DPP8 and DPP9 inhibition induces pro-caspase-1-dependent monocyte and macrophage pyroptosis

Abstract

Val-boroPro (Talabostat, PT-100), a nonselective inhibitor of post-proline cleaving serine proteases, stimulates mammalian immune systems through an unknown mechanism of action. Despite this lack of mechanistic understanding, Val-boroPro has attracted substantial interest as a potential anticancer agent, reaching phase 3 trials in humans. Here we show that Val-boroPro stimulates the immune system by triggering a proinflammatory form of cell death in monocytes and macrophages known as pyroptosis. We demonstrate that the inhibition of two serine proteases, DPP8 and DPP9, activates the pro-protein form of caspase-1 independent of the inflammasome adaptor ASC. Activated pro-caspase-1 does not efficiently process itself or IL-1β but does cleave and activate gasdermin D to induce pyroptosis. Mice lacking caspase-1 do not show immune stimulation after treatment with Val-boroPro. Our data identify what is to our knowledge the first small molecule that induces pyroptosis and reveals a new checkpoint that controls the activation of the innate immune system.

Figure 1. Val-boroPro is cytotoxic to monocytes and macrophages

a, The structure of Val-boroPro. b,c, The effects of Val-boroPro (2 μM, 24 h) on IL-1β expression and secretion in THP-1 macrophages as determined by immunoblotting (b) and ELISA (c). Full gel images are shown in Supplementary Figure 12. d, Val-boroPro induces the release of LDH from THP-1 macrophages. e, Profiles of the sensitivities of various cell lines to Val-boroPro. f, g, Val-boroPro induces LDH release from primary mouse BMDMs (f) and primary human PBMCs (g). In c-g, data are means ± SEM of three biological replicates. * p < 0.05, ** p < 0.01, ***p < 0.001 by two-sided Student’s t-test for DMSO versus Val-boroPro-treated cells. ext, extract; sup, supernatant; VbP, Val-boroPro.

Figure 2. DPP8/9 inhibition induces cell death

a, The serine hydrolase targets of Val-boroPro identified by EnPlex (10 nM to 100 μM, 10-fold dilution series). The percent inhibition at each concentration relative to DMSO controls is shown. b, Confirmation of DPP7 and DPP9 knockout in THP-1 cells by immunoblotting. c, Enrichment of active serine hydrolases in THP-1 cells with the FP-biotin activity-based probe enables DPP8 to be observed by immunoblotting and confirms DPP8 knockout. d, DPP9 knockout THP-1 cells secrete higher levels of LDH. e, Activity-based probe enrichment of serine hydrolases in THP-1 cells confirms that no active DPP9 was expressed in DPP9 knockout cells. f, DPP9 knockout cells release additional LDH after treatment with Val-boroPro (2 μM) and 1G244 (10 μM) for 24 h. g, Confirmation of DPP8/9 double knockouts in FP-biotin enriched THP-1 cell extracts by immunoblotting. h, DPP8/9 double knockouts do not respond to Val-boroPro or 1G244. Full gel images for b, c, e and g are shown in Supplementary Figure 12. In d, f, and h, data are means ± SEM of three biological replicates. *p<0.05, **p < 0.01, ***p < 0.001 by two-sided Student’s t-test for DMSO versus compound-treated cells.

Figure 3. DPP8/9 inhibitor cytotoxicity is caspase-1-dependent

a, Confirmation of caspase-1 and caspase-4 knockout in THP-1 cells by immunoblotting. Full gel images are shown in Supplementary Figure 12. b, LDH induced by Val-boroPro and LPS plus nigericin in THP-1 macrophages treated with sgRNAs to CASP1, CASP4, and CASP5. c, Cell viability of THP-1 monocytes after treatment with the indicated compounds for 48 h relative to DMSO as determined by CellTiter-Glo. The IC₅₀ values for control THP-1 cell lines (treated with an sgRNA to GFP, values in blue) and for DPP8/9 KO1 THP-1 (values in black) cells are shown. d, Viability of HT-1080 and MCF7 cells after treatment with Val-boroPro, 1G244, or L-allo-Ile-isoindoline for 48 h relative to DMSO treatment was determined by CellTiter-Glo. In b–d, data are means ± SEM of three biological replicates.

Figure 4. DPP8/9 inhibition activates pro-caspase-1 without autoproteolysis

a, Confirmation of ASC knockout in THP-1 cells by immunoblotting. b, ASC-knockout THP-1 macrophages release LDH in response to Val-boroPro but not to LPS plus nigericin. Data are means ± SEM of three biological replicates. *** p < 0.001 by two-sided Student’s t-test for GFP control versus ASC knockout cells treated with LPS plus nigericin. c,d, Immunoblots of cell extracts and supernatants from THP-1 (c) or RAW 264.7 (d) macrophages treated with Val-boroPro or LPS plus nigericin. Val-boroPro elicits exclusively pro-caspase-1. Unlike THP-1 cells, RAW 264.7 cells do not endogenously produce IL-1β and therefore no IL-1β is detected in the supernatant of Val-boroPro-treated RAW 264.7 cells. RAW 264.7 cells do not release any intracellular contents after treatment with LPS plus nigericin because these cells are ASC-deficient and are therefore unable to activate the NLRP3 inflammasome. e, Cleavage of Ac-WEHD-AFC (50 μM) was monitored in supernatants from treated THP-1 macrophages. Data are means ± SEM of four independent experiments. f, Pre-treatment (30 min) of THP-1 (left) or RAW 264.7 (right) macrophages with Ac-YVAD-CMK or Z-VAD-FMK (50 μM) blocks Val-boroPro-induced cytotoxicity at 6 h. Data are means ± SEM of three biological replicates. ** p < 0.01, ***p < 0.001 by two-sided Student’s t-test for Val-boroPro alone versus Val-boroPro plus caspase inhibitor-treated cells. Full gel images for a, c and d are shown in Supplementary Figure 12.

Figure 5. GSDMD is cleaved after DPP8/9 inhibition and contributes to cell death

a, GSDMD is cleaved in THP-1 macrophage extracts after treatment with Val-boroPro or 1G244 for 24 h, but CASP-3, CASP-7, and PARP are not. FL, full-length; CL, cleaved; * denotes non-specific bands. b, GSDMD is cleaved after Val-boroPro treatment of THP-1, RAW 264.7, J774, and primary human PBMCs as determined by immunoblotting. RAW 264.7 do not cleave Gsdmd after LPS plus nigericin treatment, as expected. c, d, The pyroptotic response in GSDMD-deficient THP-1 macrophages, which were validated by immunoblotting (c), was delayed, but not entirely prevented (d). Full gel images for a–c are shown in Supplementary Figure 12. In d, data are means ± SEM of three biological replicates.

Figure 6. Val-boroPro does not induce cytokines in caspase-1-knockout mice

a,b Val-boroPro (100 μg/mouse) induces high levels of serum G-CSF (a) and CXCL1/KC (b) after 6 h in wild-type (WT), but not Casp1^−/−, mice as measured by ELISA. p <0.0001 for G-CSF and p <0.01 for CXLCL1/KC in Val-boroPro-treated wild-type versus Val-boroPro-treated Casp1^−/− mice. Data are means ± SEM. n = 5 mice/group. Veh, vehicle. c, Model for the induction of pyroptosis by DPP8/9 inhibition.