Comparison of the ZAP70⁺ and ZAP70⁻ B-CLL Kinome: Higher Kinase Activity in ZAP70⁺ B-CLL Cells.

B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease with a highly variable clinical course. The expression of ZAP70 can serve as a novel prognostic marker in B-CLL, but the function of ZAP70 in the disease is still unknown. A high throughput analysis of protein kinases allows a comprehensive detection of signal transduction events in CLL. The goal of this study was to detect the activity of kinases in B-CLL cells and compare the kinase activity of (IgM positive) B-CLL cells immediately after stimulation through IgM. We compared 2 cases of ZAP70⁺ and 2 cases of ZAP70⁻ untreated B-CLL patients. Tumor cells were isolated from lymph node cell suspensions, purified by negative selection (> 95% purity), and cross linked with biotin labeled anti-IgM F(ab)₂ (10 μ g/mL) followed by avidin (10 μ g/mL) for 10 minutes at 37°C. Whole cell lysates in combination with ³² P-ATP were overlaid on a kinomics chip, which consists of 1024 peptides in triplicate (including 48 peptides as internal controls) and can detect the activity of 207 different kinases.

We found significant differences of the activity of 74 kinases between ZAP70⁺ and ZAP70⁻ cases, with a higher phosphorylation of 93 peptides in the ZAP-70⁺ group. Phosphorylation of ZAP70 by kinase LCK was up regulated with a factor 2.7 in the ZAP-70⁺ B-CLL group. The kinases SYK, LCK, Fyn, HPK1, CAMK2A, PRKAR1A and PKC all showed a higher activity in the ZAP70⁺ group. These kinases are mainly involved in the B cell receptor pathway and indicate that ZAP70⁺ B-CLL cells are more activated than the negative group. The kinases CDC2, CDK2, PLK, and ABL also showed a higher activity in the ZAP70⁺ B-CLL cases, indicating a higher proliferation rate. PTK6 and LIMK1 which also had a higher activity in the ZAP70⁺ group are oncogenic kinases and play a fundamental role in transformation and migration.

Crosslinking of the BCR via IgM resulted in changes in the activity of 73 kinases, 55 showing a lower activity. Since ZAP70 expression can be induced through activation in normal B-cells, it is possible that the already highly activated status of the ZAP70⁺ cells causes a fast dampening after IgM stimulation. In the ZAP70⁻ group only a minimal change was detected upon IgM cross linking. After 10 minutes stimulation with anti-IgM, the activity of ZAP70 itself as assessed by alterations of the phosphorylation status of its substrate HIP-55 was down regulated in exclusively the ZAP70⁺ cases. HIP55 possibly connects ZAP70 with both the BCR and apoptosis pathways.

In summary, ZAP70 is not only a prognosis marker of B-CLL, but might also play a key role in survival, proliferation, activation and transformation of B-CLL cells.