Published in:
Open Access
01-12-2015 | Research article
Identification of markers that functionally define a quiescent multiple myeloma cell sub-population surviving bortezomib treatment
Authors:
Alfred Adomako, Veronica Calvo, Noa Biran, Keren Osman, Ajai Chari, James C Paton, Adrienne W Paton, Kateri Moore, Denis M Schewe, Julio A Aguirre-Ghiso
Published in:
BMC Cancer
|
Issue 1/2015
Login to get access
Abstract
Background
The 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.
Methods
We 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.
Results
We 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.
Conclusions
We 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.