Targeting Hsp70 in Multiple Myeloma Induces Synergistic Cytotoxicity with Inhibitors of the Proteasome and of Hsp90
Authors: Braunstein Marc J , Scott Craig M , Behrman Shannon , Walter Peter , Wipf Peter , Smith Eric LP , Scott Sadeaqua S , Klueppelberg H. Uwe , Brodsky Jeffrey L , Batuman Olcay 
Published: April 2020

Abstract
Background: Multiple myeloma (MM) remains incurable, primarily due to the development of dose-limiting toxicity and/or resistance to previously effective drugs. A rational approach is to develop new strategies that are synergistic with existing agents. Recent research indicates that inhibition of the molecular chaperone heat shock protein (Hsp) 70 may facilitate resistance to inhibitors of cellular protein quality control such as bortezomib in MM. This is indicated by upregulation of Hsp70 expression after treatment of MM with inhibitors of the proteasome or Hsp90. Endothelial progenitor cells (EPCs) are bone marrow (BM)-derived hematopoietic precursor cells that augment tumor neovascularization and govern MM severity, suggesting that EPCs are a potential target for novel antimyeloma strategies. In this study, we examined the antimyeloma effects of MAL3-101, a member of a new class of inhibitors of Hsp 70, on MM cell lines and patient-derived MM cells and EPCs. We also determined the synergy between the antimyeloma effects of MAL3-101 and inhibition of the proteasome and Hsp90.
Methods: MM cell lines (NCI-H929, RPMI-8266, and U266), BM-derived MM cells, EPCs from untreated patients, control PBMCs, and BM cells were treated with MAL3-101, the proteasome inhibitor MG-132, the Hsp90 inhibitor 17-AAG, or DMSO, alone and in combination. Cell survival and apoptosis were assessed by the MTS assay and Annexin V-PI staining, respectively. Cell cycle progression and immunoblots were performed by standard methods. Synergistic effects were evaluated by determining the combination index (CI) using CalcuSyn software.
Results: H929 cells were most sensitive to MAL3-101, with peak cytotoxicity at 40 h (IC50 8.3 μM). In contrast, MAL3-51, a less potent Hsp70 modulator, was less effective. Furthermore, FACS analysis showed that exposure to 10 μM MAL3-101 caused a time-dependent increase in apoptosis and inhibition of cell cycle progression, indicated by a 3-fold increase in the sub-G0/G1 phase and a 2.5-fold decrease in the G2/M phase populations in H929 cells. Also, immunoblot analysis showed a time-dependent increase in the cleavage of caspase-3 and PARP by MAL3-101 in H929 cells, indicating induction of apoptosis. When H929 cells were exposed to a range of concentrations of MAL3-101 or MG-132 alone and in combination, we found that the IC50 for the agents in combination decreased to 0.008 μM. Notably, when examined alone, each compound was ineffective at the IC50 concentration of the combined compounds. The synergistic cytotoxic actions of MAL3-101 and MG-132 on H929 cells occurred over a 10-fold range of concentrations (0.01–0.1 μM) (CI &lt; 1), and also resulted in synergistic apoptosis. Similarly, we found that the combination of MAL3-101 and 17-AAG also decreased the IC50 for 17-AAG, from 0.4 μM to 0.03 μM. These results are consistent with the prediction that the antimyeloma effects of Hsp90 inhibition, which causes upregulation of Hsp70 gene expression, would be potentiated by simultaneous inhibition of Hsp70. Synergistic antimyeloma effects of MAL3-101 and MG-132 were also observed as a decrease in viability of MM cells by 33% ± 8 (mean ± SD; P=.02) and 44% ± 10 (P=.07), by MAL3-101 and MG-132, respectively, but by 75% ± 4 (P=.001) when the agents were combined. In addition, combined MAL3-101 and MG-132 reduced viability of BM EPCs, by 10% ± 14 (P=.7) and 16% ± 17 (P=.3), respectively, but in combination by 60% ± 7 (P=.001). The specificity of MAL3-101’s effect on MM cells was indicated by a lack of cytotoxicity in control cells. When intracellular and secreted IGs were quantitated, we observed that the relative amount of IG secretion was highest in H929 cells, which also demonstrated the highest sensitivity to MAL3-101-induced cytotoxicity, indicating that the sensitivity of MM cells to Hsp70 inhibition most likely arises from the added cellular stress of producing and secreting monoclonal IGs; however, there was no evidence for an upregulation of an unfolded protein response in these cells by XBP-1 mRNA splicing.
Conclusions: These results show for the first time that exposure to the Hsp70 inhibitor MAL3-101, a new Hsp70 modulator, sensitizes MM tumor and EPCs to proteasome and Hsp90 inhibition. These data support a preclinical rationale for inhibition of Hsp70 function, either alone or in combination with other agents, as a novel therapeutic strategy for MM.