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.

Liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11), has been originally identified as a tumor suppressor, whose inactivating mutations define a distinct subtype of non–small cell lung cancers (NSCLC) with poor prognosis and refractory to conventional treatments (1). Even in an era of immunotherapy practice, patients with LKB1 loss-of-function mutation still derive a limited overall response rate of less than 10% from a single programmed death (ligand)-1 [PD-(L)1] agent (2). More troublesomely, the understanding of the core issue as to how mechanistically LKB1 deficiency leads to the primary resistance to immunotherapy remains to be one of the utmost clinical challenges. Despite preliminary evidence suggesting that LKB1 deficiency impairs innate immunity (3), it is possible to debate whether the predominant immunosuppressive effect is solely attributed to this “nonspecific” immune system; a dialectic perspective of whether such an effect is also dependent on the functional interplay with the adaptive immunity is still in uncharted waters.

Paradoxically, contradictory characteristics are present in LKB1-mutant NSCLC in terms of known immunotherapeutic predictors. As LKB1 preserves genome integrity via maintaining homologous recombination (HR) function (4, 5), patients with LKB1 deficiency present an elevated tumor mutation burden (TMB) that is expected to obtain clinical benefits from checkpoint blockade (5, 6). Nevertheless, LKB1 loss of function concurrently shapes an inert tumor microenvironment (TME), characterized by decreased PD-L1 expression and reduced lymphocyte infiltration, which confers resistance to anti–PD-(L)1 agents (1, 7). Aiming at the paradoxical features, it is envisioned that antitumor activity in LKB1-mutant lung cancers could be reinvigorated from two dimensions, reversing the immunologically “cold” TME into a “hot” one (7) and inducing further DNA damage on the basis of HR defect (8). In this regard, we speculated that the strategy of reactivating the adaptive immunity while simultaneously triggering a synthetic lethal effect might be an optimal treatment paradigm to mount an immunotherapy response and transform the way we care for LKB1-deficient NSCLC populations.

Given that, we used single-cell RNA-sequencing (scRNA-seq) to depict the transcriptomic landscape of Kras/Lkb1 versus Kras-driven lung tumors in a genetically engineered mouse model, aiming at elucidating the mechanisms governing the adaptive immune resistance. Furthermore, we identified potential targets and contrapuntally proposed a novel treatment strategy capable of eliciting an antitumor activity against LKB1-mutant tumors through both threads of inducing the synthetic lethal effect and reactivating the adaptive immunity.

Animals and cell lines

Female C57BL/6 mice (5–8 weeks old) were purchased from and maintained in the specific-pathogen-free facility of the Laboratory Animal Center of Southern Medical University (Guangzhou, China). KrasG12D/+ mice were gifts from Professor Liang Chen at the Institute of Life and Health Engineering, Jinan University (Guangzhou, China).

Human lung cancer cells (H1299, A549, H460, PC9, H1944, and H1573 cells) were provided by Guangdong Lung Cancer Institute that purchased these cells from the ATCC. The Lewis lung cancer cell line (LLC1) was purchased from Guangzhou Jennio Biotech Co., Ltd. that obtained this cell line from the ATCC. All of them were matched with alleles of corresponding cells from the ATCC. All cells were maintained in a humidified incubator at 37°C with 5% CO2, and grown in DMEM supplemented with 10% FBS and 100 IU/mL penicillin/streptomycin. All cells were passaged every 2 or 3 days and used for no more than 1 month. All cell lines used were routinely assayed for Mycoplasma by MycoBlue Mycoplasma Detector (Vazyme).

Antibodies and reagents

The following antibodies were used: anti-LKB1 [27D10; Cell Signaling Technology (CST), #3050], anti-STAT1 (D1K9Y; CST, #14994), anti–phospho-Stat1 (Tyr701; D4A7; CST, #7649), anti-AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) antibody (Abcam, #ab133448), anti-Poly(ADP-ribose; Adipogen, #AG-20T-0001), anti-PARP (46D11,# 9532), anti-JAK2 (phospho Y1007 + Y1008; Abcam, #ab32101), anti–PD-L1/CD274 (Proteintech, #66248–1-Ig), anti-HLA class I ABC (Proteintech, #15240–1-AP), anti-GAPDH (DIA-AN, #2058), anti–β-actin (Fdbio Science, #FD0060–100), anti-CD8 (Abcam, #ab217344), anti-phospho-H2AX-S139 (ABclonal, #AP0687), anti-TMEM173/STING antibody (Proteintech, #19851–1-AP), anti–phospho-TMEM173/STING (Ser366) antibody (Affinity, #AF7416), anti-IRF3 antibody (Proteintech, #11312–1-AP), and anti-pIRF3 (Ser386; CST, #37829). Fluorescent secondary antibodies used were: Dylight488 (EarthOx, #E032210) and 594 (EarthOx, #E032420). Flow cytometry antibodies: FITC-conjugated CD8α, PE-conjugated NK1.1, BV605-conjugated CD45, PerCP/Cyanine5.5-conjugated CD3, APC-conjugated PD-L1, PE-conjugated PD-L1, APC/Cyanine7-conjugated CD69, these antibodies were purchased from BioLegend. The PARP inhibitor olaparib (#S1060) was obtained from Selleck. Human IFNγ (#300–02–100) was purchased from Peprotech. Mouse IFNγ (#C746) was purchased from Novoprotein.

scRNA-seq data preprocessing and analysis

scRNA-seq samples were sequenced on an Illumina Novaseq platform, paired-end mode. For the raw fastq data, we first filter low-quality data with trimomatic software, followed by computational alignment using the 10× Cell Ranger analysis (version 6.0) to map to the mm10 reference genome (GRCm38.91). Subsequently, datasets were subjected to quality control steps using “Seurat” (R package, version 3.1.5) and the expression matrices were normalized by the functions NormalizeData and ScaleData. Unique molecular identifiers from each cell were scaled and log-transformed. To identify major axes of variation within our data, the FindVariable function was applied to select the top variable genes for principal component analysis; further dimensionality reduction was conducted by Uniform Manifold Approximation and Projection (UMAP). We used unsupervised clustering using the FindClusters function with default parameters to generate cell clusters. Marker genes defining each cluster were identified using the FindAllMarkers function, which uses a Wilcoxon rank sum test to determine significant genes. These marker genes were used to assign cluster identity to individual cell types. In addition, cell clusters and markers were analyzed using the CellMarker database: http://bio-bigdata.hrbmu.edu.cn/CellMarker/index.jsp, published signatures, and existing literature.

We performed gene set variation analysis (GSVA) with the “GSVA” R package and gene set enrichment analysis with the “clusterProfiler” R package to compare immune-related gene sets between groups at the single-cell level. Enrichment levels (P values) for selected cancer hallmark signatures were calculated. The data were plotted as the −log10P values after Benjamini–Hochberg correction. The significance threshold was set at a P value of <0.05.

qRT–PCR

Cell mRNA was collected using TRIzol reagent and was reverse transcribed into complementary DNA using PrimeScript RT reagent Kit (TAKARA). qRT–PCR was performed using TB Green Premix Ex Taq (TAKARA) in a Roche LightCycler 480 System. We used the average 2−ΔΔCT to analyze the data. The bar plots and heatmaps showing the transcriptional level of the IFNγ downstream molecules were created by Prism 7 software (GraphPad Software). The primers used to amplify target genes are listed in Supplementary Table S1.

Western blot and ELISA

Western blot was used to measure intracellular proteins. Cells were solubilized using RIPA buffer (Beyotime) supplemented with protease inhibitors (cOmplete Protease Inhibitor Cocktail, Roche) and phosphatase inhibitors (PhosSTOP Phosphatase Inhibitor Cocktail, Roche). Protein concentration was quantified using a BCA kit (Thermo Fisher Scientific). For each sample, 20-μg protein lysate was loaded onto 10% or 12.5% SDS-PAGE gel. Proteins were separated and transferred to polyvinylidene difluoride membranes (Millipore). Then the membranes were incubated with appropriate antibodies. Bands were analyzed using an ECL system (BLT GelView 6000).

ELISA was used to measure secretory proteins. The concentration of medium CXCL10 was quantified by the ELISA kit (MEIMIAN, #MM-0204H1) according to the manufacturer's protocol.

Flow cytometry

To measure membrane proteins, cells were trypsinized, resuspended in PBS supplemented with 2% BSA, incubated with Fc receptor blocking agent (BioLegend, 101302), and then stained with FITC-conjugated CD8α, PE-conjugated NK1.1, BV605-conjugated CD45, PerCP/Cyanine5.5-conjugated CD3, APC-conjugated PD-L1, PE-conjugated PD-L1, APC/Cyanine7-conjugated CD69, followed by incubating on ice for 30 minutes. After washing with PBS, cells were resuspended in 0.3-mL PBS. The data were then analyzed with FlowJo software (version 10.5; Tree Star).

IHC

For histologic analysis, tumor tissues from mice were paraffin-embedded and sectioned transversely. After deparaffinization, the sections were incubated with 3% hydrogen peroxide and then were blocked in goat serum for 1 hour at room temperature. Then the slides were probed with antibodies as follows: anti-CD8α (Abcam, # ab217344), anti–PD-L1 (Abcam, # ab233482), anti-Poly (ADP-ribose; Adipogen, #AG-20T-0001), followed by treatment with secondary antibodies.

