Figure 1.
Figure 1. PRMT1 inhibition perturbs AML survival and growth. (A) Comparison of PRMT1 messenger RNA expression in mononuclear cells from BM or peripheral blood of healthy donors vs primary AML patients based on a GEO dataset (GSE7186). (B) PRMT1 protein levels in the CD34+CD38− subset from normal PBSCs (n = 8) and AML cases (n = 9), as analyzed by anti-PRMT1 intracellular staining. PRMT1 level is calculated as median fluorescence intensity of PRMT1 staining relative to immunoglobulin G (IgG) control. (C) Representative intracellular staining results are shown. (D) Western blot analysis of PRMT1 expression in primary human CD34+ cells from AML specimens (n = 18) and normal PBSC donors (n = 8). (E-F) Apoptosis of normal PBSCs (n = 4), CD34+ cells, and FLT3-ITD (n = 9) or FLT3 WT (n = 12) AML CD34+ cells transduced with ShCtrl or ShPRMT1 (targeting 3′UTR), as analyzed by Annexin V/4′,6-diamidino-2-phenylindole (DAPI) labeling. Within the FLT3-ITD+ AML and FLT3 WT AML groups, PRMT1 KD was associated with higher apoptosis levels. (E) Two-way ANOVA analyses with repeated measures revealed a statistically significant difference (P < .001) in the apoptosis increase between the 2 groups (FLT3-ITD vs FLT3 WT), indicating that PRMT1 KD induced more apoptosis in FLT3-ITD+ AML cells than in FLT3 WT AML cells. (F) Representative fluorescence-activated cell sorting plots. (G) CFC assay of FLT3-ITD (n = 5) and FLT3 WT (n = 6) AML CD34+ cells expressing ShCtrl or ShPRMT1. Colony numbers were normalized to that of ShCtrl-expressing cells. Within the FLT3-ITD+ AML and FLT3 WT AML groups, PRMT1 KD was associated with lower CFCs. Two-way ANOVA analyses with repeated measures revealed a statistically significant difference (P < .001) in the CFC decrease between the 2 groups (FLT3-ITD vs FLT3 WT). (H) CB CD34+ cells were transfected with vector control (mock), FLT3 WT, or FLT3-ITD and then further transduced with ShCtrl or ShPRMT1. Doubly transduced cells were assayed by western blotting for FLT3 and PRMT1 expression (I), for apoptosis by annexin V/DAPI labeling (J), and for CFCs (K). (L) BM cells from Mx1-Cre/PRMT1f/f or PRMT1f/f mice were transduced with a retroviral vector coexpressing MA9 plus GFP and then a lentiviral vector coexpressing FLT3-ITD or FLT3 WT plus RFP. (M) MA9, MA9/FLT3-ITD, and MA9/FLT3 WT cells, as indicated, were used to assess apoptosis in vitro after PRMT1 deletion. (N) Doubly transformed MA9/FLT3-ITD cells were transplanted into CD45.1-expressing congenic recipients to analyze leukemia progression. (O) Survival after PIPC treatment was monitored in PRMT1f/f (n = 8) and Mx1-Cre/PRMT1f/f (n = 9) groups. (P) Effects of PRMT1 deletion on splenomegaly were evaluated after the last dose of PIPC. (Q) Percentage of donor MA9/FLT3-ITD cells in BM of recipients (n = 6 per group) from the indicated group. Results represent the mean ± standard deviation. *P < .05, **P < .01, ***P < .001. NS, not statistically significant.

PRMT1 inhibition perturbs AML survival and growth. (A) Comparison of PRMT1 messenger RNA expression in mononuclear cells from BM or peripheral blood of healthy donors vs primary AML patients based on a GEO dataset (GSE7186). (B) PRMT1 protein levels in the CD34+CD38− subset from normal PBSCs (n = 8) and AML cases (n = 9), as analyzed by anti-PRMT1 intracellular staining. PRMT1 level is calculated as median fluorescence intensity of PRMT1 staining relative to immunoglobulin G (IgG) control. (C) Representative intracellular staining results are shown. (D) Western blot analysis of PRMT1 expression in primary human CD34+ cells from AML specimens (n = 18) and normal PBSC donors (n = 8). (E-F) Apoptosis of normal PBSCs (n = 4), CD34+ cells, and FLT3-ITD (n = 9) or FLT3 WT (n = 12) AML CD34+ cells transduced with ShCtrl or ShPRMT1 (targeting 3′UTR), as analyzed by Annexin V/4′,6-diamidino-2-phenylindole (DAPI) labeling. Within the FLT3-ITD+ AML and FLT3 WT AML groups, PRMT1 KD was associated with higher apoptosis levels. (E) Two-way ANOVA analyses with repeated measures revealed a statistically significant difference (P < .001) in the apoptosis increase between the 2 groups (FLT3-ITD vs FLT3 WT), indicating that PRMT1 KD induced more apoptosis in FLT3-ITD+ AML cells than in FLT3 WT AML cells. (F) Representative fluorescence-activated cell sorting plots. (G) CFC assay of FLT3-ITD (n = 5) and FLT3 WT (n = 6) AML CD34+ cells expressing ShCtrl or ShPRMT1. Colony numbers were normalized to that of ShCtrl-expressing cells. Within the FLT3-ITD+ AML and FLT3 WT AML groups, PRMT1 KD was associated with lower CFCs. Two-way ANOVA analyses with repeated measures revealed a statistically significant difference (P < .001) in the CFC decrease between the 2 groups (FLT3-ITD vs FLT3 WT). (H) CB CD34+ cells were transfected with vector control (mock), FLT3 WT, or FLT3-ITD and then further transduced with ShCtrl or ShPRMT1. Doubly transduced cells were assayed by western blotting for FLT3 and PRMT1 expression (I), for apoptosis by annexin V/DAPI labeling (J), and for CFCs (K). (L) BM cells from Mx1-Cre/PRMT1f/f or PRMT1f/f mice were transduced with a retroviral vector coexpressing MA9 plus GFP and then a lentiviral vector coexpressing FLT3-ITD or FLT3 WT plus RFP. (M) MA9, MA9/FLT3-ITD, and MA9/FLT3 WT cells, as indicated, were used to assess apoptosis in vitro after PRMT1 deletion. (N) Doubly transformed MA9/FLT3-ITD cells were transplanted into CD45.1-expressing congenic recipients to analyze leukemia progression. (O) Survival after PIPC treatment was monitored in PRMT1f/f (n = 8) and Mx1-Cre/PRMT1f/f (n = 9) groups. (P) Effects of PRMT1 deletion on splenomegaly were evaluated after the last dose of PIPC. (Q) Percentage of donor MA9/FLT3-ITD cells in BM of recipients (n = 6 per group) from the indicated group. Results represent the mean ± standard deviation. *P < .05, **P < .01, ***P < .001. NS, not statistically significant.

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