Pyrvinium modulation of WNT signaling is more effective before the onset of moderate-severe anemia. (A) Two-month-old Mx1-Cre⁻Apc^fl/+ (Cre⁻) or Mx1-Cre⁺Apc^fl/+ (Cre⁺, also referred to as Apc^del/+) recipients were treated with pIpC (to induce Apc deletion) and vehicle (DMSO) or PT 2 weeks before lethal irradiation and transplantation with WT (CD45.1) BM cells. Mice were injected twice per week with 0.01, 0.1, or 0.5 mg/kg PT or DMSO until sacrifice. Kaplan-Meier curves for overall survival show that mice treated with 0.1 mg/kg or 0.5 mg/kg PT survived about 1 to 2 months longer than vehicle-treated mice (P = .0302 and P = .0064, respectively). (B) RBC and Hb counts of DMSO and PT-treated mice at ∼100 days posttransplant (for some mice in the DMSO and 0.01 mg/kg PT groups, counts from <100 days were plotted since they died before 100 days). The RBC and Hb counts of 0.5 mg/kg PT-treated mice were higher than 0.01 mg/kg PT-treated mice, indicating the administration of 0.5 mg/kg PT delays development of anemia (P = .0018 and P = .0320). The 2 mice in the 0.1 mg/kg PT-treated group that survived beyond 200 days had noticeably higher RBC and Hb counts at 100 days (circled). (C) Cre⁺ recipients were treated with 0.5 mg/kg PT once they developed mild (Hb, 12-13.5 g/dL), moderate (Hb, 10-11.5 g/dL), or severe anemia (Hb, <10 g/dL). A Kaplan-Meier survival curve of all PT-treated mice indicates survival is extended by almost 2 months (DMSO vs PT: 104 days vs 159 days; P < .0001). (D) The average median survival of recipients from the mild, moderate, and severe anemia group vs the DMSO-treated control group is shown. A longer survival is achieved if treatment is started before the onset of severe anemia. (E) MSCs were isolated from Apc^del/+mice ∼2 months post-pIpC-induced deletion and before development of disease. MSCs were treated with or without 50 ng/mL Wnt3a ± 50 nM PT, as indicated, for 16 hours. A representative immunoblot of nuclear and cytoplasmic fractions immunoblotted with Ctnnb1, β-actin (cytoplasmic), and Hdac1 (nuclear) is shown. In 3 independent experiments, PT treatment decreased Wnt3a-mediated elevation of Ctnnb1 by 56% ± 11.5% (P = .04). (F) Apc^del/+ MSCs were stimulated in vitro ± 50 ng/mL Wnt3a ± 50 nM PT for 16 hours. RNA was isolated and transcribed to cDNA, and PCRs (run in triplicate) were quantified using Fast-SYBR Green. Gene expression was normalized to Gapdh and data are presented as mean ± SEM of 3 independent experiments. **P < .001, *P < .05. NR, not reached.