Abstract 2616: Epigenetic targeted therapies induce unique MHC-I epitopes identified by immunopeptidome analysis in low mutation-burden sarcomas

Abstract Pediatric sarcomas as a group are poorly immunogenic tumors with low mutation burdens and poor responsiveness to immunotherapies such as immune checkpoint inhibitors (ICI). We have been investigating the use of the hypomethylating agent decitabine (DAC) followed by histone deacetylase inhibitor entinostat (Enti) in our murine models of sarcoma to potentially increase the tumor immunogenicity. Using our Kras-driven murine sarcoma KP Sarc, a whole-cell vaccine using DAC and Enti treated tumor cells generates a T-cell dependent, tumor-specific immune response restricted to epigenetically regulated antigens further potentiated with addition of ICI. We showed that epigenetically regulated antigens are shared between unrelated tumors KP Sarc and sarcoma M3-9-M and allow for cross-protection after vaccination. We hypothesized that epigenetically upregulated genes are persistent in vivo and can be used as tumor-specific targets identified by immunopeptidomics. We treated KP Sarc-bearing mice in vivo with DAC or Enti for 5 days, or sequentially with DAC then Enti. Tumors harvested at the end of treatment showed upregulation of the cancer testis antigens (CTA) in DAC- and DAC+Enti-treated mice by expression density analysis and immunohistochemistry compared to no expression in untreated and Enti-treated tumors. Moreover, these changes persisted a week after DAC treatment. We examined the gene expression profile of KP Sarc by RNA sequencing (RNA-Seq). We determined that the sequential in vitro treatment of KP Sarc with DAC then Enti (DAC+Enti) leads to a higher upregulation of CTAs and inflammatory pathways than either treatment alone. We treated human rhabdomyosarcoma (RH30 and RH41) and Ewing sarcoma (CHLA-9) cell lines showing upregulation of 75 shared antigens with DAC+Enti treatment. To investigate changes induced in antigen presentation, we examined the immunopeptidome of KP Sarc under two conditions: Interferon gamma (IFN-γ) treatment alone (n=4) and DAC+Enti+IFN-γ (n=4). Following immunoprecipitation of MHC I, bound peptides were identified via liquid chromatography-mass spectrometry. We combined this analysis with whole transcriptome RNA-seq of those samples. Analysis revealed a total of 4, 171 peptides across all conditions and replicates, sourcing from 3, 393 proteins, which were matched to the mouse proteome database. On average, 3, 417 epitopes were identified in the control group compared to 3, 912 epitopes in the treated group. 526 MHC I-bound peptides were unique to the treated group, with no unique peptides observed in the control. We identified epitopes from numerous proteins beyond CTAs indicating broad changes in antigen processing and antigenic peptides in the DAC+Enti treated cells. Further studies will validate the immunogenicity and ways to increase immune responses targeting these shared epigenetically regulated epitopes in sarcomas. Citation Format: Alice Recho, Grace Huang, Himavanth R. Gatla, Erin E. Resch, Stephanie Glavaris, Maggie Phillips, Isaac Woodhouse, Pouya Faridi, Brian H. Ladle1. Epigenetic targeted therapies induce unique MHC-I epitopes identified by immunopeptidome analysis in low mutation-burden sarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2616.