Functional Effects Of NOTCH1 Mutations In Chronic Lymphocytic Leukemia Patients
Authors: Arruga Francesca , Gizdic Branimir , Serra Sara , Vaisitti Tiziana , Rossi Davide , Coscia Marta , Laurenti Luca , D'Arena Giovanni , Jaksic Ozren , Inghirami Giorgio , Gaidano Gianluca , Deaglio Silvia 
Published: October 2019

Abstract
Chronic lymphocytic leukemia (CLL), is characterized by the expansion of mature B lymphocytes present in blood, bone marrow and lymphoid organs. Clinical behavior is highly heterogeneous, thus requiring timely identification of high-risk patients.
NOTCH1 encodes a trans-membrane receptor acting as a ligand-activated transcription factor. NOTCH signaling initiates when the ligand, from either the Jagged or Delta families, binds to the receptor and induces successive proteolytic cleavages, resulting in the release and nuclear translocation of the NOTCH intra-cellular domain (NICD). Signaling is terminated by phosphorylation of the PEST domain of NOTCH1, triggering its ubiquitination and proteasomal degradation.
Whole exome sequencing approaches have revealed NOTCH1 mutations in 5-10% newly diagnosed CLL cases, with their prevalence increasing to 15-20% in progressive or relapsed patients. The most frequent mutation is 7544-7545delCT frameshift deletion in exon 34, resulting in disruption of the C-terminal PEST domain. Truncation of the PEST domain is predicted to result in NOTCH1 impaired degradation, stabilization of the active NOTCH1, and deregulated signaling.
The present study was undertaken with the aim to compare NOTCH1 expression and functional role in CLL patients harboring wild type (WT) or mutated (M) NOTCH1 gene.
NOCTH1 mRNA and surface protein were expressed at comparable high levels in peripheral blood (PB) CLL cells obtained from NOTCH1 M and WT patients, consistent with a more general requirement of NOTCH1 signaling in this leukemia. However, at a variance of NOTCH1 WT cases, NOCTH1 M patients displayed remarkable accumulation of both the intermediate molecular species of the activated NOCTH1 receptor, as well as of the active NICD. Consistently, by gene expression profiling NOCTH1 M patients displayed significantly higher levels of HES1 and DTX1, the main NOTCH1 target genes. Overall, these data suggest a more active signaling pathway in NOTCH1 M CLL than in NOTCH1 WT cases.
Expression of NOTCH1 and of its target gene (DTX1) varied across disease compartments, being higher in CLL cells obtained from the lymph nodes (LN), as compared to paired samples derived from the PB or the bone marrow (BM). By immunohistochemical analyses of primary LN tissue samples, NOTCH1 M CLL showed an intense nuclear staining as opposed to the more cytoplasmic distribution observed in NOTCH1 WT samples. These data suggest a more active NOTCH1 signaling in CLL residing in the LN microenvironment and confirm the functional effect of NOTCH1 mutations in vivo.
When PB CLL cells were cultured in vitro in the absence of any supporting layer or stimulation, they showed a rapid down regulation of the NOTCH pathway, with complete loss of NICD after 24 hours paralleled by a sharp decrease in HES1 and DTX1 transcription. Consistently, levels of presenilin-1 (PSEN1), the catalytic subunit of the g-secretase complex, were also down-regulated offering a partial mechanistic explanation for the NICD loss. NOTCH1 mRNA levels remained unchanged, with accumulation of the receptor at the plasma membrane. These effects were independent of NOTCH1 mutation status and suggested the dependence of NOTCH1 signaling activation upon in vivo microenvironmental interaction, even in NOTCH1 M CLL.
Within primary LN biopsies from CLL patients, the NOTCH1 ligand, was highly expressed on CD68+ elements of myeloid origin. This observation prompted the in vitro recreation of a lymphoid niche by co-culturing Jagged1+ nurse-like cells (NLC) with autologous CLL cells. Under these conditions, NOTCH1 activity in CLL cells was sustained over time, as shown by Q-PCR analyses of DTX1 and PSEN1. Moreover, NLCs protect NOTCH1 M CLL cells from fludarabine-induced apoptosis. This microenvironment-induced chemoresistance was prevented by pre-treatment of NOTCH1 M CLL cells with specific g-secretase inhibitors, to block NOTCH1 activation.
Taken together, these results show that the 7544-7545delCT mutation in the PEST domain of NOTCH1 has a stabilizing effect on NOTCH1 signaling pathway. They also show that micro-environmental interactions are critical in activating NOTCH1 pathway both in the M and WT patients. Lastly, these results show that NOTCH1 signals micht create local conditions that favour drug resistance, thus making NOTCH1 a potential molecular target in CLL.

Disclosures:
No relevant conflicts of interest to declare.