Abstract 5379: Inhibition of ULK1/2-mediated autophagy augments antigen processing and presentation in STK11 mutant NSCLC

Abstract

Abstract BACKGROUND: Inactivating mutations and deletions in STK11 are present in 9-20% of NSCLC and result in immunologically cold tumors that respond poorly to therapy, despite having a high tumor mutational burden and neoantigen load. STK11 mutations in NSCLC are associated with higher levels of ULK1/2-mediated macroautophagy, resulting in localization of MHC-I to autophagosomes and lysosomes leading to its degradation and decreased antigen presentation. Knockdown of ULK1/2 can restore surface MHC-I in highly autophagic cell lines. Here, we show that inhibition of ULK1/2 decreases macroautophagy, resulting in increased antigen processing and presentation (APP) and MHC-I levels in cancer cells, supporting the hypothesis that inhibition of ULK1/2 will restore antigen presentation in NSCLC STK11mut tumors. METHODS: TCGA datasets were analyzed to determine transcriptional differences in autophagy and APP between STK11 WT and mutant NSCLC tumors. CRISPR was used to stably knockout STK11 in multiple human NSCLC cell lines to generate isogenic pairs. mRNA and protein levels of APP machinery in NSCLC cell lines were assessed using qPCR, western blots, and flow cytometry. CRISPR was additionally used to knockout ULK1 and ULK2 in human STK11 mutant A549 cells. ULK1/2 tool compounds were synthesized based on existing patent literature. Effects of the combination of ULK1/2 inhibition with low levels of IFNγ on surface MHC-I levels were evaluated in several NSCLC cell lines by flow cytometry. RESULTS: NSCLC tumors with loss of function mutations or deletion of STK11 display increased transcription of genes in the ULK complex and reduced transcription of genes necessary for APP. STK11mut and KO NSCLC cell lines also display lower levels of APP at the mRNA and protein levels. Double knockout of ULK1 and ULK2 in A549 cells is not cytotoxic and results in decreased autophagy as well as increased immunoproteasome components and cell surface MHC-I. ULK inhibition reduces autophagy and restores APP at the protein level, but not at the transcriptional level, in all NSCLC cell lines evaluated. Combination of very low concentrations of IFNγ with ULK inhibition demonstrates additive increases in MHC-I greater than either single-agent treatment alone. CONCLUSIONS: STK11mut/del is associated with poor prognosis in NSCLC and leads to increased ULK-induced autophagy and decreased APP. ULK inhibition increases APP and surface MHC-I levels in STK11mut cell lines. In the presence of low concentrations of IFNγ, ULK1/2 inhibition restores APP in NSCLC STK11mut cell lines to the levels seen in NSCLC STK11 WT cell lines. The data presented here provides a rationale for targeting ULK1/2 to amplify immune recognition in immunologically cold tumors with high autophagy to increase responses to immunotherapies. Citation Format: Samantha C. Schwager, Eugene Park, Anne Van Abbema, Yihong Guan, Connor Rosen, Sean Cho, Michael Black, Inna Kurilyak, Jenna Jeffrey, Juan Jose Fung, Susan L. Paprcka, Ester Fernandez-Salas. Inhibition of ULK1/2-mediated autophagy augments antigen processing and presentation in STK11 mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5379.