Azacytidine and/or decitabine (DAC) were dramatically improved the prognosis of MDS and AML. Whereas, these drugs can be administered intravenously or subcutaneously, therefore the patients must visit a hospital every day. Aims: OR21 is a novel and potentially oral absorbable DAC prodrug. Actually, direct duodenal administration of OR21 successfully absorbed in cynomolgus monkey model. In this study, we investigated the efficacy of OR21 in MDS (MDS-L), AML cell lines (HL60, THP1, KG1a, Kasumi-1). Methods: OR21 was provided from Ohara pharmaceutical Co. MDS-L was kindly provided from Kaoru Tohyama and other cell lines were purchased from ATCC. Results: OR21 and DAC reduced DNMT protein levels and LINE-1 methylation levels in MDS and AML cell lines, using western blotting and pyrosequencing (cont, 88.9%; OR21, 74.2%; DAC, 75.2%; P < 0.05), respectively. OR21 and DAC induces cell growth inhibition and cell apoptosis in a dose dependent manner against MDS and AML. MDS and AML are characterized by hematopoietic stem cell disorder with impairment of cell differentiation. Induction of cell differentiation can be one of the therapeutic targets for MDS and AML. Up-regulated CD11b expression level was observed in OR21 or DAC treated MDS-L (cont, 17.8%; OR21, 72.0%, DAC, 70.1%; P < 0.05) and HL60 (cont, 7.1%; OR21, 42.0%; DAC, 64.2%; P < 0.05), corresponding to up-regulated CEBPE mRNA level (OR21, 14.9 fold; DAC, 21.9 fold in MDSL; OR21, 2.5 fold; DAC, 3.0 fold in HL60) which is a key late differentiation driver. These results indicate OR21 and DAC can induce cell differentiation via up-regulation of CEBPE. OR21 and DAC also upregulated tumor suppressor X (not shown) modulated proteasome pathway, inducing cell apoptosis. Finally, we used a mouse xenograft model to evaluate anti-tumor effect of OR21 in vivo. BALB/c Rag-2/JAK3 double-deficient (BRJ) mice were injected intravenously via tail vein with 5 × 10 6 HL60 cells. OR21, DAC and vehicle were administered by intraperitoneal injection at a dose of 2.7 mg/kg (OR21), 1.0 mg/kg (DAC) and 1% DMSO (vehicle) twice weekly; OR21 and DAC were equal in AUC (OR21 is readily degradation with gastric acid in mouse). Significant decreased leukemic cells in peripheral blood at 37 days (Vehicle, 2.96%; OR21, 0.75%; P = 0.049) and increased CD11b positive cells (Vehicle, 40.9%; OR21, 60.0%) was observed in OR21 treated mice. OR21 significantly prolonged survival (median survival 49 days and 44 days, P = 0.005), while DAC did not (median 46.5 days and 44 days, P = 0.164) and decreased LINE-1 methylation levels (Vehicle, 83.7%; OR21, 62.8%; P < 0.0001) were observed in OR treated mouse in bone marrow. Interestingly, OR21 treated mouse tended to be less involved in anemia than DAC treated mouse (hemoglobin; Vehicle, 17.5 g/dL; OR21, 17.1 g/dL; DAC, 15.8 g/dL). Summary/Conclusion: OR21 has anti-tumor effect agaisnt MDS and AML via cell differentiation and apoptosis induced by up-regulation of CEBPE and tumor suppressor X. OR21 has safer profile and oral absorbability than DAC, thus OR21 can b...