Runx1功能缺失突变导致eIF4E选择性翻译Bcl-2及其在AML化疗耐受中的分子机制研究

In acute myeloid leukemia (AML) treatment, Runx1 loss of function (LOF) mutation was found to be an independent prognostic factor and patients carrying this mutation were insensitive to chemotherapy. Apoptosis of tumor cells caused by chemotherapy drugs mainly depends on the regulation of Bcl-2 family proteins and high expression of Bcl-2 is usually correlated with chemo-resistance in AML patients. In our previous study using mouse models and human clinical AML samples both in vitro and in vivo, we found that Bcl-2 inhibition could significantly increase the sensitivity of cells with Runx1 LOF mutation to p53 induced apoptosis. This suggests that Bcl-2 regulates chemo-resistance in cells with Runx1 LOF mutation, yet the molecular mechanism has not been revealed. We believe that upon stress Runx1 LOF mutation cells would selectively translate Bcl-2 protein, thereby escape from apoptosis and eventually demonstrate with chemo-resistance. In this study, mouse and human hematopoietic stem cells carrying Runx1 LOF mutation will be utilized as models and the molecular mechanism of eIF4E-mediated Bcl-2 selective translation will be explored. Both mouse AML models and in-vitro test of clinic samples will be used for validation. The data of this study will provide research evidence for Runx1 LOF mutation-related cancer treatment.

Runx1功能缺失突变是急性髓系白血病（acute myeloid leukemia,AML）的一个独立预后指标，携带该突变的患者对化疗不敏感。化疗药物诱导细胞凋亡的过程主要依赖Bcl-2家族蛋白的调控,通常AML化疗耐受患者伴有Bcl-2的高表达。前期在小鼠模型和临床AML样品实验中，我们发现Runx1功能缺失细胞中抑制Bcl-2的表达能显著增加其对p53诱导的细胞凋亡的敏感性。这提示Runx1功能缺失突变通过调控Bcl-2促进细胞化疗耐受，但其分子机制尚不明确。我们推测，应激条件下Runx1功能缺失突变导致细胞通过eIF4E选择性翻译Bcl-2，从而逃逸凋亡产生化疗耐受。本课题拟以Runx1条件性敲除小鼠和人脐带血干细胞为研究模型，阐明Runx1功能缺失突变导致Bcl-2选择性翻译的分子机制，并将在动物模型和临床样本中验证相关机制。研究结果有望为相关恶性肿瘤的临床治疗提供新依据。

Runx1是造血发育过程中的关键转录因子之一，其功能缺失突变是急性髓系白血病的预后指标之一。前期实验数据提示Runx1功能缺失突变通过调控Bcl-2促进细胞化疗耐受，但分子机制尚不明确。根据Runx1功能研究，我们提出Runx1参与蛋白翻译调控，且Runx1功能缺失突变可能导致Bcl-2选择性翻译而进行凋亡逃逸。本课题构建了表达Runx1功能缺失突变体的细胞模型，从以下三方面进行了机制探索：（1）实验证明Runx1功能缺失突变导致细胞翻译水平下降，但应激条件下Runx1功能缺失突变会导致细胞eIF4E活性提高，并通过IRES序列选择性翻译Bcl-2，帮助细胞逃逸凋亡；（2）对于Runx1参与蛋白翻译的调控机制，通过构建多个Runx1截短突变体，我们利用RNA测序数据证实Runx1通过羧基末端VWRPY结构域在转录水平调控核糖体蛋白基因表达，参与蛋白翻译的整体调控；（3）我们利用质谱鉴定及免疫共沉淀技术证实：Runx1通过与翻译延长因子eEF1A1结合以参与整体细胞蛋白翻译水平的调控，同时这一过程也受到Runx1 VWRPY结构域的调节。因此，本课题数据阐明了Runx1功能缺失突变导致Bcl-2选择性翻译的分子机制，并从转录和蛋白结合两个层面探索了Runx1在蛋白翻译过程中的作用。研究结果为Bcl-2抑制剂的应用提供实验基础，同时为Runx1 VWRPY结构域功能的研究提供新思路。