DPP8/DPP9 inhibitor-induced pyroptosis for treatment of acute myeloid leukemia

Abstract

Small-molecule inhibitors of the serine dipeptidases DPP8 and DPP9 (DPP8/9) induce a lytic form of cell death called pyroptosis in mouse and human monocytes and macrophages^1,2. In mouse myeloid cells, Dpp8/9 inhibition activates the inflammasome sensor Nlrp1b, which in turn activates pro-caspase-1 to mediate cell death³, but the mechanism of DPP8/9 inhibitor-induced pyroptosis in human myeloid cells is not yet known. Here we show that the CARD-containing protein CARD8 mediates DPP8/9 inhibitor-induced pro-caspase-1-dependent pyroptosis in human myeloid cells. We further show that DPP8/9 inhibitors induce pyroptosis in the majority of human acute myeloid leukemia (AML) cell lines and primary AML samples, but not in cells from many other lineages, and that these inhibitors inhibit human AML progression in mouse models. Overall, this work identifies an activator of CARD8 in human cells and indicates that its activation by small-molecule DPP8/9 inhibitors represents a new potential therapeutic strategy for AML.

Figure 1. Val-boroPro is cytotoxic to AML cells

(a) The structure of Val-boroPro. (b,c) Dose response of cell viability of sensitive (b) and resistant (c) cell lines to treatment with Val-boroPro (24 h for MV4;11 and RS4;11 cells, 48 h for all others) relative to DMSO. (d–f) Time-course of cell viability of MV4;11 (d), OCI-AML2 (e), and THP-1 (f) cells after treatment with DMSO or Val-boroPro at the indicated concentrations. Data are means ± SEM of three biological replicates.

Figure 2. Caspase-1 is required for the sensitivity of AML cell lines to Val-boroPro

(a) Genes whose level of expression (from CCLE microarray data) distinguishes between cell lines that are sensitive and resistant to Val-boroPro. The twenty genes whose expression levels are most highly correlated with sensitivity and resistance are shown. Each row corresponds to a gene and each column corresponds to its relative expression levels in each cell line. The top ten genes are highly expressed in sensitive cells lines, and the bottom ten genes are highly expressed in resistant cell lines. (b) Immunoblots of lysates from the indicated sensitive and resistant cell lines, showing levels of CASP1, CARD8, GSDMD, and the loading control GAPDH. (c) Immunoblots of lysates from AML cell lines treated with or without Val-boroPro for 24 h, showing levels of full-length (FL) and cleaved (CL) PARP and GSDMD. See Supplementary Figure 7 for immunoblots for caspase-1 and IL-1β. Immunoblots in b and c are representative of >5 independent experiments. (d) Immunoblots of lysates from OCI-AML2 cells treated with sgRNAs to CASP1 or to GFP (control). Images are representative of 3 independent experiments. (e) Cell viability of control and caspase-1 knockout OCI-AML2 cells after treatment with the indicated concentrations of Val-boroPro (d) or L-allo-Ile-isoindoline (e) at 24 h. Data are means ± SEM of three independent biological experiments. Full gel images of cropped gels are shown in Supplementary Figure 15.

Figure 3. CARD8 mediates DPP8/9 inhibitor-induced pyroptosis in human myeloid cells

(a) Diagram of human NLRP1, mouse Nlrp1b (allele 1), and human CARD8 proteins. The lethal factor (LF) cleavage and FIIND autoproteolysis sites are indicated. The FIIND-CARD regions of CARD8 and NLRP1 are ~44% identical. The cartoon is not drawn to scale. (b–d) Top, percentage of LDH release (relative to the amount of LDH released by 0.36% Triton X-100, which lyses all cells) from CARD8 KO THP-1 (b), OCI-AML2 (c), and MV4;11 (d) cells after treatment with DMSO or Val-boroPro (VbP; 2 μM; 24 h for OCI-AML2 and MV4;11, 48 h for THP-1). Bottom, immunoblots showing levels of CARD8 and GSDMD. Antibodies that bind specifically to either the CARD8 N-terminus or the CARD8 C-terminus were used as indicated. N, N-terminus; C, C-terminus; FL, Full-length, CL, cleaved. Immunoblots in b–d are representative of 3 independent experiments. (e) Percentage of LDH release from HEK 293T cells stably expressing hCASP1 and hGSDMD that were then transiently transfected with the indicated constructs, followed 24 hours later by treatment with DMSO or Val-boroPro (2 μM) for an additional 24 hours. (f) Immunoblots of lysates of cells from e (0.05 μg transfection). Cells transiently transfected with a vector encoding RFP were used as a mock. Immunoblots in are representative of >5 independent experiments. (g) Percentage of LDH release from THP-1 or MV4;11 cells were treated for 6 h with DMSO, Val-boroPro (2 μM), bortezomib (100 nM), or both Val-boroPro and bortezomib. (g) Percentage of LDH release from HEK 293T cells or HEK 293T cells stably expressing hCASP1 and hGSDMD 24 hours after transient transfection with the indicated constructs. Data in b–e, g, and h are means ± SEM of three independent biological experiments. **p < 0.01, ***p < 0.001 by two-sided Student’s t-test. NS, not significant (p > 0.05).

Figure 4. Val-boroPro inhibits AML progression in vivo

(a–c) Bioluminescent images (a), mean bioluminescence over the time course of the study (n=9 mice per group) (b), and Kaplan-Meier survival analysis (n=6 mice per group, p=0.0005, two-sided log-rank test) (c) of mice bearing wild-type MV4;11 leukemia cells treated with vehicle (1 mM HCl/PBS) or Val-boroPro. Mouse with the median bioluminescence in each group is shown in a. (d,e) Mean bioluminescence (d) and Kaplan-Meier survival analysis (e) of mice bearing CASP1 knockout MV4;11 leukemia cells treated with vehicle or Val-boroPro (n=9 mice per group). The results for the vehicle- and drug-treated groups are not statistically different in d (p=0.68, two-sided Student’s t-test on study day 17) or e (p=0.9086, two-sided log-rank test). (f) Total number of human viable AML cells expressing CD45 and CD33 and not expressing CD3 in peripheral blood of mice bearing primary human cancer AML-10 that were treated with vehicle or Val-boroPro (n=5 mice per group). The total number of cells was determined by FACS analysis 10 days after the start of treatment. (g) Mean body weights of mice in the PDX model in f. Data in b, d, f, and g are means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by two-sided Student’s t-test.