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. 2019 Jun 19;134(6):548–560. doi: 10.1182/blood.2019001282

Figure 4.

Figure 4.

PRMT1 regulates AML cell maintenance by methylating FLT3-ITD R972/973. (A) Western blot analysis of indicated proteins in MA9-expressing murine BM cells that were further transduced with WT FLT3-ITD, FLT3-ITD Y969F, or FLT3-ITD R972/973K. (B) Following ex vivo PRMT1 deletion, Mx1-Cre/PRMT1f/f BM cells or control BM cells, both cotransduced with MA9 plus FLT3-ITD, were immunoblotted for the indicated total and phosphorylated proteins plus GAPDH. (C) The binding pose of the dimethylated peptide from molecular dynamics simulation is shown on the GRB2 SH2 domain protein (aa 56-153). Protein backbone is shown in white. Peptide backbone is shown in cyan with amino acid side chains shown in licorice mode (aa 969-974). Hydrogen atoms are omitted for clarity. Atom color: cyan, carbon; blue, nitrogen; red, oxygen. The position of the cocrystallized peptide (PDB 1BMB) is shown in tan with phospho-tyrosine shown in CPK mode. (D) MA9-expressing BM cells further transduced with FLT3-ITD, FLT3-ITD Y969F, or FLT3-ITD R972/973K were seeded for serial replating assays. (E) c-Kit+ BM cells were transduced with retroviral vectors coexpressing MLL-AF9 (MA9) plus GFP and then with lentiviral vectors coexpressing FLT3-ITD WT, FLT3-ITD Y969F, or FLT3-ITD R972/973K plus RFP. Double-positive cells were sorted and transplanted into CD45.1-expressing congenic recipients, and donor cell engraftment was analyzed 4 weeks later. (F) Representative fluorescence-activated cell sorting profile for total donor cells (GFP/RFP double-positive) and c-Kit + cells in BM of the indicated recipients. (G-H) Cumulative results are shown. (I) Survival of mice transplanted with the indicated doubly transduced murine BM cells. (J) CFC analysis of PRMT1-cKO vs PRMT1 WT doubly transformed cells. MA9-expressing BM cells from PRMT1f/f and Mx1-CRE/PRMT1f/f mice were further transduced with FLT3-ITD or FLT3-ITD R972/973K, PRMT1 deletion was induced ex vivo, and cells were seeded for CFC assays. Results represent the mean ± SD. *P < .05, **P < .01, ***P < .001.