Combination of 5-Aza with DZNep enhance prophylactic effect for acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation

Objective: Previous studies showed epigenetic prophylaxis could alleviate acute graft versus host disease (aGvHD) but affecting engraftment after allo-HSCT. Combination of two different epigenetic agents with low dose might overcome the problem. This study aims to evaluate the combined treatment of hypomethylating agent azacitidine (5-Aza) and histone H3K27 methyltransferase inhibitor 3-deazaneplanocin (DZNep) for the prophylaxis of aGvHD after allo-HSCT, and explore the possible mechanisms.Methods: First we optimized the concentration of 5-Aza and DZNep that showed no obvious toxicities on activated T cells even in combination by evaluated the T cell proliferation, activation and differentiation. Mouse model of aGvHD was then established to assess prophylactic efficacy of 5-Aza, DZNep and their combination on aGvHD. Immunomodulatory effect on T cells and hematopoietic reconstruction have been assessed. Additionally, RNA sequencing (RNA-seq) was performed to identify the underlying molecular mechanisms.Results: Compared with single treatment, in vitro application of 5-Aza with DZNep could more powerfully inhibit T cell activation, reduce Th1/Tc1 production and increase Treg production. In an allo-HSCT mouse model, in vivo application of 5-Aza with DZNep could enhance prophylactic effect for aGVHD compared with single agent. Mechanism study demonstrated that combination of 5-Aza and DZNep in vivo has an enhanced effect to inhibit Th1/Tc1 production, increase Th2/Tc2 ratio, and induce Treg production as in vitro. RNA-seq analysis revealed that cytokine and chemokine pathways as one mechanism for alleviation of aGvHD with 5-Aza and DZNep combination.Conclusion: Combination of 5-Aza with DZNep could enhance prophylactic effect aGvHD by influencing donor T cell activation and differentiation through affecting cytokine and chemokine pathways. This study shed a light on the effectively prophylactic measure for aGvHD by using different epigenetic agent combination.