e21015 Background: NSCLC Patients with LKB1 loss respond poorly to immune checkpoint inhibitors (ICI). Determining mechanisms of the underlying immune resistance and strategies to overcome it are urgent and unmet clinical needs. Methods: We re-expressed WT LKB1 into LKB1 mutant NSCLC cell lines and measured effects on immune associated phenotypes in vitro. Specifically, we evaluated response to exogenous interferon gamma (IFNG), as well T-cell mediated cytotoxicity using a T-cell co-culture assay. To identify IFNG signaling changes influenced by LKB1, we evaluated the differential effects of LKB1 on IFNG induced gene expression using RNAseq. Finally, we performed a whole genome loss of function screen using CRISPR-Cas9 library (TKOv3) to identify genes and pathways that modify susceptibility to IFNG in the LKB1 mutant and LKB1 WT state in the A549 cell line. Results: Across multiple LKB1 mutant cell lines, restoration of WT LKB1 enhanced anti-proliferative effects of IFNG in vitro. Furthermore, WT LKB1 enhanced T-cell mediated cytotoxicity in T-cell co-culture assays using anti-NY-ESO TCR and expression of NY-ESO antigen into an HLA matched NSCLC cell line, H2023 (17% vs 40% cell death; P < 0.01). IFNG treatment induced expression of a core set of interferon-driven genes in both the mutant and LKB1 WT state. However, differentially expressed gene classes included downregulation of proliferation-associated genes in the LKB1 WT state, as well as upregulation of ferroptosis genes, and enhanced IFNG-driven expression of immune evasion genes including PDL1, PDL2, TRAF1, and IDO1 in the LKB1 WT state. PDL1 expression was assessed by flow cytometry and found to be upregulated upon expression of LKB1. Our functional genomics screen identified genes whose inhibition enhanced IFNG susceptibility in the A549 mutant state, and genes whose inhibition restored IFNG resistance in the A549 LKB1 WT state. Preliminary analysis identifies candidate signaling and metabolic pathways that appear to confer IFNG sensitivity in the mutant state without significant effect on IFNG sensitivity after LKB1 add-back. These include modifiers of the Hippo/YAP pathway as well as modifiers of ferroptosis, which will form the basis for further mechanistic experiments. Conclusions: ICI resistance caused by LKB1 loss is associated with insensitivity to IFNG, and can be modified in vitro by re-expression of WT LKB1. An integrated approach to evaluate modifiers of IFNG effects identifies resistance mechanisms that may be potential therapeutic targets.
cIAP1/2 are involved in the radiosensitizing effect of birinapant on NSCLC cell line in vitro
