The NOTCH Pathway and Its Mutations in Mature B Cell Malignancies

Abstract

The systematic application of next-generation sequencing to large cohorts of oncologic samples has opened a Pandora's box full of known and novel genetic lesions implicated in different steps of cancer development and progression. Narrowing down to B cell malignancies, many previously unrecognized genes emerged as recurrently mutated. The challenge now is to determine how the mutation in a given gene affects the biology of the disease, paving the way to functional genomics studies. Mutations in NOTCH family members are shared by several disorders of the B series, even if with variable frequencies and mutational patterns. In silico predictions, revealed that mutations occurring in NOTCH receptors, despite being qualitatively different, may have similar effects on protein processing, ultimately leading to enhanced pathway activation. The discovery of mutations occurring also in downstream players, either potentiating positive signals or compromising negative regulators, indicates that multiple mechanisms in neoplastic B cells concur to activate NOTCH pathway. These findings are supported by results obtained in chronic lymphocytic leukemia and splenic marginal zone B cell lymphoma where deregulation of NOTCH signaling has been functionally characterized. The emerging picture confirms that NOTCH signaling is finely tuned in cell- and microenvironment-dependent ways. In B cell malignancies, it contributes to the regulation of proliferation, survival and migration. However, deeper biological studies are needed to pinpoint the contribution of NOTCH in the hierarchy of events driving B cells transformation, keeping in mind its role in normal B cells development. Because of its relevance in leukemia and lymphoma biology, the NOTCH pathway might represent an appealing therapeutic target: the next few years will tell whether this potential will be fulfilled.

Keywords: B cell development; NOTCH; gene mutations; mature B cell malignancies; signaling.

Figure 1

NOTCH receptors and ligands. NOTCH receptors are structurally conserved type I proteins. There are four mammalian NOTCH receptors (NOTCH1-4) that contain multiple extracellular epidermal growth factor (EGF) repeats (from 29 to 36). Specific EGF repeats mediate ligand interactions. EGF repeats are followed by the negative regulatory region (NRR), which is composed of three cysteine-rich Lin repeats (LNR) and a heterodimerization domain (HD). NOTCH also contains a transmembrane domain (TM), an RBPJk associated module (RAM) domain, a nuclear localization sequences (NLS), a seven ankyrin repeats (ANK) domain, a NOTCH cytokine response (NCR) region, a transactivation domain (TAD) and a proline-glutamic acid-serine-threonin rich (PEST) domain. Mammalian NOTCH proteins are cleaved by furin-type convertases, which convert the NOTCH polypeptide into a NOTCH extracellular domain (NECD) and NOTCH intracellular domain (NICD) heterodimer that is connected by non-covalent interactions. After ligand binding, NOTCH is cleaved by metalloproteases and γ-secretase (S1, S2, and S3). NOTCH ligands can be divided into Jagged (Jagged 1 and Jagged 2) and Delta-like (DLL1, DLL3, and DLL4) groups on the basis of their domain composition. The extracellular domain of ligands is characterized by a NOTCH ligand N-terminal domain (MNNL), a Delta/Serrate/LAG-2 (DSL) domain, EGF motifs and a cysteine-rich (Cys) domain. DLLs lack this latter domain. The intracellular portion may contain a post-synaptic density protein ligand domain (PDZL).

Figure 2

NOTCH signaling pathway. Ligand expressed on the surface of the signal-sending cell binds to NOTCH expressed on the surface of the signal-receiving cell and induces sequential cleavages by A-Disintengrin-And-Metalloprotease (ADAM) and γ-secretase, ultimately releasing the NOTCH intracellular domain (NICD) from the membrane. NICD translocates to the nucleus where it mediates the displacement of co-repressors (CoR) and Histone DeAcetylase Complex (HDAC) and directly interacts with RBPJk recruiting co-activators (CoAct), finally inducing target gene transcription. The signaling pathway is shut down by phosphorylation of the NICD subunit by C/Cyclin-dependent kinase 8 (CDK8) and subsequently poly-ubiquitinated by F-box containing protein (FBW7) and degraded via proteasome. The strength and outcome of receptor-ligand binding can be modulated by post-translational modifications of NOTCH receptors, operated by the Fringe family of enzymes.

Figure 3

NOTCH signaling in B cell development. In bone marrow-residing CLPs, NOTCH1 signaling must be switched off to allow proper B cell development. On the contrary, after migration of immature B cells to the spleen, interaction of DLL1 with NOTCH2, with the involvement of recombinant signal binding protein for immunoglobulin kJ region (RBPJk), induces NOTCH signaling in transitional B (T2) cells to specify marginal zone B cells, as opposed to follicular B cells. NOTCH2 signaling is also necessary for CLPs to differentiate toward B1 cells.

Figure 4

NOTCH1 transcriptional effects in CLL cells. NOTCH1 signaling is aberrantly activated in CLL cells, resulting in transcriptional regulation of several genes, in turn impacting different biological aspects of the neoplastic cells. Increased NOTCH1 activity contributes to leukemic cell growth, protection from apoptosis, metabolic switching toward a glycolytic metabolism, increased migration in response to specific chemokines and facilitates interactions with microenvironment. Moreover, it upregulates several genes encoding proteins insisting on the BCR signaling pathway, the driving force for CLL cells.

Figure 5

Mature B cell malignancies classification according to their normal counterpart and overview of NOTCH implications in these disorders. After antigen contact, naïve B cells are activated and enter the germinal centers. Maturation through a T cell dependent process leads to centroblast differentiation in the dark zone with high proliferation rate and somatic mutations of immunoglobulin variable regions and continue their maturation through the light zone. After that, they can become memory B cells or terminally differentiated plasmacells. On the contrary, transitional B cells are selected into a mature B cell repertoire in a T cell-independent way through the marginal zone of the spleen. B cell malignancies can arise from B lymphocytes at each maturation step. NOTCH family members have been identified as recurrently mutated in some of these disorders or to be over-expressed, ultimately resulting in an aberrant activation of the pathway.UM-CLL, IGVH unmutated chronic lymphocytic leukemia; M-CLL, mutated chronic lymphocytic leukemia; FL, follicular lymphoma; HL, Hodgkin's lymphoma; BL, Burkitt's lymphoma; DLBCL, diffuse large B cell lymphoma; MCL, mantle cell lymphoma; MZL, marginal zone lymphoma; MM, multiple myeloma; GC, germinal center.