Nucleophosmin-1 and Ribosome-Associated Components Are Constitutively Overexpressed in NOTCH1 Mutated IGHV Unmutated CLL
Authors: Dal Bo Michele , Pozzo Federico , Bomben Riccardo , Zucchetto Antonella , Tissino Erika , Benedetti Dania , Degan Massimo , Rossi Francesca Maria , Bulian Pietro , D'Agaro Tiziana , Dereani Sara , Rossi Davide , Zamò Alberto , Zaja Francesco , Pozzato Gabriele , Di Raimondo Francesco , Gaidano Gianluca , Del Poeta Giovanni , Gattei Valter 
Published: October 2019

Abstract
Abstract 3880

Background:
activating mutations of NOTCH1 have been identified in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, with a higher frequency in unmutated IGHV (IGHV-UM) CLL, chemorefractory CLL and CLL in advanced disease phases. In CLL, all NOTCH1 mutations disrupt the C-terminal PEST domain and cause an accumulation of an active NOTCH1 isoform. Notably, about 80% of NOTCH1 mutations are represented by a CT frameshift deletion at nucleotides 7544–7545 (c.7544–7545delCT). Clinically, the presence of NOTCH1 mutations is an independent predictor of overall survival in CLL and identifies a subset of patients with particularly unfavourable prognosis (Rossi et al, Blood, 119, 521, 2012).

Aim:
to identify peculiar molecular and biological features of NOTCH1 mutated CLL in the context of IGHV-UM CLL.

Methods:
the presence of the c.7544–7545delCT NOTCH1 frameshift deletion was investigated by an ad-hoc amplification refractory mutation system (ARMS) PCR set up to obtain an amplicon specific for the NOTCH1 mutated form and a second amplicon as control. The percentage of NOTCH1 DNA in the context of the CLL clone was determined by quantitative real-time PCR (QRT-PCR), calculating the ratio between the amount of the specific NOTCH1 mutated amplicon and the amount of the control amplicon, the latter representing the total amount of NOTCH1 DNA irrespective of its mutational status. Gene expression profile (GEP) was performed by a one-color labeling strategy using the 4×44K Agilent platform. The differential expression of specific genes/proteins was validated by QRT-PCR, western blotting and immunohistochemistry. A BrdU uptake assay was used to evaluate proliferation of CLL cells by CpG/IL2 stimulation.

Results:
in a cohort of 380 IGHV-UM CLL, the c.7544–7545delCT NOTCH1 mutation was found in 83/380 (21.8%) cases. QRT-PCR revealed a percentage of NOTCH1 mutated DNA ranging from 1 to 37%. CLL cases carrying the c.7544–7545delCT NOTCH1 mutation (NOTCH1-Mut) showed higher NOTCH1 protein expression than CLL cases lacking NOTCH1-Mut employing monoclonal antibodies either recognizing the trans-membrane (mean fold increase=3) or the intra-citoplasmic (mean fold increase=2.1) NOTCH1 domain. A GEP comparing RNA from purified CLL samples of 5 NOTCH1-Mut CLL and 5 CLL lacking NOTCH1-Mut was performed, selecting the 5 NOTCH1-Mut cases among those with the higher percentages of NOTCH1 mutated DNA (percentages of NOTCH1 mutated DNA ranging from 15 to 37%). This approach selected the nucleophosmin 1 gene (NPM1) and genes codifying for several ribosomal proteins (RPS6, RPS10, RPS17, RPS28, RPSA, RPL7A, RPL18) as significantly up regulated in NOTCH1-Mut CLL cases. A higher expression of the above mentioned genes in NOTCH1-Mut CLL was validated in a wider series of 34 cases (18 NOTCH1-Mut cases; NPM1, p=0.03; RPS6, p=0.045; RPS10, p=0.048; RPS17, p=0.048; RPS28, p=0.049; RPSA, p=0.048; RPL7A, p=0.039; RPL18, p=0.041, respectively). Western blot analysis in 8 cases (4 NOTCH1-Mut cases) confirmed a higher NPM1 expression in NOTCH1-Mut cases (range of fold increase from 1.6 to 5.2) also at protein level. Consistently, lymph nodes preparations from NOTCH1-Mut CLL cases revealed a strong NPM1 staining both in nucleoli and cytoplasms. Finally, when stimulated in-vitro with the CpG/IL2 combination, NOTCH1-mut IGHV-UM CLL cells proliferated, as detected by a BrdU uptake assay (>10 fold increase over control), and up-regulated NPM1 both at transcript (mean fold increase=2.02 after 18 hours of CpG exposure, p=0.001) and protein (fold increase of 1.34 after 6 hours of CpG exposure) levels.

Conclusion:
NPM1 was identified as constitutively overexpressed in NOTCH1-Mut IGHV-UM CLL together with several ribosome-associated components. These findings are suggestive for an increased activity of the ribosomal machinery in NOTCH1-Mut IGHV-UM CLL as part of the molecular processes leading to control of CLL cell growth and survival in this clinically unfavourable disease subset.

Disclosures:
No relevant conflicts of interest to declare.