The emerging roles of Notch signaling in leukemia and stem cells
© Liu et al.; licensee BioMed Central Ltd. 2013
Received: 30 June 2013
Accepted: 15 July 2013
Published: 18 July 2013
The Notch signaling pathway plays a critical role in maintaining the balance between cell proliferation, differentiation and apoptosis, and is a highly conserved signaling pathway that regulates normal development in a context- and dose-dependent manner. Dysregulation of Notch signaling has been suggested to be key events in a variety of hematological malignancies. Notch1 signaling appears to be the central oncogenic trigger in T cell acute lymphoblastic leukemia (T-ALL), in which the majority of human malignancies have acquired mutations that lead to constitutive activation of Notch1 signaling. However, emerging evidence unexpectedly demonstrates that Notch signaling can function as a potent tumor suppressor in other forms of leukemia. This minireview will summarize recent advances related to the roles of activated Notch signaling in human lymphocytic leukemia, myeloid leukemia, stem cells and stromal microenvironment, and we will discuss the perspectives of Notch signaling as a potential therapeutic target as well.
The Notch signaling pathway is highly conserved from Drosophila to human and plays an important role in the regulation of cell proliferation, differentiation and apoptosis . Moreover, it has been suggested that Notch signaling may be responsible for the development and progression of human malignancies, including leukemia.
Notch signaling pathway
Notch and the ligands
Four members of Notch proteins have been identified to date in mammals, including Notch1-4 [2–5]. The Notch proteins are single-pass transmembrane receptors, which are composed of extracellular, transmembrane and intracellular domains. The extracellular domain of all Notch proteins contain epidermal growth-factor-like repeats (EGFLR) and three LIN Notch (LNR) repeats, whereas the intracellular domain consists of the RAM23 domain (RAM) and seven Ankyrin/CDC10 repeats (ANK), necessary for protein-protein interactions. Moreover, five canonical Notch ligands have been found in mammals: Dll1 (Delta-like 1), Dll3 (Delta-like 3), Dll4 (Delta-like 4), Jagged1 and Jagged2 [2–5]. Notch ligands are transmembrane proteins of which the extracellular domain contains a characteristic number of EGF-like repeats and a cysteine rich N-terminal DSL domain, responsible for the interaction with Notch receptors.
Notch signaling activation
Target genes of the Notch signaling pathways
cyclin D1, cyclin A , p21, p27,
Cell cycle regulators
Hepatocellular cancer, renal cancer
c-myc, NF-κB2 , Akt, mTOR,
Cell proliferation and survival
Keratinocytes, liver, T-ALL,
Embryonic neural progenitor cell, human pluripotent stem cells
Osteosarcoma, endothelial and neural cells.
Invasion and metastasis
Osteosarcoma, pancreatic cancer
Notch signaling in lymphocytic leukemia
T cell lymphocytic leukemia
It has been shown that Notch signaling is abnormally regulated in many human malignancies [22, 23]. Notch1 mutations causing Notch signaling continuously activated have been found in nearly 60% of T cell acute lymphoblastic leukemia (T-ALL) patients, making Notch1 the most prominent oncogene specifically involved in the pathogenesis of T-ALL [24, 25]. The characterize mutations occur mostly in the heterodimerization (HD) domain and proline, glutamic acid, serine, threonine-rich (PEST) domain of the Notch1 receptor. HD domain mutation leads to a COOH-terminally truncated NICD, whereas PEST domain mutation results in loss of the negative regulatory domain, escaping from FBXW7-mediated degradation and prolongation of the half-life of NICD . Notch1 mutations have been shown to be an early, prenatal genetic event in T-ALL patients . In murine models of T-ALL, Notch1 activation is responsible for directly inducing leukemia and collaborating with other initiating genetic events to perpetuate leukemic growth [28, 29]. Moreover, our previous study has shown that Notch1 signaling is also required for hypoxia-induced proliferation, invasion and chemoresistance in T-ALL, suggesting that pharmacological inhibitors of Notch1 signaling may be attractive interventions for T-ALL treatment .
Additionally, other Notch signaling and target genes are also involved in the initiation and progression of T-ALL. It has been reported that Notch3 and Hes1 are highly expressed by T-ALL cells, as well as dramatically reduced or absent in remission . Downregulation of Notch3 by small hair RNA (shRNA) has been found to suppress the activity of Notch signaling, leading to growth inhibition and apoptosis induction of T-ALL cells .
