Anti-ATL effects of DAC and OR21 in an in vivo xenograft mouse model. (A) Experimental protocol for establishment of the MT-2 xenograft mouse model and treatment with DNA demethylating compounds. (B) Mean tumor volume in Balb/c Rag-2^−/− Jak3^−/− mice inoculated with MT-2 cells treated with vehicle (blue; n = 7); 1.25 mg/kg (5.5 µmol/kg) DAC (green; n = 7); 3.39 mg/kg (9.9 µmol/kg) OR21 (red; n = 7); or 1.34 mg/kg (5.5 µmol/kg) AZA (yellow; n = 7). Each compound and vehicle were injected intraperitoneally twice per week, as indicated by the black arrowheads. (C) Tumor weight and (D) DNA methylation levels at THEMIS promoter regions of xenograft tumors dissected from Balb/c Rag-2^−/−Jak3^−/− mice 22 days after first administration of compounds. Data are expressed as means with SDs (n = 7 per experimental group). Differences between subpopulations were evaluated using the Tukey-Kramer method (*P < .05). (E) Concentration of hemoglobin (HGB), complete blood counts, including white blood cells (WBC), red blood cells (RBC), platelets (PLT), and neutrophils, from BALB/c Rag2^−/−Jak3^−/− mice 22 days after first administration of compounds. (F) Experimental protocol for establishment of the patient-derived xenograft (PDX) and treatment with OR21. An ATL PDX mouse model was established by intraperitoneal inoculation of PBMCs from a patient with chronic ATL. PBMCs isolated from a patient with chronic ATL contained a much greater proportion of N subpopulation cells (77% of CD3⁺/CD4⁺ lymphocytes) than P subpopulation cells (6.5% of CD3⁺/CD4⁺ lymphocytes) determined by flow cytometric analysis. Balb/c Rag-2^−/− Jak3^−/− mice were intraperitoneally (IP) inoculated with PBMCs treated with either vehicle or 1.88 mg/kg OR21 twice per week. A relatively larger IP tumor containing human CD45⁺/mouseCD45⁻/CADM1⁺ cells (HTLV-1–infected human T cells) was formed in vehicle-treated mice ∼50 days after inoculation. (G) Body weights of individual mice and (H) Kaplan-Meier survival curves for the vehicle control (blue; n = 4) and 1.88 mg/kg OR21 (5.5 µmol/kg) (red; n = 5) treated groups. OR21 or vehicle was injected IP twice per week, as indicated by black arrowheads. Statistical significance was assessed using a 2-sided log-rank test.