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Contradictory characteristics of elevated mutational burden and a “cold” tumor microenvironment (TME) coexist in liver kinase B1 (LKB1)–mutant non–small cell lung cancers (NSCLC). The molecular basis underlying this paradox and strategies tailored to these historically difficult to treat cancers are lacking. Here, by mapping the single-cell transcriptomic landscape of genetically engineered mouse models with Kras versus Kras/Lkb1-driven lung tumors, we detected impaired tumor-intrinsic IFNγ signaling in Kras/Lkb1-driven tumors that explains the inert immune context. Mechanistic analysis showed that mutant LKB1 led to deficiency in the DNA damage repair process and abnormally activated PARP1. Hyperactivated PARP1 attenuated the IFNγ pathway by physically interacting with and enhancing the poly(ADP-ribosyl)ation of STAT1, compromising its phosphorylation and activation. Abrogation of the PARP1-driven program triggered synthetic lethality in NSCLC on the basis of the LKB1 mutation–mediated DNA repair defect, while also restoring phosphorylated STAT1 to favor an immunologically “hot” TME. Accordingly, PARP1 inhibition restored the disrupted IFNγ signaling and thus mounted an adaptive immune response to synergize with PD-1 blockade in multiple LKB1-deficient murine tumor models. Overall, this study reveals an unexplored interplay between the DNA repair process and adaptive immune response, providing a molecular basis for dual PARP1 and PD-1 inhibition in treating LKB1-mutant NSCLC.Significance:Targeting PARP exerts dual effects to overcome LKB1 loss–driven immunotherapy resistance through triggering DNA damage and adaptive immunity, providing a rationale for dual PARP and PD-1 inhibition in treating LKB1-mutant lung cancers.

Sequences for qPCR primers.

PARP1 inhibition restores phosphorylated STAT1 from poly(ADP-ribosyl)ation and triggers synthetic lethality in LKB1-deficient cells. (a) A549 lung cancer cells were transfected with lentivirus expressing the indicated genes (LV-Ctrl, LV-LKB1-WT, and LV-LKB1-Mut) followed by treatment with olaparib (5μM) for 24 hours. The expression of pSTAT1 and PARP1 was analyzed by western blotting. (b-d) LLC1-shLkb1 and LLC1-shCtrl cells were pre-treated with olaparib (5μmol/L) for 6 hours and then stimulated with IFNγ for another 24 hours. Total pSTAT1 and STAT1 were quantified using western blot (b) and cell-surface PD-L1 was measured by flow cytometry (c). Q-PCR analysis was used to detect the expression of CXCL9 and PD-L1 (d). (e, f) The three groups of A549 cells were treated with IFNγ or IFNγ/olaparib and then refreshed the culture medium. The ratio of CD8+ T (effector cells) to A549 cells (target cell) ratio was 5:1. After 6 hours of incubation, QT-PCR analysis was used to detect the expression of indicated molecules of CD8+ T cells. (g) CD8+ T cells were treated with PBS or olaparib (5μmol/L) for 24 hours, and then the cells were collected for quantification of GZMB, PRF1, TNF, and IFNγ. CD8a was selected as the reference gene. (h) LLC-LV-Ctrl cells were treated with IFNγ/anti-PD-1 antibodies (20 μg/mL) in the absence or presence of olaparib (5 µM) or BMDCs (Tumor cells: DCs= 1:1) for 24 hours. Flow cytometry assay was used for apoptosis analysis of tumor cells. The right graph showed the quantification of tumor cell apoptosis. The data are presented as the mean ± SD of triplicate experiments. Statistical significance was determined by Student's t-test; ns, not significant; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05.

