1. Identification of markers that functionally define a quiescent multiple myeloma cell sub-population surviving bortezomib treatment.
- Author
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Adomako, Alfred, Calvo, Veronica, Biran, Noa, Osman, Keren, Chari, Ajai, Paton, James, Paton, Adrienne, Moore, Kateri, Schewe, Denis, and Aguirre-Ghiso, Julio
- Subjects
Adult ,Aged ,Animals ,Apoptosis ,Azacitidine ,Bortezomib ,Cell Survival ,Cyclin-Dependent Kinase 6 ,Endoplasmic Reticulum Chaperone BiP ,Female ,Gene Expression Regulation ,Neoplastic ,Heat-Shock Proteins ,Humans ,Male ,Mice ,Middle Aged ,Multiple Myeloma ,Neoplasm Recurrence ,Local ,Xenograft Model Antitumor Assays ,p21-Activated Kinases - 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 p21(CIP1) 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 Grp78(high)/CD138+ MM cells from patients suggested that high levels correlated with progressive disease. CONCLUSIONS: We conclude that Bz-surviving MM cells display a GRP78(HIGH)/p21(HIGH)/CDK6(LOW)/P-Rb(LOW) 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.
- Published
- 2015