Identification of markers that functionally define a quiescent multiple myeloma cell sub-population surviving bortezomib treatment

Abstract

BackgroundThe mechanisms allowing residual multiple myeloma (MM) cells to persist after bortezomib (Bz) treatment remain unclear. We hypothesized that studying the biology of bortezomib-surviving cells may reveal markers to identify these cells and survival signals to target and kill residual MM cells.MethodsWe used H2B-GFP label retention, biochemical tools and in vitro and in vivo experiments to characterize growth arrest and the unfolded protein responses in quiescent Bz-surviving cells. We also tested the effect of a demethylating agent, 5-Azacytidine, on Bz-induced quiescence and whether inhibiting the chaperone GRP78/BiP (henceforth GRP78) with a specific toxin induced apoptosis in Bz-surviving cells. Finally, we used MM patient samples to test whether GRP78 levels might associate with disease progression. Statistical analysis employed t-test and Mann-Whitney tests at a 95% confidence.ResultsWe report that Bz-surviving MM cells in vitro and in vivo enter quiescence characterized by p21CIP1 upregulation. Bz-surviving MM cells also downregulated CDK6, Ki67 and P-Rb. H2B-GFP label retention showed that Bz-surviving MM cells are either slow-cycling or deeply quiescent. The Bz-induced quiescence was stabilized by low dose (500nM) of 5-azacytidine (Aza) pre-treatment, which also potentiated the initial Bz-induced apoptosis. We also found that expression of GRP78, an unfolded protein response (UPR) survival factor, persisted in MM quiescent cells. Importantly, GRP78 downregulation using a specific SubAB bacterial toxin killed Bz-surviving MM cells. Finally, quantification of Grp78high/CD138+ MM cells from patients suggested that high levels correlated with progressive disease.ConclusionsWe conclude that Bz-surviving MM cells display a GRP78HIGH/p21HIGH/CDK6LOW/P-RbLOW profile, and these markers may identify quiescent MM cells capable of fueling recurrences. We further conclude that Aza + Bz treatment of MM may represent a novel strategy to delay recurrences by enhancing Bz-induced apoptosis and quiescence stability.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1460-1) contains supplementary material, which is available to authorized users.

Resistance to proteasome inhibitors and other targeted therapies in myeloma

“…Upon accumulation of misfolded proteins in the ER, Grp78 detaches from ATF6, PERK and IRE1, enabling activation of the homeostatic UPR and chaperones the misfolded proteins for degradation by the 20S proteasome (Vincenz et al , ). In MM, Grp78 was reported to play a role in resistance to proteasome inhibitors and MM cells surviving proteasome inhibitor treatment showed increased Grp78 expression, which further increased with progressive disease (Adomako et al , ). However, this was not corroborated by others who could not demonstrate any significant differences in Grp78 expression in BM plasma cells obtained from patients with monoclonal gammopathy of undetermined significance, newly‐diagnosed MM or relapsed/refractory MM (Steiner et al , ).…”

Research progress on therapeutic targeting of quiescent cancer cells

“…In particular, these effects were further strengthened by the supplementation of conventional chemotherapy with bortezomib [61]. Administration of 5-azacytidine combined with bortezomib in multiple myeloma (MM) may present a novel therapeutic strategy to delay recurrence by potentiating bortezomib-mediated apoptosis and quiescence stability [62]. Therefore, integration of proteasome inhibitors into conventional therapeutic strategies may present a promising approach for the treatment of multiple cancer types.…”

“…Hence, increasing cellular superoxide concentrations with 2-methoxyestradiol or inhibition of autophagy/lysosomal pathways with bafilomycin A1 could sensitize quiescent fibroblasts to MG132-mediated apoptosis [5]. Furthermore, MM cells that survive bortezomib treatment exhibit a GRP78 high /p21 high /CDK6 low /pRb low profile, which may distinguish quiescent MM cells capable of spawning tumor recurrence [62]. Upregulation of the pro-survival chaperone, BiP/Grp78, an unfolded protein response (UPR) survival factor, facilitates prolonged survival of quiescent cancer cells, which may serve as a reliable target to eradicate residual tumor cells [62,64].…”

“…Furthermore, MM cells that survive bortezomib treatment exhibit a GRP78 high /p21 high /CDK6 low /pRb low profile, which may distinguish quiescent MM cells capable of spawning tumor recurrence [62]. Upregulation of the pro-survival chaperone, BiP/Grp78, an unfolded protein response (UPR) survival factor, facilitates prolonged survival of quiescent cancer cells, which may serve as a reliable target to eradicate residual tumor cells [62,64]. Subtilase cytotoxin, a bacterial AB5 toxin, specifically downregulates GRP78 and induces death of bortezomib-surviving MM cells [62].…”

“…Upregulation of the pro-survival chaperone, BiP/Grp78, an unfolded protein response (UPR) survival factor, facilitates prolonged survival of quiescent cancer cells, which may serve as a reliable target to eradicate residual tumor cells [62,64]. Subtilase cytotoxin, a bacterial AB5 toxin, specifically downregulates GRP78 and induces death of bortezomib-surviving MM cells [62]. Moreover, the Hsp90 inhibitor, IPI-504, increases bortezomib sensitivity in mantle cell lymphoma cultures and tumors via BiP/Grp78 downregulation and UPR inhibition [64].…”