(OA-34) Exploring Molecular Glues to Activate Proteasomes and Develop Shared and Personalized Neoantigen-Based Therapeutics in Multiple Myeloma

Immunotherapy has sparked a revolution in oncology by releasing the power of the human immune system. A main challenge in broadening the range of responses to immunotherapy is the limited source of novel, actionable neoantigens exclusively presented on patient cancer cells. Immune evasion is a cancer hallmark that further thwarts the efficacy of immunotherapy. The immunoproteasome is a highly specialized form of the proteasome that degrades intracellular and viral proteins to generate short peptides which associate with MHC-class I molecules to serve as antigens presented to cytotoxic CD8+ T-cells. Molecular glues (MGs) are small molecules that target currently undruggable proteins to stabilize or induce protein-protein interactions by binding to two protein surfaces, creating a ternary complex and leading to beneficial downstream events.  

Methods: High-throughput screening identified a novel compound (compound A) that specifically increased immunoproteasome activity 3-fold in MM cells. MMCLs and MM patient CD138+ cells were treated ex vivo with compound A, lysates prepared, immunoprecipitated using a pan-MHC class I Ab, bound peptides eluted at low pH and released peptides sequenced by mass spectroscopy. Cell-based, biochemical and biophysical studies further characterized the effect of compound A on the association of accessory proteins with immunoproteasomes.

Results:

Treatment of MM patient tumor cells with compound A increased the presentation of specific, individual MHC-I  antigenic peptides and neoantigens up to >100-fold relative to untreated cells. Global proteomic integral stability assays determined that compound A binds the proteasome structural subunit PSMA1 and promotes association of the proteasome activator PA28 (encoded by PSME1/PSME2) with immunoproteasomes. Genetic silencing of PSMA1, PSME1, or PSME2 as well as treatment with immunoproteasome-specific suicide inhibitors abolished the effects of compound A on antigen presentation. Biophysical studies studied the effect of compound A on the stable association of PSME1/2 with immunoproteasomes. Treatment of MMCLs and patient BM CD138+ cells with compound A significantly increased the anti-myeloma activity of allogeneic and autologous CD8+ T-cells.

Conclusions:

Taken together, our results demonstrate that the immunoproteasome represents an actionable therapeutic target that shapes the MHC-I antigenic repertoire on myeloma cells. Novel molecules that activate immunoproteasomes can overcome immune escape and promote T-cell immunity. We have identified commonly shared and individual specific MHC class I NeoAgs presented on MM patient tumor cells. Identification of shared neoantigens across patients provides a promising avenue to develop broadly applicable immunotherapies while discovery of private antigens facilitates the development of personalized therapies. Neoantigens upregulated upon immunoproteasome activation can be exploited to expand the scope of TCR-engineered T-cells, T-cell engagers and cancer vaccines.