Disruption of JNK-mediated PCK1 S151 phosphorylation using 3-MPA or JNK1/2 inhibitor elevates the anti-tumor effect of anti–PD-1 antibody. (A and B) 4T-1 cells (A) or EMT-6 cells (B) expressing mouse PCK1 shRNA with reconstituted expression of the indicated mouse PCK1 proteins were harvested for immunoblotting analyses as indicated. (C) TUNEL analyses of 4T-1 tumors expressing mouse PCK1 shRNA with reconstituted expression of Flag-rmPCK1 WT or S151A mutant were performed (left). Scale bar, 50 μm. Apoptotic cells were stained brown and quantified (right) (n = 6 per group). (D) IHC analyses of 4T-1 tumors expressing mouse PCK1 shRNA with reconstituted expression of Flag-rmPCK1 WT or S151A mutant were performed with an anti-Ki67 antibody. Representative staining images are shown (left). Scale bar, 50 μm. Ki67-positive cells were quantified (right) (n = 6 per group). (E–G) 4T-1 cells expressing mouse PCK1 shRNA with reconstituted expression of Flag-rmPCK1 WT or S151A mutant (1 × 10⁶) were injected into the mammary fat pad of female BALB/c mice treated with or without 3-MPA hydrochloride. The mice were euthanized and examined for tumor growth 30 days after injection (E), tumor volumes were calculated (F), and tumors were weighed (G) (n = 6 per group). (H and I) Representative bioluminescence images from the indicated days after the orthotopical injection of EMT-6 luciferase-expressing cells (1 × 10⁶) into the mammary fat pad of female BALB/c mice treated with control, 3-MPA, anti–PD-1 antibody, or the combination (H). The quantification of the bioluminescence imaging is shown (I) (n = 6 per group). (J and K) IHC analyses of the indicated xenograft tumor samples in H were performed with the indicated antibodies. Representative staining images are shown (J), and staining scores for the indicated tumor samples were compared (K). Scale bar, 50 μm. The indicated staining scores were quantified in n = 18 microscopic fields. (L–Q) BALB/c mice bearing 4T-1 tumors were treated with or without 3-MPA hydrochloride, anti–PD-1 antibody, or the combination. Intratumoral cells were harvested for scRNA-seq analyses. Cell density plots (L) and selected marker genes expressed by indicated sub-clustering of CD8⁺ T cells (M) were shown. Enrichment analyses of T cell cytotoxicity (N) and effector (O) signature in CD8⁺ T cell cluster and UMAP analyses of infiltrating cDC1 subpopulations (P), colored by expression module score of a DC signature (Q), were performed. (R–T) BALB/c mice bearing 4T-1 tumors treated with or without SP600125, anti–PD-1 antibody, or the combination. The mice were euthanized and examined for tumor growth 30 days after injection (R), tumor volumes were calculated (S), and tumors were weighed (T) (n = 6 per group). (U and V) IHC analyses of indicated tumor samples in R were performed with the indicated antibodies. Representative staining images are shown (U), and staining scores for the indicated tumor samples were compared (V). Scale bar, 50 μm. The indicated staining scores were quantified in n = 18 microscopic fields. (W) A schematic model showing PCK1 inhibits cGAS-STING activation by metabolic consumption of GTP to promote tumor immune evasion. Data are representative of as mean ± SD. Statistical significance was determined by two-tailed Student’s t test (C, D, G, I, K, T, and V), two-way ANOVA, followed by Tukey’s test (F and S); *P < 0.05, **P < 0.001, and ***P < 0.0001; NS, not significant. Source data are available for this figure: SourceData FS5.