Fig 2: ENPP1 dampens 2′3′-cGAMP signalingLung fibroblast cells from wild type and Enpp1-/- female mice were incubated with 2′3′-cGAMP analogs and HSV-60 at the indicated concentrations for 24 h. IFN-β in the media was measured using a B16-SEAP cell line. N=3 samples. Data are presented as mean and standard error.

Fig 3: ENPP1 is the dominant hydrolase activity for 2′3′-cGAMP(a) Knockdown of ENPP1 in MDA-MB231 cells diminished hydrolase activity. Four siRNA oligos against ENPP1 with a control siRNA sequence against PDE12 were used. 4 days after siRNA transfection, cells were lysed and assayed for activity (upper panel) and blotted for ENPP1 level (lower level). (b) Hydrolase activity in plasma from Enpp1-/- mice and their littermates. (c) Western blot characterization of Enpp1-/- mice. (d) Hydrolase activity in livers and spleens from Enpp1-/- mice and their littermates. Livers and spleens were minced and Dounce homogenized in lysis buffer: 1% NP-40, 20 mM Tris-HCl, pH 7.5, protease inhibitor cocktail. The assay was conducted in 0.2% NP-40, 20 mM Tris-HCl, 150 mM KCl, 2 mM Ca2+, 2 mM Mg2+, 200 μM Zn2+ at the indicated pH. NaOAc buffer was used for pH 5.0-6.0; PIPES buffer was used for pH 6.5 and 7.0 and 2′3′-cGAMP runs at a lower Rf in this buffer; Tris-HCl buffer was used for pH 7.5-9.0, and Borate buffer was used pH 9.5. These buffer conditions were also used in liver and spleen extract studies.

Fig 4: Development of hydrolysis resistant hSTING agonists(a) Scheme of enzymatic synthesis of 3′3′-cGAMP and 2′3′-cGAMP analogs. For 3′3′-cGAMP, 50 nM of DncV was incubated with 1 mM ATP and 1 mM GTP in 1 mL buffer containing 20 mM Tris-HCl, pH 8.0, 20 mM MgCl2 for 3 h at room temperature. For 2′3′-cGAMP analogs, 1-10 μM mouse cGAS (amino acid residues 147-507) was incubated with 1 mM ATP, 1 mM GTP, and 0.1 mg/mL HT-DNA in 1 mL of the same buffer for 12 h at room temperature. (b) Hydrolysis reactions of ENPP1 (1 nM) with the analogs (10 μM). (c) Scintillation proximity assay to measure the binding affinity of the analogs towards hSTING (amino acid residues 139-379). Biotinylated hSTING (100 nM) was immobilized onto 96-well streptavidin coated SPA plates. Neat 35S-labeled 2′3′-cGAsMP (500 pM) was used as the probe. N=3 samples. Data are presented as mean and standard error. (d) IFN-β production in THP-1 cells stimulated with the analogs. THP-1 cells were incubated with the analogs at the indicated concentrations for 24 h. IFN-β in the media was measured using a HEK-SEAP cell line. Representative data from a biological triplicate is depicted.

Fig 5: Treatment with VIR3 Enpp1 inhibitor improves tumor control by RT. (a) (i) MC38 cells were injected into wt mice and tumors were allowed to develop for 14 days. Mice were randomized to receive 21 daily doses of VIR3 or vehicle by oral gavage starting on d13, and further randomized to no further treatment or 12 Gy focal RT to the tumor on d14. (ii) Mice were followed for survival. (b) (i) Treatment as per a) but tumors were CT26 tumors injected into BALB/c mice (ii) Mice were followed for survival. Key: NS = not significant * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.