ZAP-70 Expressed in CLL Is an Hsp90 Client Protein and Its Level of Expression Can Be Downregulated by Specific Hsp90 Inhibitors.

Hsp90 is a ubiquitous molecular chaperone protein that is involved in the folding, activation and assembly of many client proteins, including mediators of signal transduction, cell-cycle control and transcriptional regulation. In order to exert its function on client proteins, Hsp90 requires the formation of an active protein complex composed of cochaperone molecules and an active ATP binding site. Proteins identified as Hsp90 client proteins include transmembrane tyrosine kinases [HER-2/neu, epidermal growth factor receptor (EGFR), MET and insulin-like growth factor-1 receptor (IGF-1R)], metastable signaling proteins (Akt, Raf-1 and IKK), mutated signaling proteins (p53, Kit, Flt3 and v-src), chimeric signaling proteins (NPM-ALK, Bcr-Abl), steroid receptors (androgen, estrogen and progesterone receptors), cell-cycle regulators (cdk4, cdk6) and apoptosis related proteins. It has been postulated that malignant progression and cancer prognosis may be associated with the presence of activated Hsp90 which exists in heightened complexes with cochaperone proteins.

The zeta-associated protein of 70 kD (ZAP-70), is a protein kinase found in a subset of patients with chronic lymphocytic leukemia (CLL) and its expression correlates with disease progression, poor clinical outcome, decreased overall survival and early requirement for treatment. ZAP-70 promotes phosphorylation of downstream signaling molecules after engagement of the B cell receptor (BCR). Since ZAP-70 is a marker of disease progression in CLL we hypothesized that its abnormal over-expression and function in CLL B cells may depend on activated Hsp90. To test this, we performed immunoprecipitation, western blot and flow cytometry analyses on CLL samples from patients that differ in their level of ZAP-70 expression (ZAP-70 positive and negative based on a cutoff expression of 20% by flow cytometry). We observed that in ZAP-70 positive CLL samples (n=4) the majority of Hsp90 present in the cytoplasm was in a complexed-activated form, whereas in ZAP-70 negative samples (n=4) most Hsp90 was found to be non-complexed. In addition, we found that ZAP-70 co-immunoprecipitated with Hsp90, suggesting that it is an Hsp90 client protein. In addition, the level of ZAP-70 expression was decreased 30–40% in cells treated for 24 hours with Hsp90 specific inhibitors at nanomolar concentrations.

Together, our data show that CLL B cells that express ZAP-70 have larger amounts of activated Hsp90 than those with negligible expression of this tyrosine kinase protein. In addition, we found that ZAP-70 is an Hsp90 client protein and its level of expression could be downregulated by using specific Hsp90 inhibitors. These findings suggest an important role for Hsp90 as a prognostic marker in CLL and provide the rationale to investigate, in clinical trials, the activity of Hsp90 inhibitors and their effect on ZAP-70 expression.