Professor Case Western Reserve University Cleveland, Ohio, United States
Introduction: Transformative advances in immunotherapy have revolutionized cancer treatment and induce durable clinical responses. However, only a small fraction of patients respond to immunotherapy and cancer cells escape immunosurveillance through molecular mechanisms that remain elusive. Proteasomes play a central role in immune surveillance mechanisms by generating peptides from intracellular proteins which are presented as antigens on the cell surface for recognition by CD8+ cytotoxic T lymphocytes (CTLs). Cancer cells employ strategies to downregulate antigen presentation and impair CTL-mediated tumor recognition and lysis. Recently, we discovered that HDAC6-specific inhibitors increased proteasome activity as well as the presentation of MHC class I antigens on tumor cells. However, current HDAC6 inhibitors have pharmacologic liabilities that limit efficacy, e.g., low potency and solubility, poor PK properties and potential genotoxicity.
Methods: We performed a high-throughput, cell-based screen of 9,600 HDAC-specific compounds to identify novel molecular entities that increased proteasome activity in MM cells. Proteasomes degrade ovalbumin to generate the neoantigenic peptide “SIINFEKL”, which is presented on the cell surface in complex with the MHC class I H-2Kb molecule and quantitated by flow cytometry. We identified and then tested a curated set of hits for the ability to increase antigen presentation on tumor cells. Finally, hits were evaluated for the ability to induce tumor lysis using T-cells engineered to express a T-cell receptor (TCR) that recognized SIINFEKL.
Results: We identified hits that increased proteasomal activity by >50%. Importantly, two novel molecules identified in the screen increased proteasome activity more potently and more rapidly than the HDAC6-specific inhibitors with comparatively low cytotoxicity. Treatment of tumor cells with the novel compounds also increased levels of the MHC-class I-SIINFEKL complex more potently and rapidly than the HDAC6 inhibitors. MM cells were then co-cultured with CTLs genetically-engineered to express a SIINFEKL-restricted TCR. Pre-treatment of lymphoma or MM cells with novel compounds and HDAC6 inhibitors significantly increased tumor lysis by CTLs that expressed the SIINFEKL-restricted TCR. CTL-mediated tumor lysis was dramatically more potent compared to known HDAC6-specific inhibitors.
Conclusions: Taken together, our results support the development of novel proteasome activators as a paradigm-shifting approach to enhance tumor antigenicity and boost CTL-mediated anti-tumor immunity. Proteasome activators represent a paradigm-shifting approach as cancer immunotherapeutics to overcome immune escape mechanisms and boost antitumor immunity. Moreover, our data provide compelling evidence to support the development of drugs that powerfully enhance proteasome proteolytic function as a therapeutic strategy to treat cancers and other disease-causing proteinopathies.
