BET蛋白抑制剂防治慢性移植物抗宿主病的作用研究

Chronic graft-versus-host disease (cGVHD) is a major complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT), while specific and effective therapy is still lacking. T cells, B cells and fibroblasts are crucial for the initiation and exacerbation of cGVHD. Epigenetic regulation is a critical form for controlling gene expression. Recent studies have demonstrated that a novel epigenetic drug, BET protein inhibitors have significant anti-tumor effect and immune-regulatory properties. Former studies from applicant showed that BET inhibitor I-BET151 could profoundly suppress T cells and dendritic cells function and reduce acute GVHD severity after allo-HSCT. However, its application on cGVHD is needed. Preliminary experiment from our group demonstrated that in murine cGVHD model, BET protein expression is increased after allo-transplant and early administration of I-BET151 can decrease clinical severity and mortality from cGVHD. On these bases, in this study, we will further explore the effect of I-BET151 on the activation, proliferation and function of allo-reactive T cells, regulatory T cells, B cells, as well as fibroblasts, eliminate related molecular mechanism, verify its application on prevention and treatment of cGVHD and influence on graft-versus-leukemia effect. Our data will provide substantial evidence that BET inhibition could be explored as a new treatment modality for cGVHD in the future.

慢性移植物抗宿主病(cGVHD)是异基因造血干细胞移植术后的重要并发症。T细胞、B细胞和成纤维细胞在cGVHD的发生发展中发挥关键作用。表观遗传修饰是细胞调控的重要形式。前期研究发现，新型表观遗传药物—BET蛋白抑制剂具有显著抗肿瘤和免疫调节作用。申请人既往研究显示BET蛋白抑制剂I-BET151能明显抑制T细胞和树突细胞功能，有效预防急性GVHD的发生，但I-BET151对cGVHD的防治作用研究尚待深入。项目组预实验证明在小鼠cGVHD模型中，BET蛋白表达水平升高，早期I-BET151治疗能明显降低cGVHD严重程度。在此基础上，本项目拟通过体内外实验进一步研究I-BET151对T细胞、B细胞和成纤维细胞的活化、增殖及功能的调控作用，阐明相关分子机制，验证I-BET151对cGVHD的防治作用和对移植物抗白血病效应的影响，以期建立以BET蛋白抑制剂为手段的cGVHD新型防治策略。

慢性移植物抗宿主病（chronic GVHD, cGVHD）是异基因造血干细胞移植术后的重要并发症，如何有效防治cGVHD提升患者生存质量是临床上面临的一个重要难题。表观遗传学修饰在免疫细胞分化、发育和功能中都发挥重要作用，有研究发现新型表观遗传药物BET蛋白抑制剂I-BET151能显著抑制T细胞和DC功能，有效预防aGVHD发生。然而，I-BET151是否能有效防治cGVHD仍不清楚。我们前期构建了cGVHD小鼠模型，发现短期应用I-BET151能显著降低小鼠cGVHD评分、减轻小鼠腹泻、改善体重减轻。HE染色结果揭示短期和长期应用I-BET151均能显著减轻cGVHD小鼠的肺、肾脏、肠道和皮肤的病理损伤情况。进一步，我们发现短期应用I-BET151明显抑制T细胞和DC的比例、降低Th1细胞。长期应用I-BET151对T细胞影响不大，而是明显降低Tfh细胞比例。同时，长期应用I-BET151能显著改善cGVHD小鼠胸腺免疫重建，胸腺内CD3T、CD4T和CD8T细胞均显著上调。机制方面，我们通过RNA seq发现长期应用I-BET151显著影响免疫系统重建，下调Ctnnbip1和Anxa1，上调CD226和CCR6，随后，我们在cGVHD小鼠体内证明I-BET151显著下调Ctnnbip1和Anxa1的水平。综上，短期应用I-BET151抑制T细胞和DC、降低Th1细胞，长期应用I-BET151降低Tfh细胞、下调Ctnnbip1和Anxa1改善胸腺免疫系统重建，抑制cGVHD的发生发展。以上研究结果为临床采用I-BET151治疗cGVHD提供重要的实验基础。