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

Viola Close, William Close, Sabrina Julia Kugler, Michaela Reichenzeller, Deyan Yordanov Yosifov, Johannes Bloehdorn, Leiling Pan, Eugen Tausch, Mike-Andrew Westhoff, Hartmut Döhner, Stephan Stilgenbauer, Franz Oswald and Daniel Mertens

Key points

  • CRISPR-induced truncation of the FBXW7 substrate binding domain identifies cleaved NOTCH1 (NICD) as a target of FBXW7 in CLL cells.

  • FBXW7 mutations in CLL correlate with increased NICD protein and elevated NOTCH1 target gene expression and thereby mimic NOTCH1 mutations.

Abstract

In chronic lymphocytic leukemia (CLL), NOTCH1 is mutated in 10% of CLL patients and is associated with poor outcome. However, NOTCH1 activation is identified in approximately half of CLL cases even in the absence of NOTCH1 mutations, hence there appears to be additional factors responsible for the impairment of NOTCH1 degradation. The E3 ubiquitin ligase FBXW7 is a negative regulator of NOTCH1 and is mutated in 2-6% of CLL patients. So far the functional consequences of these mutations are unknown. We found FBXW7 mutations in 36/905 (4%) of untreated CLL patients, of which all mutations were heterozygous. The majority were missense mutations (78%) that mostly affected the WD40 substrate binding domain, whilst 10% of mutations occurred in the first exon of the α-isoform. In aim to identify target proteins of FBXW7 in CLL, we truncated the WD40 domain in the CLL cell line HG-3 via CRISPR/Cas9. Homozygous truncation of FBXW7 resulted in increase of activated NOTCH1-NICD and c-MYC protein levels as well as elevated HIF1-α activity. In silico modeling predicted that novel mutations G423V and W425C in the FBXW7-WD40 domain change the binding of protein substrates while the mutation A503V was predicted not to be impaired. This differential binding was confirmed via co-immunoprecipitation of overexpressed FBXW7 and NOTCH1. In primary cells from CLL patients harboring FBXW7 mutations, activated NOTCH1-NICD levels were increased and remained stable upon inhibition of translation. Furthermore, FBXW7 mutations coincided with an increase in NOTCH1 target gene expression. Hence, FBXW7 mutations in CLL cells explain a proportion of patients characterized by dysregulated NOTCH1 signaling.

  • Submitted September 11, 2018.
  • Accepted November 13, 2018.