Immunofluorescence

Cells were cultured for two days on coverslips with different treatments. After washing with PBS, cells were fixed and permeabilized with paraformaldehyde for 15 minutes at room temperature. PFA-fixed cells were then permeabilized with 0.1% Saponin. Next, the samples were blocked with 10% FBS for 30 minutes at room temperature before incubation with primary antibodies overnight at 4°C. After washing three times with PBST, cells were incubated in a secondary antibody for 1 hour at room temperature. Slides were mounted on glass slides using Fluoroshield Mounting Medium with DAPI (Abcam, #ab104139).

For tumor tissue, the preliminary process was similar to that of IHC. 3% H2O2 was used to block endogenous peroxidase activity. After serum blocking, the sections were then incubated overnight at 4°C with primary antibodies. After washing with PBST, the sections were incubated with secondary antibodies for 1 hour at room temperature. Slides were then mounted on glass slides using DAPI.

Coimmunoprecipitation

Proteins were extracted by Cell Lysis Buffer for Western & IP (Beyotime, # P0013J) supplemented with phosphatase and proteinase inhibitors. Primary antibodies were added and then immunoprecipitation was performed overnight at 4°C 30-μL protein A/G beads (Santa Cruz Biotechnology, #sc-2003) were added and allowed to incubate for another 4 hours. The proteins were precipitated through centrifugation and dissolved in a loading buffer followed by Western blot analysis.

NanoLC-ESI/MS-MS analysis

The lyophilized peptide was resuspended in 2% acetonitrile containing 0.1% formic acid, and 4-μL aliquots were loaded into a ChromXP C18 (3 μmol/L, 120Å) trap column. The online chromatography separation was performed on the Ekspert NanoLC 415 system (SCIEX). The trapping and desalting procedures were carried out at a flow rate of 4 μL/min for 5 minutes with 100% solvent A (water/acetonitrile/formic acid 98/2/0.1%). Then, an elution gradient of 8%–38% solvent B (water/acetonitrile/formic acid 2/98/0.1%) was used on an analytic column (75 μm × 15 cm C18–3 μm 120Å, ChromXP, Eksigent) over 25 minutes. IDA (information-dependent acquisition) MS techniques were used to acquire tandem MS data on a Triple TOF 6600 tandem mass spectrometer (Sciex) fitted with a Nanospray III ion source. Data were acquired using an ion spray voltage of 2.4 kV, curtain gas of 35 PSI, nebulizer gas of 12 PSI, and an interface heater temperature of 150°C. The MS was operated with TOF-MS scans. For IDA, survey scans were acquired in 250 ms and up to 40 product ion scans (50 ms) were collected if a threshold of 260 cps with a charge state of 2–4 was exceeded. A rolling collision energy setting was applied to all precursor ions for collision-induced dissociation. Dynamic exclusion was set for 16s.

Coculture of peripheral blood mononuclear and A549 cells

Human peripheral blood mononuclear cells (PBMC) were isolated from the peripheral blood of our team members and then were stimulated with anti-human CD8/CD28 activation beads (BioLegend, #302914 and #317326) and human IL2 (PeproTech, #200–02) for 72 hours to induce activation of CD8+ T cells. Human A549 cells were pretreated with IFNγ or IFNγ/olaparib for 24 hours, and then the culture medium was refreshed. The ratio of PBMCs (effector cells) to A549 cells (target cell) was 5:1, as referred to a previous study (9).

To conduct the Transwell migration assay, 5 × 105 PBMCs in 200 μL complete media were loaded into the top chamber of Transwell inserts (Corning, # 3421). The bottom well was filled with a culture medium derived from IFNγ-induced A549 cells. Plates were then incubated at 37°C overnight, and cells that migrated to the lower chamber were stained by crystal violet blue to be counted under the 20× microscope with four fields of view.

Coculture of dendritic, CD8+ T, and LKB1-deficient LLC cells

Murine bone marrow (BM) cells were collected from the femurs and tibia of C57BL/6 mice and plated at a density of 4×106 /2 mL well on 6-well plates with GM-CSF (20 ng/mL, Sino Biological) in cell culture medium. BM cultures were fed on days 2, 4, and 6 with 1-mL cell culture medium containing fresh GM-CSF and IL-4 (400 U/mL, Abbkine). After 6 days of culture, lipopolysaccharide (100 ng/mL, Sigma) was added for the last 24 hours to promote bone marrow–derived DCs (BMDC) to differentiate into mature dendritic cells (DC). LKB1-deficient LLC cells were treated with IFNγ/anti–PD-1 antibodies (20 μg/mL) in the absence or presence of olaparib (5 μmol/L) or BMDCs (tumor cells: DCs = 1:1) for 24 hours. Then PBMC-derived CD8+ T cells (effector cells: tumor cells = 5:1) were added into the culture medium containing DCs and LLC cells.

Animal experiments

All the animal experimental procedures were approved by the Animal Care and Use Committee of Southern Medical University, with a project license number of SMUL2022189. To generate murine subcutaneous tumors, individual mouse was injected subcutaneously with 3 × 106 LLC1 unless otherwise specified. Seven days after injection, the tumor-bearing mice were randomly separated into four groups. Anti–PD-1 antibody (200 μg/mouse, clone RMP1–14, BioXCell) was injected intraperitoneally three times a week (days 1, 3, and 5 of a 7-day cycle). For PARP1 inhibitor (olaparib, 25 mg/kg), mice were treated intraperitoneally daily. The combination group was treated with both anti–PD-1 antibody and PARP1 inhibitor, and the control group was injected with IgG control (clone 2A3, BioXcell). The tumors were measured in two dimensions (length and width), and volume (V) was calculated as V = length × width2 × 0.5. Two weeks after treatment, tumors were collected and processed for infiltrating lymphocyte isolation or IHC.

To generate orthotopic tumors, individual mice were surgically implanted with 1×105 LLC1-shLkb1-Luc cells in 100 μL PBS-Matrigel mixture into the left chest. One week after injection, the tumor formation was visualized by BLI, and mice were randomly allocated into groups and treated with anti–PD-1 antibody (200 μg/mouse /i.p., three times weekly), olaparib (8 mg/kg/i.p., 25 mg/kg, daily), anti–PD-1 antibody plus olaparib and IgG for 2 weeks.

To establish spontaneously Kras/Lkb1-driven lung tumors, 2 × 104 pfus of pSECC-sgLkb1 or pSECC-sgTomato lentiviruses were given to genetically engineering KrasG12D/+mice by nasal inhalation. Analyses were performed after 10 weeks, as referred to the previous study (10).

In vivo immune cell depletion and cytokine studies

To study the role of CD8+ T cells and NK cells in vivo, cellular subsets were depleted by administering depleting antibody intraperitoneally twice weekly beginning 1 day before therapy, as follows: CD8+ T cells with anti-CD8α (200 μg/mouse, clone 2.43, BioXCell) and NK cells with anti-NK1.1 (250 μg/mouse, clone PK136, BioXCell).

For the cytokine study, either 1-μg IFNγ (Novoprotein, #C746) or 100-ng CXCL10 (MedChemExpress, #HY-P7227) was mixed in the tumor cell suspension before injection of the tumor. Mice then continued receiving 500-ng IFNγ or 100-ng CXCL10 intratumorally every 3 days post-tumor implantation with different treatments.

Statistical analyses

Data analyses were performed using Prism 7 software (GraphPad Software). Data are presented as mean ± SEM or SD. Statistical significance was determined by one- or two-way ANOVA, Student t test, and log-rank test and defined as a P value of less than 0.05. Levels of significance were indicated as ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Data availability

The data generated in this study are available within the article and its supplementary data files. scRNA-seq data of K versus KL tumors generated in this study are publicly available in Gene Expression Omnibus (GEO) at GSE180963. scRNA-seq data of KP versus KPL tumors were obtained from GEO at GSE194166.

LKB1 deficiency impairs the IFNγ pathway by hindering STAT1 phosphorylation

To investigate how LKB1 regulates antitumor immunity, we used CRISPR/Cas9-induced knockout of Lkb1 in the genetically engineered Kras-driven murine models to establish spontaneously Kras/Lkb1-driven lung tumors. These animals were treated with intranasal delivery of Lenti-sgLkb1 (KL) or Lenti-sgTomato (K, referred to as negative control; Fig. 1A); knockout of Lkb1 in the tumor cells was examined by immunoblot analysis (Supplementary Fig. S1A). The collected tumors were then processed for single-cell scRNA-seq (10X Genomics), where 11 major cell clusters were labeled on the basis of canonical feature cell markers (Fig. 1A). Compared with K mouse, KL mouse had a lower percentage of activated T cells and a higher percentage of exhausted T cells, indicative of an inert immune microenvironment in the LKB1-deficient context (Supplementary Fig. S1B and S1C).

Figure 1.

