The synergistic anti-tumor activity of EZH2 inhibitor SHR2554 and HDAC inhibitor chidamide through ORC1 reduction of DNA replication process in diffuse large B cell lymphoma

Background: Upregulation of H3K27me3 induced by EZH2 overexpression or somatic heterozygous mutations were implicated in lymphomagenesis. It has been demonstrated that several EZH2-target agents have notable therapeutic effects in EZH2-mutant B-cell lymphoma patients. Here we present a novel highly selective EZH2 inhibitor SHR2554 and possible combination strategy in diffuse large B cell lymphoma (DLBCL); Methods: Cell proliferation, cycle and apoptosis were analyzed by Cell Titer-Glo Luminescent Cell Viability Assay and flow cytometry. Western Blot was used to detect the regulatory protease in related signaling pathways and RNA-seq was conducted to assess transcriptome changes. Finally, CDX and PDX models were used to evaluate the synergistic anti-tumor effects of the combination in vivo; Results: The novel EZH2 inhibitor SHR2554 could inhibited proliferation, induced G1 phase arrest in EZH2-mutant DLBCL cell lines. The combination of EZH2 inhibitor SHR2554 with histone deacetylase (HDAC) inhibitor chidamide (hereafter referred as HBI8000) exerted synergistic anti-proliferative activity in vitro and in vivo. Gene expression profile analysis revealed dramatic inhibition of DNA replication process in combined treatment; Conclusions: SHR2554, a potent highly selective small molecule inhibitor of EZH2, inhibited EZH2-mutant DLBCL more significantly in vitro and in vivo. The combination of HDAC inhibitor HBI8000 with EZH2 inhibitor SHR2554 exhibited dramatically anti-tumor activity in both mutant and wild-type DLBCL, which may become potential therapeutic modality for the treatment of DLBCL patients. RNA sequencing in 2 DLBCL cell lines with SHR2554 and/or HBI8000 treatment .

背景：由增强子zeste同源物2（EZH2）过表达或体细胞杂合突变所诱导的组蛋白H3赖氨酸27三甲基化（H3K27me3）上调与淋巴瘤发生密切相关。已有研究证实，多款靶向EZH2的药物对EZH2突变型B细胞淋巴瘤患者具有显著治疗效果。本研究报道了一种新型高选择性EZH2抑制剂SHR2554，并探讨其在弥漫大B细胞淋巴瘤（diffuse large B cell lymphoma, DLBCL）中的潜在联合治疗策略。
方法：采用Cell Titer-Glo发光细胞活力分析法、流式细胞术分别检测细胞增殖、细胞周期与细胞凋亡情况；通过蛋白质印迹法（Western Blot）检测相关信号通路中的调控蛋白酶，并通过RNA测序（RNA-seq）评估转录组变化；最后采用细胞源性移植瘤（cell-derived xenograft, CDX）与患者源性移植瘤（patient-derived xenograft, PDX）模型体内评价联合用药的协同抗肿瘤活性。
结果：新型EZH2抑制剂SHR2554可抑制EZH2突变型DLBCL细胞系的增殖，并诱导其发生G1期阻滞。EZH2抑制剂SHR2554与组蛋白去乙酰化酶（HDAC）抑制剂西达本胺（chidamide，下文简称HBI8000）联合使用时，在体外与体内均展现出协同抗增殖活性。基因表达谱分析结果表明，联合给药可显著抑制DNA复制进程。
结论：SHR2554作为一种强效高选择性EZH2小分子抑制剂，在体外与体内均可更显著地抑制EZH2突变型DLBCL的生长。HDAC抑制剂HBI8000与EZH2抑制剂SHR2554的联合用药方案，对突变型与野生型DLBCL均展现出显著的抗肿瘤活性，有望成为DLBCL患者的潜在治疗手段。本研究针对2株经SHR2554单药或联合HBI8000处理的DLBCL细胞系进行了RNA测序。