Notch1 Signaling in NOTCH1-Mutated Mantle Cell Lymphoma Depends on DLL4 and Is a Potential Target for Specific Antibody Therapy

Mantle cell lymphoma (MCL) is a rare B-cell-neoplasm with an aggressive clinical course characterized by the hallmark translocation t(11;14)(q13;q32) resulting in overexpression of cyclin D1. Furthermore, genomic profiling revealed numerous secondary genetic alterations and recurrent mutations contributing to MCL pathogenesis. Among them, mutations in the NOTCH1 gene have been described with a frequency of ~10% and are associated with significantly shorter survival rates. Therefore, further investigation of the biological impact of this mutation in MCL and its potential as a target for a specific inhibitory antibody therapy is of great interest. Here we investigated the role of the Notch receptor ligands Jagged1 (JAG1), Jagged2 (JAG2), Delta-like canonical Notch ligand 1 (DLL1) and Delta-like canonical Notch ligand 4 (DLL4) in activating the Notch1-signaling pathway in NOTCH1 -mutated Mino and NOTCH1 -wt Jeko1 MCL cell lines and evaluated the effects of the novel and specific monoclonal Notch1-antibody OMP-52M51. Mino cells are characterized by a single nucleotide substitution in exon 34 of the NOTCH1 gene, leading to truncation of the C-terminal PEST-domain. These mutations are known to remove the recognition site from the ubiquitin ligase degradation complex, resulting in increased stability and transcriptional activity of Notch1 upon ligand-stimulation. We confirmed the predicted truncation of Notch1 in Mino cell lysates and demonstrated that the expression of cleaved Notch1 was potently stimulated by recombinant DLL4 protein. In contrast, in the NOTCH1- wt cell line Jeko1, no stable overexpression of cleaved Notch1 could be observed upon any ligand-stimulation. Treatment of the NOTCH1 -mutated cells with OMP-52M51 effectively prevented DLL4-dependent activation of Notch1 and suppressed transcriptional induction of well-known Notch1-target genes HES1 ( hairy and enhancer of-split 1 ), DTX1 ( deltex E3 ubiquitin ligase 1 ) and c-MYC as observed by real-time quantitative PCR. Gene expression profiling furthermore revealed that OMP-52M51 significantly impeded DLL4-induced upregulation of numerous genes involved in lymphoid biology and lymphomagenesis, as well as genes related to the cell cycle and the MAP-Kinase signaling pathway. Western blot analysis confirmed Notch1-antibody-mediated abrogation of enhanced expression of p-ERK and p-MEK upon DLL4-ligand-stimulation. Further investigations of functional effects revealed inhibitory effects of OMP-52M51 on enhanced tumor cell migration of DLL4-stimulated cells. As the DLL4-Notch-axis is known to be involved in regulating angiogenesis, we investigated the impact of activating NOTCH1 -mutations in MCL on HUVEC tube formation and observed increased vessel sprouting upon DLL4-stimulation that could be abolished by treatment with OMP-52M51. Importantly, all these effects were specific for NOTCH1 -mutated cells and did not occur in the NOTCH1 -wt cell line Jeko1. In conclusion, we demonstrate for the first time that DLL4 is the most important ligand to activate Notch1 in MCL harbouring NOTCH1 -mutations in the PEST-domain. Our findings indicate that enhanced Notch1 activity promotes a more aggressive behavior of the disease and specific antibody-inhibition of the Notch1-signaling pathway might provide an interesting therapeutic alternative for a subset of MCL patients, warranting further investigation. Disclosures No relevant conflicts of interest to declare.