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Oncogenic mechanisms of ZEB1 and LM02 in T-ALL
tions found in patients and the observed spontaneous T- cell lymphoma development in a mouse model expressing a carboxyterminal truncated form of ZEB1. This is in con- trast with the oncogenic role we have described for Zeb2 in T-ALL.21 In this study, we demonstrated that pre-leukemic T-cell differentiation is also affected by Zeb2 overexpres- sion. Maintenance of Zeb2 expression results in a partial differentiation block or delay at an early DN3C stage, coin- ciding with the T-cell developmental stage at which Zeb2 expression is normally downregulated in mice, as well as in humans. This delay could be partially rescued by decreas- ing IL7R pathway activation, suggesting that the delay is caused by an inability of the Zeb2-overexpressing thymo- cytes to downregulate IL7R expression. Interestingly, other mouse models have shown a similar block in T-cell differ- entiation at this key transition point, including mice that overexpress the IL7r.35,42 CD2-LMO2tg animals accumulate a similar aberrant DN3 population at a young age. Notably, our detailed flow cytometric analysis revealed that LMO2- overexpressing cells are delayed slightly earlier in T-cell dif- ferentiation compared to the Zeb2-overexpressing thymo- cytes, at the DN3A stage, and before T-cell rearrangement (data not shown).
As ZEB have been previously associated with the acqui- sition of cancer stem cell properties, we hypothesized that the cause of spontaneous T-ALL formation in R26-Zeb2tg could be due to acquired self-renewal of pre-leukemic thy- mocytes, as previously demonstrated for LMO2. This was supported by the fact that both proteins bind similar reg- ulatory elements, and that both factors can drive murine T-ALL development with a similar immature expression profile and increased stem cell properties. Nevertheless, using thymocyte transplantation experiments, we demon- strated that R26-Zeb2tg thymocytes are not able to recon- stitute an irradiated thymus and therefore have no pre-
leukemic self-renewal capacity. These data indicate differ- ent oncogenic mechanism for ZEB2 and LMO2. Upon transplantation, we observed that the survival of R26- Zeb2tg thymocytes was longer than that of wildtype thy- mocytes, with this difference most probably being associ- ated with increased IL7R signaling. Whether the increase in Il7r expression is involved in tumor initiation in R26- Zeb2tg animals remains to be determined.
Finally, we used a late-acting CD4-Cre line to show that Zeb2 could also transform thymic precursor cells at later stages during their development, suggesting that the delay at the DN3C stage is not essential for leukemic transfor- mation. Interestingly, a forward genetic screening used the same CD4-Cre line to activate the “sleeping beauty trans- posase” and induce oncogenic transformation specifically at later stages of T-cell development, which resulted in T- ALL with an immature expression profile.43 These data indicate that cases of immature murine T-ALL can also originate in cells beyond the DN stage. Furthermore, our data suggest that a Zeb2-transformed T-ALL with an immature/stem cell expression profile can originate from a more differentiated cell without self-renewal capacity.
In summary, we conclude that multiple oncogenes such as ZEB2 and LMO2 are able to induce subtypes of imma- ture murine T-ALL via distinct oncogenic mechanisms of action. In addition, Zeb2 can also drive T-cell transforma- tion in more differentiated T-cell precursor cells with sim- ilar kinetics.
Acknowledgments
This work was supported by the Australian NHMRC (grant 1047995 to JJH), the Worldwide Cancer Research Fund, the Swiss Bridge Foundation, Stand Up Against Cancer Fund, the Ghent University Special Research Fund and the Fund for Scientific Research-Flanders.
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