In Chronic Lymphocytic Leukemia ΔCT7544-7545 Mutant NOTCH1 Maintains Transcription Factor Activity with Longer Lasting Effects Due to Slower Degradation
Authors: Edelmann Jennifer , Klymenko Tetyana , Lu Chen , Vilventhraraja Emma , Estenfelder Sven , Tausch Eugen , Beers Stephen A , Stilgenbauer Stephan , Cragg Mark S , Ivanov Andrejs , Gribben John 
Published: October 2019

The NOTCH1 gene locusencodes a cell surface receptor which is recurrently mutated in chronic lymphocytic leukemia (CLL), with a rising incidence in higher risk cases. Upon ligand-binding two cleavage events occur. As a consequence the receptor's intracellular domain (NICD) is released and shifts to the nucleus, where it functions as a transcription factor. Since in CLL NOTCH1 mutations mainly affect the protein's PEST-domain, which is involved in NICD degradation, they were assumed to be activating mutations. However, to date this has only been proven for mutations that lead to the loss of the complete PEST-domain or major parts of it, whereas the hotspot mutation in CLL (ΔCT7544-7545; E2515fs) leads to the loss of only 39 amino acids.

To prove the activating nature of the CLL hotspot mutation, we developed a cell culture based model using lentiviral transduction of B-cell lymphoma cell lines. A full-length NOTCH1 construct was cloned into a pCCL transfer vector and the CLL hotspot mutation was introduced. The cell lines SU-DHL4, Raji and Daudi were transduced with wild-type NOTCH1 (NOTCH1WT), ΔCT-mutant NOTCH1 (NOTCH1ΔCT) and empty vector.

For validation experiments, primary CLL samples were screened for PEST-domain NOTCH1 mutations by targeted next-generation sequencing of exon 34.

Activation of NOTCH1 was induced via treatment of cells with the Ca2+ chelator EGTA (1.0 mM, 1 hour). Liberated NICD was semi-quantitatively assessed by Western Blot and image densitometry from whole cell lysates or nuclear protein fractions. In addition, the NICD was visualized by intracellular immunofluorescent staining along with nuclear staining for DAPI and histone H3K27me / histone H3K27ac. Expression of the NOTCH1 target gene HES1 was quantified using TaqMan® RT-PCR analysis.

Treatment with EGTA robustly activated NOTCH1 signalling; with the highest levels of NICD seen in the nucleus 2 to 4 hours after activation.

Immunofluorescent staining of the NICD in NOTCH1 transduced cells 3 hours after activation revealed a speckled pattern in the nucleus without differences between NICDWT and NICDΔCT. Speckles co-localized with areas of relaxed chromatin, with NICD staining coincident with histone H3K27ac staining and separate from histone H3K27me staining. Co-localization of NICD speckles with areas of relaxed chromatin was confirmed in primary CLL samples where it was again observed independent of NOTCH1 mutation status.

In Western Blot analyses, NICDWT and NICDΔCT were distinguishable due to their different molecular weight. Without prior activation, NOTCH1WT transduced cell lines displayed low levels of NICD. In contrast, NOTCH1ΔCT transduced cell lines presented with considerably higher levels of NICDΔCT despite comparable cell surface levels of transduced NOTCH1. After activation of NOTCH1WT and NOTCH1ΔCT with EGTA, the levels of NICDWT and NICDΔCT rose to a similar maximum, but a subsequent time-course showed a slower degradation of the NICDΔCT compared to the NICDWT. The slower degradation of mutant NICD was confirmed in primary CLL samples including samples with truncating mutations of the PEST-domain other than the hotspot mutation E2515fs (Q2440*, S2486*, Q2307*).

HES1 is an established NOTCH1 target gene activated by nuclear NICD. Baseline expression levels of HES1 did not differ between NOTCH1WT and NOTCH1ΔCT transduced SU-DHL4 cells. After NOTCH1 activation, HES1 expression was up-regulated ~2.9 fold compared to baseline. Peak expression levels were seen around 3 hours after activation. HES1 expression levels fell more slowly in NOTCH1ΔCT transduced SU-DHL4 cells suggesting longer lasting effects on target genes by NICDΔCT than by NICDWT.

In summary, our data demonstrates for the first time that the ΔCT7544-7545 mutant of NOTCH1 maintains transcriptional activity for longer due to slower degradation of its NICD. Our understanding about NICD target genes remains incomplete, but their identification will be crucial to understand the role of NOTCH1 mutations in CLL pathogenesis.

Disclosures

Tausch: Gilead: Other: Travel support, Speakers Bureau; Celgene: Other: Travel support; Amgen: Other: Travel support. Stilgenbauer:Pharmacyclics: Consultancy, Honoraria, Other: Travel grants , Research Funding; Boehringer Ingelheim: Consultancy, Honoraria, Other: Travel grants , Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants, Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel grants , Research Funding; Novartis: Consultancy, Honoraria, Other: Travel grants , Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grants , Research Funding; GSK: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genentech: Consultancy, Honoraria, Other: Travel grants , Research Funding; Hoffmann-La Roche: Consultancy, Honoraria, Other: Travel grants , Research Funding; Amgen: Consultancy, Honoraria, Other: Travel grants, Research Funding; Genzyme: Consultancy, Honoraria, Other: Travel grants , Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel grants , Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants , Research Funding; Celgene: Consultancy, Honoraria, Other: Travel grants , Research Funding. Cragg:Roche: Consultancy, Research Funding; GSK: Research Funding; Bioinvent International: Consultancy, Research Funding; Baxalta: Consultancy; Gilead Sciences: Research Funding.