Development of a Transgenic Mouse Model to Study the Role of ZAP-70 in the Development and Progression of Chronic Lymphocytic Leukemia

Abstract 2830

The protein tyrosine kinase ZAP-70 is an important prognostic factor in chronic lymphocytic leukemia (CLL). Patients that are considered ZAP-70-positive typically express 30–100% of the levels of ZAP-70 in T-cells, whereas in the remaining patients ZAP-70 is either not expressed or is expressed at lower levels. ZAP-70-positive patients have more aggressive disease and shorter survival than patients with low or absent ZAP-70. In vitro experiments with human lymphoma cell lines and primary CLL B-cells have shown that ZAP-70 is involved in B cell receptor (BCR) signaling, indicating that overexpression of ZAP-70 could affect the capacity of the leukemic cells to respond to antigen stimulation.

Despite the strong association between ZAP-70 expression and prognosis, it is still not clear whether ZAP-70 directly contributes to the aggressiveness of the disease or is just a marker of more aggressive CLL. To further address this issue, we generated transgenic (tg) mice that express different levels of ZAP-70 in B cells. In these mice expression of the murine ZAP-70 transgene is targeted to the B cell compartment by a VH or a CD19 promoter (VH-ZAP70 and CD19-ZAP70 tg mice, respectively). B cells in CD19-ZAP70 tg mice express the same levels of ZAP-70 as normal murine T cells, whereas the levels of ZAP-70 in B cells of VH-ZAP70 tg mice are approximately 10 times lower.

Immunophenotyping analysis of spleen and peritoneal cavity samples from wild type, VH-ZAP70 and CD19-ZAP70 tg mice did not reveal significant differences in the percentage of follicular (FO), marginal zone (MZ) and B1 B cells, indicating that ectopic expression of ZAP-70 does not affect normal B cell development and maturation. In terms of BCR signal transduction, no abnormalities were detected in VH-ZAP70 tg mice, suggesting that low levels of ZAP-70 do not affect BCR signaling. In contrast, B cells from CD19-ZAP70 tg mice showed altered phosphorylation of several molecules downstream of the BCR, such as Syk and BLNK, whereas phosphorylation of Cbl was not affected.

To investigate the impact of ZAP-70 expression on leukemia development and progression, we crossed VH-ZAP70 and CD19-ZAP70 tg mice with Eμ-TCL1 tg mice. The latter mice develop leukemias that are considered a mouse model of human CLL. These leukemias are CD5+, express unmutated IGHV genes and stereotyped polyreactive BCRs, but are always ZAP-70-negative. VH-ZAP70/Eμ-TCL1 tg mice (n=11) have been followed for over a year and did not show any differences with respect to their Eμ-TCL1 littermates (n=10). Both groups, starting from the age of 7–8 months, developed leukemias with a similar rate of progression and impact on survival, suggesting that low levels of ZAP-70 do not affect the behavior of the disease. The cohort of CD19-ZAP70/Eμ-TCL1 tg mice was more recently established. These animals are currently 4 months old and still do not show signs of leukemia development. Data from the extended follow-up of these mice will be presented at the meeting.