Our work suggests that an important function of Notch signaling in T-ALL development is to expand a pre-malignant pool of T cell clones, of which a small subset acquire additional mutations to become fully transformed and capable of leukemia propagation. In this model, Notch promotes T-ALL progression through clonal expansion. An increase in the number of pre-malignant cells greatly enhances both the likelihood and speed by which these cells accumulate the necessary mutations to become fully transformed LPCs. A role for Notch in the clonal expansion of pre-malignant T cells is in agreement with work from the Kelliher group, which utilized the Tal1/Lmo2
mouse model of T-ALL to observe that spontaneous Notch mutation in pre-leukemic animals corresponded to an expansion of DN3/DN4 clones, which gave rise to T-ALL over time (13
). Additional studies from Li et al. showed retroviral infection of murine lin
-hematopoietic precursor cells (HPCs) with NOTCH(ICD)
caused a depletion of B cells and expansion of a CD4+/CD8+ population of T cells that could spread outside of the thymus, but were non-tumorigenic (14
). Similarly, we have found that Notch signaling caused an expansion of pre-malignant clones that spread outside of the thymus, and clonal analysis based on TCRβ rearrangements demonstrated that only a small fraction of these subclones were leukemogenic when introduced into transplant recipient animals, indicating Notch signaling, alone, is not sufficient to fully transform leukemia propagating cells. Further, only 4 of 9 primary Notch
-induced T-ALL could engraft disease when transplanted into syngeneic recipient animals after >6 months, while the remaining five primary T-ALL did not engraft, irrespective of the number of T-ALL cells transplanted (ranging from 10 to 2.5×106
cells). These data are in agreement with work from Li et al
., who demonstrated that leukemia-propagating ability arose in Notch(ICD)
over-expressing murine lin
-HPCs only after the up-regulation of Akt
), suggesting that the collaboration of additional oncogenes are required for transformation of Notch expressing cells. Clappier et al.
recently demonstrated that xenograft of primary human T-ALL into immune-compromised mice selected for a small subset of clones found within the diagnosis leukemia (5
). In Clappier’s elegant experiments, transplantation likely selected for those T-ALL cells capable of leukemia propagation, and served to identify fully malignant LPCs that were able to drive relapse. Importantly, a large number of clones found in the human primary leukemia were not detected in either transplanted animals or patients at relapse. In combination with our results from the zebrafish, these data suggest that clonal expansion of pre-malignant clones is a common feature of human T-ALL and that a large portion of genetically distinct clones found within the primary leukemia likely lack self-renewal potential and cannot remake tumor.
While Notch signaling is likely required for continued tumor growth (2
), we have found that Notch may not directly promote leukemia-propagating ability. For example, Notch signaling does not collaborate with Myc to enhance the frequency of leukemia-propagating cells in T-ALL, and Notch-induced T-ALL do not have higher numbers of LPCs when compared with T-ALL that expresses Myc alone. These results are in keeping with data from McCormack et al., who showed that Notch
expression in normal thymocytes does not induce serial repopulating ability or confer self-renewing capability in thymocyte reconstitution assays. (15
). However, our work does not imply that Notch plays no role in LPC formation. While we have utilized the zebrafish T-ALL model to define the role of Notch in T-ALL development apart from Myc induction, an important corollary is that Myc is a critical target of Notch in mammalian T-ALL, and it is possible that Myc expression plays an important role in the ability of Notch to promote leukemia-propagation in human disease. For example, Li et al. has suggested that Ras over-expression resulting from activation of the Notch/Myc pathway axis likely contributes to LPC development in Notch(ICD)
expressing HPCs (14
). Further, in a recent study that utilized the TAL1/LMO1
mouse model of T-ALL, spontaneous Notch mutations were found in the Cd3ε+/+
fraction of T-ALL transplantable cells, but not the Cd3ε−/−
fraction, which were unable to engraft disease in recipient animals (44
). The collaboration of Notch
was therefore sufficient to promote LPC formation, which the authors speculate is likely through the ability of Notch to induce Myc and repress p53. In our work, 100% of Myc-expressing T-ALL subclones could initiate disease in recipient animals, with ~1% of cells capable of leukemia- propagation, suggesting the Myc pathway is a potent initiating event in T-ALL. However, hyperplastic Myc expressing thymocytes that are initially confined to the thymus are unable to induce disease in transplanted animals, suggesting that Myc alone is also not sufficient to fully transform thymocytes. Taken together, our data strongly suggests that additional collaborating genetic events are required along with Notch and/or Myc to elicit full transformation to thymocytes into T-ALL and subsequently the creation of LPCs.
In addition to identifying an important role of Notch in expanding T-ALL subclones, our experiments also highlight the use of zebrafish as a powerful model to uncover evolutionarily conserved pathways involved in T-ALL. For example, our microarray and cross-species comparisons identified common gene signatures in zebrafish, mouse, and human T-ALL that are independent of the initiating oncogene, suggesting common molecular pathways underlie T-ALL development. Mining this data set will likely uncover important genes regulating T-ALL initiation and progression. Moreover, our microarray cross-species comparisons revealed a novel Myc signature that is found in a variety of human and mouse malignancies and identified a unique Notch signature that acts independent of Myc in T-ALL. For example, we identified hes1
as Notch target genes, both of which have been shown to be important Notch targets in human T-ALL and act independently of Myc (45
). We also validated potential novel Notch target genes in human T-ALL cell lines including MYB, MEIS1 and BCL2, which have emerging roles in T-ALL malignancy but were previously not known to be regulated by Notch (26
). Future work will determine whether these and other Notch target genes contribute to pre-malignant thymocyte expansion, altered apoptotic responses, and/or T-ALL proliferation.
In summary, we have utilized the zebrafish mosaic transgenic T-ALL model to determine that the primary role of Notch signaling is to elicit the expansion of a pool of pre-malignant thymocytes, increasing the likelihood that a subset of these cells will accumulate the necessary mutations to become malignant LPCs. Because Notch-induced clonal expansion occurs even in the presence of Myc over-expression, a model that more accurately mimics the human condition, we expect that this same cellular mechanism likely drives early clonal expansion in human T-ALL.