A new genetic lesion in B‐CLL: a NOTCH1 PEST domain mutation

Abstract

We recently demonstrated that Notch1 and Notch2 constitutive activation plays a critical role in survival and apoptosis resistance of B-chronic lymphocytic leukemia (B-CLL) cells (Rosati et al, 2009). B-CLL cells also constitutively over-expressed Jagged1 and Jagged2 ligands, suggesting autocrine/paracrine loops for constitutive Notch signalling activation (Rosati et al, 2009). Notch activation mechanisms appear different in B-CLL and T-cell acute lymphoblastic leukemia (T-ALL) (Osborne, 2009). In B-CLL the altered generation of Notch1- and Notch2-intracellular (IC) domains, which is a crucial event for Notch signalling activation (Kopan, 2002), depends on ligand interactions (Rosati et al, 2009). In T-ALL, the aberrant Notch1-IC generation occurs in a ligand-independent fashion and is due to NOTCH1 activating mutations (Weng et al, 2004; O’Neil et al, 2006) which involve the heterodimerization (HD) domain, facilitating Notch1-IC proteolytic cleavage and/or the negative regulatory PEST domain, increasing Notch1-IC half-life (Monsour et al, 2006). To determine whether NOTCH1 activating mutations are present in B-CLL we studied 43 B-CLL patients (34 males/9 females, median age 59 years, range 44–79; median follow-up 84 months, range 12–204). ZAP70 expression and IGHV mutational status (Rassenti et al, 2008) were also determined (25 ZAP-70 positive; 10 ZAP-70 negative, 8 undetermined; 26 unmutated IGHV, 17 mutated IGHV) (Table I). Mutations of NOTCH1 exons 26, 27 and 34 were investigated by DNA-based polymerase chain reaction as described elsewhere (Weng et al, 2004) and frequencies were determined using the National Center for Biotechnology Information (NCBI) database. Cycling conditions were: 95°C for 5 min (one cycle); 95°C for 45 s, 58°C for 45 s, 72°C for 1 min for 36 cycles; 72°C for 6 min (one cycle). After direct sequencing from both strands, purified amplicons were compared with corresponding germline sequences, and germline polymorphisms in the NCBI SNP database were excluded. NOTCH1 sequencing did not detect mutations at the HD cleavage site. A PEST domain mutation i.e. 7544_7545 DEL (CT) was found in 2/43 patients (4.6%) (Fig 1). The mutation was confirmed by independent amplification of the amplicon. Chromatogram of NOTCH1 mutation in B-CLL as identified by direct sequencing. The arrow indicates the identified deletion (ΔCT). As suggested for T-ALL, deletions of the COOH-terminal sequences in the PEST domain, which regulates protein turnover by targeting proteins to the ubiquitin-proteosome complex for subsequent degradation, could enhance Notch-IC stability and signalling. Unlike the various mutations in T-ALL, the same NOTCH1 PEST domain mutation was found in these B-CLL patients. All had unmutated IGHV, were ZAP-70 positive and had a very poor prognosis. Both developed progressive disease from diagnosis, became resistant to more than three lines of chemotherapy and one died soon after the development of diffuse large B-cell lymphoma. In attempting to reconcile this first evidence of a NOTCH1 PEST mutation in a minority of B-CLL patients (4.6%), all with constitutively activated Notch1 and Notch2 (Rosati et al, 2009), one can only speculate that NOTCH1 mutations participate in Notch activation in B-CLL, at least in the subgroup carrying the mutation. They could be cooperating events and other genetic lesions may be involved. In fact, Notch2 plays a role in the development and maintenance of selected B cell subsets including marginal zone and B1 B cells (Witt et al, 2003) and was mutated in both HD and PEST domains in 5% of Marginal Zone Lymphoma cases (Troen et al, 2008). Additional studies are needed to clarify whether and how NOTCH mutations contribute to altered Notch signalling and oncogenesis in B-CLL. We would like to thank Dr Geraldine Anne Boyd for her help. This work was supported by ‘Associazione Umbra Leucemie e Linfomi’, Perugia Italy, by ‘Associazione Italiana Leucemie, Linfomi e Mielomi’, L’Aquila Section, L’Aquila, Italy and by ‘Fondazione Cassa di Risparmio di Perugia-2009’, Perugia, Italy.