Dysregulated Translational Control Is a Central Feature of MM Mediated By 14-3-3ε Interactome Promoting Multiple Myeloma Cell Growth and Viability-猫眼课题宝

DOI：

10.1182/blood-2018-99-118994

发表时间：

2018-11

期刊：

Blood

影响因子：

20.3

通讯作者：

中科院分区：

医学1区

文献类型：

作者：

Yan Xu;M. Fulciniti;M. Ho;M. Samur;Michael A. Lopez;Na Li;G. Bianchi;R. Szalat;Jianhong Lin;T. Perini;S. Talluri;Tengteng Yu;Kenneth Wen;Jiye Liu;Li Zhang;Liang Lin;Lijie Xing;G. An;Y. Tai;K. Anderson;L. Qiu;N. Munshi

14-3-3蛋白是分子伴侣和支架蛋白，通过与靶蛋白上的丝氨酸/苏氨酸磷酸化残基结合，迫使构象变化或影响其与其他分子的相互作用，对细胞过程施加广泛影响。改变的14-3-3表达与癌症的发展和进展相关。因此，我们评估了浆细胞疾病中所有14-3-3亚型在实验室可用的基因表达谱（GEP）数据中的状态。使用独立的患者数据集，我们观察到MM和浆细胞白血病（PCL）患者中YWHAE（亚型14-3-3ε的编码基因）的一致较高表达，而其他亚型则没有观察到一致的差异。此外，我们还证实了来自420名新诊断的MM患者的RNA-seq数据中YWHAE的较高表达，在正常浆细胞中表达相对较低。最后，还发现14-3-3ε在原代患者MM细胞和一大组MM细胞系中以蛋白质水平组成型表达，在健康供体B细胞中表达显著较低。为了评估14-3-3ε是否代表MM中的功能依赖性，我们在一组MM细胞系中进行了YWHAE的遗传扰动。使用3种不同的shRNA消耗YWHAE抑制细胞增殖，并诱导5种不同细胞系的细胞凋亡，与其遗传背景无关。我们接下来在H929和JJN 3细胞中进行了CRISPR-Cas9介导的YWHAE敲除（KO），并观察到细胞活力的显著降低和稳健的凋亡应答。用FLAG-YWHAE加回慢病毒构建体感染的H929 YWHAE KO细胞完全挽救了这种表型，证实了YWHAE的丧失是缺陷性细胞活力和凋亡表型的原因。这些观察结果通过H929 WT细胞中YWHAE的异位过表达得到证实，其显著促进MM细胞活力。为了阐明潜在的分子机制，通过质谱分析在具有14-3-3ε-FLAG加回的H929 KO细胞中与FLAG共沉淀的蛋白质。蛋白质分析显示14-3-3ε与大量蛋白质相互作用，富含mTORC 1、PI 3 K-AKT-mTOR和未折叠蛋白反应（UPR）途径相关基因。其中，TSC 2和mTORC 1蛋白被进一步研究。WB分析证实了14-3-3ε与p-mTOR（S2448）及其上游负调控因子p-TSC 2（S939）的相互作用，而mTORC 1下游靶点p-p70 S6 k和p-4 E-BP 1不与14-3-3ε相互作用。WB分析还揭示了在YWHAE KD细胞中TSC 2的活化和随后的mTORC 1的抑制（通过降低p-mTOR S2448水平）。YWHAE-FLAG addback逆转了这些效应。此外，KD细胞中的GEP数据证实了对mTORC 1通路的显著影响。重要的是，在我们的RNA-seq数据集中，YWHAE表达与mTORC 1通路中涉及的基因高度相关（R> 0.8），包括PSMC 4，COPS 5，EIF 2S 2，证明了14-3-3ε和mTORC 1在骨髓瘤背景下合作的临床意义。mTORC 1最保守的功能之一是促进翻译。因此，我们评估了YWHAE对MM细胞中整体翻译效率的影响，并观察到YWHAE调节对新生蛋白质合成的显著影响。14-3-3ε KD通过抑制mTORC 1诱导4 EBP 1去磷酸化，同时诱导EIF 2 α磷酸化。这两种效应都抑制了翻译起始复合物的形成，在机械上支持了强有力的蛋白质合成停滞。这些数据显示了响应于YWHAE调节而控制翻译起始的信号装置的几个枢纽的调节，最终产生显著的蛋白质合成抑制。翻译控制失调是MM的一个重要特征。我们的研究结果强调了YWHAE作为MM细胞存活的启动子通过调节mTOR依赖性蛋白合成和凋亡的独特功能。因此，对YWHAE/14-3-3ε的药理学抑制有可能特异性靶向翻译控制失调的恶性肿瘤，例如MM.安德森：C4 Therapeutics：股权所有权，其他：科学创始人; Millennium Takeda：咨询;吉利德：实体董事会或咨询委员会成员;布里斯托迈斯贵宝：咨询; OncoPep：股权所有权，其他：科学创始人; Celgene：咨询公司Munshi：OncoPep：其他：董事会。

