Role of Protein Arginine Methyltransferase 9 in Acute Myeloid Leukemia Maintenance

项目摘要 急性髓细胞白血病（AML）是一种致命的疾病。虽然细胞遗传学和分子风险适应 尽管这些方法可以指导治疗，但总体结果仍然很差。当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（TKI），不能消除所有白血病 克隆越来越多的证据表明，依赖癌蛋白生存的剩余细胞成为癌细胞。 复发的根源。这些癌蛋白中的许多，如Mcl-1、STAT 5和c-Myc，是短寿命的，并且它们的高表达是短寿命的。 肿瘤中的水平可能是由于翻译机制的异常激活，这是癌症的标志。因此，在本发明中， 理解翻译失调的潜在机制是对抗白血病的必要条件 坚持不懈我们最近发现PRMT 9的高表达水平，最近定义的对称- 二甲基精氨酸（SDMA）形成酶与AML患者总体生存率降低相关。我们 蛋白质组学和诱变分析揭示了一种新的机制，PRMT 9催化甲基化的 翻译延伸因子eEF 1A 1，将PRMT 9连接到主动翻译并促进AML维持。 因此，PRMT 9抑制以eEF 1A 1甲基化依赖性的方式阻断AML细胞存活/增殖。 方式，同时保留正常的造血细胞。PRMT 9/eEF 1A信号传导的抑制确实降低 蛋白质的生物合成，减少短寿命癌蛋白Mcl-1，c-Myc和STAT 5的水平，并显着 延长白血病小鼠的存活时间。因此，我们假设PRMT 9对AML发病机制至关重要， PRMT 9介导的eEF 1A甲基化促进mRNA翻译和蛋白质合成， 上维护为了验证这一假设，我们将：1）确定PRMT 9介导的eEF 1A精氨酸的功能， 使用MLL-AF 9相关的双重打击AML小鼠模型和新开发的 PRMT 9条件性敲除小鼠; 2）确定PRMT 9/eEF 1A信号转导的分子机制， 通过测试eEF 1A甲基化是否通过增强GTP/GDP结合而改变其GTP酶活性， 基于核糖体分析评估PRMT 9缺陷是否诱导AML的整体翻译变化 测序; 3）确定PRMT 9缺失或用柔红霉素/阿糖胞苷抑制的组合效应，或 AC 220治疗对鼠和人AML模型中AML根除的影响。我们的研究应该能揭示 PRMT 9和eEF 1A之间相互作用，并表明高度保守的eEF 1A R166的精氨酸甲基化 控制着癌细胞的翻译这些研究将缩小与白血病细胞如何 通过翻译机制的异常活动获得生存/生长优势，并可能显示如何 将PRMT 9的靶向与当前的治疗相结合可能代表了一种更有效的策略， AML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.