Differential Effects of Hsp70 Inhibition within the Tumor and Microenvironment in Multiple Myeloma.

Multiple myeloma (MM) remains fatal despite prolonged survival by recent advances in treatment, including the use of proteasome inhibitors. It has been proposed that proteasome inhibitors target MM by modulating the NF-κB pathway. Alternatively, proteasome inhibitors may target MM cells because the proteasome helps alleviate the unfolded protein response (UPR) that results from the accumulation of aberrant proteins in the endoplasmic reticulum (ER), which in turn may trigger apoptosis. Indeed, the UPR is induced in neoplastic plasma cells, possibly because of increased immunoglobulin (IG) synthesis that exceeds the protein folding capacity of the ER. Consequently, the degree to which inhibition of the proteasome induces apoptosis may be related to the concentration of unfolded light chains within the ER in MM. We therefore examined whether inhibition of a protein-folding mediator, the Hsp70 molecular chaperone, results in synergistic induction of MM cell apoptosis when combined with a proteasome inhibitor. To this end, the effects of MAL3-101, a novel inhibitor of Hsp70 function, both alone and in combination with a proteasome inhibitor (MG132) on three MM cell lines as well as primary patient MM cells were examined. Dose-response and time course studies in MM cell lines U266, RPMI-8226, and NCI-H929 showed increasing apoptosis and inhibition of proliferation after 16 hours of exposure to MAL3-101 (IC50: 0.9 μM) or to MG132 (IC50: 7 μM). Strikingly, when sub-effective concentrations of MG132 and MAL3-101 were combined, a strong, synergistic apoptotic response was observed in the NCI cell line after 16 hours, and synergistic effects were observed in all cell lines after 36 hours of exposure to the two drugs. Next, we studied MM cells and endothelial progenitor cells (EPCs) derived from the bone marrow of five untreated patients. These two cell populations, which were shown to bear clonotypic similarities, also showed sensitivity to dual targeting. However, these effects occurred at drug concentrations different than those found to be most potent in the cell lines. Moreover, semi-quantitative RT-PCR studies indicated that RPMI cells but not U266 cells exhibited a strong UPR induction after 16 hours of exposure to 7 μM MG132. These data correlated with the greater degree of IG secretion observed in RPMI cells compared to U266 cells as assessed by pulse-chase analysis. Taken together, these results suggest that the apoptotic response of MM cells via targeting the UPR and ER stress pathways may be dependent on basal protein production, including IG synthesis. Studies relating the secretion “index” to UPR induction and sensitivity to Hsp70 and proteasome inhibition in primary tumor cells as well as in microvascular cells from MM patients are ongoing.