Molecular Genetics of High-risk Chronic Lymphocytic Leukemia

Davide Rossi; Gianluca Gaidano

Disclosures

Expert Rev Hematol. 2012;5(6):593-602. 

In This Article

NOTCH1 Mutations

The NOTCH1 gene encodes a class I transmembrane protein functioning as a ligand-activated transcription factor. It plays an important role in a number of cellular functions during embryogenesis and in self-renewing tissues of the adult organism, including maintenance of stem cells, cell fate specification, proliferation and apoptosis.[37] A prominent role of NOTCH1 in the adult organism is the regulation of lymphoid system development and function.[38] In early differentiation stages of the lymphoid system, NOTCH1 commits hematopoietic progenitors to differentiate toward the T lineage and, in addition, it prevents B-cell development.[39] Conversely, in mature B-lymphocytes, NOTCH1 signaling promotes terminal differentiation to antibody-secreting cells.[40]

NOTCH1 is an heterodimeric complex composed of an N-terminal extracellular subunit (NEC) and a C-terminal transmembrane and intracellular subunit (NTM) (Figure 3).[41] The NEC contains the epidermal growth factor-like repeats and the Lin12/NOTCH repeats. The epidermal growth factor-like repeats are required for ligand binding. The Lin12/NOTCH repeats stabilize the NOTCH1 heterodimerization domain, which consists of the C-terminus of NEC and of the N-terminus of NTM, to prevent the spontaneous activation of the receptor in the absence of ligand.[41] The NTM subunit contains a transmembrane region followed by a series of cytoplasmic domains, including a RAM domain, a series of ankyrin repeats, a transactivator domain, and several nuclear localization signals, which collectively function as a ligand-activated transcription factor. The NTM also contains a C-terminal PEST (proline [P], glutamic acid [E], serine [S], and threonine [T]) rich domain, which is responsible for the proteosomal degradation of activated NOTCH1 (Figure 3).[41]

Figure 3.

NOTCH1 pathway. NOTCH1 is an heterodimeric complex composed of an N-terminal extracellular subunit and a C-terminal transmembrane and intracellular subunit. The NEC subunit interacts with cognate ligands (δ-like and jagged). The NTM contains a C-terminal PEST-rich domain, which is responsible for the proteosomal degradation of activated NOTCH1 in the nucleus. When membrane-bound NOTCH1 receptors interact with cognate ligands on an adjacent cell, two consecutive proteolytic cleavages of the receptor are initiated. The S2 cleavage in the HD by the ADAM10 metalloprotease generates the substrate for the S3 cleavage by the γ-secretase complex. These proteolytic cleavages allow the intracellular domain of NOTCH1 to translocate to the nucleus, thus leading to transcriptional regulation of multiple target genes.
ER: Endoplasmic reticulum; ICN: Intracellular NOTCH1; MAML: Mastermind-like protein 1; NEC: N-terminal extracellular subunit; NTM: C-terminal transmembrane and intracellular subunit; PEST: Proline, glutamic acid, serine and threonine; RBPJ: Recombination signal binding protein for immunoglobulin-κ J region.

When membrane-bound NOTCH1 receptors interact with cognate ligands on an adjacent cell, two consecutive proteolytic cleavages of the receptor are initiated (Figure 3).[37,41] The S2 cleavage in the heterodimerization domain by the ADAM10 metalloprotease generates the substrate for the S3 cleavage by the γ-secretase complex. These proteolytic cleavages allow the NOTCH1 intracellular domain to translocate to the nucleus, thus leading to transcriptional regulation of multiple target genes, including MYC activation, TP53 suppression and deregulation of genes of the NF-κB pathway.[37,41]

The first evidence for the involvement of deregulated NOTCH signaling in cancer came from T-cell acute lymphoblastic leukemia (T-ALL), in which activating NOTCH1 mutations represent the main oncogenic lesion in approximately 50% of cases.[37,41,42] Two major hotspots of mutations have been identified in T-ALL: mutations in the NOTCH1 heterodimerization domain that induce spontaneous and ligand-independent activation of the signaling and mutations in the PEST carboxy-terminal domain that increases stability of the activated intracellular NOTCH1.[37,41,42]

Recent studies have identified NOTCH1 mutations as a recurrent molecular lesion of CLL. NOTCH1 mutations recur in approximately 10% unselected newly diagnosed CLL, a prevalence that is consistent across several independent cohorts investigated for this lesion (Figure 1B).[10–12,14–16]NOTCH1 is a recurrent target of genetic alterations in specific groups of CLL patients. In fact, NOTCH1 mutations are significantly more common in the subgroup of CLL with unmutated (rather than mutated) IGHV genes and are significantly enriched in CLL harboring +12 as the sole cytogenetic abnormality.[10–12,14–16,43–45] By contrast, NOTCH1 mutations are uncommon in CLL cases in which +12 is associated with other chromosomal aberrations.[44,45] Consistently, up to 40–50% of CLL with isolated +12 and unmutated IGHV genes are characterized by NOTCH1 mutations.[43]

