NOTCH1 Mutated IGHV Unmutated Chronic Lymphocytic Leukemia Cells Are Characterized By a Constitutive Overexpression of Nucleophosmin-1 and Ribosome-Associated Components

Background: stabilizing 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), immuno-chemorefractory or advanced disease phase CLL, and have been associated with particularly unfavourable prognosis (Rossi et al, Blood, 2012; Del Poeta et al, Br J Haematol, 2013; Stingelbauer et al, Blood, 2014). In CLL, all NOTCH1 mutations disrupt the C-terminal PEST domain (about 80% of which are a 7544-7545delCT frameshift deletion) and cause an accumulation of an active NOTCH1 isoform.

Aim: to identify molecular/biological features of NOTCH1 mutated CLL.

Methods: the presence of NOTCH1 mutations was investigated by ARMS-PCR or by direct sequencing. The percentage of NOTCH1 mutated DNA in the context of the CLL clone was determined by quantitative real-time PCR (QRT-PCR) and/or by next-generation-sequencing. Gene expression profile (GEP) was performed by a one-color labeling strategy using the 4x44K platform. Specific gene/protein validations were performed by QRT-PCR, western blotting, flow cytometry and immunofluorescence. Cell proliferation was evaluated by Cell Trace Violet assay. CLL-like cell line MEC-1 was transfected with a vector either with a NOTCH1 intracellular domain (NICD) with 7544-7545delCT or with a NICD carrying a missense mutation (c.5304G>A), generating a stop codon at the beginning of the sequence, as control. Results:i) in a cohort of 588 IGHV-UM CLL, 144/588 (24.5%) cases carried NOTCH1 mutations (NOTCH1-mut cases), with a percentage of NOTCH1 mutated DNA ranging from 1% to 37%. NOTCH1-mut cases (8 cases; 11%-37% of NOTCH1 mutated DNA) showed higher NOTCH1 protein expression than NOTCH1 wild type cases (NOTCH1-wt, 11 cases) employing monoclonal antibodies either recognizing the trans-membrane (mean fold-change=3.0) or the intracellular (mean fold-change=2.1) NOTCH1 domain. ii) A GEP comparing purified cells of 5 IGHV-UM NOTCH1-mut cases (15%-37% of NOTCH1 mutated DNA) and 5 IGHV-UM NOTCH1-wt cases selected nucleophosmin-1 (NPM1) and genes codifying for several ribosomal proteins (RPS6, RPS10, RPS17, RPS28, RPSA, RPL7A, RPL18) as significantly up regulated in NOTCH1-mut CLL. A higher expression of NPM1 and of the genes codifying for 4 ribosomal proteins in NOTCH1-mut cases was validated in a wider independent series of 34 cases by QRT-PCR (18 NOTCH1-mut cases).iii) Western blot in 19 cases (8 NOTCH1-mut cases) confirmed a higher NPM1 protein expression in NOTCH1-mut cases (1.3-5.2 range of fold-change) also at protein level. When intracellular distribution and fluorescent intensity of NPM1 immunostaining were evaluated, an additional cytoplasmic staining was visible in NOTCH1-mut cases, along with a clear nucleolar staining visible in both NOTCH1-mut and NOTCH1-wt cases. NPM1 protein expression was also determined by an intra-nuclear staining by flow cytometry. By using this approach, 2 NOTCH1-mut cases were sorted according to NPM1 expression; notably, the NPM1^high subpopulation showed a higher NOTCH1 mutational load than the NPM1^low subpopulation. iv) MEC-1 cells transfected with mutated NICD showed higher NOTCH1 transcript and protein levels than MEC-1 cells transfected with the control vector (increments over the control: NICD transcript =2.2, NICD protein =1.3). These NICD mutated MEC-1 cells were characterized by higher NPM1 transcript and protein levels than the MEC-1 cells transfected with the control vector (increments over the control: NPM1 transcript =2.0, NPM1 protein =1.5). By performing a cell-trace-violet proliferation assay, NICD mutated MEC-1 cells were characterized by higher proliferation rates than MEC-1 cells transfected with the control vector (p=0.018). Finally, when the transfected MEC-1 cells were treated with 0.5 microM etoposide for 48 hours, NICD mutated MEC-1 cells presented higher viability rates than the MEC-1 cells transfected with the control vector, as evaluated by Annexin V/7-AAD staining (percentage of viable cells: 70% vs. 50%).

Conclusions: NPM1 was constitutively overexpressed in NOTCH1-mut IGHV-UM CLL together with several ribosome-associated components. An increased activity of the ribosomal machinery/DNA-repair mechanisms (Lindstrom MS. Biochem.Res.Int. 2011) in NOTCH1-mut CLL can confer proliferative advantages and resistance to chemotherapeutic agents.