LKB1 deficiency impairs the IFNγ pathway by hindering STAT1 phosphorylation. A, Schematic of the generation of genetically engineered Kras-driven mouse model to establish KrasG12D/+ mice with additional knockout of Lkb1 and UMAP plot of 14,260 cells from lung tumor samples of K and KL mouse. B, GSVA was performed in KL versus K along the tumor cells for immune-related gene sets in HALLMARK terms. C, A549 human NSCLC cell lines (LKB1-null) expressing LKB1-WT or LKB1-MUT (kinase-dead LKB1) were stimulated with IFNγ (50 ng/mL). The expression of effectors downstream of IFNγ was analyzed by Western blotting. D, Expression of pSTAT1 and IRF1 was analyzed by Western blotting. E, Expression of PD-L1 and IRF1 was analyzed by Western blotting and flow cytometry. F, Heat map of mRNA expression levels of indicated effectors in A549 cells after treatment with IFNγ (50 ng/mL) for 24 hours. G, Quantification of secretory CXCL10 in A549 cells after treatment with IFNγ (50 ng/mL) for 24 hours by ELISA analysis. H, A549 lung cancer cells were stimulated with IFNγ for 24 hours and the conditional media were collected for Transwell assay with CD8+ T cells. I, A549 cells were treated with fludarabine (Flu) at different concentrations and IFNγ (50 ng/mL) for 24 hours. The cells were then used to evaluate the expression of pSTAT1. J, A549 cells were treated with fludarabine (5 μmol/L) for 6 hours, followed by treatment with IFNγ (50 ng/mL) for 24 hours. The cells were then used to evaluate the expression of CXCL10. K and L, C57BL/6 mice bearing LLC1/LV-Lkb1 or LLC1/LV-Ctrl tumors were administered with different treatments [control immunoglobulin G (vehicle), anti–PD-1 Ab, or combination treatment; n = 6 mice/group]. Tumor size (K) and survival (L) were monitored. Results are presented as mean ± SEM. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, unpaired t tests, two-way ANOVA or log-rank tests.

Figure 1.

LKB1 deficiency impairs the IFNγ pathway by hindering STAT1 phosphorylation. A, Schematic of the generation of genetically engineered Kras-driven mouse model to establish KrasG12D/+ mice with additional knockout of Lkb1 and UMAP plot of 14,260 cells from lung tumor samples of K and KL mouse. B, GSVA was performed in KL versus K along the tumor cells for immune-related gene sets in HALLMARK terms. C, A549 human NSCLC cell lines (LKB1-null) expressing LKB1-WT or LKB1-MUT (kinase-dead LKB1) were stimulated with IFNγ (50 ng/mL). The expression of effectors downstream of IFNγ was analyzed by Western blotting. D, Expression of pSTAT1 and IRF1 was analyzed by Western blotting. E, Expression of PD-L1 and IRF1 was analyzed by Western blotting and flow cytometry. F, Heat map of mRNA expression levels of indicated effectors in A549 cells after treatment with IFNγ (50 ng/mL) for 24 hours. G, Quantification of secretory CXCL10 in A549 cells after treatment with IFNγ (50 ng/mL) for 24 hours by ELISA analysis. H, A549 lung cancer cells were stimulated with IFNγ for 24 hours and the conditional media were collected for Transwell assay with CD8+ T cells. I, A549 cells were treated with fludarabine (Flu) at different concentrations and IFNγ (50 ng/mL) for 24 hours. The cells were then used to evaluate the expression of pSTAT1. J, A549 cells were treated with fludarabine (5 μmol/L) for 6 hours, followed by treatment with IFNγ (50 ng/mL) for 24 hours. The cells were then used to evaluate the expression of CXCL10. K and L, C57BL/6 mice bearing LLC1/LV-Lkb1 or LLC1/LV-Ctrl tumors were administered with different treatments [control immunoglobulin G (vehicle), anti–PD-1 Ab, or combination treatment; n = 6 mice/group]. Tumor size (K) and survival (L) were monitored. Results are presented as mean ± SEM. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, unpaired t tests, two-way ANOVA or log-rank tests.

Close modal

Single-cell GSVA targeting cancer cell clusters revealed several cancer hallmark signatures that were defective in KL relative to K (Fig. 1B). Remarkably, IFNγ response was one of the top downregulated biological processes in KL versus K (Fig. 1B), raising the hypothesis that LKB1 deficiency impairs the IFNγ pathway to render immune escape. To further resolve the impact of LKB1 deficiency on the IFNγ signaling pathway, we also analyzed the scRNA-seq data from GEMM-derived lung tumors with Kras/Tp53 (KP) versus Kras/Tp53/Lkb1 (KPL) mutations (Supplementary Fig. S1D; ref. 11). Consistent with the results observed from K versus KL GEMMs, the tumor-intrinsic IFNγ signaling was also impaired in KPL-driven tumor cells compared with the KP counterpart (Supplementary Fig. S1E). We thus focused on investigating whether and how IFNγ response is defective in LKB1 mutant NSCLC.

On this ground, the LKB1-null human lung cancer cell line A549 was engineered to stably express wild-type or kinase-dead LKB1 (LKB1-WT or LKB1-MUT; Supplementary Fig. S1F). Upon IFNγ stimulation, the expression of phosphorylated STAT1 (pTyr701) was decreased in LKB1-deficient A549 cells compared with LKB1-WT cells, whereas neither the total lysis of JAK2 (including pTyr1022/1023) nor STAT1 was correlated with the status of LKB1 (Fig. 1C). We also found a significant decrease in the nucleus localization of pSTAT1 in LKB1-deficient A549 cells, with a resultant lower protein expression of IRF1 that is directly regulated by phosphorylated STAT1 (Fig. 1D; Supplementary Fig. S1G; ref. 12). Moreover, IFNγ-induced PD-L1, MHC class I molecular HLA-A, and T-cell attracting chemokines, including CXCL9, CXCL10, CXCL11, and CCL5, were significantly reduced in LKB1-deficient A549 cells (Fig. 1E and F). To reinforce our conclusions, we also performed parallel experiments in other human NSCLC cell lines and observed the same results, where IFNγ signaling was impaired in the LKB1-mutant/loss context (including A549, H460, H1944, and H1573) compared with the counterpart without LKB1 deficiency (H1299 and PC9) under IFNγ stimulation, represented by the downregulation of pSTAT1, PD-L1, and HLA-A (Supplementary Fig. S1H). Data from ELISA further confirmed a significant reduction of secreted CXCL10 in LKB1-mutant/loss A549 cells compared with LKB1-WT A549 cells at the protein level (Fig. 1G). Single-cell level transcriptional data of both KPL versus KP and KL versus K also showed downregulated expression of PD-L1 transcription and chemokine signature in the LKB1-deficient context (Supplementary Fig. S1I and S1J).

The regulatory role of STAT1 on these chemokines led us to assess the function of IFNγ/STAT1 in T-cell migration. Superior recruitment of CD8+ T cells by IFNγ-treated LKB1-WT versus LKB1-deficient A549 cells was observed in a Transwell assay (Fig. 1H). In addition, we explored whether the same mechanism existed in the murine lung cancer cell line LLC1, which has relatively low LKB1 expression (Supplementary Fig. S1K). Likewise, the level of phosphorylated STAT1 decreased accompanied by a reduction of IFNγ-induced PD-L1 and chemokines in LLC1-shLkb1 cells (Supplementary Fig. S1L–S1O). Altogether, these findings suggest that LKB1 deficiency impairs the IFNγ signaling pathway through downregulating phosphorylated STAT1, which subsequently enables tumor cells to evade CD8+ T-cell trafficking.

To confirm that LKB1-mediated antitumor immunity is dependent on phosphorylated STAT1, we incubated A549 cells together with IFNγ and fludarabine (Flu), a chemical inhibitor of phospho-STAT1, at different concentrations (0, 5, 10, or 20 μmol/L) for 24 hours. As shown in Fig. 1I, fludarabine caused a progressive decrease in the phosphorylation of STAT1 in a dose-dependent manner. Accordingly, IFNγ-induced mRNA expression of CXCL10 was potently downregulated after adding fludarabine compared with IFNγ treatment alone (Fig. 1J). To explore the role of IFNγ and pSTAT1 in recruiting CD8+ T cells in vivo, mice bearing LLC-lentivirus (LV)-Lkb1 tumors were divided into three groups, intratumorally injected PBS, IFNγ, or fludarabine, respectively. IHC analysis showed that there was a significant elevation of CD8+ T-cell infiltration in the IFNγ-treated group versus the PBS counterpart (Supplementary Fig. S1P). For mice treated with fludarabine that induces a low level of pSTAT1, CD8+ T-cell infiltration was remarkably decreased. Then we administered different treatments in C57BL/6 mice subcutaneously transplanted with LLC1-LV-Lkb1 or LLC1-LV-Ctrl cells in the presence or absence of an anti–PD-1 agent and fludarabine. Anti–PD-1 antibody significantly inhibited tumor growth in mice bearing LLC1-LV-Lkb1 tumors, whereas adding fludarabine reversed the immunotherapy efficacy (Fig. 1K). In addition, the involvement of fludarabine in mono-immunotherapy significantly shortened the overall survival (OS) of mice bearing LLC1-LV-Lkb1 tumors (Fig. 1L). Because fludarabine acts as a pSTAT1 inhibitor and CXCL10 was downstream of pSTAT1 in our study, we also evaluated the reversal effect of CXCL10 in mouse models-bearing LLC1-LV-Ctrl or Lkb1 tumors with different treatments. As expected, the exogenous CXCL10 significantly reduced tumor growth rate in LLC1-LV-Ctrl/Lkb1 tumors treated with anti–PD-1 antibody or combined anti–PD-1/fludarabine, respectively (Supplementary Fig. S1Q). These results demonstrate that the key element that determines the immunosuppressive function of LKB1 may lie in the IFNγ/pSTAT1 signaling pathway.

