Treatment with the Heat Shock Protein (hsp) 90 Inhibitor17-Dimethylaminoethylamino-17-Demethoxygeldanamycin (DMAG) Abrogates the Levels and Activity of the Receptor Tyrosine Kinase TrkA in Acute Leukemia Cells.
Authors: Kumaraswamy Sandhya , Lambert Que T. , Rocha Kathy , Bali Purva , Fiskus Warren , Balasis Maria , Pranpat Michael , Boyapalle Sandhya , Hannah Alison , Reuther Gary W. , Bhalla Kapil 
Published: October 2019

Nerve growth factor (NGF) mediates the phosphorylation and signaling through the receptor tyrosine kinase TrkA, which has been shown to be expressed and active in the early hemopoietic progenitor cells, as well as in the K562 and TF1 leukemia cell lines and AML-ETO-expressing human acute leukemia cells. In AML, 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 ERK1/2 and Akt activation. We have previously reported that that the ATP bound molecular chaperone hsp90 binds the leukemia associated Bcr-Abl and FLT-3 tyrosine kinases as client proteins, maintaining them in a properly folded and active conformation, and that geldanamycin analogue hsp90 inhibitors disrupt this chaperone association, resulting in polyubiquitylation and proteasomal degradation of the client proteins. In the present studies, we investigated a) whether TrkA is a client protein of hsp90 and b) the effect of the novel and highly soluble 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 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 WT 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 WT TrkA in K562 cells, suggesting that DMAG induced the polyubiquitylation and degradation of TrkA by the 26S proteasome. In K562 cells, immunoprecipitation (IP) with monoclonal anti-TrkA antibody followed by immunoblot (IB) analyses with anti-hsp90 antibody (or IP with anti-hsp90 followed by IB with anti-TrkA antibody) showed that TrkA binds to hsp90, which is inhibited by treatment with DMAG. Following suspension of K562 cells in a serum free medium containing 100 ng/ml of NGF, the levels of pTrkA, pERK1/2 and pAkt significantly increased within 5 to 10 minutes. Co-treatment with 1.0 μM DMAG inhibited pTrkA and pERK1/2 induction, suggesting that hsp90 chaperone function may be required for TrkA activity. Exposure to DMAG also depleted the levels of the other hsp90 client proteins, including c-Raf, Akt and Bcr-Abl in K562 cells, which was associated with growth arrest and apoptosis in a dose-dependent manner. These findings demonstrate that TrkA may be an hsp90 client protein, and hsp90 inhibition by treatment with DMAG would deplete WT or mutant TrkA levels and activity in human leukemia cells. These findings suggest that hsp90 inhibitors may be effective against human acute leukemia cells that may depend on the activity of mutant or WT TrkA for growth and survival.