It is well established that NOTCH signaling pathways can promote or inhibit the differentiation of lymphoid progenitors and their progeny in a lineage-dependent manner (32
). Heightened expression of NOTCH1 in hematopoietic progenitors, for example, promotes T-cell differentiation while inhibiting B-cell development (33
). In addition, DLL1, NOTCH2, CSL, and MAML1 are required for the development of marginal zone but not of follicular B cells (34
) and activated NOTCH2 promotes the development of B1 B cells (37
). Finally, DLL1-NOTCH1 interactions were shown to promote the terminal differentiation of mature B cells to antibody-secreting plasma cells (24
Importantly, aberrant expression of various NOTCH signaling molecules has also been tied to several human and mouse lymphoid malignancies. The most clearly defined role for NOTCH as an oncogene is in human T-ALL in which NOTCH1 is activated by chromosomal translocations or, more frequently, by mutations that cause either ligand-independent activation of the receptor or increased stability. Notch1
is also a frequent target of retroviral integrations in mouse B-cell lymphomas (38
Studies of possible contributions of disordered NOTCH signaling to the pathogenesis of human MM are of particular relevance to the current study of mouse PCT. In this regard, several investigations have shown that JAG1, JAG2, NOTCH1, NOTCH2, NOTCH3, NOTCH4, MIB2, and LFNG are expressed by primary tumor cells of patients with MM and/or MM cell lines and may contribute to disease initiation or progression (39
). It should be noted that other conflicting studies showed that heightened expression of NOTCH induced growth inhibition of several MM cell lines (40
). However, the mechanisms contributing to growth inhibition differ between the reports. In one report (40
), growth inhibition was associated with reduced apoptosis, whereas in the second (43
), growth inhibition was associated with increased apoptosis. In a follow-up study by Nefedova and colleagues (30
), it was shown that inhibition of NOTCH signaling was associated with increased apoptosis, but effect on cell cycle progression was not examined.
In the present study, data from qPCR array analyses of primary mouse B-cell lineage tumors established that several genes involved in NOTCH signaling were expressed at significantly higher levels by PCT than by CBL or MZL++, including Myc, Dll3, Rfng, Ncstn, Psen2, Mib1, and Dtx2. Earlier studies that identified contributions of NOTCH signaling to terminal B-cell differentiation allowed us to predict that some of these differences would be reflective of normal plasma cell differentiation but also to anticipate that others may have contributed to tumor induction, progression, or maintenance. Indeed, direct comparisons of data obtained from studies of normal plasma cells and PCT made it possible to distinguish a “differentiation” subset of genes from a candidate “cancer” subset.
Direct evidence for the involvement of NOTCH signaling in plasmacytomagenesis came from studies of cultured PCT cell lines treated with a GSI or expressing DNMAML1. Treatment with the GSI inhibited cell expansion by slowing G1
-S progression and inducing apoptosis. DNMAML1 was previously shown to bind only CSL in the presence of ICN and to interact comparably with ICN1, ICN2, ICN3, and ICN4 (15
). Consequently, DNMAML1 can be used to screen for the activities of all NOTCH receptors. We showed first that expression of a series of known NOTCH target genes was significantly reduced in cells expressing DNMAML1 and that the extent of reduction was inversely correlated with the levels of DNMAML1 transcripts. We also showed that the growth of cultured PCT cell lines expressing DNMAML1 was greatly reduced and that this was due to NOTCH promotion of cell cycle progression and survival.
Based on combined data from qPCR as well as oligomicroarrays, it seems that the NOTCH signaling pathway is activated in PCT of differing origins: pristane-treated or Abelson MuLV-infected BALB/c mice or BALB/c-IL-6 transgenic mice. PCT from almost all these mice carry balanced chromosomal translocations that activate Myc
is a transcription factor widely recognized as a master regulator of cell growth, proliferation, differentiation, metabolism, and death, which acts by occupying over 4,000 genomic loci (44
). Recent genome-wide studies designed to identify target genes regulated by NOTCH1 in T-ALL cells showed that NOTCH1 is not only a direct activator of biosynthetic pathways controlling cell growth and metabolism but also a direct regulator of MYC (25
). By reverse engineering of regulatory networks from expression profiles, it was found that NOTCH1 and MYC orchestrate two overlapping transcriptional programs with many common targets that synergize to regulate the growth of primary T-ALL cells (; ref. 25
). We suggest that a similar synergy between MYC and NOTCH is also characteristic of mouse PCT, but with Myc being activated by translocations in addition to its role as a downstream target of NOTCH (). It is noteworthy that the levels of Myc
transcripts were significantly reduced in cells expressing DNMAML1. This suggests that NOTCH continues to influence expression from the translocated Myc
gene as the other Myc
allele is silenced in tumors carrying an Ig/Myc translocation (47
). The mechanisms responsible for NOTCH activation in primary PCT, like those of BALB/c-IL-6 transgenic mice described here, await clarification. Cultured cell lines are unlikely to provide clear insights into these issues as they have had to adapt to growth in vitro
in the absence of stromal elements and soluble mediators known to influence the growth and survival of human MM and mouse PCT.
Figure 6 Integration of NOTCH and MYC signaling pathways in T-ALL of humans and mice, mouse PCT, and human MM. The results of this study and other analyses have shown that signaling pathways activated by NOTCH and MYC are common to all three types of tumors but (more ...)
Recent studies have drawn renewed attention to several similarities between mouse PCT and human MM (5
). These parallels have been extended by the demonstrations that NOTCH receptors and ligands can be identified in almost all MM cell lines and cells from primary cases (39
) and that MYC is activated by translocation, amplification, or other mechanisms in nearly all MM cell lines and in cells from more than half of primary cases. Although Myc
-activating translocations are considered to be early and perhaps initiating events in PCT, they are thought to occur during the later stages of MM progression (50
). Nonetheless, the understanding that the NOTCH and MYC signaling pathways are active in both diseases suggests that PCT might be valuable for modeling late-stage MM as it occurs in a substantial subset of patients and examining therapeutics that target both pathways.