LKB1 deficiency impairs IFNγ pathway by hindering STAT1 phosphorylation. (a) Western blotting for detection of LKB1 and pAMPK expression in K and KL lung tumors. (b) UMAP of sub-clustered CD45+ immune cells from lung tumor samples of K and KL, colored by cell types. (c) The proportion of each immune cell type in K vs KL. (d) UMAP of sub-clustered cells from lung tumor samples of KP and KPL mice, colored by their 10 major cell types. (e) Gene set enrichment analysis (GSEA) showing downregulated response to interferon-gamma in KPL compared with KP. (f) A549 lung cancer cells were transfected with lentivirus expressing the indicated genes (LV-Ctrl, LV-LKB1-WT, and LV-LKB1-Mut) followed by stimulation with IFNγ. The expression of LKB1 and pAMPK was analyzed by western blotting. (g) Expression of pSTAT1 was measured by immunofluorescent staining in the three groups of A549 cells treated with IFNγ for 24 hours. Nuclei were visualized with DAPI (blue). Scale bar = 100 μm. (h) Immunoblot of the indicated IFNγ-induced proteins in different lung cancer cells. (i) Decreased IFNγ signaling downstream molecules, PD-L1, and chemokines, in KPL vs KP malignant cells. (j) PD-L1 transcription and chemokine signature score in tumor cells of K vs KL. (k) Western blot illustrating LKB1 expression in LLC1 and CMT-167 cells respectively. (l, m) Lewis lung cancer cells (LLC1) were transfected with shRNA specific for Lkb1 (shLkb1) or noneffective scrambled shRNA (shCtrl) followed by treatment with IFNγ for 24 hours. Western blotting for the detection of pSTAT1, STAT1, LKB1, and pAMPK expression was performed. (n) Flow cytometry analysis of cell surface PD-L1 of tumor cells from mice bearing LLC1-shLkb1 and LLC1-shCtrl tumors. (o) Tumors from mice bearing LLC1-shLkb1 and LLC1-shCtrl derived tumors were analyzed by Q-PCR for detecting CXCL9, CXCL10, CXCL11 and CCL5. (p) Representative IHC staining of CD8+ T cells and quantification of infiltrated CD8+ T cells in LLC-LV-Lkb1 tumors treated with indicated treatment (n=3 mice each group). Scale bar: 100 µm. n = 5 fields from independent tumors. Error bars represent mean ± SD. Statistical analyses were performed using unpaired t-tests. (****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05). (q) LLC1/LV-Ctrl or LLC1/LV-Lkb1 tumors were administered with different treatments, anti-PD-1 monotherapy or combination treatment (n=7 mice/group). Tumor size was monitored. Results are presented as mean ± SEM; * P < 0.05; two-way ANOVA.

Olaparib-derived dual effects sensitize LKB1-deficient tumors to anti-PD-1 immunotherapy in preclinical models. (a) Mice-bearing orthotopic lung tumors were treated with different therapies. Bioluminescent images were shown. (b) Immunofluorescence staining of PD-L1 and CD8+T cells in resected lung tumors of mice bearing orthotopic tumors (n= 6 fields from independent tumors). Scale bar, 100 μm. (c). Quantification of tumor-infiltrating CD8+ T cells in immunofluorescence staining. (d, e) IHC statistical analysis of PAR and PD-L1 expression in resected lung tumors of mice bearing orthotopic tumors (n=5 fields from independent tumors). (f) Quantification of tumor-infiltrating CD8+ T cells in IHC staining in resected lung tumors of mice bearing orthotopic tumors (n= 5 fields from independent tumors). Data are represented as mean ± SD; Student's t-tests; * P < 0.05; ** P < 0.01; ***P

Olaparib promoted STING activation in LKB1-mutant/loss NSCLC cells and LLC-derived tumors. (a) Cells were pre-treated with olaparib (5 μM) for 6 hours and then stimulated with IFNγ for 24 hours. The cell lysates were then subjected to western blot analysis for key proteins in the STING signaling pathway. (b) Representative IHC staining of STING and pSTING in LLC-LV-Lkb1 tumors and LLC-LV-Ctrl tumors following different treatments. Scale bar: 200 μm. (c) IHC statistical analysis of STING and pSTING expression in LLC-LV-Ctrl/Lkb1 tumors following indicated treatment. n=5 fields from independent tumors. Data are represented as mean ± SD; Student's t-tests; ***P

LKB1-interacting proteins identified by LC-MS/MS analysis.

Anti-PD-1/olaparib treatment promotes IFNγ secretion by CD8+ T cells in the tumor microenvironment. Mice-bearing orthotopic lung tumors were treated with different therapies. Representative IHC stainings of IFNγ expression in tumor tissues were shown. Scale bar, 50 μm.The right graph showed the IHC statistical analysis of IFNγ expression in tumor tissues. n = 5 fields from independent tumors. Data are represented as mean ± SD; Student's t-tests; **P

Mechanistic link between LKB1 and PARP1. (a) A bioinformatics tool (Pepsite) was utilized to predict the possibility of PARP1 docking on the di-Arg ER retention motif of LKB1 (PRR). (b) Localization of PARP1 (green) relative to LKB1 (red) in LKB1-WT A549 cells and H1299 cells. Scale bar = 20 μm. (c) Lysates from H1299 cells were subjected to IP with anti-PARP1 and anti-LKB1 antibodies and western blot analysis with the indicated antibodies. (d) IHC statistical analysis of PAR expression in LLC-shCtrl and LLC-shLkb1 tumors. n=5 fields from independent tumors. Data are represented as mean ± SD; Student's t-tests; **, P < 0.01.

Overexpression of mutant PARP1 promotes activation of the IFNγ signaling pathway. (a, b) A549 lung cancer cells and LLC1 lung cancer cells were transfected with the catalytic mutant Flag-PARP1 (E988) followed by stimulation with IFNγ. The lysates were then subjected to western blot analysis for indicated proteins.