FBXW7 mutations reduce binding of NOTCH1, leading to cleaved NOTCH1 accumulation and target gene activation in CLL

Abstract

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 α-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-α 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.

Insights into Functional Connectivity in Mammalian Signal Transduction Pathways by Pairwise Comparison of Protein Interaction Partners of Critical Signaling Hubs

“…MYC and NOTCH1 as well as common interactor FBXW7 expression levels were significantly altered in several cancers, especially in blood cancers [67]. Consistent with the results, mutations in FBXW7 were reported in 2-6% of chronic lymphocytic leukemia (CLL) resulting in the activation of NOTCH11 intracellular domain (NCID) and MYC target genes [68]. Network visualization also revealed that RELA, GSK3B…”

FBW7 in hematological tumors (Review)

“…A previous study identified FBW7 mutations in 36 out of 905 untreated patients with CLL. All these mutations are heterozygous, missense in 78% of cases and primarily affect the substrate-binding domain of WD40 (77). Furthermore, 10% of the mutations were located on the first exon of the α-transcript.…”

“…Furthermore, 10% of the mutations were located on the first exon of the α-transcript. In order to identify the target protein of FBW7 in CLL, a WD40 domain-truncated CLL cell line (HG-3) was generated using the CRISPR/Case9 method (77). Homozygous truncation of FBW7 results in increased levels of activated Notch1-NICD and c-Myc proteins, and in enhanced activity of HIF1-α (77).…”

“…In order to identify the target protein of FBW7 in CLL, a WD40 domain-truncated CLL cell line (HG-3) was generated using the CRISPR/Case9 method (77). Homozygous truncation of FBW7 results in increased levels of activated Notch1-NICD and c-Myc proteins, and in enhanced activity of HIF1-α (77). In primary cells derived from patients with CLL carrying the FBW7 mutation, the level of activated Notch1-NICD was elevated and remained stable after inhibition of translation (77).…”

“…Homozygous truncation of FBW7 results in increased levels of activated Notch1-NICD and c-Myc proteins, and in enhanced activity of HIF1-α (77). In primary cells derived from patients with CLL carrying the FBW7 mutation, the level of activated Notch1-NICD was elevated and remained stable after inhibition of translation (77). In addition, FBW7 mutation was combined with enhanced expression of Notch1 target genes (77).…”