NOTCH1 Signaling Is Activated in CLL By Mutations of FBXW7 and Low Expression of USP28 at 11q23

Background

Oncogenic proteins can be stabilized either via genetic mutations or via defects in the ubiquitin ligating- and degradation machinery. NOTCH1 protein stability is affected by genetic mutations in approximately 10% of chronic lymphocytic leukemia (CLL) patients and mutations are associated with a worse prognosis. Yet, even in the absence of NOTCH1 mutations, NOTCH1 is activated in almost half of all CLL patients.

Aims

In order to shed light on NOTCH1 activation in CLL, we analyzed the impact of its modulator, the E3-ubiquitin ligase FBXW7 and the deubiquitinase USP28 that is a negative regulator of FBXW7 and is located in chromosomal band 11q23.

Methods

FBXW7 mutation analysis was performed via targeted next generation sequencing from a total of 905 patients. In silico modeling of potential substrate binding to the mutated FBXW7 was performed by the use of PolyPhen-2 and validated via co-immunoprecipitation of overexpressed mutated FBXW7 and NOTCH1 wild type proteins. Accumulation of FBXW7 substrates was tested via Western blot in CRISPR/Cas9 induced FBXW7 mutated HG-3 CLL cell lines and in FBXW7 mutated primary CLL cells, identifying the transcription factor NOTCH1 as FBXW7 target. Expression of NOTCH1 target genes were analyzed in FBXW7 mutated CLL cases via gene expression profiling (n=4) and RT-qPCR (n=19). In an independent cohort, USP28 and NOTCH1 target gene expression was analyzed in a total of 285 patients via gene expression profiling.

Results

Heterozygous FBXW7 mutations were identified in 41/905 (4.5%) CLL patients. The majority were missense mutations (78%) that mostly affected the WD40 substrate binding domain, while an additional 10% of mutations were located on the first exon of the abundantly expressed FBXW7 α-isoform. We identified substrate targets of FBXW7 in CLL via the generation of a truncation of the WD40 domain of FBXW7 in the CLL cell line HG-3 using CRISPR/Cas9, identifying NOTCH1 as an FBXW7 target. In silico modeling of FBXW7 mutations on protein binding predicted that novel mutations within the WD40 domain affected substrate recognition capacity of FBXW7. Interestingly, modeling predicted W425C and a new hotspot mutation G423V (found in 3/905 of CLL cases) to ablate NOTCH1 binding while the mutation A503V was predicted not to impair binding of NOTCH1 to FBXW7. The in silico modeling was confirmed by co-immunoprecipitation experiments of overexpressed NOTCH1 and FBXW7 and further revealed that mutations within the α-isoform specific N-terminus T15VR and V154I still enabled NOTCH1 binding. Intriguingly, in primary CLL cells FBXW7 mutations correlated with an increase in NOTCH1 levels that remained stable even upon inhibition of translation, underlining the enhanced protein stabilization by FBXW7 mutations. Furthermore, FBXW7 mutations in CLL resulted in an increased NOTCH1 target gene expression.

FBXW7 activity is not only modulated by gene mutations, but also by the deubiquitinase USP28 which is localized in the recurrently deleted region 11q22-q23 close to the ATM tumor suppressor gene. We hypothesized that USP28 would impact on FBXW7 activity and thus on NOTCH1 stability and activity. In line with this concept we found that in primary cells from a cohort of 285 CLL patients, low USP28 expression significantly correlated with increased NOTCH1 target gene expression, independent of the 11q deletion status of the patients where USP28 is localized.

Discussion

In CLL patient cells we have identified novel mutations within the WD40 binding domain of FBXW7 in addition to the common hot spot mutations (R465, R479, R505). Amongst these novel mutations, G423V and W425C are recurrent and result in decreased binding of the FBXW7 substrate NOTCH1 and hence in an accumulation and induction of NOTCH1 activity. Furthermore, expression of USP28, the negative regulator of FBXW7, significantly correlated with increased NOTCH1 target gene expression. Hence, our findings uncover modulation of NOTCH1 in CLL via the FBXW7-USP28-NOTCH1 axis (Figure 1) in addition to genomic NOTCH1 modification, thus explaining the high proportion of CLL cases that harbor an activation of NOTCH1 leading to more aggressive disease.

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