Bcl-2 expression is high in CLL and this appears to participate in disease progression. Zap70 expression is a poor prognostic marker for the disease whose function may be to contribute in a pathway promoting heightened bcl-2 levels. We recently identified a pathway to heightened bcl-2 transcriptional expression involving the bcl-2 promoter CRE (TGACGTCA) element and c-jun/ATF-2 downstream of JNK1. Indeed, JNK activation is a known survival pathway in B cell lymphoma (

Gururajan, et al. Blood 106: 1382, 2005
). We confirmed a strict correspondence of Zap70 expression with JNK activity in CLL cell lysates by immunoblot identification of Zap70 and of phospho-S73-c-jun; and we found bcl-2 levels to be high in cells with Zap70 and JNK activity. We uncovered an adaptor/scaffolding pathway physically linking these mediators. By immunoprecipitation from lysates in a series of Zap70+ CLL’s we found JNK1 to coimmunoprecipitate with CrkL, a JNK1 scaffolding that binds by its SH3 domain to polyproline sequences of JNK1, and with p85 and Cbl and Zap70 as well as Syk. By contrast, using GST-p85 as bait in pull-down experiments, a selective interaction was found for Zap70 with p85, particularly of the p85 C-terminal SH2 domain, in lysates with heavy Y319 phosphorylation of Zap70. In parallel pull-downs with such lysates, exclusion of Cbl N-terminal TKB domain binding from Zap70 but not Syk was observed. Zap70+ CLL cell growth stimulated by the cytokine April was inhibited by the novel JNK inhibitor, CC-401, 5 uM, without significant effect on inhibition of NFkB or AKT activations. Such inhibition of JNK1 also led to upregulation of bim mRNA and downregulation of bcl-2 mRNA, and modest-to-moderate bcl-2 protein downregulation, in cells whose nuclear proteins contained abundant phospho-ATF2/c-jun binding activity for the bcl-2 promoter CRE site indicative of a transcriptional connection. Thus, Zap70 represents a point of origin in CLL cells for parallel pathways of Zap70 and Syk signaling to JNK1, involving specific complexes of Zap70 and Syk with p85/CrkL/JNK1(Cbl), respectively. These dual routes to JNK activation promote cell survival, in part, through the ability of JNK to contribute in a bcl-2 regulatory mechanism.

Topics:
1-phosphatidylinositol 3-kinase, bcl2 gene, chronic b-cell leukemias, chronic lymphocytic leukemia, drug administration routes, zap-70 kinase, lysate, rna, messenger, b-cell lymphomas, bcl-2 protein

Disclosures: Brydon Bennett, Glenn Friedman, and Peter Worland are employees of Celgene Corporation.

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2006, The American Society of Hematology
2006
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