LKB1 loss–mediated DNA damage repair deficiency abnormally activates PARP1

To better understand the molecular mechanisms of how LKB1 regulates IFNγ-induced phosphorylation of STAT1, we first explored LKB1-interacting proteins by mass spectrometry analysis and immunoprecipitation (4). LKB1-WT or LKB1-loss A549 cells were immunoprecipitated with an anti-LKB1 antibody, and the coomassie brilliant blue assay revealed an extra band with molecular weight around 130 kDa in LKB1-WT cells (Fig. 2A). The additional band was then subjected to LC-MS/MS analysis; PARP1, a DNA damage sensor that plays a critical role in the process of DNA damage repair, was identified specifically in LKB1-WT cells with high confidence (Fig. 2B; Supplementary Table S2). Significant Gene Ontology terms showed that PARP1 expression was negatively correlated with tumor response to IFNγ (Fig. 2C). Thus, we hypothesize that LKB1 affects the IFNγ response by influencing PARP1.

Figure 2.

LKB1 loss–mediated DNA damage repair deficiency abnormally activates PARP1. A, Lysates from A549 cells (LV-Ctrl, LV-LKB1-WT) were immunoprecipitated with an anti-LKB1 antibody, followed by a Coomassie brilliant blue analysis. B, PARP1 was specifically identified in the LKB1-WT coprecipitates. The tandem spectra of the representative peptide of PARP1 are shown. C, Significantly enriched GO annotations of PARP1 in a lung adenocarcinoma cohort were identified using LinkedOmics. D and E, Lysates from IFNγ-treated A549 cells or LLC1 cells were immunoprecipitated with an anti-LKB1 antibody, followed by Western blot analysis. F, A549 lung cancer cells were stimulated with IFNγ. The levels of PARs and PARP1 were analyzed by Western blot. G, IHC staining of PAR in LLC1-shLkb1 or LLC1-shCtrl tumors. H, Immunofluorescence staining of γH2AX in LLC1-shLkb1 and LLC1-shCtrl tumors. I, Level of DNA damage among three groups of A549 cells in comet assay after the treatment with H2O2 (100 μmol/L) for 48 hours. J, The expression of γH2AX, RAD51, and pCHK1 in A549 lung cancer cells. K, The expression of γH2AX and PAR in A549 lung cancer cells after transfection with dsDNA. Statistical significance was determined by the Student t test. *, P < 0.05; **, P < 0.01.

Figure 2.

LKB1 loss–mediated DNA damage repair deficiency abnormally activates PARP1. A, Lysates from A549 cells (LV-Ctrl, LV-LKB1-WT) were immunoprecipitated with an anti-LKB1 antibody, followed by a Coomassie brilliant blue analysis. B, PARP1 was specifically identified in the LKB1-WT coprecipitates. The tandem spectra of the representative peptide of PARP1 are shown. C, Significantly enriched GO annotations of PARP1 in a lung adenocarcinoma cohort were identified using LinkedOmics. D and E, Lysates from IFNγ-treated A549 cells or LLC1 cells were immunoprecipitated with an anti-LKB1 antibody, followed by Western blot analysis. F, A549 lung cancer cells were stimulated with IFNγ. The levels of PARs and PARP1 were analyzed by Western blot. G, IHC staining of PAR in LLC1-shLkb1 or LLC1-shCtrl tumors. H, Immunofluorescence staining of γH2AX in LLC1-shLkb1 and LLC1-shCtrl tumors. I, Level of DNA damage among three groups of A549 cells in comet assay after the treatment with H2O2 (100 μmol/L) for 48 hours. J, The expression of γH2AX, RAD51, and pCHK1 in A549 lung cancer cells. K, The expression of γH2AX and PAR in A549 lung cancer cells after transfection with dsDNA. Statistical significance was determined by the Student t test. *, P < 0.05; **, P < 0.01.

Close modal

We first examined the binding relationship between LKB1 and PARP1. Bioinformatic analysis demonstrated the possibility of peptide-mediated interaction between PARP1 and LKB1 (P = 0.0309, Supplementary Fig. S2A). In addition, we performed immunofluorescence staining of LKB1 and PARP1 and observed colocalization of the two proteins in both H1299 and LKB1-WT A549 cell lines (Supplementary Fig. S2B). Moreover, as shown in Fig. 2D, LKB1 could interact with PARP1. The same phenomenon was observed in murine cell line LLC1 and human lung adenocarcinoma cell line H1299–expressing wild-type LKB1 (Fig. 2E; Supplementary Fig. S2C). Next, we examined whether LKB1 regulates the total PARP1 expression or its catalytic activity in A549 cells and LLC1-derived tumors. Intriguingly, LKB1 status exerts no effect on PARP1 expression; instead, the catalytic activity of PARP1, represented by the expression of PAR polymers (PAR), was dramatically increased in LKB1-loss and LKB1-mutant A549 cells compared with the wild-type counterpart in response to IFNγ (Fig. 2F). In addition, IHC staining of murine tumor samples implicated that knockdown of LKB1 led to an increased level of PARs in vivo (Fig. 2G; Supplementary Fig. S2D). These data confirm that LKB1 inhibits the catalytic activity of PARP1 both in vitro and in vivo.

Furthermore, we sought to elucidate how LKB1 affects PARP1 catalytic activity. Reexamination of the single-cell transcriptomic landscape of our transgenic mouse models verified a defect in the DNA repair process in KL versus K-driven lung tumors (Fig. 1B). Given that PARP1 can be activated by damaged DNA and LKB1 deficiency causes genome instability (5, 13, 14), we therefore explored whether genome instability in LKB1-deficient cells was related to abnormally activated PARP1. We found that LKB1 deficiency resulted in elevated expression of γH2AX, a DNA damage marker in vivo (Fig. 2H). To further investigate the role of LKB1 in DNA damage, we performed comet assay analysis in vitro. As shown in Fig. 2I, LKB1-loss/mutant cells displayed a significantly severe DNA damage signal upon exposure to H2O2. We also found that LKB1 deficiency resulted in elevated expression of DNA damage markers (γH2AX, RAD51, and pCHK1) in A549 cells (Fig. 2J). Most strikingly, transfection with additional dsDNA potently increased the expression of PAR and γH2AX in LKB1-WT A549 cells (Fig. 2K). Together, these results indicate that LKB1 deficiency–mediated DNA damage repair deficiency abnormally activates PARP1.

Aberrantly activated PARP1 poly(ADP-ribosyl)ates STAT1 in LKB1-deficient tumor cells

The tyrosine phosphorylation of STAT1 is required for its dimerization, nuclear translocation, and DNA binding. Because recent studies have identified tyrosine residue as an ADP-ribose acceptor site and proposed the crosstalk between poly(ADP-ribosyl)ation and phosphorylation (15, 16), we assessed whether PARP1 could poly(ADP-ribosyl)ate STAT1 to inhibit its tyrosine phosphorylation. To this end, we first conducted pulldown assays in A549 cells and LLC1 cells and confirmed a direct interaction between PARP1 and STAT1 (Fig. 3A and B). Next, we determined whether STAT1 could be poly(ADP-ribosyl)ated by endogenous PARs in A549 cells and LLC1 cells. As expected, PARs were pulled down with STAT1 (Fig. 3C and D). Poly(ADP-ribosyl)ation of STAT1 was further confirmed, as the molecular mass was markedly higher than 92kDa in the immunoprecipitation assay with PAR-specific antibodies (Fig. 3C and D). Taken together, this evidence demonstrates that STAT1 could be poly(ADP-ribosyl)ated by PARP1.

Figure 3.

Aberrantly activated PARP1 poly(ADP-ribosyl)ates STAT1 in LKB1-deficient tumor cells. A and B, A549 cells and LLC1 cells were stimulated with IFNγ (50 ng/mL) for 24 hours, and the lysates were then immunoprecipitated with an anti-PARP1 antibody, followed by immunoblotting with the STAT1 antibody. C and D, A549 cells and LLC1 cells were stimulated with IFNγ (50 ng/mL) for 24 hours, and the lysates were then immunoprecipitated with an anti-PAR antibody, followed by immunoblotting with the STAT1 antibody. E and F, A549 lung cancer cells (LV-Ctrl, LV-LKB1-WT, and LV-LKB1-MUT) and LLC1 lung cancer cells (LV-Ctrl and LV-Lkb1) were stimulated with IFNγ for 24 hours. The cell lysates were then subjected to IP assay with anti-STAT1 and Western blot analysis with indicated antibodies. G, A549 cells were transfected with siPARP1 and siRNA-NC for 24 hours and then treated with IFNγ (50 ng/mL) for 24 hours, followed by immunoprecipitation and Western blot analysis. H, H1299 cells were transfected with wild-type Flag-PARP1 or the catalytic mutant Flag-PARP1 (E988), followed by stimulation with IFNγ. The lysates were then subjected to IP assay and Western blot analysis. I and J, H1299 cells were transfected with si-LKB1 and si-NC (I), or wild-type LKB1 and empty vector (J), followed by treatment with IFNγ. The lysates were then subjected to IP assay and Western blot analysis with indicated antibodies.

