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S. Goossens et al.
ZEB2 overexpression does not induce pre-leukemic thymocyte self-renewal
For the following reasons, we subsequently hypothe- sized that thymocytes would also acquire pre-leukemic self-renewal capacity upon Zeb2 overexpression, similar to what has been observed in CD2-LMO2tg mice: (i) mice with Zeb2 overexpression phenocopy the IL7R gain-of-
function mutant and CD2-LMOtg immature T-ALL mouse models; (ii) ZEB2 and LMO2 bind similar bipartite E-box- containing regulatory elements; (iii) ZEB2 expression has been associated with acquisition of cancer stem cell prop- erties in solid tumors; and (iv) Zeb2-overexpressing T-ALL tumors and their derived cell lines have increased leukemia-initiating potential. To test our hypothesis, we
AB
C
Figure 2. Zeb2-mediated T-cell differentiation delay is independent of the thymic mirco-environment. (A,B) Flow cytometric analysis of 1- and 2-week in vitro differ- entiation cultures of E13.5 fetal liver hematopoietic progenitors on OP9-DL1 feeders. Percentages of DN1 (CD4/8- and CD44+, CD25-), DN2 (CD4/8- and CD44+, CD25+), DN3 (CD4/8-, CD44-, CD25+) and post-DN3 (sCD3 or CD8+) populations show a significant delay in differentiation upon Zeb2 overexpression (A) and the pres- ence of a cell population with intermediate CD25 expression even after 4 weeks of co-culture, as exemplified by a representative dot plot (B). (C) Lowering the con- centration of recombinant interleukin 7 (IL7) in OP9-DL1 co-cultures partially rescues the delay in T-cell differentiation of R26-Zeb2tg/tg fetal liver hematopoietic pro- genitors. Dot plots of representative cultures are shown as are the percentages of T-cell populations following addition of 5 ng/mL, 1 ng/mL and 0.2 ng/mL recom- binant IL7. *P<0.05, **P<0.01, ***P<0.001 (vs. control).
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haematologica | 2019; 104(8)