Abstract A076: Discovery and characterization of a novel, immunoproteasome activator that modulates the immunopeptidome, increases MHC class I antigenic presentation and enhances antitumor immunity

Abstract

Abstract Evasion of immune destruction is a hallmark of cancer that thwarts the efficacy of immunotherapy and reduces patient benefit. The development of immunotherapies has sparked a revolution in oncology by releasing the power of the human immune system, but the identity of novel, actionable antigens exclusively presented on cancer cells remains elusive. Immune cells contain a highly specialized variant of proteasomes, known as immunoproteasomes, in which the three constitutive catalytic subunits are replaced by cytokine-inducible, homologous catalytic subunits. Immunoproteasomes are specially adapted for a role in MHC class I antigen processing and presentation to CD8+ T-cells. Here, we performed a high-through screen to detect novel molecular entities that activated proteasomal peptide-hydrolyzing activity. We identified a curated panel of molecules which not only enhanced proteasomal hydrolysis of the cell-permeable substrate LLVY-R110 but also enhanced the presentation of MHC class I antigens. E.G7-Ova is a mouse lymphoma cell line derived by electroporation of EL4 cells (C57BL/6, H-2b, T lymphoma) with chicken ovalbumin (OVA) cDNA. Proteasomes degrade OVA to generate the peptide SIINFEKL that is presented by MHC-class I molecules. Studies demonstrated that the hits identified in the screen increased SIINFEKL presentation on E.G7-Ova cells. The lead compound, compound A, dramatically enhanced the expression of the proteasome activator PA28a/b, expressed by PSME1/2. Treatment of multiple myeloma (MM) cells with compound A enhanced the hydrolysis of Ac-ANW-MCA, which is selectively cleaved by the immunoproteasome beta5-associated catalytic activity. Treatment of MM cells followed by mass spectrometry (MS) demonstrated that compound A modulated the relative presentation of individual antigenic peptides bound to MHC class I molecules on tumor cells. The identification of actionable tumor antigens is indispensable for the development of several cancer immunotherapies, including T cell receptor (TCR)–transduced T cells and patient-specific mRNA or peptide vaccines. MS-based immunopeptidomics enables the discovery of canonical and non-canonical antigens, i.e., antigens derived from sequences outside protein-coding regions or generated by non-canonical antigen-processing mechanisms. This method enables the rapid identification of non-canonical peptides, a fraction of which may be detected exclusively by cancer cells. Novel, immunoproteasome-selective therapeutics have the potential to contribute not only to CD8+ T-cell-mediated control of human cancers, but also intracellular infections, autoimmune diseases, neurodegenerative diseases and proteinopathies that are characterized by the pathologic accumulation of toxic, proteinaceous aggregates. Citation Format: James J. Driscoll, Priyanka S. Rana, James J. Ignatz-Hoover. Discovery and characterization of a novel, immunoproteasome activator that modulates the immunopeptidome, increases MHC class I antigenic presentation and enhances antitumor immunity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A076.