Abstract LB-187: Hsp90 inhibitor SNX-2112 enhances neoantigen presentation on the surface of tumor cells

Abstract

Abstract Introduction: SNX-5422 is the orally active prodrug of SNX-2112, a potent, highly selective inhibitor of heat-shock protein 90 (Hsp90) with promising anti-tumor activity in clinical trials. SNX-5422 enhanced anti-PD-1 activity in MC38 murine model. SNX-2112 affects multiple tumor pathways, including the interferon pathway and upregulates genes associated with antigen expression, and T-cell recognition, and it interferes with tumor microenvironment immunoediting associated genes. Methods: Cells were cultured in media supplemented with 10% fetal bovine serum at 37°C, 5% CO2, and 95% humidity. The 50% effective concentration of SNX-2112 on cell viability of SkMel28, MDAMB231, SkMel2 was determined using Cell Titer-Glo® Luminescent Cell Viability Assay. Cells were treated with SNX-2112 or other anti-tumor agents, e.g., vemurafenib, to determine surface expression of MHC class I complexes by flow cytometry using MHC class I monoclonal antibody PE conjugate (Enzo-Cat# ALX-805-711R-C100) and, in parallel, induction of apoptosis by Annexin V APC. Results: At the lowest dose of SNX-2112 required to inhibit activity of model Hsp90 clients (MAPK), a 3 to 5-fold increase in MHC class I presentation was observed in all cells lines. Treatment of MDAMB231 with SNX-2112 caused a 3-fold increase in surface expression of MHC class I complexes, loss of Hsp90 client protein activity, and increased protein ubiquitination, without change in protein levels of immunoproteasome subunits LMP2 and LMP7. Increased surface expression of MHC class I complexes, loss of Hsp90 client function, increased protein ubiquitination without affecting immunoproteasome subunits were also found in both melanoma cell lines. This suggests that upregulation of cell surface MHC class I complexes is driven by increased Hsp90 client protein turnover. SNX-2112 treatment at concentrations sufficient to increase MHC class I surface expression also resulted in a small increase in apoptosis. Interestingly, treatment of SkMel28 with vemurafenib robustly induced apoptosis without any effect on surface expression of MHC class I complexes. Analysis of surface-expressed peptides by mass spectrometry revealed a change in peptide profile after treatment with SNX-2112. Conclusion: SNX-2112 increased surface expression of multiple MHC class I complexes. Results suggest that SNX-2112 can drive the selective surface presentation of Hsp90 client proteins on tumor cell surface and could improve recognition by T cells specific for various cancer-associated antigens derived from mutated genes, making it potential useful in immunotherapy of cancer. This effect of SNX-2112 is independent of the induction of apoptosis as agents like vemurafenib did not elicit the same effect. Ongoing studies are focused on identifying Hsp90 client-derived peptides in surface complexes and their role in anti-tumor immunity. Citation Format: Megan Yap, John W. Walker, Kristi Baker, Richard Fahlman, Everardus Orlemans, Paul Lapointe. Hsp90 inhibitor SNX-2112 enhances neoantigen presentation on the surface of tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-187.