherapies and cancer vaccines.