NOTCH1 Activation Negatively Impacts on Chronic Lymphocytic Leukemia Outcome and Is Not Correlated to the NOTCH1 and IGHV Mutational Status

Abstract

NOTCH1 mutations and deregulated signal have been commonly found in chronic lymphocytic leukemia (CLL) patients. Whereas the impact of NOTCH1 mutations on clinical course of CLL has been widely studied, the prognostic role of NOTCH1 activation in CLL remains to be defined. Here, we analyzed the activation of NOTCH1/NOTCH2 (ICN1/ICN2) and the expression of JAGGED1 (JAG1) in 163 CLL patients and evaluated their impact on TTFT (Time To First Treatment) and OS (Overall Survival). NOTCH1 activation (ICN1+) was found in 120/163 (73.6%) patients. Among them, 63 (52.5%) were NOTCH1 mutated (ICN1+/mutated) and 57 (47.5%) were NOTCH1 wild type (ICN1+/WT). ICN1+ patients had a significant reduction of TTFT compared to ICN1-negative (ICN1-). In the absence of NOTCH1 mutations, we found that the ICN1+/WT group had a significantly reduced TTFT compared to ICN1- patients. The analysis of IGHV mutational status showed that the distribution of the mutated/unmutated IGHV pattern was similar in ICN1+/WT and ICN1- patients. Additionally, TTFT was significantly reduced in ICN1+/ICN2+ and ICN1+/JAG1+ patients compared to ICN1-/ICN2- and ICN1-/JAG1- groups. Our data revealed for the first time that NOTCH1 activation is a negative prognosticator in CLL and is not correlated to NOTCH1 and IGHV mutational status. Activation of NOTCH2 and JAGGED1 expression might also influence clinical outcomes in this group, indicating the need for further dedicated studies. The evaluation of different NOTCH network components might represent a new approach to refine CLL risk stratification.

Keywords: IGHV mutation; NOTCH1 activation; NOTCH1 mutation; chronic lymphocytic leukemia; risk stratification.

Figure 1

Analysis of NOTCH1 activation and mutations in CLL patients. (A) Western blot analysis of NOTCH1 activation in whole cell lysates from 163 primary CLL cells. Short and long exposure of NOTCH1 activation (ICN1−/+) in 24 representative NOTCH1 WT (ΔCT−) and NOTCH1 mutated (ΔCT+, with relative AFs) CLL cases. ICN1 Val 1744 (WT) indicates the ICN1 NOTCH1 WT band, whereas ICN1 Val 1744 (Mut) indicates the ICN1 NOTCH1 mutated band (the difference in molecular weight is due to the presence of the deletion of a portion of PEST1). The Molt4 cell line was used as positive control. Protein loading was assessed by reprobing the blots with an anti-GAPDH antibody. (B) Kaplan–Meier analysis was used to determine TTFT in NOTCH1 mutated patients (NOTCH1 MUT) (n = 70) compared to NOTCH1 WT patients (n = 93) (80 vs 131 months, p = 0.04). Kaplan–Meier analysis was used to determine the influence on TTFT of the NOTCH1 activation and mutation status. Time to first treatment analysis according to: (C) NOTCH1 activation status (ICN1−, n = 43 and ICN1+, n = 120); (D) NOTCH1 activation status (ICN1−, n = 43) and NOTCH1 mutational status: ICN1+/WT (n = 57) and ICN1+/Mut (n = 63); (E) NOTCH1 activation status (ICN1−, n = 43) and NOTCH1 mutational status: ICN1+/WT (n = 57), ICN1+/Clonal mutated (n = 16) and ICN1+/Subclonal mutated (n = 47). (F) Stacked bar graph shows the IGHV mutational status in CLL patients with ICN1− compared to ICN1+/WT (Fisher’s exact Test). ***p < 0.001; **p < 0.01; *p < 0.05; and n.s., not significant.

Figure 2

Analysis of NOTCH1, NOTCH2 activation and JAGGED1 expression in CLL patients. (A) Representative Western blot analysis of NOTCH2 activation and JAGGED1 expression in whole cell lysates from 130 primary CLL cells. Protein loading was assessed by reprobing the blots with an anti-GAPDH antibody. Kaplan–Meier analysis was used to determine the influence on TTFT of the NOTCH2 activation status and JAGGED1 expression in 125 CLL patients. Time to first treatment analysis according to: (B) activation status of NOTCH1 (n = 49) compared to NOTCH2 (n = 41) activation status in NOTCH1 WT patients; (C) NOTCH1 activation status (n = 50) compared to JAGGED1 expression (n = 40) in NOTCH1 WT patients. *p < 0.05; and n.s., not significant.