At variance with T-ALL, NOTCH1 mutations in CLL are almost exclusively frameshift or nonsense events, clustering within a hotspot in exon 34, and commonly represented by one single 2-base pair (bp) deletion (c.7544_7545delCT) that accounts for approximately 80–95% of all NOTCH1 mutations in this leukemia.[10–12,14–16,43–45] By taking advantage of this mutational spectrum, PCR-based strategies not requiring DNA sequencing have been designed for the rapid detection of the c.7544_7545delCT mutation for diagnostic and prognostic purposes.[16]

The predicted functional consequence of NOTCH1 mutations in CLL is the disruption of the C-terminal PEST domain of the NOTCH1 protein. Removal of the PEST domain results in NOTCH1 impaired degradation and accumulation of an active NOTCH1 isoform sustaining deregulated signaling.[11] Consistent with this notion, a number of cellular pathways are specifically deregulated in CLL harboring NOTCH1 mutations.[11,44] The functional relevance of NOTCH1 mutations in the pathobiology of CLL is further suggested by the observation that constitutively active NOTCH1 signaling in CLL confers resistance to apoptosis through downstream activating effects on the NF-κB pathway.[46] NOTCH1 constitutive activation in CLL may rely on paracrine signals provided by specific interactions between protective microenvironmental niches and leukemic cells, as well as on autocrine loops sustained by the constitutive overexpression of NOTCH1 ligands on CLL cells. By impairing the degradation of the intracellular domain of the protein, NOTCH1 stabilizing mutations conceivably prevent a regulated off switch for NOTCH signaling, thus boosting a growth-promoting effect that is initially activated by microenvironmental interactions.

NOTCH1 is preferentially targeted in specific phases of CLL (Figures 1B & 2). The prevalence of NOTCH1 mutations increases with disease aggressiveness, being exceptional (~3%) in monoclonal B-cell lymphocytosis, a premalignant entity that often precedes CLL; rare in unselected CLL at diagnosis (10%); and frequent in relapsed and fludarabine-refractory cases (20%) and in patients who have transformed to Richter syndrome cases (30%).[10–12,14–16,47] The observation that NOTCH1 mutations accumulate in the more advanced phases of the disease suggests that they represent second-step genetic lesions progressively selected or acquired during the evolution of the clone. Consistent with this notion is the observation that clonally represented NOTCH1 mutations identified in the Richter syndrome phase are already present years before, although at a very subclonal level.[10] Initial evidence suggests that the subclones harboring the mutation are progressively selected during the CLL clinical history ending in Richter transformation.[10]

Beside their pathogenetic role, NOTCH1 mutations may also represent a new biomarker for the identification of poor risk CLL patients. NOTCH1-mutated patients have a rapidly progressive disease and a significantly shorter survival probability (~30% at 10 years) compared with NOTCH1-wild-type cases.[10,11,16] The poor prognosis associated with NOTCH1 mutations in CLL may be explained, at least in part, by a substantial risk (~50% at 15 years) of developing Richter syndrome that represents the emergence of an aggressive lymphoma in the context of CLL.[48] Within the spectrum of the various clinical phases of CLL, Richter syndrome is the most aggressive clinical phenotype because of the combined effect of chemoresistance and rapid disease kinetics. The clinical behavior of Richter syndrome is strongly related to its genetic background. The high rate of TP53 abnormalities, which occur in approximately 60% of cases, accounts for the chemoresistance profile that is very common in Richter syndrome.[24]NOTCH1 mutations and MYC abnormalities, including MYC translocations and amplifications, are the second most frequent genetic lesion in Richter syndrome, in which they occur in approximately 30% of cases.[10,24]NOTCH1 mutations are largely mutually exclusive with MYC oncogenic activation in Richter syndrome.[10] This finding is consistent with the observation that NOTCH1 directly stimulates MYC transcription and suggests that activation of oncogenic MYC may be one common final pathway selected for tumorigenesis in approximately 60% of cases of clonally related Richter syndrome.[10,37,41] In turn, MYC may account for the rapid disease kinetics observed in this very aggressive disease.

NOTCH1 mutations provide a new therapeutic target in CLL. It has been reported that treatment with γ-secretase inhibitors, by inhibiting the proteolytic system responsible for the processing and activation of NOTCH1 receptors, induces apoptosis in CLL cells.[41,42,49] However, the limitations due to toxicity of γ-secretase inhibitors in the clinical setting suggest that alternative strategies may be needed for the therapeutic targeting of NOTCH1.[41,42] A number of compounds directed against the NOTCH pathway are under preclinical development, including metalloproteinase inhibitors blocking the proteolytic process at a different position, antibodies directed against the extracellular domains of NOTCH1 that block its interaction with the ligands by capping the receptor, and antagonists that act in the nucleus by directly targeting the NOTCH transactivation complex.[49]

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