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.

Aberrant Notch signaling plays a pivotal role in T-cell acute lymphoblastic leukemia (T-ALL) and chronic lymphocytic leukemia (CLL). Amplitude and duration of the Notch response is controlled by ubiquitin-dependent proteasomal degradation of the Notch1 intracellular domain (NICD1), a hallmark of the leukemogenic process. Here, we show that HDAC3 controls NICD1 acetylation levels directly affecting NICD1 protein stability. Either genetic loss-of-function of HDAC3 or nanomolar concentrations of HDAC inhibitor apicidin lead to downregulation of Notch target genes accompanied by a local reduction of histone acetylation. Importantly, an HDAC3-insensitive NICD1 mutant is more stable but biologically less active. Collectively, these data show a new HDAC3- and acetylation-dependent mechanism that may be exploited to treat Notch1-dependent leukemias.

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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. Disclosures Tausch: AbbVie: Consultancy, Other: Travel grants; Celgene: Consultancy, Other: Travel grants; Gilead: Consultancy, Other: Travel grants. Döhner:AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celator: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; AROG Pharmaceuticals: Research Funding; Astellas: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Agios: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding. Stilgenbauer:Novartis: Consultancy, Honoraria, Other: travel support, Research Funding; Roche: Consultancy, Honoraria, Other: travel support, Research Funding; Genetech: Consultancy, Honoraria, Other: travel support, Research Funding; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: travel support, Research Funding; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding; Janssen: Consultancy, Honoraria, Other: travel support, Research Funding; Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding.

Chronic lymphocytic leukemia (CLL) is known for its strong dependency on the tumor microenvironment. We found progranulin (GRN), a protein that has been linked to inflammation and cancer, to be upregulated in the serum of CLL patients compared to healthy controls, and increased GRN levels to be associated with an increased hazard for disease progression and death. This raised the question of whether GRN is a functional driver of CLL. We observed that recombinant GRN did not directly affect viability, activation, or proliferation of primary CLL cells in vitro. However, GRN secretion was induced in co-cultures of CLL cells with stromal cells that enhanced CLL cell survival. Gene expression profiling and protein analyses revealed that primary mesenchymal stromal cells (MSCs) in co-culture with CLL cells acquire a cancer-associated fibroblast-like phenotype. Despite its upregulation in the co-cultures, GRN treatment of MSCs did not mimic this effect. To test the relevance of GRN for CLL in vivo, we made use of the Eμ-TCL1 CLL mouse model. As we detected strong GRN expression in myeloid cells, we performed adoptive transfer of Eμ-TCL1 leukemia cells to bone marrow chimeric Grn−/− mice that lack GRN in hematopoietic cells. Thereby, we observed that CLL-like disease developed comparable in Grn−/− chimeras and respective control mice. In conclusion, serum GRN is found to be strongly upregulated in CLL, which indicates potential use as a prognostic marker, but there is no evidence that elevated GRN functionally drives the disease.

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