Purpose

The t(4;11)(q21;q23) translocation characterizes a form of acute lymphoblastic leukemia with a poor prognosis. It results in a fusion gene encoding a chimeric transcription factor, MLL-AF4, that deregulates gene expression through a variety of still controversial mechanisms. To provide new insights into these mechanisms, we examined the interaction between AF4, the most common MLL fusion partner, and the scaffold protein 14-3-3θ, in the context of t(4;11)-positive leukemia.

Methods

Protein-protein interactions were analyzed using immunoprecipitation and in vitro binding assays, and by fluorescence microscopy in t(4;11)-positive RS4;11 and MV4–11 leukemia cells and in HEK293 cells. Protein and mRNA expression levels were determined by Western blotting and RT-qPCR, respectively. A 5-bromo-2′-deoxyuridine assay and an annexin V/propidium iodide assay were used to assess proliferation and apoptosis rates, respectively, in t(4;11)-positive and control cells. Chromatin immunoprecipitation was performed to assess binding of 14-3-3θ and AF4 to a specific promoter element.

Results

We found that AF4 and 14-3-3θ are nuclear interactors, that 14-3-3θ binds Ser⁵⁸⁸ of AF4 and that 14-3-3θ forms a complex with MLL-AF4. In addition, we found that in t(4;11)-positive cells, 14-3-3θ knockdown decreased the expression of MLL-AF4 target genes, induced apoptosis and hampered cell proliferation. Moreover, we found that 14-3-3θ knockdown impaired the recruitment of AF4, but not of MLL-AF4, to target chromatin. Overall, our data indicate that the activity of the chimeric transcription factor MLL-AF4 depends on the cellular availability of 14-3-3θ, which triggers the transactivating function and subsequent degradation of AF4.

Conclusions

From our data we conclude that the scaffold protein 14-3-3θ enhances the aberrant activity of the chimeric transcription factor MLL-AF4 and, therefore, represents a new player in the molecular pathogenesis of t(4;11)-positive leukemia and a new promising therapeutic target.

中文翻译：

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14-3-3θ 和 AF4 之间的串扰增强 MLL-AF4 活性并促进白血病细胞增殖。

目的

t(4;11)(q21;q23) 易位是一种预后不良的急性淋巴细胞白血病。它产生了一种编码嵌合转录因子 MLL-AF4 的融合基因，该基因通过各种仍有争议的机制解除对基因表达的调控。为了提供对这些机制的新见解，我们在 t(4;11) 阳性白血病的背景下检查了 AF4（最常见的 MLL 融合伙伴）和支架蛋白 14-3-3θ 之间的相互作用。

方法

在 t(4;11) 阳性 RS4;11 和 MV4-11 白血病细胞和 HEK293 细胞中使用免疫沉淀和体外结合测定法分析蛋白质-蛋白质相互作用。蛋白质和 mRNA 表达水平分别通过蛋白质印迹和 RT-qPCR 确定。5-溴-2'-脱氧尿苷测定和膜联蛋白 V/碘化丙啶测定分别用于评估 t(4;11) 阳性和对照细胞的增殖和凋亡率。进行染色质免疫沉淀以评估 14-3-3θ 和 AF4 与特定启动子元件的结合。

结果

我们发现 AF4 和 14-3-3θ 是核相互作用物，14-3-3θ 与AF4 的Ser ⁵⁸⁸结合，14-3-3θ 与 MLL-AF4 形成复合物。此外，我们发现在 t(4;11) 阳性细胞中，14-3-3θ 敲低降低了 MLL-AF4 靶基因的表达，诱导细胞凋亡并阻碍细胞增殖。此外，我们发现 14-3-3θ 敲低会损害 AF4 的募集，但不会损害 MLL-AF4 靶向染色质的募集。总的来说，我们的数据表明嵌合转录因子 MLL-AF4 的活性取决于 14-3-3θ 的细胞可用性，这会触发 AF4 的反式激活功能和随后的降解。

结论

根据我们的数据，我们得出结论，支架蛋白 14-3-3θ 增强了嵌合转录因子 MLL-AF4 的异常活性，因此，它代表了 t(4;11) 阳性白血病分子发病机制的新参与者和新的有希望的治疗靶点。