Validation of epigenetic mechanisms regulating gene expression in canine B-cell lymphoma: An in vitro and in vivo approach

Despite canine B-cell Lymphoma (BCL) representing the most common haematological tumour, epigenetic events driving development and progression are scarcely known. Recently, canine Diffuse Large BCL (DLBCL) DNA methylome by genome-wide CpG microarray has identified genes and pathways associated to pathogenesis. To validate data previously obtained by array analysis, the CLBL-1 cell line was used and the HOXD10, FGFR2, ITIH5 and RASAL3 genes were selected. CLBL-1 cells were treated with two hypomethylating drugs (HDs; IC50, 50% inhibitory concentration), i.e. azacytidine and decitabine (DEC), either alone or in combination with three histone deacetylase inhibitors (HDACis; IC20), i.e. valproic acid, trichostatin and vorinostat. Following the incubation with both HDs, an overall decrease of promoter methylation was highlighted, thus confirming target genes hypermethylation. The highest mRNA restoration was observed following the exposure to HDs combined with HDACis, and mostly with valproic acid. Contrasting results were only obtained for RASAL3. An in vivo confirmation was finally attempted treating Nod-Scid mice engrafted with CLBL-1 cells with DEC. Although DEC did not arrest tumour growth, target genes promoter methylation was significantly reduced in DEC-treated mice vs controls. Overall, this work demonstrates that CLBL-1 cell line represents a reliable in vitro model to validate the methylation-dependent silencing of key genes for BCL; moreover, it may be useful for xenograft models in mice, despite its aggressive behaviour. In future, functional studies will be performed to deepen the role of selected genes on BCL pathogenesis and progression, and their methylation-dependent mechanism of regulation.犬B细胞淋巴瘤（canine B-cell Lymphoma, BCL）是临床最为常见的血液系统肿瘤，但其驱动发生与进展的表观遗传调控机制仍鲜为人知。近期，研究人员通过全基因组CpG芯片分析犬弥漫大B细胞淋巴瘤（Diffuse Large BCL, DLBCL）的DNA甲基化组，已成功鉴定出与该病发病机制相关的基因及信号通路。为验证前期芯片分析所得的实验数据，本研究选用CLBL-1细胞系，并选定HOXD10、FGFR2、ITIH5及RASAL3四个基因开展后续验证实验。将CLBL-1细胞分别经两种去甲基化药物（HDs；IC50即50%抑制浓度）——阿扎胞苷与地西他滨（DEC）——单独处理，或与三种组蛋白去乙酰化酶抑制剂（HDACis；IC20即20%抑制浓度）——丙戊酸、曲古抑菌素与伏立诺他——联合孵育后，经去甲基化药物处理的细胞整体呈现启动子甲基化水平显著降低的特征，证实了靶基因确实存在高甲基化修饰。当去甲基化药物与组蛋白去乙酰化酶抑制剂联合处理时，靶基因的mRNA恢复表达水平最高，其中尤以与丙戊酸联合处理时效果最为显著；但RASAL3的检测结果与此截然相反。最后，本研究尝试通过体内实验验证上述结论：向移植了CLBL-1细胞的Nod-Scid小鼠（Nod-Scid mice）施以地西他滨处理。尽管地西他滨未能抑制肿瘤生长，但与对照组相比，给药组小鼠体内靶基因启动子的甲基化水平显著降低。综上，本研究证实CLBL-1细胞系是验证B细胞淋巴瘤关键基因甲基化依赖型沉默的可靠体外模型；尽管该细胞系具有侵袭性表型，但其仍可用于小鼠异种移植模型的构建。未来，本团队将开展功能学研究，进一步阐明所选基因在B细胞淋巴瘤发生发展中的作用，以及其甲基化依赖的调控机制。