Dual inhibition of EZH2 and G9a suppresses multiple myeloma cell proliferation by regulating interferon signal and IRF4-MYC axis. Dual inhibition of EZH2 and G9a suppresses multiple myeloma cell proliferation by regulating interferon signal and IRF4-MYC axis

Epigenetic mechanisms including histone modifications play key roles in the pathogenesis of multiple myeloma (MM). We have previously shown that a histone H3 lysine 27 (H3K27) methyltransferase EZH2 and a H3K9 methyltransferase G9a are potential therapeutic targets in MM. Recent studies suggested that EZH2 and G9a cooperate to regulate gene expression. We thus aimed to evaluate the anti-tumor effect by dual targeting of EZH2 and G9a in MM. A combination of an EZH2 inhibitor and a G9a inhibitor strongly suppressed myeloma cell proliferation through inducing cell cycle arrest and apoptosis in vitro. Dual inhibition of EZH2 and G9a also repressed xenograft formation by myeloma cells in vivo. Higher expression levels of EZH2 and EHMT2, which encodes G9a, are significantly associated with poorer prognosis in MM patients, respectively. Microarray analysis revealed that EZH2/G9a inhibition significantly upregulated interferon (IFN)-stimulated genes and suppressed IRF4-MYC axis genes in myeloma cells. Notably, we found increased expression and reduced H3K27/H3K9 methylation levels of endogenous retrovirus (ERV) genes in myeloma cells with the dual inhibition, suggesting that activation of the ERV genes may cause the IFN response. These results suggest that dual targeting of EZH2 and G9a may be a novel therapeutic strategy in MM. Overall design: RPMI-8226 and MM.1S cells treated with GSK126 (1 μM), UNC0638 (1 μM) or their combination (1 μM each) for 6 days.

表观遗传机制（包括组蛋白修饰）在多发性骨髓瘤（multiple myeloma, MM）的发病机制中发挥关键作用。我们前期研究证实，组蛋白H3赖氨酸27（H3K27）甲基转移酶EZH2与组蛋白H3赖氨酸9（H3K9）甲基转移酶G9a均可作为MM的潜在治疗靶点。近期研究表明，EZH2与G9a可协同调控基因表达。据此，本研究旨在评估同时靶向EZH2与G9a的抗肿瘤效应。体外实验中，EZH2抑制剂与G9a抑制剂联合给药可通过诱导细胞周期阻滞与细胞凋亡，显著抑制骨髓瘤细胞增殖。体内实验显示，双重抑制EZH2与G9a可有效抑制骨髓瘤细胞的异种移植瘤形成。EZH2与编码G9a的EHMT2基因的高表达，分别与MM患者较差的预后显著相关。微阵列分析结果显示，同时抑制EZH2与G9a可显著上调骨髓瘤细胞中干扰素（interferon, IFN）刺激基因的表达，并抑制IRF4-MYC轴相关基因的表达。值得注意的是，本研究发现经双重抑制处理的骨髓瘤细胞中，内源性逆转录病毒（endogenous retrovirus, ERV）基因的表达水平升高，而其H3K27/H3K9甲基化水平降低，提示ERV基因的激活可能介导了干扰素应答反应。上述研究结果表明，同时靶向EZH2与G9a或可成为MM的新型治疗策略。整体实验设计：将RPMI-8226与MM.1S细胞分别用GSK126（1 μM）、UNC0638（1 μM）或二者联合（各1 μM）处理6天。