Fig. 4: Identification of a PRMT9 inhibitor.
a, Screening pipeline. HT, high throughput. b, Docking pose of the top 30 hits. c, Effects of the top 20 compounds on Molm13 viability. d, Screening of nine compounds using the R493 methylation assay. Catalytic activity was assessed using a dot blot assay with an anti-R493-specific antibody. No. 1: LD2. e, Three-dimensional docking model. Left: LD2 in the pocket. Right: LD2 binding sites. f, CPMG NMR for 40 μM LD2 (blue); LD2 in the presence of PRMT9. g, STD NMR. (i) Reference (blue) and saturated (red) spectra. (ii) STD spectrum showing the difference between reference and saturated spectra. Asterisk denotes impurity. h,i, Thermal shift assay (h) and relative PRMT9 protein (i) of WT mutant PRMT9 from Molm13 cells treated with 2.5 μM LD2. The catalysis inhibition curves are based on the gray intensity of blots normalized to intensity at 37 °C (n = 3 independent experiments). A comparison was made between LD2-treated PRMT9 WT versus LD2-treated PRMT9 mutant. i, Data (n = 3 independent replicates) are represented as the mean ± s.d. P values were determined using a two-way ANOVA. j, Half-maximal inhibitory concentration (IC50) of LD2 in the indicated cells. Cells were treated for 4 days with LD2. MV4-11 (n = 6), NB4 (n = 3), U937 (n = 3), PBSC CD34+ (n = 6), MA9.6ITD (n = 3), Molm13 (n = 3) and THP1 (n = 4); data are the mean ± s.d. n indicates independent experiments and represents the number of independent experiments. k, Protein synthesis in the indicated cells after treatment with 2.5 μM LD2, based on an OPP assay. Right: results in vehicle versus LD2 (n = 3 independent experiments). Data are presented as the mean ± s.d. l, R493 methylation of Molm13 cells treated as indicated (n = 3 independent experiments). m, CyTOF of AML MNCs after 4 days of treatment with LD2 (2.5 μM). The frequency of T cells and CD34+CD45dim AML blast cells was noted. Color bar: CD34 intensity. n, Flow plots showing T cell and AML populations in the AML01, before and after T cell depletion. o, T cell depleted or bulk MNCs (n = 3 patients) were treated with LD2 (2.5 μM). AML blasts were determined using flow cytometry. Data are the mean ± s.d. from three independent experiments. p, Frequency of PRMT9hi (n = 43) versus low (n = 67) AML samples displaying the CTL score high versus low signatures in GSE12417. The P value was determined using a two-sided Fisher exact test. n represents the number of patients.
