The Geldanamycin Analogue Heat Shock Protein 90 (HSP90) Inhibitor 17-AAG in Lymphomas: Molecular Mechanisms of 17-AAG-Induced Cell Death in Hodgkin Disease and Non- Hodgkin Lymphoma Cell Lines.

Abstract

Heat Shock Proteins (HSP) are stress-induced proteins that act as molecular chaperones in all benign and malignant cells. HSPs are important for maintaining the function of several client proteins involved in transcriptional regulation, signal transduction, and cell-cycle control. HSP90 has been recently identified as a potential therapeutic target in several types of tumors. However, the effect HSP90 inhibition in Hodgkin disease (HD) and non-Hodgkin lymphomas (NHL) remains unknown. The geldanamycin analogue 17-allylamino-17-demethoxy-geldanamycin (17-AAG) is a small molecule inhibitor of HSP90, which interferes with ATP-dependent chaperones activity, with subsequent degredation of client proteins by the proteasome. We studied the activity of 17-AAG in a panel of cell lines of HD (HD-MyZ, HD-LM2, L-428, KM-H2) and NHL (SUDHL1, and Jurkat) origin. HSP90 was expressed in all HD cell lines but not in normal peripheral blood lymphocytes of healthy donors. 17-AAG demonstrated a significant antiproliferative effect in a dose (01 micromolar - 5 micromolar) and time (24 hours to 72 hours) dependent manner in all cells lines, but was most prominant in the HD-LM2 cells. In all HD-derived cell lines, 17-AAG had more than 50% inhibitory effect over control cells. This effect was most prominant after 72 hours of incubation. Subsequently, we examined the molecular mechanisms responsible for this antiproliferative effect. Both cell cycle arrest and induction of apoptosis were observed. In the HD-LM2 cell line, 17-AAG predominantly induced a G0/G1 cell cycle phase arrest, while in the L-428 cell line there was a G2/M phase arrest (as determined by propidium iodide staining and flow-cytometric analysis). This effect was associated with alterations in MDM2 and cyclin D1 proteins. Cell cycle arrest was followed by apoptosis as determined by Annexin-V binding assay, which was predominantly mediated by caspase 9 activation. 17-AAG also reduced the cellular content of Bcl-2, cFLIP, and AKT, but had no significant effect on XIAP or cIAP2. Furthermore, 17-AAG decreased the total ERK1/2 which has been shown to play an important role in HRS cell survival. 17-AAG-induced apoptosis was partially reversed by the pancaspase inhibitor ZVAD-FMK. Preliminary data suggested that 17-AAG did not enhance the effect of doxoorubicin or gemcitabine chemotherapy. These data suggest a first proof-of-principle that inhibition of HSP90 might be of therapeutic value in HD and NHL.