Abstract 7242: Inhibition of ULK1/2-mediated autophagy augments antigen processing and presentation promoting increased antigen specific CD8+ T-cell cytotoxicity

Abstract BACKGROUND: High levels of ULK1/2-mediated macroautophagy (autophagy) are associated with immunologically cold tumors that respond poorly to therapy. Increased autophagy results in decreased antigen processing and presentation (APP) mediated by autophagosomal degradation of neoantigen-generating immunoproteasomes and MHC-I. Here, we demonstrate that inhibition of ULK1/2-mediated autophagy restores APP and increases antigen-specific CD8+ T-cell cytotoxicity. METHODS: Levels of APP machinery were assessed at baseline and after ULK1/2 inhibition using qPCR, western blot, and/or flow cytometry in both murine and human cancer cell lines. Autophagic flux was evaluated using mCherry-GFP-LC3B reporter cell lines imaged in Incucyte. Localization of MHC-I and LC3B following inhibition of ULK1/2 were assessed by immunofluorescence via microscopy. Levels of peptide-loaded MHC-I were assessed by flow cytometry using antibodies that detected either OVA loaded MHC I or KRAS-peptide loaded MHC I. Antigen-specific CD8+ T-cell cytotoxicity was evaluated utilizing MC38-OVA-mCherry tumor cells and OT-1 CD8+ T-cells in the absence or presence of ULK1/2 inhibition. RESULTS: Inhibition of ULK1/2 decreases basal levels of autophagy in multiple cancer cell lines. Subcellular localization of MHC-I is dramatically altered by inhibition of ULK1/2 as evidenced by decreases in co-localization of MHC-I with the autophagosome marker LC3B and increases in both total and surface levels of MHC-I. Moreover, levels of peptide-loaded MHC-I increased in multiple cancer cell lines upon ULK1/2 inhibition demonstrating that inhibition of autophagy restores antigen processing and presentation. To determine the functional impact of these changes to AAP, we utilized the OT-1 OVA co-culture system. Inhibition of ULK1/2 results in significantly enhanced killing of the MC38-OVA tumor cells by OT-1 CD8+ T cells. Importantly, the killing is antigen specific since ULK1/2 inhibition did not result in increased killing of non-OVA expressing tumor cells by OT-1 CD8+ T cells. CONCLUSIONS: Tumors with elevated levels of autophagy are associated with reduced antigen processing and presentation resulting in low immune infiltration and poor responses to therapy. Decreasing autophagy by inhibiting ULK1/2 results in increased antigen processing and presentation, leading to enhanced antigen specific CD8+ T-cell cytotoxicity. Together, these results demonstrate that targeting ULK1/2 enhances antigen presentation and antigen-specific T cell mediated killing of cancer cells. Citation Format: Jhansi L. Leslie, Umida Djakbarova, Samantha S. Hodge, Logan Chinn, Natalie Hill, Kayla Walzer, Hyunyoung Moon, Xuelei Yan, Anne Van Abbema, Yihong Guan, Connor Rosen, Jenna L. Jeffrey, Ester Fernandez-Salas, Susan L. Paprcka. Inhibition of ULK1/2-mediated autophagy augments antigen processing and presentation promoting increased antigen specific CD8+ T-cell cytotoxicity [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 7242.