Role of Protein Arginine Methyltransferase 9 in Acute Myeloid Leukemia Maintenance

项目摘要 急性髓系白血病(AML)是一种致命的疾病。尽管细胞遗传学和分子风险适应 方法可以指导治疗，但总体结果仍然很差。目前可用的基于细胞周期的 化疗或靶向治疗，如使用酪氨酸激酶抑制剂(TKIs)，不能消除所有白血病 克隆人。越来越多的证据表明，依赖癌蛋白生存的剩余细胞成为 复发的源头。其中许多癌蛋白，如Mcl-1，STAT5和c-Myc，都是短暂的，它们的高表达 肿瘤中的水平可能是由于翻译机制的异常激活，这是癌症的一个标志。因此， 了解异常翻译的机制对于对抗白血病是必要的 坚持不懈。我们最近发现，PRMT9的高表达水平，最新定义的对称- 二甲基精氨酸(SDMA)形成酶与AML患者总体存活率的降低有关。我们的 蛋白质组学和突变分析揭示了PRMT9催化甲基化的新机制 翻译延伸系数eEF1A1，将PRMT9与主动翻译联系起来，并促进AML的维护。 因此，抑制PRMT9在eEF1A1甲基化依赖的情况下阻止了AML细胞的生存/增殖 方式，同时保留正常的造血细胞。对PRMT9/eEF1A信号的抑制确实减少了 蛋白质生物合成，降低短命癌蛋白Mcl-1，c-Myc和STAT5的水平，并显著 延长白血病小鼠的存活期。因此，我们假设PRMT9在AML的发病机制中起关键作用，并且 PRMT9介导的eEF1A甲基化促进mRNA翻译和蛋白质合成使AML成为可能 维修。为了验证这一假设，我们将：1)定义PRMT9介导的eEF1a精氨酸的功能 利用MLL-AF9相关的双重打击AML小鼠模型和新开发的甲基化在AML发病机制中的作用 PRMT9条件性基因敲除小鼠；2)定义PRMT9/eEF1A信号转导的分子机制 通过检测eEF1A甲基化是否通过增强GTP/GDP结合而改变其GTP酶活性和 基于核糖体图谱评估PRMT9缺乏是否导致急性髓系白血病整体翻译改变 测序；3)确定PRMT9缺失或抑制与柔红霉素/阿糖胞苷或 AC220治疗对小鼠和人急性髓系白血病模型的根除作用。我们的研究应该揭示出 PRMT9与eEF1A的相互作用及高度保守的eEF1AR166的精氨酸甲基化 控制癌细胞中的翻译。这些研究将填补与白血病细胞如何 通过翻译机器的异常活动获得生存/增长优势，并可能展示如何 将靶向PRMT9与目前的治疗相结合可能是一种更有效的策略来消除 急性髓系白血病细胞。

Project Summary Acute myeloid leukemia (AML) is a deadly disease. Although cytogenetic and molecular risk-adapted approaches can guide treatment, overall outcomes remain poor. Currently available cell cycle-based chemotherapy or targeted therapy, such as use of tyrosine kinase inhibitors (TKIs), cannot eliminate all leukemia clones. Mounting evidence suggests that remaining cells that depend on oncoproteins for survival become a source of relapse. Many of these oncoproteins, such as Mcl-1, STAT5, and c-Myc, are short-lived, and their high levels in tumors are likely due to aberrant activation of the translation machinery, a hallmark of cancer. Thus, understanding mechanisms underlying dysregulated translation is necessary to antagonize leukemia persistence. We recently found that higher expression levels of PRMT9, the most recently defined symmetric- dimethylarginine (SDMA)-forming enzyme, are associated with decreased overall AML patient survival. Our proteomics and mutagenesis analysis revealed a novel mechanism whereby PRMT9 catalyzes methylation of translation elongation factor eEF1A1, linking PRMT9 to active translation and promoting AML maintenance. Accordingly, PRMT9 inhibition blocked AML cell survival/proliferation in an eEF1A1 methylation-dependent manner, while sparing normal hematopoietic cells. Inhibition of PRMT9/eEF1A signaling indeed decreased protein biosynthesis, reducing levels of the short-lived oncoproteins Mcl-1, c-Myc, and STAT5 and significantly extending leukemic mouse survival. Thus, we hypothesize that PRMT9 is critical for AML pathogenesis and that PRMT9-mediated eEF1A methylation promotes mRNA translation and protein synthesis to enable AML maintenance. To test this hypothesis, we will: 1) define the function of PRMT9-mediated eEF1A arginine methylation in AML pathogenesis using a MLL-AF9-related doubly-hit AML mouse model and a newly developed PRMT9 conditional knockout mouse; 2) define molecular mechanisms underlying PRMT9/eEF1A signaling in AML by testing whether eEF1A methylation alters its GTPase activity by enhancing GTP/GDP binding and assessing whether PRMT9 deficiency induces global translation changes in AML based on ribosome profiling sequencing; 3) determine combined effects of PRMT9 deletion or inhibition with daunorubicin/cytarabine or AC220 treatments on AML eradication in murine and human AML models. Our studies should reveal functional interaction between PRMT9 and eEF1A and indicate how arginine methylation of highly conserved eEF1A R166 governs translation in cancer cells. These studies will close the knowledge gap relevant to how leukemia cells acquire a survival/growth advantage through aberrant activity of the translation machinery and may show how combining targeting of PRMT9 with current treatments could represent a more effective strategy to eliminate AML cells.