Page 117 - 2019_08-Haematologica-web

Oncogenic mechanisms of ZEB1 and LM02 in T-ALL
at later stages of T-cell differentiation.5,6 These widely expressed basic helix-loop-helix (bHLH) transcription fac- tors cooperate with more tissue/lineage-restricted bHLH proteins, such as LYL1 and SCL/TAL1, to form het- erodimers that recognize a single 5’-CANNTG-3’ E-box motif in their target promoters and regulatory elements. As such, E-proteins regulate essential T-cell fate-determin- ing factors including Rag1, Notch1, and the interleukin-7 receptor (Il7r). Only in the presence of the LIM-domain- only (LMO) proteins and LMO-binding protein 1 (LDB1) can bHLH heterodimers form larger multiprotein com- plexes with a second bHLH heterodimer or with other transcription factors, such as GATA proteins. LMO pro- teins do not have intrinsic DNA-binding capacity and act solely as essential scaffolding proteins for this multipro- tein complex formation to bridge bipartite DNA motifs, e.g. two E-boxes.7,8
Activation of LMO1 and LMO2 genes, which results from chromosomal aberrations such as translocations, deletions or insertions in regulatory elements and promot- ers, has been recurrently observed in patients with T- ALL.9-11 Mouse models overexpressing Lmo1 and Lmo2 have demonstrated that both are potent oncogenic drivers within the T-cell lineage.12,13 From 5 months of age, CD2- LMO2tg mice spontaneously develop T-ALL with an immature Lyl1+ expression profile.14 Unlike the thymus of normal mice, which is continously replenished by progen- itors from the bone marrow, the pre-leukemic thymus of CD2-LMO2tg transgenic mice is self-sustaining.15,16 Similar to other long-lived pre-leukemic stem cells, Lmo2-overex- pressing thymocyte precursors retain the ability to differ- entiate into the full spectrum of mature daughter cells, but in addition, their stem cell properties allow clonal expan- sion and subsequent acquisition of extra oncogenic driver mutations, eventually leading to the onset of a fully trans- formed leukemia. The pre-leukemic self-renewal capacity of CD2-LMO2tg thymocytes is restricted to the CD4-CD8- double-negative (DN) precursor T cells, more specifically the CD4-CD8-CD44-CD25+ (DN3) subpopulation, and strictly depends on the expression of the bHLH protein LYL1.17
Zinc finger E-box binding homeobox transcription fac- tors, ZEB1 and ZEB2, recognize a similar bipartite E-box motif in their target promoters and regulatory elements.18,19 and as such regulate epithelial-to-mesenchymal transition in the context of progression of solid tumors. In addition, ZEB expression has been correlated with the acquisition of cancer stem cell properties.20,21 Using loss-of-function mouse models, it was previously demonstrated that both ZEB proteins are also essential hematopoietic transcrip- tion factors that play pivotal roles at various cell fate deci- sion check points during hematopoiesis,22-24 including the T-cell lineage.24-27 In addition, we recently showed that Zeb2 overexpression can result in spontaneous develop- ment of T-ALL with an immature Lyl1+ expression profile21 and a latency similar to that in CD2-LMO2tg/+ mice,14 sug- gesting a common oncogenic mechanism of action. In these mouse T-ALL, Zeb2 overexpression drives increased expression of Il7r and aberrant activation of the IL7R- JAK/STAT signaling pathway.21 Activating IL7R mutations are also recurrently found in T-ALL patients.28 Interestingly, overexpression of gain-of-function mutant variants of IL7R in p19(Arf)-/- mouse hematopoietic pro- genitors resulted in a similar T-ALL formation with an immature Lyl1+ expression profile, and high levels of
Lmo2.29 Based on the high Lmo2 expression in the IL7R mutant tumors and phenotypic similarities with the CD2- LMOtg mouse models, the authors suggested that T-ALL initiation in both models might act via converging down- stream signaling pathways that result in aberrant pre- leukemic thymocyte self-renewal.29
In this study, we used transgenic mouse models to fur- ther analyze the effects of Zeb1 and Zeb2 overexpression on pre-leukemic T-cell differentiation. In contrast to Zeb1, Zeb2 overexpression resulted in a partial cell-autonomous differentiation delay and accumulation of a DN3 precursor T-cell population, similar to what has been described in the CD2-LMO2tg and IL7R mutant mouse models. However, Zeb2 overexpression was not associated with gain of pre-leukemic self-renewal capacity. Finally, using a late-acting Cre line, we demonstrated that the early T-cell differentiation defects are not essential for Zeb2-mediated T-ALL initiation. Collectively, our data indicate that Zeb2 and Lmo2 drive a similar immature T-ALL subtype, but via distinct oncogenic mechanisms.
Methods
Animal experimentation and handling
All experiments were performed according to the regulations and guidelines of the ethics committee for care and use of labora- tory animals of Ghent University and Monash University.
For thymocyte transplantation experiments, donor thymi were dissected under aseptic conditions. Single cells were prepared in cold phosphate-buffered saline using a 40 μM cell strainer. Cell concentrations were measured using a Burker cell counter cham- ber. Thymocytes (1x 107) were intravenously injected into 6- to 10- week old syngeneic Ly5.1 recipients that were irradiated with a sublethal (550 Rad) dose 4 h before the transplant. One day before and 14 days after the irradiation, mice were kept on neomycin prophylaxis delivered at a dose of 1.7 mg/mL in acidified (pH 2.5) drinking water.
Histology
Tissue samples were fixed in formalin, embedded in paraffin, sectioned at 6 μm and stained with hematoxylin and eosin (H&E) for histopathological examination as described in detail elsewhere.21
Flow cytometry
Both lobes of thymi were carefully dissected in 1 mL of cold phosphate-buffered saline. Single-cell preparations were made using a 40 μm cell strainer and cell numbers were quantified using a Burker cell counting chamber. Cells were stained and analyzed either by LSRII (BD Biosciences) or FACSAria II (BD Biosciences), and FACSDiva or FlowJo software (BD Biosciences). Cell debris and cell aggregates were gated out and dead cells were discarded using the fixable Viability Dye eFluro506 (eBioscience). The anti- bodies used for flow cytometry are listed in Online Supplementary Table S1. Intracellular staining was done using a BD Cytofix/Cytoperm kit (BD Bioscience) according to the manufac- turer’s guidelines.
In vitro differentiation of fetal hematopoietic progenitors towards the T-cell lineage
The differentiation experiments were performed as described previously,30 plating 1 x 104 Lin-cKit+ hematopoietic progenitor cells per 24-well plates of OP9-DL1 feeders in OP9 culture medium supplemented with 5 ng/mL Flt3 ligand (R&D Systems) and 5
haematologica | 2019; 104(8)
1609