Fig 2: LAMTOR1 mediates DNA fragments-induced cGAS degradation. (A) Immunoblot analysis of cGAS in LAMTOR1 knockdown 4T1 tumors treated with 5-FU (100 mg/kg) or vehicle. (B) Immunoblot analysis of cGAS in LAMTOR1 knockdown HeLa cells exposed to 5-FU (100 μM) for indicated days. (C) Immunoblot analysis of cGAS in LAMTOR1 knockdown and control MC38 (above) or HeLa (below) cells. (D) Immunoblot analysis of cGAS in HeLa (Left) and MCF-7 (Right) cells transfected with gradient amounts of LAMTOR1-Flag plasmids. (E and F) Immunoblot analysis (E) and quantification (F) of cGAS in LAMTOR1 knockdown MC38 cells treated with cycloheximide (CHX) (25 μg/mL) for indicated hours. n = 3; one-way ANOVA was used. (G) Lysosome isolation and immunoblot analysis of cGAS and LAMTOR1 in TMEM192-3HA stably expressed MC38 tumors treated with 5-FU (100 mg/kg) or vehicle for three times and released for indicated days. (H) qPCR analysis of LAMTOR1 mRNA in 4T1 tumors treated with 5-FU (100 mg/kg) or vehicle. n = 6; a two-tailed unpaired t test was used. (I) qPCR analysis of LAMTOR1 mRNA in MC38 tumors treated with 5-FU (100 mg/kg) or vehicle. n = 6; a two-tailed unpaired t test was used. (J) qPCR analysis of cGAS mRNA in rectal tumors before or after chemotherapy. n = 6; a two-tailed unpaired t test was used. (K) Immunoblot analysis of cGAS and LAMTOR1 in B16F10 (Left) and HeLa (Right) cells treated with 5-FU (100 μM) for indicated days. (L and M) Representative images (L) and statistical analysis (M) of immunohistochemical staining for LAMTOR1 and cGAS in the same clinical colorectal cancer samples. Scale bars, 5×, 600 μm and 40×, 75 μm. n = 55. (N) Immunoblot analysis of cGAS and LAMTOR1 in clinical colorectal tumors. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent SEM.

Fig 3: Loss of LAMTOR1 enhances the cGAS–STING–IFN-β pathway. (A) qPCR analysis of IFN-β mRNA by in HeLa cells transfected with LAMTOR1-Flag or vector for 24 h followed by transfection with HT-DNA (1 μg/mL) for another 12 h. n = 3; a two-tailed unpaired t test was used. (B) Immunoblot analysis of cGAS signaling in HeLa cells transfected with LAMTOR1-Flag or vector for 24 h followed by transfection with HT-DNA (1 μg/mL) for indicated hours. (C) qPCR analysis of IFN-β (Left) and CXCL10 (Right) mRNA in LAMTOR1 knockdown MC38 cells transfected with HT-DNA (1 μg/mL) for 12 h. n = 3; one-way ANOVA was used. (D) Immunoblot analysis of cGAS signaling in LAMTOR1 knockdown HeLa cells transfected with HT-DNA (1 μg/mL) for 6 h. (E) Immunoblot analysis of cGAS signaling in LAMTOR1 knockdown B16F10 cells transfected with HT-DNA (1 μg/mL) for indicated hours. (F) Immunoblot analysis of cGAS signaling in LAMTOR1 knockdown HeLa cells treated with 5-FU (100 μM) for 24 h and released 24 h. (G) Immunoblot analysis of cGAS signaling in LAMTOR1 knockdown B16F10 cells treated with 5-FU (100 μM) for 24 h and released 24 h. (H) qPCR analysis of IFN-β mRNA in LAMTOR1 knockdown HeLa cells treated with 5-FU (100 μM), etoposide (20 μM), or cDDP (4 μM) for 24 h. n = 3; one-way ANOVA was used. (I) qPCR analysis of IFN-β mRNA in LAMTOR1 knockdown B16F10 cells treated with 5-FU (100 μM), etoposide (20 μM), or cDDP (4 μM) for 24 h. n = 3; one-way ANOVA was used. (J and K) Representative images (J) and statistical analysis (K) of immunofluorescent imaging of cGAS foci in LAMTOR1 knockdown HeLa cells transfected with Cy5-ISD (2 μg/mL) for 8 h. Scale bars, 20 μm. n = 4; one-way ANOVA was used. (L) Immunoblot analysis of cGAS signaling in cGAS KO together with LAMTOR1 knockdown HeLa cells transfected with HT-DNA (1 μg/mL) for 6 h. (M) Quantitative PCR assay of IFN-β (Left) and CXCL10 (Right) mRNA in HeLa cells were transfected with HT-DNA (1 μg/mL) for 12 h and treated with chloroquine (CQ, 100 μM) or Bafilomycin A1 (Baf A1, 200 nM) for another 8 h. n = 3; one-way ANOVA was used. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars represent SEM.