Figure 3.

Aberrantly activated PARP1 poly(ADP-ribosyl)ates STAT1 in LKB1-deficient tumor cells. A and B, A549 cells and LLC1 cells were stimulated with IFNγ (50 ng/mL) for 24 hours, and the lysates were then immunoprecipitated with an anti-PARP1 antibody, followed by immunoblotting with the STAT1 antibody. C and D, A549 cells and LLC1 cells were stimulated with IFNγ (50 ng/mL) for 24 hours, and the lysates were then immunoprecipitated with an anti-PAR antibody, followed by immunoblotting with the STAT1 antibody. E and F, A549 lung cancer cells (LV-Ctrl, LV-LKB1-WT, and LV-LKB1-MUT) and LLC1 lung cancer cells (LV-Ctrl and LV-Lkb1) were stimulated with IFNγ for 24 hours. The cell lysates were then subjected to IP assay with anti-STAT1 and Western blot analysis with indicated antibodies. G, A549 cells were transfected with siPARP1 and siRNA-NC for 24 hours and then treated with IFNγ (50 ng/mL) for 24 hours, followed by immunoprecipitation and Western blot analysis. H, H1299 cells were transfected with wild-type Flag-PARP1 or the catalytic mutant Flag-PARP1 (E988), followed by stimulation with IFNγ. The lysates were then subjected to IP assay and Western blot analysis. I and J, H1299 cells were transfected with si-LKB1 and si-NC (I), or wild-type LKB1 and empty vector (J), followed by treatment with IFNγ. The lysates were then subjected to IP assay and Western blot analysis with indicated antibodies.

Close modal

Then we analyzed the balance between poly(ADP-ribosly)ation and phosphorylation of STAT1 in A549 cells and LLC1 cells. Under IFNγ stimulation, a higher level of poly(ADP-ribosly)ation of STAT1 was observed in LKB1 loss or mutant cells compared with LKB1-WT cells (Fig. 3E and F). Knockdown of PARP1 markedly increased the IFNγ-induced phosphorylation of STAT1 in A549 cells through attenuating poly(ADP-ribosly)ation of STAT1 (Fig. 3G). To confirm whether inhibition of phosphorylated STAT1 was dependent on the catalytic activity of PARP1, we introduced a cell model with catalytic mutant PARP1 (E988). Under IFNγ stimulation, poly(ADP-ribosly)ation of STAT1 was significantly decreased accompanied by upregulated expression of pSTAT1 and PD-L1 in H1299 cells transfected with MUT-PARP1 (Fig. 3H). We further compared the activation of the IFNγ signaling pathway using A549 and LLC1 in regard of mutant PARP1 introduction. Similar results were obtained in both LKB1-deficient cell lines; under IFNγ stimulation, cells transfected with Mut-PARP1 exhibited decreased expression of PAR (PARP1 activity) and upregulated expression of pSTAT1 and PD-L1 compared with the control counterpart. These results suggested that mutant PARP1 impairs the PARP1 activity thereby enhancing the downstream IFNγ signaling (Supplementary Fig. S3A and S3B).

We next sought to investigate the functional interplay among LKB1, PARP1, and STAT1. Combined with our previous results (Fig. 2), we conjectured that LKB1 promotes IFNγ-induced phosphorylation of STAT1 by suppressing poly(ADP-ribosly)ation of STAT1. By silencing or overexpressing LKB1 in H1299 cells expressing wild-type LKB1, we observed that knockdown of LKB1 dramatically enhanced the poly(ADP-ribosly)ation of STAT1 and increased intracellular PARs while simultaneously reducing phosphorylated STAT1 and PD-L1, which was reversed by overexpressing LKB1 (Fig. 3I and J). Collectively, these findings suggest that aberrant activation of PARP1 in LKB1-defective cells leads to a potent poly(ADP-ribosyl)ation of STAT1, which inhibits its phosphorylation.

PARP1 inhibition restores phosphorylated STAT1 from poly(ADP-ribosyl)ation and triggers synthetic lethality in LKB1-deficient context

To test whether PARP1 inhibition restored the intracellular IFNγ pathway by promoting activation of STAT1 in LKB1-deficient cells, a clinically approved PARP1 inhibitor, olaparib (17), was selected for further study. Under IFNγ stimulation, the phosphorylation of STAT1 was restored along with a significant decrease of its PARylation level in LKB1-deficient cells treated with olaparib (Fig. 4A), whereas olaparib alone exerts no effect on STAT1 activation in the absence of IFNγ stimulation (Supplementary Fig. S4A). Consistently, total and nuclear pSTAT1 protein was upregulated, concomitant with nuclear IRF1, in IFNγ-induced LKB1-loss and LKB1-mutant cells following olaparib treatment (Fig. 4B).

Figure 4.

PARP1 inhibition restores phosphorylated STAT1 from poly(ADP-ribosyl)ation and triggers synthetic lethality in LKB1-deficient cells. A, Cells were pretreated with olaparib (5μmol/L) for 6 hours and then stimulated with IFNγ for 24 hours. The cell lysates were then subjected to IP assay and Western blot. BE, A549 cells (LV-Ctrl, LV-LKB1-WT, and LV-LKB1-Mut) were pretreated with olaparib and then stimulated with IFNγ. Total pSTAT1 and STAT1 as well as nuclear pSTAT1 and IRF1 were quantified using Western blot (B). Cell-surface PD-L1 was measured by flow cytometry (C and D). The mRNA expression of CXCL9, CXCL10, CCL5, and HLA-A was measured by qRT-PCR (E). F, Level of DNA damage among three groups of A549 cells in comet assay after the treatment with IFNγ and olaparib for 48 hours. G, A549 cells were treated with IFNγ alone or in combination with olaparib for 48 hours and then the cells were collected for apoptosis assay using flow cytometry. Data are presented as the mean ± SEM of three independent experiments. Statistical significance was determined by the Student t test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 4.

PARP1 inhibition restores phosphorylated STAT1 from poly(ADP-ribosyl)ation and triggers synthetic lethality in LKB1-deficient cells. A, Cells were pretreated with olaparib (5μmol/L) for 6 hours and then stimulated with IFNγ for 24 hours. The cell lysates were then subjected to IP assay and Western blot. BE, A549 cells (LV-Ctrl, LV-LKB1-WT, and LV-LKB1-Mut) were pretreated with olaparib and then stimulated with IFNγ. Total pSTAT1 and STAT1 as well as nuclear pSTAT1 and IRF1 were quantified using Western blot (B). Cell-surface PD-L1 was measured by flow cytometry (C and D). The mRNA expression of CXCL9, CXCL10, CCL5, and HLA-A was measured by qRT-PCR (E). F, Level of DNA damage among three groups of A549 cells in comet assay after the treatment with IFNγ and olaparib for 48 hours. G, A549 cells were treated with IFNγ alone or in combination with olaparib for 48 hours and then the cells were collected for apoptosis assay using flow cytometry. Data are presented as the mean ± SEM of three independent experiments. Statistical significance was determined by the Student t test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Close modal

We further found that olaparib restored IFNγ-induced PD-L1 surface expression, and simultaneously upregulated Th1-type chemokines and HLA-A expression in LKB1-deficient cells (Fig. 4CE). These results were orthogonally validated in LLC1-shLkb1 cells (Supplementary Fig. S4B–S4D). To examine the effect of olaparib on the tumor immune microenvironment, the activation level of CD8+ T cells was assessed after coculturing with tumor cells (LKB1-deficient cells versus LKB1-WT cells) pretreated with IFNγ alone or in combination with olaparib. Results showed that the expression levels of cytokines and effector molecules secreted by cytotoxic CD8+ T cells were significantly upregulated when cocultured with olaparib-treated tumor cells, especially in the LKB1-deficient context (Supplementary Fig. S4E and S4F). In addition, we also evaluated the direct effect of olaparib on T-cell function; CD8+ T cells were stimulated with anti-CD8/CD28 followed by treatment with PBS or olaparib for 24 hours. Examination of the effector molecules secreted by cytotoxic CD8+ T cells showed a very modest or insignificant effect of olaparib on CD8+ T cells (Supplementary Fig. S4G). In view that IFNγ-induced antigen presentation molecules were reduced in LKB1-mutant NSCLC cells, we next explored the role of DCs in mediating the therapeutic responses of combined PARP1 and PD-1 inhibitors. We extracted DCs to coculture with CD8+ T cells and LKB1-loss LLC1 cells pretreated with IFNγ in the presence or absence of olaparib. Notably, apoptosis of tumor cells was significantly increased after coculturing with DCs, and the degree of improvement was even greater under olaparib treatment (Supplementary Fig. S4H), supporting the role of DCs in mediating the antitumor efficacy of the combined therapy in LKB1-mutant NSCLC.

In parallel, based upon the prior knowledge of LKB1 in maintaining HR and the clinically established role of olaparib in targeting cancers with HR defect, we hypothesized that the deficiency of LKB1 might confer sensitivity of cancer cells to olaparib through the synthetic lethal effect. We confirmed that olaparib treatment upregulated the percentage of DNA in the tail, which was more evident in LKB1-loss/mutant cancer cells than in the LKB1-WT counterpart (Fig. 4F). Supporting the comet assay data, measurement of cell apoptosis by the flow cytometry showed that the addition of olaparib resulted in enhanced cytotoxicity in LKB1-loss/mutant cancer cells (Fig. 4G). Together with the finding that olaparib ameliorates the impaired IFNγ signaling, these results indicate that PARP1 inhibition might serve as an optimal combination candidate for PD-1 blockade particularly for the treatment of LKB1-deficient lung tumors by reigniting the adaptive immunity while simultaneously triggering synthetic lethality of tumor cells.

