Modeling Myeloma Treatment through Epigenetic Modification: Synergistic Combination of EZH2 and Histone Deacetylase Inhibitors

Abstract

Multiple Myeloma (MM) management has been dramatically improved over the last decade through the clinical application of new agents including proteasome inhibitors (PIs). Despite the initial efficacy of new agents, MM remains mostly incurable. Heterogeneity in patient drug response, tumor sub-clonal heterogeneity and the eventual emergence drug-resistant relapse in most patients all greatly support the need for novel therapeutic strategies that overcome drug resistance and improve the efficacy of existing treatments. The epigenetic regulator enhancer of zeste homolog 2 (EZH2) methylates histone H3 at lysine 27 (H3K27) to silence gene transcription and regulate multiple cell functions including differentiation and stem cell maintenance. Aberrant expression and activity of EZH2 has been observed in many cancers as a facilitator of oncogenisis, proliferation, migration and invasion. Accumulating evidence suggests that EZH2 is also aberrantly expressed in MM. We have previously demonstrated that EZH2 acts as an oncogene in human myeloma cell lines (HCMLs) and that siRNA depletion of EZH2 is sufficient to arrest HCML proliferation (Croonquist & Van Ness, Oncogene 24.41, 2005). Recently, specific EZH2 chemical inhibitors (EZH2i’s) have been developed and shown efficacy in lymphoma; however, no published studies have evaluated the efficacy of these inhibitors in MM.We tested the single agent cytotoxicity of the specific EZH2i’s GSK-126 and EPZ-6438 in several HMCLs using high-throughput Cell-Titer Glow (Promega) viability assays. We found that both GSK-126 and EPZ-6438 demethylate H3K27 in HCMLs at concentrations and rates previously described in other cancer types (demonstrated through western blot), however these concentrations and treatment schedules failed to induced cytotoxicity in HMCLs alone. While neither drug demonstrated consistent double-agent synergy with PIs (Bortezomib, Carfilzomib, Ixazomib and Oprozomib), both GSK-126 and EPZ-6438 demonstrated significant synergistic cytotoxicity with the histone deacetylase inhibitor (HDACi) panobinostat in all HCMLs tested (n=7). This double agent synergy was effective when HCMLs were pre-treated with EZH2i’s for three days prior to panobinostat treatment and in some cases induced up to an 85% increase in cytotoxicity at concentrations that do not induce a significant reduction in cell viability as single agents. We conclude that GSK-126 and EPZ-6438 pre-treatment is a potent enhancer of panobinostat cytotoxicity in HMCLs. HDACi’s have been shown to act synergistically with current MM chemotherapeutics in pre-clinical models. These data may demonstrate an additional agent to increase HDAC efficacy in MM treatment. Specific gene targets of EZH2 inhibition are being investigated by kinetic gene expression profiles in HMCLs after treatment and linked to mechanisms further promoted by HDAC inhibitors. These results will provide a comprehensive pre-clinical analysis of epigenetic modification as a therapeutic strategy in treating myeloma. DisclosuresNo relevant conflicts of interest to declare.