Journal: Cancer Research
Article Title: PARP Inhibition Induces Synthetic Lethality and Adaptive Immunity in LKB1-Mutant Lung Cancer
doi: 10.1158/0008-5472.can-22-1740
Figure Lengend Snippet: Figure 1. LKB1 deficiency impairs the IFNg pathway by hindering STAT1 phosphorylation. A, Schematicof the generation of genetically engineered Kras-driven mouse model to establish KrasG12D/þ 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 IFNg (50 ng/mL). The expression of effectors downstream of IFNg 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 IFNg (50 ng/mL) for 24 hours. G, Quantification of secretory CXCL10 in A549 cells after treatment with IFNg (50 ng/mL) for 24 hours by ELISA analysis. H, A549 lung cancer cells were stimulated with IFNg for 24 hours and the conditional media were collected for Transwell assay with CD8þ T cells. I, A549 cells were treated with fludarabine (Flu) at different concentrations and IFNg (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 mmol/L) for 6 hours, followed by treatment with IFNg (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 ¼ 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.
Article Snippet: Cancer Res; 83(4) February 15, 2023 CANCER RESEARCH570 D ow nloaded from http://aacrjournals.org/cancerres/article-pdf/83/4/568/3268535/568.pdf by guest on 02 M arch 2024 For the cytokine study, either 1-mg IFNg (Novoprotein, #C746) or 100-ng CXCL10 (MedChemExpress, #HY-P7227) was mixed in the tumor cell suspension before injection of the tumor.
Techniques: Phospho-proteomics, Knock-Out, Expressing, Western Blot, Cytometry, Enzyme-linked Immunosorbent Assay, Transwell Assay, Control
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![Figure 1. LKB1 deficiency impairs the IFNg pathway by hindering STAT1 phosphorylation. A, Schematicof the generation of genetically engineered Kras-driven mouse model to establish KrasG12D/þ 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 IFNg (50 ng/mL). The expression of effectors downstream of IFNg 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 IFNg (50 ng/mL) for 24 hours. G, Quantification of secretory <t>CXCL10</t> in A549 cells after treatment with IFNg (50 ng/mL) for 24 hours by ELISA analysis. H, A549 lung cancer cells were stimulated with IFNg for 24 hours and the conditional media were collected for Transwell assay with CD8þ T cells. I, A549 cells were treated with fludarabine (Flu) at different concentrations and IFNg (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 mmol/L) for 6 hours, followed by treatment with IFNg (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 ¼ 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.](https://doi-unpaywalled-images-cdn.bioz.com/8849/10__1158_slash_0008___5472__can___22___1740/10__1158_slash_0008___5472__can___22___1740____page5_image1.jpg)