14-3-3 proteins are chaperone and scaffold proteins that exert a widespread influence on cellular processes through binding to serine/threonine-phosphorylated residues on target proteins, forcing conformational changes or influencing their interactions with other molecules. Altered 14-3-3 expression is associated with development and progression of cancer. We therefore evaluated the status of all 14-3-3 isoforms in plasma cells disorders in publically available gene expression profiling (GEP) data. Using independent patient datasets, we observed a consistent higher expression of YWHAE (coding gene for the isoform 14-3-3ε) in MM and plasma cell leukemia (PCL) patients, while no consistent differences were observed with the other isoforms. Moreover, we also confirmed higher expression of YWHAE in our RNA-seq data from 420 newly-diagnosed MM patients, with relatively low expression in normal plasma cells. Finally, 14-3-3ε was also found to be constitutively expressed at protein level in primary patient MM cells and in a large panel of MM cell lines, with significantly lower expression in healthy donor B cells. To evaluate if 14-3-3ε represents a functional dependency in MM, we performed genetic perturbation of YWHAE in a panel of MM cell lines. Depletion of YWHAE using 3 different shRNA inhibited cell proliferation and induced cell apoptosis across 5 different cell lines, independently of their genetic background. We next performed CRISPR-Cas9-mediated YWHAE knock out (KO) in H929 and JJN3 cells and observed a significant decrease in cell viability and a robust apoptotic response. H929 YWHAE KO cells infected with FLAG-YWHAE addback lentiviral construct completely rescued this phenotype, confirming that loss of YWHAE is responsible for the defective cell viability and apoptotic phenotype. These observations were corroborated by ectopic overexpression of YWHAE in H929 WT cells that significantly promoted MM cell viability. To elucidate the underlying molecular mechanisms, proteins immunocomplexed co-precipitated with FLAG in H929 KO cells with 14-3-3ε-FLAG addback were analyzed by mass spectrometry. Protein analysis revealed interaction of 14-3-3ε with a large number of proteins, enriched in mTORC1, PI3K-AKT-mTOR and unfolded protein response (UPR) pathway-related genes. Among these, TSC2 and mTORC1 proteins were further studied. WB analysis confirmed interaction of 14-3-3ε with p-mTOR (S2448) and its upstream negative regulator p-TSC2 (S939), while mTORC1 downstream targets, p-p70 S6k and p-4E-BP1, did not interact with 14-3-3ε. WB analysis also revealed activation of TSC2 and consequent inhibition of mTORC1 (via decrease of p-mTOR S2448 levels) in YWHAE KD cells. YWHAE-FLAG addback reversed these effects. Additionally, GEP data in KD cells confirmed a significant impact on mTORC1 pathways. Importantly, YWHAE expression highly correlated (R> 0.8) with genes involved in the mTORC1 pathway, including PSMC4, COPS5, EIF2S2, in our RNA-seq dataset, demonstrating a clinical significance of 14-3-3ε and mTORC1 cooperation in the context of myeloma. One of the most conserved functions of mTORC1 is to promote translation. We therefore assessed the impact of YWHAE on global translational efficiencies in MM cells, and observed significant impact on nascent protein synthesis by YWHAE modulation. 14-3-3ε KD induced 4EBP1 de-phosphorylation through inhibited mTORC1, and concomitantly induced EIF2α phosphorylation. Both effects inhibited translation initiation complex formation, mechanistically supporting a strong protein synthesis arrest. These data show the modulation of several hubs of the signaling apparatus controlling translation initiation in response to YWHAE modulation, ultimately producing a marked protein synthesis inhibition. Deregulated translational control is a central feature of MM. Our findings highlight a unique function for YWHAE as promoter of MM cell survival through regulation of mTOR-dependent protein synthesis and apoptosis. Pharmacological inhibition of YWHAE/14-3-3ε is therefore a possibility to specifically target malignancies with deregulated translational control such as MM. Anderson: C4 Therapeutics: Equity Ownership, Other: Scientific founder; Millennium Takeda: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; OncoPep: Equity Ownership, Other: Scientific founder; Celgene: Consultancy. Munshi:OncoPep: Other: Board of director.

Dysregulated Translational Control Is a Central Feature of MM Mediated By 14-3-3ε Interactome Promoting Multiple Myeloma Cell Growth and Viability

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