B cell lymphocytic leukemia
In contrast with B-ALL, Notch signaling could maintain B cell chronic lymphoblastic leukemia (B-CLL) cell survival and apoptosis resistance, undoubtedly indicating an oncogenic role in B-CLL. Emerging evidence suggests that the Notch signaling network is frequently deregulated in human B-CLL with up-regulated expression of Notch1 and Notch2 as well as their ligands Jagged1 and Jagged2 . Moreover, Notch signaling inhibition by the gamma-secretase inhibitors (GSIs) and the specific Notch2 down-regulation using small interfering RNA (siRNA) could promote B-CLL cell apoptosis [38, 42]. It has been also reported that Notch2 is not only overexpressed in B-CLL cells but also might be related to the failure of apoptosis-oriented treatment for this disease and deregulation of Notch2 signaling is involved in the aberrant expression of CD23 in B-CLL [39–41]. Taken together, these results suggest that Notch signaling is constitutively activated in B-CLL cells, and can sustain the survival of these cells.
Notch signaling in myeloid leukemia
Notch in myeloid leukemia
B cell lymphoma 2 (BCL2) loss and enhanced p53/p21 expression
Jagged1 and Dll1 were overexpressed. GSIs could reduce self-renewal and colony formation of Kit + Lin-Sca1+ cell
Notch1–3−/− or Ncstn−/− mice developed CMML-like disease
Inhibition of proliferation
Notch signaling appears to play a tumor suppressive role in chronic myeloid leukemia (CML). It is reported that overexpression of the active form of Notch1 or Notch2 in K562 cells resulted in the inhibition of proliferation, accompanied by increased Hes1 mRNA level [54, 55]. On the other hand, attenuation of Notch signaling by overexpression of a dominant-negative RBP-J calledRBP-JR218H led to the increased proliferation of K562 cells. Moreover, activation of Notch signaling was found to inhibit the colony-forming activity of K562 cells while repression of Notch signaling played the opposite role . These results provide evidence that Notch signaling might play a role as a tumor suppressor in CML.
Notch signaling in leukemia stem cells
Leukemia stem cells (LSCs) arise either from corrupted HSCs or from more differentiated and committed progenitors that acquire self-renewal potential [56–58]. Therefore, targeting this unique property of LSCs—self-renewal capacity—is thought to be a promising way to eradicate disease if one can determine which pathways are critical for LSC, but not HSC. Notch signaling is active in HSCs in vivo and downregulated as HSCs differentiated. Inhibition of Notch signaling could lead to accelerated differentiation of HSCs in vitro and depletion of HSCs in vivo[59, 60]. Furthermore, Notch1 drives cell fate decision (the choice between TCRγ/δ orα/β and between CD4+ or CD8+) by inductive interactions from thymic stromal cells [61, 62], suggesting that Notch1 expression is finely regulated during T-cell lineage development . Notch1 is also reported to plays a role in rescuing T cells from apoptosis .
Activation of Notch signaling by stromal microenvironment
Leukemia cell survival relies on leukemic microenvironment, which is composed of bone marrow stromal cells (BMSCs), endothelial cells and other factors. Accumulating evidence emphasized the importance of Notch signaling in the cross-talk between leukemia cells and their stromal microenvironment. BMSCs were shown to induce upregulation of Notch signaling molecules, such as Notch1, Notch3 and4 or Jagged1/2 and Dll1 [40, 66]. Moreover, activation of Notch signaling by stromal microenvironment were necessary for leukemia cell survival by preventing blast cell apoptosis and favoring their reciprocal interactions and cross-talk with bone marrow microenvironment [66–68]. Our previous study reported that Notch-1 activation was induced by coculture with BMSCs and down-regulation of Notch-1 increased cocultured Jurkat cell sensitivity to chemotherapy [40, 66]. Florence et al. also found that coculture of primary human T-ALL with a mouse stromal cell line expressing the Dll1 reproducibly allowed maintenance of T-LiC and long-term growth of blast cells through rescuing from apoptosis . The molecular mechanisms of apoptosis resistance may be associated with a variety of cytokines, such as IL-7 [70, 71], lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) . Inactivation of Notch signaling resulted in the decrease of leukemia cell survival, either cultured alone or cocultured in presence of stromal cells from normal donors and leukemia patients . In addition, previous in vitro studies have demonstrated that endothelial cells enhance proliferation and survival of AML cells . Our study showed a bidirectional cross-talk between endothelial and AML cells that had a promoting effect on endothelial cell function, and elucidated a novel mechanism by which the interplay between AML and endothelial cells promotes angiogenesis through VEGF activation of the Notch/Dll4 pathway .
Inhibitors of Notch signaling and the potential clinical application
Clinical research of GSIs in the treatment of leukemia
8 mice (leukemia models) were used in each control or treatment group.
Six adult and two pediatric patients with leukemia (seven with T-ALL and one with AML) received MK-0752
Percentages of human CD45+ cells were determined
No significance in event-free survival 
Limited antitumor activity and major gastrointestinal toxicity
Controversy will remain, as we do not understand the complexity of the Notch pathway and tools to specifically modulate the Notch pathway are still limited. Further studies assessing the levels of Notch activation and inhibition in leukemia still need to be carried out. Further advancement in understanding the molecular events of Notch signaling can potentially lead to further clinical benefit.
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