Abstract
We found that expression of the zeta-associated protein of 70 kD (ZAP-70) by chronic lymphocytic leukemia (CLL) B cells enhanced IgM-receptor signaling, even though such CLL cells also expressed a highly-related and more active tyrosine kinase, p72Syk. To investigate whether the kinase activity of ZAP-70 is necessary for this effect, we transfected ZAP-70-negative primary leukemia cells with expression vectors encoding either wild-type ZAP-70 or ZAP-70-KI, a mutant ZAP-70 that has an inactivating point mutation in the ATP-binding site (Lys369-Ala) and that lacks tyrosine kinase activity. We achieved high-level expression of ZAP-70 or ZAP-70-KI in transfected CLL cell samples and compared these cells with each other and with the same CLL cell samples that had been mock transfected with a control vector and that remained negative for expression of ZAP-70 (n = 7). To examine the B-cell-receptor (BCR) signaling potential of these cells we assessed the extent of tyrosine phosphorylation of p72Syk, B-cell linker protein (BLNK), and phospholipase C gamma (PLC-γ), and measured intracellular calcium flux ([Ca2+]I) before and 5–10 minutes after surface IgM ligation with F(ab)2 anti- μantibody. Treatment of control mock-transfected ZAP-70-negative CLL cells with anti-μ resulted in negligible-to-minimal increases in phosphorylation of these cytosolic proteins, with mean increases in phosphorylation of p72Syk, BLNK, and PLC-γ of only 46% ± 47% (S.D), 173% ± 112%, and 73% ± 76%, respectively. CLL cells engineered to express non-mutated ZAP-70 experienced significantly higher levels of protein tyrosine phosphorylation following treatment with anti-μ, with mean increases in phosphorylation of p72Syk, BLNK, and PLC-γ of 186% ± 102%, 490% ± 323%, and 722% ± 836%, respectively, as had been noted in earlier studies. Surprisingly, CLL cells made to express the tyrosine-kinase-defective mutant ZAP-70KI also experienced significantly higher levels of protein tyrosine phosphorylation following treatment with anti-μ than did the mock-transfected CLL cells, with mean increases in phosphorylation of p72Syk, BLNK, and PLC-γ of 172% ± 94%, 431% ± 261%, and 759% ± 637%, respectively. These values were similar to those noted for anti-μ treated CLL cells that expressed the wild-type ZAP-70 protein. These results were reflected also in the ([Ca2+]I) induced by anti-μ in each of the three groups of CLL cells. Whereas anti-μ treatment of mock-transfected CLL cells resulted in negligible-to-minimal increases in ([Ca2+]I) of 0.25 units ± 0.19, anti-μ treatment of ZAP-70-transfected or ZAP-70KI-transfected CLL cells resulted in increases in ([Ca2+]I) of 1.05 units ± 0.53 and 0.95 units ± 0.46, respectively. These values each were significantly higher than that noted for mock-transfected CLL cells. We conclude that the tyrosine-kinase activity of ZAP-70 is not required for ZAP-70 to enhance BCR signaling in CLL cells.
The Bruton Tyrosine Kinase (BTK) Inhibitor Acalabrutinib Demonstrates Potent On-Target Effects and Efficacy in Two Mouse Models of Chronic Lymphocytic Leukemia