Olaparib-derived dual effects sensitize LKB1-deficient tumors to anti–PD-1 immunotherapy in preclinical models

On the basis of the findings in vitro, we further assessed whether PARP1 inhibitors could reverse resistance to immunotherapies using multiple LKB1-deficient mouse models administered with different treatments: vehicle, anti–PD-1 antibody, olaparib, or their combination. We observed that the combination of olaparib and anti–PD-1 antibody controlled tumor growth in our genetically engineered mouse models with Kras/Lkb1-driven lung tumors (Fig. 5A), and increased tumor PD-L1 expression and CD8+ T-cell infiltration (Fig. 5B). Similar results were observed in the orthotopic models after the implantation of LLC1-shLkb1-Luc cells in situ (Fig. 5C; Supplementary Fig. S5A). Concordantly, olaparib significantly increased tumor PD-L1 expression whereas the combination treatment simultaneously augmented CD8+ T-cell infiltration, and this effect was accompanied with a decrease in tumor-intrinsic PARylation level (Fig. 5D and E; Supplementary Fig. S5B–S5F). As expected, olaparib induced DNA damage, indicated by a higher γH2AX level in the LKB1-deficient orthotopic models treated with olaparib or combination therapy (Fig. 5F). Moderate treatment benefits were observed in mice receiving olaparib compared with both vehicle and anti–PD-1 monotherapy according to tumor burden, indicative of the synthetic lethal effect (Fig. 5G and H). Still, olaparib alone was not sufficient to restrict tumor growth and failed to yield significant long-term survival benefits (Fig. 5I). Notably, the combination of olaparib with an anti–PD-1 antibody substantially restricted tumor growth and prolonged the OS compared with both monotherapies, indicative of the long-term benefit derived from the reinvigoration of the adaptive immunity by olaparib (Fig. 5GI; Supplementary Fig. S5G). Collectively, these preclinical results indicate that targeting PARP1 by olaparib overcomes the primary resistance of PD-1 blockade immunotherapy in LKB1-deficient lung cancer by eliciting synthetic lethality and adaptive immunity.

Figure 5.

Olaparib-derived dual effects sensitize LKB1-deficient tumors to anti–PD-1 immunotherapy in preclinical models. A, Representative coronal sections of MRI images of genetically engineered Kras-driven mouse model with additional deletion of Lkb1 lung tumors before treatment and after two weeks of treatment (vehicle, anti–PD-1 Ab, and combination treatment). The contours of lung tumors were sketched. B, Immunofluorescence analysis of PD-L1+ cells (green) and CD8+ T cells (red) in resected KL GEMM samples following different arms of treatment. Scale bar, 100 μm. C, Mice-bearing orthotopic lung tumors were treated with different therapies. Representative bioluminescent images are shown. DF, Representative images of immunofluorescence staining of PD-L1+ cells (green) and CD8+ T cells (red; D), γH2AX+ cells (red; F), and images of PAR, CD8+ T, and PD-L1 IHC staining (E) in resected lung tumors of mice bearing orthotopic tumors. Scale bar, 100 μm (D), 50 μm (E), and 20 μm (F). GI, Mice bearing subcutaneous lung tumors were treated with different therapies (n = 5∼7 mice/group). Tumor size (G and H) and survival (I) in different treatment arms were monitored. Results are presented as mean ± SEM. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, two-way ANOVA or log-rank tests.

Figure 5.

Olaparib-derived dual effects sensitize LKB1-deficient tumors to anti–PD-1 immunotherapy in preclinical models. A, Representative coronal sections of MRI images of genetically engineered Kras-driven mouse model with additional deletion of Lkb1 lung tumors before treatment and after two weeks of treatment (vehicle, anti–PD-1 Ab, and combination treatment). The contours of lung tumors were sketched. B, Immunofluorescence analysis of PD-L1+ cells (green) and CD8+ T cells (red) in resected KL GEMM samples following different arms of treatment. Scale bar, 100 μm. C, Mice-bearing orthotopic lung tumors were treated with different therapies. Representative bioluminescent images are shown. DF, Representative images of immunofluorescence staining of PD-L1+ cells (green) and CD8+ T cells (red; D), γH2AX+ cells (red; F), and images of PAR, CD8+ T, and PD-L1 IHC staining (E) in resected lung tumors of mice bearing orthotopic tumors. Scale bar, 100 μm (D), 50 μm (E), and 20 μm (F). GI, Mice bearing subcutaneous lung tumors were treated with different therapies (n = 5∼7 mice/group). Tumor size (G and H) and survival (I) in different treatment arms were monitored. Results are presented as mean ± SEM. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, two-way ANOVA or log-rank tests.

Close modal

Rejuvenation of adaptive immunity by olaparib yields the long-term immunotherapeutic benefit of LKB1-deficient tumors

To determine whether the long-term benefit of combined olaparib/PD-1 blockade was mediated by the reactivation of the IFNγ signaling pathway in LKB1-deficient tumors, we characterized the alterations of the TME at the end of the treatment. Following our in vivo observations that a combination of olaparib and anti–PD-1 antibody significantly facilitated CD8+ T-cell infiltration, IFNγ secreted by activated T cells was confirmed and IFNγ was increased in tumors from mice following anti–PD-1/olaparib treatment by using histochemical staining (Supplementary Fig. S6). Simultaneously, IFNγ-induced production of chemokines, such as CXCL9, CXCL10, CXCL11, and CCL5, were significantly elevated following olaparib/anti–PD-1 treatment compared with monotherapy or vehicle, indicative of a more proinflammatory landscape within TME (Fig. 6A). In addition, combination therapy resulted in a significantly upregulated expression level of PD-L1 on tumor cells, which was similarly observed in the olaparib group (Fig. 6B).

Figure 6.

Rejuvenation of adaptive immunity by olaparib yields the long-term immunotherapeutic benefit of LKB1-deficient tumors. AG, Tumors from mice bearing LLC1-shLkb1 tumors with different therapies [immunoglobulin G (IgG), anti–PD-1 Ab or olaparib (OLA), or combination treatment] were harvested after a 2-week treatment. Tumor cytokine/chemokine was quantified by qRT-PCR. Results are presented as mean ± SEM; Student t tests; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (A). Quantification of surface expression of PD-L1 on tumors (B) and tumor-infiltrating immune cells was analyzed by flow cytometry (CE). Expressions of CD69 and PD-1 on tumor-infiltrating CD8+ T cells were quantified by flow cytometry (F). Results are presented as mean ± SEM; Student t tests; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Expression of CD8+ T cells secreted IFNγ and TNF was analyzed by qRT-PCR (G). Results are presented as mean ± SEM. Student t tests; ***, P < 0.001; ****, P < 0.0001. H and I, C57BL/6 mice bearing LLC1-shLkb1 tumors were treated with depleting antibodies before treatment with anti–PD-1 Ab (200 μg/mouse, three times weekly) plus olaparib (25 mg/kg/i.p., daily) for 2 weeks (n = 6 mice/group). Tumor volume (H) or survival (I) was assayed. Results are presented as mean ± SEM; two-way ANOVA or log-rank tests; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6.

Rejuvenation of adaptive immunity by olaparib yields the long-term immunotherapeutic benefit of LKB1-deficient tumors. AG, Tumors from mice bearing LLC1-shLkb1 tumors with different therapies [immunoglobulin G (IgG), anti–PD-1 Ab or olaparib (OLA), or combination treatment] were harvested after a 2-week treatment. Tumor cytokine/chemokine was quantified by qRT-PCR. Results are presented as mean ± SEM; Student t tests; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (A). Quantification of surface expression of PD-L1 on tumors (B) and tumor-infiltrating immune cells was analyzed by flow cytometry (CE). Expressions of CD69 and PD-1 on tumor-infiltrating CD8+ T cells were quantified by flow cytometry (F). Results are presented as mean ± SEM; Student t tests; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Expression of CD8+ T cells secreted IFNγ and TNF was analyzed by qRT-PCR (G). Results are presented as mean ± SEM. Student t tests; ***, P < 0.001; ****, P < 0.0001. H and I, C57BL/6 mice bearing LLC1-shLkb1 tumors were treated with depleting antibodies before treatment with anti–PD-1 Ab (200 μg/mouse, three times weekly) plus olaparib (25 mg/kg/i.p., daily) for 2 weeks (n = 6 mice/group). Tumor volume (H) or survival (I) was assayed. Results are presented as mean ± SEM; two-way ANOVA or log-rank tests; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Close modal

Consistent with our findings in vitro, olaparib induced activation of tumor IFNγ signaling pathway and led to an immunologically “hot” landscape characterized by an enhanced migration of immune cells into TME, especially cytotoxic lymphocytes, such as CD8+ T cells and natural killer (NK) cells, which was most pronounced in the combination treatment group (Fig. 6CE). Apart from the quantity, the function of TILs was also assessed. Simultaneously, a more activated status of CD8+ T cells was observed in the combination group, as indicated by the upregulation of activation markers (CD69 and PD-1) and functional effector cytokines (IFNγ and TNF; Fig. 6F and G). To further demonstrate the antitumor efficacy of the combination treatment is dependent on CD8+ T cells and NK cells, depletion antibodies were used accordingly. Compared with the control group, olaparib/anti–PD-1–mediated tumor suppression was reversed by adding anti-CD8 and anti-NK1.1 antibodies, and the survival time was also shortened (Fig. 6H and I). Collectively, these results confirm that PARP1 inhibition promotes the efficacy of anti–PD-1 immunotherapy through the IFNγ signaling pathway, which is thereby dependent on CD8+ T cells and NK cells.

