Levels and activity of nerve growth factor (NGF) receptor tyrosine kinase TrkA, a heat shock protein (hsp) 90 chaperoned client protein, are abrogated in human acute leukemia cells by hsp90 inhibitor17-dimethylaminoethylamino-17-demethoxygeldanamycin (DMAG).

5512 Nerve growth factor (NGF) mediates the phosphorylation and signaling through the receptor tyrosine kinase TrkA, which is expressed and active in the early hemopoietic progenitor cells, as well as in the K562 and TF1 human leukemia cell lines and AML-ETO-expressing primary human acute myelogenous leukemia (AML) cells. In primary AML cells, a 75-amino acid deletion mutant of TrkA (ΔTrkA) has also been demonstrated to be constitutively active as a pro-growth and pro-survival protein through mechanisms involving ERK1/2 and Akt activities. In previous reports, we have shown that the acute leukemia associated Bcr-Abl and mutant FLT-3 are chaperoned by hsp90 and the geldanamycin analogue hsp90 inhibitors induce misfolding, polyubiquitylation and proteasomal degradation of Bcr-Abl and mutant FLT-3. In the present studies, we determined whether TrkA is a hsp90 client protein. We also determined the effect of the novel hsp90 inhibitor DMAG (Kosan Biosciences Inc.) on TrkA levels and activity in mouse myeloid 32D cells with or without the ectopic expression of ΔTrkA (32D/ΔTrkA cells), as well as on the endogenous levels of wild-type (WT) TrkA in K562 and TF1 cells. Exposure to 0.25 or 1.0 μM DMAG attenuated the levels of TrkA in K562, TF1 and 32D, as well as ΔTrkA in 32D/ΔTrkA cells. Co-treatment with the proteasome inhibitor bortezomib (100 nM) restored DMAG mediated depletion of TrkA in K562 cells. In K562 cells, immunoprecipitation (IP) with monoclonal anti-TrkA antibody followed by immunoblot (IB) analyses with anti-hsp90, anti-p23 and anti-cdc37 antibodies showed that TrkA binds to hsp90 and its co-chaperones, p23 and cdc37. This association is inhibited by treatment with DMAG. Following suspension of K562 cells in a serum free medium containing 100 ng/ml of NGF, within 5 to 10 minutes the levels of pTrkA, pERK1/2 and pAkt were significantly upregulated. Co-treatment with 1.0 μM DMAG inhibited the induction of pTrkA and pERK1/2. Exposure of K562 cells to DMAG also depleted the levels of other hsp90 client proteins, including c-Raf, Akt and Bcr-Abl in K562 cells. This was associated with growth arrest and apoptosis in a dose-dependent manner. Additionally in the rat pheochromocytoma PC-12 cells, NGF mediated neurite formation was significantly inhibited by co-treatment with 17-DMAG. In two samples of primary CML cells, treatment with DMAG attenuated the levels of TrkA, which was also associated with loss of survival. These findings demonstrate that TrkA is an hsp90 client protein, and hsp90 inhibition by treatment with DMAG depletes WT or mutant TrkA levels and activity in acute leukemia cells. These findings suggest that hsp90 inhibitors may be effective treatment against human acute leukemia cells in which TrkA mediated signaling supports growth and survival.