Abstract A101: Inhibition of ULK1/2-Mediated Autophagy Enhances Responses to RAS Inhibition and Augments Antigen-Specific T Cell Cytotoxicity

Abstract Introduction Autophagy is a cellular degradation pathway used to adapt to changing nutrient conditions and stress. Cytoprotective autophagy is induced by RAS inhibition in (K)RAS mutant tumors, resulting in therapeutic resistance. Elevated levels of autophagy are also associated with reduced antigen presentation and low immune infiltration. The ULK1 and ULK2 kinases (ULK1/2) are critical for the initiation of autophagy. Here we demonstrate that inhibition of ULK1/2 enhances the efficacy of multiple RAS inhibitors, increases antigen presentation and augments antigen-specific T-cell cytotoxicity in vitro and in vivo, resulting in strong tumor growth inhibition. Methods An autophagy gene signature was derived from multiple public datasets and resources, was validated and refined using in-house and published in vitro cell line data and applied to clinical datasets. Autophagic flux was evaluated using multiple mCherry-GFP-LC3B reporter cell lines imaged by Incucyte. Cell viability or death was evaluated by CellTiterGlo, Caspase3/7 and CellTox. Localization of MHC-I and LC3B was assessed by immunofluorescence via microscopy. Levels of peptide-loaded MHC-I were assessed by flow cytometry using antibodies detecting OVA-loaded MHC I or KRAS-peptide loaded MHC I. Antigen-specific CD8+ T-cell cytotoxicity was evaluated using MC38-OVA-mCherry tumor cells and OT-1 CD8+ T-cells. In vivo efficacy and survival were evaluated in H1373 human xenograft and CMT-167 KRAS G12C knock-in (KI) syngeneic models. Tumor pharmacodynamics were evaluated by Western blot and IHC. Results Autophagy-related gene expression is rapidly induced in (K)RAS-mutant patient tumors in response to RAS inhibition. Increased levels of autophagy are maintained in the setting of therapeutic resistance with no reactivating MAPK mutations. Inhibition of ULK1/2 prevents RAS inhibitor-induced autophagy in multiple cell lines and increases the efficacy of multiple classes of RAS inhibitors, including mutation-specific, pan-RAS, “ON” and “OFF”. Importantly, combined inhibition of ULK1/2 and RAS significantly increases cancer cell death. Inhibition of ULK1/2 with adagrasib results in a significant reduction of tumor growth in H1373 human xenografts as well as CMT-167 KRAS G12C KI syngeneic models compared to single agent adagrasib. ULK1/2 inhibition also enhanced cancer cell immunogenicity via increased localization of MHC-I to the cell surface and antigen presentation, resulting in significantly greater antigen-specific T-cell activation. Inhibition of ULK1/2 alone resulted in increased tumor CD8+ T cell infiltration in the CMT-167 KRAS G12C KI tumors. ULK1/2 inhibition combined with adagrasib and anti-PD-1 results in significantly greater tumor growth inhibition and survival. Conclusion Inhibition of ULK1/2-mediated autophagy enhances responses to RAS inhibition by disabling a rapid mechanism of resistance, restoring neoantigen presentation, and enhancing T cell-mediated cytotoxicity. Citation Format: Umida Djakbarova, Jhansi L. Leslie, Jenna Pappalardo, Yihong Guan, Ferdie Soriano, Jamison Buendia, Xuelei Yan, Hyunyoung Moon, Jenna L. Jeffrey, Ester Fernandez-Salas, Susan L. Paprcka. Inhibition of ULK1/2-Mediated Autophagy Enhances Responses to RAS Inhibition and Augments Antigen-Specific T Cell Cytotoxicity [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A101.