In Vitro Antimyeloma Effects of Inhibitors of the Heat Shock Protein 70 (Hsp70) Molecular Chaperone.
Authors: Braunstein M.J. , Scott C.M. , Berman S. , Walter P. , Wipf P. , Brodsky J.L. , Batuman O. 
Published: October 2019

Abstract
Background: Multiple myeloma (MM) remains incurable, primarily due to drug resistance; recent research indicates that Hsp70 induction may accompany this process. Cytoplasmic Hsp70 is upregulated in MM, particularly after proteasome or Hsp90 inhibition. Hsp70 homolog in the ER helps fold immunoglobulins whose overexpression and misassembly trigger the unfolded protein response (UPR). We thus explored the antimyeloma effects of MAL3-101, a recently developed Hsp70 inhibitor on MM cell lines, primary MM cells, and endothelial progenitor cells (EPCs), a key MM microenvironment component; we also determined its synergy with proteasome and Hsp90 inhibition.
Methods: MM cells (NCI-H929, RPMI-8266, and U266) were treated with MAL3–101, proteasome inhibitor MG-132, or Hsp90 inhibitor 17-AAG, alone and in combination. MTS assay and Annexin V-PI were used to detect survival and apoptosis, respectively. Primary MM cells and EPCs were outgrown from bone marrows of untreated patients. Synergistic effects were determined by Chou-Talalay combination index (CI).
Results: Dose-effect and time course studies showed that MAL3–101 was most effective in NCI-H929 cells and induced maximum cytotoxicity at 40h (IC50 of 8.3μM). In contrast, MAL3–51, a less potent Hsp70 modulator, was less effective, suggesting that MAL3–101’s effects are due to Hsp70 inhibition. Next, MAL3–101 or MG-132 were added alone or combined at 0.01–20μM. MG-132 decreased NCI-H929 cell survival (IC50 of 1.7μM); at 0.01–0.10 μM, both drugs were ineffective alone, whereas combined, a >60% decrease in survival resulted with CIs ≤0.01. Synergy was also observed in primary MM cells, reducing survival by 75±10%, and in EPCs by 60±16%. Induction of Hsp70 may provide a means of resisting apoptosis induced by Hsp90 inhibition. We thus exposed NCI-H929 cells to MAL3–101 with 17-AAG (0.025–10μM) for 40h. IC50 for 17-AAG decreased from 0.4μM to 0.3 and 0.03μM in the presence of either 5 or 10μM of MAL3–101, respectively, with CIs <1. We next examined the effect of MAL3–101 on UPR induction as indicated by XBP-1 splicing. None of the drugs, alone or in combination, resulted in XBP-1 splicing, while, as expected, tunicamycin induced XBP-1 splicing, in these cells. Levels of BiP, an ER lumenal chaperone whose synthesis is enhanced by UPR activation, also did not change. These data suggest that MAL3–101-mediated apoptosis may not occur via UPR activation. Whether ERAD might still be functional even though a significant proportion of the proteasome and/or Hsp70 is inhibited or whether certain MM cells might lack the ability to sufficiently induce the UPR are being studied.
Conclusions: These results show for the first time that Hsp70 inhibition may provide a viable means of enhancing the antimyeloma effects of both established (i.e., proteasome inhibition) and experimental (i.e., Hsp90 inhibition) MM treatments. MAL3–101, a recently described Hsp70 modulator effective in killing breast cancer cells, sensitizes MM cells to proteasome and Hsp90 inhibition. These data also suggest that the previously reported upregulation of Hsp70 in MM cells is required to promote the growth and survival of MM tumors and its microvasculature. Finally, because Hsp upregulation may occur in response to the apoptotic effects of other MM-modulators, such as HDAC and tubulin inhibitors, we suggest that Hsp70 inhibition might provide a means to overcome drug resistance in MM.