Fig 4: DNA fragments enhance cGAS–LAMTOR1 interaction. (A) Silver staining of cGAS interactors by immunoprecipitating with M2 beads in HEK 293T cells treated with 5-FU (100 μM) or DMSO. (B) Overlapped proteins of annotated Lysosome gene sets and cGAS interactors whose amounts increased upon 5-FU (100 μM) treatment (ratio > 1.5). (C) Endogenous coimmunoprecipitation of cGAS and LAMTOR1 in HeLa cells. (D) GST pulldown assay of cGAS-Flag and GST-LAMTOR1 with full-length or different truncates as indicated. (E) GST pulldown assay of LAMTOR1-Flag and GST-cGAS with full-length or different truncates as indicated. (F and G) Representative images (F) and quantification (G) of colocalization for cGAS and HA (LAMTOR1) in HeLa cells treated with DMSO or 5-FU (100 μM) for indicated days. Scale bars, 5 μm. n = 4; one-way ANOVA was used. (H and I) Representative images (H) and quantification (I) of immunofluorescent imaging for cGAS and Flag (LAMTOR1) in HeLa cells transfected with HT-DNA (1 μg/mL) for 8 h. Scale bars, 10 μm. n = 4; a two-tailed unpaired t test was used. (J) Coimmunoprecipitation of cGAS-HA and LAMTOR1-Flag in HEK 293T upon treatment with 5-FU (100 μM), etoposide (20 μM), or DMSO for indicated days. NS, not significant, *P < 0.05, **P < 0.01, ****P < 0.0001. Error bars represent SEM.

Fig 5: LAMTOR1 promotes tumor growth via impeding antitumor immunity. (A and B) Tumor volume (A) and tumor weight (B) of LAMTOR1 knockdown B16F10 tumors. n = 6; one-way ANOVA was used. (C) The survival time of C57BL/6J mice inoculated with LAMTOR1 knockdown B16F10 cells. n = 7, log-rank (Mantel-Cox) test was used. (D) ELISA of cGAMP in LAMTOR1 knockdown B16F10 tumors after 20 d post inoculation. n = 6; one-way ANOVA was used. (E and F) Tumor volume (E) and tumor weight (F) of LAMTOR1 knockdown B16F10 tumors together with cGAS knock-out. n = 6; one-way ANOVA was used. (G) LAMTOR1 expression level in normal and tumor tissue in COAD and READ by bioinformatics analysis of TCGA and GTEx database. A two-tailed unpaired t test was used. (H and I) Representative image (H) and statistical analysis (I) of immunohistochemical staining for LAMTOR1 in the paired cancer and paracancer tissues. Scale bars, 5×, 500 μm and 40×, 50 μm. n = 58. (J and K) Representative images (J) and quantification (K) of immunofluorescent imaging for markers of effective T lymphocytes, CD3, CD4, or CD8 in LAMTOR1 knockdown B16F10 tumors after 20 d post inoculation. Scale bars, 50 μm. n = 8-12; one-way ANOVA was used. (L) Quantification of infiltrating CD3+, CD4+, CD8+ T cells in LAMTOR1 knockdown B16F10 tumors via flow cytometry after 20 d post inoculation. n = 6; one-way ANOVA was used. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars represent SEM.