Figure 5
Figure 5. Effect of R723 on BM of V617F-TG mice. (A) BM progenitors in V617F-TG mice and effect of in vivo R723 treatment. V617F-TG mice were dosed orally with R723 at 70 mg/kg or vehicle for 4 weeks. BM cells were collected, and the proportion of CMPs (Lin−Sca-1−cKit+CD34+ FcγRlo), GMPs (Lin−Sca-1−cKit+CD34+FcγRhi), MEPs (Lin−Sca-1−cKit+CD34−FcγR−/lo), and MKPs (Lin−cKit+CD9+FcγRlo CD41+) was determined by FACS. In V617F-TG mice, the proportion of GMPs increased, and those of CMPs, MEPs, and MKPs were comparable to those in WT mice. Four weeks of R723 treatment in V617F-TG mice normalized the proportion of GMPs. MEPs and MKPs were increased compared with WT mice or V617F-TG mice treated with vehicle. These results are representative of 6 independent experiments. (B) Changes in BM progenitor cells after R723 treatment. The numbers of CFU-GEMM and CFU-GM colonies increased and the numbers of CFU-E colonies decreased in the BM from 18-week-old TG-vehicle mice compared with those in the BM from the age-matched WT-vehicle mice (†P < .05; ‡P < .01). R723 treatment for 6 weeks suppressed CFU-GM and BFU-E colonies (*P < .05). In contrast, CFU-E colonies increased in V617F-TG mice after R723 treatment (**P < .01). Data are presented as means ± SE. (C) FACS analysis of BM cells. The percentage of Mac-1/Gr-1+ myeloid cells increased and that of B220+ B cells decreased in 18-week-old V617F-TG mice treated with vehicle (TG-vehicle) compared with the age-matched WT mice treated with vehicle (WT-vehicle) (‡P < .01). R723 treatment for 6 weeks had little effect on the proportion of BM myeloid or B cells in V617F-TG mice (top panel). The proportion of CD71/TER119 double-positive erythroblasts and TER119 single-positive erythroblasts decreased in TG-vehicle mice compared with that in WT-vehicle mice (†P < .05; ‡P < .01). Significant restoration of CD71/TER119-positive erythroblasts was observed in V617F-TG mice after R723 treatment for 6 weeks (*P < .05). TER119 single-positive erythroblasts were not restored by R723 treatment (bottom panel). Data are presented as means ± SE. (D) Changes in EPO-independent CFU-E colonies after in vivo R723 treatment. The number of EPO-independent CFU-E colonies increased in BM cells from 18-week-old TG-vehicle mice compared with that in BM cells from the age-matched WT-vehicle mice (†P < .01). In vivo R723 treatment for 6 weeks increased the number of EPO-independent CFU-E colonies (*P < .01). Data are presented as means ± SE. Whereas EPO-dependent CFU-E colonies increased in V617F-TG mice after in vivo R723 treatment (panel B), the proportion of EPO-independent/EPO-dependent CFU-E colonies remained the same.

Effect of R723 on BM of V617F-TG mice. (A) BM progenitors in V617F-TG mice and effect of in vivo R723 treatment. V617F-TG mice were dosed orally with R723 at 70 mg/kg or vehicle for 4 weeks. BM cells were collected, and the proportion of CMPs (Lin−Sca-1−cKit+CD34+ FcγRlo), GMPs (Lin−Sca-1−cKit+CD34+FcγRhi), MEPs (Lin−Sca-1−cKit+CD34−FcγR−/lo), and MKPs (Lin−cKit+CD9+FcγRlo CD41+) was determined by FACS. In V617F-TG mice, the proportion of GMPs increased, and those of CMPs, MEPs, and MKPs were comparable to those in WT mice. Four weeks of R723 treatment in V617F-TG mice normalized the proportion of GMPs. MEPs and MKPs were increased compared with WT mice or V617F-TG mice treated with vehicle. These results are representative of 6 independent experiments. (B) Changes in BM progenitor cells after R723 treatment. The numbers of CFU-GEMM and CFU-GM colonies increased and the numbers of CFU-E colonies decreased in the BM from 18-week-old TG-vehicle mice compared with those in the BM from the age-matched WT-vehicle mice (†P < .05; ‡P < .01). R723 treatment for 6 weeks suppressed CFU-GM and BFU-E colonies (*P < .05). In contrast, CFU-E colonies increased in V617F-TG mice after R723 treatment (**P < .01). Data are presented as means ± SE. (C) FACS analysis of BM cells. The percentage of Mac-1/Gr-1+ myeloid cells increased and that of B220+ B cells decreased in 18-week-old V617F-TG mice treated with vehicle (TG-vehicle) compared with the age-matched WT mice treated with vehicle (WT-vehicle) (‡P < .01). R723 treatment for 6 weeks had little effect on the proportion of BM myeloid or B cells in V617F-TG mice (top panel). The proportion of CD71/TER119 double-positive erythroblasts and TER119 single-positive erythroblasts decreased in TG-vehicle mice compared with that in WT-vehicle mice (†P < .05; ‡P < .01). Significant restoration of CD71/TER119-positive erythroblasts was observed in V617F-TG mice after R723 treatment for 6 weeks (*P < .05). TER119 single-positive erythroblasts were not restored by R723 treatment (bottom panel). Data are presented as means ± SE. (D) Changes in EPO-independent CFU-E colonies after in vivo R723 treatment. The number of EPO-independent CFU-E colonies increased in BM cells from 18-week-old TG-vehicle mice compared with that in BM cells from the age-matched WT-vehicle mice (†P < .01). In vivo R723 treatment for 6 weeks increased the number of EPO-independent CFU-E colonies (*P < .01). Data are presented as means ± SE. Whereas EPO-dependent CFU-E colonies increased in V617F-TG mice after in vivo R723 treatment (panel B), the proportion of EPO-independent/EPO-dependent CFU-E colonies remained the same.

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