LKB1 deficiency leads to primary resistance to anti–PD-1 therapy in NSCLC (2). In this work, we reveal an aberrant PARP1 function underlying the adaptive immune evasion mechanism in LKB1-deficient lung cancers and accordingly propose the use of PARP1 inhibitors in overcoming immunotherapy resistance (Fig. 7). Our current findings expand the role of LKB1 in adaptive immunity via the PARP1/STAT1/IFNγ axis, which enriches the theory system of immunotherapy resistance mediated by LKB1 deficiency. Contemporaneous with the study on the interplay between the LKB1 and the cGAS-STING pathway (3), the mechanistic basis for the impaired immunotherapeutic effect of LKB1 deficiency can be comprehensively interpreted from both dimensions of innate and adaptive immunity.

Figure 7.

A mechanistic schematic diagram showing PARP1-mediated poly(ADP-ribosyl)ation of STAT1 as a key step in inhibiting the adaptive immune response mediated by IFNγ signal pathway in LKB1-mutant NSCLC. The utilization of the inhibitor olaparib, which inhibits PARP1 activity, can overcome the immunotherapy resistance by simultaneously triggering a synthetic lethal effect and reactivating the adaptive immunity by restoring phosphorylated STAT1 from poly(ADP-ribosyl)ation.

Figure 7.

A mechanistic schematic diagram showing PARP1-mediated poly(ADP-ribosyl)ation of STAT1 as a key step in inhibiting the adaptive immune response mediated by IFNγ signal pathway in LKB1-mutant NSCLC. The utilization of the inhibitor olaparib, which inhibits PARP1 activity, can overcome the immunotherapy resistance by simultaneously triggering a synthetic lethal effect and reactivating the adaptive immunity by restoring phosphorylated STAT1 from poly(ADP-ribosyl)ation.

Close modal

As bulk sequencing of biopsies has a limited resolution in dissecting intratumoral subclonal hierarchy (18), which may obscure cell type–specific expression programs, our study gains deeper insights, into the single-cell resolution, into the mechanisms of LKB1 deficiency–induced resistance through using scRNA-seq technology with genetically engineered mouse models. On this basis, we provide evidence that LKB1 deficiency restrains immunotherapy efficacy due to the compromised IFNγ response in tumor cells. It is the downregulation of the IFNγ signaling pathway that leads to decreased expression of chemokines/cytokines, PD-L1, and MHC class I molecules in LKB1-deficient tumors, consequently forming an immune-suppressive TME characterized by reduced recruitment and impaired activation of effector immune cells.

Further screens from mass spectrometry and protein interaction analyses indicate that PARP1 serves as an intermediary for the communication between LKB1 and the IFNγ signaling. Although PAPR1 was traditionally characterized to play a pivotal role in DNA repair and modification of chromatin structure (19), increasing evidence has revealed that PAPR1 can also exert marked effects on a wide range of biological processes through poly(ADP-ribosyl)ation of the corresponding target proteins (20). Several theories regarding the precise molecular mechanisms have been proposed, most notably the functional interplay between poly(ADP-ribosyl)ation and phosphorylation (20, 21). In the present study, we provide novel insights into the adaptive immunity regulation of PARP1-mediated poly(ADP-ribosyl)ation of STAT1, a key molecule of IFNγ signaling, in the context of LKB1-deficient NSCLC. Mechanistically, LKB1 deficiency-mediated HR defect gives rise to the secondary activation of catalytic activity of PARP1, which provokes the poly(ADP-ribosyl)ation of STAT1 and replaces its phosphorylation (the activated form), thereby blocking the downstream effector molecules of IFNγ signaling. As LKB1 loss of function causes genomic instability due to HR defect (4, 5), nonsynonymous mutations are generated accompanied with the secondarily activated PARP1; thus, our findings explain the seemingly contradictory but mechanistically uniform coexistence of high TMB and poor immunotherapeutic efficacy in LKB1-deficient NSCLC.

In light of the aforementioned contradictory unity of characteristics specific to LKB1 deficiency, this property is exploited to propose the tailored combination strategy of PARP1 and PD-1 inhibitions. Compared with other combination strategies, PARP1 inhibitors play a dual role in restoring the antitumor efficacy of PD-1 blockade in the context of LKB1-deficient NSCLC. On one side, PARP1 inhibitors have been traditionally used for tumors with HR defects based on the genetic concept of synthetic lethality over the decades (8, 22); this makes sense as LKB1 has been identified as a critical maintainer of HR (4, 5). On the other, which is even more pronounced, PARP1 inhibitors can reverse the LKB1-deficiency–mediated immunosuppressive phenotype by reactivating the IFNγ signaling pathway through restoring the phosphorylation of STAT1. Our preclinical mouse models demonstrate that the monotherapy of the PARP1 inhibitor, olaparib, slows the tumor growth though not predominant visibly, potentially representing the synthetic lethality effect; meanwhile, the addition of the anti–PD-1 agent to olaparib exhibits a significantly enhanced antitumor activity, representing the adaptive immune activation effect of PARP1 inhibitors (Fig. 5F). This evidence lends a mechanistic basis to the combination of PARP1 and PD-1 inhibitors specifically for those NSCLC patients with LKB1 loss of function.

Previous studies have indicated that LKB1 mutations cause innate immunity deficiency by silencing STING and PARP1 inhibition could remarkably potentiate the antitumor effect of PD-L1 blockade and augmented cytotoxic T-cell infiltration by activating the STING pathway (23, 24). We also confirmed that PARP1 inhibition could not only restores the IFNγ signaling pathway in LKB1-mutant NSCLC but also triggers STING pathway activation (Supplementary Fig. S7A–S7C). The result is consistent with the prior knowledge that PARP1 inhibition compromises the ability of tumor cells to repair DNA single-strand breaks, resulting in the accumulation of dsDNA, whereas STING can be activated by the aberrant dsDNA (25). Complementary to these findings, our study further revealed a more direct regulatory mechanism between LKB1 and STAT1, through their interplay with PARP1 independent of the STING pathway. Given the role of LKB1 in maintaining HR and PARP1 in impairing STAT1 activity through poly(ADP-ribosyl)ation observed in our study, we proposed the olaparib-derived dual effects in overcoming LKB1 loss-driven immunotherapy resistance by harnessing the PARP1 inhibition-mediated crosstalk between synthetic lethality and adaptive immunity, which might transform the way we care for these difficult-to-treat patients.

Limitations existed in the present study. First of all, the established GEMMs of our study were K versus KL lung tumors. Because KRAS mutation-driven lung cancers frequently harbored concurrent inactivated TP53 and/or LKB1 (2, 26, 27), the conclusion would be more convincing if parallel experiments were conducted in the context of an oncogenotype combination of KRAS, TP53, and LKB1 mutations; despite that, we used the scRNA-seq data of KP versus KPL-driven lung tumors and to some extents verified the findings observed in K versus KL models. In addition, the histopathological classification was different between the lung tumor cell lines used in our study, including A549 and LLC1 (28, 29); therefore, our conclusions are not limited to a specific pathological subgroup of NSCLC.

In summary, the present study expands the theory of LKB1 deficiency–mediated immunotherapy resistance to the adaptive immune regulation of the PARP1/STAT1/IFNγ axis. Importantly, our results suggest a new strategy to reverse primary immunotherapy resistance by combining PARP1 inhibitors with anti–PD-1 antibodies in the context of LKB1-deficient lung tumors. This tailored approach elicits antitumor activity against LKB1-deficient tumors through both threads of inducing the synthetic lethal effect and reactivating the adaptive immunity. More clinical studies should be conducted to further validate the effectiveness of this combination therapy.

X. Pan reports grants from Natural Science Foundation of Guangdong Province during the conduct of the study. No disclosures were reported by the other authors.

L.-L. Long: Conceptualization, data curation, formal analysis, validation, investigation, visualization, methodology, writing–original draft. S.-C. Ma: Conceptualization, formal analysis, validation, investigation, visualization, methodology, writing–original draft, writing–review and editing. Z.Q. Guo: Software, formal analysis, validation, investigation. Y.P. Zhang: Data curation, formal analysis, investigation, writing–review and editing. Z. Fan: Resources. L.-J. Liu: Software. L. Liu: Supervision, visualization. D.-D. Han: Software, validation. M.-X. Leng: Formal analysis. J. Wang: Visualization. X.-J. Guo: Methodology. J.-L. Tan: Methodology. X.-T. Cai: Investigation. Y. Lin: Visualization. X. Pan: Resources. D.-H. Wu: Resources, supervision, funding acquisition, writing–review and editing. X. Bai: Supervision, funding acquisition, validation, writing–review and editing. Z.-Y. Dong: Conceptualization, resources, supervision, funding acquisition, validation, project administration, writing–review and editing.

