FBXW7 mutations reduce binding of NOTCH1, leading to cleaved NOTCH1 accumulation and target gene activation in CLL
FBXW7 mutations reduce binding of NOTCH1, leading to cleaved NOTCH1 accumulation and target gene activation in CLL
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DOI：
10.1182/blood-2018-09-874529
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发表时间：
2019-02-21
期刊：
影响因子：
20.3
通讯作者：
Mertens, Daniel
中科院分区：
文献类型：
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作者：
Close, Viola;Close, William;Mertens, Daniel
NOTCH1 在 10% 的慢性淋巴细胞白血病 (CLL) 患者中发生突变，并且与不良预后相关。然而，即使在没有 NOTCH1 突变的情况下，大约一半的 CLL 病例也会发现 NOTCH1 激活。因此，似乎还有其他因素导致 NOTCH1 降解受损。 E3-泛素连接酶 F-box 和 WD40 重复结构域包含 7 (FBXW7)（NOTCH1 的负调节因子）在 2% 至 6% 的 CLL 患者中发生突变。 CLL 中这些突变的功能后果尚不清楚。我们在 905 名未经治疗的 CLL 患者中发现了 36 名 (4%) 存在杂合 FBXW7 突变。大多数是错义突变 (78%)，主要影响 WD40 底物结合域； 10% 的突变发生在 α-异构体的第一个外显子中。为了鉴定 CLL 中 FBXW7 的靶蛋白，我们通过成簇规则间隔的短回文重复序列 (CRISPR)/CRISPR 相关蛋白 9 (Cas9) 截断了 CLL 细胞系 HG-3 中的 WD40 结构域。 FBXW7 的纯合截短导致激活的 NOTCH1 胞内结构域 (NICD) 和 c-MYC 蛋白水平增加，以及缺氧诱导因子 1-α 活性升高。计算机模拟预测 FBXW7-WD40 结构域中的新突变 G423V 和 W425C 会改变蛋白质底物的结合。通过过表达的 FBXW7 和 NOTCH1 的免疫共沉淀证实了这种差异结合。在携带 FBXW7 突变的原代 CLL 细胞中，激活的 NICD 水平升高，并在翻译抑制后保持稳定。 FBXW7 突变与 NOTCH1 靶基因表达增加同时发生，并解释了一部分以 NOTCH1 信号传导失调为特征的患者。
NOTCH1 is mutated in 10% of chronic lymphocytic leukemia (CLL) patients and is associated with poor outcome. However, NOTCH1 activation is identified in approximately one-half of CLL cases even in the absence of NOTCH1 mutations. Hence, there appear to be additional factors responsible for the impairment of NOTCH1 degradation. E3-ubiquitin ligase F-box and WD40 repeat domain containing-7 (FBXW7), a negative regulator of NOTCH1, is mutated in 2% to 6% of CLL patients. The functional consequences of these mutations in CLL are unknown. We found heterozygous FBXW7 mutations in 36 of 905 (4%) untreated CLL patients. The majority were missense mutations (78%) that mostly affected the WD40 substrate binding domain; 10% of mutations occurred in the first exon of the alpha-isoform. To identify target proteins of FBXW7 in CLL, we truncated the WD40 domain in CLL cell line HG-3 via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9). Homozygous truncation of FBXW7 resulted in an increase of activated NOTCH1 intracellular domain (NICD) and c-MYC protein levels as well as elevated hypoxia-inducible factor 1-alpha activity. In silico modeling predicted that novel mutations G423V and W425C in the FBXW7-WD40 domain change the binding of protein substrates. This differential binding was confirmed via coimmunoprecipitation of overexpressed FBXW7 and NOTCH1. In primary CLL cells harboring FBXW7 mutations, activated NICD levels were increased and remained stable upon translation inhibition. FBXW7 mutations coincided with an increase in NOTCH1 target gene expression and explain a proportion of patients characterized by dysregulated NOTCH1 signaling.