Expression of ZAP-70 Does Not Accelerate Leukemia Development and Progression in the Eμ-TCL1 Transgenic Mouse Model of Chronic Lymphocytic Leukemia
Authors: Gobessi Stefania , Belfiore Francesca , Bennardo Sara , Doe Brendan , Laurenti Luca , Efremov Dimitar G 
Published: October 2019

Abstract
Abstract 925
One of the most relevant prognostic factors in chronic lymphocytic leukemia (CLL) is expression of the protein tyrosine kinase ZAP-70. Typically, patients whose leukemic cells express ZAP-70 at 30–100% of the levels in normal T cells have aggressive disease, whereas patients whose leukemic cells do not express ZAP-70 or express only low levels of this protein have indolent disease. Previously, we and others demonstrated that ZAP-70 modulates B-cell receptor signaling and thus affects the capacity of the leukemic cells to respond to antigen stimulation. However, a direct link between an altered antigen response and CLL pathogenesis has still not been established and, more importantly, the question whether ZAP-70 directly contributes to the aggressiveness of the disease or is just a marker of aggressive CLL still remains to be answered.
We have now addressed these issues by analyzing in vivo the impact of forced expression of ZAP-70 on the development and behavior of leukemias that arise in the Eμ-TCL1 transgenic (tg) mouse model of CLL. This model is characterized by the development of antigen-driven leukemias that resemble human CLL in many aspects but are always ZAP-70-negative. To force the expression of ZAP-70 in TCL1 leukemias, we generated two tg lines with targeted expression of ZAP-70 in the B cell compartment (ZAP70high and ZAP70low) and crossed them with Eμ-TCL1 tg mice. B cells in ZAP70high tg mice express similar levels of ZAP-70 as normal mouse T cells, whereas the levels of ZAP-70 in B cells of ZAP70lowtg mice are approximately 10 times lower.
Both cohorts of Eμ-TCL1/ZAP70 double tg mice developed characteristic TCL1 leukemias. Eμ-TCL1/ZAP70low tg mice developed leukemias with onset and rate of progression similar to their ZAP-70-negative littermates, indicating that low levels of ZAP-70 do not alter the development and behavior of the disease. Surprisingly, Eμ-TCL1/ZAP70high tg mice developed leukemias with an approximately 2 month delay compared to their ZAP-70-negative Eμ-TCL1 tg littermates, which was contrary to the expectation that high ZAP-70 expression will accelerate leukemia development. The delay in leukemia development was especially evident at 6 months of age, when leukemic cells could be detected in the PB of 77% (10/13) of Eμ-TCL1 tg mice and only 24% (4/17) of Eμ-TCL1/ZAP70hightg mice (P=0.011).
Since ZAP-70 expression can affect the migratory and adhesion capacity of human CLL cells in vitro, we first investigated if the delayed appearance of leukemic cells in the PB of Eμ-TCL1/ZAP70high tg mice could be due to increased retention of the leukemic cells in the lymphoid tissues. Assessment of tumor burden in the spleen, peritoneal cavity (PC), bone marrow and PB of 7 months old mice showed that the number of tumor cells in each compartment was significantly lower in Eμ-TCL1/ZAP70hightg mice than their Eμ-TCL1 littermates, suggesting that the delay in leukemia appearance is not caused by increased tissue retention but rather by reduced tumor growth.
To investigate if ZAP-70 impairs tumor growth by affecting proliferation, we performed in vivo BrdU incorporation analysis of leukemic cells from spleen and PC of Eμ-TCL1 and Eμ-TCL1/ZAP70high tg mice. Spleen and PC samples were analyzed because they are the major sites of leukemia proliferation in Eμ-TCL1 tg mice. Interestingly, while the percentage of proliferating leukemic cells in the spleens of Eμ-TCL1 and Eμ-TCL1/ZAP70high tg mice was similar (mean % of BrdU+ cells ±SD: 6.81 ±1.67 and 6.15 ±2.92, respectively; P=n.s.), the percentage of proliferating leukemic cells in the PC of Eμ-TCL1/ZAP70high tg mice was significantly lower (mean % of BrdU+cells ±SD: 1.74 ±1.05 and 0.56 ±0.39, respectively; P=0.024).
In summary, this study shows that ZAP-70 expression, per se, is unable to accelerate leukemia development and progression in an established in vivo model of CLL and suggests that ZAP-70 is not directly responsible for the greater disease severity in the poor prognosis subset of CLL. In addition, this study reveals that ZAP-70 in certain tissue environments can function as a negative regulator of leukemic cell proliferation, contrary to the widespread perception of ZAP-70 as a positive regulator of leukemic cell responses.

Disclosures:
No relevant conflicts of interest to declare.