The authors are deeply grateful to Prof. Liang Chen (Institute of Life and Health Engineering, Jinan University, Guangzhou, China) for providing gifts of KrasG12D/+ mice. This study was supported by the National Natural Science Foundation of China (grant no. 82272820 and 82272731), the National Natural Science Foundation for Young Scientists of China (grant no. 81802863 and 81902353), and the Outstanding Youths Development Scheme of Nanfang Hospital, Southern Medical University (grant no. 2017J003 and 2020J011).

The publication costs of this article were defrayed in part by the payment of publication fees. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

1.
Skoulidis
F
,
Byers
LA
,
Diao
L
,
Papadimitrakopoulou
VA
,
Tong
P
,
Izzo
J
, et al
.
Co-occurring genomic alterations define major subsets of KRAS-mutant lung adenocarcinoma with distinct biology, immune profiles, and therapeutic vulnerabilities
.
Cancer Discov
2015
;
5
:
860
77
.
2.
Skoulidis
F
,
Goldberg
ME
,
Greenawalt
DM
,
Hellmann
MD
,
Awad
MM
,
Gainor
JF
, et al
.
STK11/LKB1 mutations and PD-1 inhibitor resistance in KRAS-mutant lung adenocarcinoma
.
Cancer Discov
2018
;
8
:
822
35
.
3.
Kitajima
S
,
Ivanova
E
,
Guo
S
,
Yoshida
R
,
Campisi
M
,
Sundararaman
SK
, et al
.
Suppression of STING associated with LKB1 loss in KRAS-driven lung cancer
.
Cancer Discov
2019
;
9
:
34
45
.
4.
Deng
J
,
Thennavan
A
,
Dolgalev
I
,
Chen
T
,
Li
J
,
Marzio
A
, et al
.
ULK1 inhibition overcomes compromised antigen presentation and restores antitumor immunity in LKB1 mutant lung cancer
.
Nat Cancer
2021
;
2
:
503
14
.
5.
Gupta
R
,
Liu
AY
,
Glazer
PM
,
Wajapeyee
N
.
LKB1 preserves genome integrity by stimulating BRCA1 expression
.
Nucleic Acids Res
2015
;
43
:
259
71
.
6.
Mandal
R
,
Samstein
RM
,
Lee
KW
,
Havel
JJ
,
Wang
H
,
Krishna
C
, et al
.
Genetic diversity of tumors with mismatch repair deficiency influences anti–PD-1 immunotherapy response
.
Science
2019
;
364
:
485
91
.
7.
Binnewies
M
,
Roberts
EW
,
Kersten
K
,
Chan
V
,
Fearon
DF
,
Merad
M
, et al
.
Understanding the tumor immune microenvironment (TIME) for effective therapy
.
Nat Med
2018
;
24
:
541
50
.
8.
O'Neil
NJ
,
Bailey
ML
,
Hieter
P
.
Synthetic lethality and cancer
.
Nat Rev Genet
2017
;
18
:
613
23
.
9.
Doumba
PP
,
Nikolopoulou
M
,
Gomatos
IP
,
Konstadoulakis
MM
,
Koskinas
J
.
Co-culture of primary human tumor hepatocytes from patients with hepatocellular carcinoma with autologous peripheral blood mononuclear cells: study of their in vitro immunological interactions
.
BMC Gastroenterol
2013
;
13
:
17
.
10.
Wu
Q
,
Tian
Y
,
Zhang
J
,
Tong
X
,
Huang
H
,
Li
S
, et al
.
In vivo CRISPR screening unveils histone demethylase UTX as an important epigenetic regulator in lung tumorigenesis
.
Proc Natl Acad Sci U S A
2018
;
115
:
E3978
-
e86
.
11.
Li
H
,
Liu
Z
,
Liu
L
,
Zhang
H
,
Han
C
,
Girard
L
, et al
.
AXL targeting restores PD-1 blockade sensitivity of STK11/LKB1 mutant NSCLC through expansion of TCF1(+) CD8 T cells
.
Cell Rep Med
2022
;
3
:
100554
.
12.
Blazanin
N
,
Cheng
T
,
Carbajal
S
,
DiGiovanni
J
.
Activation of a protumorigenic IFNγ/STAT1/IRF-1 signaling pathway in keratinocytes following exposure to solar ultraviolet light
.
Mol Carcinog
2019
;
58
:
1656
69
.
13.
Wattenberg
MM
,
Reiss
KA
.
Determinants of homologous recombination deficiency in pancreatic cancer
.
Cancers
2021
;
13
:
4716
.
14.
Chaudhuri
AR
,
Nussenzweig
A
.
The multifaceted roles of PARP1 in DNA repair and chromatin remodelling
.
Nat Rev Mol Cell Biol
2017
;
18
:
610
21
.
15.
Pedrioli
DML
,
Leutert
M
,
Bilan
V
,
Nowak
K
,
Gunasekera
K
,
Ferrari
E
, et al
.
Comprehensive ADP-ribosylome analysis identifies tyrosine as an ADP-ribose acceptor site
.
EMBO Rep
2018
;
19
:
e45310
.
16.
Huang
D
,
Camacho
CV
,
Setlem
R
,
Ryu
KW
,
Parameswaran
B
,
Gupta
RK
, et al
.
Functional interplay between histone H2B ADP-ribosylation and phosphorylation controls adipogenesis
.
Mol Cell
2020
;
79
:
934
49
.
17.
Santiago-O'Farrill
JM
,
Weroha
SJ
,
Hou
X
,
Oberg
AL
,
Heinzen
EP
,
Maurer
MJ
, et al
.
Poly(adenosine diphosphate ribose) polymerase inhibitors induce autophagy-mediated drug resistance in ovarian cancer cells, xenografts, and patient-derived xenograft models
.
Cancer
2020
;
126
:
894
907
.
18.
Levitin
HM
,
Yuan
J
,
Sims
PA
.
Single-cell transcriptomic analysis of tumor heterogeneity
.
Trends Cancer
2018
;
4
:
264
8
.
19.
Krishnakumar
R
,
Kraus
WL
.
The PARP side of the nucleus: molecular actions, physiological outcomes, and clinical targets
.
Mol Cell
2010
;
39
:
8
24
.
20.
Alemasova
EE
,
Lavrik
OI
.
Poly(ADP-ribosyl)ation by PARP1: reaction mechanism and regulatory proteins
.
Nucleic Acids Res
2019
;
47
:
3811
27
.
21.
Ding
L
,
Chen
X
,
Xu
X
,
Qian
Y
,
Liang
G
,
Yao
F
, et al
.
PARP1 suppresses the transcription of PD-L1 by Poly(ADP-Ribosyl)ating STAT3
.
Cancer Immunol Res
2019
;
7
:
136
49
.
22.
Mateo
J
,
Lord
CJ
,
Serra
V
,
Tutt
A
,
Balmaña
J
,
Castroviejo-Bermejo
M
, et al
.
A decade of clinical development of PARP inhibitors in perspective
.
Ann Oncol
2019
;
30
:
1437
47
.
23.
Kitajima
S
,
Ivanova
E
,
Guo
S
,
Yoshida
R
,
Campisi
M
,
Sundararaman
SK
, et al
.
Suppression of STING associated with LKB1 loss in KRAS-driven lung cancer
.
Cancer Discov
2019
;
9
:
34
45
.
24.
Sen
T
,
Rodriguez
BL
,
Chen
L
,
Corte
CMD
,
Morikawa
N
,
Fujimoto
J
, et al
.
Targeting DNA damage response promotes antitumor immunity through STING-mediated T-cell activation in small-cell lung cancer
.
Cancer Discov
2019
;
9
:
646
61
.
25.
Kwon
J
,
Bakhoum
SF
.
The cytosolic DNA-sensing cGAS–STING pathway in cancer
.
Cancer Discov
2020
;
10
:
26
39
.
26.
Kadara
H
,
Choi
M
,
Zhang
J
,
Parra
ER
,
Rodriguez-Canales
J
,
Gaffney
SG
, et al
.
Whole-exome sequencing and immune profiling of early-stage lung adenocarcinoma with fully annotated clinical follow-up
.
Ann Oncol
2017
;
28
:
75
82
.
27.
Rizvi
H
,
Sanchez-Vega
F
,
La
K
,
Chatila
W
,
Jonsson
P
,
Halpenny
D
, et al
.
Molecular determinants of response to anti-programmed cell death (PD)-1 and anti-programmed Death-ligand 1 (PD-L1) blockade in patients with non–small cell lung cancer profiled with targeted next-generation sequencing
.
J Clin Oncol
2018
;
36
:
633
41
.
28.
Yu
M
,
Qi
B
,
Xiaoxiang
W
,
Xu
J
,
Liu
X
.
Baicalein increases cisplatin sensitivity of A549 lung adenocarcinoma cells via PI3K/Akt/NF-κB pathway
.
Biomed Pharmacother
2017
;
90
:
677
85
.
29.
Liu
P
,
Zhao
L
,
Senovilla
L
,
Kepp
O
,
Kroemer
G
.
In vivo imaging of orthotopic lung cancer models in mice
.
Methods Mol Biol
2021
;
2279
:
199
212
.

Supplementary data