The cytosolic HSP90 has a diverse list of client proteins that participate in pathways linked to all six hallmarks of cancer development proposed by Weinberg [
5,
20]. A number of ATP-competitive inhibitors, including the geldanamycin derived 17-AAG, 17-DMAG and IPI-504, have been developed and investigated for the treatment of both solid and hematologic malignancies. The geldanamycin derivatives have demonstrated significant dose limiting non-hematopoietic toxicities, limiting their clinical efficacy [
5]. This has led to development of purine scaffold, non-benzoquinone HSP90 inhibitors [
21]. Preclinical studies with PU-H71 in triple-negative breast cancer [
6] and diffuse large B-cell lymphoma [
7] have demonstrated considerable efficacy with limited hematopoietic or non-hematopoietic toxicity. However, the efficacy of PU-H71 has not been investigated in multiple myeloma. We exposed a panel of human multiple myeloma cell lines to escalating doses of PU-H71 and discovered it to be highly active
in vitro. The anti-tumor activity was observed in both drug-sensitive and drug-resistant cell lines. PU-H71 showed comparable anti-myeloma activity in IL-6 dependent cell line INA-6 and the other IL-6 independent cell lines. PU-H71 also demonstrated
in vitro synergy with other conventional anti-myeloma drugs. Mechanistically, we found that PU-H71 led to activation of the UPR and apoptosis via the caspase-dependent pathway.
The past decade brought forth valuable insights on the endoplasmic reticulum HSP90 family member, i.e., gp96 (HSP90B1, grp94). On the one hand, because of the nature of gp96 to chaperone antigenic peptides to antigen-cross-presentation pathway [
22], vaccinations with purified tumor-derived gp96-peptide complex have demonstrated promising pre-clinical and clinical results in a number of malignancies, including multiple myeloma [
23]. On the other hand, endogenous gp96 is the master chaperone for most integrins (including α4, αV and αL) [
14] and is one of the key mediators in the unfolded protein response. gp96 blockade, therefore, can (a) disrupt the integrin-dependent cell adhesion mediated drug resistance in multiple myeloma; (b) upset protein homeostasis and compromise the efficiency of the unfolded protein response. In this study, we established a stable gp96 knockdown human myeloma cell line to evaluate the
in vitro activity of anti-myeloma drugs in the absence of gp96. We found, as predicted, that gp96 knockdown cells were more sensitive to bortezomib which further compromises protein homeostasis. gp96 knockdown cells were also more susceptive to killing mediated by genotoxic drug melphalan and doxorubicin, and to thalidomide that has a yet not fully understood mechanism of action. The anti-myeloma activity of PU-H71 and the geldanamycin analogues (17-AAG, 17-DMAG), which are all pan-HSP90 inhibitors, was, however, significantly reduced in the absence of gp96. This data suggests that HSP90 inhibitors exert their acute cytotoxic activity via inhibiting not only cytosolic HSP90, but also gp96. The interaction between HSP90 inhibitors and gp96 might trigger apoptosis that is dependent on the unfolded protein response pathway. In the absence or low level of gp96 in the chronic setting (as in the case of shRNA knockdown), an unknown compensatory mechanism might develop to overcome the defective protein quality control associated with gp96 loss. Such a mechanism might render cells less dependent on even the cytosolic HSP90. Interestingly, dexamethasone also had reduced efficacy in the absence of gp96, suggesting that TLR-dependent signaling might participate in the glucocorticoid-mediated apoptosis. Further study is necessary to clarify this point.