低剂量地西他滨上调白血病细胞表达GADD45A介导预处理增敏对难治复发AML移植后生存改善的作用机制研究

It's a great challenge to overcome drug resistance and improve the conditioning regimen in order to prevent relapse post allogeneic hematopoietic stem cell transplantation. Our recent studies demonstrated that low dose decitabine (DAC) could sensitize and enhance the efficacy of BUCY as a conditioning regimen in the treatment of acute myeloid leukemia patients and it significantly reduces the cumulative incidence of relapse and improve 2 years overall survival, especially for relapse and refractory AML patients. However, the underlying mechanism is yet to know. We further found that DAC could induce G2-arrest and increase DNA damage caused by BuCy conditioning regimen. Moreover, we found, in a bioinformatic analysis, that DAC could up-regulate the expression of GADD45A, a key gene in cell cycle checkpoint and in response to DNA damage. The preliminary results predicts a hypothesis that there is a DAC/GADD45A axle which results in AML cell cycle arrest, DNA damage and apoptosis. Based on these findings, we in the present proposal, therefore, will elucidate the mechanisms of action that DAC promotes the expression of GADD45A through demethylation at cellular and molecular levels, and will analyze the effect of GADD45A expression on the cell cycle arrest by gene overexpression and gene knockdown protocols. Furthermore, we will explore whether DAC chemosensitizes AML patients through the DAC/GADD45A axle to reduce the relapse rate after transplantation by using BU-resistant AML cell lines, deletion or overexpression of GADD45A in AML cell lines and corresponding animal models. This study will help us to explore the mechanisms of the new conditioning regimens and to develop the novel conditioning regimens to prevent the relapse post transplantation.

克服耐药和改进预处理方案以预防异基因造血干细胞移植后复发是移植领域研究的热点。我们的临床研究表明低剂量地西他滨（DAC）可增敏BUCY预处理方案，显著降低难治复发急性髓细胞白血病患者（r/r AML）复发率，提高移植后生存，然而机制不明。我们进一步发现DAC能阻滞细胞周期于G2期，并增加BUCY造成的DNA损伤；生物信息分析发现DAC能够上调细胞周期相关基因GADD45A表达。鉴于此，本项目拟从细胞和分子不同水平阐明DAC上调GADD45A的机制，采用基因过表达和敲除技术，分析GADD45A对细胞周期的作用；运用BU耐药AML细胞系及敲除或高表达GADD45AAML细胞系和相应动物模型，证实DAC是否通过“DAC/去甲基化/上调GADD45A/G2期阻滞/DNA损伤”这一信号轴发挥预处理增敏，降低移植后复发率，为阐明新型预处理方案的机制和优化预处理新方案奠定理论基础。

异基因造血干细胞移植（allo-HSCT）后复发是难治复发急性髓系白血病（R/R AML）患者移植后死亡的主要原因。本课题组创新性地运用地西他滨（DAC）联合改良BuCy方案治疗R/R AML患者，长期生存率优于改良BuCy方案，其机制尚未有研究报道。本研究通过前瞻性临床研究、体外实验和动物模型证实：1. DAC联合改良BuCy组在长期生存上明显优于改良BuCy组（3年总生存率：81.1% vs 56.4%，P=0.0257；3年无病生存率：73.0% vs 46.1%，P=0.0258）。2.成功建立了经免疫表型证实的MLL-AF9原代小鼠AML模型。3.体外实验发现，DAC和马利兰（BU）对AML发挥协同杀伤作用，与对照组相比，降低白血病干细胞（LSCs）残留率（8.87±2.55% vs 29.15±6.89%，P=0.009），促进LSCs凋亡（6.84±0.67% vs 2.43±0.28%，P<0.001），促进LSCs从G0期进入G2/M期（G0期：11.72±1.47% vs 16.69±1.32%，P<0.001；G2/M期：42.32±0.94% vs 23.69±1.39%，P<0.001），抑制LSCs克隆形成能力（90.0±5.57% vs 141.3±7.51%，P<0.001）。4.体内实验证实，DAC和BU序贯给药可以通过促进凋亡、促进G0期细胞进入G2/M期来降低小鼠AML和LSCs残留率，从而延长小鼠生存（34天 vs 30天，P<0.001）。5.染色质开放性测序和转录组测序联合分析表明，4个与细胞周期和代谢相关的基因在DAC处理后染色质开放，基因表达上调。综上研究表明，DAC通过开放周期相关基因染色质，上调基因表达，促进AML和LSCs从G0期进入G2/M期，从而增敏BU的杀伤作用。