GSK-3β Inhibitors In Overcoming Chemoresistance In Multiple Myeloma

Abstract 3009

Despite recent advances in targeted therapies for myeloma, resistance to therapy, either primary or after initial therapy, is a significant issue. This is especially problematic in myeloma because time to progression is only a modest 6 months and long term maintenance is limited by the toxicity profiles of available drugs. In an effort to identify a non-neurotoxic and steroid-sparing combination of drugs to overcome chemoresistance in myeloma, here we report on the synergy between inhibitors of glycogen synthase kinase-3 beta (GSK-3β) and histone deacetylase inhibitors (HDACi) in reducing the viability of myeloma cells. Our prior studies identified potentiation of apoptosis by HDACi and doxorubicin in a panel of five myeloma cell lines independent of p53 and caspase 3 activation. At clinically relevant concentrations, HDACi and doxorubicin alone had only a marginal effect on the survival of fresh myeloma cells (CD138⁺) derived from bone marrow aspirates of 18 myeloma patients. Combining HDACi (sodium butyrate and vorinostat) with doxorubicin augmented their antimyeloma activity with a significant reduction in the survival of fresh myeloma samples (p≤0.01, in one-way repeated measures of ANOVA). However, a subset of fresh myeloma samples was resistant to the treatments, and HDACi plus doxorubicin did not attain IC₅₀ in these samples (8/18 with sodium butyrate plus doxorubicin or 6/9 with vorinostat plus doxorubicin).

To identify the potential survival, antiapoptotic and drug resistance genes that may confer resistance to vorinostat and doxorubicin combinations, basal gene expression of resistant (n=4) and sensitive (n=4) cohorts were profiled. Non-parametric analysis of gene expression array results identified significantly increased expression (>7.5×, p≤0.05) of several metallothioneins in resistant cohort (MT1M - 67.7×, MT1F - 47.1×, MT1G - 39.8×, MT1H - 28.1×, MT1× -12.4× and MT2A -11.4×) as well as Cyclin D1 (13.2×), Hey1 (9.1×) and JNK3 (7.5×). The ability of metallothioneins to protect cells from metal toxicities and oxidative stress suggested that elevated levels of metallothioneins might have antagonized the cytotoxic activity of both vorinostat and doxorubicin by scavenging the reactive oxygen species (ROS) leading to resistance. Overexpression of multiple metallothioneins with redundant function suggested that inhibition of a specific metallothionein might not be a viable strategy to overcome resistance. Metallothioneins are a family of cysteine-rich proteins that are regulated at transcription by metal regulatory transcription factor 1 (MTF1). The activity of MTF1 is suppressed by HDACs whereas phosphorylated CCAAT/enhancer-binding protein alpha (CEBP-α) is indispensible for its activation. The phosphorylation of CEBP-α is regulated by GSK-3β, a multifunctional serine/threonine kinase which plays a critical role in the survival of myeloma cells. Therefore, we hypothesized that inhibition of GSK-3β would sensitize myeloma cells to therapies including HDACi by silencing the expression of metallothioneins. To test this hypothesis, NCI H929, RPMI 8226 and U266 cells were treated with sub-IC₅₀ concentrations of a cell permeable non-competitive GSK-3β inhibitor TDZD-8 (0.25, 0.5 and 1 μM), vorinostat (75, 150 and 300 nM) or with their combinations for 72 hrs. Results of the viability assays and Chou and Talalay analysis identified strong synergy (combination indices of <1) between the combinations of vorinostat and GSK-3β inhibitor. Further demonstration of the synergy between these two classes of drugs in fresh myeloma cells and in mouse xenograft models will provide a rationale for combining GSK-3β inhibitors that are currently under clinical trials for other indications with existing antimyeloma agents or with HDACi for relapsed or refractory myeloma.