Early work on Notch signaling in B lineage cells focused on its antagonism of B cell development. Strong gain-of-function NOTCH1
alleles skews the differentiation of hematopoietic progenitors towards T cell fate and away from B cell fate [4
], and leads to growth arrest or apoptosis of several B cell neoplasms [5
]. By contrast, notch2 is essential for murine splenic marginal zone B cell development [6
], and notch1 has been implicated in B cell activation [7
] and Ig secretion [8
]. The possible ability of Notch to antagonize early steps in B cell development yet act positively at subsequent stages is reminiscent of the complex role of Notch in tissues such as the peripheral nervous system and the eye of Drosophila
that arise through successive hierarchical cell fate decisions. It is thus possible that Notch may have additional uncharacterized roles in the development or function of various B cell subtypes.
Chronic lymphocytic leukemia (CLL) is an indolent but incurable neoplasm with a gene expression signature resembling that of normal memory B cells [9
]. Several past reports have suggested a role for Notch signaling in CLL, but genetic evidence of Notch involvement was lacking. This changed in 2009, when focused resequencing of 43 cases of CLL by Falzetti’s group identified 2 cases with NOTCH1
exon 34 mutations leading to PEST degron deletions [10
]. Subsequent study of 133 newly diagnosed CLL cases by the same group confirmed a low frequency of Notch1 PEST domain mutations (5.3%) [11
]. In 2011, two unbiased whole exome studies of large European CLL cohorts also identified Notch1 gain-of-function mutations [12
]. In combination, these studies detected Notch1 mutations in 56 of 561 (10.0%) of newly diagnosed tumors. Notch1 mutations were associated with a worse outcome in all three of these series, and in the series from the Gaidano group [13
] were associated with disease progression to large cell lymphoma and refractoriness to chemotherapy. Notch1 mutations were also frequently subclonal or undetectable at diagnosis and clonal in transformed tumors from the same patient, providing additional evidence of a link between Notch1 signaling and disease progression. Conversely, Notch1 mutations were identified in only 2 of 63 monoclonal CD5-positive B cell proliferations [14
], an early stage of CLL development. Several groups have recently also reported that Notch1 mutations are enriched in CLLs associated with trisomy 12 [15
], a common cytogenetic aberration in CLL.
Subsequently, Gascoyne’s group found NOTCH1
gain-of-function mutations in approximately 12% of mantle cell lymphoma (MCL) [18
], an aggressive neoplasm usually derived from immunologically naïve mature B cells. As in CLL, most of these mutations lead to PEST degron deletions. Prior studies indicated that the NOTCH1
locus is frequently hypomethylated in MCL cells [19
], but otherwise there was little reason to suspect a role for Notch signaling in this disease. Beyond MCL, Gaidano’s group also identified several diffuse large B cell lymphomas with Notch1 PEST domain mutations [13
], and a recent report from Japan described a handful of diffuse large B cell lymphomas with Notch2 PEST domain mutations [20
]. It is thus increasingly evident that Notch signaling has an oncogenic role in a number of B cell malignancies.
Several features of Notch1 mutations in CLL and MCL differ from those in T-ALL (). Virtually all of the mutations are PEST deletions, and of these roughly 90% in CLL and >50% in MCL consist of a del(CT) mutation involving codon 2514. This contrasts sharply with human and murine T-ALL, in which PEST mutations occur across a wide region (). Furthermore, while the NRR is the most common site of mutations in human T-ALL, and >95% of CLL and MCL have wild type NRR sequences. The Notch1 mutations in CLL and MCL thus raise several questions: 1) why are del(CT) mutations so prevalent (relative to T-ALL)?; 2) how is ICN1 generated in the absence of NRR mutations?; and 3) do Notch1 mutations reflect some normal role for Notch1 in mature human B cells?
Figure 2 Distribution of Notch1 PEST domain mutations in lymphoid cancers. Positions of nonsense and frameshift mutations in human chronic lymphocytic leukemia (CLL) [11–13], human mantle cell lymphoma (MCL), and human [43–46] and murine T-cell (more ...)
Structure/function analyses suggest that the functional effect of the codon 2514del(CT) mutation is unlikely to differ from other PEST mutations, and indeed other Notch1 PEST mutations occur in CLL and MCL. A more likely possibility is that codon 2514 is embedded within a DNA context that is prone to spontaneous microdeletion. Of note, codon 2514 lies within the first of two 9 base pair direct tandem repeats (), which may make this sequence susceptible to “slippage” during DNA replication. If this is correct, codon 2514del(CT) mutations should be relatively common in all human cancers in which there is selective pressure for Notch1 gain-of-function. Consistent with this prediction, codon2514 del(CT) is the most common NOTCH1 mutation in human T-ALL as well. The idea that DNA sequence influences the distribution of PEST mutations is also supported by a comparison of human and murine T-ALL (). In contrast to human T-ALL, in murine T-ALL the most common PEST mutations are generated by insertions or deletions centered on codon 2361 (codon 2372 in human NOTCH1), which in the mouse (but not in the human) is framed by two direct 7 base pair tandem repeats that may promote illegitimate recombination events. Del(CT) mutations also occur in murine T-ALL, but at lower frequency than in human disease, possibly because of a single base substitution in the first of the two direct repeats ().
How ICN1 is generated in CLL and MCL is of biologic and therapeutic importance, since the mechanism will dictate the choice of targeting strategies. CLL cells proliferate in a specialized niche found mainly in lymph nodes that is defined by the presence of “nurse cells”, which are known to express membrane-bound factors such as BAFF that stimulate CLL growth and survival; one attractive possibility is that these cells also express Notch ligands. A second possibility is that the region encoding the Notch1 NRR may be disrupted by a mutation that might be missed by whole exome sequencing, such as a deletion or rearrangement; as mentioned, this type of mutation is common in murine T-ALL and also occurs in human breast cancer (described later). Finally, genetic studies in invertebrates have shown that aberrant Notch trafficking into late endosomes leads to ligand-independent receptor activation; the contribution of this mechanism to oncogenic Notch signaling in mammalian cancer is unknown.
It will also be of interest to compare and contrast the genes and pathways that are upregulated by Notch1 in B-cell tumors and T-ALL. Major oncogenic targets of Notch1 in T-ALL include MYC
, the PI3K/Akt pathway, and possibly the NF-kB pathway as well. Of possible relevance, EBNA2, an Epstein-Barr virus (EBV) protein that binds CSL and functionally resembles ICN, directly upregulates MYC
in EBV-transformed B cells [21
]. Expression profiling suggests that MYC
is also a direct target of Notch1 in MCL cell lines [18
], supporting the idea that Notch signaling promotes the proliferation of transformed B cells. Proliferation index predicts outcome in MCL [22
], bolstering the rationale for targeting Notch1 in patients with this disease.