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Oncogenic mechanisms of ZEB1 and LM02 in T-ALL
backcrossed our R26-Zeb2tg model for ten generations to pure C57BL/6 background (CD45.2+) mice to enable thy- mocyte transplantation experiments. We injected 1x107 thymocytes from 8-week old R26-Zeb2tg/tg mice or litter- mate controls into the tail vein of sublethally irradiated syngeneic Ly5.1 (CD45.1+) recipients and used CD2- LMO2tg thymocytes as the positive control. Via flow cyto- metric analysis of CD45.1 versus CD45.2, we analyzed how many of the donor versus recipient cells contributed to the repopulation of the thymus at 4 and 6 weeks after transplantation. Four weeks after transplantation, no CD45.2+ wildtype thymocytes could be detected, while varying low numbers of CD45.2+ Zeb2-overexpressing thymocytes were consistently seen at this time point. However, and in contrast to the CD2-LMO2tg thymocytes, none of the Zeb2-overexpressing CD45.2 thymocytes could be detected 6 weeks after transplantation (Figure 3). These data demonstrate that Zeb2-overexpressing thymo- cytes have prolonged survival capacity compared to that of wildtype thymocytes, but that they do not have long- term repopulation or self-renewal capacity.
The differentiation delay is not essential for the formation of Zeb2-mediated T-cell acute lymphoblastic leukemia
Finally, we wondered whether the observed T-cell dif- ferentiation defect in Zeb2-overexpressing mice is essen- tial for Zeb2-mediated T-ALL formation. To this end, we crossed our R26-Zeb2tg mice with the CD4-cre, a late-act- ing T-cell-specific Cre line that is only active after the above-described DN3C block/delay (Figure 4A). No thy- mocyte hypoplasia (Figure 4B), or early T-cell differentia- tion defects could be observed in 8-week old CD4-cre, R26-Zeb2tg/tg mice versus control littermates. Irrespective of the lack of this early T-cell differentiation block, these mice developed leukemia with approximately the same latency and same penetrance (Figure 4C) as observed with the early acting Tie2-cre line. Necropsy and histopatho- logical examination was done on eight Tie2-cre,R26- Zeb2tg/tg and five CD4-cre,R26-Zeb2tg/tg mice, as described in our previous study.21 All leukemic mice were diagnosed
with precursor T-cell lymphoblastic leukemia. In general, no immunophenotypic differences were observed between the leukemias developing in the early- versus late- acting Cre line. However, we noted that while all exam- ined Tie2-cre,R26-Zeb2tg/tg mice had large mediastinal mass- es, two out of the five CD4-cre,R26-Zeb2tg/tg mice had no mediastinal mass. Furthermore, in these two leukemic mice, the neoplastic cells appeared smaller. Since the phe- notype in both models is not fully penetrant and we observed large variation in leukemia onset and phenotype, only a limited number (8 vs. 5) leukemic mice could be analyzed fully and we cannot, therefore, draw strong con- clusions from our observations.
Discussion
In various solid cancer types, ZEB expression has been correlated with both poor prognosis and patients’ out- comes. Indeed, ZEB1 and ZEB2 have been demonstrated to increase cancer cell invasion and dissemination via the regulation of epithelial-to-mesenchymal transition. In addition, as for other regulators of epithelial-to-mesenchy- mal transition, their expression has been associated with the acquisition of cancer stem cell properties and therapy resistance.36
It has been debated whether these structurally very related family members compensate for each other or whether they also have unique functions. The differences in phenotypes between Zeb1 and Zeb2 knockout mice can be explained by their complementary expression patterns.37 Studies with compound Zeb1/Zeb2 double knockouts have shown that they have at least partly over- lapping, compensatory functions, but could not rule out that they also have unique functions. Overexpression of ZEB1/ZEB2 in various epithelial cancer cell lines catalyzed similar phenotypes, with overlapping downstream tar- gets.18,38,39 Mild differences between ZEB1/2-mediated epithelial-to-mesenchymal transition were often reported, but these could be attributed to differences in overexpres- sion levels and/or the cellular context. Here, we provide
Figure 3. Zeb2 overexpression does not induce pre-leukemic thymocyte self-renewal. Percentages of CD45.2+ donor-derived thymocytes that contribute to the repop- ulation of sublethally irradiated syngeneic CD45.1+ recipients 4 or 6 weeks after transplantation.
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