Fig. 6: Immunity after PRMT9 inhibition requires cGAS activity.
a, Upregulated ISGs in MA9 cells. b, GSEA of DEGs in Prmt9 KD MA9 cells. c, Overlapped DEGs in the indicated cells (fold change > 2). NES, normalized enrichment score. d, ISG15 expression in Molm13 cells with endogenous PRMT9 KD and after rescuing with PRMT9 WT or a catalytically dead mutant (n = 5 independent experiments). Data are the mean ± s.d. The P value was determined using a one-way ANOVA. e, ISG levels in AML CD34+ cells. Data are the mean ± s.d. from three independent experiments. f, Luciferase activity of THP1-IRF cells engineered as indicated (n = 5 independent experiments). Data are the mean ± s.d. The P value was determined using a one-way ANOVA. g, cGAMP levels in engineered THP1 supernatant (n = 3 independent experiments). Data are the mean ± s.d. h, Left: immunostaining for γH2AX in THP1 cells. Right: γH2AX intensity (n = 100 cells per group). Scale bars, 10 μm. The P value was determined using an unpaired two-sided t-test. i, dsDNA using immunostaining in THP1 cells. Right: dsDNA intensity (n = 50 cells per group). Scale bar, 10 μm. The P value was determined using an unpaired two-sided t-test. j, MA9/OVA cells (Ctrl, n = 5 mice), Prmt9 KD (n = 7 mice), Ctrl + cGAS KO (n = 5 mice) and Prmt9 KD + cGAS WT (n = 5 mice)) were transplanted to establish AML. Prmt9 KD was induced. The Kaplan–Meier curves show the survival. P values were determined using a log-rank (Mantel–Cox) test. k, cGAS KO MA9 cells were transduced with inducible HA-tagged cGAS WT or ΔN. Exogenous cGAS was then assessed (n = 1). l,m, cGAS KO (n = 5 mice), cGAS WT (n = 5 mice) or cGAS-ΔN MA9 (n = 7 mice) cells were transplanted. DOX was given to induce expression of cGAS variants. l, AML engraftment was assessed. Data are the mean ± s.e.m. P values were determined using a one-way ANOVA. m, For another cohort, Kaplan–Meier curves show survival. P values were determined using a log-rank (Mantel–Cox) test. n, cGAS levels in BEAT AML cases (n = 451 patients) and healthy donors (n = 19). The P value was determined using an unpaired two-sided t-test. o, cGAMP levels in the bone marrow of mice (n = 3 mice per group). Data are the mean ± s.d. p, Expression of Cd80, Cd86 and H2-ab1 in the DCs of the scRNA-seq of Ctrl (n = 108 cells) and Prmt9 KD (n = 57 cells) bone marrow. P values were determined using an unpaired two-sided t-test. q,r, LD2-pretreated MA9/OVA cells were cocultured with bone marrow-derived DCs. DCs were then cocultured with OT-I transgenic CD8+ T cells. q, IFN-γ production by CD8+ T cells. r, IFN-β production by DCs. n = 3 independent experiments. Data are the mean ± s.d. s, MA9 AML cells were implanted into Batf3 WT or KO mice: (1) Prmt9 KD/Batf3 KO (n = 5 mice), (2) Prmt9 KD/Batf3 WT (n = 7 mice) and (3) Prmt9 WT/Batf3 WT (n = 7 mice). Kaplan–Meier curves show survival. P values were determined using a log-rank (Mantel–Cox) test.
