NOTCH1 Stabilization By PEST Mutations Enhances IgM-Mediated Activity in Chronic Lymphocytic Leukemia
Authors: Arruga Francesca , Bracciamà Valeria , Yeomans Alison , D'Avola Annalisa , Coscia Marta , D'Arena Giovanni Francesco , Gaidano Gianluca , Allan John N. , Furman Richard R. , Packham Graham , Deaglio Silvia , Forconi Francesco 
Published: October 2019

Abstract
BACKGROUND. Mutations in NOTCH1 PEST domain (NOTCH1-M) are present in ~10% of Chronic Lymphocytic Leukemia (CLL) patients, result in accumulation of more stable NOTCH1 protein, and associate with poorer prognosis. NOTCH1-M are enriched in unmutated (U) immunoglobulin gene heavy-chain variable region (IGHV) CLL, which show high surface IgM (sIgM) expression and signaling capacity. mRNA translation is a prominent response to B cell receptor (BCR) engagement, increased in U-CLL, and for which therapeutic inhibitors are under active development. In CLL, c-MYC is an essential mediator of BCR-driven translation and direct target of NOTCH1, suggesting the impact of NOTCH1 on anti-IgM-mediated cell growth via MYC.
AIMS AND METHODS. Our aim was to investigate the functional role of NOTCH1-M on anti-IgM-mediated signaling, compared to wild-type (WT) NOTCH1. The impact on global mRNA translation was studied using a flow cytometry-based O-propargyl-puromycin (OPP) incorporation assay and polysome fractionation assays. The effects of stabilized vs WT NOTCH1 were measured after 24-hour cultures of CLL cells, when data demonstrate differences in the expression of the two forms. Two cohorts of U-CLLs were compared: i) a subset of samples carrying NOTCH1-M [variant allele frequency (VAF) ≥30%, n=21] and ii) a cohort of samples with WT NOTCH1 (VAF<1%, n=23). In both subsets no additional cytogenetic lesions other than 13q deletion were present.
RESULTS. sIgM levels and signaling capacity (measured by anti-IgM mediated iCa2+ mobilization) were higher in NOTCH1-M than in -WT samples, consistent with previous observations (1). Conceivably, anti-IgM-mediated phosphorylation of PLCg2 and ERK1/2 was stronger in M than in WT CLLs. In keeping with these results, expression of downstream targets as MYC and CCL3 was also induced at higher levels in M samples. Interestingly, inhibition of NOTCH1 with g-secretase inhibitor (DAPT) significantly decreased BCR target genes induction in M cells, reducing the differences with WT samples, and further enhanced the effects of ibrutinib when used in combination. In order to investigate the impact of NOTCH1 on IgM-mediated CLL cell growth, anti-IgM-induced global mRNA translation was compared in the two cohorts. Consistent with the higher MYC mRNA and protein levels, anti-IgM led to higher global mRNA translation in NOTCH1-M than in -WT cells. DAPT inhibited it in both CLL subsets, while ibrutinib led to complete inhibition of mRNA translation only in the -WT subset, suggesting a major contribution of NOTCH1 to the process. Consistently, the combination of DAPT+ibrutinib abrogated the difference between M and WT CLL cells.
Importantly, MYC (but not translation initiation factors eIF4G, eIF4A or eIF3b) was already induced at 6 hours following anti-IgM stimulation and was maintained at high levels at 24 hours, while up-regulation of eIF4G, eIF4A and eIF3b was evident only at 24 hours, supporting the hypothesis of a direct MYC-dependent regulation of the translation machinery (2).
NOTCH1 itself was post-transcriptionally regulated upon BCR ligation, as we observed increased NOTCH1 mRNA in polysome-enriched actively translated fractions and increased protein levels on the surface of anti-IgM stimulated cells, specifically inhibited by ibrutinib. Consequently, NOTCH1 pathway was significantly more activated upon anti-IgM stimulation in M than WT cells, as determined by qPCR of NOTCH1 target genes. Both Ibrutinib and DAPT significantly prevented NOTCH1 activation upon BCR triggering, with the drug combination being the most effective treatment. Moreover, in line with data showing NOTCH1-dependent regulation of a B cell gene signature, expression of BTK, LYN and BLNK was significantly increased in anti-IgM activated NOTCH1-M samples, an effect prevented by DAPT.
CONCLUSIONS. These data indicate that NOTCH1 stabilization associates with stronger IgM signaling capacity and suggest an interplay between BCR and NOTCH1 pathway, with the former promoting NOTCH1 expression and activation. The evidence that NOTCH1 pathway inhibition reverts this difference suggests a direct effect of NOTCH1 on IgM signaling. In this scenario, stabilizing NOTCH1 mutations may enhance BCR signaling by boosting translation through MYC induction and by directly regulating expression of BCR cascade elements.
NOTES. SD and FF share senior authorshipD'Avola, Blood 2016Ruggero, Cancer Res 2009

Disclosures
Coscia: Abbvie, Gilead, Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen, Karyopharm: Research Funding. Gaidano:Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Morphosys: Honoraria; Amgen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Allan:Genentech: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Sunesis: Membership on an entity's Board of Directors or advisory committees; Acerta: Consultancy; Verastem: Membership on an entity's Board of Directors or advisory committees. Furman:Gilead: Consultancy; AbbVie: Consultancy; Verastem: Consultancy; Janssen: Consultancy; Genentech: Consultancy; Incyte: Consultancy, Other: DSMB; Loxo Oncology: Consultancy; TG Therapeutics: Consultancy; Sunesis: Consultancy; Acerta: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy. Packham:Aquinox: Research Funding. Deaglio:iTeos therapeutics: Research Funding; VelosBio inc: Research Funding; Verastem: Research Funding. Forconi:Abbvie: Consultancy; Janssen-Cilag: Consultancy.